Lack of evidence for metabolism of p-phenylenediamine by human hepatic cytochrome P450 enzymes

International Nuclear Information System (INIS)

Stanley, Lesley A.; Skare, Julie A.; Doyle, Edward; Powrie, Robert; D'Angelo, Diane; Elcombe, Clifford R.

2005-01-01

p-Phenylenediamine (PPD) is a widely used ingredient in permanent hair dyes; however, little has been published on its metabolism, especially with respect to hepatic cytochrome P450 (CYP)-mediated oxidation. This is regarded as a key step in the activation of carcinogenic arylamines that ultimately leads to the development of bladder cancer. Most epidemiology studies show no significant association between personal use of hair dyes and bladder cancer, but one recent study reported an increased risk of bladder cancer in women who were frequent users of permanent hair dyes. The aim of the present study was to use intact human hepatocytes, human liver microsomes, and heterologously expressed human CYPs to determine whether PPD is metabolised by hepatic CYPs to form an N-hydroxylamine. p-Phenylenediamine was N-acetylated by human hepatocytes to form N-acetylated metabolites, but there was no evidence for the formation of mono-oxygenated metabolites or for enzyme-mediated covalent binding of 14 C-PPD to microsomal protein. In contrast, 2-aminofluorene underwent CYP-mediated metabolism to ≥4 different hydroxylated metabolites. The lack of evidence for hepatic CYP-mediated metabolism of PPD is inconsistent with the hypothesis that this compound plays a causal role in the development of bladder cancer via a mode of action involving hepatic metabolism to an N-hydroxyarylamine

Microsomal UDP-glucuronyltransferase-catalyzed bilirubin diglucuronide formation in human liver

NARCIS (Netherlands)

Peters, W. H.; Jansen, P. L.

1986-01-01

Human liver microsomal bilirubin UDP-glucuronyltransferase catalyzes formation of bilirubin mono- and diglucuronide. KmUDPGA and Vmax of the enzyme are 0.6 mM and 1.69 nmol/mg protein X min. In vitro, bilirubin readily dissolves in the microsomal lipid phase. Taking this into account a Kmbilirubin

Determination of the 4-monohydroxy metabolites of perhexiline in human plasma, urine and liver microsomes by liquid chromatography.

Science.gov (United States)

Davies, Benjamin J; Herbert, Megan K; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

2006-11-07

The use of perhexiline (PHX) is limited by hepatic and neurological toxicity associated with elevated concentrations in plasma that are the result of polymorphism of the cytochrome P450 2D6 isoform (CYP2D6). PHX is cleared by hepatic oxidation that produces three 4-monohydroxy metabolites: cis-OH-PHX, trans1-OH-PHX and trans2-OH-PHX. The current study describes an HPLC-fluorescent method utilising pre-column derivatization with dansyl chloride. Following derivatization, the metabolites were resolved on a C18 column with a gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of the metabolites in human plasma and urine following clinical doses and for kinetic studies using human liver microsomes. The method demonstrates sufficient sensitivity, accuracy and precision between 5.0 and 0.01, 50.0 and 0.2 and 1.0 and 0.005 mg/l in human plasma, urine and liver microsomes, respectively, with intra-assay coefficients of variation and bias D6 extensive metaboliser (EM) patients at steady state with respect to PHX dosing determined that the mean (+/-S.D.) renal clearances of trans1-OH-PHX and cis-OH-PHX were 1.58+/-0.35 and 0.16+/-0.06l/h, respectively. The mean (+/-S.D.) dose recovered in urine as free and glucuronidated 4-monohydroxy PHX metabolites was 20.6+/-11.6%.

In vitro metabolism of [14C]-toluene by human and rat liver microsomes and liver slices

International Nuclear Information System (INIS)

Chapman, D.E.; Moore, T.J.; Michener, S.R.; Powis, G.

1990-01-01

Toluene metabolites produced by liver microsomes from six human donors included benzylalcohol (Balc), benzaldehyde (Bald) and benzoic acid (Bacid). Microsomes from only one human donor metabolized toluene to p-cresol and o-cresol. Human liver microsomes also metabolized Balc to Bald. Balc metabolism required NADPH, was inhibited by carbon monoxide, and was decreased at a buffer pH of 10. Balc metabolism was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P450 is responsible for the in vitro metabolism of Balc by human liver microsomes. Toluene metabolites formed by human liver slices and released into the incubation media included hippuric acid, and Bacid. Cresols or cresol-conjugates were not detected in liver slice incubation media from any human donor. Toluene metabolism by human liver was compared to metabolism by comparable liver preparations from male Fischer F344 rats. Rates of toluene metabolism by human liver microsomes and liver slices were 9-fold and 1.3-fold greater than for rat liver, respectively. Covalent binding of toluene to human liver microsomes and liver slices was 21-fold and 4-fold greater than for comparable rat liver preparations. Covalent binding of toluene to human microsomes required NADPH, was significantly decreased by coincubation with 4 mM cysteine or 4 mM glutathione, and radioactivity associated with microsomes was decreased by subsequent digestion of microsomes with protease. These results suggest that toluene metabolism and covalent binding of toluene are underestimated if the male Fischer 344 rat is used as a model for human toluene metabolism

Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam

Energy Technology Data Exchange (ETDEWEB)

Thomassin, J.; Tephly, T.R. (Univ. of Iowa, Iowa City (USA))

1990-09-01

Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of (3H)flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with (3H) flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000).

In vitro inactivation of hepatic microsomal phospholipase A2 by the marine natural product manoalide

International Nuclear Information System (INIS)

Master, M.M.; Jacobs, R.S.

1986-01-01

The effects of manoalide (MLD) and several analogs (isolated from the sponge Luffariella variabilis) on mouse hepatic microsomal phospholipase A 2 (PLA 2 ) activity was investigated. Microsomal PLA 2 , a membrane bound, Ca ++ dependent enzyme with an alkaline pH optimum, functions in intracellular phospholipid turnover. In vitro PLA 2 activity was assayed by preincubating MLD or analogs (2.5-100μM) with microsomes for 60 min. at 37 0 C, combining this mixture with 14 C-phosphatidylcholine and CaCl 2 , and incubating at 37 0 C for 40 minutes. Enzyme activity was quantitated by measurement of the extracted 14 C-arachidonic acid product. MLD inhibited PLA 2 in a dose-dependent manner, with an IC 50 = 94μM. Lineweaver-Burk analysis suggests that MLD inhibits PLA 2 noncompetitively. One of the analogs, producing a comparable dose-response curve to MLD, was found to be more potent (IC 50 = 33μM). Another analog facilitated PLA 2 activity (15%) at 25μM, followed by inactivation at higher doses (IC 50 > 100 μM). Facilitation of PLA 2 activity was seen with concentrations as low as 2.5μM of a third analog, and significant inactivation of PLA 2 was evident. These results indicate that MLD is not as potent against microsomal PLA 2 as has been shown with purified bee venom and cobra venom PLA 2 's

Cranberry juice suppressed the diclofenac metabolism by human liver microsomes, but not in healthy human subjects

Science.gov (United States)

Ushijima, Kentarou; Tsuruoka, Shu-ichi; Tsuda, Hidetoshi; Hasegawa, Gohki; Obi, Yuri; Kaneda, Tae; Takahashi, Masaki; Maekawa, Tomohiro; Sasaki, Tomohiro; Koshimizu, Taka-aki; Fujimura, Akio

2009-01-01

AIM To investigate a potential interaction between cranberry juice and diclofenac, a substrate of CYP2C9. METHODS The inhibitory effect of cranberry juice on diclofenac metabolism was determined using human liver microsome assay. Subsequently, we performed a clinical trial in healthy human subjects to determine whether the repeated consumption of cranberry juice changed the diclofenac pharmacokinetics. RESULTS Cranberry juice significantly suppressed diclofenac metabolism by human liver microsomes. On the other hand, repeated consumption of cranberry juice did not influence the diclofenac pharmacokinetics in human subjects. CONCLUSIONS Cranberry juice inhibited diclofenac metabolism by human liver microsomes, but not in human subjects. Based on the present and previous findings, we think that although cranberry juice inhibits CYP2C9 activity in vitro, it does not change the pharmacokinetics of medications metabolized by CYP2C9 in clinical situations. PMID:19694738

Evidence of a genetic basis for the different geographic occurrences of liver/kidney microsomal antibody type 1 in hepatitis C.

Science.gov (United States)

Muratori, Paolo; Czaja, Albert J; Muratori, Luigi; Granito, Alessandro; Guidi, Marcello; Ferri, Silvia; Volta, Umberto; Mantovani, Wilma; Pappas, Georgios; Cassani, Fabio; Lenzi, Marco; Bianchi, Francesco B

2007-01-01

Antibodies to liver/kidney microsome type 1 occur in Italian patients with hepatitis C, but rarely develop in North American patients. Our goals were to compare the frequencies of the HLA markers associated with autoimmune expression in Italian and North American patients with chronic hepatitis C and to determine genetic bases for regional differences in antibody production. HLA B8, DR3, DR4, DR7, DR11, DR13, DQ2, and the B8-DR3-DQ2 haplotype were determined by microlymphocytotoxicity and polymerase chain reaction in 105 Italian patients (50 with microsomal antibodies), 100 North American patients (none with microsomal antibodies), and Italian and North American healthy control subjects. Italian patients with microsomal antibodies differed from North American patients without these antibodies by having a higher frequency of HLA DR7 (54% vs. 27%, P=0.002). HLA DR7 occurred more frequently in seropositive Italian patients than in seronegative counterparts (54% vs. 11% P < 0.0001), Italian healthy control subjects (54% vs. 29%, P=0.0009), and North American healthy control subjects (54% vs. 19%, P < 0.0001). The frequency of HLA DR7 was similar in North American patients and controls (27% vs. 19%, P=0.2), but it was lower than in Italian controls (19% vs. 29%, P=0.059). Seropositive Italian patients had a lower frequency of HLA DR11 than seronegative Italian patients and Italian controls (18% vs. 34%, P=0.07, and 18% vs. 35%, P=0.02, respectively). In contrast to seropositive Italian patients, North American patients had HLA DR4 (30% vs. 12%, P=0.02), HLA DR13 (29% vs. 10%, P=0.01), and the B8-DR3-DQ2 haplotype (23% vs. 6%, P=0.01) more often. Similarly, HLA DR4 and the B8-DR3-DQ2 phenotype were more frequent in North American patients than in Italian controls (30% vs. 16%, P=0.005, and 23% vs. 7%, P=0.00002, respectively). HLA DR7 is associated with the development of microsomal antibodies in Italian patients with chronic hepatitis C. The lower frequency of HLA DR7

Binding of bilirubin and its structural analogues to hepatic microsomal bilirubin UDP glucuronyltransferase

International Nuclear Information System (INIS)

Vanstapel, F.; Blanckaert, N.

1987-01-01

Hepatic glucuronidation of the asymmetrical natural bilirubin molecule results in formation of two different positional isomers, bilirubin C-8 monoglucuronide and bilirubin C-12 monoglucuronide. In view of the existence of multiple isoforms of UDPglucuronyltransferase, which is the microsomal enzyme system responsible for bilirubin esterification, the authors performed kinetic analysis of microsomal glucuronidation of bilirubin and a number of its structural congeners to determine whether synthesis of the two monoglucuronide isomers involved two distinct substrate-binding sites or reflected two different modes of binding to a single catalytic site. Both isomers were found in all tested species (man, rat, guinea pig, sheep), but there were marked species differences in the C-8/C-12 ratio of monoglucuronide found in bile or formed by liver microsomes. Correspondence between in vivo and in vitro results for such regioselectivity of glucuronidation was excellent in each species. On the basis of these results of kinetic analysis of bilirubin esterification at variable pigment substrate concentrations and inhibition studies with alternative substrates, the authors postulate that both natural monoglucuronide isomers are synthesized at a single binding site. Possible mechanisms responsible for the markedly regioselective esterification of bilirubin by rat and sheep liver were investigated by study of glucuronidation of selected structural analgoues of the pigment. Collectively, their findings suggest that the molecular from(s) of bilirubin able to engage in catalytically effective binding to UDPglucuronyltransferase does (do) not correspond with intramolecularly hydrogen-bonded conformers and that the nature of the β-substituents of the outer pyrromethenone rings is a key determinant of glucuronidation rate

Frequency and significance of antibodies to liver/kidney microsome type 1 in adults with chronic active hepatitis.

Science.gov (United States)

Czaja, A J; Manns, M P; Homburger, H A

1992-10-01

To assess the frequency of antibodies to liver/kidney microsome type 1 (anti-LKM1) in patients with chronic active hepatitis, 131 such patients were tested by an indirect immunofluorescence assay. Of 62 patients with type 1 autoimmune hepatitis, none were seropositive. In contrast, 3 of 11 patients with autoimmune hepatitis and antimitochondrial antibodies (27%) were seropositive for anti-LKM1. Each had responded to corticosteroid therapy, and retesting of sera confirmed that each had been misclassified as antimitochondrial antibody positive. None of the patients with chronic active hepatitis B (14 patients) or C (24 patients) had anti-LKM1. Similarly, none of the 20 patients with cryptogenic disease had these antibodies. It is concluded that anti-LKM1 is specific for type 2 autoimmune hepatitis and is infrequent in adult patients seen at a referral center in the United States for chronic active hepatitis. Anti-LKM1 reactivity may be misinterpreted as antimitochondrial antibody reactivity by indirect immunofluorescence. Chronic hepatitis B and C virus infections are not important stimuli for the production of anti-LKM1, and testing for anti-LKM 1 is unlikely to clarify the nature of cryptogenic disease.

In vitro inactivation of hepatic microsomal phospholipase A/sub 2/ by the marine natural product manoalide

Energy Technology Data Exchange (ETDEWEB)

Master, M.M.; Jacobs, R.S.

1986-03-01

The effects of manoalide (MLD) and several analogs (isolated from the sponge Luffariella variabilis) on mouse hepatic microsomal phospholipase A/sub 2/ (PLA/sub 2/) activity was investigated. Microsomal PLA/sub 2/, a membrane bound, Ca/sup + +/ dependent enzyme with an alkaline pH optimum, functions in intracellular phospholipid turnover. In vitro PLA/sub 2/ activity was assayed by preincubating MLD or analogs (2.5-100..mu..M) with microsomes for 60 min. at 37/sup 0/C, combining this mixture with /sup 14/C-phosphatidylcholine and CaCl/sub 2/, and incubating at 37/sup 0/C for 40 minutes. Enzyme activity was quantitated by measurement of the extracted /sup 14/C-arachidonic acid product. MLD inhibited PLA/sub 2/ in a dose-dependent manner, with an IC/sub 50/ = 94..mu..M. Lineweaver-Burk analysis suggests that MLD inhibits PLA/sub 2/ noncompetitively. One of the analogs, producing a comparable dose-response curve to MLD, was found to be more potent (IC/sub 50/ = 33..mu..M). Another analog facilitated PLA/sub 2/ activity (15%) at 25..mu..M, followed by inactivation at higher doses (IC/sub 50/ > 100 ..mu..M). Facilitation of PLA/sub 2/ activity was seen with concentrations as low as 2.5..mu..M of a third analog, and significant inactivation of PLA/sub 2/ was evident. These results indicate that MLD is not as potent against microsomal PLA/sub 2/ as has been shown with purified bee venom and cobra venom PLA/sub 2/'s.

Comparative Metabolism Study of Five Protoberberine Alkaloids in Liver Microsomes from Rat, Rhesus Monkey, and Human.

Science.gov (United States)

Li, Yan; Zhou, Yanyan; Si, Nan; Han, Lingyu; Ren, Wei; Xin, Shaokun; Wang, Hongjie; Zuo, Ran; Wei, Xiaolu; Yang, Jian; Zhao, Haiyu; Bian, Baolin

2017-11-01

Protoberberine alkaloids including berberine, palmatine, jatrorrhizine, coptisine, and epiberberine are major components in many medicinal plants. They have been widely used for the treatment of cancer, inflammation, diabetes, depression, hypertension, and various infectious areas. However, the metabolism of five protoberberine alkaloids among different species has not been clarified previously. In order to elaborate on the in vitro metabolism of them, a comparative analysis of their metabolic profile in rat, rhesus monkey, and human liver microsomes was carried out using ultrahigh-performance liquid chromatography coupled with a high-resolution linear trap quadrupole-Orbitrap mass spectrometer (UHPLC-electrospray ionization-Orbitrap MS) for the first time. Each metabolite was identified and semiquantified by its accurate mass data and peak area. Fifteen metabolites were characterized based on accurate MS/MS spectra and the proposed MS/MS fragmentation pathways including demethylation, hydroxylation, and methyl reduction. Among them, the content of berberine metabolites in human liver microsomes was similar with those in rhesus monkey liver microsomes, whereas berberine in rat liver microsomes showed no demethylation metabolites and the content of metabolites showed significant differences with that in human liver microsomes. On the contrary, the metabolism of palmatine in rat liver microsomes resembled that in human liver microsomes. The content of jatrorrhizine metabolites presented obvious differences in all species. The HR-ESI-MS/MS fragmentation behavior of protoberberine alkaloids and their metabolic profile in rat, rhesus monkey, and human liver microsomes were investigated for the first time. The results demonstrated that the biotransformation characteristics of protoberberine alkaloids among different species had similarities as well differences that would be beneficial for us to better understand the pharmacological activities of protoberberine alkaloids

Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

Science.gov (United States)

Klette, K L; Anderson, C J; Poch, G K; Nimrod, A C; ElSohly, M A

2000-10-01

The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

Hepatic drug clearance following traumatic injury.

Science.gov (United States)

Slaughter, R L; Hassett, J M

1985-11-01

Trauma is a complex disease state associated with physiologic changes that have the potential to alter hepatic drug clearance mechanisms. These responses include alterations in hepatic blood flow, reduction in hepatic microsomal activity, reduction in hepatic excretion processes, and changes in protein binding. Hepatic blood flow is influenced by sympathomimetic activity. Both animal and human studies demonstrate an initial reduction and subsequent increase in hepatic blood flow, which coincides with an observed increase and subsequent return to normal in serum catecholamine concentrations. Unfortunately, there are no human studies that address the importance these findings may have to the clearance processes of high intrinsic clearance compounds. Animal studies of trauma indicate that hepatic microsomal activity is depressed during the post-traumatic period. Reduction in the hepatic clearance of antipyrine, a model low intrinsic compound, has also been demonstrated in animal models of trauma. In addition to these effects, hepatic excretion of substances such as indocyanine green and bilirubin have been demonstrated to be impaired in both traumatized animals and humans. Finally, substantial increases in the serum concentration of the binding protein alpha 1-acid glycoprotein occur in trauma patients. This has been reported to be associated with subsequent decreases in the free fraction of lidocaine and quinidine. In addition to changing serum drug concentration/response relationships, the pharmacokinetic behavior of drugs bound to alpha 1-acid glycoprotein should also change. Preliminary observations in our laboratory in a dog model of surgically-induced trauma have shown a reduction in the total clearance of lidocaine and reduction in free lidocaine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Overlapping but distinct specificities of anti-liver-kidney microsome antibodies in autoimmune hepatitis type II and hepatitis C revealed by recombinant native CYP2D6 and novel peptide epitopes

Science.gov (United States)

Klein, R; Zanger, U M; Berg, T; Hopf, U; Berg, P A

1999-01-01

Anti-liver-kidney microsome antibodies (anti-LKM) occur in autoimmune hepatitis (AIH) type II and in a subset of patients with hepatitis C. Anti-LKM1 in AIH are directed against cytochrome P4502D6 (CYP2D6), but conflicting data exist concerning the specificity of anti-LKM in hepatitis C. The aim of this study was to evaluate binding specificities of anti-LKM antibodies in both diseases using novel test antigens as well as their inhibitory capacity on CYP2D6 enzyme activity. Sera from 22 patients with AIH type II and 17 patients with hepatitis C being anti-LKM-positive in the immunofluorescence test were investigated for binding to native recombinant CYP2D6 and liver microsomes by ELISA and immunoblotting, and to synthetic peptides covering the region 254–339 (254–273, 257–269, 270–294, 291–310, 307–324, 321–339, 373–389) as well as the novel peptide 196–218 by ELISA. Furthermore, all sera were tested for inhibition of CYP2D6-dependent bufuralol 1′-hydroxylase activity. Twenty of the 22 AIH type II sera (91%) and nine of the 17 hepatitis C sera (53%) were positive for CYP2D6 by ELISA and/or immunoblotting. The previously described major peptide epitope comprising CYP2D6 amino acids 257–269 was recognized by 16 of the 22 AIH sera but by only one hepatitis C serum. A further epitope, 196–218, could be defined for the first time as another immunodominant epitope for AIH because it was recognized by 15 of the 22 AIH (68%) but only three of the 17 hepatitis C sera (18%). With the exception of the peptide 254–273, the other peptides showed no significant reactivity. Analysing the inhibitory properties of anti-LKM antibodies it emerged that 95% of AIH sera and 88% of hepatitis C sera inhibited enzyme function. These data indicate that anti-LKM antibodies in AIH and hepatitis C react with CYP2D6, as shown by their inhibitory activity, and that besides the known epitope 257–269 a further immunodominant epitope exists on CYP2D6 which is recognized

Liver/kidney microsomal antibody type 1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis

OpenAIRE

Muratori, L; Cataleta, M; Muratori, P; Lenzi, M; Bianchi, F

1998-01-01

Background—Liver/kidney microsomal antibody type 1 (LKM1) and liver cytosol antibody type 1 (LC1) are the serological markers of type 2 autoimmune hepatitis (AIH). 
Aims—Since LKM1 and LC1 react against two distinct liver specific autoantigens (cytochrome P450IID6 (CYP2D6) and a 58 kDa cytosolic polypeptide respectively), the aim was to see whether LKM1 and LC1 concentrations correlate with liver disease activity. 
Patients—Twenty one patients with type 2 AIH were studied. 
Methods—A...

Inhibitory effects of cytostatically active 6-aminobenzo[c]phenanthridines on cytochrome P450 enzymes in human hepatic microsomes.

Science.gov (United States)

Zebothsen, Inga; Kunze, Thomas; Clement, Bernd

2006-07-01

Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(+)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 microM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.

Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

Science.gov (United States)

Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

2013-09-05

The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Clearance Prediction Methodology Needs Fundamental Improvement: Trends Common to Rat and Human Hepatocytes/Microsomes and Implications for Experimental Methodology.

Science.gov (United States)

Wood, F L; Houston, J B; Hallifax, D

2017-11-01

Although prediction of clearance using hepatocytes and liver microsomes has long played a decisive role in drug discovery, it is widely acknowledged that reliably accurate prediction is not yet achievable despite the predominance of hepatically cleared drugs. Physiologically mechanistic methodology tends to underpredict clearance by several fold, and empirical correction of this bias is confounded by imprecision across drugs. Understanding the causes of prediction uncertainty has been slow, possibly reflecting poor resolution of variables associated with donor source and experimental methods, particularly for the human situation. It has been reported that among published human hepatocyte predictions there was a tendency for underprediction to increase with increasing in vivo intrinsic clearance, suggesting an inherent limitation using this particular system. This implied an artifactual rate limitation in vitro, although preparative effects on cell stability and performance were not yet resolved from assay design limitations. Here, to resolve these issues further, we present an up-to-date and comprehensive examination of predictions from published rat as well as human studies (where n = 128 and 101 hepatocytes and n = 71 and 83 microsomes, respectively) to assess system performance more independently. We report a clear trend of increasing underprediction with increasing in vivo intrinsic clearance, which is similar both between species and between in vitro systems. Hence, prior concerns arising specifically from human in vitro systems may be unfounded and the focus of investigation in the future should be to minimize the potential in vitro assay limitations common to whole cells and subcellular fractions. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes

Energy Technology Data Exchange (ETDEWEB)

Smith, Jordan N.; Mehinagic, Denis; Nag, Subhasree; Crowell, Susan R.; Corley, Richard A.

2017-03-01

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase I metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14 µM), and higher intrinsic clearance at lower substrate concentrations (<0.07 µM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.

Clinical features and effect of antiviral therapy on anti-liver/kidney microsomal antibody type 1 positive chronic hepatitis C.

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Ferri, Silvia; Muratori, Luigi; Quarneti, Chiara; Muratori, Paolo; Menichella, Rita; Pappas, Georgios; Granito, Alessandro; Ballardini, Giorgio; Bianchi, Francesco B; Lenzi, Marco

2009-06-01

Anti-liver/kidney microsomal antibody type 1 (anti-LKM1), a serological marker of type 2 autoimmune hepatitis, is also detected in a small proportion of patients with hepatitis C. This study aimed to evaluate clinical features and effect of antiviral therapy in patients with hepatitis C who are anti-LKM1 positive. Sixty consecutive anti-LKM1 positive and 120 age and sex-matched anti-LKM1 negative chronic hepatitis C patients were assessed at diagnosis and during follow-up. Of these, 26 anti-LKM1 positive and 72 anti-LKM1 negative received antiviral therapy. Anti-LKM1 was detected by indirect immunofluorescence and immunoblot. Number of HCV-infected hepatocytes and intrahepatic CD8+ lymphocytes was determined by immunohistochemistry. At diagnosis anti-LKM1 positive patients had higher IgG levels and more intrahepatic CD8+ lymphocytes (p 0.022 and 0.046, respectively). Viral genotypes distribution and response to therapy were identical. Hepatic flares during antiviral treatment only occurred in a minority of patients in concomitance with anti-LKM1 positivity. Immune system activation is more pronounced in anti-LKM1 positive patients with hepatitis C, possibly representing the expression of autoimmune mechanisms of liver damage. Antiviral treatment is as beneficial in these patients as in anti-LKM1 negative patients, and the rare necroinflammatory flares are effectively controlled by corticosteroids, allowing subsequent resumption of antiviral therapy.

Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

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Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

1989-03-15

Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

Metabolic activation of 2-methylfuran by rat microsomal systems

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Ravindranath, V.; Boyd, M.R.

1985-01-01

2-Methylfuran (2-MF), a constituent of cigarette smoke and coffee, causes necrosis of liver, lungs, and kidneys in rodents. 2-MF is metabolically activated by mixed-function oxidases to acetylacrolein, a reactive metabolite that binds covalently to microsomal protein. The hepatic microsomal metabolism of 2-MF to reactive metabolite required the presence of NADPH and oxygen and was dependent on incubation time and substrate concentration. The microsomal metabolism of 2-MF was inducible by pretreatment of rats with phenobarbital and was inhibited by piperonyl butoxide and N-octyl imidazole, which indicates that the metabolism of 2-MF may be mediated by cytochrome P-450. Acetylacrolein was a potent inhibitor of mixed-function oxidase and completely inhibited the microsomal metabolism of 2-MF, indicating that 2-MF is a suicide substrate for the enzyme. The sulfhydryl nucleophile cysteine was a better trapping agent of the reactive metabolite of 2-MF than N-acetylcysteine or glutathione. Lysine decreased the covalent binding of 2-MF metabolites, presumably by reacting with the aldehyde group of acetylacrolein. In addition, in the presence of NADPH, 2-MF was bioactivated by both pulmonary and renal cortical microsomes to reactive metabolites that were covalently bound to microsomal proteins

CHARACTERIZATION OF HUMAN LIVER MICROSOMAL UDP-GLYCOSYLTRANSFERASES USING PHOTOAFFINITY ANALOGS

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LITTLE, JM; DRAKE, RR; VONK, R; KUIPERS, F; LESTER, R; RADOMINSKA, A

The photoaffinity analogs [beta-P-32]5-azido-UDP-glucuronic acid ([P-32]5N3UDP-GlcUA) and [beta-P-32]5-azido-UDP-glucose ([P-32]5N(3)UDP-Glc) were used to characterize UDP-glycosyl-transferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to

Characterization of in vitro glucuronidation clearance of a range of drugs in human kidney microsomes: comparison with liver and intestinal glucuronidation and impact of albumin.

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Gill, Katherine L; Houston, J Brian; Galetin, Aleksandra

2012-04-01

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL(int, UGT)) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL(int, UGT) on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL(int, UGT) in different tissues. Although BSA increased CL(int, UGT) in all tissues, the extent was tissue- and drug-dependent. Scaled CL(int, UGT) in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min(-1) · g tissue(-1) in liver, kidney, and intestinal microsomes. Renal CL(int, UGT) (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL(int, UGT) for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CL(int, UGT) (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL(int, UGT) is particularly important for UGT1A9 substrates.

Enantioselective metabolism of hydroxychloroquine employing rats and mice hepatic microsomes

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Carmem Dickow Cardoso

2009-12-01

Full Text Available Hydroxychloroquine (HCQ is an important chiral drug used, mainly, in the treatment of rheumatoid arthritis, systemic lupus erythematosus and malaria, and whose pharmacokinetic and pharmacodynamic properties look to be stereoselective. Respecting the pharmacokinetic properties, some previous studies indicate that the stereoselectivity could express itself in the processes of metabolism, distribution and excretion and that the stereoselective metabolism looks to be a function of the studied species. So, the in vitro metabolism of HCQ was investigated using hepatic microsomes of rats and mice. The microsomal fraction of livers of Wistar rats and Balb-C mice was separated by ultracentrifugation and 500 μL were incubated for 180 minutes with 10 μL of racemic HCQ 1000 μg mL-1. Two stereospecific analytical methods, high performance liquid chromatography (HPLC and capillary electrophoresis (CE, were used to separate and quantify the formed metabolites. It was verified that the main formed metabolite is the (--(R-desethyl hydroxychloroquine for both animal species.A hidroxicloroquina (HCQ é um importante fármaco quiral usado, principalmente, no tratamento de artrite reumatóide, lupus eritematoso sistêmico e malária e cujas propriedades farmacocinéticas e farmacodinâmicas parecem ser estereosseletivas. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que a estereosseletividade pode se expressar nos processos de metabolismo, distribuição e excreção e que o metabolismo estereosseletivo parece ser função da espécie estudada. Sendo assim, o metabolismo in vitro da HCQ foi investigado usando microssomas de fígado de ratos e de camundongos. A fração microssômica de fígados de ratos Wistar e de camundongos Balb-C foi isolada por ultracentrifugação e 500 μL foram incubados por 180 minutos com 10 μL de HCQ racêmica 1000 μg mL-1. Dois métodos analíticos estereoespecíficos, por cromatografia líquida de

Immunological cross-reactivity to multiple autoantigens in patients with liver kidney microsomal type 1 autoimmune hepatitis.

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Choudhuri, K; Gregorio, G V; Mieli-Vergani, G; Vergani, D

1998-11-01

We describe two patients with liver kidney microsomal antibody type 1 (LKM1)-positive autoimmune hepatitis (AIH) with associated endocrinopathies. The first patient had insulin-dependent diabetes (IDDM), and the second patient had Addison's disease and hypoparathyroidism, and is also positive for islet cell antibodies, without overt diabetes. To account for the existence of multiple endocrinopathy in these patients, we investigated whether there is sequence similarity between the target of LKM1 antibodies, cytochrome P4502D6 (CYP2D6), and other human proteins, and if so, whether this structural similarity produces a detectable cross-reactive immune response. Our database search identified two proteins, carboxypeptidase H, an autoantigen in insulin-dependent diabetes, and 21-hydroxylase, the major autoantigen in Addison's disease, that share sequence similarity to the second major LKM1 epitope on CYP2D6. We tested the reactivity of sera from these patients to the homologous regions of the three autoantigens using an enzyme-linked immunosorbent assay (ELISA). The cut-off for positivity was established by testing sera from 22 healthy children. To determine the significance of reactivity to the peptide homologues of the three autoantigens, we investigated 16 additional patients with LKM1 AIH and 20 children with chronic hepatitis B virus infection as pathological controls. We found that reactivity to the second major epitope of CYP2D6 is significantly associated with reactivity to the homologous regions of carboxypeptidase H (CPH) and 21-hydroxylase (21-OHase) in patients with LKM1 AIH, and that this simultaneous recognition is cross-reactive. We suggest that a cross-reactive immune response between homologous autoantigens may contribute to the development of multiple endocrinopathies in LKM1 AIH.

The tobacco carcinogen NNK is stereoselectively reduced by human pancreatic microsomes and cytosols.

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Trushin, Neil; Leder, Gerhard; El-Bayoumy, Karam; Hoffmann, Dietrich; Beger, Hans G; Henne-Bruns, Doris; Ramadani, Marco; Prokopczyk, Bogdan

2008-07-01

Cigarette smoking increases the risk of cancer of the pancreas. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the only known environmental compound that induces pancreatic cancer in laboratory animals. Concentrations of NNK are significantly higher in the pancreatic juice of smokers than in that of nonsmokers. The chiral NNK metabolite, (R,S)-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is itself a potent pancreatic carcinogen in rats. The carcinogenicity of NNAL is related to its stereochemistry; (S)-NNAL is a more potent lung tumorigen in the A/J mouse than is (R)-NNAL. In this study, we determined the potential of the human pancreas to convert NNK into NNAL. Human pancreatic microsomes and cytosols were incubated with [5-(3)H]NNK, and the metabolic products were determined by high-performance liquid chromatography (HPLC). (S)-NNAL was the predominant isomer formed in all cytosolic incubations. In ten microsomal samples, NNAL was formed at an average rate of 3.8 +/- 1.6 pmol/mg/min; (R)-NNAL was the predominant isomer in this group. The average rate of NNAL formation in 18 other microsomal samples was significantly lower, 0.13 +/- 0.12 pmol/mg/min (p < 0.001); (S)-NNAL was the predominant isomer formed in this group. In human pancreatic tissues, there is intraindividual variability regarding the capacity for, and stereoselectivity of, carbonyl reduction of NNK.

Sera of children with hepatitis C infection and anti-liver-kidney microsome-1 antibodies recognize different CYP2D6 epitopes than adults with LKM+/HCV+ sera.

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Herzog, D; Yamamoto, A M; Jara, P; Maggiore, G; Sarles, J; Alvarez, F

1999-11-01

Liver-kidney microsome type 1 (LKM1) antibodies are specific markers of autoimmune hepatitis (AIH) type 2. Antibodies to LKM1 have been found in 2% to 3% of adults infected with hepatitis C virus (HCV) without AIH. Thirty percent of these antibodies are directed against linear sequences of CYP2D6 protein. LKM1 antibodies in HCV+/LKM1+ sera and in sera of AIH patients do not recognize the same CYP2D6 epitopes. The current study was conducted to determine whether LKM1 antibodies in HCV+/LKM1+ children's sera are the result of the same immune response as the antibodies described in AIH type 2 and in HCV+/LKM1+ adult patients. Sera from 10 HCV+/LKM1+ children were tested against human liver microsomal and cytosolic proteins by Western blot analysis and against synthetic peptides of the CYP2D6 sequence between amino acids 200 and 429 by dot blot. The same sera were tested against radiolabeled CYP2D6 by immunoprecipitation. Four of 10 sera tested by Western blot analysis showed immunoglobulin (Ig) G-type antibodies against CYP2D6, and 2 had antibodies against proteins of 58, 66, and 84 kDa. One of the sera also contained IgM-type anti-66-kDa and 84-kDa proteins. The radioligand test detected anti-CYP2D6 antibodies in 9 of 10 patients. Five of the anti-CYP2D6-positive sera recognized a peptide between amino acids 200 and 429 including amino acids 254-271. Most HCV+/LKM1+ sera from children recognize conformational epitopes of the CYP2D6 antigen, and half recognize linear epitopes. Some HCV+/LKM1+ sera demonstrated antibodies against the AIH type 2 main antigenic site of the CYP2D6. Screening of HCV RNA should be performed before starting treatment of presumed autoimmune hepatitis associated with LKM1.

In vitro hepatic microsomal metabolism of meloxicam in koalas (Phascolarctos cinereus), brushtail possums (Trichosurus vulpecula), ringtail possums (Pseudocheirus peregrinus), rats (Rattus norvegicus) and dogs (Canis lupus familiaris).

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Kimble, B; Li, K M; Valtchev, P; Higgins, D P; Krockenberger, M B; Govendir, M

2014-04-01

Quantitative and qualitative aspects of in vitro metabolism of the non-steroidal anti-inflammatory drug meloxicam, mediated via hepatic microsomes of specialized foliage (Eucalyptus) eating marsupials (koalas and ringtail possums), a generalized foliage eating marsupial (brushtail possum), rats, and dogs, are described. Using a substrate depletion method, intrinsic hepatic clearance (in vitro Clint) was determined. Significantly, rates of oxidative transformation of meloxicam, likely mediated via cytochromes P450 (CYP), were higher in marsupials compared to rats or dogs. The rank order of apparent in vitro Clint was brushtail possums (n=3) (mean: 394μL/min/mg protein), >koalas (n=6) (50), >ringtail possums (n=2) (36) (with no significant difference between koalas and ringtail possums), >pooled rats (3.2)>pooled dogs (in which the rate of depletion, as calculated by the ratio of the substrate remaining was <20% and too slow to determine). During the depletion of meloxicam, at a first-order rate constant, 5-hydroxymethyl metabolite (M1) was identified in the brushtail possums and the rat as the major metabolite. However, multiple hydroxyl metabolites were observed in the koala (M1, M2, and M3) and the ringtail possum (M1 and M3) indicating that these specialized foliage-eating marsupials have diverse oxidation capacity to metabolize meloxicam. Using a well-stirred model, the apparent in vitro Clint of meloxicam for koalas and the rat was further scaled to compare with published in vivo Cl. The closest in vivo Cl prediction from in vitro data of koalas was demonstrated with scaled hepatic Cl(total) (average fold error=1.9) excluding unbound fractions in the blood and microsome values; whereas for rats, the in-vitro scaled hepatic Cl fu(blood, mic), corrected with unbound fractions in the blood and microsome values, provided the best prediction (fold error=1.86). This study indicates that eutherians such as rats or dogs serve as inadequate models for dosage

[The effect of alpha-tocopherol and ionol on the physical structure of the membranes of rat liver microsomes under conditions of antioxidant insufficiency].

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Gubskiĭ, Iu I; Boldeskul, A E; Primak, R G; Zadorina, O V

1989-01-01

Physiochemical conformity of the alpha-tocopherol interaction with hepatic microsomal membranes has been studied by means of fluorescent probes (pyrene and 1-anilinonaphthalene-8-sulphonate). The microsomal membrane microviscosity is shown to sharply decrease under conditions of the antioxidant deficiency with vitamin E expelled into animals normalizes microviscosity, but feebly influences the microsomal surface charge. Microcalorimetry has been used to establish that penetration of tocopherol into microsomal membranes was accompanied by the exothermic effect.

Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity

International Nuclear Information System (INIS)

Chen, Jin-Yan; Chen, Wan-Nan; Jiao, Bo-Yan; Lin, Wan-Song; Wu, Yun-Li; Liu, Ling-Ling; Lin, Xu

2014-01-01

The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity

Influence of whole body irradiation on induction of the hepatic microsomal system metabolizing drugs

International Nuclear Information System (INIS)

Szyszko, A.; Bitny-Szlachto, S.

1977-01-01

Effects of whole body irradiation (600 R) on rat liver aminophenazone demethylase activities of the liver homogenate 10,000 X g supernatant and its microsomal fraction were compared. Either activities were found to be decreased by irradiation by some 35%. The phenobarbital treatment (3 x 100 mg/kg i.p.) has turned out to provide higher relative augmentation of the liver demethylase activity in irradiated than in unirradiated rats. The cytoplasmic activity was found to be augmented by phenobarbital treatment 2,21-fold in unirradiated, and 3,20-fold in irradiated rats, and the microsomal activity increased 3,28-fold and 3,77-fold, respectively. Microsomal levels of cytochrome P-450 were found to be not affected by irradiation. (author)

Hepatic microsomal metabolism of BDE-47 and BDE-99 by lesser snow geese and Japanese quail.

Science.gov (United States)

Krieger, Lisa K; Szeitz, András; Bandiera, Stelvio M

2017-09-01

In the present study, we investigated the oxidative biotransformation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) by liver microsomes from wild lesser snow geese (Chen caerulescens caerulescens) and domesticated Japanese quail (Coturnix japonica). Formation of hydroxy-metabolites was analyzed using an ultra-high performance liquid chromatography-tandem mass spectrometry-based method. Incubation of BDE-47 with avian liver microsomes produced sixteen hydroxy-metabolites, eight of which were identified using authentic standards. The major metabolites formed by liver microsomes from individual lesser snow geese were 4-hydroxy-2,2',3,4'-tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47), and 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether (4'-OH-BDE-49). By comparison, 4-OH-BDE-42 and 4'-OH-BDE-49, but not 3-OH-BDE-47, were major metabolites of Japanese quail liver microsomes. Unidentified metabolites included monohydroxy- and dihydroxy-tetrabromodiphenyl ethers. Incubation of BDE-99 with avian liver microsomes produced seventeen hydroxy-metabolites, twelve of which were identified using authentic standards. The major metabolites formed by lesser snow goose liver microsomes were 2,4,5-tribromophenol, 3-OH-BDE-47, 4'-OH-BDE-49, 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90), and 5'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (5'-OH-BDE-99). By comparison, the major metabolites produced by liver microsomes from Japanese quail included 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47) and 2-hydroxy-2',3,4,4',5-pentabromodiphenyl ether (2-OH-BDE-123), but not 3-OH-BDE-47. Unidentified metabolites consisted of monohydroxy-pentabromodiphenyl ethers, monohydroxy-tetrabromodiphenyl ethers and dihydroxy-tetrabromodiphenyl ethers. Another difference between the two species was that formation rates of BDE-47 and BDE-99 metabolites were greater with liver

The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes

International Nuclear Information System (INIS)

Parkinson, Andrew; Mudra, Daniel R.; Johnson, Cory; Dwyer, Anne; Carroll, Kathleen M.

2004-01-01

We have measured cytochrome P450 (CYP) activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). In human liver microsomes, there were no statistically significant differences (P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6β-hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with β-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in

Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity.

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Naiman, Karel; Martínková, Markéta; Schmeiser, Heinz H; Frei, Eva; Stiborová, Marie

2011-12-24

N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ∼6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

Antioxidant Capacity of Flavonoids in Hepatic Microsomes Is not Reflected by Antioxidant Effects In Vivo

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Garry Duthie

2012-01-01

Full Text Available Flavonoids are polyphenolic compounds with potential antioxidant activity via multiple reduction capacities. Oxidation of cellular lipids has been implicated in many diseases. Consequently, this study has assessed the ability of several dietary flavonoid aglycones to suppress lipid peroxidation of hepatic microsomes derived from rats deficient in the major lipid soluble antioxidant, dα-tocopherol. Antioxidant effectiveness was galangin > quercetin > kaempferol > fisetin > myricetin > morin > catechin > apigenin. However, none of the flavonoids were as effective as dα-tocopherol, particularly at the lowest concentrations used. In addition, there appears to be an important distinction between the in vitro antioxidant effectiveness of flavonoids and their ability to suppress indices of oxidation in vivo. Compared with dα-tocopherol, repletion of vitamin E deficient rats with quercetin, kaempferol, or myricetin did not significantly affect indices of lipid peroxidation and tissue damage. Direct antioxidant effect of flavonoids in vivo was not apparent probably due to low bioavailability although indirect redox effects through stimulation of the antioxidant response element cannot be excluded.

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

International Nuclear Information System (INIS)

Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

2008-01-01

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

Metabolism of UV-filter benzophenone-3 by rat and human liver microsomes and its effect on endocrine-disrupting activity

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Watanabe, Yoko, E-mail: y-watanabe@nichiyaku.ac.jp [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Kojima, Hiroyuki; Takeuchi, Shinji [Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819 (Japan); Uramaru, Naoto [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Sanoh, Seigo [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Sugihara, Kazumi [Faculty of Pharmaceutical Science, Hiroshima International University, Koshingai 5-1-1, Kure, Hiroshima 737-0112 (Japan); Kitamura, Shigeyuki [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Ohta, Shigeru [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan)

2015-01-15

Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes. - Highlights: • Metabolic modification of the endocrine-disrupting activity of BP-3 was examined. • 2,4,5-TriOH BP and 3-OH BP-3 were identified as new BP-3 metabolites. • 2,4-DiOH BP and 2,3,4-triOH BP exhibited high or similar estrogenic activities. • Estrogenic activity of BP-3 was enhanced by incubation with rat liver

Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes.

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Gates, Leah A; Lu, Ding; Peterson, Lisa A

2012-03-01

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.

Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes.

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Yamazaki, H; Mimura, M; Oda, Y; Inui, Y; Shiraga, T; Iwasaki, K; Guengerich, F P; Shimada, T

1993-07-01

We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in O-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki, H. and Shimada, T., Biochem. Pharmacol., 44, 913-920, 1992). Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains. Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S. typhimurium NM2009 than in the original strain of S. typhimurium TA1535/pSK1002. The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4. Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-amino-chrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction. Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization. Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG. alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes. Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the

Liver/kidney microsomal antibody type 1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis.

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Muratori, L; Cataleta, M; Muratori, P; Lenzi, M; Bianchi, F B

1998-05-01

Liver/kidney microsomal antibody type 1 (LKM1) and liver cytosol antibody type 1 (LC1) are the serological markers of type 2 autoimmune hepatitis (AIH). Since LKM1 and LC1 react against two distinct liver specific autoantigens (cytochrome P450IID6 (CYP2D6) and a 58 kDa cytosolic polypeptide respectively), the aim was to see whether LKM1 and LC1 concentrations correlate with liver disease activity. Twenty one patients with type 2 AIH were studied. All sera were tested by indirect immunofluorescence, counterimmunoelectrophoresis, and immunoblotting visualised by enhanced chemiluminescence. To evaluate LKM1 and LC1 levels, the 50 kDa microsomal reactivity (corresponding to CYP2D6) and the 58 kDa cytosolic reactivity were quantified by densitometric analysis. Seven patients were positive for LKM1, nine for LC1, and five for both. Serial serum samples at onset and during immunosuppressive treatment were analysed in 13 patients (four positive for LKM1, six positive for LC1 and three positive for both). During remission, LKM1 concentration remained essentially unchanged in six of seven patients, and decreased in only one. Conversely, in two of nine patients, LC1 was completely lost, and, in the remaining seven, LC1 concentration was reduced by more than 50%. After immunosuppression tapering or withdrawal, flare ups of liver necrosis ensued with increasing LC1 concentration, but not LKM1. LC1 concentration, at variance with that of LKM1, parallels liver disease activity, and its participation in the pathogenic mechanisms of liver injury can be hypothesised.

JTT-130, a microsomal triglyceride transfer protein (MTP inhibitor lowers plasma triglycerides and LDL cholesterol concentrations without increasing hepatic triglycerides in guinea pigs

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Shrestha Sudeep

2005-09-01

Full Text Available Abstract Background Microsomal transfer protein inhibitors (MTPi have the potential to be used as a drug to lower plasma lipids, mainly plasma triglycerides (TG. However, studies with animal models have indicated that MTPi treatment results in the accumulation of hepatic TG. The purpose of this study was to evaluate whether JTT-130, a unique MTPi, targeted to the intestine, would effectively reduce plasma lipids without inducing a fatty liver. Methods Male guinea pigs (n = 10 per group were used for this experiment. Initially all guinea pigs were fed a hypercholesterolemic diet containing 0.08 g/100 g dietary cholesterol for 3 wk. After this period, animals were randomly assigned to diets containing 0 (control, 0.0005 or 0.0015 g/100 g of MTPi for 4 wk. A diet containing 0.05 g/100 g of atorvastatin, an HMG-CoA reductase inhibitor was used as the positive control. At the end of the 7th week, guinea pigs were sacrificed to assess drug effects on plasma and hepatic lipids, composition of LDL and VLDL, hepatic cholesterol and lipoprotein metabolism. Results Plasma LDL cholesterol and TG were 25 and 30% lower in guinea pigs treated with MTPi compared to controls (P Conclusion These results suggest that JTT-130 could have potential clinical applications due to its plasma lipid lowering effects with no alterations in hepatic lipid concentrations.

A reliable radiochromatographic assay technique for hepatic microsomal 16α-hydroxylase activity towards oestrone 3-sulphate

International Nuclear Information System (INIS)

Tsoutsoulis, C.J.; Hobkirk, R.

1980-01-01

A reliable procedure for the assay of liver microsomal 16α-hydroxylation of oestrone 3-sulphate has been developed for the guinea pig. It is based on the rapid, quantitative separation of oestradiol and oestriol by Sephadex LH-20 columns after the chemical reduction and enzymic hydrolysis of the incubation products. Microsomal preparations and incubation conditions that optimized 16α-hydroxylation of oestrone 3-sulphate were employed. Under these circumstances, reduction of the substrate at C-17 and hydrolysis of the sulphate were minimized. Conditions were established that yielded reaction linearity with respect to time and microsomal concentration. This hydroxylation had an absolute requirement for NADPH, which could not be satisfied by NADH. Apparent Ksub(m) values for oestrone 3-sulphate and NADPH, under the conditions used, were 14μM and 0.17mM respectively. 16α-hydroxylase activity was present in the liver microsomal fraction from heavily pigmented, female English Shorthaired guinea pigs. Much lower activity was detected in mature pigmented males and albino females. No activity could be demonstrated in mature, albino males. (author)

The iron-responsive microsomal proteome of Aspergillus fumigatus.

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Moloney, Nicola M; Owens, Rebecca A; Meleady, Paula; Henry, Michael; Dolan, Stephen K; Mulvihill, Eoin; Clynes, Martin; Doyle, Sean

2016-03-16

siderophore as a 'Trojan horse' to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome. Copyright © 2016 Elsevier B.V. All rights reserved.

Covalent modification of hepatic microsomal lipids of rats by carbon tetrachloride

International Nuclear Information System (INIS)

Kaphalia, B.S.; Ansari, G.A.

1989-01-01

The present study was undertaken to isolate and identify various lipids bound to 14C label during hepatic microsomal metabolism of 14CCl4 in vitro under anaerobic conditions and in vivo in rats. The two major radioactive fractions identified by thin-layer chromatography each for neutral lipids and phospholipids from in vitro and in vivo experiments corresponded to fatty acids and triglycerides and to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. Approximately 89% of the radioactivity associated with phospholipids was found in PC and PE fractions. Hydrolysis of PC and PE with phospholipase A2 released about 50% of the total radioactivity as lipid moieties corresponding to fatty acids. The radioactive neutral lipids and the lipid moieties hydrolyzed from PC and PE were methylated with boron trifluoride in methanol. These methylated lipids were separated by reversed-phase high-performance liquid chromatography (HPLC), and the elution profiles of 14C label found for the lipids obtained from in vitro experiments were similar to those from in vivo. The major radioactive fractions eluted immediately after methyl oleate were identified as trichloromethyloctadecenoic and trichloromethyleicosatrienoic acid methyl esters by chemical ionization mass spectrometry. The mass spectral analysis of these fractions also indicated the formation of dichlorocarbene adduct of oleic acid. However, similar mass spectrometric detection of trichloromethylated lipids was not evident in neutral lipids and phospholipids isolated from in vivo studies. The 14C-labeled lipids eluted as a nonpolar fraction exhibited a high molecular weight containing more than three chlorines. Dimerization and cross-linking of trichloromethylated lipids based on HPLC and mass spectral analysis are also discussed in this paper

A murine model of type 2 autoimmune hepatitis: Xenoimmunization with human antigens.

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Lapierre, Pascal; Djilali-Saiah, Idriss; Vitozzi, Susana; Alvarez, Fernando

2004-04-01

Autoimmune hepatitis (AIH) is characterized by an immune-mediated injury of the hepatic parenchyma of unknown pathogenesis. Type 2 AIH is identified by the presence of anti-liver-kidney microsomes type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) autoantibodies. The current study shows that a murine model of AIH can be generated by DNA immunization against type 2 AIH self-antigens (P450 2D6 and formiminotransferase-cyclodeaminase). A pCMV plasmid containing the N-terminal region of mouse CTLA-4 and the antigenic region of human CYP2D6 (672-1,377 bp) and human formiminotransferase cyclodeaminase (FTCD; 1,232-1,668 bp) was used for DNA immunization of C57BL/6 female mice. Immunized mice showed elevated levels of alanine aminotransferase (ALT), with peaks at 4 and 7 months postinjection. Periportal, portal, and intralobular liver inflammatory infiltrates were observed at histology. Mainly CD4+ lymphocytes, but also CD8+ and B lymphocytes, were found in the liver. Cytotoxic-specific T cells were found in both the liver and spleen of these animals. Mice developed anti-LKM1 and anti-LC1 antibodies of immunoglobulin G2 (IgG2) subclass, against specific mouse autoantigens. The ALT levels correlated with both the presence of anti-LKM1/anti-LC1 antibodies and the presence of liver necroinflammation. In conclusion, in mice, DNA immunization against human autoantigens breaks tolerance and induces an autoimmune liver disease. Molecular mimicry between foreign and self-antigens explains the liver injury. This model of AIH resembles human type 2 AIH and will be helpful for the study of its pathogenesis.

Microsomal metabolism of trenbolone acetate metabolites ...

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Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbolone, 17-trenbolone and trendione), little is known about other metabolites formed in vivo and subsequently discharged into the environment, with some evidence suggesting these unknown metabolites comprise a majority of the TBA mass dosed to the animal. Here, we explored the metabolism of the three known TBA metabolites using rat liver microsome studies. All TBA metabolites are transformed into a complex mixture of monohydroxylated products. Based on product characterization, the majority are more polar than the parent metabolites but maintain their characteristic trienone backbone. A minor degree of interconversion between known metabolites was also observed, as were higher order hydroxylated products with a greater extent of reaction. Notably, the distribution and yield of products were generally comparable across a series of variably induced rat liver microsomes, as well as during additional studies with human and bovine liver microsomes. Bioassays conducted with mixtures of these transformation products suggest that androgen receptor (AR) binding activity is diminished as a result of the microsomal treatment, suggesting that the transformation products are generally less potent than

Coupled motions direct electrons along human microsomal P450 Chains.

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Christopher R Pudney

2011-12-01

Full Text Available Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR to all microsomal cytochrome P450s (CYPs. Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron

Establishment of a novel radioligand assay using eukaryotically expressed cytochrome P4502D6 for the measurement of liver kidney microsomal type 1 antibody in patients with autoimmune hepatitis and hepatitis C virus infection.

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Ma, Y; Gregorio, G; Gäken, J; Muratori, L; Bianchi, F B; Mieli-Vergani, G; Vergani, D

1997-06-01

Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an in vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection), 128 LKM1-negative patients and 57 normal controls were tested. Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls (pLKM1 (r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.

Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection.

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Lenzi, M; Manotti, P; Muratori, L; Cataleta, M; Ballardini, G; Cassani, F; Bianchi, F B

1995-01-01

Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7797126

Theories about evolutionary origins of human hepatitis B virus in primates and humans

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Breno Frederico de Carvalho Dominguez Souza

2014-09-01

Conclusion: Some hypotheses about the evolutionary origins of human hepatitis B virus have been debated since the ‘90s. One theory suggested a New World origin because of the phylogenetic co-segregation between some New World human hepatitis B virus genotypes F and H and woolly monkey human hepatitis B virus in basal sister-relationship to the Old World non-human primates and human hepatitis B virus variants. Another theory suggests an Old World origin of human hepatitis B virus, and that it would have been spread following prehistoric human migrations over 100,000 years ago. A third theory suggests a co-speciation of human hepatitis B virus in non-human primate hosts because of the proximity between the phylogeny of Old and New World non-human primate and their human hepatitis B virus variants. The importance of further research, related to the subject in South American wild fauna, is paramount and highly relevant for understanding the origin of human hepatitis B virus.

Lipid peroxidation in microsomes of murine bone marrow after low-dose γ-irradiation

International Nuclear Information System (INIS)

Schwenke, K.; Coslar, S.; Muehlensiepen, H.; Altman, K.I.; Feinendegen, L.E.

1994-01-01

The principal aim of the study was to investigate the effect of low-dose γ-irradiation on lipid peroxidation (LPO) in murine bone marrow. To this end, the degree of LPO in suspensions of microsomes of murine bone marrow cells (BMC) was determined in terms of malondialdehyde (MDA) formation after whole-body or in vitro exposure to various doses of γ-radiation. These effects were compared to some extent with similar effects in liver and spleen preparations. As to the effect of γ-irradiation on LPO in BMC, the response depends on the dose level and on whether whole-body or in vitro exposures are involved. Whole-body irradiation did not result in an increase in LPO in BMC microsomes, even at such high doses as 15 Gy, although hepatic microsomes showed a marked increase. In contrast, in vitro irradiation of BMC microsomes with 0.1, 10 and 50 Gy brought about an increase in LPO. This increase was already significant (P < 0.05) at 0.1 Gy following a post-irradiation incubation and substantial at 50 Gy, even without subsequent incubation. The results show that low doses of γ-irradiation are able to induce an elevation of LPO in murine BMC microsomes, but only after in vitro irradiation. In the case of whole-body irradiation cellular radical scavengers and other metabolic reactions may prevent a measurable increase in LPO. This is partly illustrated by the case of vitamin-E deficiency, where a substantial increase in LPO in BMC microsomes is observed even without γ-irradiation in comparison with euvitaminotic mice because normally occurring radicals are not scavenged sufficiently. (orig.)

A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1.

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Kerkar, N; Ma, Y; Hussain, M; Muratori, L; Targett, C; Williams, R; Bianchi, F B; Mieli-Vergani, G; Vergani, D

1999-03-04

Liver Kidney Microsomal type 1 (LKM1) antibody, the diagnostic marker of autoimmune hepatitis type 2, is also found in a proportion of patients with hepatitis C virus infection (HCV). It is detected conventionally by the subjective immunofluorescence technique. Our aim was to establish a simple and objective enzyme-linked immunosorbent assay (ELISA) that measures antibodies to cytochrome P4502D6 (CYP2D6), the target of LKM1. An indirect ELISA using eukaryotically expressed CYP2D6 was designed. Absorbance values obtained against a reference microsomal preparation were subtracted from those obtained against a microsomal preparation over-expressing CYP2D6, thus removing the non-CYP2D6-specific reaction. Sera from 51 LKM1 positive patients (21 autoimmune hepatitis and 30 with HCV infection), 111 LKM1 negative patients with chronic liver disease (including 20 with HCV infection) and 43 healthy controls were tested. Of 51 patients positive by immunofluorescence, 48 were also positive by ELISA while all the 154 LKM1 negative subjects were also negative by ELISA. There was a high degree of association between IFL and ELISA as demonstrated by a kappa reliability value of 0.96. The absorbance values by ELISA correlated with immunofluorescence LKM1 titres both in autoimmune hepatitis (r = 0.74, p < 0.001) and HCV infection (r = 0.67, p < 0.001). The simple, objective ELISA described has the potential to replace the standard immunofluorescence technique.

Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

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Ju-Hyun Kim

2017-05-01

Full Text Available Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP enzyme activities in human liver microsomes were evaluated using liquid chromatography–tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4’-hydroxylation with a Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4’-hydroxylation (Ki, 3.7 μM; kinact, 0.102 min−1, CYP2C8-catalyzed amodiaquine N-deethylation (Ki, 10.7 μM; kinact, 0.082 min−1, and CYP3A4-catalyzed midazolam 1’-hydroxylation (Ki, 23.0 μM; kinact, 0.050 min−1 in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1’-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.

CYP2B6, CYP2D6, and CYP3A4 catalyze the primary oxidative metabolism of perhexiline enantiomers by human liver microsomes.

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Davies, Benjamin J; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

2007-01-01

The cytochrome P450 (P450)-mediated 4-monohydroxylations of the individual enantiomers of the racemic antianginal agent perhexiline (PHX) were investigated in human liver microsomes (HLMs) to identify stereoselective differences in metabolism and to determine the contribution of the polymorphic enzyme CYP2D6 and other P450s to the intrinsic clearance of each enantiomer. The cis-, trans1-, and trans2-4-monohydroxylation rates of (+)- and (-)-PHX by human liver microsomes from three extensive metabolizers (EMs), two intermediate metabolizers (IMs), and two poor metabolizers (PMs) of CYP2D6 were measured with a high-performance liquid chromatography assay. P450 isoform-specific inhibitors, monoclonal antibodies directed against P450 isoforms, and recombinantly expressed human P450 enzymes were used to define the P450 isoform profile of PHX 4-monohydroxylations. The total in vitro intrinsic clearance values (mean +/- S.D.) of (+)- and (-)-PHX were 1376 +/- 330 and 2475 +/- 321, 230 +/- 225 and 482 +/- 437, and 63.4 +/- 1.6 and 54.6 +/- 1.2 microl/min/mg for the EM, IM, and PM HLMs, respectively. CYP2D6 catalyzes the formation of cis-OH-(+)-PHX and trans1-OH-(+)-PHX from (+)-PHX and cis-OH-(-)-PHX from (-)-PHX with high affinity. CYP2B6 and CYP3A4 each catalyze the trans1- and trans2-4-monohydroxylation of both (+)- and (-)-PHX with low affinity. Both enantiomers of PHX are subject to significant polymorphic metabolism by CYP2D6, although this enzyme exhibits distinct stereoselectivity with respect to the conformation of metabolites and the rate at which they are formed. CYP2B6 and CYP3A4 are minor contributors to the intrinsic P450-mediated hepatic clearance of both enantiomers of PHX, except in CYP2D6 PMs.

Grapefruit juice intake does not enhance but rather protects against aflatoxin B1-induced liver DNA damage through a reduction in hepatic CYP3A activity.

Science.gov (United States)

Miyata, Masaaki; Takano, Hiroki; Guo, Lian Q; Nagata, Kiyoshi; Yamazoe, Yasushi

2004-02-01

Influence of grapefruit juice intake on aflatoxin B1 (AFB1)-induced liver DNA damage was examined using a Comet assay in F344 rats given 5 mg/kg AFB1 by gavage. Rats allowed free access to grapefruit juice for 5 days prior to AFB1 administration resulted in clearly reduced DNA damage in liver, to 65% of the level in rats that did not receive grapefruit juice. Furthermore, rats treated with grapefruit juice extract (100 mg/kg per os) for 5 days prior to AFB1 treatment also reduced the DNA damage to 74% of the level in rats that did not receive grapefruit juice. No significant differences in the portal blood and liver concentrations of AFB1 were observed between grapefruit juice intake rats and the controls. In an Ames assay with AFB1 using Salmonella typhimurium TA98, lower numbers of revertant colonies were detected with hepatic microsomes prepared from rats administered grapefruit juice, compared with those from control rats. Microsomal testosterone 6beta-hydroxylation was also lower with rats given grapefruit juice than with control rats. Immunoblot analyses showed a significant decrease in hepatic CYP3A content, but not CYP1A and CYP2C content, in microsomes of grapefruit juice-treated rats than in non-treated rats. No significant difference in hepatic glutathione S-transferase (GST) activity and glutathione content was observed in the two groups. GSTA5 protein was not detected in hepatic cytosol of the two groups. In microsomal systems, grapefruit juice extract inhibited AFB1-induced mutagenesis in the presence of a microsomal activation system from livers of humans as well as rats. These results suggest that grapefruit juice intake suppresses AFB1-induced liver DNA damage through inactivation of the metabolic activation potency for AFB1 in rat liver.

Metabolism of ginger component [6]-shogaol in liver microsomes from mouse, rat, dog, monkey, and human.

Science.gov (United States)

Chen, Huadong; Soroka, Dominique; Zhu, Yingdong; Sang, Shengmin

2013-05-01

There are limited data on the metabolism of [6]-shogaol (6S), a major bioactive component of ginger. This study demonstrates metabolism of 6S in liver microsomes from mouse, rat, dog, monkey, and human. The in vitro metabolism of 6S was compared among five species using liver microsomes from mouse, rat, dog, monkey, and human. Following incubations with 6S, three major reductive metabolites 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-ol (M9), and 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), as well as two new oxidative metabolites (1E,4E)-1-(4'-hydroxy-3'-methoxyphenyl)-deca-1,4-dien-3-one (M14) and (E)-1-(4'-hydroxy-3'-methoxyphenyl)-dec-1-en-3-one (M15) were found in all species. The kinetic parameters of M6 in liver microsomes from each respective species were quantified using Michaelis-Menten theory. A broad CYP-450 inhibitor, 1-aminobenzotriazole, precluded the formation of oxidative metabolites, M14 and M15, and 18β-glycyrrhetinic acid, an aldo-keto reductase inhibitor, eradicated the formation of the reductive metabolites M6, M9, and M11 in all species. Metabolites M14 and M15 were tested for cancer cell growth inhibition and induction of apoptosis and both showed substantial activity, with M14 displaying greater potency than 6S. We conclude that 6S is metabolized extensively in mammalian species mouse, rat, dog, monkey, and human, and that there are significant interspecies differences to consider when planning preclinical trials toward 6S chemoprevention. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice.

Science.gov (United States)

Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E

1991-02-01

A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

OpenAIRE

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confo...

Theories about evolutionary origins of human hepatitis B virus in primates and humans.

Science.gov (United States)

Souza, Breno Frederico de Carvalho Dominguez; Drexler, Jan Felix; Lima, Renato Santos de; Rosário, Mila de Oliveira Hughes Veiga do; Netto, Eduardo Martins

2014-01-01

The human hepatitis B virus causes acute and chronic hepatitis and is considered one of the most serious human health issues by the World Health Organization, causing thousands of deaths per year. There are similar viruses belonging to the Hepadnaviridae family that infect non-human primates and other mammals as well as some birds. The majority of non-human primate virus isolates were phylogenetically close to the human hepatitis B virus, but like the human genotypes, the origins of these viruses remain controversial. However, there is a possibility that human hepatitis B virus originated in primates. Knowing whether these viruses might be common to humans and primates is crucial in order to reduce the risk to humans. To review the existing knowledge about the evolutionary origins of viruses of the Hepadnaviridae family in primates. This review was done by reading several articles that provide information about the Hepadnaviridae virus family in non-human primates and humans and the possible origins and evolution of these viruses. The evolutionary origin of viruses of the Hepadnaviridae family in primates has been dated back to several thousand years; however, recent analyses of genomic fossils of avihepadnaviruses integrated into the genomes of several avian species have suggested a much older origin of this genus. Some hypotheses about the evolutionary origins of human hepatitis B virus have been debated since the '90s. One theory suggested a New World origin because of the phylogenetic co-segregation between some New World human hepatitis B virus genotypes F and H and woolly monkey human hepatitis B virus in basal sister-relationship to the Old World non-human primates and human hepatitis B virus variants. Another theory suggests an Old World origin of human hepatitis B virus, and that it would have been spread following prehistoric human migrations over 100,000 years ago. A third theory suggests a co-speciation of human hepatitis B virus in non-human primate

Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency ...

African Journals Online (AJOL)

Background: The epidemiology of viral hepatitis and Human immunodeficiency virus (HIV) during pregnancy is of great importance for health planners and program managers. However, few published data on viral hepatitis and HIV are available in Sudan especially during pregnancy. Objectives: The current study was ...

The Metabolism of Separase Inhibitor Sepin-1 in Human, Mouse, and Rat Liver Microsomes

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Feng Li

2018-05-01

Full Text Available Separase, a known oncogene, is widely overexpressed in numerous human tumors of breast, bone, brain, blood, and prostate. Separase is an emerging target for cancer therapy, and separase enzymatic inhibitors such as sepin-1 are currently being developed to treat separase-overexpressed tumors. Drug metabolism plays a critical role in the efficacy and safety of drug development, as well as possible drug–drug interactions. In this study, we investigated the in vitro metabolism of sepin-1 in human, mouse, and rat liver microsomes (RLM using metabolomic approaches. In human liver microsomes (HLM, we identified seven metabolites including one cysteine–sepin-1 adduct and one glutathione–sepin-1 adduct. All the sepin-1 metabolites in HLM were also found in both mouse and RLM. Using recombinant CYP450 isoenzymes, we demonstrated that multiple enzymes contributed to the metabolism of sepin-1, including CYP2D6 and CYP3A4 as the major metabolizing enzymes. Inhibitory effects of sepin-1 on seven major CYP450s were also evaluated using the corresponding substrates recommended by the US Food and Drug Administration. Our studies indicated that sepin-1 moderately inhibits CYP1A2, CYP2C19, and CYP3A4 with IC50 < 10 μM but weakly inhibits CYP2B6, CYP2C8/9, and CYP2D6 with IC50 > 10 μM. This information can be used to optimize the structures of sepin-1 for more suitable pharmacological properties and to predict the possible sepin-1 interactions with other chemotherapeutic drugs.

Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

International Nuclear Information System (INIS)

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2007-01-01

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are ∼ 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (∼ 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected

Effect of radio-detoxified endotoxin on the liver microsomal drug metabolizing enzyme system in rats

International Nuclear Information System (INIS)

Bertok, L.; Szeberenyi, S.

1983-01-01

E. coli endotoxin (LPS) depresses the hepatic microsomal mono-oxygenase activity. Radio-detoxified LPS (TOLERIN: 60 Co irradiated endotoxin preparation) decreases this biotransforming activity to a smaller extent. Phenobarbital, an inducer of this mono-oxygenase system, failed to induce in LPS-treated animals. In radio-detoxified LPS-treated rats, phenobarbital induced the mono-oxygenase and almost fully restored the biotransformation

Biotransformation of a novel antimitotic agent, I-387, by mouse, rat, dog, monkey, and human liver microsomes and in vivo pharmacokinetics in mice.

Science.gov (United States)

Ahn, Sunjoo; Kearbey, Jeffrey D; Li, Chien-Ming; Duke, Charles B; Miller, Duane D; Dalton, James T

2011-04-01

3-(1H-Indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel indole compound with antitubulin action and potent antitumor activity in various preclinical models. I-387 avoids drug resistance mediated by P-glycoprotein and showed less neurotoxicity than vinca alkaloids during in vivo studies. We examined the pharmacokinetics and metabolism of I-387 in mice as a component of our preclinical development of this compound and continued interest in structure-activity relationships for antitubulin agents. After a 1 mg/kg intravenous dose, noncompartmental pharmacokinetic analysis in plasma showed that clearance (CL), volume of distribution at steady state (Vd(ss)), and terminal half-life (t(1/2)) of I-387 were 27 ml per min/kg, 5.3 l/kg, and 7 h, respectively. In the in vitro metabolic stability study, half-lives of I-387 were between 10 and 54 min by mouse, rat, dog, monkey, and human liver microsomes in the presence of NADPH, demonstrating interspecies variability. I-387 was most stable in rat liver microsomes and degraded quickly in monkey liver microsomes. Liquid chromatography-tandem mass spectrometry was used to identify phase I metabolites. Hydroxylation, reduction of a ketone group, and O-demethylation were the major metabolites formed by the liver microsomes of the five species. The carbonyl group of I-387 was reduced and identified as the most labile site in human liver microsomes. The results of these drug metabolism and pharmacokinetic studies provide the foundation for future structural modification of this pharmacophore to improve stability of drugs with potent anticancer effects in cancer patients.

Microsomal aryl hydrocarbon hydroxylase comparison of the direct, indirect and radiometric assays

International Nuclear Information System (INIS)

Denison, M.S.; Murray, M.; Wilkinson, C.F.

1983-01-01

The direct fluorometric assay of aryl hydrocarbon hydroxlyase has been compared to the more commonly used indirect fluorometric and radiometric assays. Although rat hepatic microsomal activities measured by the direct assay were consistently higher than those obtained by the other assays, the relative changes in activity following enzyme induction and/or inhibition were similar. The direct assay provides an accurate and rapid measure of aryl hydrocarbon hydroxylase activity and avoids several problems inherent in the indirect and radiometric assays. 2 tables

[Autoimmune hepatitis in a girl with presence of anti-LKM1 antibodies].

Science.gov (United States)

Larraín, F; Miquel, J F; González, S

1997-06-01

Autoimmune hepatitis is an inflammatory liver disease characterized by dense mononuclear cell infiltrate in the portal tract, and serologically by the presence of non-organ and liver-specific autoantibodies and increased levels of gammaglobulins in the absence of a known etiology. Three subgroups of autoimmune hepatitis have been recognized, depending on the nature of the autoantibody present in the serum: Type 1 autoimmune hepatitis, associated with smooth-muscle (SMA) or antinuclear antibody (ANA) seropositivity; type 2, with anti-liver/kidney microsome antibody (anti-LKM1), and type 3, with the absence of ANA, SMA and anti-LKM1 and presence of other autoantibodies such as anti-soluble liver antigen (SLA). Subtypes of chronic autoimmune hepatitis have clinically different features and prognoses. An 8 year old female patient presented mild jaundice of insidious onset. The liver was tender and enlarged. Serologic markers for A, B, C, E, Epstein Barr and cytomegalovirus were negative. The liver biopsy showed a histological picture consistent with chronic active hepatitis. High titers of anti-liver/kidney-microsome antibody were found by indirect immunofluorescence test, and this finding was confirmed by Western blot against specific liver microsome antigens. Therapy with prednisolone induced a clinical and biochemical remission after four weeks. The suspension of therapy under strict medical control produced a rapid relapse of clinical and biochemical features. The reinitiation of prednisolone was successful, and an alternate-day program was started and maintained until 8 months follow-up.

Prevalence of hepatitis B surface antigen, hepatitis C and Human ...

African Journals Online (AJOL)

Objective: Human immunodeficiency virus (HIV), hepatitis B virus, and hepatitis C viruses (HCV) are major causes of mortality and morbidity worldwide. They are also among the commonest transfusiontransmissible infectious agents. Students of higher institutions are often used as voluntary unpaid donors by many ...

In vitro modulatory effects of Terminalia arjuna, arjunic acid, arjunetin and arjungenin on CYP3A4, CYP2D6 and CYP2C9 enzyme activity in human liver microsomes

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Alice Varghese

2015-01-01

Full Text Available Terminalia arjuna is a tree having an extensive medicinal potential in cardiovascular disorders. Triterpenoids are mainly responsible for cardiovascular properties. Alcoholic and aqueous bark extracts of T. arjuna, arjunic acid, arjunetin and arjungenin were evaluated for their potential to inhibit CYP3A4, CYP2D6 and CYP2C9 enzymes in human liver microsomes. We have demonstrated that alcoholic and aqueous bark extract of T. arjuna showed potent inhibition of all three enzymes in human liver microsomes with IC50 values less than 50 μg/mL. Arjunic acid, arjunetin and arjungenin did not show significant inhibition of CYP enzymes in human liver microsomes. Enzyme kinetics studies suggested that the extracts of arjuna showed reversible non-competitive inhibition of all the three enzymes in human liver microsomes. Our findings suggest strongly that arjuna extracts significantly inhibit the activity of CYP3A4, CYP2D6 and CYP2C9 enzymes, which is likely to cause clinically significant drug–drug interactions mediated via inhibition of the major CYP isozymes.

Auto-immune hepatitis following delivery.

Science.gov (United States)

Saini, Vandana; Gupta, Mamta; Mishra, S K

2013-05-01

Auto-immune hepatitis first presenting in the early postpartum period is rare. Immunosuppressive effects of pregnancy result in delayed manifestation of auto-immune hepatitis, and in established cases, the spontaneous improvements are there. Auto-immune hepatitis should be considered in the differential diagnosis of liver dysfunction first presenting in the early postpartum period. A case of postpartum hepatitis of auto-immune aetiology is being presented here. It is disease of unknown aetiology, characterised by inflammation of liver (as evidenced by raised serum transaminases, presence of interface hepatitis on histological examination), hypergammaglobulinaemia (> 1.5 times normal), presence of auto-antibodies [(antinuclear antibodies (ANA)], smooth muscle antibody (SMA) and antibody to liver-kidney microsome type 1 (LKM1) in the absence of viral markers ie, hepatitis B (HBsAg) and C (AntiHCV) and excellent response to corticosteroid therapy.

Hepatitis C virus infection can mimic type 1 (antinuclear antibody positive) autoimmune chronic active hepatitis.

Science.gov (United States)

Pawlotsky, J M; Deforges, L; Bretagne, S; André, C; Métreau, J M; Thiers, V; Zafrani, E S; Goossens, M; Duval, J; Mavier, J P

1993-01-01

Hepatitis C virus (HCV) has been shown to induce anti-liver-kidney microsomal-1 (LKM1) antibody positive chronic active hepatitis, simulating type 2 autoimmune chronic active hepatitis. The cases of five patients presenting with features of type 1 (antinuclear antibody positive) autoimmune chronic active hepatitis and extrahepatic autoimmune manifestations, in whom immunosuppressive treatment had no effect on liver disease are presented. In these patients, HCV infection could be shown by the presence in serum of anti-HCV antibodies and HCV-RNA detected by polymerase chain reaction. These cases suggest the following: (a) chronic HCV infection can mimic type 1, as well as type 2, autoimmune chronic active hepatitis; (b) HCV infection might be systematically sought in patients presenting with features of type 1 autoimmune chronic active hepatitis, with special care in patients who are unresponsive to immunosuppressive treatment. Images Figure PMID:7686122

The induction of autoimmune hepatitis in the human leucocyte antigen-DR4 non-obese diabetic mice autoimmune hepatitis mouse model.

Science.gov (United States)

Yuksel, M; Xiao, X; Tai, N; Vijay, G M; Gülden, E; Beland, K; Lapierre, P; Alvarez, F; Hu, Z; Colle, I; Ma, Y; Wen, L

2016-11-01

Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti-smooth muscle actin and/or anti-nuclear, anti-liver kidney microsomal type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)-DR3, -DR7 and -DR13. HLA-DR4 has the second strongest association with adult AIH, after HLA-DR3. We investigated the role of HLA-DR4 in the development of AIH by immunization of HLA-DR4 (DR4) transgenic non-obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti-LKM1/anti-LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (T regs ), which had decreased programmed death (PD)-1 expression. Splenic T regs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8 + T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild-type (WT) NOD mice. Our results demonstrate that HLA-DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of T regs and reduced PD-1 expression may result in spontaneous activation of key immune cell subsets, such as antigen-presenting cells and CD8 + T effectors, facilitating the induction of AIH and persistent liver damage. © 2016 British Society for Immunology.

The induction of autoimmune hepatitis in the human leucocyte antigen‐DR4 non‐obese diabetic mice autoimmune hepatitis mouse model

Science.gov (United States)

Yuksel, M.; Xiao, X.; Tai, N.; Vijay, G. M.; Gülden, E.; Beland, K.; Lapierre, P.; Alvarez, F.; Hu, Z.; Colle, I.; Ma, Y.

2016-01-01

Summary Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti‐smooth muscle actin and/or anti‐nuclear, anti‐liver kidney microsomal type 1 (anti‐LKM1) and anti‐liver cytosol type 1 (anti‐LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)‐DR3, ‐DR7 and ‐DR13. HLA‐DR4 has the second strongest association with adult AIH, after HLA‐DR3. We investigated the role of HLA‐DR4 in the development of AIH by immunization of HLA‐DR4 (DR4) transgenic non‐obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti‐LKM1/anti‐LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (Tregs), which had decreased programmed death (PD)‐1 expression. Splenic Tregs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8+ T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild‐type (WT) NOD mice. Our results demonstrate that HLA‐DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of Tregs and reduced PD‐1 expression may result in spontaneous activation of key immune cell subsets, such as antigen‐presenting cells and CD8+ T effectors, facilitating the induction of AIH and persistent liver damage. PMID:27414259

The influence of single application of paracetamol and/or N-acetylcysteine on rats subchronic exposed to trichloroethylene vapours. I. Effect on hepatic moonooxygenase system dependent of cytochrome P450

Directory of Open Access Journals (Sweden)

Andrzej Plewka

2012-06-01

Full Text Available Background: There is a number of factors which potentially affect occurrence of toxic change in liver after overdosing of paracetamol. Hepatic metabolism of trichloroethylene has primary impact on hepatotoxic effect of this solvent. This means that the combined exposure to these xenobiotics can be particularly harmful for human. The influence of N-acetylcysteine (NAC as a protective factor after paracetamol intoxication was studies. Materials and method: Tests were carried out on rats which were treated with trichloroethylene, paracetamol and/or N-acetylcysteine. In the hepatic microsomal fraction activity of the components of cytochrome P450- dependent monooxygenases was determined Results: Paracetamol slightly stimulated cytochrome P450 having no effect on reductase activity cooperating with it. Cytochrome b5 and its reductase were inhibited by this compound. Trichloroethylene was the inhibitor of compounds of II microsomal electron transport chain. N-acetylcysteine inhibited activity of reductase of NADH-cytochrome b5. Conclusions: Tested doses of the xenobiotics influenced on II microsomal electron transport chain. Protective influence of N-acetylcysteine was better if this compound was applied 2 hours after exposure on xenobiotics

[Detection and the production mechanism of antinuclear antibodies (ANA) and anti-liver/kidney microsomal tpe 1 antibodies (anti-LKM1) in patients with chronic hepatitis C].

Science.gov (United States)

Bai, Li; Lu, Hai-Ying; Feng, Zhen-Ru; Yu, Min; Li, Wen-Gang; Gong, Wei-Bo; Zhao, Nu-en-ji-ya; Xu, Xiao-Yuan

2009-08-01

To investigate the prevalence of antinuclear antibodies (ANA) and anti-liver/ kidney microsomal type 1 antibodies (anti-LKM1) in patients with chronic hepatitis C (CHC)and to explore the mechanism of production of these autoantibodies. Serum samples were collected from 360 patients with CHC (case group), 69 patients with chronic hepatitis B (CHB) and 69 patients with autoimmune hepatitis (AIH) (control group). Serum ANA and anti-LKM1 were detected by indirect immunofluorescence (HF) technique and enzyme-linked immunosorbent assay (ELISA), respectively. Multi-factor analysis was performed to explore the correlations of the production of autoantibodies with some factors such as age, sex, viral loads, HCV genotype, biochemical parameters and clinical characteristics. Fifty-four (15%) of 360 patients infected with HCV were positive in autoantibodies. The prevalence of ANA and anti-LKM1 were 12.5% (45/360) and 2.5% (9/ 360), respectively. The positive rate of autoantibodies in patients with CHC was significantly higher than that in patients with CHB (15% vs 2.9%, P = 0.006), but significantly lower than that in patients with AIH (15% vs 47.9%, P 0.05). Autoantibodies related to AIH can be detected in CHC patients; interferon may not induce the production of autoantibodies; it is very likely that HCV infection induces the autoimmune reaction and the production of autoantibodies.

Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms.

Science.gov (United States)

Brill, Shirley S; Furimsky, Anna M; Ho, Mark N; Furniss, Michael J; Li, Yi; Green, Adam G; Bradford, Wallace W; Green, Carol E; Kapetanovic, Izet M; Iyer, Lalitha V

2006-04-01

Resveratrol (trans-resveratrol, trans-3,5,4'-trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3-O-glucuronide and resveratrol 4'-O-glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3-O-glucuronide (Km = 149 microM) and 4'-O-glucuronide (Km = 365 microM), respectively. The glucuronide conjugates were formed at higher levels (up to 10-fold) by intestinal rather than liver microsomes. Resveratrol was co-incubated with substrates of UGT1A1 (bilirubin and 7-ethyl-10-hydroxycamptothecin (SN-38)) and UGT1A9 (7-hydroxytrifluoromethyl coumarin (7-HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN-38 (Ki = 6.2 +/- 2.1 microM) and 7-HFC (Ki = 0.6 +/- 0.2 microM). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.

Non-organ specific autoantibodies in children with chronic hepatitis C.

Science.gov (United States)

Bortolotti, F; Vajro, P; Balli, F; Giacchino, R; Crivellaro, C; Barbera, C; Cataleta, M; Muratori, L; Pontisso, P; Nebbia, G; Zancan, L; Bertolini, A; Alberti, A; Bianchi, F

1996-11-01

Recent studies in adult patients have established a relationship between hepatitis C virus infection and the presence of liver-kidney microsomal autoantibody type 1 (LKM1). Conversely, little is known regarding the relationship between hepatitis C and autoimmunity in children. In this study, we investigated non-organ specific autoantibodies in 40 otherwise healthy Italian children with chronic hepatitis C. All but four patients included in the study were asymptomatic. Liver histology, obtained in 35, showed features ranging from minimal to mild chronic hepatitis. Autoantibodies were investigated by indirect immunofluorescence. HCV RNA was assayed by the polymerase chain reaction in 34 cases and viral genotypes were determined. Antinuclear antibodies were detected in three (7.5%) cases, one with a homogeneous pattern; smooth muscle autoantibodies in seven (17.5%) cases, always with V (vessels only) specificity and LKM1 in four (10%), at titers ranging from 1:20 and 1:2560. Clinical and virologic features did not significantly differ between autoantibody positive and negative cases, although infections with HCV genotypes 1a and 2 were more frequent in LKM1-positive patients. During observation, the child with the highest LKM1 titre was unsuccessfully treated with alpha interferon but responded to steroids. Twelve LKM1 negative children were also treated with interferon and one developed low LKM1 titers concomitant with an alanine aminotransferase flare. The sera of the five LKM1-positive children with investigated by immunoblotting with a human microsomal fraction and peptide 257-269 of cytochrome P450IID6. Only the serum of the child with the highest LKM1 titers was reactive. These results show that a consistent proportion of children with chronic hepatitis C circulate non-organ specific autoantibodies. The prevalence of LKM1 is greater than in adults and this could raise problems for the treatment of the disease with interferon. The analysis of LKM1 target antigens

In vitro metabolism and bioactivation of 1,2,3-trichloropropane.

Science.gov (United States)

Weber, G L; Sipes, I G

1992-03-01

In vitro studies using rat and human hepatic microsomes have shown that the halogenated hydrocarbon 1,2,3-trichloropropane (TCP) is bioactivated to the direct acting mutagen 1,3-dichloroacetone (DCA). The presence of DCA in microsomal incubations was confirmed by gas chromatography-mass spectrometry. DCA formation was totally dependent on the presence of NADPH. The rate of DCA formation using rat and human microsomes was 0.268 +/- 0.029 and 0.026 +/- 0.006 nmol/min/mg protein +/- SE, respectively. When hepatic microsomes were isolated from rats pretreated with the cytochrome P-450 inducers, phenobarbital, and dexamethasone, 24- and 2.5-fold increases, respectively, in the rate of DCA production, were observed. Pretreatment with beta-naphthoflavone resulted in a 50% inhibition in DCA formation. The inhibitors of cytochromes P-450, SKF 525-A and 1-aminobenzotriazol, produced 85 and 70% inhibitions of DCA formation, respectively. When alcohol dehydrogenase and NADH were added to microsomal incubations, two TCP-related alcohols, 1,3-dichloro-2-propanol and 2,3-dichloropropanol, were formed. These alcohols are products of the initial microsomal metabolites, DCA and 2,3-dichloropropanal. [14C]TCP equivalents bound covalently to rat hepatic microsomal protein. This binding was increased 8-fold when hepatic microsomes from phenobarbital pretreated rats were used. The addition of either glutathione or N-acetylcysteine to the incubations completely inhibited this binding. In the presence of N-acetylcysteine, 1,3-(2-propanone)-bis-S-(N-acetylcysteine) (PDM) was the only N-acetylcysteine conjugate detected. It represented 87% of TCP microsomal metabolism. The formation of PDM implicates DCA as the major microsomal protein-binding metabolite of TCP. The formation of DCA, a direct-acting mutagen, may be responsible for the mutagenicity of TCP in systems using rat hepatic microsomes. Its role in the tumorigenicity and carcinogenicity of TCP remains to be established.

Autoantibodies in Autoimmune Hepatitis.

Science.gov (United States)

Muratori, Luigi; Deleonardi, Gaia; Lalanne, Claudine; Barbato, Erica; Tovoli, Alessandra; Libra, Alessia; Lenzi, Marco; Cassani, Fabio; Muratori, Paolo

2015-01-01

The detection of diagnostic autoantibodies such as antinuclear antibodies (ANA), anti-smooth muscle antibodies (SMA), anti-liver/kidney microsomal type 1 (anti-LKM1), anti-liver cytosol type 1 (anti-LC1) and anti-soluble liver antigen (anti-SLA) is historically associated with the diagnosis of autoimmune hepatitis. When autoimmune hepatitis is suspected, the detection of one or any combination of diagnostic autoantibodies, by indirect immunofluorescence or immuno-enzymatic techniques with recombinant antigens, is a pivotal step to reach a diagnostic score of probable or definite autoimmune hepatitis. Diagnostic autoantibodies (ANA, SMA, anti-LKM1, anti-LC1, anti-SLA) are a cornerstone in the diagnosis of autoimmune hepatitis. Other ancillary autoantibodies, associated with peculiar clinical correlations, appear to be assay-dependent and institution-specific, and validation studies are needed. © 2015 S. Karger AG, Basel.

Anticuerpos anti LKM-1 y crioglobulinemia en hepatitis crónica autoinmune y por virus C de la hepatitis

OpenAIRE

Jirón V,M. Isabel; Ardiles S,Adriana; Parra B,M Adriana; Orellana V,Juana

2000-01-01

Background: Anti liver kidney microsome antibodies (LKM-1) have been recently incorporated to the study and classification of chronic autoimmune hepatitis (HC-A1). The presence of anti LKM-1 antibodies and essential cryoglobulinemia is frequent in virus C associated chronic hepatitis (HC-VC). Aim: To study the frequency of anti LKM-1 antibodies and cryoglobulin levels in patients with HC-AI, HC-VC and cryptogenic cirrhosis. Patients and methods: Forty two patients were studied. Nineteen adult...

Influence of cadmium on ketamine-induced anesthesia and brain microsomal Na[sup +], K[sup +]-ATPase in mice

Energy Technology Data Exchange (ETDEWEB)

Shen, Y.; Sangiah, S. (Oklahoma State Univ., Stillwater, OK (United States))

1994-10-01

Cadmium is a rare metallic element, present in almost all types of food. Shellfish, wheat and rice accumulate very high amounts. Occupational and environmental pollutants are the main sources of cadmium exposure. Cadmium has a very long biologic half-life. Exposure to Cadmium causes anemia, hypertension, hepatic, renal, pulmonary and cardiovascular disorders as well as being a possible mutagen, teratogen and carcinogen. Acute cadmium treatment increased the hexobarbital sleeping time and inhibited hepatic microsomal drug metabolism due to a decrease in cytochrome P[sub 450] content. Cadmium potentiated ethanol-induced sleep in a dose-dependent manner. Cadmium has been shown to inhibit brain microsomal Na[sup +], K[sup +]-ATPase activity in vitro and in vivo. Cadmium and ethanol additively inhibited brain Na[sup +], K[sup +]-ATPase. This might be a direct interaction between cadmium and ethanol in the central nervous system. Ketamine is an intravenous anesthetic agent. It acts on central nervous system and produces [open quotes]dissociative anaesthesia.[close quotes] Ketamine provides adequate surgical anesthesia and is used alone in humans and/or combination with xylazine, an [alpha][sub 2]-adrenergic agonist in animals. It produces CNS depression, analgesia, amnesia, immobility and a feeling of dissociation from the environment. Ketamine is a non-competitive antagonist of the NMDA subset of the glutamate receptor. This perhaps results in an increase in neuronal activity leading to disorganization of normal neurotransmission and produces dissociative anesthetic state. Because it is different from most other anesthetics, ketamine may be expected to have a unique effect on brain biochemical parameters and enzymes. The purpose of this study was to examine the interactions between cadmium and ketamine on the central nervous system and ATPase, in an attempt to further understand the mechanism of action. 12 refs., 3 figs.

Hepatic Diacylglycerol-Associated Protein Kinase Cε Translocation Links Hepatic Steatosis to Hepatic Insulin Resistance in Humans

NARCIS (Netherlands)

ter Horst, Kasper W.; Gilijamse, Pim W.; Versteeg, Ruth I.; Ackermans, Mariette T.; Nederveen, Aart J.; la Fleur, Susanne E.; Romijn, Johannes A.; Nieuwdorp, Max; Zhang, Dongyan; Samuel, Varman T.; Vatner, Daniel F.; Petersen, Kitt F.; Shulman, Gerald I.; Serlie, Mireille J.

2017-01-01

Hepatic lipid accumulation has been implicated in the development of insulin resistance, but translational evidence in humans is limited. We investigated the relationship between liver fat and tissue-specific insulin sensitivity in 133 obese subjects. Although the presence of hepatic steatosis in

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

Science.gov (United States)

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy PMID:10716687

Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

Science.gov (United States)

Gates, Simon; Miners, John O

1999-01-01

Aims The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. Methods The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. Results The CYP1A2 inhibitor furafylline variably inhibited (0–65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by ≈55–60%, 35–55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. Conclusions Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans. PMID:10215755

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis

OpenAIRE

Rigopoulou, Eirini I; Mytilinaiou, Maria; Romanidou, Ourania; Liaskos, Christos; Dalekos, George N

2007-01-01

Background Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody – the serological marker of type 2 autoimmune hepatitis (AIH-2)- is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1) – either in association with anti-LKM1, or in isolation- and anti-soluble liver antigen antibodies (anti-SLA) have been considered as us...

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

Science.gov (United States)

Kobayashi, Misato; Hoshinaga, Yukiko; Miura, Natsuko; Tokuda, Yuki; Shigeoka, Shigeru; Murai, Atsushi; Horio, Fumihiko

2014-01-01

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

Methodological approaches to disinfection of human hepatitis B virus.

OpenAIRE

Prince, D L; Prince, H N; Thraenhart, O; Muchmore, E; Bonder, E; Pugh, J

1993-01-01

Three commercial disinfectants (two quaternary formulations and one phenolic) were tested against human hepatitis B virus (HHBV). The treated virus was assayed for infectivity by the chimpanzee assay and for morphological alteration by the Morphological Alteration and Disintegration Test. The same agents were tested against duck hepatitis B virus in a duck hepatocyte infectivity assay. It is apparent that human and duck hepatitis viruses were relatively susceptible to disinfection, becoming n...

[Hepatitis: a longstanding companion in human history].

Science.gov (United States)

Craxi, Lucia

2012-03-01

Hepatitis has gone along with human history since its origins, due to its prompt identifiability linked to jaundice as a symptom. Written evidence of outbreaks of epidemic jaundice can be tracked back a few millenniums before Christ. Unavoidable confusion arises due to the overlap of different sources possibly linked to different aetiologies, identified over time as epidemic jaundice (HAV or HEV hepatitis?) and serum hepatitis (HBV or HCV hepatitis?). The journey that brought to recognize viruses as the main cause of jaundice was long and started midway during the last century, when the infectious hypothesis, which had taken place step by step, was finally confirmed by epidemiological investigations of an outbreak occurring in the US army in 1942, after a yellow fever immunization campaign. Further research identified two clinically different types of hepatitis, called for the first time hepatitis A and hepatitis B.

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

Directory of Open Access Journals (Sweden)

Menéndez R.

2000-01-01

Full Text Available The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS. When D002 (5-100 mg/kg body weight was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46% occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40% and brain (28-44% microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Antithyroid microsomal antibody

Science.gov (United States)

Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

Effects of model traumatic injury on hepatic drug metabolism in the rat. IV. Glucuronidation.

Science.gov (United States)

Griffeth, L K; Rosen, G M; Rauckman, E J

1985-01-01

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on hepatic glucuronidation activity. As was previously observed with hepatic oxidative drug metabolism, model trauma resulted in a significant decrease in the in vivo glucuronidation of chloramphenicol, with a 23% drop in clearance of this drug. The effect on in vivo pharmacokinetics appeared to result from a complex interaction between trauma's differential influences on conjugating enzyme(s), deconjugating enzyme(s), and hepatic UDP-glucuronic acid levels, as well as the relative physiological importance of these variables. Hepatic UDP-glucuronyltransferase activities towards both p-nitrophenol and chloramphenicol were elevated (44-54%) after model injury when measured in native hepatic microsomes. However, microsomes which had been "activated" by treatment with Triton X-100 showed no significant difference between control and traumatized animals. Serum beta-glucuronidase activities were elevated by 58%, while hepatic beta-glucuronidase rose by about 16%. Nevertheless, in vivo deconjugation showed no significant change. Model trauma also resulted in a 46% decrease in hepatic UDP-glucuronic acid content. Thus, the observed post-traumatic depression of in vivo chloramphenicol glucuronidation could be due either to a diminished availability of a necessary cofactor (UDP-glucuronic acid) or to an alteration in enzyme kinetics or function in vivo.

Features of Hepatitis in Hepatitis-associated Aplastic Anemia: Clinical and Histopathologic Study.

Science.gov (United States)

Patel, Kalyani R; Bertuch, Alison; Sasa, Ghadir S; Himes, Ryan W; Wu, Hao

2017-01-01

Hepatitis-associated aplastic anemia (HAA) is a rare variant of aplastic anemia in which patients present with severe pancytopenia after an episode of acute hepatitis. The marrow failure is often rapid, severe, and usually fatal if untreated. The preceding hepatitis is largely under-studied. Retrospective study of the clinical and histopathologic features of hepatitis in pediatric patients who subsequently developed aplastic anemia and comparison with consecutive cases of acute liver failure and random cases of autoimmune hepatitis during the same time frame. All 7 patients of HAA had significant elevations in aminotransferases and conjugated hyperbilirubinemia at initial presentation. Echoing liver function indices, cholestatic hepatitis with sinusoidal obstruction-type endothelial injury was seen histomorphologically. Autoimmune hepatitis serology such as anti-F-actin, anti-liver/kidney microsome, and hypergammaglobulinemia was negative in all patients. Five of 7 patients (71.4%) had, however, elevated antinuclear antibody, all with a speckled pattern. Hepatitis virus serology was negative in all patients. By immunohistochemical staining, the lobular CD8/CD4 lymphocyte ratio was markedly elevated in all of the initial samples with significant reduction in this ratio (P = 0.03) in 3 patients post treatment (ursodiol, antibiotics, and/or immunosuppressive therapy). Hepatitis preceding HAA is characterized by marked elevation of aminotransferases, conjugated hyperbilirubinemia, elevated antinuclear antibody with a speckled pattern, cholestatic hepatitis with sinusoidal obstruction morphology, and CD8 dominant lobular infiltrates. The present study suggests HAA may result from cytotoxic T-cell-mediated sinusoidal endothelial and hepatocytic injury.

Etiology of fatty liver in dairy cattle: effects of nutritional and hormonal status on hepatic microsomal triglyceride transfer protein.

Science.gov (United States)

Bremmer, D R; Trower, S L; Bertics, S J; Besong, S A; Bernabucci, U; Grummer, R R

2000-10-01

We conducted three experiments to determine the effects of nutritional and hormonal status on microsomal triglyceride transfer protein (MTP) activity and mass. In experiment 1, 18 nonlactating Holstein cows, 75 d before expected calving date, in their second gestation or greater were monitored from d 75 to 55 prepartum. Cows were fed a control diet from d 75 to 62 prepartum for covariable measurements. From d 61 to 55 prepartum, six cows continued to receive the control diet, six cows were restricted to 2.3 kg of grass hay/d, and six cows were fed the control diet plus 1.8 kg of concentrate/d and 500 ml of propylene glycol given 2 times/d as an oral drench. Plasma glucose and serum insulin concentrations were highest in cows that received propylene glycol and lowest in feed restricted cows. Plasma nonesterified fatty acids (NEFA) and liver triglyceride (TG) concentrations were highest in feed restricted cows and not different between cows that received the control diet and cows that received propylene glycol. Hepatic MTP activity and mass were not affected by treatment in experiment 1. In experiment 2, bovine hepatocytes isolated from the caudate process of five preruminating Holstein bull calves were incubated with either 0, 0.5, 1.0, or 2.0 mM NEFA for 48 h. Intracellular TG increased linearly as NEFA concentration in the media increased. Concentration of NEFA in the incubation media had no effect on MTP activity or mass. There was a quadratic effect of concentration of NEFA in the incubation media on MTP mRNA. In experiment 3, bovine hepatocytes isolated from the caudate process of five preruminating Holstein bull calves were incubated with 2 mM [1-14C]oleate for 24 h to accumulate TG, followed by a 36-h period of TG depletion, during which hepatocytes were incubated with no hormone, 10 nM insulin, or 10 nM glucagon. There was no effect of insulin or glucagon on intracellular TG, MTP activity or mass. Cells incubated with no hormone had higher levels of MTP m

Hepatic cholesterol ester hydrolase in human liver disease.

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Simon, J B; Poon, R W

1978-09-01

Human liver contains an acid cholesterol ester hydrolase (CEH) of presumed lysosomal origin, but its significance is unknown. We developed a modified CEH radioassay suitable for needle biopsy specimens and measured hepatic activity of this enzyme in 69 patients undergoing percutaneous liver biopsy. Histologically normal livers hydrolyzed 5.80 +/- 0.78 SEM mumoles of cholesterol ester per hr per g of liver protein (n, 10). Values were similar in alcoholic liver disease (n, 17), obstructive jaundice (n, 9), and miscellaneous hepatic disorders (n, 21). In contrast, mean hepatic CEH activity was more than 3-fold elevated in 12 patients with acute hepatitis, 21.05 +/- 2.45 SEM mumoles per hr per g of protein (P less than 0.01). In 2 patients studied serially, CEH returned to normal as hepatitis resolved. CEH activity in all patients paralleled SGOT levels (r, 0.84; P less than 0.01). There was no correlation with serum levels of free or esterified cholesterol nor with serum activity of lecithin-cholesterol acyltransferase, the enzyme responsible for cholesterol esterification in plasma. These studies confirm the presence of CEH activity in human liver and show markedly increased activity in acute hepatitis. The pathogenesis and clinical significance of altered hepatic CEH activity in liver disease require further study.

Autoimmunity and Extrahepatic Manifestations in Treatment-Naïve Children with Chronic Hepatitis C Virus Infection

Directory of Open Access Journals (Sweden)

Giuseppe Indolfi

2012-01-01

Full Text Available Hepatitis C virus (HCV infection has been associated with autoimmunity and extrahepatic manifestations in adults. Few data are available on these topics in children. Nonorgan specific auto-antibodies development is part of the natural course of chronic hepatitis C in children. Smooth muscle autoantibody is the most common autoantibody found, while liver-kidney microsomal type-1 antibody positivity is the most peculiar autoimmune feature of children with HCV infection. The clinical significance of non-organ specific autoantibodies in the course of paediatric chronic hepatitis C is still debated. Autoantibody positivity can be considered neutral for most patients, while it can be associated with negative connotations for others, especially those positive for liver-kidney microsomal type-1 autoantibody. Subclinical hypothyroidism but not autoimmune thyroiditis has been demonstrated in HCV infection in children, while only few cases of HCV-associated membranoproliferative glomerulonephritis have been described. Single reports are available in the literature reporting the anecdotal association between chronic hepatitis C and other extrahepatic manifestations such as myopathy and opsoclonus-myoclonus syndrome. Despite the low incidence of extrahepatic manifestations of chronic hepatitis C in children, overall, available data suggest a careful monitoring.

Multiple viral/self immunological cross-reactivity in liver kidney microsomal antibody positive hepatitis C virus infected patients is associated with the possession of HLA B51.

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Bogdanos, D-P; Lenzi, M; Okamoto, M; Rigopoulou, E I; Muratori, P; Ma, Y; Muratori, L; Tsantoulas, D; Mieli- Vergani, G; Bianchi, F B; Vergani, D

2004-01-01

Liver Kidney Microsomal autoantibody type 1(LKM1) directed to cytochrome P4502D6 (CYP2D6) characterises autoimmune hepatitis type-2 (AIH-2), but is also found in a proportion of chronic hepatitis C virus (HCV) infected patients, CYP2D6252-271 being a major B- cell autoepitope. Molecular mimicry and immunological cross-reactivity between CYP2D6252-271, HCV polyprotein and the infected cell protein 4 (ICP4) of herpes simplex virus type 1 (HSV-1) have been suggested as triggers for the induction of LKM1, but reactivity and cross-reactivity to the relevant sequences have not been investigated experimentally. CYP2D6252-271 and its viral homologues were constructed and tested by ELISA in the sera of 46 chronically infected HCV patients, 23 of whom were LKM1 positive. Reactivity to the E1 HCV and ICP4 HSV1 mimics was frequently found in HCV infected patients irrespectively of their LKM1 status; viral/self cross-reactivity (as indicated by inhibition studies), however, was present in the only 2 of the 23 LKM1 seropositive HCV patients, who possessed the HLA allotype B51. Our results indicate that in HCV infected patients virus/self cross-reactivity is dependent on a specific immunogenetic background, a finding awaiting confirmation by studies in larger series of patients.

Hepatic Flavin-Containing Monooxygenase 3 Enzyme Suppressed by Type 1 Allergy-Produced Nitric Oxide.

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Tanino, Tadatoshi; Bando, Toru; Komada, Akira; Nojiri, Yukie; Okada, Yuna; Ueda, Yukari; Sakurai, Eiichi

2017-11-01

Flavin-containing monooxygenases (FMOs) are major mammalian non-cytochrome P450 oxidative enzymes. T helper 2 cell-activated allergic diseases produce excess levels of nitric oxide (NO) that modify the functions of proteins. However, it remains unclear whether allergy-induced NO affects the pharmacokinetics of drugs metabolized by FMOs. This study investigated alterations of hepatic microsomal FMO1 and FMO3 activities in type 1 allergic mice and further examined the interaction of FMO1 and FMO3 with allergy-induced NO. Imipramine (IMP; FMO1 substrate) N- oxidation activity was not altered in allergic mice with high serum NO and immunoglobulin E levels. At 7 days after primary sensitization (PS7) or secondary sensitization (SS7), benzydamine (BDZ; FMO1 and FMO3 substrate) N- oxygenation was significantly decreased to 70% of individual controls. The expression levels of FMO1 and FMO3 proteins were not significantly changed in the sensitized mice. Hepatic inducible NO synthase (iNOS) mRNA level increased 5-fold and 15-fold in PS7 and SS7 mice, respectively, and hepatic tumor necrosis factor- α levels were greatly enhanced. When a selective iNOS inhibitor was injected into allergic mice, serum NO levels and BDZ N- oxygenation activity returned to control levels. NO directly suppressed BDZ N- oxygenation, which was probably related to FMO3-dependent metabolism in comparison with IMP N- oxidation. In hepatic microsomes from PS7 and SS7 mice, the suppression of BDZ N- oxygenation was restored by ascorbate. Therefore, type 1 allergic mice had differentially suppressed FMO3-dependent BDZ N- oxygenation. The suppression of FMO3 metabolism related to reversible S- nitrosyl modifications of iNOS-derived NO. NO is expected to alter FMO3-metabolic capacity-limited drug pharmacokinetics in humans. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Identification of AKB-48 and 5F-AKB-48 Metabolites in Authentic Human Urine Samples Using Human Liver Microsomes and Time of Flight Mass Spectrometry

OpenAIRE

Vikingsson, Svante; Josefsson, Martin; Green, Henrik

2015-01-01

The occurrence of structurally related synthetic cannabinoids makes the identification of unique markers of drug intake particularly challenging. The aim of this study was to identify unique and abundant metabolites of AKB-48 and 5F-AKB-48 for toxicological screening in urine. Investigations of authentic urine samples from forensic cases in combination with human liver microsome (HLM) experiments were used for identification of metabolites. HLM incubations of AKB-48 and 5F-AKB-48 along with 3...

Hepatitis B, C and Human Immunodeficiency Virus (HIV) Co ...

African Journals Online (AJOL)

TNHJOURNALPH

BACKGROUND. Nigeria which has one of the world's highest burden of children living with. Sickle cell anaemia is also endemic for hepatitis B, C and the Human immunodeficiency virus (HIV). This study set out to determine the prevalence of. Hepatitis B surface antigen (HBsAg), antibodies to Hepatitis C Virus (HCV) and.

Effect of Curcuma longa on CYP2D6- and CYP3A4-mediated metabolism of dextromethorphan in human liver microsomes and healthy human subjects.

Science.gov (United States)

Al-Jenoobi, Fahad Ibrahim; Al-Thukair, Areej A; Alam, Mohd Aftab; Abbas, Fawkeya A; Al-Mohizea, Abdullah M; Alkharfy, Khalid M; Al-Suwayeh, Saleh A

2015-03-01

Effect of Curcuma longa rhizome powder and its ethanolic extract on CYP2D6 and CYP3A4 metabolic activity was investigated in vitro using human liver microsomes and clinically in healthy human subjects. Dextromethorphan (DEX) was used as common probe for CYP2D6 and CYP3A4 enzymes. Metabolic activity of CYP2D6 and CYP3A4 was evaluated through in vitro study; where microsomes were incubated with NADPH in presence and absence of Curcuma extract. In clinical study phase-I, six healthy human subjects received a single dose (30 mg) of DEX syrup, and in phase-II DEX syrup was administered with Curcuma powder. The enzyme CYP2D6 and CYP3A4 mediated O- and N-demethylation of dextromethorphan into dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. Curcuma extract significantly inhibited the formation of DOR and 3-MM, in a dose-dependent and linear fashion. The 100 μg/ml dose of curcuma extract produced highest inhibition, which was about 70 % for DOR and 80 % for 3-MM. Curcuma significantly increases the urine metabolic ratio of DEX/DOR but the change in DEX/3-MM ratio was statistically insignificant. Present findings suggested that curcuma significantly inhibits the activity of CYP2D6 in in vitro as well as in vivo; which indicates that curcuma has potential to interact with CYP2D6 substrates.

Four stages of hepatic hematopoiesis in human embryos and fetuses.

Science.gov (United States)

Fanni, D; Angotzi, F; Lai, F; Gerosa, C; Senes, G; Fanos, V; Faa, G

2018-03-01

The liver is a major hematopoietic organ during embryonic and fetal development in humans. Its hematopoietic activity starts during the first weeks of gestation and continues until birth. During this period the liver is colonized by undifferentiated hematopoietic stem cells (HSCs) that gradually differentiate and once mature, enter the circulatory system through the hepatic sinusoids, this process is called hepatic hematopoiesis. The morphology of hepatic hematopoiesis, has been studied in humans through the years, and led to a characterization of all the cell types that make up these phenomena. Studies on murine models also helped to describe the extent of hepatic hematopoiesis at different gestational ages. Using this knowledge, we attempted to describe how hepatic hematopoiesis morphologically evolves as gestation progresses, in human embryos and fetuses. Thus, we observed a total of 32 tissue specimens obtained from the livers of embryos and fetuses at different gestational ages. Basing our observations on the four stages of liver hematopoiesis identified by Sasaki and Sonoda in mice, we also described four consecutive stages of liver hematopoiesis in humans, which resulted to be highly similar to those described in murine models.

Seroprevalence of Hepatitis A virus infection in non-human primates in Assam, India

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B.G. Nath

2013-08-01

Full Text Available The present study investigated 37 serum samples of non-human primates in Assam State Zoo and the Department of Forest and Environment, Govt. of Assam for seroprevalence of hepatitis A virus infection during the period from December, 2007 to November, 2009. Four serum samples were also collected from animal keepers of the zoo to investigate transmission of the disease to the attendants working with these primates. Competitive ELISA was performed using hepatitis A virus ELISA kit (Wanti Hep. AV to detect hepatitis A virus antibody in serum samples. Ten (27.21% of the non-human primate samples and three (75% human samples had detectable anti-hepatitis A virus antibodies. Living status of the non-human primates (Free living was a high potential risk for hepatitis A virus infection. Seroprevalence of hepatitis A virus infection had significant difference between free living non-human primates and captive non-human primates (P less than 0.05. No significant difference (p=0.86 was seen between male and female non-human primates

Variability of human hepatic UDP-glucuronosyltransferase activity

NARCIS (Netherlands)

Little, JM; Lester, R; Kuipers, F; Vonk, R; Mackenzie, PI; Drake, RR; Frame, L; Radominska-Pandya, A

1999-01-01

The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases

LKM3 autoantibodies in hepatitis C cirrhosis: a further phenomenon of the HCV-induced autoimmunity.

Science.gov (United States)

Csepregi, A; Nemesánszky, E; Luettig, B; Obermayer-Straub, P; Manns, M P

2001-03-01

Chronic hepatitis C is frequently associated with laboratory markers-including LKM1 autoantibodies--of autoimmunity. A 62-yr-old woman with hepatitis C cirrhosis presented autoantibodies against liver and kidney microsomal proteins. By further evaluation of autoantibodies using ELISA and immunoblotting LKM1 and LKM3 autoantibodies could be revealed. The target antigen of LKM3 autoantibodies proved to be UGT-1.1 isoenzyme. In the absence of chronic hepatitis D infection or autoimmune hepatitis type 2, this is the first case that reports the occurrence of LKM3 autoantibodies in HCV-induced chronic liver disease.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is Enantioselectively Oxidized to Hydroxylated Metabolites by Rat Liver Microsomes

Science.gov (United States)

Wu, Xianai; Pramanik, Ananya; Duffel, Michael W.; Hrycay, Eugene G.; Bandiera, Stelvio M.; Lehmler, Hans-Joachim; Kania-Korwel, Izabela

2011-01-01

Developmental exposure to multiple-ortho substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB-), dexamethasone (DEX-) and corn oil (VEH-)treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzymes. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer didn't affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of

Non-organ-specific autoantibodies in children with chronic hepatitis C: clinical significance and impact on interferon treatment.

Science.gov (United States)

Muratori, Paolo; Muratori, Luigi; Verucchi, Gabriella; Attard, Luciano; Bianchi, Francesco B; Lenzi, Marco

2003-11-15

We evaluated the prevalence and clinical significance of non-organ-specific autoantibodies (NOSAs) in 47 hepatitis C virus (HCV)-positive children with abnormal alanine transaminase levels and analyzed the association between NOSAs and virus level, genotype, human leukocyte antigen status, and interferon (IFN) response. Forty-two hepatitis B virus (HBV)-positive children and 25 age- and sex-matched healthy children served as control subjects. NOSAs were found in 34% of the HCV-positive children, 12% of the HBV-positive controls, and none of the healthy control subjects. Liver-kidney microsomal antibody type 1 (LKM1) was detected in 11% of the HCV-positive children but in none of the controls. The HCV load was significantly higher in NOSA-negative than in NOSA-positive children. HCV genotype distribution and human leukocyte antigen alleles were similar, irrespective of NOSA status. Long-term response to IFN therapy was achieved by 18% of the NOSA-positive and 55% of the NOSA-negative subjects. Two LKM1-positive children developed acute, self-limited hepatocellular necrosis while receiving IFN therapy. NOSAs are frequently present in children with hepatitis C, who are less likely to benefit from IFN therapy.

Chromatographic separation of piracetam and its metabolite in a mixture of microsomal preparations, followed by an MS/MS analysis.

Science.gov (United States)

Sahu, Kapendra; Siddiqui, Anees A; Shaharyar, Mohammad; Ahmad, Niyaz; Anwar, Mohammad; Ahmad, Farhan J

2013-07-01

A rapid bioanalytical method was evaluated for the simultaneous determination of piracetam and its metabolite (M1) in human microsomal preparations by fast ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). In addition, a validated method of M1 in rat plasma was developed and successfully applied on pharmacokinetic studies. The present study was carried out to determine the metabolic pathways of piracetam for phase I metabolism and used cytochrome P450 isoforms responsible for the piracetam metabolism in human liver microsomes (HLMs). While additional potential metabolites of piracetam were suggested by computer-modeling. The resulting 2-(2-oxopyrrolidin-1-yl) acetic acid was the sole metabolite detected after the microsomal treatment. The amide hydrolysis mainly underwent to form a metabolite i.e., 2-(2-oxopyrrolidin-1-yl) acetic acid (M1). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Prevalence of human immunodeficiency virus, hepatitis C virus ...

African Journals Online (AJOL)

Background. Human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis remain major infections around the world. In Angola, about 166 000 individuals are living with HIV, representing a prevalence of 1.98% in adults between 15 and 49 years of age. In a 2003 study in Luanda, 4.5% ...

Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2.

Science.gov (United States)

Martini, E; Abuaf, N; Cavalli, F; Durand, V; Johanet, C; Homberg, J C

1988-01-01

A new autoantibody was detected by immunoprecipitation in the serum of 21 patients with chronic active hepatitis. The antibody reacted against a soluble cytosolic antigen in liver. The antibody was organ specific but not species specific and was therefore called anti-liver cytosol antibody Type 1 (anti-LC1). In seven of 21 cases, no other autoantibody was found; the remaining 14 cases had anti-liver/kidney microsome antibody Type 1 (anti-LKM1). With indirect immunofluorescence, a distinctive staining pattern was observed with the seven sera with anti-LC1 and without anti-LKM1. The antibody stained the cytoplasm of hepatocytes from four different animal species and spared the cellular layer around the central veins of mouse and rat liver that we have called juxtavenous hepatocytes. The immunofluorescence pattern disappeared after absorption of sera by a liver cytosol fraction. The 14 sera with both antibodies displayed anti-LC1 immunofluorescent pattern after absorption of anti-LKM1 by the liver microsomal fraction. The anti-LC1 was found in the serum only in patients with chronic active hepatitis of unknown cause. Anti-LC1 antibody was not found in sera from 100 patients with chronic active hepatitis associated with anti-actin antibody classic chronic active hepatitis Type 1, 100 patients with primary biliary cirrhosis, 157 patients with drug-induced hepatitis and a large number of patients with liver and nonliver diseases. This new antibody was considered a second marker of chronic active hepatitis associated with anti-LKM1 (anti-LKM1 chronic active hepatitis) or autoimmune chronic active hepatitis Type 2.

A novel “humanized mouse” model for autoimmune hepatitis and the association of gut microbiota with liver inflammation

Science.gov (United States)

Yuksel, Muhammed; Wang, Yipeng; Tai, Ningwen; Peng, Jian; Guo, Junhua; Beland, Kathie; Lapierre, Pascal; David, Chella; Alvarez, Fernando; Colle, Isabelle; Yan, Huiping; Mieli-Vergani, Giorgina; Vergani, Diego; Ma, Yun; Wen, Li

2016-01-01

Background Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease, characterized by interface hepatitis, the presence of circulating autoantibodies and hyper-gammaglobulinemia. There are two types of AIH, type-1 (AIH-1) and type-2 (AIH-2) characterized by distinct autoimmune serology. Patients with AIH-1 are positive for anti-smooth muscle and/or anti-nuclear (SMA/ANA) autoantibodies whereas patients with AIH-2 have anti-liver kidney microsomal type 1 (anti-LKM1) and/or anti-liver cytosol type 1 (anti-LC1) autoantibodies. Cytochrome P4502D6 (CYP2D6) is the antigenic target of anti-LKM1 and formiminotransferase cyclodeaminase (FTCD) is the antigenic target of anti-LC1. It is known that AIH, both type-1 and type-2, is strongly linked to the Human Leukocyte Antigen (HLA) alleles -DR3, -DR4 and -DR7. However, the direct evidence of the association of HLA with AIH is lacking. Methods We developed a novel mouse model of AIH using the HLA-DR3 transgenic mouse on the non-obese diabetic (NOD) background (HLA-DR3 NOD) by immunization of HLA-DR3− and HLA-DR3+ NOD mice with a DNA plasmid, coding for human CYP2D6/FTCD fusion protein. Results Immunization with CYP2D6/FTCD leads to a sustained elevation of alanine aminotransferase (ALT), development of ANA and anti-LKM1/anti-LC1 autoantibodies, chronic immune cell infiltration and parenchymal fibrosis on liver histology in HLA-DR3+ mice. Immunized mice also showed an enhanced Th1 immune response and paucity of the frequency of regulatory T-cell (Treg) in the liver. Moreover, HLA-DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. PMID:26185095

RATE AND CAPACITY OF HEPATIC MICROSOMAL RING HYDROXYLATION OF PHENOL TO HYDROQUINONE AND CATECHOL IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

Science.gov (United States)

Rainbow trout liver microsomes were used to study the rate of ring-hydroxylation of phenol (PH) by directly measuring the production of hydroquinone (HQ), the primary metabolite, and catechol (CAT), a secondary metabolite. An HPLC method with integrated ultroviolet (UV) and elect...

Automation of metabolic stability studies in microsomes, cytosol and plasma using a 215 Gilson liquid handler.

Science.gov (United States)

Linget, J M; du Vignaud, P

1999-05-01

A 215 Gilson liquid handler was used to automate enzymatic incubations using microsomes, cytosol and plasma. The design of automated protocols are described. They were based on the use of 96 deep well plates and on HPLC-based methods for assaying the substrate. The assessment of those protocols was made with comparison between manual and automated incubations, reliability and reproducibility of automated incubations in microsomes and cytosol. Examples of the use of those programs in metabolic studies in drug research, i.e. metabolic screening in microsomes and plasma were shown. Even rapid processes (with disappearance half lives as low as 1 min) can be analysed. This work demonstrates how stability studies can be automated to save time, render experiments involving human biological media less hazardous and may be improve inter-laboratory reproducibility.

AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

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Ju-Hyun Kim

2017-03-01

Full Text Available AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP or uridine 5′-diphospho-glucuronosyltransferase (UGT enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7 enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate.

Science.gov (United States)

Venkatakrishnan, K; von Moltke, L L; Greenblatt, D J

2001-04-01

The relative activity factor (RAF) approach is being increasingly used in the quantitative phenotyping of multienzyme drug biotransformations. Using lymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressant amitriptyline as a model substrate, we have tested the hypothesis that the human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the biotransformation catalyzed by individual recombinant CYPs using the RAF approach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurements in heterologous expression systems. In addition, we have compared the results of two widely used methods of quantitative reaction phenotyping, namely, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-demethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicted biotransformation rates in microsomes from a panel of 12 human livers determined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance via N-demethylation was 53% lower than the previously reported in vivo pharmacokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correlated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhibitors used were specific to their target CYP isoforms.

The use of non-human primates as animal models for the study of hepatitis viruses

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C.L. Vitral

1998-08-01

Full Text Available Hepatitis viruses belong to different families and have in common a striking hepatotropism and restrictions for propagation in cell culture. The transmissibility of hepatitis is in great part limited to non-human primates. Enterically transmitted hepatitis viruses (hepatitis A virus and hepatitis E virus can induce hepatitis in a number of Old World and New World monkey species, while the host range of non-human primates susceptible to hepatitis viruses transmitted by the parenteral route (hepatitis B virus, hepatitis C virus and hepatitis delta virus is restricted to few species of Old World monkeys, especially the chimpanzee. Experimental studies on non-human primates have provided an invaluable source of information regarding the biology and pathogenesis of these viruses, and represent a still indispensable tool for vaccine and drug testing.

Diagnosis and therapy of autoimmune hepatitis.

Science.gov (United States)

Granito, Alessandro; Muratori, Paolo; Ferri, Silvia; Pappas, Georgios; Quarneti, Chiara; Lenzi, Marco; Bianchi, Francesco B; Muratori, Luigi

2009-06-01

Autoimmune hepatitis (AIH) is a chronic progressive hepatitis, characterized by interface hepatitis with lymphoplasmacellular infiltrates on liver biopsy, high serum globulin level and circulating autoantibodies. It is classified into two types, according to autoantibody profile: type 1 is characterized by anti-nuclear (ANA) and/or anti-smooth muscle (SMA) antibodies; type 2 by anti-liver kidney microsomal type 1 (anti-LKM-1) antibodies. AIH affects all ages, may be asymptomatic, frequently has an acute onset, and can present as fulminant hepatitis. The diagnosis of AIH is based on a scoring system codified by an international consensus. Corticosteroids alone or in conjunction with azathioprine is the treatment of choice in patients with AIH and results in remission induction in over 80% of patients. Alternative proposed strategies in patients who have failed to achieve remission on standard therapy or patients with drug toxicity include the use of cyclosporine, tacrolimus, budesonide or mycophenolate mofetil. Liver transplantation is the treatment of choice in managing decompensated disease, however AIH can recur or develop de novo after liver transplantation.

The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3 radical

International Nuclear Information System (INIS)

Srivastava, S.P.; Chen, N.Q.; Holtzman, J.L.

1990-01-01

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3 radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals and are toxic to cultured hepatocytes. The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4

High affinity binding of [3H]cocaine to rat liver microsomes

International Nuclear Information System (INIS)

El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

1988-01-01

] 3 H]cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in [ 3 H]cocaine binding. On the other hand, chronic administration of cocaine reduced [ 3 H]cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of [ 3 H]cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced [ 3 H]cocaine binding to liver with a different rank order of potency than their displacement of [ 3 H]cocaine binding to striatum. This high affinity [ 3 H]cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

Antiviral treatment for chronic hepatitis C in patients with human immunodeficiency virus

DEFF Research Database (Denmark)

Iorio, Alfonso; Marchesini, Emanuela; Awad, Tahany

2010-01-01

Antiviral treatment for chronic hepatitis C may be less effective if patients are co-infected with human immunodeficiency virus (HIV).......Antiviral treatment for chronic hepatitis C may be less effective if patients are co-infected with human immunodeficiency virus (HIV)....

Dietary saturated and monounsaturated fats protect against acute acetaminophen hepatotoxicity by altering fatty acid composition of liver microsomal membrane in rats

Directory of Open Access Journals (Sweden)

Shim Eugene

2011-10-01

Full Text Available Abstract Background Dietary polyunsaturated fats increase liver injury in response to ethanol feeding. We evaluated the effect of dietary corn oil (CO, olive oil (OO, and beef tallow (BT on fatty acid composition of liver microsomal membrane and acute acetaminophen hepatotoxicity. Methods Male Sprague-Dawley rats were fed 15% (wt/wt CO, OO or BT for 6 weeks. After treatment with acetaminophen (600 mg/kg, samples of plasma and liver were taken for analyses of the fatty acid composition and toxicity. Results Treatment with acetaminophen significantly elevated levels of plasma GOT and GPT as well as hepatic TBARS but reduced hepatic GSH levels in CO compared to OO and BT groups. Acetaminophen significantly induced protein expression of cytochrome P450 2E1 in the CO group. In comparison with the CO diet, lower levels of linoleic acid, higher levels of oleic acids and therefore much lower ratios of linoleic to oleic acid were detected in rats fed OO and BT diets. Conclusions Dietary OO and BT produces similar liver microsomal fatty acid composition and may account for less severe liver injury after acetaminophen treatment compared to animals fed diets with CO rich in linoleic acid. These findings imply that types of dietary fat may be important in the nutritional management of drug-induced hepatotoxicity.

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay

International Nuclear Information System (INIS)

Greaves, Alana K.; Su, Guanyong; Letcher, Robert J.

2016-01-01

The in vitro biotransformation and kinetics of six organophosphate triester (OPE) flame retardants were investigated in herring gulls (Larus argentatus) from the Great Lakes using a hepatic microsomal metabolism assay. Administration of each individual OPE (tri-n-butyl phosphate (TNBP), tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), triethyl phosphate (TEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-chloroisopropyl) phosphate (TCIPP)) to the in vitro assay (concentration range 0.01 to 10 μM) resulted in rapid depletion with the exception of TEP. Following the Michaelis-Menten enzyme kinetics model, a preliminary 2-minute incubation period was used to estimate the V max (± SE) values (i.e., the maximal rate of reaction for a saturated enzyme system), which ranged from 5.0 ± 0.4 (TPHP) to 29 ± 18 pmol/min/mg protein (TBOEP), as well as the K M (± SE) values (i.e., the OPE concentration corresponding to one half of the V max ), which ranged from 9.8 ± 1 (TPHP) to 189 ± 135 nM (TBOEP). Biotransformation assays over a 100-minute incubation period revealed that TNBP was metabolized most rapidly (with a depletion rate of 73 ± 4 pmol/min/mg protein), followed by TBOEP (53 ± 8 pmol/min/mg), TCIPP (27 ± 1 pmol/min/mg), TPHP (22 ± 2 pmol/min/mg) and TDCIPP (8 ± 1 pmol/min/mg). In vitro biotransformation of OP triesters was clearly structure-dependent where non-halogenated alkyl OP triesters were metabolized more rapidly than halogenated alkyl triesters. Halogenated OP triesters were transformed to their respective diesters more efficiently relative to non-halogenated OP triesters. To our knowledge, this is the first study to investigate OP triester metabolism and OP diester formation in an avian or wildlife model system, which is important to understand the fate and biological activity of OPEs in an exposed organism. - Highlights: • The metabolism and kinetics of 6 OPEs were examined in herring gull liver microsomes. • The

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay

Energy Technology Data Exchange (ETDEWEB)

Greaves, Alana K. [Wildlife and Landscape Directorate, Science and Technology Branch, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON K1A 0H3 (Canada); Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6 (Canada); Su, Guanyong, E-mail: guanyong.su85@gmail.com [Wildlife and Landscape Directorate, Science and Technology Branch, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON K1A 0H3 (Canada); Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6 (Canada); Letcher, Robert J., E-mail: robert.letcher@canada.ca [Wildlife and Landscape Directorate, Science and Technology Branch, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON K1A 0H3 (Canada); Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6 (Canada)

2016-10-01

The in vitro biotransformation and kinetics of six organophosphate triester (OPE) flame retardants were investigated in herring gulls (Larus argentatus) from the Great Lakes using a hepatic microsomal metabolism assay. Administration of each individual OPE (tri-n-butyl phosphate (TNBP), tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), triethyl phosphate (TEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-chloroisopropyl) phosphate (TCIPP)) to the in vitro assay (concentration range 0.01 to 10 μM) resulted in rapid depletion with the exception of TEP. Following the Michaelis-Menten enzyme kinetics model, a preliminary 2-minute incubation period was used to estimate the V{sub max} (± SE) values (i.e., the maximal rate of reaction for a saturated enzyme system), which ranged from 5.0 ± 0.4 (TPHP) to 29 ± 18 pmol/min/mg protein (TBOEP), as well as the K{sub M} (± SE) values (i.e., the OPE concentration corresponding to one half of the V{sub max}), which ranged from 9.8 ± 1 (TPHP) to 189 ± 135 nM (TBOEP). Biotransformation assays over a 100-minute incubation period revealed that TNBP was metabolized most rapidly (with a depletion rate of 73 ± 4 pmol/min/mg protein), followed by TBOEP (53 ± 8 pmol/min/mg), TCIPP (27 ± 1 pmol/min/mg), TPHP (22 ± 2 pmol/min/mg) and TDCIPP (8 ± 1 pmol/min/mg). In vitro biotransformation of OP triesters was clearly structure-dependent where non-halogenated alkyl OP triesters were metabolized more rapidly than halogenated alkyl triesters. Halogenated OP triesters were transformed to their respective diesters more efficiently relative to non-halogenated OP triesters. To our knowledge, this is the first study to investigate OP triester metabolism and OP diester formation in an avian or wildlife model system, which is important to understand the fate and biological activity of OPEs in an exposed organism. - Highlights: • The metabolism and kinetics of 6 OPEs were examined in herring gull liver

Central effects of humanin on hepatic triglyceride secretion.

Science.gov (United States)

Gong, Zhenwei; Su, Kai; Cui, Lingguang; Tas, Emir; Zhang, Ting; Dong, H Henry; Yakar, Shoshana; Muzumdar, Radhika H

2015-08-01

Humanin (HN) is an endogenous mitochondria-associated peptide that has been shown to protect against various Alzheimer's disease-associated insults, myocardial ischemia-reperfusion injury, and reactive oxygen species-induced cell death. We have shown previously that HN improves whole body glucose homeostasis by improving insulin sensitivity and increasing glucose-stimulated insulin secretion (GSIS) from the β-cells. Here, we report that intraperitoneal treatment with one of HN analogs, HNG, decreases body weight gain, visceral fat, and hepatic triglyceride (TG) accumulation in high-fat diet-fed mice. The decrease in hepatic TG accumulation is due to increased activity of hepatic microsomal triglyceride transfer protein (MTTP) and increased hepatic TG secretion. Both intravenous (iv) and intracerebroventricular (icv) infusion of HNG acutely increase TG secretion from the liver. Vagotomy blocks the effect on both iv and icv HNG on TG secretion, suggesting that the effects of HNG on hepatic TG flux are centrally mediated. Our data suggest that HN is a new player in central regulation of peripheral lipid metabolism. Copyright © 2015 the American Physiological Society.

Fenproporex N-dealkylation to amphetamine--enantioselective in vitro studies in human liver microsomes as well as enantioselective in vivo studies in Wistar and Dark Agouti rats.

Science.gov (United States)

Kraemer, Thomas; Pflugmann, Thomas; Bossmann, Michael; Kneller, Nicole M; Peters, Frank T; Paul, Liane D; Springer, Dietmar; Staack, Roland F; Maurer, Hans H

2004-09-01

Fenproporex (FP) is known to be N-dealkylated to R(-)-amphetamine (AM) and S(+)-amphetamine. Involvement of the polymorphic cytochrome P450 (CYP) isoform CYP2D6 in metabolism of such amphetamine precursors is discussed controversially in literature. In this study, the human hepatic CYPs involved in FP dealkylation were identified using recombinant CYPs and human liver microsomes (HLM). These studies revealed that not only CYP2D6 but also CYP1A2, CYP2B6 and CYP3A4 catalyzed this metabolic reaction for both enantiomers with slight preference for the S(+)-enantiomer. Formation of amphetamine was not significantly changed by quinidine and was not different in poor metabolizer HLM compared to pooled HLM. As in vivo experiments, blood levels of R(-)-amphetamine and S(+)-amphetamine formed after administration of FP were determined in female Dark Agouti rats (fDA), a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats (mDA), an intermediate model, and in male Wistar rats (WI), a model of the human CYP2D6 extensive metabolizer phenotype. Analysis of the plasma samples showed that fDA exhibited significantly higher plasma levels of both amphetamine enantiomers compared to those of WI. Corresponding plasma levels in mDA were between those in fDA and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher amphetamine plasma levels, which did not significantly differ from those in fDA. The in vivo studies suggested that CYP2D6 is not crucial to the N-dealkylation but to another metabolic step, most probably to the ring hydroxylation. Further studies are necessary for elucidating the role of CYP2D6 in FP hydroxylation.

Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

International Nuclear Information System (INIS)

Meier, U.T.; Meyer, U.A.

1987-01-01

The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single [ 125 I]-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme

Seroprevalence of the Hepatitis B, Hepatitis C, and Human Immunodeficiency Viruses and Treponema pallidum at the Beijing General Hospital from 2010 to 2014: A Cross-Sectional Study.

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Shaoxia Xu

Full Text Available The hepatitis B, hepatitis C, human immunodeficiency viruses and Treponema pallidum are important causes of infectious diseases concern to public health.Between 2010 and 2014, we used an automated chemiluminescence microparticle immunoassay to detect the hepatitis B, hepatitis C, and human immunodeficiency viruses as well as Treponema pallidum (the rapid plasma regain test was used in 2010-2011. Positive human immunodeficiency virus tests were confirmed via western blotting.Among 416,130 subjects, the seroprevalences for hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and Treponema pallidum were 5.72%, 1.23%, 0.196%, and 0.76%, respectively. Among 671 patients with positive human immunodeficiency virus results, 392 cases were confirmed via western blotting. Hepatitis B and human immunodeficiency virus infections were more frequent in men (7.78% and 0.26%, respectively than in women (4.45% and 0.021%, respectively. The hepatitis B and C virus seroprevalences decreased from 6.21% and 1.58%, respectively, in 2010, to 5.37% and 0.988%, respectively, in 2014. The human immunodeficiency virus seroprevalence increased from 0.04% in 2010 to 0.17% in 2014, and was elevated in the Infectious Disease (2.65%, Emergency (1.71%, and Dermatology and Sexually Transmitted Diseases (1.12% departments. The specificity of the human immunodeficiency virus screening was 71.4%. The false positive rates for the Treponema pallidum screening tests increased in patients who were 60-70 years old. The co-infection rates for the hepatitis C and human immunodeficiency viruses were 0.47% in hepatitis C virus-positive patients and 7.33% in human immunodeficiency virus-positive patients.During 2010-2014, hepatitis B virus and human immunodeficiency virus infections were more frequent among men at our institution. Although the seroprevalences of hepatitis B and C viruses decreased, the seroprevalence of human immunodeficiency virus infection increased (with

Microsomal receptor for steroid hormones: functional implications for nuclear activity.

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Muldoon, T G; Watson, G H; Evans, A C; Steinsapir, J

1988-01-01

Target tissues for steroid hormones are responsive by virtue of and to the extent of their content of functional intracellular receptors. Recent years have seen a shift in considerations of the cellular dynamics and distribution of these receptors, with current views favoring predominant intranuclear localization in the intact cell. This paper summarizes our analyses of the microsomal estrogen and androgen binding capability of rat uterine and ventral prostate tissue, respectively; these studies have revealed a set of high affinity sites that may act as a conduit for estrogen traversing the cell en route to the nucleus. These sites have many properties in common with cytosolic receptors, with the salient difference of a failure to activate to a more avid DNA-binding form under conditions which permit such activation of cytosolic receptors. The microsomal estrogen-binding proteins also have appreciable affinity for progesterone, another distinction from other known cellular estrogen receptor species. Various experimental approaches were employed to demonstrate that the microsomal receptors were not simply cytosol contaminants; the most convincing evidence is the recent successful separation of the cytosolic and microsomal forms by differential ammonium sulfate precipitation. Discrete subfractionation of subcellular components on successive sucrose gradients, with simultaneous assessments of binding capability and marker enzyme concentrations, indicates that the major portion of the binding is localized within the vesicles of the endoplasmic reticulum free of significant plasma membrane contamination. The microsomal receptors are readily solubilized by extraction with high- or low-salt-containing buffers or with steroid. The residual microsomes following such extraction have the characteristics of saturable acceptor sites for cytosolic estrogen-receptor complexes. The extent to which these sites will accept the cytosolic complexes is equal to the concentration of

Formation of glutathione conjugates by reactive metabolites of vinylidene chloride in microsomes and isolated hepatocytes

International Nuclear Information System (INIS)

Liebler, D.C.; Meredith, M.J.; Guengerich, F.P.

1985-01-01

Oxidation of the vinyl halide carcinogen and hepatotoxin vinylidene chloride (VDC) by microsomal cytochrome P-450 yields 2,2-dichloroacetaldehyde, 2-chloroacetyl chloride, 2-chloroacetic acid, and 1,1-dichloroethylene oxide. The roles of these metabolites in covalent modification of proteins and reduced glutathione (GSH) were examined. 2-Chloroacetyl chloride reacted with model thiols at least 10(3)-fold faster than did 1,1-dichloroethylene oxide and at least 10(5)-fold faster than did 2,2-dichloroacetaldehyde or 2-chloroacetic acid. Microsomal covalent binding of [ 14 C]VDC was inhibited by GSH but not by lysine, suggesting that protein thiols, rather than amino groups, are major targets. Liver microsomes catalyzed the formation of three GSH:VDC metabolite conjugates, identified as S-(2,2-dichloro-1-hydroxy)ethylglutathione, 2-(S-glutathionyl)acetate, and S-(2-glutathionyl)acetylglutathione, a novel conjugate containing both stable (thioether) and labile (thioester) linkages. The latter two conjugates also were formed in isolated rat hepatocytes and measurable amounts of 2-(S-glutathionyl)acetate were released into the incubation medium. Both 2-(S-glutathionyl)acetate and S-(2-glutathionyl)acetylglutathione were formed with [ 35 S]GSH added to the hepatic medium, indicating that reactive VDC metabolites are capable of crossing the plasma membrane to react with extracellular targets. Unlabeled S-(2-glutathionyl)-acetylglutathione underwent carbonyl substitution with added [ 35 S]GSH, suggesting that this conjugate may participate in modification of protein thiols. This conjugate also underwent hydrolysis with a half-life of approximately 3 hr. GSH:VDC metabolite conjugates may serve as accessible models for labile covalent adducts formed between VDC metabolites and protein thiols

Effects of sh-reagents on rat hepatic aldehyde dehydrogenase activity

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Konoplitskaya, K.L.; Kuz' mina, G.I.; Grigor' yeva, M.V.; Poznyakova, T.N.

The liver serves as the primary organ for the oxidation of ingested ethanol via a pathway involving alcohol- and aldehyde dehydrogenase. In view of the problem of alcoholism, three enzymes are of particular interest in understanding the biochemical mechanism that may be involved in alcohol addiction and in the formulation of therapeutic approaches. While alcohol dehydrogenase has been studied in considerable detail, current attention is centered on aldehyde dehydrogenase. A comparative analysis of the effects of a series of SH-active reagents - tetraethylthiuram disulfide (TETD), 5,5-dithiobisnitrobenzoic acid (DTNB), p-chloromercurybenzoate (PCMB), and N-ethylmaleimide (NEM) - were tested for their effects on the activity of aldehyde dehydrogenase of the hepatic mitochondrial (isozymes I and II) and microsomal (isozyme II) fractions of outbred albino rats. DTNB was found to be inhibited by 100 and 50% mitochondrial isozymes I and II, respectively, and by 20%, the microsomal enzyme under the conditions employed. DTNB and NEM inhibited by 30 and 50% isozymes I and II of the mitochondria, but had no effect on the microsomal isozyme. 24 references, 3 figures.

Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

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Russell, Julie S; Halbrook, Richard S; Woolf, Alan; French, John B; Melancon, Mark J

2004-08-01

We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

Increased hepatic nicotine elimination after phenobarbital induction in the conscious rat

International Nuclear Information System (INIS)

Foth, H.; Walther, U.I.; Kahl, G.F.

1990-01-01

Elimination parameters of [14C]nicotine in conscious rats receiving nicotine (0.3 mg/kg) either intravenously or orally were studied. The oral availability of unchanged nicotine, derived by comparison of the respective areas under the concentration vs time curves (AUC), was 89%, indicating low hepatic extraction ratios of about 10%. Pretreatment of rats with phenobarbital (PB) markedly increased hepatic first-pass extraction of nicotine. The oral availability of unchanged nicotine in plasma dropped to 1.4% of the corresponding values obtained from PB-treated rats receiving nicotine iv. After PB pretreatment, the clearance of iv nicotine was increased approximately twofold over controls, much less than the observed more than ninefold increase of hepatic first-pass extraction. It is assumed that extrahepatic metabolism contributed significantly to the rapid removal of nicotine from the plasma. The elimination of cotinine, originating from nicotine administered either po or iv, was significantly increased by PB pretreatment, as determined by the ratio of corresponding AUCs. The pattern of nicotine metabolites in urine also indicated an increase in the rate of cotinine metabolic turnover. The amount of norcotinine in the organic extract of urine paralleled PB microsomal enzyme induction. The ratio between urinary concentrations of the normetabolite and cotinine correlated strongly with the PB-induced state of rat liver. This may be a suitable indicator of PB-inducible hepatic cytochrome P450 isoenzyme(s). Since smoking habits in man are feedback-regulated by nicotine plasma concentrations, a similar increase of nicotine elimination by microsomal enzyme induction in man may be of relevance for tobacco consumption

Mutagenic activation and detoxification of benzo[a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals.

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Darwish, W; Ikenaka, Y; Eldaly, E; Ishizuka, M

2010-01-01

The mutagenic activation activity of hepatic microsomes from three meat-producing animals (cattle, deer and horses) was compared with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using alpha-naphthoflavone, anti-rat CYP1A1, CYP3A2 and CYP2E1 antibodies suggests that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucoronosyl transferase and cytosolic glutathione-S-transferase, reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. This report is the first to investigate the mutagenic activation activity of the hepatic microsomes and the role of phase II enzymes against this activity in meat-producing animals. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Soybean meal fermented by Aspergillus awamori increases the cytochrome P-450 content of the liver microsomes of mice.

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Kishida, T; Ataki, H; Takebe, M; Ebihara, K

2000-04-01

The effect of soybean meal fermented by Aspergillus awamori on the acute lethality of acetaldehyde, pentobarbital sleeping time, and cytochrome P-450 content of the hepatic microsomes was studied in mice. Most of the daidzin and genistin in soybean meal (SBM) were converted into the respective aglycones, daidzein and genistein, by fermentation. In experiment 1, mice were fed isonitrogenic test diets with one of the following five protein sources for 28 d: casein, SBM, fermented and hot-air-dried SBM (FSBM-HD), fermented and freeze-dried SBM (FSBM-FD), or methanol-extracted FSBM-FD (FSMB-FD-R). The acute lethality of acetaldehyde in mice fed the FSBM-FD diet was significantly lower than that in mice fed the SBM, FSBM-HD, or FSBM-FD-R diet. In experiments 2 and 3, mice were fed isonitrogenic test diets with one of the following four protein sources for 28 d: casein, SBM, FSBM-FD, and FSBM-FD-R. The pentobarbital sleeping time was significantly shorter and the cytochrome P-450 content was significantly higher in the mice fed the FSBM-FD diet than the respective value in mice fed the other test diets. In experiment 4, mice were fed one of eight diets which contained different levels of aglycone obtained by varying the proportion of FSBM-FD and FSBM-FD-R, for 28 d. The cytochrome P-450 content in hepatic microsomes increased as the dietary level of isoflavonoid aglycones increased, but there was a saturation phenomenon. These results suggest that soy isoflavonoid aglycones are more potent inducers of cytochrome P-450 than isoflavonoid glycosides.

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

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Kukiełka, E; Cederbaum, A I

1995-04-15

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

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Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

2012-04-01

Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

Urinary Elimination of Bile Acid Glucuronides under Severe Cholestatic Situations: Contribution of Hepatic and Renal Glucuronidation Reactions

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Martin Perreault

2018-01-01

Full Text Available Biliary obstruction, a severe cholestatic complication, causes accumulation of toxic bile acids (BAs in liver cells. Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT enzymes, detoxifies cholestatic BAs. Using liquid chromatography coupled to tandem mass spectrometry, 11 BA glucuronide (-G species were quantified in prebiliary and postbiliary stenting serum and urine samples from 17 patients with biliary obstruction. Stenting caused glucuronide- and fluid-specific changes in BA-G levels and BA-G/BA metabolic ratios. In vitro glucuronidation assays with human liver and kidney microsomes revealed that even if renal enzymes generally displayed lower KM values, the two tissues shared similar glucuronidation capacities for BAs. By contrast, major differences between the two tissues were observed when four human BA-conjugating UGTs 1A3, 1A4, 2B4, and 2B7 were analyzed for mRNA and protein levels. Notably, the BA-24G producing UGT1A3 enzyme, abundant in the liver, was not detected in kidney microsomes. In conclusion, the circulating and urinary BA-G profiles are hugely impacted under severe cholestasis. The similar BA-glucuronidating abilities of hepatic and renal extracts suggest that both the liver and kidney may contribute to the urine BA-G pool.

Autoimmune hepatitis.

Science.gov (United States)

Vergani, D; Mieli-Vergani, G

2004-06-01

Autoimmune hepatitis (AIH) is characterised histologically by interface hepatitis, and serologically by the presence of non-organ and liver specific autoantibodies and increased levels of immunoglobulin G. Its onset is often ill-defined, frequently mimicing acute hepatitis. AIH usually responds to immunosuppressive treatment, which should be instituted as soon as diagnosis is made. Two types of AIH are recognized according to seropositivity for smooth muscle and/or antinuclear antibody (SMA/ANA, type 1 AIH) or liver kidney microsomal type 1 antibody (LKM1, type 2 AIH). There is a female predominance in both. LKM1 positive patients tend to present more acutely, at a younger age and commonly have immunoglobulin A deficiency, while duration of symptoms before diagnosis, clinical signs, family history of autoimmunity, presence of associated autoimmune disorders, response to treatment and long-term prognosis are similar in the 2 groups. Susceptibility to AIH type 1 is conferred by possession of HLA DR3 and DR4, while to AIH type 2 by possession of HLA DR7. Liver damage is likely to derive from an immune reaction to liver cell antigens, possibly triggered by a mechanism of molecular mimicry, where immune responses to external pathogens, e.g. viruses, become directed towards structurally similar self-components. In AIH this process would be perpetuated by impairment in immune regulation.

Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

International Nuclear Information System (INIS)

Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

1985-01-01

The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H 2 O 2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization

An integrated study for the utilization of anthraquinone compounds extract “Heshouwu” In vivo and their comparative metabolism in liver microsomes using UPLC-ESI-Q-TOF/MSn

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Sha Chen

2018-01-01

Full Text Available Objective: Anthraquinone (AQ, a major bioactive component of the traditional Chinese medicine HeShouWu, has widespread applications in industry and medicine. The objective of the current study is to explore the differences in the bioavailability of anthraquinones in vivo and the metabolism in liver microsomes. Materials and Methods: In vivo, we used a reliable UPLC-ESI-QqQ-MS/MS method to measure seven AQ compounds in the jugular vein plasma of rats following oral administration of HeShouWu. Furthermore, in order to quantify the bioavailability of AQs in vivo and to further understand the metabolism of these compounds, we compared the in vitro metabolism of AQ in different species with respect to metabolic profiles, the enzymes involved, and catalytic efficiency using liver microsomes from human (HLM, mouse (MLM, rat (RLM, and beagle dog (DLM. Results: We identified two metabolic pathways, including the hydroxylation and glucuronidation of AQ, in the liver microsomes of humans and other species using UPLC-ESI-Q-TOF. We found that substitutions on the AQ ring were crucial to the activity and regioselectivity of its hydroxylation. In general, hydroxylation activity decreased greatly with β-COOH (rhein and enhanced dramatically with β-OH (emodin. We also found that glucuronidation of the compound emodin-8-O-β-D-glucoside acts as the main isoform in AQ hydroxylation in HLM and DLM. Total microsomal intrinsic clearance values for AQ were greatest in mouse microsomes, followed by those in dog, human, and rat microsomes. Conclusion: The absorption of different anthrquinone compounds varied based on the compound structure, the metabolism types and products of anthraquinones in liver microsomes were different in different species. These findings provide vital information for a deeper unuunderstanding of the metabolism of AQs.

Human Placenta Extract Therapy for Feline Hepatic Lipidosis

OpenAIRE

2018-01-01

Feline hepatic lipidosis (HL), the most common hepatobiliary disease in cats, is characterized by the accumulation of excessive triglycerides (TGs) in more than 80% of hepatocytes. Forced oral feeding is recommended as the only therapy for this disease but the prognosis is often poor. As human placenta extract (Laennec) has been used to improve hepatic metabolism, we investigated the efficacy of this drug for the treatment of cats with HL. Ten cats diagnosed with HL in this study were treated...

Microsomal lipid peroxidation as a mechanism of cellular damage. [Dissertation

Energy Technology Data Exchange (ETDEWEB)

Kornbrust, D.J.

1979-01-01

The NADPH/iron-dependent peroxidation of lipids in rat liver microsomes was found to be dependent on the presence of free ferrous ion and maintains iron in the reduced Fe/sup 2 +/ state. Chelation of iron by EDTA inhibited peroxidation. Addition of iron, after preincubation of microsomes in the absence of iron, did not enhance the rate of peroxidation suggesting that iron acts by initiating peroxidative decomposition of membrane lipids rather than by catalyzing the breakdown of pre-formed hydroperoxides. Liposomes also underwent peroxidation in the presence of ferrous iron at a rate comparable to intact microsomes and was stimulated by ascorbate. Carbon tetrachloride initiated lipid peroxidation in the absence of free metal ions. Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely between different tissues and species. The effects of paraquat on lipid peroxidation was also studied. (DC)

Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes.

Science.gov (United States)

Yang, Fang; Yang, Hong; Ramesh, Aramandla; Goodwin, J Shawn; Okoro, Emmanuel U; Guo, ZhongMao

2016-01-01

We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and

Tribbles-1: a novel regulator of hepatic lipid metabolism in humans.

Science.gov (United States)

Bauer, Robert C; Yenilmez, Batuhan O; Rader, Daniel J

2015-10-01

The protein tribbles-1, encoded by the gene TRIB1, is increasingly recognized as a major regulator of multiple cellular and physiological processes in humans. Recent human genetic studies, as well as molecular biological approaches, have implicated this intriguing protein in the aetiology of multiple human diseases, including myeloid leukaemia, Crohn's disease, non-alcoholic fatty liver disease (NAFLD), dyslipidaemia and coronary artery disease (CAD). Genome-wide association studies (GWAS) have repeatedly identified variants at the genomic TRIB1 locus as being significantly associated with multiple plasma lipid traits and cardiovascular disease (CVD) in humans. The involvement of TRIB1 in hepatic lipid metabolism has been validated through viral-mediated hepatic overexpression of the gene in mice; increasing levels of TRIB1 decreased plasma lipids in a dose-dependent manner. Additional studies have implicated TRIB1 in the regulation of hepatic lipogenesis and NAFLD. The exact mechanisms of TRIB1 regulation of both plasma lipids and hepatic lipogenesis remain undetermined, although multiple signalling pathways and transcription factors have been implicated in tribbles-1 function. Recent reports have been aimed at developing TRIB1-based lipid therapeutics. In summary, tribbles-1 is an important modulator of human energy metabolism and metabolic syndromes and worthy of future studies aimed at investigating its potential as a therapeutic target. © 2015 Authors; published by Portland Press Limited.

Feline hepatic biotransformation of diazepam: Differences between cats and dogs.

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van Beusekom, Cyrina D; van den Heuvel, Jeroen J M W; Koenderink, Jan B; Russel, Frans G M; Schrickx, Johannes A

2015-12-01

In contrast to humans and dogs, diazepam has been reported to induce severe hepatic side effects in cats, particularly after repeated dosing. With the aim to elucidate the mechanisms underlying this apparent sensitivity of cats to drug-induced liver injury, in a series of in vitro experiments, the feline-specific biotransformation of diazepam was studied with liver microsomes obtained from cats and dogs and the possible inhibition of the bile salt export pump (Bsep) was measured in isolated membrane vesicles overexpressing feline and canine Bsep. In line with previous in vivo studies, the phase I metabolites nordiazepam, temazepam and oxazepam were measurable in microsomal incubations, although enzyme velocity of demethylases and hydroxylases differed significantly between cats and dogs. In cats, the main metabolite was temazepam, which also could be glucuronidated. In contrast to dogs, no other glucuronidated metabolites could be observed. In addition, in the membrane vesicles an inhibition of the transport of the Bsep substrate taurocholic acid could be observed in the presence of diazepam and its metabolites. It was concluded that both mechanisms, the slow biotransformation of diazepam as well the inhibition of the bile acid efflux that results in an accumulation of bile acids in the hepatocytes, seem to contribute to the liver injury observed in cats following repetitive treatment with diazepam. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mechanism of microsomal metabolism of benzene to phenol

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Hinson, J.A.; Freeman, J.P.; Potter, D.W.; Mitchum, R.K.; Evans, F.E.

1985-05-01

The mechanism of microsomal hydroxylation of benzene to phenol has been studied by examining the microsomal metabolism of the specifically deuterated derivative 1,3,5-(/sub 2/H/sup 3/)benzene. Evidence for the formation of the following four products was obtained: 2,3,5-(/sub 2/H/sup 3/)phenol, 3,5-(/sub 2/H/sup 2/)phenol, 2,4,6-(/sub 2/H/sup 3/)phenol, and 2,4-(/sub 2/H/sup 2/)phenol. The presence of 2,3,5-(2H3)phenol and 2,4-(/sub 2/H/sup 2/)phenol shows that, in the microsomal metabolism of benzene to phenol, a NIH shift had occurred. A deuterium isotope effect (kH/kD) of approximately 4 was detected in both the meta- and para-deuterated phenols. This finding indicates that cyclohexadienone, formed either by isomerization of the epoxide or directly from the enzyme-substrate complex, is a major intermediate in the metabolism of benzene to phenol.

Human plasma concentrations of tolbutamide and acetaminophen extrapolated from in vivo animal pharmacokinetics using in vitro human hepatic clearances and simple physiologically based pharmacokinetic modeling for radio-labeled microdose clinical studies

International Nuclear Information System (INIS)

Yamazaki, Hiroshi; Kunikane, Eriko; Nishiyama, Sayako; Murayama, Norie; Shimizu, Makiko; Sugiyama, Yuichi; Chiba, Koji; Ikeda, Toshihiko

2015-01-01

The aim of the current study was to extrapolate the pharmacokinetics of drug substances orally administered in humans from rat pharmacokinetic data using tolbutamide and acetaminophen as model compounds. Adjusted animal biomonitoring equivalents from rat studies based on reported plasma concentrations were scaled to human biomonitoring equivalents using known species allometric scaling factors. In this extrapolation, in vitro metabolic clearance data were obtained using liver preparations. Rates of tolbutamide elimination were roughly similar in rat and human liver microsome experiments, but acetaminophen elimination by rat liver microsomes and cytosolic preparations showed a tendency to be faster than those in humans. Using a simple physiologically based pharmacokinetic (PBPK) model, estimated human plasma concentrations of tolbutamide and acetaminophen were consistent with reported concentrations. Tolbutamide cleared in a roughly similar manner in humans and rats, but medical-dose levels of acetaminophen cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in rats. The data presented here illustrate how pharmacokinetic data in combination with a simple PBPK model can be used to assist evaluations of the pharmacological/toxicological potential of new drug substances and for estimating human radiation exposures from radio-labeled drugs when planning human studies. (author)

ROLE OF LEPTIN ON CYTOCHROME P-450 AND SOME LIVER MICROSOMAL ENZYMES ACTIVITIES IN THE OBESE AND LEAN MICE

International Nuclear Information System (INIS)

HEBEISHY, M.I.A.; MAZEN, G.M.A.; SHAHIN, M.I

2008-01-01

Leptin is a hormone that is secreted by adipocytes and regulates body weight through its effect on satiety and energy metabolism. The obese mouse is deficient in this protein and is characterized by obesity and other metabolic disorders. This study investigated the alterations of several hepatic cytochrome P 4 -5 0 (CYP), conjugation and antioxidant enzymes in lean and obese mice and the role of leptin in the modulation of these enzymes. Lean and obese male mice were injected with leptin (100 μg / rat) for 15 days. The obtained results revealed that administration of leptin to lean mice caused a significant elevation in the level of blood glucose, serum insulin, 6α, 6β, 16α- hydroxylation of testosterone, the activity of CYP 1 A 1 , CYP 4 A and GSH reductase in liver microsomes while serum corticosterone and the activity of total GSH were significantly decreased when compared to lean control mice. Moreover, obese mice treated with leptin recorded significant reduction in body weight, blood glucose concentration, serum levels of insulin and corticosterone, 7α and 16α- hydroxylation of testosterone, the activity of CYP 1A 1, CYP 2 B 1 and CYP 4 A and GST in liver microsomes. On the other hand, 6α, 6β-hydroxylation of testosterone, the activity of CYP 2 E 1 and GSH reductase in liver microsome were significantly increased when compared to obese control mice. The mechanism for the observed alterations may be due to direct leptin effects or via indirect alterations in insulin, corticosterone and/or growth hormone

Human Interferon Alpha2a as Anti Hepatitis B and C

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Ratih A. Ningrum

2017-12-01

Full Text Available Hepatitis is an inflammation of the liver mainly caused by hepatitis viruses. There are 5 different types of hepatitis based on the infecting virus; A, B, C, D and E. Hepatitis B and C are chronic diseases that potentially develop into hepatocarcinoma and cirrhosis on unappropriate treatments. World Health Organization (WHO stated that currently 350 million people worldwide are living with chronic hepatitis B and 150 million people are living with Hepatitis C. The mortality rate in the world due to hepatitis is about 1.5 million people per year. The human interferon alpha2a (hIFNα2a is a therapeutic protein used as therapeutic protein for hepatitis B and C. This review discusses the hepatitis B (HBV and C (HCV viruses, mechanisms of hIFNα2a as antivirus through signal transduction pathway and improvement of hIFNα2a properties by protein modification. The application of recombinant hIFNα2a (rhIFNα2a in the treatment of hepatitis B and C that recommended by European Association for The Study of Liver (EASL and the viral resistance mechanism are also included. The status of hepatitis B and C and the development of rhIFNα2a is also described as well.

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity.

Science.gov (United States)

Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Fallon, John K; Barber, Jill; Smith, Philip C; Rostami-Hodjegan, Amin; Goosen, Theunis C

2017-10-01

Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes ( n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.).

Science.gov (United States)

Guo, Dianjing; Chen, Fang; Dixon, Richard A

2002-11-01

Microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.) contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic acid 3-O-methyltransferase (COMT), and catalyzed the S-adenosyl L-methionine (SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was 5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of monolignol precursors was observed in microsomal preparations from transgenic alfalfa down-regulated in COMT expression. In most microsomal preparations, the level of the metabolic conversions was independent of added recombinant COMT. Taken together, the data provide only limited support for the concept of metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.

Up-regulation of hepatic Acyl CoA: Diacylglycerol acyltransferase-1 (DGAT-1) expression in nephrotic syndrome.

Science.gov (United States)

Vaziri, Nosratola D; Kim, Choong H; Phan, Dennis; Kim, Sara; Liang, Kaihui

2004-07-01

Nephrotic syndrome is associated with hypercholesterolemia, hypertriglyceridemia, and marked elevations of plasma low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Hypertriglyceridemia in nephrotic syndrome is accompanied by increased hepatic fatty acid synthesis, elevated triglyceride secretion, as well as lipoprotein lipase, VLDL-receptor, and hepatic triglyceride lipase deficiencies, which lead to impaired clearance of triglyceride-rich lipoproteins. Acyl CoA: diacylglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl CoA to 1, 2-diacylglycerol to form triglyceride. Two distinct DGATs (DGAT-1 and DGAT2) have recently been identified in the liver and other tissues. The present study tested the hypothesis that the reported increase in hepatic triglyceride secretion in nephrotic syndrome may be caused by up-regulation of DGAT. Male Sprague-Dawley rats were rendered nephrotic by two sequential injections of puromycin aminonucleoside (130 mg/kg on day 1 and 60 mg/kg on day 14) and studied on day 30. Placebo-treated rats served as controls. Hepatic DGAT-1 and DGAT-2 mRNA abundance and enzymatic activity were measured. The nephrotic group exhibited heavy proteinuria, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, and marked elevation of VLDL concentration. Hepatic DGAT-1 mRNA, DGAT-1, and total DGAT activity were significantly increased, whereas DGAT-2 mRNA abundance and activity were unchanged in the nephrotic rats compared to the control animals. The functional significance of elevation of DGAT activity was illustrated by the reduction in microsomal free fatty acid concentration in the liver of nephrotic animals. Nephrotic syndrome results in up-regulation of hepatic DGAT-1 expression and activity, which can potentially contribute to the associated hypertriglyceridemia by enhancing triglyceride synthesis. Thus, it appears that both depressed catabolism and increased synthetic capacity contribute to

AUTOIMMUNE HEPATITIS

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Yusri Dianne Jurnalis

2010-05-01

Full Text Available AbstrakHepatitis autoimun merupakan penyakit inflamasi hati yang berat dengan penyebab pasti yang tidak diketahui yang mengakibatkan morbiditas dan mortalitas yang tinggi. Semua usia dan jenis kelamin dapat dikenai dengan insiden tertinggi pada anak perempuan usia prepubertas, meskipun dapat didiagnosis pada usia 6 bulan. Hepatitis autoimun dapat diklasifikasikan menjadi 2 bagian berdasarkan adanya antibodi spesifik: Smooth Muscle Antibody (SMA dengan anti-actin specificity dan/atau Anti Nuclear Antibody (ANA pada tipe 1 dan Liver-Kidney Microsome antibody (LKM1 dan/atau anti-liver cytosol pada tipe 2. Gambaran histologisnya berupa “interface hepatitis”, dengan infiltrasi sel mononuklear pada saluran portal, berbagai tingkat nekrosis, dan fibrosis yang progresf. Penyakit berjalan secara kronik tetapi keadaan yang berat biasanya menjadi sirosis dan gagal hati.Tipe onset yang paling sering sama dengan hepatitis virus akut dengan gagal hati akut pada beberapa pasien; sekitar sepertiga pasien dengan onset tersembunyi dengan kelemahan dan ikterik progresif ketika 10-15% asimptomatik dan mendadak ditemukan hepatomegali dan/atau peningkatan kadar aminotransferase serum. Adanya predominasi perempuan pada kedua tipe. Pasien LKM1 positif menunjukkan keadaan lebih akut, pada usia yang lebih muda, dan biasanya dengan defisiensi Immunoglobulin A (IgA, dengan durasi gejala sebelum diagnosis, tanda klinis, riwayat penyakit autoimun pada keluarga, adanya kaitan dengan gangguan autoimun, respon pengobatan dan prognosis jangka panjang sama pada kedua tipe.Kortikosteroid yang digunakan secara tunggal atau kombinasi azathioprine merupakan terapi pilihan yang dapat menimbulkan remisi pada lebih dari 90% kasus. Strategi terapi alternatif adalah cyclosporine. Penurunan imunosupresi dikaitkan dengan tingginya relap. Transplantasi hati dianjurkan pada penyakit hati dekom-pensata yang tidak respon dengan pengobatan medis lainnya.Kata kunci : hepatitis Autoimmune

Hepatic differentiation potential of commercially available human mesenchymal stem cells.

Science.gov (United States)

Ong, Shin-Yeu; Dai, Hui; Leong, Kam W

2006-12-01

The ready availability and low immunogenicity of commercially available mesenchymal stem cells (MSC) render them a potential cell source for the development of therapeutic products. With cell source a major bottleneck in hepatic tissue engineering, we investigated whether commercially available human MSC (hMSC) can transdifferentiate into the hepatic lineage. Based on previous studies that find rapid gain of hepatic genes in bone marrow-derived stem cells cocultured with liver tissue, we used a similar approach to drive hepatic differentiation by coculturing the hMSC with rat livers treated or untreated with gadolinium chloride (GdCl(3)). After a 24-hour coculture period with liver tissue injured by GdCl(3) in a Transwell configuration, approximately 34% of the cells differentiated into albumin-expressing cells. Cocultured cells were subsequently maintained with growth factors to complete the hepatic differentiation. Cocultured cells expressed more hepatic gene markers, and had higher metabolic functions and P450 activity than cells that were only differentiated with growth factors. In conclusion, commercially available hMSC do show hepatic differentiation potential, and a liver microenvironment in culture can provide potent cues to accelerate and deepen the differentiation. The ability to generate hepatocyte-like cells from a commercially available cell source would find interesting applications in liver tissue engineering.

Sero-prevalence of Human Immunodeficiency Virus and hepatitis ...

African Journals Online (AJOL)

Sero-prevalence of Human Immunodeficiency Virus and hepatitis viruses and their correlation with CD4 T-cell lymphocyte counts in pregnant women in the Buea Health District of Cameroon. Rebecca Enow Tanjong, Pride Teyim, Henry Lucien Kamga, Edwin Suh Neba, Theresia Nkuo-Akenji ...

Prevalence of hepatitis C Antibody in Human Immunodeficiency ...

African Journals Online (AJOL)

Background: Hepatitis C virus (HCV) is a major public health problem for Human Immunodeficiency virus (HIV) infected population. Both infections share same routes of transmission, and quite often co-exist, with dual infections associated with reciprocal and mutually more rapid progression than either infection alone.

One window-period donation in two years of individual donor-nucleic acid test screening for hepatitis B, hepatitis C and human immunodeficiency virus

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Jose Eduardo Levi

2013-06-01

Full Text Available Objective: To describe general data on nucleic acid/serology testing and report the first hepatitis B-nucleic acid testing yield case of an immunized donor in Brazil. Methods: A total of 24,441 donations collected in 2010 and 2011 were submitted to individual nucleic acid testing for hepatitis B, hepatitis C and human immunodeficiency virus using the TaqMan® MPX kit (Roche on the Cobas s201 platform, in addition to routine screening for serological markers. Nucleic acid testing-reactive donations were further evaluated by real-time polymerase chain reaction using Cobas AmpliPrep/Cobas TaqMan hepatitis B virus, hepatitis C virus and human immunodeficiency virus tests. Results: Thirty-two donations were reactive by nucleic acid testing, 31 were also serologically reactive and one first-time donor was identified as having hepatitis B in the window period. Follow-up samples showed increasing titers of anti-HBs rising from 19 UI/mL in the index donation to 109 IU/mL seven months later attributable to his vaccination history. Curiously, this donor was never reactive for HbsAg nor for anti-HBc. In the yield donation, he was concomitantly reactive for syphilis (enzyme immunoassay and fluorescent treponemal antibody-absorption; venereal disease research laboratory non-reactive. Overall, six donors (0.02% were characterized as occult hepatitis B. A total of 35% of the confirmed (recombinant immunoblot assay positive hepatitis C donations were nucleic acid testing non-reactive and no human immunodeficiency virus "elite controller" was identified. Conclusion: The yield rate (1:24,441; 95% confidence interval: 1:9,537 - 1:89,717 contrasts to the North American rate (1:410,540 donations and strongly advocates the adoption of nucleic acid testing for hepatitis B in Brazil despite the increasing rate of anti-HBs reactive subjects due to the successful immunization program.

Radioimmunoassay and some properties of human antibodies to hepatitis B core antigen

Energy Technology Data Exchange (ETDEWEB)

Neurath, A R; Szmuness, W; Stevens, C E; Strick, N; Harley, E J [New York Blood Center, N.Y. (USA)

1978-03-01

A solid-phase radioimmunoassay for antibodies to hepatitis B core antigen (anti-HBsub(c)) is described. Polystyrene beads coated with anti-HBsub(c), hepatitis B core antigen prepared from pooled sera of humans infected with hepatitis B virus (HBV) and /sup 125/I-labelled anti-HBsub(c) were used for the test. Distinct patterns of development and changes of anti-HBsub(c) and their immunological properties are all related to variations of other markers specific for HBV infections. Knowledge concerning the detailed features of the immune response to hepatitis B core antigen may provide deeper insight into the pathogenesis of HBV infections.

Role of CYP2B6 and CYP3A4 in the in vitro N-dechloroethylation of (R)- and (S)-ifosfamide in human liver microsomes.

Science.gov (United States)

Granvil, C P; Madan, A; Sharkawi, M; Parkinson, A; Wainer, I W

1999-04-01

The central nervous system toxicity of ifosfamide (IFF), a chiral antineoplastic agent, is thought to be dependent on its N-dechloroethylation by hepatic cytochrome P-450 (CYP) enzymes. The purpose of this study was to identify the human CYPs responsible for IFF-N-dechloroethylation and their corresponding regio- and enantioselectivities. IFF exists in two enantiomeric forms, (R) - and (S)-IFF, which can be dechloroethylated at either the N2 or N3 positions, producing the corresponding (R,S)-2-dechloroethyl-IFF [(R, S)-2-DCE-IFF] and (R,S)-3-dechloroethyl-IFF [(R,S)-3-DCE-IFF]. The results of the present study suggest that the production of (R)-2-DCE-IFF and (S)-3-DCE-IFF from (R)-IFF is catalyzed by different CYPs as is the production of (S)-2-DCE-IFF and (R)-3-DCE-IFF from (S)-IFF. In vitro studies with a bank of human liver microsomes revealed that the sample-to-sample variation in the production of (S)-3-DCE-IFF from (R)-IFF and (S)-2-DCE-IFF from (S)-IFF was highly correlated with the levels of (S)-mephenytoin N-demethylation (CYP2B6), whereas (R)-2-DCE-IFF production from (R)-IFF and (R)-3-DCE-IFF production from (S)-IFF were both correlated with the activity of testosterone 6beta-hydroxylation (CYP3A4/5). Experiments with cDNA-expressed P-450 and antibody and chemical inhibition studies supported the conclusion that the formation of (S)-3-DCE-IFF and (S)-2-DCE-IFF is catalyzed primarily by CYP2B6, whereas (R)-2-DCE-IFF and (R)-3-DCE-IFF are primarily the result of CYP3A4/5 activity.

Human immunodeficiency virus (HIV) seropositivity and hepatitis B ...

African Journals Online (AJOL)

Method: A total of 130 donors comprising 120 commercial donors and 10 voluntary donors were tested for antibodies to human immunodeficiency virus and hepatitis B surface antigen in Benin city using Immunocomb HIV - 1 and 2 Biospot kit and Quimica Clinica Aplicada direct latex agglutination method respectively.

[Comparative metabolism of three amide alkaloids from Piper longum in five different species of liver microsomes].

Science.gov (United States)

He, Huan; Guo, Wei-Wei; Chen, Xiao-Qing; Zhao, Hai-Yu; Wu, Xia

2016-08-01

Piperine, piperlonguminine and pellitorine are three major amide alkaloids from Piper longum, showing a variety of pharmacological activities. In order to investigate the different metabolism pathways of these compounds in five species of liver microsomes in vitro, the data of full mass spectrum, and MS2, MS3 spectra of these three alkaloids were collected and analyzed by using ultra-high-performance liquid chromatography coupled with a LTQ-orbitrap mass spectrometer (UHPLC-LTQ-Orbitrap MS); gragment ion information was collected and combined with fragmentation regularities of mass spectra and accurate mass spectrometry data of metabolites, to compare the metabolism difference of three amide alkaloids in liver microsomes of human, rhesus monkey, Beagle dogs, rats and mice. 3 metabolites of piperine, 2 metabolites of piperlonguminine and 1 metabolite of pellitorine were identified quickly. The results showed that the major metabolic pathways of these amide alkaloids in liver microsomes were methylenedioxy group demethylation and oxidation reaction, and metabolic rates were different between species. This study provides basis for further research on in vivo metabolism of piperine analogues from Piper longum. Copyright© by the Chinese Pharmaceutical Association.

Counter-attack on viral hepatitis. [Hepatitis A; Hepatitis B

Energy Technology Data Exchange (ETDEWEB)

Prozesky, O W [Pretoria Univ. (South Africa). Dept. of Medical Virology; Jupp, P G; Joubert, J J; Taylor, M B; Grabow, W O.K.

1985-07-01

The most highly developed radioimmunoassay test system in medical virology is proving of exceptional value in research aimed at controlling and eventually eradicating the scourge of human hepatitis. The use of radioimmunoassay in detecting hepatitis A (HAV) and hepatitis B (HBV) viruses is discussed. The hepatitis A virus is an enterovirus which infects the gastrointestinal tract and is usually transmitted by contaminated food, milk or water. Hepatitis B spreads mainly by the parenteral rate. Bedbugs and ticks are considered as possible transmitters of HBV. Another important contribution of radioimmunoassay is the ability to monitor the immune response of persons at risk who are vaccinated against hepatitis B.

Structure of the human hepatic triglyceride lipase gene

International Nuclear Information System (INIS)

Cai, Shengjian; Wong, D.M.; Chen, Sanhwan; Chan, L.

1989-01-01

The structure of the human hepatic triglyceride lipase gene was determined from multiple cosmid clones. All the exons, exon-intron junctions, and 845 bp of the 5' and 254 bp of the 3' flanking DNA were sequenced. Comparison of the exon sequences to three previously published cDNA sequences revealed differences in the sequence of the codons for residue 133, 193, 202, and 234 that may represent sequence polymorphisms. By primer extension, hepatic lipase mRNA initiates at an adenine 77 bases upstream of the translation initiation site. The hepatic lipase gene spans over 60 kb containing 9 exons and 8 introns, the latter being all located within the region encoding the mature protein. The exons are all of average size (118-234 bp). Exon 1 encodes the signal peptide, exon 4, a region that binds to the lipoprotein substrate, and exon 5, an evolutionarily highly conserved region of potential catalytic function, and exons 6 and 9 encode sequences rich in basic amino acids thought to be important in anchoring the enzyme to the endothelial surface by interacting with acidic domains of the surface glycosaminoglycans. The human lipoprotein lipase gene has been recently reported to have an identical exon-intron organization containing the analogous structural domains. The observations strongly support the common evolutionary origin of these two lipolytic enzymes

Prevalence of hepatitis C Antibody in Human Immunodeficiency ...

African Journals Online (AJOL)

2015-10-25

Oct 25, 2015 ... Abstract: Background: Hepatitis. C virus (HCV) is a major public health problem for Human Immu- nodeficiency virus (HIV) infected population. Both infections share same routes of transmission, and quite often co-exist, with dual infections associated with recipro- cal and mutually more rapid pro- gression ...

Human hepatic lipase overexpression in mice induces hepatic steatosis and obesity through promoting hepatic lipogenesis and white adipose tissue lipolysis and fatty acid uptake.

Directory of Open Access Journals (Sweden)

Lídia Cedó

Full Text Available Human hepatic lipase (hHL is mainly localized on the hepatocyte cell surface where it hydrolyzes lipids from remnant lipoproteins and high density lipoproteins and promotes their hepatic selective uptake. Furthermore, hepatic lipase (HL is closely associated with obesity in multiple studies. Therefore, HL may play a key role on lipid homeostasis in liver and white adipose tissue (WAT. In the present study, we aimed to evaluate the effects of hHL expression on hepatic and white adipose triglyceride metabolism in vivo. Experiments were carried out in hHL transgenic and wild-type mice fed a Western-type diet. Triglyceride metabolism studies included β-oxidation and de novo lipogenesis in liver and WAT, hepatic triglyceride secretion, and adipose lipoprotein lipase (LPL-mediated free fatty acid (FFA lipolysis and influx. The expression of hHL promoted hepatic triglyceride accumulation and de novo lipogenesis without affecting triglyceride secretion, and this was associated with an upregulation of Srebf1 as well as the main genes controlling the synthesis of fatty acids. Transgenic mice also exhibited more adiposity and an increased LPL-mediated FFA influx into the WAT without affecting glucose tolerance. Our results demonstrate that hHL promoted hepatic steatosis in mice mainly by upregulating de novo lipogenesis. HL also upregulated WAT LPL and promoted triglyceride-rich lipoprotein hydrolysis and adipose FFA uptake. These data support the important role of hHL in regulating hepatic lipid homeostasis and confirm the broad cardiometabolic role of HL.

Human hepatic lipase overexpression in mice induces hepatic steatosis and obesity through promoting hepatic lipogenesis and white adipose tissue lipolysis and fatty acid uptake.

Science.gov (United States)

Cedó, Lídia; Santos, David; Roglans, Núria; Julve, Josep; Pallarès, Victor; Rivas-Urbina, Andrea; Llorente-Cortes, Vicenta; Laguna, Joan Carles; Blanco-Vaca, Francisco; Escolà-Gil, Joan Carles

2017-01-01

Human hepatic lipase (hHL) is mainly localized on the hepatocyte cell surface where it hydrolyzes lipids from remnant lipoproteins and high density lipoproteins and promotes their hepatic selective uptake. Furthermore, hepatic lipase (HL) is closely associated with obesity in multiple studies. Therefore, HL may play a key role on lipid homeostasis in liver and white adipose tissue (WAT). In the present study, we aimed to evaluate the effects of hHL expression on hepatic and white adipose triglyceride metabolism in vivo. Experiments were carried out in hHL transgenic and wild-type mice fed a Western-type diet. Triglyceride metabolism studies included β-oxidation and de novo lipogenesis in liver and WAT, hepatic triglyceride secretion, and adipose lipoprotein lipase (LPL)-mediated free fatty acid (FFA) lipolysis and influx. The expression of hHL promoted hepatic triglyceride accumulation and de novo lipogenesis without affecting triglyceride secretion, and this was associated with an upregulation of Srebf1 as well as the main genes controlling the synthesis of fatty acids. Transgenic mice also exhibited more adiposity and an increased LPL-mediated FFA influx into the WAT without affecting glucose tolerance. Our results demonstrate that hHL promoted hepatic steatosis in mice mainly by upregulating de novo lipogenesis. HL also upregulated WAT LPL and promoted triglyceride-rich lipoprotein hydrolysis and adipose FFA uptake. These data support the important role of hHL in regulating hepatic lipid homeostasis and confirm the broad cardiometabolic role of HL.

Oxidation of esterified arachidonate by rat liver microsomes

International Nuclear Information System (INIS)

Davis, H.W.; Suzuki, T.; Schenkman, J.B.

1986-01-01

The authors have previously demonstrated a relationship between phospholipid arachidonate in liver microsomes and malondialdehyde (MDA) formation during lipid peroxidation. In this study arachidonic acid (U- 14 C) was incorporated into rat liver microsomes and NADPH-supported peroxidation was carried out at 37 0 C for 15 minutes. The microsomes were pelleted by centrifugation and the labeled products in the supernatant were isolated by a solid phase method. Pellets were hydrolyzed with phospholipase A 2 and extracted with diethyl ether and the products from both fractions were separated by reverse phase HPLC. The results show that (1) oxidation occurs in all of the major phospholipids but that phosphatidylethanolamine is the most susceptible; (2) a linear correlation exists between MDA formation and supernatant radioactivity; (3) several different polar products are found in both the supernatant and the hydrolyzed pellet but that the ratios of product peaks in HPLC do not change during the peroxidation, indicating no secondary metabolism or propagation; and (4) cytochrome P-450 is not involved in the peroxidative reactions since no oxidation occurs in the absence of Fe 3+ and since product formation is unaffected in the presence of carbon monoxide

Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

Directory of Open Access Journals (Sweden)

Liu W

2015-10-01

Full Text Available Wenqin Liu,1,2,* Jian Shi,1,2,* Lijun Zhu,2 Lingna Dong,1 Feifei Luo,2 Min Zhao,2 Ying Wang,2 Ming Hu,2,3 Linlin Lu,2 Zhongqiu Liu1,2 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China; 2International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 3Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, USA *These authors contributed equally to this work Abstract: Oxymatrine (OMT is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT, and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs and human intestinal microsomes (HIMs and the cytochrome P450 (CYP isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT

Anti-LC1 autoantibodies in patients with chronic hepatitis C virus infection.

Science.gov (United States)

Béland, Kathie; Lapierre, Pascal; Marceau, Gabriel; Alvarez, Fernando

2004-03-01

Various autoantibodies have been reported in patients chronically infected by hepatitis C virus. 2% to 10% of theses patients have anti-liver-kidney microsome type 1 (anti-LKM1) autoantibodies. In type 2 autoimmune hepatitis, anti-LKM1 autoantibodies are frequently associated with anti-liver-cytosol type 1 (anti-LC1) autoantibodies. To determine the prevalence of anti-LC1 autoantibodies in a hepatitis C-positive population and characterize their reactivity. 146 patients suffering from liver diseases, of which 99 were chronically infected by hepatitis C virus, were tested by Western blotting and immunoprecipitation to detect and characterize anti-LC1 autoantibodies. 12% of this hepatitis C population had anti-LC1 autoantibodies. LC1 positivity by Western blotting was 30% of LC1+ sera. Epitopes were found throughout the protein but linear epitopes were situated in the 395-541 amino acid region of formiminotransferase cyclodeaminase. Three putative conformational epitopes were identified by phage display. Anti-LC1 autoantibodies are as prevalent as anti-LKM1 autoantibodies in patients infected with hepatitis C virus and their production is not dependent of anti-LKM1 autoantibodies formation. Autoantibody reactivity against the anti-LC1 antigen is different in hepatitis C than in type 2 autoimmune hepatitis. Anti-LC1 autoantibodies can now be regarded as a serological marker of autoimmunity in chronic hepatitis C infection.

Changes induced by gamma radiation in microsomal membranes of storage of garlic

International Nuclear Information System (INIS)

Perez, M.B.; Croci, C.A.; Aveldano, M.I.

2003-01-01

This study evaluates the effects of the radio inhibition process on garlic bulbs in terms of phase properties of microsomal membranes and their lipid and fatty acid composition. Garlic bulbs were irradiated with an average dose of 60 Gy of 60 Co gamma rays 30-40 days after harvest. The treatment was carried out in the facilities of the National Atomic Energy Commission (CNEA). Rough and smooth microsomal membranes were isolated by ultracentrifugation from tissues of irradiated and non-irradiated storage leaves. Wide angle X-ray diffractograms of both fractions were recorded along 270 days of storage. Lipids were separated by thin layer chromatography. The fatty acid composition of major lipid fractions was studied by gas-liquid chromatography. The diffractograms featured peaks at Bragg spacing of 4.15 Armstrong and 3.75 Armstrong, revealing the presence of a gel (crystalline) phase, while the characteristic peak of the liquid-crystalline phase (4.6 Armstrong) was not observed in both sorts of membranes. Irradiation was found to bring about modifications in the intensity of 4.15 Armstrong and 3.75 Armstrong peaks from smooth microsomal membranes, but not in the behaviour along the studied period. Data from the rough microsomal fraction were erratic. Parallel to these changes, radiation induced significant modifications in the level of smooth microsomal membrane triacylglycerols in relation to phospholipids and their fatty acids. These findings indicate that the storage leaf tissues of garlic are radiosensitive both in terms of physical and chemical properties of their microsomal membranes. From the practical point of view, these results could be the basis for the development of techniques to be applied to storage garlic to evaluate if it was irradiated. (author)

Effect of ethylene glycol monomethyl ether and diethylene glycol monomethyl ether on hepatic metabolizing enzymes.

Science.gov (United States)

Kawamoto, T; Matsuno, K; Kayama, F; Hirai, M; Arashidani, K; Yoshikawa, M; Kodama, Y

1990-06-01

Glycol ethers have been extensively used in industry over the past 40-50 years. Numerous studies on the toxicity of glycol ethers have been performed, however, the effects of glycol ethers on the hepatic drug metabolizing enzymes are still unknown. We studied the changes of the putative metabolic enzymes, that is, the hepatic microsomal mixed function oxidase system and cytosolic alcohol dehydrogenase, by the oral administration of diEGME and EGME. Adult male Wistar rats were used. DiEGME was administered orally; 500, 1000, 2000 mg/kg for 1, 2, 5 or 20 days and EGME was 100, 300 mg/kg for 1, 2, 5 or 20 days. Decreases in liver weights were produced by highest doses of diEGME (2000 mg/kg body wt/day for 20 days) and EGME (300 mg/kg body wt/day for 20 days). DiEGME increased hepatic microsomal protein contents and induced cytochrome P-450, but not cytochrome b5 or NADPH-cytochrome c reductase. The activity of cytosolic ADH was not affected by diEGME administration. On the other hand, EGME did not change cytochrome P-450, cytochrome b5 or NADPH-cytochrome c reductase. The activity of cytosolic ADH was increased by repeated EGME treatment. Therefore it is suspected that the enzyme which takes part in the metabolism of diEGME is different from that of EGME, although diEGME is a structural homologue of EGME.

The antibiotic tiamulin is a potent inducer and inhibitor of cytochrome P4503A via the formation of a stable metabolic intermediate complex. Studies in primary hepatocyte cultures and liver microsomes of the pig.

Science.gov (United States)

Witkamp, R F; Nijmeijer, S M; Monshouwer, M; Van Miert, A S

1995-05-01

Tiamulin is a semisynthetic antibiotic frequently used in agricultural animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds that are simultaneously administered. To explain this, it has been suggested that tiamulin selectively inhibits oxidative drug metabolism via the formation of a cytochrome P450 metabolic intermediate complex. The aim of the present study was to provide further support for this hypothesis. When hepatic microsomes and cultured primary pig hepatocytes were incubated with tiamulin, a maximum in the absorbance spectrum at 455 nm was observed, which disappeared after adding KFe(CN)6. When hepatocytes were incubated with tiamulin for 72 hr, cytochrome P450 content and cytochrome P4503A apoprotein levels were increased. Tiamulin strongly inhibited and concentration dependently inhibited the hydroxylation rate of testosterone at the 6 beta-position in both microsomes and hepatocytes, and the microsomal N-demethylation rate of ethylmorphine. Other testosterone hydroxylations were inhibited to a lesser extent or not affected. The relative inhibition of the hydroxylation of testosterone at the 6 beta-position was more pronounced in microsomes from rifampicin- and triacetyloleandomycin-treated pigs. The results indicate that cytochrome P450 complex formation can at least partly explain the interactions observed with tiamulin. Tiamulin seems to be a strong, probably selective, inhibitor of the cytochrome P4503A subfamily and an interesting tool for further research.

Human Immunodeficiency Virus and Hepatitis C Virus Co-infection ...

African Journals Online (AJOL)

Human Immunodeficiency Virus and Hepatitis C Virus Co-infection in Cameroon: Investigation of the Genetic Diversity and Virulent ... AFRICAN JOURNALS ONLINE (AJOL) · Journals · Advanced Search · USING AJOL · RESOURCES ... DNA sequencing, and bioinformatics tools for sequence management and analysis.

Pharmacokinetic study of isocorynoxeine metabolites mediated by cytochrome P450 enzymes in rat and human liver microsomes.

Science.gov (United States)

Zhao, Lizhu; Zang, Bin; Qi, Wen; Chen, Fangfang; Wang, Haibo; Kano, Yoshihiro; Yuan, Dan

2016-06-01

Isocorynoxeine (ICN) is one of the major bioactive tetracyclic oxindole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks. that is widely used for the treatment of hypertension, vascular dementia, and stroke. The present study was undertaken to assess the plasma pharmacokinetic characteristics of major ICN metabolites, and the role of simulated gastric and intestinal fluid (SGF and SIF), human and rat liver microsomes (HLMs and RLMs), and seven recombinant human CYP enzymes in the major metabolic pathway of ICN. A rapid, sensitive and accurate UHPLC/Q-TOF MS method was validated for the simultaneous determination of ICN and its seven metabolites in rat plasma after oral administration of ICN at 40mg/kg. It was found that 18.19-dehydrocorynoxinic acid (DCA) and 5-oxoisocorynoxeinic acid (5-O-ICA) were both key and predominant metabolites, rather than ICN itself, due to the rapid and extensive metabolism of ICN in vivo. The further study indicated that ICN was mainly metabolized in human or rat liver, and CYPs 2C19, 3A4 and 2D6 were the major enzymes responsible for the biotransformation of ICN to DCA and 5-O-ICA in human. These findings are of significance in understanding of the pharmacokinetic nature of tetracyclic oxindole alkaloids, and provide helpful information for the clinical co-administration of the herbal preparations containing U. rhynchophylla with antihypertensive drugs that are mainly metabolized by CYP3A4 and CYP2C19. Copyright © 2016 Elsevier B.V. All rights reserved.

Hepatitis A virus infection and hepatitis A vaccination in human immunodeficiency virus-positive patients: A review.

Science.gov (United States)

Lin, Kuan-Yin; Chen, Guan-Jhou; Lee, Yu-Lin; Huang, Yi-Chia; Cheng, Aristine; Sun, Hsin-Yun; Chang, Sui-Yuan; Liu, Chun-Eng; Hung, Chien-Ching

2017-05-28

Hepatitis A virus (HAV) is one of the most common infectious etiologies of acute hepatitis worldwide. The virus is known to be transmitted fecal-orally, resulting in symptoms ranging from asymptomatic infection to fulminant hepatitis. HAV can also be transmitted through oral-anal sex. Residents from regions of low endemicity for HAV infection often remain susceptible in their adulthood. Therefore, clustered HAV infections or outbreaks of acute hepatitis A among men who have sex with men and injecting drug users have been reported in countries of low endemicity for HAV infection. The duration of HAV viremia and stool shedding of HAV may be longer in human immunodeficiency virus (HIV)-positive individuals compared to HIV-negative individuals with acute hepatitis A. Current guidelines recommend HAV vaccination for individuals with increased risks of exposure to HAV (such as from injecting drug use, oral-anal sex, travel to or residence in endemic areas, frequent clotting factor or blood transfusions) or with increased risks of fulminant disease (such as those with chronic hepatitis). The seroconversion rates following the recommended standard adult dosing schedule (2 doses of HAVRIX 1440 U or VAQTA 50 U administered 6-12 mo apart) are lower among HIV-positive individuals compared to HIV-negative individuals. While the response rates may be augmented by adding a booster dose at week 4 sandwiched between the first dose and the 6-mo dose, the need of booster vaccination remain less clear among HIV-positive individuals who have lost anti-HAV antibodies.

CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

Science.gov (United States)

Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

2013-11-01

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

Low prevalence of liver-kidney microsomal autoantibodies of type 1 (LKM1) in hepatitis C seropositive subjects on Crete, Greece.

Science.gov (United States)

Drygiannakis, D; Lionis, C; Drygiannakis, I; Pappas, G; Kouroumalis, E

2001-01-01

Hepatitis C is a serious problem on the Greek island of Crete, where a high prevalence of antibodies against hepatitis C (anti-HCV) has recently been reported. This article reports the findings of a study carried out in Crete, which investigated the prevalence of serum autoantibodies in patients with chronic hepatitis C. One hundred and forty two patients (59 men and 83 women), who were found anti-HCV seropositive in two hospitals and two Primary Health Care Centres in Crete, were eligible. Sixty healthy blood donors (46 men, 14 women), which were negative to anti-HCV, were used as the control group. They were randomly selected from those attending Rethymnon Hospital. Autoantibodies were identified using the indirect immunofluorescence (IFL) technique on human epithelial cells from larynx cancer (HEp-2 cells), rat liver-kidney-stomach substrate (CT3) and Chrithidia Luciliae (CL). Serum autoantibodies were detected in 104 HCV patients, yielding an overall prevalence of 73.2%. The most frequent autoantibodies were antinuclear antibodies (ANA), positive in 72 patients (50.7%). Anti-smooth muscle antibodies (ASMA) were detected in 33 patients (23.2%). Only one patient was positive for LKM1 autoantibodies. No autoantibodies were found in 38 patients (26.7%). Autoantibodies were also found in 5 out of the 60 examined healthy blood donors (8.3%). Autoantibodies, mainly ANA and ASMA are very common in HCV seropositive patients from Crete. By contrast LKM1 autoantibodies are exceptionally rare in these patients.

Bioenergetic Changes during Differentiation of Human Embryonic Stem Cells along the Hepatic Lineage

DEFF Research Database (Denmark)

Hopkinson, Branden M; Madsen, Claus Desler; Kalisz, Mark

2017-01-01

Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless, a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells...... of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction...

Hepatic arachidonic acid metabolism is disrupted after hexachlorobenzene treatment

International Nuclear Information System (INIS)

Billi de Catabbi, Silvia C.; Faletti, Alicia; Fuentes, Federico; San Martin de Viale, Leonor C.; Cochon, Adriana C.

2005-01-01

Hexaclorobenzene (HCB), one of the most persistent environmental pollutants, can cause a wide range of toxic effects including cancer in animals, and hepatotoxicity and porphyria both in humans and animals. In the present study, liver microsomal cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolism, hepatic PGE production, and cytosolic phospholipase A 2 (cPLA 2 ) activity were investigated in an experimental model of porphyria cutanea tarda induced by HCB. Female Wistar rats were treated with a single daily dose of HCB (100 mg kg -1 body weight) for 5 days and were sacrificed 3, 10, 17, and 52 days after the last dose. HCB treatment induced the accumulation of hepatic porhyrins from day 17 and increased the activities of liver ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and aminopyrine N-demethylase (APND) from day 3 after the last dose. Liver microsomes from control and HCB-treated rats generated, in the presence of NADPH, hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), 11,12-Di HETE, and ω-OH/ω-1-OH AA. HCB treatment caused an increase in total NADPH CYP-dependent AA metabolism, with a higher response at 3 days after the last HCB dose than at the other time points studied. In addition, HCB treatment markedly enhanced PGE production and release in liver slices. This HCB effect was time dependent and reached its highest level after 10 days. At this time cPLA 2 activity was shown to be increased. Unexpectedly, HCB produced a significant decrease in cPLA 2 activity on the 17th and 52nd day. Our results demonstrated for the first time that HCB induces both the cyclooxygenase and CYP-dependent AA metabolism. The effects of HCB on AA metabolism were previous to the onset of a marked porphyria and might contribute to different aspects of HCB-induced liver toxicity such as alterations of membrane fluidity and membrane-bound protein function. Observations also suggested that a possible role of cPLA 2 in

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

OpenAIRE

Kukiełka, E; Cederbaum, A I

1995-01-01

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid...

The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus.

Science.gov (United States)

Flisiak, Robert; Horban, Andrzej; Gallay, Philippe; Bobardt, Michael; Selvarajah, Suganya; Wiercinska-Drapalo, Alicja; Siwak, Ewa; Cielniak, Iwona; Higersberger, Jozef; Kierkus, Jarek; Aeschlimann, Christian; Grosgurin, Pierre; Nicolas-Métral, Valérie; Dumont, Jean-Maurice; Porchet, Hervé; Crabbé, Raf; Scalfaro, Pietro

2008-03-01

Debio-025 is an oral cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus activity in vitro. Its effect on viral load as well as its influence on intracellular Cyp levels was investigated in a randomized, double-blind, placebo-controlled study. Mean hepatitis C viral load decreased significantly by 3.6 log(10) after a 14-day oral treatment with 1200 mg twice daily (P CypB) levels in peripheral blood mononuclear cells decreased from 67 +/- 6 (standard error) ng/mg protein (baseline) to 5 +/- 1 ng/mg protein at day 15 (P CypB levels, coinciding with the decrease in hepatitis C viral load. These are the first preliminary human data supporting the hypothesis that CypB may play an important role in hepatitis C virus replication and that Cyp inhibition is a valid target for the development of anti-hepatitis C drugs.

Epidemiological studies on viral infections and co-infections : Human immunodeficiency virus, hepatitis C virus and human papillomavirus

NARCIS (Netherlands)

van Santen, D.K.

2018-01-01

The research described in this thesis aimed to increase our understanding of the incidence, disease progression and treatment of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and human papillomavirus (HPV) infections and co-infections in key populations. Chapter 1 contains an overview

Thyroid hormone levels and hepatic enzyme activity in lactating dams after gestational exposure to low dose PBDE 47

Energy Technology Data Exchange (ETDEWEB)

Kuriyama, S.N.; Grande, S.W.; Akkoc, Z.; Souza, C.A.M. de; Chahoud, I. [Charite Univ. Medical School Berlin (Germany). Inst. of Clinical Pharmacology and Toxicology, Dept. Toxicology, Campus Benjamin Franklin; Fidalgo-Neto, A.A. [Oswaldo Cruz Foundation, Rio de Janeiro (Brazil). Lab. of Environmental Toxicology

2004-09-15

Polybrominated diphenyl ethers (PBDEs), a class of widely used flame retardants, are found extensively in the environment (shown by several studies on sentinel animal species), as well as in humans. In rodents, technical commercial PBDE mixtures and individual congeners have shown to interfere with thyroid hormone homeostasis, produce a mix-type induction of hepatic microsomal enzymes, disrupt spontaneous behaviour, impair learning and memory and alter the cholinergic transmitter system. In rat and mice, some technical PBDE commercial mixtures such as DE-71 and Bromkal 70 and the congener PBDE 47 have shown to decrease circulating thyroid hormone levels. PBDEs are also able to induce both hepatic phase I and phase II detoxification enzymes, demonstrated by several investigations in laboratory animals. For example, induction of ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-Odespenthylase (PROD) and uridinediphospho-glucuronosyltransferase (UDPGT) has been shown in rodents and cell lines after exposure to technical mixtures or individual congeners. However, these studies deal with doses much higher than that found in human tissues, highlighting the importance of assessing the adverse effects of doses close to human exposure levels. PBDE 47 is the most predominant congener found in environmental and human samples (including human milk) and, therefore, hazard identification is extremely important for human risk assessment. We administered a single dose to gravid dams on gestation day 6 of either 140 {mu}g/kg BW or 700 {mu}g/kg BW of the congener, 2,2'4,4'-tetrabromo diphenyl ether (PBDE 47). These doses are pertinent to human exposure levels because a study by She et al. found a mean level of 33.3 {mu}g PBDE 47 /kg fat in human breast adipose tissue with a range from 7.01 to 196 {mu}g PBDE 47 /kg fat. In this study, thyroid hormone levels and hepatic enzyme activity were evaluated in lactating dams after in utero administration of low dose PBDE 47.

Prevalence of hepatitis B, hepatitis C and human immunodeficiency ...

African Journals Online (AJOL)

Background. Hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV are common blood-borne infections unevenly distributed across regions in Nigeria. Few population-based prevalence studies have been done in Nigeria. Objective. To determine the prevalence of HBV, HCV and HIV and risk factors for infection with ...

Long-term culture of human liver tissue with advanced hepatic functions.

Science.gov (United States)

Ng, Soon Seng; Xiong, Anming; Nguyen, Khanh; Masek, Marilyn; No, Da Yoon; Elazar, Menashe; Shteyer, Eyal; Winters, Mark A; Voedisch, Amy; Shaw, Kate; Rashid, Sheikh Tamir; Frank, Curtis W; Cho, Nam Joon; Glenn, Jeffrey S

2017-06-02

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

International Nuclear Information System (INIS)

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.; Selvapandiyan, Angamuthu; Hewlett, Indira; Duncan, Robert; Puri, Raj K.; Nakhasi, Hira L.; Kaplan, Gerardo G.

2007-01-01

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients

Low prevalence of liver-kidney microsomal autoantibodies of type 1 (LKM1 in hepatitis C seropositive subjects on Crete, Greece

Directory of Open Access Journals (Sweden)

Pappas Georgios

2001-06-01

Full Text Available Abstract Background Hepatitis C is a serious problem on the Greek island of Crete, where a high prevalence of antibodies against hepatitis C (anti-HCV has recently been reported. This article reports the findings of a study carried out in Crete, which investigated the prevalence of serum autoantibodies in patients with chronic hepatitis C. Patients and Methods One hundred and forty two patients (59 men and 83 women, who were found anti-HCV seropositive in two hospitals and two Primary Health Care Centres in Crete, were eligible. Sixty healthy blood donors (46 men, 14 women, which were negative to anti-HCV, were used as the control group. They were randomly selected from those attending Rethymnon Hospital. Autoantibodies were identified using the indirect immunofluorescence (IFL technique on human epithelial cells from larynx cancer (HEp-2 cells, rat liver-kidney-stomach substrate (CT3 and Chrithidia Luciliae (CL. Results Serum autoantibodies were detected in 104 HCV patients, yielding an overall prevalence of 73.2%. The most frequent autoantibodies were antinuclear antibodies (ANA, positive in 72 patients (50.7%. Anti-smooth muscle antibodies (ASMA were detected in 33 patients (23.2%. Only one patient was positive for LKM1 autoantibodies. No autoantibodies were found in 38 patients (26.7%. Autoantibodies were also found in 5 out of the 60 examined healthy blood donors (8.3%. Conclusions Autoantibodies, mainly ANA and ASMA are very common in HCV seropositive patients from Crete. By contrast LKM1 autoantibodies are exceptionally rare in these patients.

Modification of radiation-induced oxidative damage in liposomal and microsomal membrane by eugenol

Energy Technology Data Exchange (ETDEWEB)

Pandey, B.N. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Lathika, K.M. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Mishra, K.P. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)]. E-mail: kpm@magnum.barc.ernet.in

2006-03-15

Radiation-induced membrane oxidative damage, and their modification by eugenol, a natural antioxidant, was investigated in liposomes and microsomes. Liposomes prepared with DPH showed decrease in fluorescence after {gamma}-irradiation, which was prevented significantly by eugenol and correlated with magnitude of oxidation of phospholipids. Presence of eugenol resulted in substantial inhibition in MDA formation in irradiated liposomes/microsomes, which was less effective when added after irradiation. Similarly, the increase in phospholipase C activity observed after irradiation in microsomes was inhibited in samples pre-treated with eugenol. Results suggest association of radio- oxidative membrane damage with alterations in signaling molecules, and eugenol significantly prevented these membrane damaging events.

Committee Opinion No. 655 Summary: Hepatitis B, Hepatitis C, and Human Immunodeficiency Virus Infections in Obstetrician-Gynecologists.

Science.gov (United States)

2016-02-01

To prevent transmission of bloodborne pathogens, it is important that health care providers adhere to standard precautions, follow fundamental infection-control principles, and use appropriate procedural techniques. All obstetrician-gynecologists who provide clinical care should receive the hepatitis B virus vaccine series. The Society for Healthcare Epidemiology of America has established guidelines for the management of health care providers who are infected with hepatitis B virus, hepatitis C virus, or human immunodeficiency virus (HIV). The guidelines categorize representative obstetric and gynecologic procedures according to level of risk of bloodborne pathogen transmission and include recommendations for health care provider clinical activities, based on these categories and viral burden. It is important to note that when no restrictions are recommended, careful supervision should be carried out as highlighted. These recommendations provide a framework within which to consider such cases; however, each case should be independently considered in context by the expert review panel.

Hepatitis c and human rights: comparison of legal experience of Ukraine and Georgia.

Science.gov (United States)

Senyuta, Iryna Y

2018-01-01

A comparative legal research of human rights provision in Ukraine and Georgia, in the aspect of combating viral HCV, was conducted. Ukrainian advocacy experience and Georgian strategic litigation experience with regard to human rights and HCV was analyzed. Key international instruments, which lay the conceptual foundations as well as outline the measures, which are directed at human rights in patient care provision and fighting viral hepatitis, were elucidated. Attention was paid to the Global health sector strategy. Viral hepatitis, 2016 - 2021 [1], which, for the first time, defined a global strategy on fighting viral hepatitis, in particular HCV and envisaged the advocacy vectors. The frames of interaction of the human rights in patient care concept and public health, which consists in realization of certain human rights were elucidated and the necessity to embody the human rights in patient care concept into the state policy in the field of public health was determined. It was found out that a common international problem in combating HCV is a deficiency of financial resources, which are necessary for effective fighting the epidemics and guarantee equal access to treatment for every person. The international community outlined five most important spheres, which require investments and will catalyze the measures, which need to be taken in order to fight hepatitis. Analysis of the Ukrainian experience was focused on the issue of donated blood safety and successful advocacy campaigns, which were carried out in order to promote the adoption of programs on prophylactics, diagnostics and treatment of HCV both on national and regional levels. Examples of ensuring the rights of the marginalized groups during HCV treatment, in particular of the people who inject drugs, people living with HIV, participants of the antiterrorist operation were provided. Interesting and important is the experience of Georgia concerning human rights protection in the ECtHR, which has a legal

Photoeffects of near ultraviolet light upon a polycyclic aromatic hydrocarbon exposed to mouse skin microsomes

International Nuclear Information System (INIS)

Peirano, W.B.

1991-01-01

Near ultraviolet (UV) light has been reported to both enhance and inhibit the tumor incidence in mice dermally exposed to benzo(a)pyrene (BaP) or polycyclic aromatic hydrocarbon (PAH) mixtures. Near UV light interacts with PAHs producing a variety of oxygenated products such as phenols, endoperoxides and quinones. However, little is known about BaP products formed from near UV irradiation of BaP-exposed mouse skin. Therefore, 14 C-BaP was incubated with 3-methylcholanthrene (3-MC) induced C 3 H/HeJ and DBA/2J mouse skin microsomes with or without a 365 nm light source. The results indicated that the concurrent 365 nm light irradiation of induced mouse skin microsomes and BaP greatly enhanced the total conversion of BaP to its products, approximately 3-fold for the C 3 H/HeJ and approximately 7-fold for the DBA/2J mouse microsomes, compared to the induced mouse skin microsomes and BaP alone. HPLC analyses of organic extracts indicated a more than additive enhancement of the formation of most of the individual cochromatographed BaP metabolites due to the combined interaction of 365 nm light with BaP and skin microsomes. Similar interactions were observed using benz(a)anthracene (BaA) in this system. These data show that near UV light alters the metabolic profile of PAHs produced by mouse skin microsomes

Predicting Human Clearance of OATP substrates using Cynomolgus monkey: In vitro-in vivo scaling of hepatic uptake clearance.

Science.gov (United States)

de Bruyn, Tom; Ufuk, Ayse; Cantrill, Carina; Kosa, Rachel E; Bi, Yi-An; Niosi, Mark; Modi, Sweta; Rodrigues, A David; Tremaine, Larry M; Varma, Manthena Vs; Galetin, Aleksandra; Houston, J Brian

2018-05-02

This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) transporters. Nine OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin, bosentan and valsartan) were investigated in plated cynomolgus monkey and human hepatocytes. Total uptake clearance and passive diffusion were measured in vitro from initial rates in the absence and presence of the OATP inhibitor rifamycin SV, respectively. Total uptake clearance values in plated hepatocytes ranged over three orders of magnitude in both species with a similar rank order and good agreement in the relative contribution of active transport to total uptake between cynomolgus monkey and human. In vivo hepatic clearance for these nine drugs was determined in cynomolgus monkey after intravenous dosing. Hepatic clearances showed a similar range to human parameters and good predictions from respective hepatocyte parameters (with 2.7 and 3.8-fold bias on average, respectively). The use of cross species empirical scaling factors (based on either dataset average or individual drug scaling factor from cynomolgus monkey data) improved prediction (less bias, better concordance) of human hepatic clearance from human hepatocyte data alone. In vitro intracellular binding in hepatocytes also correlated well between species. It is concluded that the minimal species differences observed for the current dataset between cynomolgus monkey and human hepatocyte uptake, both in vitro and in vivo, support future use of this preclinical model to delineate drug hepatic uptake and enable prediction of human in vivo intrinsic hepatic clearance. The American Society for Pharmacology and Experimental Therapeutics.

Abetalipoproteinemia: A novel mutation of microsomal triglyceride ...

African Journals Online (AJOL)

Hager Barakizou

2016-01-25

Jan 25, 2016 ... Abetalipoproteinemia: A novel mutation of microsomal triglyceride transfer protein (MTP) gene in a young Tunisian patient. Hager Barakizou a,. *, Souha Gannouni a. , Khalil Messaoui a. , Mathilde Difilippo b. ,. Agne`s Sassolas b. , Fethi Bayoudh a a Department of Pediatrics, Military Hospital of Tunis, ...

Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

International Nuclear Information System (INIS)

Bennett, W.M.; Mela-Riker, L.M.; Houghton, D.C.; Gilbert, D.N.; Buss, W.C.

1988-01-01

Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. [3H]leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

Energy Technology Data Exchange (ETDEWEB)

Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cawley, George F.; Ardoin, Taylor G. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Backes, Wayne L. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

2014-06-01

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

International Nuclear Information System (INIS)

Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.

2014-01-01

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

Chlordecone, a mixed pregnane X receptor (PXR) and estrogen receptor alpha (ERα) agonist, alters cholesterol homeostasis and lipoprotein metabolism in C57BL/6 mice

International Nuclear Information System (INIS)

Lee, Junga; Scheri, Richard C.; Zhang Yuan; Curtis, Lawrence R.

2008-01-01

Chlordecone (CD) is one of many banned organochlorine (OC) insecticides that are widespread persistent organic pollutants. OC insecticides alter lipid homeostasis in rodents at doses that are not neurotoxic or carcinogenic. Pretreatment of mice or rats with CD altered tissue distribution of a subsequent dose of [ 14 C]CD or [ 14 C]cholesterol (CH). Nuclear receptors regulate expression of genes important in the homeostasis of CH and other lipids. In this study, we report that CD suppresses in vitro reporter systems for human liver X receptors (LXRs) and activates those for human farnesoid X receptor (FXR), pregnane X receptor (PXR) and estrogen receptor α (ERα) in a concentration-dependent manner (0-50 μM). Consistent with human PXR activation in vitro, three days after a single dose of CD (15 mg/kg) hepatic microsomal CYP3A11 protein increases in C57BL/6 mice. CD decreases hepatic CH ester content without altering total CH concentration. Apolipoprotein A-I (apoA-I) contents of hepatic lipoprotein-rich and microsomal fractions of CD-treated mice are higher than controls. There is a significant reduction in non-high density lipoprotein CH but not apolipoprotein B-48/100 (apoB-48/100) in plasma from CD-treated mice after a 4 h fast. At 14 days after 15 mg CD/kg apoA-I and apoB-100 proteins but not CYP3A11 protein in hepatic microsomes are similar to controls. This work indicates that altered CH homeostasis is a mode of OC insecticide action of relevance after a single dose. This at least partially explains altered CH tissue distribution in CD-pretreated mice

Strategy for Hepatotoxicity Prediction Induced by Drug Reactive Metabolites Using Human Liver Microsome and Online 2D-Nano-LC-MS Analysis.

Science.gov (United States)

Zhuo, Yue; Wu, Jian-Lin; Yan, Xiaojing; Guo, Ming-Quan; Liu, Ning; Zhou, Hua; Liu, Liang; Li, Na

2017-12-19

Hepatotoxicity is a leading cause of drug withdrawal from the market; thus, the assessment of potential drug induced liver injury (DILI) in preclinical trials is necessary. More and more research has shown that the covalent modification of drug reactive metabolites (RMs) for cellular proteins is a possible reason for DILI. Unfortunately, so far no appropriate method can be employed to evaluate this kind of DILI due to the low abundance of RM-protein adducts in complex biological samples. In this study, we proposed a mechanism-based strategy to solve this problem using human liver microsomes (HLMs) and online 2D nano-LC-MS analysis. First, RM modification patterns and potential modified AA residues are determined using HLM and model amino acids (AAs) by UHPLC-Q-TOF-MS. Then, a new online 2D-nano-LC-Q-TOF-MS method is established and applied to separate the digested modified microsomal peptides from high abundance peptides followed by identification of RM-modified proteins using Mascot, in which RM modification patterns on specific AA residues are added. Finally, the functions and relationship with hepatotoxicity of the RM-modified proteins are investigated using ingenuity pathway analysis (IPA) to predict the possible DILI. Using this strategy, 21 proteins were found to be modified by RMs of toosendanin, a hepatotoxic drug with complex structure, and some of them have been reported to be associated with hepatotoxicity. This strategy emphasizes the identification of drug RM-modified proteins in complex biological samples, and no pretreatment is required for the drugs. Consequently, it may serve as a valuable method to predict potential DILI, especially for complex compounds.

Co–inection of hepatitis B and C viruses among human ...

African Journals Online (AJOL)

Introduction: The co–infection of Human immunodeficiency virus (HIV), Hepatitis B and C viruses remains a public health problem particularly in resource limited setting like Nigeria. Studies on these co–infections have been done principally among adult and pregnant women with limited information on the pediatric ...

Hepatitis E Virus Genotype 3 in Humans and Swine, Bolivia

Science.gov (United States)

Cavallo, Annalisa; Gonzales, José Luis; Bonelli, Sara Irene; Valda, Ybar; Pieri, Angela; Segundo, Higinio; Ibañez, Ramón; Mantella, Antonia; Bartalesi, Filippo; Tolari, Francesco; Bartoloni, Alessandro

2011-01-01

We determined the seroprevalence of hepatitis E virus (HEV) in persons in 2 rural communities in southeastern Bolivia and the presence of HEV in human and swine fecal samples. HEV seroprevalence was 6.3%, and HEV genotype 3 strains with high sequence homology were detected. PMID:21801630

The role of microsomal enzyme inducers in the reduction of misonidazole neurotoxicity

International Nuclear Information System (INIS)

Jones, D.H.; Bleehen, N.M.; Workman, P.; Smith, N.C.

1983-01-01

It has been shown that phenytoin, 300 mg daily for one week, produces consistent hepatic microsomal enzyme induction, resulting in a decrease of 25% in misonidazole half-life, without causing any toxicity per se. A longer period of administration gives only a slightly greater induction. Phenobarbitone in a daily dose of 90 mg causes a reduction of 18% and 23% in misonidazole half-life after 1 and 2 weeks' pre-treatment respectively, but is less suitable clinically because of its sedative effect. A further series of studies using phenytoin as the inducing agent has shown that, despite adequate enzyme induction and increased misonidazole metabolism, it is impossible to increase the total dose of misonidazole beyond the usually accepted value of 12 g/m 2 because of unacceptable neuropathy (a rate of 50% at a dose of 14 g/m 2 over three weeks). In single doses of above 3.0-4.0 g of misonidazole, severe nausea and vomiting are prominent, so that this side effect is a determining factor in the treatment fractionation. Audiometric studies show no correlation between the incidence of peripheral neuropathy and abnormal audiograms, and have no value in the early prediction of neurotoxicity. (author)

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

Science.gov (United States)

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques

NARCIS (Netherlands)

Moonen, H.; Bosch, H. van den

1979-01-01

1. 1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate

Incubation of 14C-trichloroethylene vapor with rat liver microsomes: uptake of radioactivity and covalent protein binding of metabolites

International Nuclear Information System (INIS)

Bolt, H.M.; Wolowski, L.; Buchter, A.; Bolt, W.; Gil, D.L.

1977-01-01

Microsomal uptake irreversible protein binding of labelled trichloroehtylene was measured following incubation with rat liver microsomes in an all-glass vacuum system. If the cofactor for oxidative metabolism, NADPH, is not added, the gaseous trichloroethylene rapidly equilibrates with the microsomal suspension. Addition of NADPH results in a further uptake of 14 C-trichloroethylene from the gas phase, linearly with time, which is due to enzymic metabolism. This part of uptake is inhibited by some arylimidazoles and 1.2.3-benzothiadiazoles. The compounds of greatest inhibitory potency were 6-chloro-1.2.3-benzothiadiazole and 5.6-dimethyl-1.2.3-benzothiadiazole. Part of the metabolites of 14 C-trichloroethylene formed by rat liver microsomes were irreversibly bound to microsomal protein, amounting up to 1 nmol per mg microsomal protein per hour. Model experiments on uptake of 14 C-trichloroethylene from the gas phase by albumin solutions and liposomal suspensions (from lecithin) showed a rapid equilibration of trichloroethylene also with these systems. Comparison with previous analogous data on vinyl chloride revealed an about 10 times higher affinity of trichloroethylene to albumin and lipid, consistent with the behaviour of both compounds in the rat liver microsomal system. (orig.) [de

Hepatitis A Virus and Hepatitis E Virus: Emerging and Re-Emerging Enterically Transmitted Hepatitis Viruses.

Science.gov (United States)

Lemon, Stanley M; Walker, Christopher M

2018-05-07

Over the past two decades, progress in understanding human infections with hepatitis A virus (HAV) and hepatitis E virus (HEV) has been eclipsed by the priority of combating persistent hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. During that time, the global burden of liver disease caused by enteric hepatitis viruses has not abated. Because of vaccines, hepatitis A has become increasingly a disease of adults instead of early childhood in many regions of the world, resulting in an age-related shift toward more severe disease. HEV has remained endemic in many developing countries, and in well-developed, economically advanced countries it is now recognized as a cause of chronic, progressive liver disease in individuals with compromised immunity. The goal of this collection of articles is to review recent progress and to shine a bright light on gaps in our understanding of how these viruses replicate, cause disease, interact with the liver and host immune system, and are transmitted, along with prospects for improved control in human populations. Renewed efforts to study and compare HAV and HEV biology in humans and animal models have high potential to enhance our understanding of host-pathogen balance in the liver, and may contribute ultimately to the control of other infectious diseases of the liver. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

[Immunosuppressant effect of cyclophosphamide activated in vitro by liver microsomes from different strains of mice].

Science.gov (United States)

Telegin, L Iu; Zhirnov, G F; Mazurov, A V; Pevnitskiĭ, L A

1981-07-01

The paper is concerned with activation of cyclophosphamide by mouse liver microsomes in vitro. Liver microsomes from BALB/c mice metabolize cyclophosphamide more effectively as compared with those from DBA/2 mice, which manifested by a more intense output of products having alkylating or immunodepressant properties. This seems likely to be a consequence of the increased P-450 cytochrome content in liver microsomes from BALB/c mice, as well as of its structural characteristics in the mouse. The relationship between the immunodepressant effect of cyclophosphamide in vivo and in vitro in mice of varied genotypes is discussed.

Glutathione delays varies as-tocopherol oxidation and subsequent lipid peroxidation in rat liver microsomes

International Nuclear Information System (INIS)

Robey, S.; Mavis, R.

1986-01-01

A method has been developed for in vitro trace radiolabeling of rat liver microsomes with 3 H-α-tocopherol (αT*) which allows virtually complete oxidation of the αT* under oxidizing conditions. The supernatant of a 16,000 xg centrifugation of homogenized rat liver, containing the cytosolic rat liver vitamin E (VE) transfer protein, was incubated with an ethanolic solution of αT* for 10 minutes at 37 0 C. Labeled microsomes were collected in the washed 100,000 xg pellet. Microsomes were then incubated with 30 μM Fe 2+ in an NADPH-generating system, and both production of malondialdehyde (MDA) (a product of lipid peroxidation) and oxidation of αT* were monitored over a time course in the presence and absence of glutathione (GSH). The results indicate virtually complete oxidation of αT* precedes significant membrane lipid peroxidation, and that addition of 5 mM GSH delays both αT* oxidation and subsequent MDA production. This suggests that the previously observed VE-dependent heat labile inhibition of microsomal lipid peroxidation by GSH involves maintaining membrane levels of α-tocopherol

Role of microsomal enzyme inducers in the reduction of misonidazole neurotoxicity

Energy Technology Data Exchange (ETDEWEB)

Jones, D.H.; Bleehen, N.M.; Workman, P.; Smith, N.C. (Cambridge Univ. (UK). Dept. of Clinical Oncology and Radiotherapeutics; Addenbrooke' s Hospital, Cambridge (UK))

1983-11-01

It has been shown that phenytoin, 300 mg daily for one week, produces consistent hepatic microsomal enzyme induction, resulting in a decrease of 25% in misonidazole half-life, without causing any toxicity per se. A longer period of administration gives only a slightly greater induction. Phenobarbitone in a daily dose of 90 mg causes a reduction of 18% and 23% in misonidazole half-life after 1 and 2 weeks' pre-treatment respectively, but is less suitable clinically because of its sedative effect. A further series of studies using phenytoin as the inducing agent has shown that, despite adequate enzyme induction and increased misonidazole metabolism, it is impossible to increase the total dose of misonidazole beyond the usually accepted value of 12 g/m/sup 2/ because of unacceptable neuropathy (a rate of 50% at a dose of 14 g/m/sup 2/ over three weeks). In single doses of above 3.0-4.0 g of misonidazole, severe nausea and vomiting are prominent, so that this side effect is a determining factor in the treatment fractionation. Audiometric studies show no correlation between the incidence of peripheral neuropathy and abnormal audiograms, and have no value in the early prediction of neurotoxicity.

Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

International Nuclear Information System (INIS)

Thierry-Palmer, M.; Cullins, S.; Rashada, S.; Gray, T.K.; Free, A.

1986-01-01

The authors have determined the ontogeny of vitamin D 3 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM 3 H-vitamin D 3 in the presence of an NADPH generating system, oxygen, KCl, and MgCl 2 . Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D 3 (250HD 3 ) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD 3 in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D 3 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D 3 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D 3 25-hydroxylase

Human gamma interferon production by cytotoxic T lymphocytes sensitized during hepatitis A virus infection

International Nuclear Information System (INIS)

Maier, K.; Gabriel, P.; Koscielniak, E.; Stierhof, Y.D.; Wiedmann, K.H.; Flehmig, B.; Vallbracht, A.

1988-01-01

The production of interferon (IFN) during a chromium-51 release assay with hepatitis A virus (HAV)-infected fibroblasts and autologous peripheral blood lymphocytes from patients with acute HAV infection was studied to determine whether IFN plays a role in immunopathogenesis of hepatitis A infection in humans. Skin fibroblasts of eight patients after acute HAV infection and from two control persons without history of current of past HAV infection were infected with HAV. Peripheral blood lymphocytes were collected at different times after the onset of icterus and tested in a chromium-51 release assay against autologous HAV-infected skin fibroblasts for their cytolytic and IFN-producing activity. The IFN produced during the assay was characterized and found to have the properties of human gamma IFN. Cytotoxicity and gamma IFN release were virus specific. The cell types responsible for both functions were characterized and found to be in the HLA-dependent T8 + lymphocyte subset. Considering that gamma IFN has an antiviral effect on persistent HAV infection in vitro and that it probably accounts for stimulation of HLA class I antigen expression on hepatocytes, these experimental results presented here demonstrate that human gamma IFN produced by HAV-specific T cells may participate in pathogenesis of hepatitis A infection in humans

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes

International Nuclear Information System (INIS)

Liang, T.; Cheung, A.H.; Reynolds, G.F.; Rasmusson, G.H.

1985-01-01

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a K/sub i/ value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [ 3 H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([ 3 H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2- 3 H]Diazo-MAPD binds to a single high affinity site in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000

Hepatitis C virus infection in the human immunodeficiency virus infected patient

DEFF Research Database (Denmark)

Clausen, Louise Nygaard; Lundbo, Lene Fogt; Benfield, Thomas

2014-01-01

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) share the same transmission routes; therefore, coinfection is frequent. An estimated 5-10 million individuals alone in the western world are infected with both viruses. The majority of people acquire HCV by injection drug use and...

Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: An in vitro comparative study

Energy Technology Data Exchange (ETDEWEB)

Buc-Calderon, P.; Roberfroid, M. (Universite Catholique de Louvain, Brussels (Belgium))

1989-09-01

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.

Effects of 1,2-dibromo-3-chloropropane on hepatic heme synthesis

International Nuclear Information System (INIS)

Moody, D.E.; Clawson, G.A.; Piper, W.N.; Smuckler, E.A.

1984-01-01

Previous studies showed that 1,2-dibromo-3-chloropropane (DBCP) caused a decrease in hepatic microsomal cytochrome P-450 suggesting that hepatic heme metabolism may be affected by DBCP treatment. Various parameters of hepatic heme synthesis were measured at intervals ranging from 0 to 72 hr in male Sprague-Dawley rats given a single oral dose (200 mg/kg) of DBCP. Incorporation of the radiolabeled heme precursor [delta-14C]aminolevulinic acid (14C-ALA) into liver, protein, extracted heme, and subcellular fractions of liver homogenates was significantly decreased to 75, 58, and 81% of controls, respectively, at 24 hr. At 48 and 72 hr after DBCP treatment, the accumulation of 14C-ALA label after 4 hr in liver homogenates and subcellular fractions was significantly increased in comparison to controls. These changes in 14C-ALA uptake were accompanied by decreases in total liver and microsomal heme, but not mitochondrial heme. Decreases were found in the spectral content of two heme proteins, cytochromes P-450 and b5, and the activity of another heme protein, catalase. Heme oxygenase activity increased to 130, 151, 209, and 186% of control values at 12, 24, 48, and 72 hr after DBCP, respectively. A slight, but significant, increase in ALA-synthetase to 112% of controls occurred at 24 hr, and slight, but significant, decreases in ALA-dehydratase to 90 and 80% of control occurred at 12 and 24 hr, respectively. No significant changes in uroporphyrinogen-1-synthetase or ferrochelatase at the time points tested was noted. The porphyrin content of liver was increased to 130% of control, while the serum and urine porphyrin levels were decreased to 30% of the control values at 24 hr. Liver ALA content was not significantly altered through the time period studied, but serum and urine levels were increased at 24 hr to 176 and 130% of the control values, respectively. In conclusion, the decreases in liver heme proteins following a single oral dose of DBCP are accompanied by

Genetic Polymorphisms in Organic Cation Transporter 1 Attenuates Hepatic Metformin Exposure in Humans

DEFF Research Database (Denmark)

Sundelin, E. I.O.; Gormsen, Lars C; Jensen, J. B.

2017-01-01

the transporter protein OCT1, affect the hepatic distribution of metformin in humans. We performed noninvasive 11C-metformin positron emission tomography (PET)/computed tomography (CT) to determine hepatic exposure in 12 subjects genotyped for variants in SLC22A1. Hepatic distribution of metformin...... was significantly reduced after oral intake in carriers of M420del and R61C variants in SLC22A1 without being associated with changes in circulating levels of metformin. Our data show that genetic polymorphisms in transporter proteins cause variation in hepatic exposure to metformin, and it demonstrates......Metformin has been used successfully to treat type 2 diabetes for decades. However, the efficacy of the drug varies considerably from patient to patient and this may in part be due to its pharmacokinetic properties. The aim of this study was to examine if common polymorphisms in SLC22A1, encoding...

Immunological disorders in chronic hepatitis C Egyptian patients.

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Shaker, M K; Fahmy, H M

1997-01-01

It is known that hepatitis C virus (HCV) related to chronic liver disease may be associated with various immunological disorders, among these disorders are mixed cryoglobulinemia, serum antinuclear antibodies, antismooth muscle antibodies and liver/kidney microsomal antibody type 1 (LKM1). However, the actual prevalence and pathogenic role of these disorders in patients with chronic hepatitis C are unclear. It was our aim to estimate the prevalence of different autoimmune antibodies in cases of hepatitis C chronic liver disease and to assess if such changes have any clinical significance. A total of 30 chronic hepatitis C patients (22 males and 8 females) with a mean age of 43.5 +/- 6.7 years, all patients were ELISA II positive, HCV RNA PCR positive and HbsAg negative, with elevated ALT more than 2 folds of the normal, in addition to 20 healthy controls of matched age and sex were tested for rheumatoid factor, cryoglobulin, antinuclear antibody, antismooth muscle antibody, antimitochondrial antibody and LKM1. The rheumatoid factor was present in 18 (60%) of the HCV Ab +ve patients and in 1 (5%) of the controls (p 0.24), antinuclear antibody was positive in 4 (13.3%) of the patients and in 1 (5%) of the controls (p > 0.6), antismooth muscle antibody was positive in 1 (3.3%) of the patients and not detected in any of the controls, the antimitochondrial antibody and LKM1 were not detected in both the patients and the controls. In conclusion, we can see that chronic hepatitis C patients show prevalence of some autoimmune antibodies and their presence is not associated with any implication on the clinical presentation.

In vitro biotransformation of flavonoids by rat liver microsomes

DEFF Research Database (Denmark)

Nielsen, S. E.; Breinholt, V.; Justesen, U.

1998-01-01

1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver...... pathway leading to the corresponding 3',4'-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B......-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C-4'-, and not at the C-3'-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P...

Modulation of trichloroethylene in vitro metabolism by different drugs in human.

Science.gov (United States)

Cheikh Rouhou, Mouna; Haddad, Sami

2014-08-01

Toxicological interactions with drugs have the potential to modulate the toxicity of trichloroethylene (TCE). Our objective is to identify metabolic interactions between TCE and 14 widely used drugs in human suspended hepatocytes and characterize the strongest using microsomal assays. Changes in concentrations of TCE and its metabolites were measured by headspace GC-MS. Results with hepatocytes show that amoxicillin, cimetidine, ibuprofen, mefenamic acid and ranitidine caused no significant interactions. Naproxen and salicylic acid showed to increase both TCE metabolites levels, whereas acetaminophen, carbamazepine and erythromycin rather decreased them. Finally, diclofenac, gliclazide, sulphasalazine and valproic acid had an impact on the levels of only one metabolite. Among the 14 tested drugs, 5 presented the most potent interactions and were selected for confirmation with microsomes, namely naproxen, salicylic acid, acetaminophen, carbamazepine and valproic acid. Characterization in human microsomes confirmed interaction with naproxen by competitively inhibiting trichloroethanol (TCOH) glucuronidation (Ki=2.329 mM). Inhibition of TCOH formation was also confirmed for carbamazepine (partial non-competitive with Ki=70 μM). Interactions with human microsomes were not observed with salicylic acid and acetaminophen, similar to prior results in rat material. For valproic acid, interactions with microsomes were observed in rat but not in human. Inhibition patterns were shown to be similar in human and rat hepatocytes, but some differences in mechanisms were noted in microsomal material between species. Next research efforts will focus on determining the adequacy between in vitro observations and the in vivo situation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dried blood spots, valid screening for viral hepatitis and human immunodeficiency virus in real-life

DEFF Research Database (Denmark)

Mössner, Belinda K; Staugaard, Benjamin; Jensen, Janne

2016-01-01

AIM: To detect chronic hepatitis B (CHB), chronic hepatitis C (CHC) and human immunodeficiency virus (HIV) infections in dried blood spot (DBS) and compare these samples to venous blood sampling in real-life. METHODS: We included prospective patients with known viral infections from drug treatment......, but correctly classified 95% of the anti-HCV-positive patients with chronic and past infections. Anti-HBc and anti-HBS showed low sensitivity in DBS (68% and 42%). CONCLUSION: DBS sampling, combined with an automated analysis system, is a feasible screening method to diagnose chronic viral hepatitis and HIV...

Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells

Directory of Open Access Journals (Sweden)

Joseph Ignatius Irudayam

2015-12-01

Full Text Available Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5, hepatoblast (day 15 and hepatocyte-like cells (day 21 were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21 had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.

Regulation of hepatic level of fatty-acid-binding protein by hormones and clofibric acid in the rat.

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Nakagawa, S; Kawashima, Y; Hirose, A; Kozuka, H

1994-01-01

Regulation of the hepatic level of fatty-acid-binding protein (FABP) by hormones and p-chlorophenoxyisobutyric acid (clofibric acid) was studied. The hepatic level of FABP, measured as the oleic acid-binding capacity of the cytosolic FABP fraction, was decreased in streptozotocin-diabetic rats. The level of FABP was markedly increased in adrenalectomized rats, and the elevation was prevented by the administration of dexamethasone. Hypothyroidism decreased the level of FABP and hyperthyroidism increased it. A high correlation between the incorporation of [14C]oleic acid in vivo into hepatic triacylglycerol and the level of FABP was found for normal, diabetic and adrenalectomized rats. The level of FABP was increased by administration of clofibric acid to rats in any altered hormonal states, as was microsomal 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, a peroxisome-proliferator-responsive parameter. These results suggest that the hepatic level of FABP is under regulation by multiple hormones and that clofibric acid induces FABP and 1-acyl-GPC acyltransferase by a mechanism which may be distinct from that by which hormones regulate the level of FABP. PMID:8110197

Metabolism of bilirubin by human cytochrome P450 2A6

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Abu-Bakar, A' edah, E-mail: a.abubakar@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Arthur, Dionne M. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Wikman, Anna S. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Department of Pharmaceutical Biosciences, Uppsala University, SE-75123 Uppsala (Sweden); Rahnasto, Minna; Juvonen, Risto O.; Vepsäläinen, Jouko; Raunio, Hannu [School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, POB 1627, 70211 Kuopio (Finland); Ng, Jack C. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Lang, Matti A. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia)

2012-05-15

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K{sub i} of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic

Metabolism of bilirubin by human cytochrome P450 2A6

International Nuclear Information System (INIS)

Abu-Bakar, A'edah; Arthur, Dionne M.; Wikman, Anna S.; Rahnasto, Minna; Juvonen, Risto O.; Vepsäläinen, Jouko; Raunio, Hannu; Ng, Jack C.; Lang, Matti A.

2012-01-01

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K i of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic CYP2A6

Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

Science.gov (United States)

Geldof, Lore; Lootens, Leen; Polet, Michael; Eichner, Daniel; Campbell, Thane; Nair, Vinod; Botrè, Francesco; Meuleman, Philip; Leroux-Roels, Geert; Deventer, Koen; Eenoo, Peter Van

2014-07-01

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method. Copyright © 2014 John Wiley & Sons, Ltd.

Selective inhibition of CYP2C8 by fisetin and its methylated metabolite, geraldol, in human liver microsomes.

Science.gov (United States)

Shrestha, Riya; Kim, Ju-Hyun; Nam, Wongshik; Lee, Hye Suk; Lee, Jae-Mok; Lee, Sangkyu

2018-04-01

Fisetin is a flavonol compound commonly found in edible vegetables and fruits. It has anti-tumor, antioxidant, and anti-inflammatory effects. Geraldol, the O-methyl metabolite of fisetin in mice, is reported to suppress endothelial cell migration and proliferation. Although the in vivo and in vitro effects of fisetin and its metabolites are frequently reported, studies on herb-drug interactions have not yet been performed. This study was designed to investigate the inhibitory effect of fisetin and geraldol on eight isoforms of human cytochrome P450 (CYP) by using cocktail assay and LC-MS/MS analysis. The selective inhibition of CYP2C8-catalyzed paclitaxel hydroxylation by fisetin and geraldol were confirmed in pooled human liver microsomes (HLMs). In addition, an IC 50 shift assay under different pre-incubation conditions confirmed that fisetin and geraldol shows a reversible concentration-dependent, but not mechanism-based, inhibition of CYP2C8. Moreover, Michaelis-Menten, Lineweaver-burk plots, Dixon and Eadie-Hofstee showed a non-competitive inhibition mode with an equilibrium dissociation constant of 4.1 μM for fisetin and 11.5 μM for geraldol, determined from secondary plot of the Lineweaver-Burk plot. In conclusion, our results indicate that fisetin showed selective reversible and non-competitive inhibition of CYP2C8 more than its main metabolite, geraldol, in HLMs. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

Directory of Open Access Journals (Sweden)

Ruchi Sharma

2010-01-01

Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

Science.gov (United States)

Attar, Mayssa; Dong, Dahai; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

2003-04-01

Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

Coinfection of Hepatic Cell Lines with Human Immunodeficiency Virus and Hepatitis B Virus Leads to an Increase in Intracellular Hepatitis B Surface Antigen▿

Science.gov (United States)

Iser, David M.; Warner, Nadia; Revill, Peter A.; Solomon, Ajantha; Wightman, Fiona; Saleh, Suha; Crane, Megan; Cameron, Paul U.; Bowden, Scott; Nguyen, Tin; Pereira, Cândida F.; Desmond, Paul V.; Locarnini, Stephen A.; Lewin, Sharon R.

2010-01-01

Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals. PMID:20357083

Coinfection of hepatic cell lines with human immunodeficiency virus and hepatitis B virus leads to an increase in intracellular hepatitis B surface antigen.

Science.gov (United States)

Iser, David M; Warner, Nadia; Revill, Peter A; Solomon, Ajantha; Wightman, Fiona; Saleh, Suha; Crane, Megan; Cameron, Paul U; Bowden, Scott; Nguyen, Tin; Pereira, Cândida F; Desmond, Paul V; Locarnini, Stephen A; Lewin, Sharon R

2010-06-01

Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals.

Comparative Pathology of Hepatitis A Virus and Hepatitis E Virus Infection.

Science.gov (United States)

Cullen, John M; Lemon, Stanley M

2018-04-30

Hepatitis A virus (HAV) and hepatitis E virus (HEV) cause acute, self-limiting hepatic infections that are usually spread by the fecal-oral route in humans. Naturally occurring and experimental infections are possible in a variety of nonhuman primates and, in the case of HEV, a number of other species. Many advances in understanding the pathogenesis of these viruses have come from studies in experimental animals. In general, animals infected with these viruses recapitulate the histologic lesions seen in infected humans, but typically with less severe clinical and histopathological manifestations. This review describes the histopathologic changes associated with HAV and HEV infection in humans and experimental animals. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Occurrence of water-borne enteric viruses in two settlements based in Eastern Chad: analysis of hepatitis E virus, hepatitis A virus and human adenovirus in water sources.

Science.gov (United States)

Guerrero-Latorre, Laura; Carratala, Anna; Rodriguez-Manzano, Jesus; Calgua, Byron; Hundesa, Ayalkibet; Girones, Rosina

2011-09-01

Hepatitis E virus (HEV) is a common cause of water-borne acute hepatitis in areas with poor sanitation. In 2004 an outbreak of HEV infection affected around 2,000 people in Eastern Chad (Dar Sila). This paper describes the decrease in the incidence of acute jaundice syndrome (AJS) from 2004 until 2009 when a mean incidence of 0.48 cases/1,000 people/year was recorded in the region. Outbreaks of AJS were identified in some of the camps in 2007 and 2008. Moreover, water samples from drinking water sources were screened for human adenoviruses considered as viral indicators and for hepatitis A virus and HEV. Screening of faecal samples from donkeys for HEV gave negative results. Some of the samples were also analysed for faecal coliforms showing values before disinfection treatment between 3 and >50 colony forming units per 100 mL. All water samples tested were negative for HEV and HAV; however, the presence of low levels of human adenoviruses in 4 out of 16 samples analysed indicates possible human faecal contamination of groundwater. Consequently, breakdowns in the treatment of drinking water and/or increased excretion of hepatitis viruses, which could be related to the arrival of a new population, could spread future outbreaks through drinking water.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

Science.gov (United States)

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M P; Albano, E; Bianchi, F B

2000-04-01

Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

Methadone hydrochloride: acute administration, disposition and effects on hepatic function in guinea pigs.

Science.gov (United States)

Pak, R C; Ecobichon, D J

1981-01-01

d,1-Methadone hydrochloride was administered orally to adult female albino guinea pigs at a dose of 25 mg/kg body weight every 12 h for 10 consecutive days. Twelve hours after a dose, subgroups of animals were sacrificed at 2, 5 and 10 days for tissue (blood plasma, brain, liver and kidney) methadone residue analysis and the in vitro measurement of hepatic microsomal p-nitroanisole O-demethylase (OD), aniline hydroxylase (AH) and glucuronosyltransferase (GT) activities. No overt toxicity was observed during treatment other than a decrease in body weight. Withdrawal signs were absent during the 14-day post-treatment regression period. Tissue methadone levels were constant except for a decreased concentration in the liver at 5 and 10 days. No effect on hepatic OD and AH was observed during treatment but a significant decrease in GT activity was measured which returned to normal values 14 days after terminating treatment.

Human hepatic carbohydrate metabolism. Dynamic observation using 13C MRS without proton decoupling

International Nuclear Information System (INIS)

Ikehira, H.; Obata, T.; Koga, M.; Yoshida, K.

1997-01-01

Purpose: Dynamic natural-abundance 13 C MR spectroscopy (MRS) studies without proton decoupling were performed in the human liver using commercial 1.5 T MR equipment. Material and methods: A single tuned custom-made circular surface coil with an OD of 20 cm operating at 16.04 MHz was used for the 13 C study. Seventy-five grams of glucose dissolved in water was administered for the natural-abundance 13 C-MRS dynamic study which lasted for approximately 40 to 60 min. Data acquisition was broken into 20-min and 1.7-min blocks. Localized proton shimming with a whole-body coil was performed with sufficient volume to include the observing area of the surface coil; the line width of the water signal was less than 20 Hz. Results and Conclusion: The glucose and glycogen spectra were clearly visible at 80 to 120 ppm after oral administration of the glucose solution. These data demonstrate that dynamic hepatic carbohydrate metabolism can be observed with commercially available MR equipment. Given that the human hepatic glycogen pool reaches maximum level within less than 10 min, this technique should provide a direct diagnosis of hepatic carbohydrate metabolic disorders. (orig.)

Immunoglobulin GM and KM Allotypes and Prevalence of Anti-LKM1 Autoantibodies in Patients with Hepatitis C Virus Infection

OpenAIRE

Muratori, Paolo; Sutherland, Susan E.; Muratori, Luigi; Granito, Alessandro; Guidi, Marcello; Pappas, Georges; Lenzi, Marco; Bianchi, Francesco B.; Pandey, Janardan P.

2006-01-01

GM and KM allotypes—genetic markers of immunoglobulin (Ig) γ and κ chains, respectively—are associated with humoral immunity to several infection- and autoimmunity-related epitopes. We hypothesized that GM and KM allotypes contribute to the generation of autoantibodies to liver/kidney microsomal antigen 1 (LKM1) in hepatitis C virus (HCV)-infected persons. To test this hypothesis, we characterized 129 persons with persistent HCV infection for several GM and KM markers and for anti-LKM1 antibo...

The role of CYP2D6 in primary and secondary oxidative metabolism of dextromethorphan: in vitro studies using human liver microsomes.

Science.gov (United States)

Kerry, N L; Somogyi, A A; Bochner, F; Mikus, G

1994-01-01

1. The enzyme kinetics of dextromethorphan O-demethylation in liver microsomes from three extensive metabolisers (EM) with respect to CYP2D6 indicated high (Km1 2.2-9.4 microM) and low (Km2 55.5-307.3 microM) affinity sites whereas microsomes from two poor metabolisers (PM) indicated a single site (Km 560 and 157 microM). Similar differences were shown for 3-methoxymorphinan O-demethylation to 3-hydroxymorphinan (Km 6.9-9.6 microM in EM subjects; Km 307 and 213 microM in PM subjects). 2. Dextromethorphan O-demethylation was inhibited competitively by quinidine (Ki 0.1 microM), rac-perhexiline (Ki 0.4 microM), dextropropoxyphene (Ki 6 microM), rac-methadone (Ki 8 microM), and 3-methoxymorphinan (Ki 15 microM). These compounds were also potent inhibitors of 3-methoxymorphinan O-demethylation with IC50 values ranging from 0.02-12 microM. Anti-LKM1 serum inhibited both dextromethorphan and 3-methoxymorphinan O-demethylations in a titre-dependent manner. 3. The Michaelis-Menten constant for dextromethorphan N-demethylation to 3-methoxymorphinan (Km 632-977 microM) and dextrorphan N-demethylation to 3-hydroxymorphinan (Km 1571-4286 microM) did not differ between EM and PM microsomes. These N-demethylation reactions were not inhibited by quinidine and rac-methadone or LKM1 antibodies. 4. Dextromethorphan and 3-methoxymorphinan are metabolised by the same P450 isoform, CYP2D6, whereas the N-demethylation reactions are not carried out by CYP2D6. PMID:7826826

Prevalence of nucleic acid sequences specific for human parvoviruses, hepatitis A and hepatitis E viruses in coagulation factor concentrates.

Science.gov (United States)

Modrow, S; Wenzel, J J; Schimanski, S; Schwarzbeck, J; Rothe, U; Oldenburg, J; Jilg, W; Eis-Hübinger, A M

2011-05-01

Due to their high resistance to inactivation procedures, nonenveloped viruses such as parvovirus B19, human bocavirus (HBoV), human parvovirus 4 (PARV4), hepatitis A (HAV) and hepatitis E virus (HEV) pose a particular threat to blood products. Virus transmission to patients treated with blood products presents an additional burden to disease. We determined the frequency and the amount of nucleic acid specific for nonenveloped viruses in recently manufactured preparations of commercial coagulation factor concentrates. At least three different batches of each of 13 different plasma-derived and recombinant coagulation factor products were tested for the presence and the amount of nucleic acid for parvovirus B19, HBoV, human parvovirus 4, hepatitis A virus and HEV by using quantitative polymerase chain reaction. Whereas none of the recombinant products tested positive for any of these viruses, parvovirus B19 DNA with amounts ranging between 2×10(1) and 1.3×10(3) genome equivalents/ml was detected in five plasma-derived products. In addition to parvovirus B19 genotype 1, genotypes 2 and 3 were observed in two batches of a factor VIII/von-Willebrand factor product. In two products (one factor VIII concentrate and one activated prothrombin complex concentrate), a combination of both genotypes 1 and 2 of parvovirus B19 was detected. The data show that nucleic acids from several relevant nonenveloped viruses are not found at detectable levels in coagulation factor concentrates. In some cases, parvovirus B19 DNA was detectable at low levels. Testing of the plasma pools for the full range of parvovirus genotypes is advocated for ensuring product safety. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

Calmodulin stimulation of calcium transport in carrot microsomal vesicles

International Nuclear Information System (INIS)

Pierce, W.S.; Sze, H.

1987-01-01

ATP-dependent 45 Ca 2+ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca 2+ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca 2+ uptake (at 1.17 g/cc) was not affected by oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca 2+ . These results are consistent with the ER being an important site of intracellular Ca 2+ regulation

Correlation between the dielectric properties and biological activities of human ex vivo hepatic tissue

International Nuclear Information System (INIS)

Wang, Hang; You, Fusheng; Fu, Feng; Dong, Xiuzhen; Shi, Xuetao; He, Yong; Yang, Min; Yan, Qingguo

2015-01-01

Dielectric properties are vital biophysical features of biological tissues, and biological activity is an index to ascertain the active state of tissues. This study investigated the potential correlation between the dielectric properties and biological activities of human hepatic tissue with prolonged ex vivo time through correlation and regression analyses. The dielectric properties of 26 cases of normal human hepatic tissue at 10 Hz to 100 MHz were measured from 15 min after isolation to 24 h at 37 °C with 90% humidity. Cell morphologies, including nucleus area (NA) and alteration rate of intercellular area (ICAR), were analyzed as indicators of biological activities. Conductivity, complex resistivity, and NA exhibited opposing changes 1 h after isolation. Relative permittivity and ex vivo time were not closely correlated (p > 0.05). The dielectric properties measured at low frequencies (i.e. <1 MHz) were more sensitive than those measured at high frequencies in reflecting the biological activity of ex vivo tissue. Highly significant correlations were found between conductivity, resistivity and the ex vivo time (p < 0.05) as well as conductivity and the cell morphology (p < 0.05). The findings indicated that establishing the correlation between the dielectric properties and biological activities of human hepatic tissue is of great significance for promoting the role of dielectric properties in biological science, particularly in human biology. (paper)

Using Bovine Viral Diarrhea Virus (BVDV) As Surrogate for Human Hepatitis C Virus

Science.gov (United States)

This test is designed to validate virucidal effectiveness claims for a product to be registered as a virucide. It determines the potential of the test agent to disinfect hard surfaces contaminated with human Hepatitis C virus (HCV).

Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

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Andreas Bitter

2015-01-01

Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

Identification of cytochrome P450s involved in the metabolism of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) using human recombinant enzymes and rat liver microsomes in vitro.

Science.gov (United States)

Lu, Ying-Yuan; Cheng, Hai-Xu; Wang, Xin; Wang, Xiao-Wei; Liu, Jun-Yi; Li, Pu; Lou, Ya-Qing; Li, Jun; Lu, Chuang; Zhang, Guo-Liang

2017-08-01

1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and human in vitro. 2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). 3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including K m , V max , and CL int were 2.3 μM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4. 4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug-drug interactions.

Peginterferon plus ribavirin for chronic hepatitis C in patients with human immunodeficiency virus

DEFF Research Database (Denmark)

Gluud, Lise Lotte; Marchesini, Emanuela; Iorio, Alfonso

2009-01-01

OBJECTIVES: The aim of this study was to assess the effects of peginterferon plus ribavirin for chronic hepatitis C in patients with human immunodeficiency virus (HIV). METHODS: Trials were identified through manual and electronic searches. Randomized trials comparing peginterferon plus ribavirin...... with other antiviral treatments for patients with chronic hepatitis C and HIV were included. The primary outcome measure was virological response at the end of treatment and after > or =6 months (sustained). Intention-to-treat meta-analyses including data on all patients who were randomized were carried out....... RESULTS: Seven randomized trials were eligible for inclusion. The patients included had chronic hepatitis C and stable HIV and were not previously treated with interferon or ribavirin (treatment naive). The mean dosages were 180 or 1.5 microg/kg once weekly for peginterferon and 800 mg daily for ribavirin...

Distinct ontogenic patterns of overt and latent DGAT activities of rat liver microsomes.

Science.gov (United States)

Waterman, Ian J; Price, Nigel T; Zammit, Victor A

2002-09-01

We have studied the ontogeny of the two functional diacylglycerol acyltransferase (DGAT) activities (overt and latent) during postnatal development in rat liver. We find that the ontogenic patterns of the two are highly distinct. Overt DGAT shows a transient rise in activity up to day 4 postnatally, after which it declines until weaning; thereafter, it increases steadily to reach high adult values that may contribute to the high rates of turnover of cytosolic triacylglycerol (TAG). By contrast, latent DGAT activity increases continuously during the suckling period but falls sharply upon weaning onto chow but not onto a high-fat diet. Rates of TAG secretion by hepatocytes are higher than in the adult during the first 7 days after birth, and are largely dependent on the mobilization of the abundant intrahepatocyte TAG as a source of acyl moieties. When the hepatic steatosis is cleared (after day 7) the TAG secretion rate declines by 80% to reach adult values. Quantification of the content of mRNA for the DGAT1 and DGAT2 genes does not show correlation with either of the DGAT activities. We conclude that post-translational modification may play an important role in the overt and latent distribution of DGAT activity in the liver microsomal membrane.

Chlordecone altered hepatic disposition of [14C]cholesterol and plasma cholesterol distribution but not SR-BI or ABCG8 proteins in livers of C57BL/6 mice

International Nuclear Information System (INIS)

Lee, Junga; Scheri, Richard C.; Curtis, Lawrence R.

2008-01-01

Organochlorine (OC) insecticides continue to occur in tissues of humans and wildlife throughout the world although they were banned in the United States a few decades ago. Low doses of the OC insecticide chlordecone (CD) alter hepatic disposition of lipophilic xenobiotics and perturb lipid homeostasis in rainbow trout, mice and rats. CD pretreatment altered tissue and hepatic subcellular distribution of exogenous [ 14 C]cholesterol (CH) equivalents 4 and 16 h after a bolus intraperitoneal (ip) injection of 5 ml corn oil/kg that contained 10 mg CH/kg. CD pretreatment altered tissue distribution of exogenously administered [ 14 C]CH by decreased hepatic and renal accumulation, and increased biliary excretion up to 300%. Biliary excretion of polar [ 14 C]CH metabolites was not altered by CD. CD pretreatment decreased subcellular distribution of [ 14 C]CH equivalents in hepatic cytosol and microsomes and lipoprotein-rich fraction-to-homogenate ratio. CD pretreatment increased the ratio of [ 14 C]CH equivalents in high density lipoprotein (HDL) to that in plasma and reduced [ 14 C]CH equivalents in the non-HDL fraction 4 h after a bolus lipid dose. CD pretreatment increased plasma non-HDL total CH by 80% 4 h after a bolus lipid dose. Scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G8 (ABCG8) proteins were quantified by western blotting in hepatic membranes from control and CD treated mice. Liver membrane contents of SR-BI and ABCG8 proteins were unchanged by CD pretreatment. The data demonstrated that a single dose of CD altered CH homeostasis and lipoprotein metabolism

Chlordecone altered hepatic disposition of [14C]cholesterol and plasma cholesterol distribution but not SR-BI or ABCG8 proteins in livers of C57BL/6 mice.

Science.gov (United States)

Lee, Junga; Scheri, Richard C; Curtis, Lawrence R

2008-06-15

Organochlorine (OC) insecticides continue to occur in tissues of humans and wildlife throughout the world although they were banned in the United States a few decades ago. Low doses of the OC insecticide chlordecone (CD) alter hepatic disposition of lipophilic xenobiotics and perturb lipid homeostasis in rainbow trout, mice and rats. CD pretreatment altered tissue and hepatic subcellular distribution of exogenous [(14)C]cholesterol (CH) equivalents 4 and 16 h after a bolus intraperitoneal (ip) injection of 5 ml corn oil/kg that contained 10 mg CH/kg. CD pretreatment altered tissue distribution of exogenously administered [(14)C]CH by decreased hepatic and renal accumulation, and increased biliary excretion up to 300%. Biliary excretion of polar [(14)C]CH metabolites was not altered by CD. CD pretreatment decreased subcellular distribution of [(14)C]CH equivalents in hepatic cytosol and microsomes and lipoprotein-rich fraction-to-homogenate ratio. CD pretreatment increased the ratio of [(14)C]CH equivalents in high density lipoprotein (HDL) to that in plasma and reduced [(14)C]CH equivalents in the non-HDL fraction 4 h after a bolus lipid dose. CD pretreatment increased plasma non-HDL total CH by 80% 4 h after a bolus lipid dose. Scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G8 (ABCG8) proteins were quantified by western blotting in hepatic membranes from control and CD treated mice. Liver membrane contents of SR-BI and ABCG8 proteins were unchanged by CD pretreatment. The data demonstrated that a single dose of CD altered CH homeostasis and lipoprotein metabolism.

PKCδ regulates hepatic insulin sensitivity and hepatosteatosis in mice and humans

DEFF Research Database (Denmark)

Bezy, Olivier; Tran, Thien T; Pihlajamäki, Jussi

2011-01-01

C57BL/6J and 129S6/Sv (B6 and 129) mice differ dramatically in their susceptibility to developing diabetes in response to diet- or genetically induced insulin resistance. A major locus contributing to this difference has been mapped to a region on mouse chromosome 14 that contains the gene encoding...... tolerance, and reduced hepatosteatosis with aging. Conversely, mice with liver-specific overexpression of PKCδ developed hepatic insulin resistance characterized by decreased insulin signaling, enhanced lipogenic gene expression, and hepatosteatosis. Therefore, changes in the expression and regulation...... of PKCδ between strains of mice and in obese humans play an important role in the genetic risk of hepatic insulin resistance, glucose intolerance, and hepatosteatosis; and thus PKCδ may be a potential target in the treatment of metabolic syndrome....

Susceptibility to thyroid disorders in hepatitis C.

Science.gov (United States)

Muratori, Luigi; Bogdanos, Dimitrios P; Muratori, Paolo; Lenzi, Marco; Granito, Alessandro; Ma, Yun; Mieli-Vergani, Giorgina; Bianchi, Francesco B; Vergani, Diego

2005-06-01

Autoimmune thyroid disorders (AITDs) are reported, especially during interferon treatment, in chronic HCV infection, in which non-organ-specific autoantibodies (NOSAs) are common. We wondered whether seropositivity for NOSA is associated with susceptibility to AITDs. We evaluated thyroid function and antithyroglobulin and antithyroperoxidase antibodies in 348 Italian patients with chronic hepatitis C (34% NOSA-positive), 196 patients (33% NOSA-positive) of whom received interferon treatment. At baseline, thyroid disorders were significantly more frequent in liver/kidney microsomal antibody type 1 (LKM1)-positive patients (29% vs 9%, P LKM1-positive patients (50% vs 3%, P LKM1 positivity were predictors of AITD, but only the latter remained significant after logistic regression analysis. Cross-reactivity to all 7 linear epitopes encoding homologous amino acid sequences shared by the HCV polyprotein, CYP2D6 (the LKM1 autoantigen), and thyroperoxidase was detected in 86% LKM1-positive HCV patients with clinical thyroid disorders, but in none of the LKM1-positive or negative HCV patients without thyroid disease, and none of an HCV-negative control group comprising subjects with LKM1-positive autoimmune hepatitis or AITD without liver disease ( P LKM1 are susceptible to develop AITDs, in association with treatment. Molecular mimicry and epitope spreading are potential pathogenic mechanisms.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

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Siqueira, E.R.F. [Departamento de Gastroenterologia, LIM-07, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Departamento de Bioquímica, Faculdade de Medicina, Universidade de Pernambuco, Recife, PE (Brazil); Departamento de Gastroenterologia, Faculdade de Medicina, Universidade de Pernambuco, Recife, PE (Brazil); Oliveira, C.P.M.S. [Departamento de Gastroenterologia, LIM-07, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Correa-Giannella, M.L. [Laboratório de Endocrinologia Celular e Molecular, LIM-25, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Stefano, J.T. [Departamento de Gastroenterologia, LIM-07, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Cavaleiro, A.M.; Fortes, M.A.H.Z. [Laboratório de Endocrinologia Celular e Molecular, LIM-25, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Muniz, M.T.C.; Silva, F.S. [Departamento de Bioquímica, Faculdade de Medicina, Universidade de Pernambuco, Recife, PE (Brazil); Pereira, L.M.M.B. [Departamento de Gastroenterologia, Faculdade de Medicina, Universidade de Pernambuco, Recife, PE (Brazil); Instituto do Fígado de Pernambuco, Recife, PE (Brazil); Carrilho, F.J. [Departamento de Gastroenterologia, LIM-07, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil)

2011-12-09

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson's disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

International Nuclear Information System (INIS)

Siqueira, E.R.F.; Oliveira, C.P.M.S.; Correa-Giannella, M.L.; Stefano, J.T.; Cavaleiro, A.M.; Fortes, M.A.H.Z.; Muniz, M.T.C.; Silva, F.S.; Pereira, L.M.M.B.; Carrilho, F.J.

2011-01-01

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson's disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients

Transfusion-associated hepatitis before the screening of blood for hepatitis risk factors

DEFF Research Database (Denmark)

Engle, Ronald E; Bukh, Jens; Alter, Harvey J

2014-01-01

%) with HBV alone, and one (3%) with both viruses. Overall, 100% of patients with hepatitis and 39% of those without hepatitis were infected with HBV and/or HCV; one patient was also infected with hepatitis E virus. The donor carrier rate for HBV and/or HCV was estimated to be more than 6%; contemporaneously......%) developed biochemical evidence of hepatitis; of these, 20 (67%) were infected with hepatitis C virus (HCV) alone, four (13%) with hepatitis B virus (HBV) alone, and six (20%) with both viruses. Among the 36 patients who did not develop hepatitis, four (11%) were newly infected with HCV alone, nine (25...... prepared pooled normal human plasma was also contaminated with multiple hepatitis viruses. CONCLUSION: TAH virus infections were a larger problem than perceived 50 years ago and HCV was the predominant agent transmitted. All hepatitis cases could be attributed to HCV and/or HBV and hence...

[Significance of non-organ-specific autoantibodies in HCV-related chronic hepatitis].

Science.gov (United States)

Guidi, Marcello; Muratori, Paolo; Granito, Alessandro; Muratori, Luigi; Pappas, Georgios; Bianchi, Francesco B

2005-12-01

The preliminary question regarding the clinical issue of the antiviral therapy in the HCV related chronic hepatitis patients is: is it mandatory the research for the autoantibodies in the eligible patients for the antiviral treatment? This issue is of particular interest at the light of the the reported cases of HCV positive patients with positivity for liver kidney microsome type 1 antibody who developed a hepatitic flare during the antiviral treatment. The data from literature about the efficacy and safety on the antiviral treatment in patients with autoantibodies are few and controversial, particularly if the ones regarding antiviral drugs and more recent treatment regimens are taking into account (peg-interferon, combined therapy of interferon and ribavirin). Large and prospective studies are needed for a thorough evaluation about the potential impact of autoantibodies reactivity on the therapeutic outcome. To date, it must be confirmed that a strict monitoring of hepatic parameters is to recommend during the whole treatment phase. This in the light of a potential appearance of significant flares of aminotransferases, particularly in subjects with anti LKM-1 autoantibodies, during interferon therapy.

Autoantibody detection in type 2 autoimmune hepatitis using a chimera recombinant protein.

Science.gov (United States)

Vitozzi, Susana; Lapierre, Pascal; Djilali-Saiah, Idriss; Alvarez, Fernando

2002-04-01

Autoantibodies against cytochrome P450 2D6 (CYP2D6), known as anti-liver/kidney microsome type 1 (LKM1) and/or anti-human formiminotransferase cyclodeaminase, formally known as anti-liver cytosol type 1 (LC1) define type 2 autoimmune hepatitis (AIH). The aims of this work are to develop a sensitive and specific test to detect anti-LKM1 and/or anti-LC1 autoantibodies and to establish the prevalence of anti-LC1. Sera from children with type 2 AIH (n=48) and those from a control group (n=100) were evaluated for anti-LKM1 and anti-LC1 by Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. Each serum sample was assayed for reactivity against formiminotransferase cyclodeaminase and CYP2D6 alone or as part of a recombinant chimera protein. By ELISA with recombinant chimera protein, 50 serum samples were positive, 48 from patients with type 2 AIH and 2 from patients with chronic hepatitis C. Twenty-five of 48 (52%) patients studied were positive for both CYP2D6 and LC1 autoantibodies. Anti-LC1, either as the only marker or associated with anti-LKM1, was positive in 34/48 (71%). By Western blotting, anti-LC1 was found in 27/48 (56%) patients. This ELISA technique has proven to be antigen-specific and more sensitive than Western blot for the detection of anti-LC1 and anti-LKM1 autoantibodies. The prevalence of anti-LC1 (71%) confirms it as an important immunomarker in type 2 AIH.

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

Science.gov (United States)

Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

2013-07-19

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Hepatitis E virus and fulminant hepatitis--a virus or host-specific pathology?

Science.gov (United States)

Smith, Donald B; Simmonds, Peter

2015-04-01

Fulminant hepatitis is a rare outcome of infection with hepatitis E virus. Several recent reports suggest that virus variation is an important determinant of disease progression. To critically examine the evidence that virus-specific factors underlie the development of fulminant hepatitis following hepatitis E virus infection. Published sequence information of hepatitis E virus isolates from patients with and without fulminant hepatitis was collected and analysed using statistical tests to identify associations between virus polymorphisms and disease outcome. Fulminant hepatitis has been reported following infection with all four hepatitis E virus genotypes that infect humans comprising multiple phylogenetic lineages within genotypes 1, 3 and 4. Analysis of virus sequences from individuals infected by a common source did not detect any common substitutions associated with progression to fulminant hepatitis. Re-analysis of previously reported associations between virus substitutions and fulminant hepatitis suggests that these were probably the result of sampling biases. Host-specific factors rather than virus genotype, variants or specific substitutions appear to be responsible for the development of fulminant hepatitis. © 2014 The Authors. Liver International Published by John Wiley & Sons Ltd.

Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

Science.gov (United States)

Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

2012-11-01

Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats.

α-Tocopherol incorporation in mitochondria and microsomes upon supranutritional vitamin E supplementation

DEFF Research Database (Denmark)

Lauridsen, Charlotte; Jensen, Søren Krogh

2012-01-01

Vitamin E (α-tocopherol) is a major lipid-soluble chain-breaking antioxidant in humans and mammals and plays an important role in normal development and physiology. The localization of α-tocopherol within the highly unsaturated phospholipid bilayer of cell membranes provides a means of controlling...... cellular compartments is important in order to maintain oxidative stability of the membrane-bound lipids and prevent damage from the reactive oxygen species. Many studies regarding mitochondrial disease and dysfunction have been performed in relation to deficiency of vitamin E and other antioxidants...... with antioxidants on their incorporation into mitochondria and other cellular membranes. The purpose of this review is therefore to briefly summarize experimental data performed with dietary vitamin E treatments in relation to the deposition of α-tocopherol in mitochondria and microsomes....

Detection on immunoblot of new proteins from the soluble fraction of the cell recognized either by anti-liver-kidney microsome antibodies type 1 or by anti-liver cytosol antibodies type 1--relationship with hepatitis C virus infection.

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Ballot, E; Desbos, A; Monier, J C

1996-09-01

Antibodies directed against liver cytosol protein, called anti-liver cytosol type 1 (LC1 Ab), have been described by both immunofluorescence (IF) and immunodiffusion techniques in sera from patients with autoimmune hepatitis (AIH). They have never been found in association with antibodies directed against the hepatitis C virus (HCV), unlike the anti-liver-kidney microsome antibodies type 1 (LKM1 Ab), the serological marker of AIH type 2. This suggests that there are two subgroups of AIH type 2, i.e., HCV-related and non-HCV-related. In this study, immunoblotting experiments were performed using proteins from the soluble phase of the rat liver cell; 141 sera which tested positive for LKM1 Ab by IF, 24 identified as having LC1 Ab by IF, and 50 from blood donors as controls were analyzed. Three bands were stained by LC1 Ab sera more often than by the control sera, and with a statistically significant frequency. These 3 proteins were located at apparent Mr 50,000, 55,000, and 60,000. The LKM1 Ab-positive sera as defined by IF stained six bands with a statistically significant frequency compared to the controls. Their apparent Mr were 35,000, 39,000, 47,000, 50,000, 55,000, and 60,000. LKM1 Ab-positive sera which were anti-HCV negative recognized a 60,000 protein belonging to the soluble phase of the cell, with a statistically significant frequency compared to LKM1 Ab-positive sera which were anti-HCV positive. This 60,000 protein was also recognized by LC1 Ab-positive sera, which were almost always anti-HCV negative. The presence of antibodies against a 60,000 protein from the soluble phase of the cell is discussed in terms of the anti-HCV serological markers found in the sera from patients with AIH.

Hepatitis C virus nonstructural protein 5A favors upregulation of gluconeogenic and lipogenic gene expression leading towards insulin resistance: a metabolic syndrome.

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Parvaiz, Fahed; Manzoor, Sobia; Iqbal, Jawed; McRae, Steven; Javed, Farrakh; Ahmed, Qazi Laeeque; Waris, Gulam

2014-05-01

Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.

Gene expression data from acetaminophen-induced toxicity in human hepatic in vitro systems and clinical liver samples

Directory of Open Access Journals (Sweden)

Robim M. Rodrigues

2016-06-01

Full Text Available This data set is composed of transcriptomics analyses of (i liver samples from patients suffering from acetaminophen-induced acute liver failure (ALF and (ii hepatic cell systems exposed to acetaminophen and their respective controls. The in vitro systems include widely employed cell lines i.e. HepaRG and HepG2 cells as well as a novel stem cell-derived model i.e. human skin-precursors-derived hepatocyte-like cells (hSKP-HPC. Data from primary human hepatocytes was also added to the data set “Open TG-GATEs: a large-scale toxicogenomics database” (Igarashi et al., 2015 [1]. Changes in gene expression due to acetaminophen intoxication as well as comparative information between human in vivo and in vitro samples are provided. The microarray data have been deposited in NCBI׳s Gene Expression Omnibus and are accessible through GEO Series accession number GEO: GSE74000. The provided data is used to evaluate the predictive capacity of each hepatic in vitro system and can be directly compared with large-scale publically available toxicogenomics databases. Further interpretation and discussion of these data feature in the corresponding research article “Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems” (Rodrigues et al., 2016 [2].

Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes.

Science.gov (United States)

Kot, Marta; Daniel, Władysława A

2009-01-01

The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.

Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro

International Nuclear Information System (INIS)

Uehleke, H.; Poplawski-Tabarelli, S.

1977-01-01

1) 14 C-labelled trichloroethylene was injected i.p. into male mice (10 μmole/g of b.w.). The radioactivity irreversibly bound to hepatic protein reached highest levels after 6 h : 2 nmole/mg in cytosol protein, 4.4 nmole/mg in mitochondrial protein, and 7.6 nmole/mg in microsomal protein. 2) The commercial trichloroethylene contained radioactive impurities binding to proteins without metabolic activation. Purification by various extraction removed 60-70% of those materials. In aerobic incubates of mice hepatic microsomes and NADPH the covalent binding rate of the purified trichloroethylene was 1.4 nmole/mg protein in 60 min. The activity of rat liver microsomes was approximately 40% less. Covalent binding increased 2-fold with microsomes of mice pretreated with phenobarbital. (orig.) [de

Modulation of catechol estrogen synthesis by rat liver microsomes: effects of treatment with growth hormone or testosterone

International Nuclear Information System (INIS)

Quail, J.A.; Jellinck, P.H.

1987-01-01

The ability of GH from various mammalian species, administered to normal mature male rats by constant infusion, to decrease the hepatic 2-hydroxylation of estradiol (E2) to female levels, as measured by the release of 3 H 2 O from [2-3H]E2, was determined. Rat and human GH (hGH) showed the highest activity while ovine GH was inactive. PRL (0.6 IU/h X kg) administered together with hGH (0.02 IU/h X kg) did not antagonize the feminizing action of GH. Infusion of hGH into male rats decreased the affinity of estradiol 2-hydroxylase for its steroid substrate and altered the linear Lineweaver-Burk plot towards a nonlinear hyperbolic plot characteristic of the female. The apparent Michaelis-Menten constant (Km) for the reaction was 1.69 microM for males and 2.75 microM for testosterone-treated ovariectomized females. An equal mixture of liver microsomes from male and female rats gave kinetic values similar to those observed with males alone. Neonatal imprinting with androgen did not alter the magnitude of the response of female rats to treatment with testosterone and/or GH at maturity and the androgen effect could only be shown in ovariectomized animals. The results with rats of different endocrine status were corroborated by the kinetic data and by the pattern of metabolites obtained with [4- 14 C]E2 when examined by TLC and autoradiography. The hormonal control of estradiol 2-hydroxylase, the key enzyme in catechol estrogen formation, and the contribution of sex-specific multiple forms of the enzyme to this reaction are discussed

Studies on the metabolism of chlorotrianisene to a reactive intermediate and subsequent covalent binding to microsomal proteins

International Nuclear Information System (INIS)

Juedes, M.J.

1989-01-01

The studies on chlorotrianisene were conducted to determine whether metabolism of chlorotrianisene occurs via the cytochrome P450 monooxygenase system and whether a reactive intermediate is being formed that is capable of binding covalently to microsomal proteins. [ 3 H]-chlorotrianisene was incubated with liver microsomes supplemented with NADPH. At the termination of the incubation, the protein was trapped on a glass filter and the unbound chlorotrianisene was removed by extensive washing of the protein with organic solvent. A dramatic stimulation of covalent binding was demonstrated in microsomes from rats treated with methylcholanthrene (60 fold increase) versus control or phenobarbital treatment. Verification of covalent binding was achieved by localization of radiolabeled bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the macromolecules in the incubation mixture. Further analysis of the radiolabeled macromolecules separated on SDS-PAGE revealed that these macromolecules were degraded by protease degradation indicating that the macromolecules were proteins. Further investigations were done to determine the cause of the dramatic stimulation of covalent binding detected in microsomes from methylcholanthrene treated rats versus control or phenobarbital treated rats. Further evidence for the participation of P-450c was obtained with a reconstituted cytochrome P-450 system. Incubations of chlorotrianisene with reconstituted P-450c and NADPH-cytochrome P-450 reductase exhibited covalent binding characteristics comparable to those seen in microsomal incubations. Investigations into the nature of the binding site and the reactive intermediate are currently being conducted. By analyzing the BSA adduct, the author intends to isolate the specific amino acid binding site(s)

Molecular Pathogenesis of Liver Steatosis Induced by Hepatitis C Virus

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Cheng Jun

2012-09-01

Full Text Available Liver steatosis is a pathological hallmark in patients with chronic hepatitis C (CHC. Increased lipid uptake, decreased lipid secretion, increased lipid synthesis and decreased lipid degradation are all involved in pathogenesis of steatosis induced by hepatitic C virus (HCV infection. Level of low density lipoprotein receptor (LDL-R and activity of peroxisome proliferator-activated receptor (PPAR α is related to liver uptake of lipid from circulation, and affected by HCV. Secretion via microsomal triglyceride transfer protein (MTTP, and formation of very low density lipoprotein (VLDL have been hampered by HCV infection. Up-regulation of lipid synthesis related genes, such as sterol regulatory element-binding protein (SREBP-1, SREBP-2, SREBP-1c, fatty acid synthase (FASN, HMG CoA reductase (HMGCR, liver X receptor (LXR, acetyl-CoA carboxylase 1 (ACC1, hepatic CB (1 receptors, retinoid X receptor (RXR α, were the main stay of liver steatosis pathogenesis. Degradation of lipid in liver is decreased in patients with CHC. There is strong evidence that heterogeneity of HCV core genes of different genotypes affect their effects of liver steatosis induction. A mechanism in which steatosis is involved in HCV life cycle is emerging.

A simple assay method for omega-oxidation of lauric acid by hepatic enzymes

International Nuclear Information System (INIS)

Giera, D.D.; van Lier, R.B.L.

1990-01-01

Routine assessment of hepatic ω-oxidation of fatty acids in toxicology studies requires a simpler method of enzymatic analysis than HPLC or TLC. A method depending upon selective solvent separation of 14 C-lauric acid and 14 C-11/12-hydroxy lauric acid was developed. Following enzymatic incubation and addition of 15% methanol to the acidified incubation mixtures, partitioning with an alkane solvent such as iso-octane, cyclohexane, or n-hexane separated the lauric acid substrate and ω-hydroxylated products into two immiscible phases. Approximately 98% of the substrate partitioned into the organic phase, and approximately 83% of the hydroxylated products partitioned into the aqueous phase. Subsequent quantitation of the enzymatic activity required only liquid scintillation counting of the aqueous phase. Hepatic homogenates from male rats treated with 0.01, 0.05, 0.125, and 0.25% clofibrate in the diet for 7 days had enzyme levels 1.3, 6.1, 11.1, and 15.9 times control values, respectively, when assayed by conventional TLC methods, and 1.3, 5.3, 12.3, and 15.3 times control values when assayed by the solvent extraction method. The data indicate that the selective solvent partitioning yields comparable precision and sensitivity to the more conventional TLC method when studying induction of hepatic microsomal enzymes

Inhibition of rat mammary microsomal oxidation of ethanol to acetaldehyde by plant polyphenols.

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Maciel, María Eugenia; Castro, José Alberto; Castro, Gerardo Daniel

2011-07-01

We previously reported that the microsomal fraction from rat mammary tissue is able to oxidize ethanol to acetaldehyde, a mutagenic-carcinogenic metabolite, depending on the presence of NADPH and oxygen but not inhibited by carbon monoxide or other cytochrome P450 inhibitors. The process was strongly inhibited by diphenyleneiodonium, a known inhibitor of NADPH oxidase, and by nordihydroguaiaretic acid, an inhibitor of lipoxygenases. This led us to suggest that both enzymes could be involved. With the purpose of identifying natural compounds present in food with the ability to decrease the production of acetaldehyde in mammary tissue, in the present studies, several plant polyphenols having inhibitory effects on lipoxygenases and of antioxidant nature were tested as potential inhibitors of the rat mammary tissue microsomal pathway of ethanol oxidation. We included in the present screening study 32 polyphenols having ready availability and that were also tested against the rat mammary tissue cytosolic metabolism of ethanol to acetaldehyde. Several polyphenols were also able to inhibit the microsomal ethanol oxidation at concentrations as low was 10-50 μM. The results of these screening experiments suggest the potential of several plant polyphenols to prevent in vivo production and accumulation of acetaldehyde in mammary tissue.

Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats.

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Vilaseca, Marina; García-Calderó, Héctor; Lafoz, Erica; Ruart, Maria; López-Sanjurjo, Cristina Isabel; Murphy, Michael P; Deulofeu, Ramon; Bosch, Jaume; Hernández-Gea, Virginia; Gracia-Sancho, Jordi; García-Pagán, Juan Carlos

2017-07-01

In cirrhosis, activated hepatic stellate cells (HSC) play a major role in increasing intrahepatic vascular resistance and developing portal hypertension. We have shown that cirrhotic livers have increased reactive oxygen species (ROS), and that antioxidant therapy decreases portal pressure. Considering that mitochondria produce many of these ROS, our aim was to assess the effects of the oral mitochondria-targeted antioxidant mitoquinone on hepatic oxidative stress, HSC phenotype, liver fibrosis and portal hypertension. Ex vivo: Hepatic stellate cells phenotype was analysed in human precision-cut liver slices in response to mitoquinone or vehicle. In vitro: Mitochondrial oxidative stress was analysed in different cell type of livers from control and cirrhotic rats. HSC phenotype, proliferation and viability were assessed in LX2, and in primary human and rat HSC treated with mitoquinone or vehicle. In vivo: CCl 4 - and thioacetamide-cirrhotic rats were treated with mitoquinone (5 mg/kg/day) or the vehicle compound, DecylTPP, for 2 weeks, followed by measurement of oxidative stress, systemic and hepatic haemodynamic, liver fibrosis, HSC phenotype and liver inflammation. Mitoquinone deactivated human and rat HSC, decreased their proliferation but with no effects on viability. In CCl 4 -cirrhotic rats, mitoquinone decreased hepatic oxidative stress, improved HSC phenotype, reduced intrahepatic vascular resistance and diminished liver fibrosis. These effects were associated with a significant reduction in portal pressure without changes in arterial pressure. These results were further confirmed in the thioacetamide-cirrhotic model. We propose mitochondria-targeted antioxidants as a novel treatment approach against portal hypertension and cirrhosis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Carbonyl Reduction of NNK by Recombinant Human Lung Enzymes. Identification of HSD17β12 as the Reductase important in (R)-NNAL formation in Human Lung.

Science.gov (United States)

Ashmore, Joseph H; Luo, Shaman; Watson, Christy J W; Lazarus, Philip

2018-05-17

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of the present study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17β6, HSD17β12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels >HSD11β1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17β12 the most highly expressed. Of these lung-expressing enzymes, only HSD17β12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knockdown of HSD17β12 resulted in significant decreases in (R)-NNAL formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17β12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung.

Prevalence of human immunodeficiency virus, syphilis, hepatitis B and C in blood donations in Namibia.

Science.gov (United States)

Mavenyengwa, Rooyen T; Mukesi, Munyaradzi; Chipare, Israel; Shoombe, Esra

2014-05-05

Transfusion Transmissible Infections (TTIs) such as Human Immunodeficiency Virus (HIV), syphilis, hepatitis B virus (HBV) and hepatitis C virus (HCV) are infections which are common in some communities in Southern Africa. It is important to screen blood donations for these infections. This is a retrospective study which involved reviewing of previous blood donation records for the year 2012 in Namibia. The records were analyzed to determine the prevalence of HIV, syphilis, Hepatitis B and C among blood donations with regard to gender, age and geographical region of the donors. The findings indicated a significantly low prevalence of HIV, syphilis, HBsAg and anti-Hepatitis C among the blood donations. A low infection rate of 1.3% by any of the four tested TTIs was found among the blood donations given by the donor population in Namibia in 2012. The blood donations given by the donor population in Namibia has a low infection rate with the HIV, syphilis, HBsAg and anti-HCV. A strict screening regime must continue to be used as the infections are still present albeit in small numbers.

Counter-attack on virol hepatitis

International Nuclear Information System (INIS)

Prozesky, O.W.; Jupp, P.G.; Joubert, J.J.; Taylor, M.B.; Grabow, W.O.K.

1985-01-01

The most highly developed radioimmunoassay test system in medical virology is proving of exceptional value in research aimed at controlling and eventually eradicating the scourge of human hepatitis. The use of radioimmunoassay in detecting hepatitis A (HAV) and hepatitis B (HBV) viruses is discussed. The hepatitis A virus is an enterovirus which infects the gastrointestinal tract and is usually transmitted by contaminated food, milk or water. Hepatitis B spreads mainly by the parenteral rate. Bedbugs and ticks are considered as possible transmitters of HBV. Another important contribution of radioimmunoassay is the ability to monitor the immune response of persons at risk who are vaccinated against hepatitis B

Oxidative metabolism of BDE-47, BDE-99, and HBCDs by cat liver microsomes: Implications of cats as sentinel species to monitor human exposure to environmental pollutants.

Science.gov (United States)

Zheng, Xiaobo; Erratico, Claudio; Luo, Xiaojun; Mai, Bixian; Covaci, Adrian

2016-05-01

BDE-99 is different by cat and human liver microsomes. This suggests that cats are not a suitable sentinel to represent internal exposure of PBDEs for humans, but is likely a promising sentinel for internal HBCDs exposure for humans. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Peroxide modification of membranes and isomorphic composition of cytochrome P-450 of rat liver microsomes during antioxidant deficiency].

Science.gov (United States)

Gubskiy, Iu I; Paramonova, G I; Boldeskul, A E; Primak, R G; Bogdanova, L A; Zadorina, O V; Litvinova, N V

1992-01-01

Lipid peroxidation (LPO), physico-chemical properties of the membranes and isoformic composition of microsomal cytochrome P-450 from the rat liver were studied under conditions of antioxidant insufficiency (AOI) which was modelled by exclusion of alpha-tocopherol from the animals' ration. An insignificant accumulation of microsomal diene conjugates and schiff bases against a sharp increase of the ability to the prooxidant stimulated LPO in vitro took place. A significant decrease of membrane lipid microviscosity and a change in surface properties of microsomal membranes of rats with AOI was determined. Absence of alpha-tocopherol in the ration was accompanied by a significant change in the content of separate isoforms of cytochrome P-450 exhibited in growth of a polypeptide with m. w. 54 kDa and the lowering of proteins with m. w. 48 and 50 kDa. Less intensive quenching of tryptophan fluorescence by acrylamide was also revealed, which testified to a lower accessibility of the quencher to membrane proteins or their fluorophore sites. Modification of lipid composition and of physicochemical properties of the rat liver membrane microsomes which was observed at AOI was significantly correlated by pretreatment with the antioxidant 4-methyl-2,6-ditretbutylphenol (ionol).

A preliminary discussion of angiographic anatomy and variations of rabbit hepatic vessels and catheterization methods of hepatic artery

International Nuclear Information System (INIS)

Wang Diaodong; Yang Renjie; Zhang Hongzhi; Sun Hongliang

2006-01-01

Objective: To study the normal angiographic anatomy and variations of rabbit hepatic vessels, and explore the optimal method for hepatic artery catheterization. Methods: 30 rabbits were divided into two groups randomly. Modified surgical method and interventional method were used to catheterize hepatic artery respectively, and followed by angiography to demonstrate the normal anatomy and variations of rabbit celiac artery, hepatic artery and portal vein. Results: The route and distribution of rabbit celiac artery and hepatic artery were very different from human's. The commonly seen variation showed the differences in branching bifurcation of hepatic-gastric artery, with the incidence of 13.3%. The rates of successfully hepatic artery catheterization with surgical and interventional methods were 86.6%(13/15) and 80%(12/15) respectively (P>0.05). The surgical method will not be successful, whenever there's variation. Conclusion: The normal anatomy and variation of rabbit celiac artery and hepatic artery are quite different from human's. Both surgical and interventional catheterizations could be rather successful but possessing advantages and disadvantages of each its own. (authors)

[History of viral hepatitis].

Science.gov (United States)

Fonseca, José Carlos Ferraz da

2010-01-01

The history of viral hepatitis goes back thousands of years and is a fascinating one. When humans were first infected by such agents, a natural repetitive cycle began, with the capacity to infect billions of humans, thus decimating the population and causing sequelae in thousands of lives. This article reviews the available scientific information on the history of viral hepatitis. All the information was obtained through extensive bibliographic review, including original and review articles and consultations on the internet. There are reports on outbreaks of jaundice epidemics in China 5,000 years ago and in Babylon more than 2,500 years ago. The catastrophic history of great jaundice epidemics and pandemics is well known and generally associated with major wars. In the American Civil War, 40,000 cases occurred among Union troops. In 1885, an outbreak of catarrhal jaundice affected 191 workers at the Bremen shipyard (Germany) after vaccination against smallpox. In 1942, 28,585 soldiers became infected with hepatitis after inoculation with the yellow fever vaccine. The number of cases of hepatitis during the Second World War was estimated to be 16 million. Only in the twentieth century were the main agents causing viral hepatitis identified. The hepatitis B virus was the first to be discovered. In this paper, through reviewing the history of major epidemics caused by hepatitis viruses and the history of discovery of these agents, singular peculiarities were revealed. Examples of this include the accidental or chance discovery of the hepatitis B and D viruses.

Autoimmune hepatitis in children: what is different from adult AIH?

Science.gov (United States)

Mieli-Vergani, Giorgina; Vergani, Diego

2009-08-01

Autoimmune hepatitis (AIH) is characterized by inflammatory liver histology, circulating non-organ-specific autoantibodies, and increased levels of immunoglobulin (Ig) G in the absence of a known etiology. Two types of childhood AIH are recognized according to seropositivity: smooth muscle antibody (SMA) and/or antinuclear antibody (ANA), which is AIH type 1; and antibodies to liver-kidney microsome type 1 (anti-LKM1), which is AIH type 2. There is a female predominance in both. Autoimmune hepatitis type 2 presents more acutely, at a younger age, and commonly with IgA deficiency; however, duration of symptoms before diagnosis, clinical signs, family history of autoimmunity, presence of associated autoimmune disorders, response to treatment, and long-term prognosis are similar in the two groups. Immunosuppressive treatment with steroids and azathioprine, which should be instituted promptly to avoid progression to cirrhosis, induces remission in 80% of cases. Relapses are common, often due to nonadherence. Drugs effective in refractory cases include cyclosporine and mycophenolate mofetil. Long-term treatment is usually required, with only some 20% of AIH type 1 patients able to discontinue therapy successfully. In childhood, sclerosing cholangitis with strong autoimmune features, including interface hepatitis and serological features identical to AIH type 1, is as prevalent as AIH, but it affects boys and girls equally. The differential diagnosis relies on cholangiographic studies. In autoimmune sclerosing cholangitis, liver parenchymal damage responds satisfactorily to immunosuppressive treatment, whereas bile duct disease tends to progress. Copyright Thieme Medical Publishers.

Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle.

Science.gov (United States)

Ballent, M; Virkel, G; Maté, L; Viviani, P; Lanusse, C; Lifschitz, A

2016-10-01

Monepantel (MNP) is a new amino-acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO2 ) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40 μm of MNP. Heat pretreatment (50 °C for 2 min) of liver microsomes was performed for inactivation of the flavin-monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80 μm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well-known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO2 was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO2 was fivefold higher (P ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive degree of adsorption (83% to 90%) to the solid phase of the ruminal content. Overall, these results are a further contribution to the understanding of the metabolic fate of this anthelmintic drug in ruminants. © 2016 John Wiley & Sons Ltd.

Dietary sea cucumber cerebroside alleviates orotic acid-induced excess hepatic adipopexis in rats

Science.gov (United States)

2012-01-01

Background Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease in industrialized countries. The present study was undertaken to explore the preventive effect of dietary sea cucumber cerebroside (SCC) extracted from Acaudina molpadioides in fatty liver rats. Methods Male Wistar rats were randomly divided into four groups including normal control group, NAFLD model group, and two SCC-treated groups with SCC at 0.006% and 0.03% respectively. The fatty liver model was established by administration of 1% orotic acid (OA) to the rats. After 10d, serum and hepatic lipid levels were detected. And the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were also determined. Besides, to gain the potential mechanism, the changes of key enzymes and gene expressions related to the hepatic lipid metabolism were measured. Results Dietary SCC at the level of 0.006% and 0.03% ameliorated the hepatic lipid accumulation in fatty liver rats. SCC administration elevated the serum triglyceride (TG) level and the ALT, AST activities in OA-fed rats. The activities of hepatic lipogenic enzymes including fatty acid synthase (FAS), malic enzyme (ME) and glucose-6-phosphatedehydrogenase (G6PDH) were inhibited by SCC treatment. And the gene expressions of FAS, ME, G6PDH and sterol-regulatory element binding protein (SREBP-1c) were also reduced in rats fed SCC. However, dietary SCC didn't affect the activity and mRNA expression of carnitine palmitoyltransferase (CPT) in liver. Besides, suppression of microsomal triglyceride transfer protein (MTP) activity was observed in SCC-feeding rats. Conclusions These results suggested that dietary SCC could attenuate hepatic steatosis due to its inhibition of hepatic lipogenic gene expression and enzyme activity and the enhancement of TG secretion from liver. PMID:22569330

Dietary sea cucumber cerebroside alleviates orotic acid-induced excess hepatic adipopexis in rats

Directory of Open Access Journals (Sweden)

Zhang Bei

2012-05-01

Full Text Available Abstract Background Nonalcoholic fatty liver disease (NAFLD is a prevalent chronic liver disease in industrialized countries. The present study was undertaken to explore the preventive effect of dietary sea cucumber cerebroside (SCC extracted from Acaudina molpadioides in fatty liver rats. Methods Male Wistar rats were randomly divided into four groups including normal control group, NAFLD model group, and two SCC-treated groups with SCC at 0.006% and 0.03% respectively. The fatty liver model was established by administration of 1% orotic acid (OA to the rats. After 10d, serum and hepatic lipid levels were detected. And the serum alanine aminotransferase (ALT and aspartate aminotransferase (AST activities were also determined. Besides, to gain the potential mechanism, the changes of key enzymes and gene expressions related to the hepatic lipid metabolism were measured. Results Dietary SCC at the level of 0.006% and 0.03% ameliorated the hepatic lipid accumulation in fatty liver rats. SCC administration elevated the serum triglyceride (TG level and the ALT, AST activities in OA-fed rats. The activities of hepatic lipogenic enzymes including fatty acid synthase (FAS, malic enzyme (ME and glucose-6-phosphatedehydrogenase (G6PDH were inhibited by SCC treatment. And the gene expressions of FAS, ME, G6PDH and sterol-regulatory element binding protein (SREBP-1c were also reduced in rats fed SCC. However, dietary SCC didn't affect the activity and mRNA expression of carnitine palmitoyltransferase (CPT in liver. Besides, suppression of microsomal triglyceride transfer protein (MTP activity was observed in SCC-feeding rats. Conclusions These results suggested that dietary SCC could attenuate hepatic steatosis due to its inhibition of hepatic lipogenic gene expression and enzyme activity and the enhancement of TG secretion from liver.

The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes

DEFF Research Database (Denmark)

Perry, Rachel J; Samuel, Varman T.; Petersen, Kitt Mia Falck

2014-01-01

Non-alcoholic fatty liver disease and its downstream sequelae, hepatic insulin resistance and type 2 diabetes, are rapidly growing epidemics, which lead to increased morbidity and mortality rates, and soaring health-care costs. Developing interventions requires a comprehensive understanding...... of the mechanisms by which excess hepatic lipid develops and causes hepatic insulin resistance and type 2 diabetes. Proposed mechanisms implicate various lipid species, inflammatory signalling and other cellular modifications. Studies in mice and humans have elucidated a key role for hepatic diacylglycerol...... activation of protein kinase Cε in triggering hepatic insulin resistance. Therapeutic approaches based on this mechanism could alleviate the related epidemics of non-alcoholic fatty liver disease and type 2 diabetes....

Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery.

Science.gov (United States)

Naritomi, Yoichi; Sanoh, Seigo; Ohta, Shigeru

2018-02-01

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus

OpenAIRE

El-Bacha , Tatiana; Midlej , Victor; Silva , Ana Paula Pereira Da; Costa , Leandro Silva Da; Benchimol , Marlene; Galina , Antonio; Poian , Andrea T. Da

2007-01-01

Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus correspondence: Corresponding author. Fax: +55 21 22708647. (El-Bacha, Tatiana) (El-Bacha, Tatiana) Laboratorio de Bioquimica de Virus, Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro - RJ-Brasil--> , Av. Bauhinia n? 400 ? CCS Bloco H 2? andar--> , sala 22. Ilha do Governador--> ...

[Study of enzymes of xenobiotic metabolism in the evaluation of quality of protein-containing wheat germ flakes and wallpaper flour].

Science.gov (United States)

Martinchuk, A N; E En Gyn; Safronova, A M; Peskova, E V

1991-01-01

Intake of wheat upholstery meal by growing rats was attended by a sharp decrease in the content and activity of xenobiotic metabolism enzymes in the hepatic microsomes, that was caused by the low biological value of the meal proteins. Hepatic microsomes of the rats that were fed with wheat germ flakes showed increased specific content of cytochromes P-450 and b5, but the total blood protein content per 100 g of body mass was lower than during casein consumption. No significant changes were detected in hydroxylation rate of benz(a)pyrene, aniline and ethylmorphine. During consumption of wheat germ flakes induction of UDP-glucuronide-transferase was detected in hepatic microsomes. Wheat germ flakes induced a 5-fold increase of Se-dependent glutathione peroxidase activity. Wheat germ flakes produced no significant effect on glutathione-S-aryltransferase and glutathione reductase activity.

Unanticipated increases in hepatic steatosis among human immunodeficiency virus patients receiving mineralocorticoid receptor antagonist eplerenone for non-alcoholic fatty liver disease.

Science.gov (United States)

Chaudhury, Chloe S; Purdy, Julia B; Liu, Chia-Ying; Morse, Caryn G; Stanley, Takara L; Kleiner, David; Hadigan, Colleen

2018-05-01

Non-alcoholic fatty liver disease is common in human immunodeficiency virus, but there are no approved therapies. The aim of this open-label proof-of-concept study was to determine the effect of the mineralocorticoid receptor antagonist eplerenone on hepatic fat in human immunodeficiency virus-infected patients with hepatic fat ≥5% by magnetic resonance spectroscopy. Five subjects received eplerenone (25 mg daily × 1 week followed by 50 mg daily × 23 weeks). Laboratory tests were done at each visit, and the primary endpoint, change in hepatic fat content, was determined by MRI spectroscopy at baseline and week 24. The study was stopped early after observing unexpected significant increases in hepatic fat at week 24 (mean increase 13.0 ± 7.3%, P = .02). The increases in steatosis were accompanied by a tendency for transaminase values to decrease (alanine aminotransferase mean change -14 ± 16 IU/L, P = .14). There were no consistent changes in other metabolic parameters or blood pressure. Repeat assessment of hepatic steatosis 1-2 months after stopping study medication revealed improvements in steatosis towards baseline values. The unexpected observation of increased hepatic steatosis with the administration of eplerenone led to early termination of the investigation. While limited because of the small number of participants and the open-label design, this study provides data to suggest that mineralocorticoid receptor antagonism with eplerenone may not be an effective approach to treat hepatic steatosis in human immunodeficiency virus or the general population. Additional research is needed to determine the pathophysiological mechanism behind these unanticipated observations. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

Science.gov (United States)

Pu, Qiang-Hong; Shi, Liang; Yu, Chao

2015-03-01

1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

Age-Dependent Human Hepatic Carboxylesterase 1 (Ces1) and Carboxylesterase 2 (Ces2) Postnatal Ontogeny

Science.gov (United States)

Human hepatic carboxylesterase 1 and 2 (CES1 and CES2) are important for ester- and amide- bond containing pharmaceutical and environmental chemical disposition. Despite concern regarding juvenile sensitivity to such compounds, CES1 and CES2 ontogeny has not been well characteriz...

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin administration and high-fat diet on the body weight and hepatic estrogen metabolism in female C3H/HeN mice

International Nuclear Information System (INIS)

Zhu Baoting; Gallo, Michael A.; Burger, Conney W.; Meeker, Robert J.; Cai, May Xiaoxin; Xu Shiyao; Conney, Allan H.

2008-01-01

We studied the effect of administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by i.p. injection once every 2 weeks in combination with a high-fat (HF) diet for 8 or 16 weeks on the body and organ weight changes as well as on the hepatic enzyme activity for estrogen metabolism in C3H/HeN female mice. Administration of TCDD at 100 μg/kg b.w. once every 2 weeks for 8 weeks increased the body weight by 46% in the HF diet-fed animals, but not in the regular diet-fed animals. This is the first observation suggesting that TCDD at a high dose (100 μg/kg b.w.), but not at lower doses (1 or 10 μg/kg b.w.), may have a strong obesity-inducing effect in C3H/HeN mice fed an HF diet. While TCDD increased liver weight and decreased thymus weight in animals, these effects were enhanced by feeding animals an HF diet. Metabolism studies showed that TCDD administration for 8 or 16 weeks increased the liver microsomal activity for the 2- and 4-hydroxylation of 17β-estradiol in animals fed a control diet, but surprisingly not in animals fed an HF diet. Treatment with TCDD dose-dependently increased the hepatic activity for the O-methylation of catechol estrogens in both control and HF diet-fed animals, and it also decreased the levels of liver microsomal sulfatase activity for hydrolysis of estrone-3-sulfate. TCDD did not significantly affect the hepatic enzyme activity for the glucuronidation or esterification of endogenous estrogens. It is suggested that enhanced metabolic inactivation of endogenous estrogens by hepatic estrogen-metabolizing enzymes in TCDD-treated, control diet-fed animals contributes importantly to the reduced incidence of estrogen-associated tumors in animals treated with TCDD

Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions

Czech Academy of Sciences Publication Activity Database

Svobodová, Kateřina; Mikesková, Hana; Petráčková, Denisa

2013-01-01

Roč. 97, č. 24 (2013), s. 10263-10273 ISSN 0175-7598 R&D Projects: GA TA ČR TE01020218 Institutional support: RVO:61388971 Keywords : Fungal microsomes * Cytochrome P450 * Biodegradation Subject RIV: EE - Microbiology, Virology Impact factor: 3.811, year: 2013

Seroprevalence of hepatitis and human immuno-deficiency virus in multitransfused patients from a pediatric hematology clinic

Directory of Open Access Journals (Sweden)

Suar Çakı Kılıç

2008-12-01

Full Text Available OBJECTIVE: Transfusion transmitted hepatitis has been a severe problem in Turkey in pediatric cancer patients and in chronic congenital anemia. The aim of the present study was to investigate the prevalence of hepatitis B, hepatitis C and human immunodeficiency virus infections in these patients in a University Hospital. METHODS: Multi-transfused 66 children (59 acute leukemia, 6 thalassemia major, 1 severe hereditary spherocytosis diagnosed and followed-up between May, 2000 and December, 2006 were evaluated. Screening of all the patients for HbsAg, anti-HBs, anti-HBc, anti-HCV and anti-HIV was performed at presentation and during the last follow-up. Serologic studies of leukemic patients were also repeated at the end of the chemotherapy. Hepatitis B vaccination was administered to unvaccinated patients with anemia. All blood products were provided by Blood Bank of the Center. RESULTS: No patient was found HBsAg, anti-HCV or anti-HIV positive at diagnosis and at the end of the therapy. There was history of hepatitis B vaccination in only 42% of the patients at diagnosis due to administration of this vaccine to newborns since 1998. At the beginning of the study, 45 % (n=27 of the leukemic patients were immune for hepatitis B, but after completion of the intensive chemotherapy seropositivity persisted in only 28.8 % (n=17. CONCLUSION: Transmission of these viruses is no longer a real problem even in multitransfused immunosuppressed children in Pediatric Hematology Units as a result of the improvements in screening of voluntary blood donors, administration of disposable material in clinics and vaccination by hepatitis B.

Optical diagnostic of hepatitis B (HBV) and C (HCV) from human blood serum using Raman spectroscopy

International Nuclear Information System (INIS)

Anwar, Shahzad; Firdous, Shamaraz

2015-01-01

Hepatitis is the second most common disease worldwide with half of the cases arising in the developing world. The mortality associated with hepatitis B and C can be reduced if the disease is detected at the early stages of development. The aim of this study was to investigate the potential of Raman spectroscopy as a diagnostic tool to detect biochemical changes accompanying hepatitis progression. Raman spectra were acquired from 20 individuals with six hepatitis B infected patients, six hepatitis C infected patients and eight healthy patients in order to gain an insight into the determination of biochemical changes for early diagnostic. The human blood serum was examined at a 532 nm excitation laser source. Raman characteristic peaks were observed in normal sera at 1006, 1157 and 1513 cm −1 , while in the case of hepatitis B and C these peaks were found to be blue shifted with decreased intensity. New Raman peaks appeared in HBV and HCV infected sera at 1194, 1302, 844, 905, 1065 and 1303 cm −1 respectively. A Mat lab subroutine and frequency domain filter program is developed and applied to signal processing of Raman scattering data. The algorithms have been successfully applied to remove the signal noise found in experimental scattering signals. The results show that Raman spectroscopy displays a high sensitivity to biochemical changes in blood sera during disease progression resulting in exceptional prediction accuracy when discriminating between normal and malignant. Raman spectroscopy shows enormous clinical potential as a rapid non-invasive diagnostic tool for hepatitis and other infectious diseases. (letter)

Optical diagnostic of hepatitis B (HBV) and C (HCV) from human blood serum using Raman spectroscopy

Science.gov (United States)

Anwar, Shahzad; Firdous, Shamaraz

2015-06-01

Hepatitis is the second most common disease worldwide with half of the cases arising in the developing world. The mortality associated with hepatitis B and C can be reduced if the disease is detected at the early stages of development. The aim of this study was to investigate the potential of Raman spectroscopy as a diagnostic tool to detect biochemical changes accompanying hepatitis progression. Raman spectra were acquired from 20 individuals with six hepatitis B infected patients, six hepatitis C infected patients and eight healthy patients in order to gain an insight into the determination of biochemical changes for early diagnostic. The human blood serum was examined at a 532 nm excitation laser source. Raman characteristic peaks were observed in normal sera at 1006, 1157 and 1513 cm-1, while in the case of hepatitis B and C these peaks were found to be blue shifted with decreased intensity. New Raman peaks appeared in HBV and HCV infected sera at 1194, 1302, 844, 905, 1065 and 1303 cm-1 respectively. A Mat lab subroutine and frequency domain filter program is developed and applied to signal processing of Raman scattering data. The algorithms have been successfully applied to remove the signal noise found in experimental scattering signals. The results show that Raman spectroscopy displays a high sensitivity to biochemical changes in blood sera during disease progression resulting in exceptional prediction accuracy when discriminating between normal and malignant. Raman spectroscopy shows enormous clinical potential as a rapid non-invasive diagnostic tool for hepatitis and other infectious diseases.

Hepatitis B and C virus co-infections in human immunodeficiency virus positive North Indian patients

Science.gov (United States)

Gupta, Swati; Singh, Sarman

2006-01-01

AIM: To determine the prevalence of hepatitis B and C virus infections in human immunodeficiency virus (HIV) -positive patients at a tertiary care hospital in New Delhi, India. METHODS: Serum samples from 451 HIV positive patients were analyzed for HBsAg and HCV antibodies during three years (Jan 2003-Dec 2005). The control group comprised of apparently healthy bone-marrow and renal donors. RESULTS: The study population comprised essentially of heterosexually transmitted HIV infection. The prevalence rate of HBsAg in this population was 5.3% as compared to 1.4% in apparently healthy donors (P < 0.001). Though prevalence of HCV co-infection (2.43%) was lower than HBV in this group of HIV positive patients, the prevalence was significantly higher (P < 0.05) than controls (0.7%). Triple infection of HIV, HBV and HCV was not detected in any patient. CONCLUSION: Our study shows a significantly high prevalence of hepatitis virus infections in HIV infected patients. Hepatitis viruses in HIV may lead to faster progression to liver cirrhosis and a higher risk of antiretroviral therapy induced hepatotoxicity. Therefore, it would be advisable to detect hepatitis virus co-infections in these patients at the earliest. PMID:17106941

In vivo activity of a mixture of two human monoclonal antibodies (anti-HBs) in a chronic hepatitis B virus carrier chimpanzee

NARCIS (Netherlands)

R. Heijtink; W. Paulij; P.A.C. van Bergen (Patrick); M.H. van Roosmalen (Mark); D. Rohm; B. Eichentopf (Bertram); E. Muchmore; A.D.M.E. Osterhaus (Albert); R.A. de Man (Robert)

1999-01-01

textabstractA 35-year-old female hepatitis B virus carrier chimpanzee was infused with one dose of a mixture of human monoclonal antibodies 9H9 and 4-7B (antibodies against hepatitis B virus surface antigen; HBsAg). Blood samples were taken before and up to 3 weeks

Relationship between the murine Ah locus and 2,3,7,8-tetrachlorodibenzo-p-dioxin hepatic metabolism, enzyme induction, and toxicity

International Nuclear Information System (INIS)

Shen, E.S.

1988-01-01

The influence of the Ah locus and hepatic microsomal enzyme induction on 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) metabolism and hepatotoxicity was investigated using C57BL/6J (C57) and DBA/2J (DBA) mice. C57 mice are more sensitive to toxic and enzyme inductive effects of 2,3,7,8-TCDD than DBA mice. Characterization of interstrain differences in hepatic enzyme induction, 2,3,7,8-TCDD metabolism, and hepatotoxicity may aid in identifying the mechanism(s) of 2,3,7,8-TCDD toxicity. The hepatic uptake and metabolism of [ 14 C]2,3,7,8-TCDD were studied using isolated hepatocytes from control and 2,3,7,8-TCDD-pretreated C57 and DBA mice. Pretreated mice were injected with 2,3,7,8-TCDD at doses that maximally induce aryl hydrocarbon hydroxylase activity or at doses that approach the LD 50 value. Despite the induction of hepatic 7-ethoxyresorufin O-deethylase activity and benzo[a]pyrene metabolism, all 2,3,7,8-TCDD pretreatment doses failed to increase the rate of [ 14 C]2,3,7,8-TCDD metabolism for both C57 and DBA mice. These results suggest that the uptake and rate of hepatic metabolism of 2,3,7,8-TCDD do not correlate with genetic differences at the murine Ah locus

Age-dependent Hepatic UDP-glucuronosyltransferase Gene Expression and Activity in Children

Directory of Open Access Journals (Sweden)

Elizabeth Neumann

2016-11-01

Full Text Available ABSTRACTUDP-glucuronosyltransferases (UGTs are important phase II drug metabolism enzymes. The aim of this study was to explore the relationship between age and changes in mRNA expression and activity of major human hepatic UGTs, as well as to understand the potential regulatory mechanism underlying this relationship. Using previously generated data, we investigated age-dependent mRNA expression levels of 11 hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17 and 16 transcription factors (AHR, AR, CAR, ESR2, FXR, GCCR, HNF1a, HNF3a, HNF3b, HNF4a, PPARA, PPARG, PPARGC, PXR, SP1, and STAT3 in liver tissue of donors (n = 38 ranging from 0 to 25 years of age. We also examined the correlation between age and microsomal activities using 14 known UGT drug substrates in the liver samples (n = 19 of children donors. We found a statistically significant increase (nominal p < 0.05 in the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT2B7 and UGT2B17, as well as glucuronidation activities of serotonin, testosterone, and vorinostat during the first 25 years of life. Expression of estrogen receptor 1 (ESR1 and pregnane X receptor (PXR, two strong UGT transcriptional regulators, were significantly correlated with both age and UGT mRNA expression (p ≤ 0.05. These results suggest that both UGT expression and activity increase during childhood and adolescence, possibly driven in part by hormonal signaling. Our findings may help explain inter-patient variability in response to medications among children.

Identification of a tryptanthrin metabolite in rat liver microsomes by liquid chromatography/electrospray ionization-tandem mass spectrometry.

Science.gov (United States)

Lee, Sang Kyu; Kim, Ghee Hwan; Kim, Dong Hyeon; Kim, Dong Hyun; Jahng, Yurngdong; Jeong, Tae Cheon

2007-10-01

Tryptanthrin originally isolated from Isatis tinctoria L. has been characterized to have anti-inflammatory activities through the dual inhibition of cyclooxygenase-2 and 5-lipoxygenase mediated prostaglandin and leukotriene syntheses. To characterize phase I metabolite(s), tryptanthrin was incubated with rat liver microsomes in the presence of NADPH-generating system. One metabolite was identified by liquid chromatography/electrospray ionization-tandem mass spectrometry. M1 could be identified as a metabolite mono-hydroxylated on the aromatic ring of indole moiety from the MS(2) spectra of protonated tryptanthrin and M1. The structure of metabolite was confirmed as 8-hydroxytryptanthrin with a chemically synthesized authentic standard. The formation of M1 was NADPH-dependent and was inhibited by SKF-525A, a general CYP-inhibitor, indicating the cytochrome P450 (CYP)-mediated reaction. In addition, it was proposed that M1 might be formed by CYP 1A in rat liver microsomes from the experiments with enriched rat liver microsomes.

Stereoselective in vitro metabolism of rhynchophylline and isorhynchophylline epimers of Uncaria rhynchophylla in rat liver microsomes.

Science.gov (United States)

Wang, Xin; Qiao, Zhou; Liu, Jia; Zheng, Mei; Liu, Wenyuan; Wu, Chunyong

2017-11-10

1. The objective was to investigate the underlying mechanism of the stereoselectivity in the metabolism of rhynchophylline (RIN) and isorhynchophylline (IRN) epimers in rat liver microsomes (RLM). 2. After incubation, eight metabolites of RIN (M1-5) and IRN (M6-8) reacted at A- and C-ring were identified using LC-Q-TOF/MS. Metabolic pathways included oxidation, hydroxylation, N-oxidation and dehydrogenation. In addition, hydroxylation at A-ring was the major metabolic pathway for RIN whereas the oxidation at C-ring was the major one for IRN. 3. Enzyme kinetics showed that the intrinsic clearance (CL int ) for IRN elimination was 1.9-fold higher than RIN and the degradation half-life (T 1/2 ) of RIN was 4.7-fold higher than that of IRN, indicating IRN was more favorable to be metabolized than RIN in RLM. 4. Data from chemical inhibition study demonstrated CYP3A was the predominant isoform involved in the metabolic elimination of both epimers, as well as the formation of M1-8. 5. In conclusion, data revealed that due to the spatial configurations at C-7 position, RIN and IRN epimers possessed different hepatic metabolic pathways and elimination rates which were mainly mediated by CYP3A.

Suppression of cytochrome P450 reductase (POR) expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic POR-null mice.

Science.gov (United States)

Porter, Todd D; Banerjee, Subhashis; Stolarczyk, Elzbieta I; Zou, Ling

2011-06-01

Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

The prevalence of autoantibody and its relationship with genotypes of hepatitis C virus in patients with chronic hepatitis C virus infection.

Science.gov (United States)

Kirdar, Sevİn; Sener, Asli Gamze; Cengİz, Merve; Aydin, Nerİman

2016-11-01

The prevalence of autoantibody in the patients with chronic hepatitis C infection, and the relationship between the autoantibodies and HCV genotypes were investigated in this study. One hundred and eight anti-HCV positive and 86 anti-HCV negative patients were included in the study. Anti-HCV were studied by enzyme immunassay (EIA). HCV RNA was determined by real time polymerase chain reaction (PCR) and HCV genotypes were determined by a reverse-line blot hybridization. Anti-nuclear antibodies (ANA), anti-smooth muscle antibodies (ASMA), Anti-mitochondrial antibodies (AMA), liver kidney microsomal antibodies (LKM) were detected by indirect immunofluorescence assay. Among patients, 13 (12.03%) of 108 were positive for at least one autoantibody. The positivity was not observed in control group. The most prevalent autoantibody in anti-HCV positive group was ANA. ANA was positive in six HCV patients with genotype 1. In HCV patients with genotype 1, the frequencies of ANA, ASMA, AMA and LKM1 were six, two, three and one, respectively. In HCV patients with genotype 2, ANA was positive one patient and ASMA, AMA and LKM1 were not detected in HCV patients with genotype 2. In conclusion, the autoantibodies in patients with chronic hepatitis C in the study were low as compared to those reported in previous studies. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

International Nuclear Information System (INIS)

Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.; Smith, W.C.; Talamantes, F.

1990-01-01

[ 125 I]Iodomouse GH [( 125 I]iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operated pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse

Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5′-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

Directory of Open Access Journals (Sweden)

Soon-Sang Kwon

2016-04-01

Full Text Available Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca2+-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP and uridine 5′-diphospho-glucuronosyltransferase (UGT enzymes of human liver microsomes to determine if mechanistic aschantin–enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1′-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.

Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

Energy Technology Data Exchange (ETDEWEB)

Choi, Hye Jin [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Lee, Dong-Hyung [Department of Obstetrics and Gynecology, Medical Research Institute, Pusan National University, Busan (Korea, Republic of); Park, Seong-Hwan; Kim, Juil; Do, Kee Hun [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); An, Tae Jin; Ahn, Young Sup; Park, Chung Berm [Department of Herbal Crop Research, NIHHS, RDA, Eumseong (Korea, Republic of); Moon, Yuseok, E-mail: moon@pnu.edu [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Medical Research Institute and Research Institute for Basic Sciences, Pusan National University, Busan (Korea, Republic of)

2011-09-30

Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

DDT increases hepatic testosterone metabolism in rats

Energy Technology Data Exchange (ETDEWEB)

Sierra-Santoyo, Adolfo; Albores, Arnulfo; Cebrian, Mariano E. [Cinvestav-IPN, Seccion de Toxicologia, Mexico (Mexico); Hernandez, Manuel [Cinvestav-IPN, Departamento de Biologia Celular (Mexico)

2005-01-01

DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-{sup 14}C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2{alpha}-hydroxytestosterone (OHT), and 16{alpha}-OHT but higher 6{beta}-OHT whereas treated females produced less 7{alpha}-OHT and AD but higher 6{beta}-OHT and 6{alpha}-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6{beta}-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6{alpha}-/15{alpha}-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6{alpha}-/16{alpha}-OHT and 6-dehydrotestosterone/16{alpha}-OHT ratios followed a similar tendency, with the ratio 6{alpha}-/16{alpha}-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorphism in testosterone metabolism and decreased the metabolic differences between male and female rats. (orig.)

Microsomal prostaglandin E synthase-1 in rheumatic diseases

Directory of Open Access Journals (Sweden)

Marina eKorotkova

2011-01-01

Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

Interaction of rocuronium with human liver cytochromes P450

OpenAIRE

Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-01-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

Changes in distribution of hepatic blood flow induced by intra-arterial infusion of angiotensin II in human hepatic cancer

International Nuclear Information System (INIS)

Sasaki, Y.; Imaoka, S.; Hasegawa, Y.

1985-01-01

Changes in the distribution of the hepatic blood flow induced by intra-arterial infusion of angiotensin II (AT-II) were studied in human hepatic cancers using extremely short-lived radioisotope (RI) (krypton 81 m [/sup 81m/Kr]; half-life, 13 seconds). After the start of continuous infusion of AT-II, the radioactivity of the tumor showed about a two-fold increase, whereas that of the nontumor region decreased to about one half as much as the level before the infusion. Consequently, the mean ratio of the arterial blood flow in the tumor region to that in the nontumor region (T/N ratio) increased to 3.30 (P less than 0.001). The T/N ratio showed a peak before the peripheral blood pressure reached the maximum, and thereafter tended to decrease. Intra-arterial infusion of AT-II raised the T/N ratio more obviously than did intravenous infusion of the drug, with less rise in the peripheral blood pressure. It is believed that intra-arterial infusion chemotherapy with local use of AT-II enables better accessibility of chemotherapeutic drugs to tumors

Natural history and treatment of hepatitis B virus and hepatitis C virus coinfection

Directory of Open Access Journals (Sweden)

Keeffe Emmet B

2005-09-01

Full Text Available Abstract Hepatitis B virus (HBV and hepatitis C virus (HCV coinfection is not uncommon as a result of similar routes of infection. Patients who are coinfected represent a unique group with diverse serologic profiles. Combined chronic hepatitis B and C leads to more severe liver disease and an increased risk of hepatocellular carcinoma. Furthermore, coinfected patients represent a treatment challenge. No standard recommendations exist for treatment of viral hepatitis due to dual HBV/HCV infection, and therefore treatment must be individualized based on patient variables such as serologic and virologic profiles, patient's prior exposure to antiviral treatment, and the presence of other parenterally transmitted viruses such as hepatitis D virus and human immunodeficiency virus. The natural history and treatment of patients with HBV and HCV coinfection is reviewed.

Role of metabolic activation by cytochrome P-450 in covalent binding of VP 16-213 to rat liver and HeLa cell microsomal proteins

Energy Technology Data Exchange (ETDEWEB)

van Maanen, J.M.; de Ruiter, C.; de Vries, J.; Kootstra, P.R.; Gobas, F.; Pinedo, H.M.

1985-09-01

Covalent binding of /sup 3/H-labeled VP 16-213 to rat liver and HeLa cell microsomal proteins was studied in vitro. Metabolic activation by cytochrome P-450 was found to play a role in the covalent binding of VP 16-213 to rat liver microsomal proteins, as shown by the need of NADPH cofactor, the increased binding after phenobarbital pretreatment and the inhibition by SFK-525A. Addition of ascorbic acid or alpha-phenyl-N-tert. butylnitrone to the incubation mixture depressed covalent binding by about 85%, suggesting that formation of a reactive metabolite from the phenolic structure may be involved in the binding process. VP 16-213 did not inhibit aminopyrine N-demethylase at the concentration used in the binding experiments (17 microM), indicating that metabolism of its methylenedioxy group does not play a role in binding to microsomal proteins. HeLa cell microsomes were found to possess aminopyrine N-demethylase activity. Covalent binding of radiolabeled VP 16-213 to HeLa cell microsomes decreased by about 64% if NADPH was omitted.

Detection of Osteopontin in the pericyst of human hepatic Echinococcus granulosus.

Science.gov (United States)

Peng, Xinyu; Li, Jianhui; Wu, Xiangwei; Zhang, Shijie; Niu, Jianhua; Chen, Xiaoping; Yao, Jin; Sun, Hong

2006-12-01

It aims at investigating the expression and distribution of the Osteopontin (OPN) in the pericyst of human hepatic Echinococcus granulosus and their related significances. Sixty pericysts excised by "sub-adventitial cystectomy" were studied. OPN was detected in 80% (48/60) of cysts by Western blotting and distributed in the side of "exocyst" layer directing to the parasite, also macrophages were identified in the vicinity of OPN by immunohistochemistry staining. The coexpression of OPN and CD68 was observed by immunofluorescence double labeling and analyzed by Image-Pro Plus 5.1; with special stain techniques, variable degrees of calcium deposits were observed in 80% (48/60) cysts, and the calcium deposits concurrencely found with the OPN expression. The selective distribution of OPN, calcium in the "exocyst" provides a new pathological evidence for the "sub-adventitial cystectomy" we developed. The pericyst of hepatic E. granulosus consists of two detachable layers with different formative mechanisms: the "exocyst" layer directing towards the cyst of parasite was the result of granulomatous reaction; also the results suggest OPN is one regulator in the granulomatous reaction and calcification of "exocyst".

Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

DEFF Research Database (Denmark)

Danielsen, E M; Norén, Ove; Sjöström, H

1983-01-01

-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000...

Effects of tin-protoporphyrin administration on hepatic xenobiotic metabolizing enzymes in the juvenile rat

International Nuclear Information System (INIS)

Stout, D.L.; Becker, F.F.

1988-01-01

The heme analogue tin-protoporphyrin IX (SnP) is a potent inhibitor of microsomal heme oxygenase. Administration of SnP to neonatal rats can prevent hyperbilirubinemia by blocking the postnatal increase of heme oxygenase activity. Apparently innocuous at therapeutic doses, it is of potential clinical value for chemoprevention of neonatal jaundice. We found that when 50-g male Sprague-Dawley rats were treated daily with 50 mumol of SnP/kg sc for 6 days, hepatic microsomal cytochromes b5 and P-450 were significantly diminished. Cytochrome P-450 reductase, two P-450-dependent monooxygenases, aminopyrine demethylase and benzo(a)pyrene hydroxylase, and catalase, a peroxisomal hemoprotein, were also significantly diminished. These results suggested that SnP might significantly affect the metabolism of other xenobiotics. This possibility was confirmed by the finding that hexobarbital-induced sleep lasted 4 times longer in SnP-treated rats than in controls. Inhibition of protein synthesis by SnP was ruled out as the cause of hemoprotein loss when administration of [ 3 H]leucine to SnP-treated and control rats demonstrated that proteins of the microsomal, cytosolic, and plasma membrane fractions of the livers from both groups incorporated similar levels of leucine. When 55 FeCl 3 and [2- 14 C]glycine were administered to measure heme synthesis, heme extract from the livers of SnP-treated rats contained 4 times more label from iron and glycine than did heme from control livers. Despite the apparent increased rate of heme synthesis in SnP-treated rats, each of the three cell fractions demonstrated a significant loss of heme but contained sizable amounts of SnP. These findings suggest that SnP causes a decrease of functional hemoprotein and partial loss of enzymic activity by displacing intracellular heme

Giving It Our Best Shot? Human Papillomavirus and Hepatitis B Virus Immunization Among Refugees, Massachusetts, 2011-2013.

Science.gov (United States)

Berman, Rachel Stein; Smock, Laura; Bair-Merritt, Megan H; Cochran, Jennifer; Geltman, Paul L

2017-06-22

The receipt rate of hepatitis B virus vaccine among adolescents in the United States is high, while the receipt rate of human papillomavirus vaccine is low. Rates have not been closely studied among refugees, whose home countries have high rates of disease caused by these viruses. We examined human papillomavirus and hepatitis B virus immunization rates among 2,269 refugees aged 9 to 26 years who resettled in Massachusetts from 2011 through 2013. This was a secondary analysis of data from their medical screenings. We used binary logistic regression to assess characteristics associated with immunization and bivariate analyses to compare refugee immunization rates with those of the general US population. Forty-five percent of US adolescents aged 13 to 17 years received 1 dose of human papillomavirus vaccine, compared with 68% of similarly aged refugees. Males (adjusted odds ratio [aOR], 0.62; 95% confidence interval [CI], 0.52-0.74), refugees older than 13 years (aOR, 0.74; 95% CI, 0.60-0.93), and refugees not from Sub-Saharan Africa (aOR, 0.74; 95% CI, 0.59-0.92) were less likely to receive human papillomavirus vaccine, while arrivals in 2012 through 2013 were more likely (aOR, 1.6; 95% CI, 1.3-1.9) than those arriving in 2011. Refugees older than 13 years were less likely to receive 2 doses of hepatitis B virus vaccine (aOR, 0.49; 95% CI, 0.37-0.63) than older refugees. Specialized post-arrival health assessment may improve refugees' immunization rates.

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

Science.gov (United States)

Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Stereospecificity (ST) of the microsomal ethanol oxidizing system (MEOS)

International Nuclear Information System (INIS)

Alderman, J.; Kato, S.; Lasker, J.; Lieber, C.S.

1987-01-01

The ST of MEOS for the ethanol 1R hydrogen has been variously reported as absolute, partial or absent, with free radical involvement postulated in the latter case. To determine both the ST of MEOS and the participation of free radicals in the reaction, they investigated MEOS ST using 1R[1- 3 H] ethanol as substrate. ST is expressed as the fraction of 3 H labeling in acetaldehyde formed, relative to that in ethanol, and ranges from 0.5 to 0. Partial ST was observed using liver microsomes from both rats and hamsters; it significantly decreased after ethanol feeding. 0.1 mM desferrioxamine (dfx) did not increase ST in any of these microsomal preparations while ferric EDTA decreased it, suggesting that ethanol treatment induces a cytochrome P-450 with lower ST rather than increasing free radical involvement. This is supported by a virtual absence of ST observed in a reconstituted system containing purified hamster P-450/sub ALC/, a liver cytochrome P-450 isozyme induced in hamsters by ethanol treatment. Their results indicate that, unlike other enzymes that oxidize ethanol, MEOS has only partial ST. Thus, ST alone cannot be used as an index of free radical involvement but, when evaluated with the response of ST to dfx, it indicated that MEOS is unlikely to involve free radical attack on ethanol in solution

Kinetics of naphthalene metabolism in target and non-target tissues of rodents and in nasal and airway microsomes from the Rhesus monkey

Energy Technology Data Exchange (ETDEWEB)

Buckpitt, Alan, E-mail: arbuckpitt@ucdavis.edu [Department of Molecular Biosciences, School of Veterinary Medicine, UC Davis, Davis, CA 95616 (United States); Morin, Dexter [Department of Molecular Biosciences, School of Veterinary Medicine, UC Davis, Davis, CA 95616 (United States); Murphy, Shannon; Edwards, Patricia; Van Winkle, Laura [Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, UC Davis, Davis, CA 95616 (United States); Center for Health and the Environment, UC Davis, Davis, CA 95616 United States (United States)

2013-07-15

Naphthalene produces species and cell selective injury to respiratory tract epithelial cells of rodents. In these studies we determined the apparent K{sub m}, V{sub max}, and catalytic efficiency (V{sub max}/K{sub m}) for naphthalene metabolism in microsomal preparations from subcompartments of the respiratory tract of rodents and non-human primates. In tissues with high substrate turnover, major metabolites were derived directly from naphthalene oxide with smaller amounts from conjugates of diol epoxide, diepoxide, and 1,2- and 1,4-naphthoquinones. In some tissues, different enzymes with dissimilar K{sub m} and V{sub max} appeared to metabolize naphthalene. The rank order of V{sub max} (rat olfactory epithelium > mouse olfactory epithelium > murine airways ≫ rat airways) correlated well with tissue susceptibility to naphthalene. The V{sub max} in monkey alveolar subcompartment was 2% that in rat nasal olfactory epithelium. Rates of metabolism in nasal compartments of the monkey were low. The catalytic efficiencies of microsomes from known susceptible tissues/subcompartments are 10 and 250 fold higher than in rat airway and monkey alveolar subcompartments, respectively. Although the strong correlations between catalytic efficiencies and tissue susceptibility suggest that non-human primate tissues are unlikely to generate metabolites at a rate sufficient to produce cellular injury, other studies showing high levels of formation of protein adducts support the need for additional studies. - Highlights: • Naphthalene is metabolized with high catalytic efficiency in susceptible tissue. • Naphthalene is metabolized at low catalytic efficiency in non-susceptible tissue. • Respiratory tissues of the non human primate metabolize naphthalene slowly.

Randomized Trial: Immunogenicity and Safety of Coadministered Human Papillomavirus-16/18 AS04-Adjuvanted Vaccine and Combined Hepatitis A and B Vaccine in Girls

DEFF Research Database (Denmark)

Pedersen, Court; Breindahl, Morten; Aggarwal, Naresh

2012-01-01

This randomized, open, controlled, multicenter study (110886/NCT00578227) evaluated human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine (HPV-16/18 vaccine) coadministered with inactivated hepatitis A and B (HAB) vaccine. Coprimary objectives were to demonstrate noninferiority of hepatitis A......, hepatitis B, and HPV-16/18 immune responses at month 7 when vaccines were coadministered, compared with the same vaccines administered alone....

In vitro enantioselective human liver microsomal metabolism and prediction of in vivo pharmacokinetic parameters of tetrabenazine by DLLME-CE.

Science.gov (United States)

Bocato, Mariana Zuccherato; de Lima Moreira, Fernanda; de Albuquerque, Nayara Cristina Perez; de Gaitani, Cristiane Masetto; de Oliveira, Anderson Rodrigo Moraes

2016-09-05

A new capillary electrophoresis method for the enantioselective analysis of cis- and trans- dihydrotetrabenazine (diHTBZ) after in vitro metabolism by human liver microsomes (HLMs) was developed. The chiral electrophoretic separations were performed by using tris-phosphate buffer (pH 2.5) containing 1% (w/v) carboxymethyl-β-CD as background electrolyte with an applied voltage of +15kV and capillary temperature kept at 15°C. Dispersive liquid-liquid microextraction was employed to extract the analytes from HLMs. Dichloromethane was used as extraction solvent (75μL) and acetone as disperser solvent (150μL). The method was validated according to official guidelines and showed to be linear over the concentration range of 0.29-19.57μmolL(-1) (r=0.9955) for each metabolite enantiomer. Within- and between-day precision and accuracy evaluated by relative standard deviation and relative error were lower than 15% for all enantiomers. The stability assay showed that the analytes kept stable under handling, storage and in metabolism conditions. After method validation, an enantioselective in vitro metabolism and in vivo pharmacokinetic prediction was carried out. This study showed a stereoselective metabolism and the observed kinetic profile indicated a substrate inhibition behavior. DiHTBZ enantiomers were catalyzed mainly by CYP2C19 and the predicted clearance suggests that liver metabolism is the main route for TBZ elimination which supports the literature data. Copyright © 2016 Elsevier B.V. All rights reserved.

Human immunodeficiency virus (HIV) seropositivity and hepatitis B surface antigenemia (HBSAG) among blood donors in Benin city, Edo state, Nigeria.

Science.gov (United States)

Umolu, Patience Idia; Okoror, Lawrence Ehis; Orhue, Philip

2005-03-01

Human Immunodeficiency Virus and Hepatitis B virus are blood borne pathogens that can be transmitted through blood transfusion and could pose a huge problem in areas where mechanisms of ensuring blood safety are suspect. This study became necessary in a population where most of the blood for transfusion is from commercial blood donors. A total of 130 donors comprising 120 commercial donors and 10 voluntary donors were tested for antibodies to human immunodeficiency virus and hepatitis B surface antigen in Benin city using Immunocomb HIV - 1 and 2 Biospot kit and Quimica Clinica Aplicada direct latex agglutination method respectively. Thirteen (10%) samples were HIV seropositive and 7(5.8%) were HBsAg positive. The age bracket 18 - 25years had the highest numbers of donors and also had the highest number of HBsAg positive cases (7.8%) while the age group 29 - 38years had highest number of HIV seropositive cases. High prevalence of HIV antibodies and Hepatitis B surface antigen was found among commercial blood donors. Appropriate and compulsory screening of blood donors using sensitive methods, must be ensured to prevent post transfusion hepatitis and HIV.

Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans.

Science.gov (United States)

Heppard, E P; Kinney, A J; Stecca, K L; Miao, G H

1996-01-01

The polyunsaturated fatty acid content is one of the major factors influencing the quality of vegetable oils. Edible oils rich in monounsaturated fatty acid provide improved oil stability, flavor, and nutrition for human and animal consumption. In plants, the microsomal omega-6 desaturase-catalyzed pathway is the primary route of production of polyunsaturated lipids. We report the isolation of two different cDNA sequences, FAD2-1 and FAD2-2, encoding microsomal omega-6 desaturase in soybeans and the characterization of their developmental and temperature regulation. The FAD2-1 gene is strongly expressed in developing seeds, whereas the FAD2-2 gene is constitutively expressed in both vegetative tissues and developing seeds. Thus, the FAD2-2 gene-encoded omega-6 desaturase appears to be responsible for production of polyunsaturated fatty acids within membrane lipids in both vegetative tissues and developing seeds. The seed-specifically expressed FAD2-1 gene is likely to play a major role in controlling conversion of oleic acid to linoleic acid within storage lipids during seed development. In both soybean seed and leaf tissues, linoleic acid and linolenic acid levels gradually increase as temperature decreases. However, the levels of transcripts for FAD2-1, FAD2-2, and the plastidial omega-6 desaturase gene (FAD 6) do not increase at low temperature. These results suggest that the elevated polyunsaturated fatty acid levels in developing soybean seeds grown at low temperature are not due to the enhanced expression of omega-6 desaturase genes. PMID:8587990

Human leukocyte antigen-e alleles are associated with hepatitis c virus, torque teno virus, and toxoplasma co-infections but are not associated with hepatitis b virus, hepatitis d virus, and GB virus c co-infections in human immunodeficiency virus patients

Directory of Open Access Journals (Sweden)

Afiono Agung Prasetyo

2016-01-01

Full Text Available Context: Data regarding the distribution of Human Leukocyte Antigen (HLA-E alleles and their association with blood-borne pathogen infections/co-infections are limited for many populations, including Indonesia. Aims: The aim of this study was to analyze the association between HLA-E allelic variants and infection with blood-borne pathogens such as hepatitis B virus (HBV, hepatitis C virus (HCV, hepatitis D virus (HDV, torque teno virus (TTV, GB virus C (GBV-C, and Toxoplasma gondii (T. gondii in Indonesian Javanese human immunodeficiency virus (HIV patients. Settings and Design: A total of 320 anti-HIV-positive blood samples were analyzed for HBV, HCV, HDV, TTV, GBV-C, and T. gondii infection status and its association with HLA-E allelic variants. Materials and Methods: Nucleic acid was extracted from plasma samples and used for the molecular detection of HBV DNA, HCV RNA, HDV RNA, TTV DNA, and GBV-C RNA, whereas hepatitis B surface antigen, anti-HCV, immunoglobulin M and G (IgM and IgG anti-T. gondii were detected through serological testing. The blood samples were genotyped for HLA-E loci using a sequence-specific primer-polymerase chain reaction. Statistical Analysis Used: Either the Chi-square or Fisher′s exact test was performed to analyze the frequency of HLA-E alleles and blood-borne pathogen infections in the population. Odds ratios (ORs were calculated to measure the association between the antibodies found and the participants′ possible risk behaviors. A logistic regression analysis was used to assess the associations. Results: HLA-EFNx010101/0101 was associated with HCV/TTV co-infection (adjusted OR [aOR]: 3.5; 95% confidence interval [CI]: 1.156-10.734; P = 0.027 and IgM/IgG anti-Toxo positivity (aOR: 27.0; 95% CI: 3.626-200.472; P = 0.001. HLA-EFNx010103/0103 was associated with TTV co-infection (aOR: 2.7; 95% CI: 1.509-4.796; P = 0.001. Conclusions: HLA-E alleles in Indonesian Javanese HIV patients were found to be associated

Current Knowledge on Hepatitis E.

Science.gov (United States)

Pérez-Gracia, María Teresa; García, Mario; Suay, Beatriz; Mateos-Lindemann, María Luisa

2015-06-28

Although only a single serotype of hepatitis E virus (HEV), the causative agent of hepatitis E, has been identified, there is great genetic variation among the different HEV isolates reported. There are at least four major recognized genotypes of HEV: genotypes 1 and 2 are mainly restricted to humans and linked to epidemic outbreaks in nonindustrialized countries, whereas genotypes 3 and 4 are zoonotic in both developing and industrialized countries. Besides human strains, genotype 3 and 4 strains of HEV have been genetically characterized from swine, sika deer, mongooses, sheep, and rabbits. Currently, there are approximately 11,000 human and animal sequences of HEV available at the International Nucleotide Sequence Database Collaboration. HEV is the major cause of waterborne outbreaks of hepatitis in areas of poor sanitation. Additionally, it is responsible for sporadic cases of viral hepatitis in not only endemic but industrialized countries as well. Transmission of HEV occurs predominantly by the fecal-oral route, although parenteral and perinatal routes have been reported. HEV infection develops in most individuals as a self-limiting, acute, icteric hepatitis; with mortality rates around 1%. However, some affected individuals will develop fulminant hepatic failure, a serious condition that is frequently fatal without a liver transplant. This complication is particularly common when the infection occurs in pregnant women, where mortality rates rise dramatically to up to 25%. Among the preventive measures available to avoid HEV infection, two separate subunit vaccines containing recombinant truncated capsid proteins of HEV have been shown to be highly effective in the prevention of disease. One of them, HEV 239, was approved in China, and its commercialization by Innovax began in November 2012 under the name Hecolin(®).

Identification of AKB-48 and 5F-AKB-48 Metabolites in Authentic Human Urine Samples Using Human Liver Microsomes and Time of Flight Mass Spectrometry.

Science.gov (United States)

Vikingsson, Svante; Josefsson, Martin; Gréen, Henrik

2015-01-01

The occurrence of structurally related synthetic cannabinoids makes the identification of unique markers of drug intake particularly challenging. The aim of this study was to identify unique and abundant metabolites of AKB-48 and 5F-AKB-48 for toxicological screening in urine. Investigations of authentic urine samples from forensic cases in combination with human liver microsome (HLM) experiments were used for identification of metabolites. HLM incubations of AKB-48 and 5F-AKB-48 along with 35 urine samples from authentic cases were analyzed with liquid chromatography quadrupole tandem time of flight mass spectrometry. Using HLMs 41 metabolites of AKB-48 and 37 metabolites of 5F-AKB-48 were identified, principally represented by hydroxylation but also ketone formation and dealkylation. Monohydroxylated metabolites were replaced by di- and trihydroxylated metabolites within 30 min. The metabolites from the HLM incubations accounted for on average 84% (range, 67-100) and 91% (range, 71-100) of the combined area in the case samples for AKB-48 and 5F-AKB-48, respectively. While defluorinated metabolites accounted for on average 74% of the combined area after a 5F-AKB-48 intake only a few identified metabolites were shared between AKB-48 and 5F-AKB-48, illustrating the need for a systematic approach to identify unique metabolites. HLMs in combination with case samples seem suitable for this purpose. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Loss of miR-141/200c ameliorates hepatic steatosis and inflammation by reprogramming multiple signaling pathways in NASH

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Tran, Melanie; Lee, Sang-Min; Shin, Dong-Ju

2017-01-01

Accumulation of lipid droplets and inflammatory cell infiltration is the hallmark of nonalcoholic steatohepatitis (NASH). The roles of noncoding RNAs in NASH are less known. We aim to elucidate the function of miR-141/200c in diet-induced NASH. WT and miR-141/200c–/– mice were fed a methionine and choline deficient (MCD) diet for 2 weeks to assess markers of steatosis, liver injury, and inflammation. Hepatic miR-141 and miR-200c RNA levels were highly induced in human patients with NASH fatty liver and in WT MCD mice. miR-141/200c–/– MCD mice had reduced liver weights and triglyceride (TG) levels, which was associated with increased microsomal TG transfer protein (MTTP) and PPARα but reduced SREBP1c and FAS expression. Inflammation was attenuated and F4/80 macrophage activation was suppressed in miR-141/200c–/– mice, as evidenced by decreased serum aminotransferases and IL-6 and reduced hepatic proinflammatory, neutrophil, and profibrotic genes. Treatment with LPS in BM-derived macrophages isolated from miR-200c/141–/– mice polarized macrophages toward the M2 antiinflammatory state by increasing Arg1 and IL-10 levels while decreasing the M1 marker iNOS. In addition, elevated phosphorylated AMPK (p-AMPK), p-AKT, and p-GSK3β and diminished TLR4 and p-mTOR/p-4EBP1 proteins were observed. Lipidomics and metabolomics revealed alterations of TG and phosphatidylcholine (PC) lipid species by miR-141/200c deficiency. In summary, miR-141/200c deficiency diminished NASH-associated hepatic steatosis and inflammation by reprogramming lipid and inflammation signaling pathways. PMID:29093267

Gene expression variability in human hepatic drug metabolizing enzymes and transporters.

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Lun Yang

Full Text Available Interindividual variability in the expression of drug-metabolizing enzymes and transporters (DMETs in human liver may contribute to interindividual differences in drug efficacy and adverse reactions. Published studies that analyzed variability in the expression of DMET genes were limited by sample sizes and the number of genes profiled. We systematically analyzed the expression of 374 DMETs from a microarray data set consisting of gene expression profiles derived from 427 human liver samples. The standard deviation of interindividual expression for DMET genes was much higher than that for non-DMET genes. The 20 DMET genes with the largest variability in the expression provided examples of the interindividual variation. Gene expression data were also analyzed using network analysis methods, which delineates the similarities of biological functionalities and regulation mechanisms for these highly variable DMET genes. Expression variability of human hepatic DMET genes may affect drug-gene interactions and disease susceptibility, with concomitant clinical implications.

Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1

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Wang, Ai-Guo; Seo, Sang-Beom; Moon, Hyung-Bae; Shin, Hye-Jun; Kim, Dong Hoon; Kim, Jin-Man; Lee, Tae-Hoon; Kwon, Ho Jeong; Yu, Dae-Yeul; Lee, Dong-Seok

2005-01-01

It is generally thought that histone deacetylases (HDACs) play important roles in the transcriptional regulation of genes. However, little information is available concerning the specific functions of individual HDACs in disease states. In this study, two transgenic mice lines were established which harbored the human HDAC1 gene. Overexpressed HDAC1 was detected in the nuclei of transgenic liver cells, and HDAC1 enzymatic activity was significantly higher in the transgenic mice than in control littermates. The HDAC1 transgenic mice exhibited a high incidence of hepatic steatosis and nuclear pleomorphism. Molecular studies showed that HDAC1 may contribute to nuclear pleomorphism through the p53/p21 signaling pathway

Sensitive genotyping of foodborne-associated human noroviruses and hepatitis A virus using an array-based platform

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The viral pathogens, human norovirus (NoV) and hepatitis A virus (HAV), are significant contributors of foodborne associated outbreaks. To develop a typing tool for foodborne viruses, a focused, low-density DNA microarray was developed in conjunction with a rapid and high-throughput fluorescent meth...

Stereo-selectivity and regio-selectivity in the metabolism of 7,8-dihydrobenzo[a]pyrene by cytochrome P450, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

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Adams, J D; Yagi, H; Levin, W; Jerina, D M

1995-03-30

The active site of cytochrome P450 1A1 has been probed with the substrate 7,8-dihydrobenzo[a]pyrene using a purified, reconstituted system composed of cytochrome P450 1A1, NADPH-cytochrome c reductase and lipid in the presence or absence of epoxide hydrolase. The turnover of the substrate was found to be 38 nmol/nmol of cytochrome P450/min. The metabolic products that were identified are: a phenolic 7,8-dihydrobenzo[a]pyrene (20-29%); 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (17-28%); benzo[a]pyrene (12-19%); 7-hydroxy-7,8-dihydrobenzo[a]pyrene (13-16%); 8-hydroxy-7,8-dihydrobenzo[a]pyrene (7-15%); 3-hydroxybenzo[a]pyrene (7-15%); 4,5-epoxy-4,5,7,8-tetrahydrobenzo[a]pyrene (0-4%); and a triol of 7,8,9,10-tetrahydrobenzo[a]pyrene (0-4%). 9,10-Epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene undergoes rapid hydrolysis to cis- and trans-9,10-dihydroxy-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (2:1) by benzylic attack of water at C-10. Approximately 71% of the trans diols are derived from (+)-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, indicating that cytochrome P450 1A1 has more than a 2:1 preference for selective epoxidation of an enantiotopic face of 7,8-dihydrobenzo[a]pyrene. This stereo-selectivity agrees with the postulated stereo-selectivity predicted by a previously described active site model for cytochrome P450 1A1. Epoxide hydrolase in pure form or in hepatic microsomes catalyzes the hydrolysis of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, which is inhibited by 1,1,1-trichloropropane 2,3-oxide. The (+)-(9S,10R)-isomer of the epoxide is slightly preferred as a substrate over its enantiomer and is cleaved by benzylic and nonbenzylic attack. Only benzylic attack was found with (-)-(9R,10S)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

Plasticizers May Activate Human Hepatic Peroxisome Proliferator-Activated Receptor α Less Than That of a Mouse but May Activate Constitutive Androstane Receptor in Liver

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Ito, Yuki; Nakamura, Toshiki; Yanagiba, Yukie; Ramdhan, Doni Hikmat; Yamagishi, Nozomi; Naito, Hisao; Kamijima, Michihiro; Gonzalez, Frank J.; Nakajima, Tamie

2012-01-01

Dibutylphthalate (DBP), di(2-ethylhexyl)phthalate (DEHP), and di(2-ethylhexyl)adipate (DEHA) are used as plasticizers. Their metabolites activate peroxisome proliferator-activated receptor (PPAR) α, which may be related to their toxicities. However, species differences in the receptor functions between rodents and human make it difficult to precisely extrapolate their toxicity from animal studies to human. In this paper, we compared the species differences in the activation of mouse and human hepatic PPARα by these plasticizers using wild-type (mPPARα) and humanized PPARα (hPPARα) mice. At 12 weeks old, each genotyped male mouse was classified into three groups, and fed daily for 2 weeks per os with corn oil (vehicle control), 2.5 or 5.0 mmol/kg DBP (696, 1392 mg/kg), DEHP (977, 1953 mg/kg), and DEHA (926, 1853 mg/kg), respectively. Generally, hepatic PPARα of mPPARα mice was more strongly activated than that of hPPARα mice when several target genes involving β-oxidation of fatty acids were evaluated. Interestingly, all plasticizers also activated hepatic constitutive androstane receptor (CAR) more in hPPARα mice than in mPPARα mice. Taken together, these plasticizers activated mouse and human hepatic PPARα as well as CAR. The activation of PPARα was stronger in mPPARα mice than in hPPARα mice, while the opposite was true of CAR. PMID:22792086

Plasticizers May Activate Human Hepatic Peroxisome Proliferator-Activated Receptor α Less Than That of a Mouse but May Activate Constitutive Androstane Receptor in Liver

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Yuki Ito

2012-01-01

Full Text Available Dibutylphthalate (DBP, di(2-ethylhexylphthalate (DEHP, and di(2-ethylhexyladipate (DEHA are used as plasticizers. Their metabolites activate peroxisome proliferator-activated receptor (PPAR α, which may be related to their toxicities. However, species differences in the receptor functions between rodents and human make it difficult to precisely extrapolate their toxicity from animal studies to human. In this paper, we compared the species differences in the activation of mouse and human hepatic PPARα by these plasticizers using wild-type (mPPARα and humanized PPARα (hPPARα mice. At 12 weeks old, each genotyped male mouse was classified into three groups, and fed daily for 2 weeks per os with corn oil (vehicle control, 2.5 or 5.0 mmol/kg DBP (696, 1392 mg/kg, DEHP (977, 1953 mg/kg, and DEHA (926, 1853 mg/kg, respectively. Generally, hepatic PPARα of mPPARα mice was more strongly activated than that of hPPARα mice when several target genes involving β-oxidation of fatty acids were evaluated. Interestingly, all plasticizers also activated hepatic constitutive androstane receptor (CAR more in hPPARα mice than in mPPARα mice. Taken together, these plasticizers activated mouse and human hepatic PPARα as well as CAR. The activation of PPARα was stronger in mPPARα mice than in hPPARα mice, while the opposite was true of CAR.

Reduction of VLDL secretion decreases cholesterol excretion in niemann-pick C1-like 1 hepatic transgenic mice.

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Stephanie M Marshall

Full Text Available An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL support TICE, antisense oligonucleotides (ASO were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP, which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg mice, which predominantly excrete cholesterol via TICE, and wild type (WT littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.

Mode of action analysis for the synthetic pyrethroid metofluthrin-induced rat liver tumors: evidence for hepatic CYP2B induction and hepatocyte proliferation.

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Deguchi, Yoshihito; Yamada, Tomoya; Hirose, Yukihiro; Nagahori, Hirohisa; Kushida, Masahiko; Sumida, Kayo; Sukata, Tokuo; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Okuno, Yasuyoshi

2009-03-01

Two-year treatment with high doses of Metofluthrin produced hepatocellular tumors in both sexes of Wistar rats. To understand the mode of action (MOA) by which the tumors are produced, a series of studies examined the effects of Metofluthrin on hepatic microsomal cytochrome P450 (CYP) content, hepatocellular proliferation, hepatic gap junctional intercellular communication (GJIC), oxidative stress and apoptosis was conducted after one or two weeks of treatment. The global gene expression profile indicated that most genes with upregulated expression with Metofluthrin were metabolic enzymes that were also upregulated with phenobarbital. Metofluthrin induced CYP2B and increased liver weights associated with centrilobular hepatocyte hypertrophy (increased smooth endoplasmic reticulum [SER]), and induction of increased hepatocellular DNA replication. CYP2B1 mRNA induction by Metofluthrin was not observed in CAR knockdown rat hepatocytes using the RNA interference technique, demonstrating that Metofluthrin induces CYP2B1 through CAR activation. Metofluthrin also suppressed hepatic GJIC and induced oxidative stress and increased antioxidant enzymes, but showed no alteration in apoptosis. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. All of these effects were reversible upon cessation of treatment. Metofluthrin did not cause cytotoxicity or peroxisome proliferation. Thus, it is highly likely that the MOA for Metofluthrin-induced liver tumors in rats is through CYP induction and increased hepatocyte proliferation, similar to that seen for phenobarbital. Based on analysis with the International Life Sciences Institute/Risk Science Institute MOA framework, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans, at least at expected levels of exposure.

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis.

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Rigopoulou, Eirini I; Mytilinaiou, Maria; Romanidou, Ourania; Liaskos, Christos; Dalekos, George N

2007-02-04

Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody--the serological marker of type 2 autoimmune hepatitis (AIH-2)--is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1)--either in association with anti-LKM1, or in isolation--and anti-soluble liver antigen antibodies (anti-SLA) have been considered as useful and specific diagnostic markers for AIH. However, their specificity for AIH has been questioned by some recent studies, which have shown the detection of anti-LC1 and anti-SLA by immunoprecipitation assays in HCV patients irrespective of their anti-LKM1 status. The aim of the present study was to test the anti-LC1 and anti-SLA presence by specific enzyme linked immunosorbent assays (ELISAs), in a large group of Greek HCV-infected patients with or without anti-LKM1 reactivity as firstly, immunoprecipitation assays are limited to few specialized laboratories worldwide and cannot be used routinely and secondly, to assess whether application of such tests has any relevance in the context of patients with viral hepatitis since antibody detection based on such ELISAs has not been described in detail in large groups of HCV patients. One hundred and thirty eight consecutive HCV patients (120 anti-LKM1 negative and 18 anti-LKM1 positive) were investigated for the presence of anti-LC1 and anti-SLA by commercial ELISAs. A similar number (120) of chronic hepatitis B virus (HBV) infected patients seronegative for anti-LKM1 was also tested as pathological controls. Six out of 18 (33%) anti-LKM(pos)/HCV(pos) patients tested positive for anti-LC1 compared to 1/120 (0.83%) anti-LKM(neg)/HCV(pos) patients and 0/120 (0%) of the anti-LKM1(neg)/HBV(pos) patients (p LKM1) or HBV-infected patients. We showed that anti-LC1 and anti-SLA autoantibodies are not detected by conventional assays in a large group of

Drug-binding ability of human serum albumin at children with chronic virus hepatitis radiochemical definition method

International Nuclear Information System (INIS)

Kim, A.A.; Dadakhanov, J.A.; Djuraeva, G.T.; Shukurov, B.V.; Mavlyanov, I.R.

2006-01-01

Full text: The chronic virus hepatitis produces numerous abnormalities of liver function. The viruses of B, C, D, F and G hepatitis possess the ability to cause chronically proceeding diseases. Earlier we have found that binding ability of serum albumin at patients with acute forms of virus hepatitis is authentically reduced in comparison with the given parameters of control group. At an acute virus hepatitis B with middle severity the reducing of binding ability of serum albumin was observed at 70 % of patients. At an acute virus hepatitis A the reduce of binding ability of serum albumin is less expressed than at acute virus hepatitis B. At of chronic virus intoxication in human organism there is a formation and accumulation of toxic compounds in the excessive concentrations, which are not inherent to a normal metabolism. One of universal mechanisms of reaction of an organism on the increasing concentration of metabolism products is formation of complexes of various compounds with blood plasma proteins. The formation in an organism of endo- and exotoxins excessive concentrations results in blocking the binding centers of albumin molecule that causes the change of its complexing ability. The purpose of the present research: investigation of binding ability of serum albumin with use of radiochemical method at children with a chronic virus hepatitis B and C. Materials and methods. Under clinical observation there were 52 children in the age from 3 till 14 years. From them at 32 the chronic virus hepatitis B was confirmed, at 20 chronic virus - hepatitis C. Etiological diagnostics was carried out by definition of specific markers of a hepatitis B and C method IFA and PCR. Binding ability of serum albumin was defined by radiochemical method with use of the tritium labeled no-spa (drotaverine hydrochloride). The control group consists from 10 conditionally health children of similar age. Results and their discussion. The results of investigation have shown, that at a

Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b/sub 5/ and cytochrome P-450/sub cam/

Energy Technology Data Exchange (ETDEWEB)

Vickery, L; Salmon, A; Sauer, K

1975-01-01

Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats. The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b/sub 5/ and P-450, which dominate the spectra. The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome b/sub 5/ from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450/sub cam/ from Pseudomonas putida. The magnetic circular dichroism spectra of the membrane bound cytochrome b/sub 5/ are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure. The soluble bacterial cytochrome P-450/sub cam/ also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes. No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed.

[Epidemiologic aspects of human immunodeficiency virus and hepatitis virus infections].

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Diarra, M; Konate, A; Minta, D; Sounko, A; Dembele, M; Toure, C S; Kalle, A; Traore, H H; Maiga, M Y

2006-01-01

In order to determinate the prevalence of hepatitis B virus and hepatitis C virus among patients infected by the HIV, We realized a transverse survey case--control in hepato-gastro-enterological ward and serology unity of National Institute of Research in Public health (INRSP). Our sample was constituted with 100 patients HIV positive compared to 100 controls HIV negative. The viral markers research has been made by methods immuno-enzymatiqueses of ELISA 3rd generation. Tests permitted to get the following results: Hepatitis B surface antigen (HBs Ag) was positive among 21% with patients HIV positive versus 23% among control (p = 0,732); Antibody to hepatitis C virus (anti-HCV ab) was present among 23% with patients HIV positive versus 0% among control (p <0,05). Female was predominant among co-infections patient, but without statistic link (p = 0,9 and p = 0,45); The co-infection HBV- HCV was significatively linked to age beyond 40 years (p = 0,0005). Co-infections with HIV infection and hepatitis virus are not rare and deserve to be investigated.

Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts.

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Rajalakshmy, A R; Malathi, J; Madhavan, H N; Bhaskar, S; Iyer, G K

2016-01-01

Recent studies indicate that hepatitis C virus (HCV) proteins can mediate innate immune response and inflammation in conjunctival fibroblasts which contributes to the pathology of dry eye condition associated with chronic HCV infection. The present study investigates the phagocytic potential of human conjunctival fibroblasts (HCFj) for HCV core protein. HCFj cells were incubated with HCV core antigen for different periods of time, and fluorescent micrographs were taken to observe protein internalisation. HCFj cells were capable of internalising HCV core antigen within 1 h; this gives an insight into another molecular mechanism which may contribute towards HCV-associated conjunctival inflammation.

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

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Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

2013-10-15

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

Links between human LINE-1 retrotransposons and hepatitis virus-related hepatocellular carcinoma

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Honda, Tomoyuki

2016-05-01

Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancers, the third most frequent cause of cancer mortality. The most prevalent risk factors for HCC are infections by hepatitis B or hepatitis C virus. Findings suggest that hepatitis virus-related HCC might be a cancer in which LINE-1 retrotransposons, often termed L1, activity plays a potential role. Firstly, hepatitis viruses can suppress host defense factors that also control L1 mobilization. Secondly, many recent studies also have indicated that hypomethylation of L1 affects the prognosis of HCC patients. Thirdly, endogenous L1 retrotransposition was demonstrated to activate oncogenic pathways in HCC. Fourthly, several L1 chimeric transcripts with host or viral genes are found in hepatitis virus-related HCC. Such lines of evidence suggest a linkage between L1 retrotransposons and hepatitis virus-related HCC. Here, I briefly summarize current understandings of the association between hepatitis virus-related HCC and L1. Then, I discuss potential mechanisms of how hepatitis viruses drive the development of HCC via L1 retrotransposons. An increased understanding of the contribution of L1 to hepatitis virus-related HCC may provide unique insights related to the development of novel therapeutics for this disease.

Discovery of a novel hepatovirus (Phopivirus of seals) related to human Hepatitis A Virus

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Anthony. S.J.,; St. Leger, J.A; Liang, E.; Hicks, A.L.; Sanchez-Leon, M.D; Ip, Hon S.; Jain, K.; Lefkowitch, J. H.; Navarrete-Macias, I.; Knowles, N.; Goldstein, T.; Pugliares, K.; Rowles, T.; Lipkin, W.I.

2015-01-01

Describing the viral diversity of wildlife can provide interesting and useful insights into the natural history of established human pathogens. In this study, we describe a previously unknown picornavirus in harbor seals (tentatively named phopivirus) that is related to human hepatitis A virus (HAV). We show that phopivirus shares several genetic and phenotypic characteristics with HAV, including phylogenetic relatedness across the genome, a specific and seemingly quiescent tropism for hepatocytes, structural conservation in a key functional region of the type III internal ribosomal entry site (IRES), and a codon usage bias consistent with that of HAV.

Factors in enhancing blood safety by nucleic acid technology testing for human immunodeficiency virus, hepatitis C virus and hepatitis B virus.

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Shyamala, Venkatakrishna

2014-01-01

In the last few decades through an awareness of transfusion transmitted infections (TTI), a majority of countries have mandated serology based blood screening assays for Human immunodeficiency virus (HIV), Hepatitis C virus (HCV), and Hepatitis B virus (HBV). However, despite improved serology assays, the transfusion transmission of HIV, HCV, and HBV continues, primarily due to release of serology negative units that are infectious because of the window period (WP) and occult HBV infections (OBI). Effective mode of nucleic acid technology (NAT) testing of the viruses can be used to minimize the risk of TTIs. This review compiles the examples of NAT testing failures for all three viruses; analyzes the causes for failure, and the suggestions from retrospective studies to minimize such failures. The results suggest the safest path to be individual donation testing (ID) format for highest sensitivity, and detection of multiple regions for rapidly mutating and recombining viruses. The role of blood screening in the context of the donation and transfusion practices in India, the donor population, and the epidemiology is also discussed. World wide, as the public awareness of TTIs increases, as the recipient rights for safe blood are legally upheld, as the possibility to manage diseases such as hepatitis through expensive and prolonged treatment becomes accessible, and the societal responsibility to shoulder the health costs as in the case for HIV becomes routine, there is much to gain by preventing infections than treating diseases.

Factors in enhancing blood safety by nucleic acid technology testing for human immunodeficiency virus, hepatitis C virus and hepatitis B virus

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Venkatakrishna Shyamala

2014-01-01

Full Text Available In the last few decades through an awareness of transfusion transmitted infections (TTI, a majority of countries have mandated serology based blood screening assays for Human immunodeficiency virus (HIV, Hepatitis C virus (HCV, and Hepatitis B virus (HBV. However, despite improved serology assays, the transfusion transmission of HIV, HCV, and HBV continues, primarily due to release of serology negative units that are infectious because of the window period (WP and occult HBV infections (OBI. Effective mode of nucleic acid technology (NAT testing of the viruses can be used to minimize the risk of TTIs. This review compiles the examples of NAT testing failures for all three viruses; analyzes the causes for failure, and the suggestions from retrospective studies to minimize such failures. The results suggest the safest path to be individual donation testing (ID format for highest sensitivity, and detection of multiple regions for rapidly mutating and recombining viruses. The role of blood screening in the context of the donation and transfusion practices in India, the donor population, and the epidemiology is also discussed. World wide, as the public awareness of TTIs increases, as the recipient rights for safe blood are legally upheld, as the possibility to manage diseases such as hepatitis through expensive and prolonged treatment becomes accessible, and the societal responsibility to shoulder the health costs as in the case for HIV becomes routine, there is much to gain by preventing infections than treating diseases.

Induction of hepatic aryl hydrocarbon hydroxylase and epoxide hydrase in Wistar rats pretreated with oral methadone hydrochloride.

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Bellward, G D; Gontovnick, L S; Otten, M

1977-01-01

Methadone-HCl added to the drinking water of adult female Wistar rats for 4 weeks produced an increase in the aryl hydrocarbon hydroxylase activity of the hepatic microsomal fraction to 222% of control levels. No change was seen in epoxide hydrase activity. In contrast, when male rats were treated similarly, there was an increase in epoxide hydrase activity to 212% of controls with no change in aryl hydrocarbon hydroxylase activity. No such changes were observed when the subcutaneous route of administration or chronic, low-dose, intraperitoneal injections were used. There were no differences in hepatic cytochrome P-450 or protein concentrations in treated animals as compared to their respective control groups. Control studies were carried out with quinine sulfate in the drinking water to decrease water intake to the level of the methadone-treated group. No elevation in either enzyme activity occurred in this control group. Similarly, paired-feeding studies showed the elevation of enzyme activity to be due to the methadone, not food deprivation. The effects of concurrent therapy of methadone with phenobarbital sodium or 3-methylcholanthrene were compared.

Increase in covalent binding of 5-hydroxydiclofenac to hepatic tissues in rats co-treated with lipopolysaccharide and diclofenac: involvement in the onset of diclofenac-induced idiosyncratic hepatotoxicity.

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Kishida, Tomoyuki; Onozato, Tomoya; Kanazawa, Toru; Tanaka, Satoru; Kuroda, Junji

2012-01-01

Diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is well known to induce idiosyncratic hepatotoxicity. Although there remains much to be elucidated about its onset mechanism, it is widely accepted as a hypothesis that idiosyncratic hepatotoxicity arises from a specific immune response to a hapten formed by covalent binding of drugs or their reactive metabolites to hepatic tissues. In this study, we investigated the effects of covalent binding of DCF reactive metabolites to hepatic tissues using a rat model of liver injury induced by co-treatment with lipopolysaccharide (LPS) at a non-hepatotoxic dose. In studies done in vitro using hepatic microsomes prepared from rats treated with LPS alone, 4'- and 5-hydroxylation activities on DCF metabolism and adducts of reactive metabolites to dansyl glutathione (dGSH) were markedly decreased associated with a decrease in total P450 content. However, in studies done in vivo, the LPS/DCF co-treatment significantly increased adducts of 5-hydroxydiclofenac (5-OH-DCF) to rat hepatic tissues and delayed the elimination of 5-OH-DCF from plasma. Furthermore, we investigated the effects of co-treatment on hepatic GSH level in rats. A decrease of hepatic GSH was observed with the LPS/DCF co-treatment but not with LPS or DCF alone. The results suggest that covalent binding of reactive metabolites via 5-OH-DCF to hepatic tissues may play an important role in the onset of DCF-induced idiosyncratic hepatotoxicity, especially under decreased GSH conditions.

Clinical significance of autoantibodies in autoimmune hepatitis.

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Liberal, Rodrigo; Mieli-Vergani, Giorgina; Vergani, Diego

2013-10-01

The accurate diagnosis and classification of autoimmune hepatitis (AIH) rely upon the detection of characteristic autoantibodies. Positivity for anti-nuclear (ANA) and/or anti-smooth muscle (SMA) autoantibodies defines AIH type 1 (AIH-1), whereas anti-liver kidney microsomal type 1 (anti-LKM1) and/or anti-liver cytosol type 1 (anti-LC1) define AIH type 2 (AIH-2). ANA and SMA, and less commonly anti-LKM1, have also been detected in de-novo autoimmune hepatitis developing after liver transplantation, a condition that may affect patients transplanted for non-autoimmune liver disease. The diagnostic autoantibodies associated with AIH-1 are also detected in the paediatric AIH/sclerosing cholangitis overlap syndrome, referred to as autoimmune sclerosing cholangitis (ASC). ASC, like adult primary sclerosing cholangitis, is often associated with atypical perinuclear anti-neutrophil cytoplasmic autoantibodies (p-ANCA), although p-ANCA are also detected in other autoimmune liver diseases. These associations highlight the necessity for simple and prompt diagnostic autoantibody testing, and the requirement for the accurate interpretation of the results of the tests in the clinical context. Fine-mapping of antigenic autoantibody targets has facilitated the development of rapid molecular assays that have the potential to revolutionise the field if properly standardised and when used in combination with classical immunofluorescence. Despite their diagnostic significance, the pathogenic role of the various autoantibodies and the mechanisms by which they can potentially inflict damage onto the liver cell remain a topic for further research. Copyright © 2013 Elsevier Ltd. All rights reserved.

A consensus for occupational health management of healthcare workers infected with human immunodeficiency virus, hepatitis B virus, and / or hepatitis C virus.

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Ishimaru, Tomohiro; Wada, Koji; Smith, Derek R

2017-05-25

Occupational health management plays an important role in the prevention of provider-to-patient transmission in healthcare workers infected with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and/or hepatitis C virus (HCV). Therefore, the Japan Society for Occupational Health's Research Group on Occupational Health for Health Care Workers has proposed a consensus for the management of healthcare workers infected with HIV, HBV, and/or HCV based on recent evidence for each concerned group. The consensus recommends that: (1) employers in medical institutions should establish a policy of respecting the human rights of healthcare workers, management strategies for occupational blood exposure, and occupational health consultation; (2) occupational health staff should appropriately assess the risk of provider-to-patient transmission of HIV, HBV, and/or HCV infection and rearrange their tasks if necessary. When conducting risk assessment, occupational health staff should obtain informed consent and then cooperate with the physician in charge as well as infection control experts in the workplace; (3) healthcare workers infected with HIV, HBV, and/or HCV should disclose their employment to their treating physician and consult with their doctor regarding the need for special considerations at work; and (4) supervisors and colleagues in medical institutions should correctly understand the risks of HIV, HBV, and HCV infection and should not engage in any behavior that leads to discrimination against colleagues infected with HIV, HBV, and/or HCV.

PHOTOMETRIC EVIDENCE FOR THE OSMOTIC BEHAVIOR OF RAT LIVER MICROSOMES

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Tedeschi, Henry; James, Joseph M.; Anthony, William

1963-01-01

Electron microscope observations are consistent with the interpretation that the elements of the endoplasmic reticulum are osmotically active in situ as well as after isolation. More recently, it has been reported that microsomal suspensions equilibrate almost completely with added C14-sucrose and that no osmotic behavior is evident from photometric data. These findings were considered at variance with the electron microscope data. However, equilibration with added label simply attests to a relatively high permeability, and, in addition, the photometric data need not be critical. Osmotic volume changes, measured photometrically, may be masked by concomitant events (e.g., changes in the refractive index of the test solutions at varying osmotic pressures, breakdown of the particles, and agglutination). For these reasons the photometric experiments were repeated. In this work, the reciprocal of optical density of microsomal suspensions was found to vary linearly with the reciprocal of concentration of the medium at constant refractive index. These changes probably correspond to osmotic volume changes, since the effect was found to be (a) independent of substance used and (b) osmotically reversible. The transmission of the suspension was found to vary with the refractive index of the medium, the concentration of particles, and the wavelength of incident light, according to relationships that are similar to or identical with those obtained for mitochondrial suspensions. PMID:14064105

Coordinated defects in hepatic long chain fatty acid metabolism and triglyceride accumulation contribute to insulin resistance in non-human primates.

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Subhash Kamath

Full Text Available Non-alcoholic fatty liver disease (NAFLD is characterized by accumulation of triglycerides (TG in hepatocytes, which may also trigger cirrhosis. The mechanisms of NAFLD are not fully understood, but insulin resistance has been proposed as a key determinant.To determine the TG content and long chain fatty acyl CoA composition profile in liver from obese non-diabetic insulin resistant (IR and lean insulin sensitive (IS baboons in relation with hepatic and peripheral insulin sensitivity.Twenty baboons with varying grades of adiposity were studied. Hepatic (liver and peripheral (mainly muscle insulin sensitivity was measured with a euglycemic clamp and QUICKI. Liver biopsies were performed at baseline for TG content and LCFA profile by mass spectrometry, and histological analysis. Findings were correlated with clinical and biochemical markers of adiposity and insulin resistance.Obese IR baboons had elevated liver TG content compared to IS. Furthermore, the concentration of unsaturated (LC-UFA was greater than saturated (LC-SFA fatty acyl CoA in the liver. Interestingly, LC-FA UFA and SFA correlated with waist, BMI, insulin, NEFA, TG, QUICKI, but not M/I. Histological findings of NAFLD ranging from focal to diffuse hepatic steatosis were found in obese IR baboons.Liver TG content is closely related with both hepatic and peripheral IR, whereas liver LC-UFA and LC-SFA are closely related only with hepatic IR in non-human primates. Mechanisms leading to the accumulation of TG, LC-UFA and an altered UFA: LC-SFA ratio may play an important role in the pathophysiology of fatty liver disease in humans.

Seroprevalence of Hepatitis B surface antigen, antibodies to the Hepatitis C virus, and human immunodeficiency virus in a hospital-based population in Jaipur, Rajasthan

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Sood Smita

2010-01-01

Full Text Available Background: Hepatitis B, hepatitis C, and HIV infections are a serious global and public health problem. To assess the magnitude and dynamics of disease transmission and for its prevention and control, the study of its seroprevalence is important. A private hospital catering to the needs of a large population represents an important center for serological surveys. Available data, at Rajasthan state level, on the seroprevalence of these bloodborne pathogens is also very limited. Objective: A study was undertaken to estimate the seroprevalence of hepatitis B surface antigen (HBsAg and antibodies to hepatitis C (anti-HCV Ab and human immunodeficiency virus (anti-HIV Ab in both the sexes and different age groups in a hospital-based population in Jaipur, Rajasthan. Materials and Methods: Serum samples collected over a period of 14 months from patients attending OPDs and admitted to various IPDs of Fortis Escorts Hospital, Jaipur, were subjected within the hospital-based lab for the detection of HBsAg and anti-HCV Ab and anti-HIV Ab using rapid card tests. This was followed by further confirmation of all reactive samples by a microparticle enzyme immunoassay (Abbott AxSYM at Super Religare Laboratories (formerly SRL Ranbaxy Reference Lab, Mumbai. Results: The seroprevalence of HBsAg was found to be 0.87%, of anti-HCV Ab as 0.28%, and of anti-HIV Ab as 0.35%. Conclusion: The study throws light on the magnitude of viral transmission in the community in the state of Rajasthan and provides a reference for future studies.

Hepatitis B virus, hepatitis C virus and human immunodeficiency virus infection in undocumented migrants and refugees in southern Italy, January 2012 to June 2013.

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Coppola, Nicola; Alessio, Loredana; Gualdieri, Luciano; Pisaturo, Mariantonietta; Sagnelli, Caterina; Caprio, Nunzio; Maffei, Rita; Starace, Mario; Angelillo, Italo Francesco; Pasquale, Giuseppe; Sagnelli, Evangelista

2015-01-01

Screening of undocumented migrants or refugees for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections has been offered free of charge and free from bureaucratic procedures since 2012 at four primary-level clinical centres in Naples and Caserta, Italy. Of 926 undocumented migrants and refugees visiting one of the primary-level clinical centres from January 2012 to June 2013, 882 (95%) were screened for hepatitis B surface antigen (HBsAg), total hepatitis B core antibody (anti-HBc) and antibodies against HCV and HIV. Of the 882 individuals enrolled, 78 (9%) were HBsAg positive, 35 (4%) anti-HCV positive and 11 (1%) anti-HIV positive (single infections); seven (1%) had more than one infection (three were HBsAg positive). Of the 801 HBsAg-negative patients, 373 (47%) were anti-HBc positive. The HBsAg-positivity rate was high (14%; 62/444) in individuals from sub-Saharan Africa and intermediate in those from eastern Europe (6%; 12/198), northern Africa (2%; 2/80) and Bangladesh, India, Pakistan and Sri Lanka (the 'India-Pakistan area') (3%; 4/126). Anti-HCV was detected in 9/126 (7%) individuals originating from the India-Pakistan area, in 12/198 (6%) from eastern Europe, in 17/444 (4%) from sub-Saharan and in 2/80 (2%) from northern Africa. The HBV, HCV and HIV infections in the undocumented migrants and refugees screened serve as a reminder to the Italian healthcare authorities to carry out extensive screening and educational programmes for these populations.

Hepatic Metabolism of Sakuranetin and Its Modulating Effects on Cytochrome P450s and UDP-Glucuronosyltransferases

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Hyesoo Jeong

2018-06-01

Full Text Available Sakuranetin (SKN, found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug–herb interactions through the modulation of drug metabolizing enzymes (DMEs. HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP 3A4 gene.

High-fructose corn syrup-55 consumption alters hepatic lipid metabolism and promotes triglyceride accumulation.

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Mock, Kaitlin; Lateef, Sundus; Benedito, Vagner A; Tou, Janet C

2017-01-01

High-fructose corn syrup-55 (HFCS-55) has been suggested to be more lipogenic than sucrose, which increases the risk for nonalcoholic fatty liver disease (NAFLD) and dyslipidemia. The study objectives were to determine the effects of drinking different sugar-sweetened solutions on hepatic gene expression in relation to liver fatty acid composition and risk of NAFLD. Female rats were randomly assigned (n=7 rats/group) to drink water or water sweetened with 13% (w/v) HFCS-55, sucrose or fructose for 8 weeks. Rats drinking HFCS-55 solution had the highest (P=.03) hepatic total lipid and triglyceride content and histological evidence of fat infiltration. Rats drinking HFCS-55 solution had the highest hepatic de novo lipogenesis indicated by the up-regulation of stearoyl-CoA desaturase-1 and the highest (Ptriglyceride-rich lipoprotein from the liver was increased as shown by up-regulation of gene expression of microsomal triglyceride transfer protein in rats drinking sucrose, but not HFCS-55 solution. The observed lipogenic effects were attributed to the slightly higher fructose content of HFCS-55 solution in the absence of differences in macronutrient and total caloric intake between rats drinking HFCS-55 and sucrose solution. Results from gene expression and fatty acid composition analysis showed that, in a hypercaloric state, some types of sugars are more detrimental to the liver. Based on these preclinical study results, excess consumption of caloric sweetened beverage, particularly HFCS-sweetened beverages, should be limited. Published by Elsevier Inc.

Mutagenicity of anthraquinone and hydroxylated anthraquinones in the Ames/Salmonella microsome system.

Science.gov (United States)

Liberman, D F; Fink, R C; Schaefer, F L; Mulcahy, R J; Stark, A A

1982-01-01

The mutagenicity of anthracene, anthraquinone, and four structurally similar compounds of each was evaluated in the Ames/Salmonella microsome assay. Anthraquinone was shown to be mutagenic for strains TA1537, TA1538, and TA98 in the absence of rat liver homogenate. The four anthraquinone derivatives tested were mutagenic for TA1537 exclusively. None of the anthracenes exhibited mutagenic activity. PMID:7103489

Mutagenicity of anthraquinone and hydroxylated anthraquinones in the Ames/Salmonella microsome system.

OpenAIRE

Liberman, D F; Fink, R C; Schaefer, F L; Mulcahy, R J; Stark, A A

1982-01-01

The mutagenicity of anthracene, anthraquinone, and four structurally similar compounds of each was evaluated in the Ames/Salmonella microsome assay. Anthraquinone was shown to be mutagenic for strains TA1537, TA1538, and TA98 in the absence of rat liver homogenate. The four anthraquinone derivatives tested were mutagenic for TA1537 exclusively. None of the anthracenes exhibited mutagenic activity.

An implantable vascularized protein gel construct that supports human fetal hepatoblast survival and infection by hepatitis C virus in mice.

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Martha J Harding

2010-04-01

Full Text Available Widely accessible small animal models suitable for the study of hepatitis C virus (HCV in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice.We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH within a vascularized rat collagen type I/human fibronectin (rCI/hFN gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC in severe combined immunodeficient X beige (SCID/bg mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7 mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4alpha (HNF4alpha mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM components and/or hepatocyte growth factor (HGF-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM analyses of gel tissue.The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.

Studies on the propagation in cell culture and the infectivity for baboons of human hepatitis A virus

International Nuclear Information System (INIS)

Taylor, M.B.

1985-05-01

Current aspects of hepatitis A and hepatitis A virus (HAV) research and the techniques used for the propagation and monitoring of HAV and HAV antigen (HA Ag) production in vitro and HAV infection in vivo, and its sequelae are reviewed. Radioimmunoassay, immunofluorescence and electron microscopic techniques for the demonstration of HA Ag were adapted for this investigation. The cell-adapted strain of HAV(MBB) was successfully propagated in the human hepatoma cell line PLC/PRF/5 at 32 degrees Celsius. A crystalline structure was demonstrated in the cytoplasm of HAV-infected cells by thin-section electron microscopy. The origin and significance of this structure is uncertain. A possible temperature variant of HAV (strain MBB) or an HAV-related baboon virus was detected in PLC/PRF/5 cells maintained at 37 degrees Celsius after infection with a faecal extract prepared from baboons which had been infected with the cell-cultured HAV. Baboons, both free-ranging and in captivity, were found to have antibodies to HAV, which suggests susceptibility to human HAV or another cross-reacting virus. The experimental infection of the Cape baboon orally, intravenously or by both routes with HAV were investigated. The results of the study suggest reasons for the presence of anti-HAV antibodies in certain baboon populations and show that the baboon is not an ideal model for hepatitis A investigations

Studies on the propagation in cell culture and the infectivity for baboons of human hepatitis A virus

Energy Technology Data Exchange (ETDEWEB)

Taylor, M B

1985-01-01

Current aspects of hepatitis A and hepatitis A virus (HAV) research and the techniques used for the propagation and monitoring of HAV and HAV antigen (HA Ag) production in vitro and HAV infection in vivo, and its sequelae are reviewed. Radioimmunoassay, immunofluorescence and electron microscopic techniques for the demonstration of HA Ag were adapted for this investigation. The cell-adapted strain of HAV(MBB) was successfully propagated in the human hepatoma cell line PLC/PRF/5 at 32 degrees Celsius. A crystalline structure was demonstrated in the cytoplasm of HAV-infected cells by thin-section electron microscopy. The origin and significance of this structure is uncertain. A possible temperature variant of HAV (strain MBB) or an HAV-related baboon virus was detected in PLC/PRF/5 cells maintained at 37 degrees Celsius after infection with a faecal extract prepared from baboons which had been infected with the cell-cultured HAV. Baboons, both free-ranging and in captivity, were found to have antibodies to HAV, which suggests susceptibility to human HAV or another cross-reacting virus. The experimental infection of the Cape baboon orally, intravenously or by both routes with HAV were investigated. The results of the study suggest reasons for the presence of anti-HAV antibodies in certain baboon populations and show that the baboon is not an ideal model for hepatitis A investigations.

Hepatitis C, human immunodeficiency virus and metabolic syndrome: interactions.

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Kotler, Donald P

2009-03-01

Significant concerns have been raised about the metabolic effects of antiretroviral medication, including the classic triad of dyslipidaemia, insulin resistance (IR) and characteristic alterations in fat distribution (lipoatrophy and lipohypertrophy). Co-infection with hepatitis C appears to exacerbate IR, reduce serum lipids and induce prothrombotic changes in the treated human immunodeficiency virus patient. The effects of co-infection are complex. While combination antiretroviral therapy has been shown to be associated with an increased risk of cardiovascular events through promotion of dyslipidaemia, IR and fat redistribution, co-infection exacerbates IR while reducing serum lipids. Co-infection also promotes a prothrombotic state characterized by endothelial dysfunction and platelet activation, which may enhance risk for cardiovascular disease. Consideration must be given to selection of appropriate treatment regimens and timing of therapy in co-infected patients to minimize metabolic derangements and, ultimately, reduce cardiovascular risk.

Clinical assessment of hepatic functional reserve using 99mTc DTPA galactosyl human serum albumin SPECT to prognosticate chronic hepatic diseases. Validation of the use of SPECT and a new indicator

International Nuclear Information System (INIS)

Onodera, Yuya; Tamaki, Nagara; Miyasaka, Kazuo; Takahashi, Kazuei; Sugai, Yukio; Togashi, Tadashi

2003-01-01

It is generally known that scintigraphy of 99m Tc diethylenetriamine pentaacetic acid-galactosyl human serum albumin ( 99m Tc-GSA) is useful for assessing hepatic functional reserve. For hepatic functional indicators, the index of the calculated planar image has been used in previous studies. However, there have been few reports that suggest that the indicators calculated from static SPECT data would be useful for the assessment of hepatic function. The aims of this study were to establish a simple method for assessing hepatic functional reserve using the liver SPECT of 99m Tc-GSA and to apply this method for rich stratification in patients with chronic hepatic diseases. A liver phantom (a 50% concentration of 99m Tc solution) was used to compare the planar and SPECT methods. According to the definition of the new indicator, the liver SPECT of 99m Tc-GSA was divided by a syringe SPECT of 99m Tc-GSA and was called the liver uptake ratio (LUR). We correlated the LUR and the liver uptake ratio calculated according to the blood-sampling method. 99m Tc-GSA SPECT was performed in 137 patients with hepatic diseases, including chronic hepatic diseases, and 20 healthy volunteers. The LUR was correlated between the formed subtypes for all subjects. The acquired phantom-count ratio calculated by the SPECT method was more accurate than that acquired by the planar method. A good correlation was obtained between the LUR and the blood-sampling method (r=0.971). The LUR was significantly lower in subjects with severe cirrhosis than in healthy subjects or those with chronic hepatitis and mild cirrhosis, and it was significantly lower in subjects with chronic hepatitis and mild cirrhosis than in healthy subjects. The LUR was significantly correlated with other hepatic function tests. Based on LUR, the chronic hepatic diseases were divided into two groups: Group A, with LURs 30% and higher, and Group B, with LURs below 30%. An LUR of 30% marked the 25th percentile of the mild

Detection and Characterization of Homologues of Human Hepatitis Viruses and Pegiviruses in Rodents and Bats in Vietnam.

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Van Nguyen, Dung; Van Nguyen, Cuong; Bonsall, David; Ngo, Tue Tri; Carrique-Mas, Juan; Pham, Anh Hong; Bryant, Juliet E; Thwaites, Guy; Baker, Stephen; Woolhouse, Mark; Simmonds, Peter

2018-02-28

Rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. Numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. In this study of pegivirus and human hepatitis-related viruses, liver and serum samples from Vietnamese rodents and bats were examined by PCR and sequencing. Nucleic acids homologous to human hepatitis B, C, E viruses were detected in liver samples of 2 (1.3%) of 157 bats, 38 (8.1%), and 14 (3%) of 470 rodents, respectively. Hepacivirus-like viruses were frequently detected (42.7%) in the bamboo rat, Rhizomys pruinosus , while pegivirus RNA was only evident in 2 (0.3%) of 638 rodent serum samples. Complete or near-complete genome sequences of HBV, HEV and pegivirus homologues closely resembled those previously reported from rodents and bats. However, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the Hepacivirus genus. Of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined.

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

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Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

2018-05-01

The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

Immunoglobulin GM and KM allotypes and prevalence of anti-LKM1 autoantibodies in patients with hepatitis C virus infection.

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Muratori, Paolo; Sutherland, Susan E; Muratori, Luigi; Granito, Alessandro; Guidi, Marcello; Pappas, Georges; Lenzi, Marco; Bianchi, Francesco B; Pandey, Janardan P

2006-05-01

GM and KM allotypes-genetic markers of immunoglobulin (Ig) gamma and kappa chains, respectively-are associated with humoral immunity to several infection- and autoimmunity-related epitopes. We hypothesized that GM and KM allotypes contribute to the generation of autoantibodies to liver/kidney microsomal antigen 1 (LKM1) in hepatitis C virus (HCV)-infected persons. To test this hypothesis, we characterized 129 persons with persistent HCV infection for several GM and KM markers and for anti-LKM1 antibodies. The heterozygous GM 1,3,17 23 5,13,21 phenotype was significantly associated with the prevalence of anti-LKM1 antibodies (odds ratio, 5.13; P=0.002), suggesting its involvement in this autoimmune phenomenon in HCV infection.

Nonhuman Primate Models of Hepatitis A Virus and Hepatitis E Virus Infections.

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Lanford, Robert E; Walker, Christopher M; Lemon, Stanley M

2018-04-23

Although phylogenetically unrelated, human hepatitis viruses share an exclusive or near exclusive tropism for replication in differentiated hepatocytes. This narrow tissue tropism may contribute to the restriction of the host ranges of these viruses to relatively few host species, mostly nonhuman primates. Nonhuman primate models thus figure prominently in our current understanding of the replication and pathogenesis of these viruses, including the enterically transmitted hepatitis A virus (HAV) and hepatitis E virus (HEV), and have also played major roles in vaccine development. This review draws comparisons of HAV and HEV infection from studies conducted in nonhuman primates, and describes how such studies have contributed to our current understanding of the biology of these viruses. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Interaction of rocuronium with human liver cytochromes P450.

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Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-02-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

LKM1 autoantibodies in chronic hepatitis C infection: a case of molecular mimicry?

Science.gov (United States)

Marceau, Gabriel; Lapierre, Pascal; Béland, Kathie; Soudeyns, Hugo; Alvarez, Fernando

2005-09-01

Anti-liver-kidney microsome type 1 (LKM1) autoantibodies directed against the cytochrome P450 2D6 (CYP2D6) are considered specific markers of type 2 autoimmune hepatitis, but are also found in 5% of sera from patients chronically infected by hepatitis C virus (HCV). Molecular mimicry between HCV proteins and CYP2D6 has been proposed to explain the emergence of these autoantibodies. Anti-LKM1 autoantibodies from hepatitis C-infected patients were affinity-purified against immobilized CYP2D6 protein and used to screen a phage display library. CYP2D6 conformational epitopes were identified using phage display analysis and the identification of statistically significant pairs (SSPs). Cross-reactivity between CYP2D6 and HCV protein candidates was tested by immunoprecipitation. Nineteen different clones were isolated, and their sequencing resulted in the mapping of a conformational epitope to the region of amino acids 254-288 of CYP2D6. Candidate HCV proteins for molecular mimicry included: core, E2, NS3 and NS5a. Affinity-purified autoantibodies from HCV+/LKM1+ patients immunoprecipitated either NS3, NS5a, or both, and these reactivities were specifically inhibited by immobilized CYP2D6. In conclusion, HCV+/LKM1+ sera recognize a specific conformational epitope on CYP2D6 between amino acids 254 to 288, the region that contains the major linear epitope in type 2 autoimmune hepatitis patients. Cross-reactivity due to molecular mimicry at the B-cell level was shown between the CYP2D6 and the HCV NS3 and NS5a proteins and could explain the presence of anti-LKM1 in patients chronically infected with HCV. Further investigation of the role played by this molecular mimicry in HCV-infected patients may lead to more specific strategies for diagnosis and treatment.

Organophosphorothionate pesticides inhibit the bioactivation of imipramine by human hepatic cytochrome P450s

International Nuclear Information System (INIS)

Di Consiglio, Emma; Meneguz, Annarita; Testai, Emanuela

2005-01-01

The drug-toxicant interaction between the antidepressant imipramine (IMI) and three organophosphorothionate pesticides (OPTs), to which humans may be chronically and simultaneously exposed, has been investigated in vitro. Concentrations of IMI (2-400 μM) and OPTs (≤10 μM) representative of actual human exposure have been tested with recombinant human CYPs and human liver microsomes (HLM). The different CYPs involved in IMI demethylation to the pharmacologically active metabolite desipramine (DES) were CYP2C19 > CYP1A2 > CYP3A4. The OPTs significantly inhibited (up to >80%) IMI bioactivation catalyzed by the recombinant CYPs tested, except CYP2D6, and by HLM; the inhibition was dose-dependent and started at low pesticide concentrations (0.25-2.5 μM). The OPTs, having lower K m values, efficiently competed with IMI for the enzyme active site, as in the case of CYP2C19. However, with CYP1A2 and CYP3A4, a time- and NADPH-dependent mechanism-based inactivation also occurred, consistently with irreversible inhibition due to the release of the sulfur atom, binding to the active CYP during OPT desulfuration. At low IMI and OPT concentrations, lower IC50 values have been obtained with recombinant CYP1A2 (0.7-1.1 μM) or with HLM rich in 1A2-related activity (2-10.8 μM). The K i values (2-14 μM), independent on substrate concentrations, were quite low and similar for the three pesticides. Exposure to OPTs during IMI therapeutic treatments may lead to decreased DES formation, resulting in high plasma levels of the parent drug, eventual impairment of its pharmacological action and possible onset of adverse drug reactions (ADRs)

Regulation of rat liver cytochrome P450j, a high affinity N-nitrosodimethylamine demethylase (NDMAD)

International Nuclear Information System (INIS)

Thomas, P.E.; Bandiera, S.; Maines, S.L.; Ryan, D.E.; Levin, W.

1987-01-01

Purified IgG from sera of rabbits immunized with homogeneous P450j was absorbed to produce monospecific anti-P450j. Results using anti-P450j in ELISA show that rat liver microsomal P450j content decreases between 3 and 6 wks of age in both sexes. Several xenobiotics (Aroclor 1254, mirex and 3-methylcholanthrene) repressed P450j levels when administered to male rats. In contrast, hepatic levels of P450j were induced by isoniazid, dimethylsulfoxide, pyrazole, 4-methylpyrazole, ethanol and chemically-induced diabetes. P450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries and testes; however, extra-hepatic P450j was inducible by isoniazid. Between 80-90% of microsomal NDMAD was inhibited by anti-P450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of P450j. Results obtained with the reconstituted system suggest that the remaining microsomal NDMAD resistant to antibody inhibition is the result of the inaccessibility of a certain proportion of P450j due to interference by NADPH-P450 reductase. P450j content and NDMAD activity correlated well in microsomes from rats of all treatment groups. The evidence indicates that P450j is the primary, and possibly only, microsomal catalyst of NDMAD at substrate concentrations relevant to hepatocarcinogenesis induced by NDMA

Tiamulin selectively inhibits oxidative hepatic steroid and drug metabolism in vitro in the pig.

Science.gov (United States)

Witkamp, R F; Nijmeijer, S M; Csikó, G; van Miert, A S

1994-08-01

The simultaneous use of the antibiotic tiamulin with certain ionophoric antibiotics (monensin, salinomycin) may give rise to a toxic interaction in pigs and poultry. In the present study, effects of tiamulin on hepatic cytochrome P450 activities in vitro were studied using pig liver microsomes. When tiamulin was added to the incubation medium the N-demethylation rate of ethylmorphine and the hydroxylation of testosterone at the 6 beta- and 11 alpha-positions was strongly inhibited. Tiamulin inhibited these activities more than SKF525A or cimetidine, but less than ketoconazole. The microsomal N-demethylation rate of erythromycin and the hydroxylation of testosterone at the 2 beta-position were inhibited to a lesser degree, whereas the ethoxyresorufin-O-deethylation, aniline hydroxylation and testosterone hydroxylations at the 15 alpha- and 15 beta-positions were not affected by tiamulin. No in vitro complexation by tiamulin of cytochrome P450 resulting in a loss of CO-binding capacity could be demonstrated. Results from the present study suggest a selective inhibition of cytochrome P450 enzymes in pigs, probably belonging to the P4503A subfamily. The mechanism of this interaction is still unclear. However, interactions between tiamulin and those veterinary drugs or endogenous compounds which undergo oxidative metabolism by P450 enzymes must be considered. More research is needed to reveal which of the P450 enzymes are affected by tiamulin in order to improve the understanding and probably the predictability of this interaction.

Recent advances in Hepatitis E virus.

Science.gov (United States)

Meng, X J

2010-03-01

Hepatitis E virus (HEV), the causative agent of hepatitis E, belongs to the family Hepeviridae. At least four major genotypes of HEV have been recognized: genotypes 1 and 2 are restricted to humans and associated with epidemics in developing countries, whereas genotypes 3 and 4 are zoonotic and infect humans and several other animals in both developing and industrialized countries. Besides humans, strains of HEV have been genetically identified from swine, chickens, sika deer, mongeese, and rabbits. The genome of HEV consists of three open reading frames (ORFs): ORF1 codes for nonstructural proteins, ORF2 codes for capsid protein, and ORF3 codes for a small multifunctional protein. The ORF2 and ORF3 proteins are translated from a single bicistronic mRNA and overlap each other but neither overlaps ORF1. The recent determination of the 3D crystal structure of the HEV capsid protein should facilitate the development of vaccines and antivirals. The identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by swine HEV raise public health concerns for zoonosis. Accumulating evidence indicated that hepatitis E is a zoonotic disease and pigs and more likely other animal species are reservoirs for HEV. This article provides an overview of the recent advances in hepatitis E and its causative agent, including nomenclature and genomic organization, gene expression and functions, 3D structure of the virions, changing perspectives on higher mortality during pregnancy and chronic hepatitis E, animal reservoirs, zoonotic risk, food safety, and novel animal models.

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

Directory of Open Access Journals (Sweden)

Ayaka Yanagida

Full Text Available Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(highCD133(+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632, individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein and a cholangiocytic marker gene (cytokeratin 7, and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that

Hepatic uptake and storage of warfarin. The relation with the target enzyme vitamin K 2,3-epoxide reductase

International Nuclear Information System (INIS)

Thijssen, H.H.; Baars, L.G.

1987-01-01

The mechanisms of the reported dose-dependent warfarin pharmacokinetics were investigated using [ 14 C]warfarin. When administered in microdoses (9 micrograms i.v.) to rats (male Wistars, 270-300 g), a steep distribution phase (T1/2 = 0.25 hr) was followed by a relatively slow beta-phase (T1/2 = 40 hr). The observed volume of distribution was 390 ml. This pharmacokinetic behavior contrasted highly with the one seen for higher (greater than 0.2 mg/kg) doses (unlabeled) warfarin; volume of distribution = 45 ml, T1/2 = 12.5 hr. If a macrodose (0.2 mg/kg) preceded (16 hr) the microdose, normal pharmacokinetics were observed for the latter, suggesting a saturable deep compartment. The administration of 4-hydroxycoumarins (i.e., acenocoumarol, phenprocoumon and warfarin) after the microdose of [ 14 C]warfarin was in its beta-phase caused a rapid rise of plasma [ 14 C]warfarin indicating [ 14 C]warfarin to be displaced from the deep compartment. The rate of appearance of [ 14 C]warfarin was 0.3 hr-1 irrespective the 4-hydroxycoumarin used. The hepatic distribution of [ 14 C]warfarin was investigated and the effect of a displacer thereupon. Fifty-three hours after the [ 14 C]warfarin administration, the liver contained about 40% of the dose; 45% of it was bound to microsomes. The administration of acenocoumarol (0.2 mg/kg) at 48 hr, halved the liver content. [ 14 C]warfarin was redistributed from microsomes (-65%) and from the 10,000 X g pellet (-65%) into the cytosol (+260%) and the plasma (+320%). Microsomal bound [ 14 C]warfarin in vitro could not be washed out or be displaced unless dithiothreitol (50 mM) was included in the washing buffers

Effects of phenobarbital pretreatment on the in vivo metabolism of carbaryl in rats

International Nuclear Information System (INIS)

Knight, E.V.; Alvares, A.P.; Chin, B.H.

1987-01-01

Phenobarbital (PB) pretreatment of animals is known to induce the activity of drug-metabolizing enzymes in liver microsomes. Previous studies showed that incubation of carbaryl with microsomes obtained from livers of untreated or PB-treated rats resulted in little or no oxidative metabolism of the substrate. In addition, no spectral interactions were observed when carbaryl was added to hepatic microsomal suspensions. The present study was carried out to determine the effect of PB pretreatment on the in vivo metabolism of carbaryl in rats

Systemic excretion of benzo(a)pyrene in the control and microsomally induced rat: the influence of plasma lipoproteins and albumin as carrier molecules

International Nuclear Information System (INIS)

Shu, H.P.; Bymun, E.N.

1983-01-01

In vitro studies have previously indicated that benzo(a)pyrene distributes primarily into the plasma lipoprotein fraction when incubated with whole plasma. Hydroxylated metabolites of benzo(a)pyrene distribute increasingly into the albumin fraction as the degree of metabolite hydroxylation increases. This report assesses the influence of plasma lipoproteins and albumin as carriers for benzo(a)pyrene on carcinogen excretion in the control and microsomally induced rat. Male Sprague-Dawley rats cannulated in the bile duct received i.v. injections of radiolabeled benzo(a)pyrene noncovalently bound to the very-low-density, low-density, or high-density lipoproteins in equimolar amounts. Bile was collected and measured for radioactivity. Cumulative biliary excretions of benzo(a)pyrene complexed with rat lipoproteins were 39.6 +/- 9.7 (S.D.), 24.6 +/- 1.3, and 21.2 +/- 8.8% for very low-density, low-density, and high-density lipoprotein, respectively. Values for excretion of benzo(a)pyrene complexed with rat or human lipoproteins were comparable. These data suggest that the transport molecule can effect a 2-fold difference in benzo(a)pyrene excretion under conditions of the present study. Thus, excretion increased as the degree of benzo(a)pyrene hydroxylation increased. The effect of microsomal enzyme induction on excretion of lipoprotein-bound benzo(a)pyrene was also assessed. Contrary to expectation, excretion of benzo(a)pyrene bound to the very-low-density, low-density, or high-density lipoproteins in Aroclor-induced rats was not greater than that of control animals. Hence, under the conditions of the present study, 60 to 80% of the injected benzo(a)pyrene and 50 to 60% of the injected benzo(a)pyrene metabolites were not excreted immediately in control or microsomally induced animals. This benzo(a)pyrene may represent a carcinogen pool that is slowly excreted

Dual Infection with Hepatitis B and Epstein-Barr Virus Presenting with Severe Jaundice, Coagulopathy, and Hepatitis B Virus Chronicity Outcome

OpenAIRE

Rao, Sirish C.; Ashraf, Imran; Mir, Fazia; Samiullah, Sami; Ibdah, Jamal A.; Tahan, Veysel

2017-01-01

Patient: Female, 34 Final Diagnosis: HBV and EBV dual infection Symptoms: Jaundice ? fatigue ? anorexia ? subjective weight loss Medication: ? Clinical Procedure: ? Specialty: Gastroenterology and Hepatology Objective: Rare co-existance of disease or pathology Background: Hepatitis B virus (HBV) has been reported as a coinfection with hepatitis C virus (HCV), hepatitis D virus (HDV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV). Case Report: A 34-year-old female presented to ...

Acetyl CoA Carboxylase Inhibition Reduces Hepatic Steatosis but Elevates Plasma Triglycerides in Mice and Humans: A Bedside to Bench Investigation.

Science.gov (United States)

Kim, Chai-Wan; Addy, Carol; Kusunoki, Jun; Anderson, Norma N; Deja, Stanislaw; Fu, Xiaorong; Burgess, Shawn C; Li, Cai; Ruddy, Marcie; Chakravarthy, Manu; Previs, Steve; Milstein, Stuart; Fitzgerald, Kevin; Kelley, David E; Horton, Jay D

2017-08-01

Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion. Copyright © 2017 Elsevier Inc. All rights reserved.

Hepatitis B virus evasion from cGAS sensing in human hepatocytes.

Science.gov (United States)

Verrier, Eloi R; Yim, Seung-Ae; Heydmann, Laura; El Saghire, Houssein; Bach, Charlotte; Turon-Lagot, Vincent; Mailly, Laurent; Durand, Sarah C; Lucifora, Julie; Durantel, David; Pessaux, Patrick; Manel, Nicolas; Hirsch, Ivan; Zeisel, Mirjam B; Pochet, Nathalie; Schuster, Catherine; Baumert, Thomas F

2018-04-20

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this study, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes and HBV-infected human liver chimeric mice. Here we show that cGAS is expressed in the human liver, primary human hepatocytes and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral cccDNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Dyslipidaemia--hepatic and intestinal cross-talk.

LENUS (Irish Health Repository)

Tomkin, Gerald H

2010-06-01

Cholesterol metabolism is tightly regulated with the majority of de novo cholesterol synthesis occurring in the liver and intestine. 3 Hydroxy-3-methylglutaryl coenzyme A reductase, a major enzyme involved in cholesterol synthesis, is raised in both liver and intestine in diabetic animals. Niemann PickC1-like1 protein regulates cholesterol absorption in the intestine and facilitates cholesterol transport through the liver. There is evidence to suggest that the effect of inhibition of Niemann PickC1-like1 lowers cholesterol through its effect not only in the intestine but also in the liver. ATP binding cassette proteins G5\\/G8 regulate cholesterol re-excretion in the intestine and in the liver, cholesterol excretion into the bile. Diabetes is associated with reduced ATP binding cassette protein G5\\/G8 expression in both the liver and intestine in animal models. Microsomal triglyceride transfer protein is central to the formation of the chylomicron in the intestine and VLDL in the liver. Microsomal triglyceride transfer protein mRNA is increased in diabetes in both the intestine and liver. Cross-talk between the intestine and liver is poorly documented in humans due to the difficulty in obtaining liver biopsies but animal studies are fairly consistent in showing relationships that explain in part mechanisms involved in cholesterol homeostasis.

Enantioselective N-demethylation and hydroxylation of sibutramine in human liver microsomes and recombinant cytochrome p-450 isoforms.

Science.gov (United States)

Shinde, Dhananjay D; Kim, Min-Jung; Jeong, Eun-Sook; Kim, Yang-Weon; Lee, Ji-Woo; Shin, Jae-Gook; Kim, Dong-Hyun

2014-01-01

The enantioselective metabolism of sibutramine was examined using human liver microsomes (HLM) and recombinant cytochrome P-450 (CYP) isoforms. This drug is metabolized to N-mono-desmethyl- (M1) and N,N-di-desmethylsibutramine (M2), and subsequent hydroxylation results in hydroxyl M1 (HM1) and hydroxyl M2 (HM2). No significant difference was noted in formation of M1from sibutramine between R- and S-sibutramine in HLM. However, S-enantiomers of M1 and M2 were preferentially metabolized to M2, HM1, and HM2compared to R-enantiomers in HLM, and intrinsic clearance (Clint) ratios of S-enantiomers/R-enantiomers were 1.97, 4.83, and 9.94 for M2, HM1, and HM2, respectively. CYP3A4 and CYP3A5 were only involved in the formation of M1, whereas CYP2B6 and CYP2C19 were responsible for all metabolic reactions of sibutramine. CYP2C19 and CYP3A5 displayed catalytic preference for S-sibutramine to S-M1, whereas CYP2B6 and CYP3A4 showed little or no stereoselectivity in metabolism of sibutramine to M1. In the case of M2 formation, CYP2B6 metabolized S-M1 more rapidly than R-M1 with a Clint ratio of 2.14. However, CYP2C19 catalyzed less S-M1 than R-M1 and the Clint ratio of S-M1 to R-M1 was 0.65. The most significant enantioselectivity was observed in formation of HM1 from M1, and HM2 from M2. CYP2B6 and CYP2C19 exhibited preferential catalysis of formation of hydroxyl metabolites from S-enantiomers rather than R-enantiomers. These results indicate that S-sibutramine was more rapidly metabolized by CYP isoforms than R-sibutramine, and that enantioselective metabolism needs to be considered in drug interactions involving sibutramine and co-administered drugs.

The intraportal injection model: A practical animal model for hepatic metastases and tumor cell dissemination in human colon cancer

International Nuclear Information System (INIS)

Thalheimer, Andreas; Waaga-Gasser, Ana M; Otto, Christoph; Bueter, Marco; Illert, Bertram; Gattenlohner, Stefan; Gasser, Martin; Meyer, Detlef; Fein, Martin; Germer, Christoph T

2009-01-01

The development of new therapeutic strategies for treatment of metastasized colorectal carcinoma requires biologically relevant and adequate animal models that generate both reproducible metastasis and the dissemination of tumor cells in the form of so-called minimal residual disease (MRD), an expression of the systemic character of neoplastic disease. We injected immunoincompetent nude mice intraportally with different numbers (1 × 10 5 , 1 × 10 6 and 5 × 10 6 cells) of the human colon carcinoma cell lines HT-29 and SW-620 and investigated by histological studies and CK-20 RT-PCR the occurrence of hematogenous metastases and the dissemination of human tumor cells in bone marrow. Only the injection of 1 × 10 6 cells of each colon carcinoma cell line produced acceptable perioperative mortality with reproducible induction of hepatic metastases in up to 89% of all animals. The injection of 1 × 10 6 cells also generated tumor cell dissemination in the bone marrow in up to 63% of animals with hepatic metastases. The present intraportal injection model in immunoincompetent nude mice represents a biologically relevant and adequate animal model for the induction of both reproducible hepatic metastasis and tumor cell dissemination in the bone marrow as a sign of MRD

A physiologically based toxicokinetic model for inhaled ethylene and ethylene oxide in mouse, rat, and human.

Science.gov (United States)

Filser, Johannes Georg; Klein, Dominik

2018-04-01

Ethylene (ET) is the largest volume organic chemical. Mammals metabolize the olefin to ethylene oxide (EO), another important industrial chemical. The epoxide alkylates macromolecules and has mutagenic and carcinogenic properties. In order to estimate the EO burden in mice, rats, and humans resulting from inhalation exposure to gaseous ET or EO, a physiological toxicokinetic model was developed. It consists of the compartments lung, richly perfused tissues, kidneys, muscle, fat, arterial blood, venous blood, and liver containing the sub-compartment endoplasmic reticulum. Modeled ET metabolism is mediated by hepatic cytochrome P450 2E1, EO metabolism by hepatic microsomal epoxide hydrolase or cytosolic glutathione S-transferase in various tissues. EO is also spontaneously hydrolyzed or conjugated with glutathione. The model was validated on experimental data collected in mice, rats, and humans. Modeled were uptake by inhalation, wash-in-wash-out effect in the upper respiratory airways, distribution into tissues and organs, elimination via exhalation and metabolism, and formation of 2-hydroxyethyl adducts with hemoglobin and DNA. Simulated concentration-time courses of ET or EO in inhaled (gas uptake studies) or exhaled air, and of EO in blood during exposures to ET or EO agreed excellently with measured data. Predicted levels of adducts with DNA and hemoglobin, induced by ET or EO, agreed with reported levels. Exposures to 10000 ppm ET were predicted to induce the same adduct levels as EO exposures to 3.95 (mice), 5.67 (rats), or 0.313 ppm (humans). The model is concluded to be applicable for assessing health risks from inhalation exposure to ET or EO. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Epidemiology of hepatitis B and hepatitis C in Lebanon.

Science.gov (United States)

Abou Rached, Antoine; Abou Kheir, Selim; Saba, Jowana; Ammar, Walid

2016-03-01

Hepatitis B and C are two potentially life threatening liver infections. Lebanon is ranked as a zone of moderate endemicity. This study aimed to determine the prevalence of hepatitis B and C in Lebanon and their distribution according to age, region and sex. This national prospective cross-sectional study was conducted from January 2011 till December 2012 in the six Lebanese Governorates in collaboration with municipalities, the Ministry of Public Health, Health Centres and dispensaries. An upcoming screening for hepatitis B and C was announced? in different districts of each Governorate. All individuals presenting to local laboratory, not known to have chronic hepatitis, were asked for a blood sample and answered a questionnaire addressing sex, age, place of birth and residence. Screening tests were "Abbots" for hepatitis B and "Human Hexagon" for hepatitis C. PCR testing was used to confirm the positivity of the previous tests. Of 31147 individuals screened, 542 had a rapid test positive for HBV (prevalence 1.74%, 95% CI 1.6-1.89) with a male to female ratio of 1.08. This prevalence was higher in the South and Nabatieh (1.9%) compared to Beirut (0.73%). Of 31,147 individuals screened, 64 had a rapid test positive for HCV (prevalence 0.21%, 95% CI 0.16-0.27) with a male to female ratio of 0.85. This prevalence was higher in Nabatieh (0.61%) compared to Mount Lebanon (0.08%). The prevalence of HBV and HCV in Lebanon is 1.74% and 0.21%, respectively with a higher prevalence in South and Nabatieh districts. These data rank Lebanon amongst countries with low endemicity for both viruses. Decrease in the prevalence of HBV is due to awareness campaign as well as success of the MOPH National Hepatitis Program in vaccinating all new born since 1998 and in screening and vaccinating high risk groups. Copyright © 2016 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.

Autoantibody profile in individuals with chronic hepatitis C.

Science.gov (United States)

Marconcini, Maíra Luciana; Fayad, Leonardo; Shiozawa, Maria Beatriz Cacese; Dantas-Correa, Esther Buzaglo; Lucca Schiavon, Leonardo de; Narciso-Schiavon, Janaína Luz

2013-01-01

Autoantibodies are often produced during infection with chronic hepatitis C virus (HCV), but it remains controversial whether they influence the biochemical profile and histological features of this disease. Therefore, this current study sought to describe these autoantibodies and evaluate their impact on the clinical and histological presentation of hepatitis C. This cross-sectional analytical study assessed patients with HCV (RNA+) from October 2011 to July 2012. This study included 66 patients, with a mean age of 53.2±10.5 years. Of these patients, 60.6% were male, and 54.3% presented with genotype 1. Non-organ-specific autoantibodies (NOSA) were detected in 24% of the patients; of these, 7.6% were anti-mitochondrial antibodies (AMA+), 26.7% were anti-smooth muscle antibodies (SMA+) and 6.8% were liver kidney microsomal type 1 antibodies (LKM1+). With respect to the thyroid autoantibodies, 7.4% were anti-peroxidase (ATPO+) antibodies, and none were anti-thyroglobulin (ATG+) antibodies. Regarding celiac disease autoantibodies, 5.8% were endomysial antibodies (EMA+), and no transglutaminase (TTG+) antibodies were detected. Cryoglobulins were found in 2.1% of patients. When NOSA+ individuals were compared to patients without the presence of NOSAs, they exhibited higher median alkaline phosphatase (0.7 vs. 0.6 xULN; p=0.041), lower median platelet counts (141,500.0 vs. 180,500.0/mm 3 ; p=0.036), lower mean prothrombin activity (72.6±11.5% vs. 82.2±16.0%; p=0.012) and an increased prevalence of significant fibrosis (E≥2) (45.5% vs. 18.2%; p=0.012). There was also a tendency for a greater proportion of NOSA+ cases to have marked periportal activity (APP≥3) (44.5% vs. 15.6%; p=0.087). In addition to the high prevalence of autoantibodies associated with HCV infection, it was observed that NOSA positivity was associated with a more severe histological and biochemical profile of hepatitis C infection.

Autoantibody profile in individuals with chronic hepatitis C

Directory of Open Access Journals (Sweden)

Maíra Luciana Marconcini

2013-04-01

Full Text Available Introduction Autoantibodies are often produced during infection with chronic hepatitis C virus (HCV, but it remains controversial whether they influence the biochemical profile and histological features of this disease. Therefore, this current study sought to describe these autoantibodies and evaluate their impact on the clinical and histological presentation of hepatitis C. Methods This cross-sectional analytical study assessed patients with HCV (RNA+ from October 2011 to July 2012. Results This study included 66 patients, with a mean age of 53.2±10.5 years. Of these patients, 60.6% were male, and 54.3% presented with genotype 1. Non-organ-specific autoantibodies (NOSA were detected in 24% of the patients; of these, 7.6% were anti-mitochondrial antibodies (AMA+, 26.7% were anti-smooth muscle antibodies (SMA+ and 6.8% were liver kidney microsomal type 1 antibodies (LKM1+. With respect to the thyroid autoantibodies, 7.4% were anti-peroxidase (ATPO+ antibodies, and none were anti-thyroglobulin (ATG+ antibodies. Regarding celiac disease autoantibodies, 5.8% were endomysial antibodies (EMA+, and no transglutaminase (TTG+ antibodies were detected. Cryoglobulins were found in 2.1% of patients. When NOSA+ individuals were compared to patients without the presence of NOSAs, they exhibited higher median alkaline phosphatase (0.7 vs. 0.6 xULN; p=0.041, lower median platelet counts (141,500.0 vs. 180,500.0/mm 3 ; p=0.036, lower mean prothrombin activity (72.6±11.5% vs. 82.2±16.0%; p=0.012 and an increased prevalence of significant fibrosis (E≥2 (45.5% vs. 18.2%; p=0.012. There was also a tendency for a greater proportion of NOSA+ cases to have marked periportal activity (APP≥3 (44.5% vs. 15.6%; p=0.087. Conclusions In addition to the high prevalence of autoantibodies associated with HCV infection, it was observed that NOSA positivity was associated with a more severe histological and biochemical profile of hepatitis C infection.

Making the invisible visible: searching for human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2 in Brazilian patients with viral hepatitis B and C

Directory of Open Access Journals (Sweden)

Adele Caterino-de-Araujo

Full Text Available With this study, the authors hope to alert clinicians regarding the presence of human T-cell lymphotropic virus type 1 and 2 (HTLV-1/-2 infections in patients with viral hepatitis B and C in Brazil. HTLV-1/-2 were detected in 1.3% of hepatitis B virus (HBV- and 5.3% of hepatitis C virus (HCV-infected blood samples sent for laboratory viral load measurements. A partial association of human immunodeficiency virus (HIV-1 and HTLV-1/-2 infection was detected in patients with HCV (HIV+, 27.3%, whereas this association was almost 100% in HBV-infected patients (HIV+, all except one. The high prevalence of HTLV-1/-2 infection among patients with hepatitis C was of concern, as HTLV-1/-2 could change the natural course of subsequent liver disease. The authors suggest including HTLV-1/-2 serology in the battery of tests used when following patients with viral hepatitis in Brazil, regardless of the HIV status.

Making the invisible visible: searching for human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) in Brazilian patients with viral hepatitis B and C.

Science.gov (United States)

Caterino-de-Araujo, Adele; Alves, Fabiana Aparecida; Campos, Karoline Rodrigues; Lemos, Marcílio Figueiredo; Moreira, Regina Célia

2018-02-01

With this study, the authors hope to alert clinicians regarding the presence of human T-cell lymphotropic virus type 1 and 2 (HTLV-1/-2) infections in patients with viral hepatitis B and C in Brazil. HTLV-1/-2 were detected in 1.3% of hepatitis B virus (HBV)- and 5.3% of hepatitis C virus (HCV)-infected blood samples sent for laboratory viral load measurements. A partial association of human immunodeficiency virus (HIV)-1 and HTLV-1/-2 infection was detected in patients with HCV (HIV+, 27.3%), whereas this association was almost 100% in HBV-infected patients (HIV+, all except one). The high prevalence of HTLV-1/-2 infection among patients with hepatitis C was of concern, as HTLV-1/-2 could change the natural course of subsequent liver disease. The authors suggest including HTLV-1/-2 serology in the battery of tests used when following patients with viral hepatitis in Brazil, regardless of the HIV status.

Long-term Therapy With Tenofovir Is Effective for Patients Co-Infected With Human Immunodeficiency Virus and Hepatitis B Virus

NARCIS (Netherlands)

de Vries-Sluijs, Theodora E. M. S.; Reijnders, Jurriën G. P.; Hansen, Bettina E.; Zaaijer, Hans L.; Prins, Jan M.; Pas, Suzan D.; Schutten, Martin; Hoepelman, Andy I. M.; Richter, Clemens; Mulder, Jan W.; de Man, Rob A.; Janssen, Harry L. A.; van der Ende, Marchina E.

2010-01-01

BACKGROUND & AIMS: We investigated the long-term efficacy and renal safety of tenofovir disoproxil fumarate (TDF), administered to patients co-infected with human immunodeficiency virus and hepatitis B virus (HBV) as part of an antiretroviral therapy. METHODS: We performed a multicenter, prospective

Identification of rodent homologs of hepatitis C virus and pegiviruses

DEFF Research Database (Denmark)

Kapoor, Amit; Simmonds, Peter; Scheel, Troels K H

2013-01-01

UNLABELLED: Hepatitis C virus (HCV) and human pegivirus (HPgV or GB virus C) are globally distributed and infect 2 to 5% of the human population. The lack of tractable-animal models for these viruses, in particular for HCV, has hampered the study of infection, transmission, virulence, immunity...... into the origins of human infections and enhances our ability to study their pathogenesis and explore preventive and therapeutic interventions. Horses are the only reported host of nonprimate homologs of hepatitis C virus (HCV). Here, we report the discovery of HCV-like viruses in wild rodents. The majority of HCV...... of small-animal models for HCV, the most common infectious cause of liver failure and hepatocellular carcinoma after hepatitis B virus, and help to explore the health relevance of the highly prevalent human pegiviruses....

Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes.

Science.gov (United States)

Shi, Rong; Ma, Bingliang; Wu, Jiasheng; Wang, Tianming; Ma, Yueming

2015-10-01

The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high-throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin-O deethylation, coumarin-7 hydroxylation, bupropion hydroxylation, taxol-6 hydroxylation, omeprazole-5 hydroxylation, dextromethorphan-O demethylation, tolbutamide-4 hydroxylation, chlorzoxazone-6 hydroxylation, testosterone-6β hydroxylation, and midazolam-1 hydroxylation in rat liver microsomes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

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Richter, Lilian H J; Kaminski, Yeda Rumi; Noor, Fozia; Meyer, Markus R; Maurer, Hans H

2016-09-01

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

Hepatitis E as a Zoonosis.

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Widén, Frederik

2016-01-01

Hepatitis E (HE) virus infection is not limited to spread from human to human but also occurs between animals and more importantly as zoonotic spread from animals to humans. Genotyping of strains from hepatitis E virus-infected patients has revealed that these infections are not all caused by genotypes 1 or 2 but often by genotypes 3 or 4. Therefore, it is important to understand the striking difference between the spread of genotypes 1 and 2 in countries with poor sanitary standards and the spread of genotypes 3 and 4 in countries with good sanitary standards. The number of animal species known to be infected with HEV is expanding rapidly. The finding of HEV in new host species always raises the question regarding the zoonotic potential of these newfound strains. However, as new strains are found, the complexity increases.Certain genotypes are known to have the ability of zoonotic spread from certain animal species and these animals may even constitute an infection reservoir. Some animal species may contribute to zoonotic infections albeit on a smaller scale, while others are believed to be of minor or no importance at all. This chapter reviews possible sources of zoonotic hepatitis E virus infection.

Seroprevalence of Human Immunodeficiency Virus, Hepatitis B ...

African Journals Online (AJOL)

surface antigen (HBsAg), syphilis and HCV from the antenatal records. The data were extracted by two trained assistants. Hepatitis B surface antigen and antibodies to HCV were determined using Clinotech diagnostic enzyme linked immunosorbent assay (ELISA) test kits (Clinotech Laboratories,. USA; batch/lot no. for ...

A Microsomal Proteomics View of H2O2- and ABA-Dependent Responses

KAUST Repository

Alquraishi, May Majed; Thomas, Ludivine; Gehring, Chris; Marondedze, Claudius

2017-01-01

The plant hormone abscisic acid (ABA) modulates a number of plant developmental processes and responses to stress. In planta, ABA has been shown to induce reactive oxygen species (ROS) production through the action of plasma membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases. Although quantitative proteomics studies have been performed to identify ABA- or hydrogen peroxide (H₂O₂)-dependent proteins, little is known about the ABA- and H₂O₂-dependent microsomal proteome changes. Here, we examined the effect of 50 µM of either H₂O₂ or ABA on the Arabidopsis microsomal proteome using tandem mass spectrometry and identified 86 specifically H₂O₂-dependent, and 52 specifically ABA-dependent proteins that are differentially expressed. We observed differential accumulation of proteins involved in the tricarboxylic acid (TCA) cycle notably in response to H₂O₂. Of these, aconitase 3 responded to both H₂O₂ and ABA. Additionally, over 30 proteins linked to RNA biology responded significantly to both treatments. Gene ontology categories such as 'response to stress' and 'transport' were enriched, suggesting that H₂O₂ or ABA directly and/or indirectly cause complex and partly overlapping cellular responses. Data are available via ProteomeXchange with identifier PXD006513.

A Microsomal Proteomics View of H2O2- and ABA-Dependent Responses

KAUST Repository

Alquraishi, May Majed

2017-08-21

The plant hormone abscisic acid (ABA) modulates a number of plant developmental processes and responses to stress. In planta, ABA has been shown to induce reactive oxygen species (ROS) production through the action of plasma membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases. Although quantitative proteomics studies have been performed to identify ABA- or hydrogen peroxide (H₂O₂)-dependent proteins, little is known about the ABA- and H₂O₂-dependent microsomal proteome changes. Here, we examined the effect of 50 µM of either H₂O₂ or ABA on the Arabidopsis microsomal proteome using tandem mass spectrometry and identified 86 specifically H₂O₂-dependent, and 52 specifically ABA-dependent proteins that are differentially expressed. We observed differential accumulation of proteins involved in the tricarboxylic acid (TCA) cycle notably in response to H₂O₂. Of these, aconitase 3 responded to both H₂O₂ and ABA. Additionally, over 30 proteins linked to RNA biology responded significantly to both treatments. Gene ontology categories such as \\'response to stress\\' and \\'transport\\' were enriched, suggesting that H₂O₂ or ABA directly and/or indirectly cause complex and partly overlapping cellular responses. Data are available via ProteomeXchange with identifier PXD006513.

Metabolism and interactions of pesticides in human and animal in vitro hepatic models

OpenAIRE

Abass, K. M. (Khaled M.)

2010-01-01

Abstract Risk assessment of chemicals needs reliable scientific information and one source of information is the characterization of the metabolic fate and toxicokinetics of a chemical. Metabolism is often the most important factor contributing to toxicokinetics. Cytochrome P450 (CYP) enzymes are a superfamily of microsomal proteins playing a pivotal role in xenobiotic metabolism. In the present study, pesticides were used as representative xenobiotics since exposure to pesticides is ...

Nanovaccine for immunotherapy and reduced hepatitis-B virus in humanized model.

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Dewangan, Hitesh Kumar; Pandey, Tarun; Singh, Sanjay

2017-11-27

Chronic Hepatitis B Virus (HBV) infections are severe with weak antiviral immune responses. The lack of an appropriate small animal model for chronic hepatitis, a major hurdle for studying the immunotolerance and immunopathogenesis induced by hepatitis B viral (HBV) infection. In this study, for enhancing the antibody production efficiency the prepared polymeric HBsAg-loaded nanoparticles (nanovaccine) will be tested in immune-deficit mice, which suffer from chronic Hepatitis B virus. Vaccination of Balb/c mice by this prepared nanoparticles that were engrafted with peripheral blood mononuclear cells (PBMCs), which was already lethally irradiated and transplanted by the bone marrow of NOD (knockout mice) mice. In the present study, after the vaccination detected the high frequencies of immunoglobulin G (IgG)-secreting B cells and mitogen-responsive interferon-Y (IFN-Y) secreting T cells in serum, determined by specific ELISA technique. During the entire observation period, unvaccinated animals showed lower concentration of specific IgG secreting B cells and IFN-Y secreting T cells found in comparison to vaccinated mice group. Chronic HBV carrier PBMCs transplanted into the chimera failed to produce antigen and increased the antibodies production due to vaccination. Furthermore, another advantage was that the viral gene expression and viral DNA replication was no longer observed in vaccinated group. This prepared nanovaccine formulations is better for the cure of Hepatitis B viral infection carrier. Therefore, specific memory responses were elicited by vaccination with Hepatitis B virus surface (HBsAg) antigen of chimeric mice transplanted with PBMCs derived from HBV donors.

Carcinogen-Induced Hepatic Tumors in KLF6+/- Mice Recapitulate Aggressive Human Hepatocellular Carcinoma Associated with p53 Pathway Deregulation

NARCIS (Netherlands)

Tarocchi, Mirko; Hannivoort, Rebekka; Hoshida, Yujin; Lee, Ursula E.; Vetter, Diana; Narla, Goutham; Villanueva, Augusto; Oren, Moshe; Llovet, Josep M.; Friedman, Scott L.

Inactivation of KLF6 is common in hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) infection, thereby abrogating its normal antiproliferative activity in liver cells. The aim of the study was to evaluate the impact of KLF6 depletion on human HCC and experimental

Inhibition of the human liver microsomal and human cytochrome P450 1A2 and 3A4 metabolism of estradiol by deployment-related and other chemicals.

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Usmani, Khawja A; Cho, Taehyeon M; Rose, Randy L; Hodgson, Ernest

2006-09-01

Cytochromes P450 (P450s) are major catalysts in the metabolism of xenobiotics and endogenous substrates such as estradiol (E2). It has previously been shown that E2 is predominantly metabolized in humans by CYP1A2 and CYP3A4 with 2-hydroxyestradiol (2-OHE2) the major metabolite. This study examines effects of deployment-related and other chemicals on E2 metabolism by human liver microsomes (HLM) and individual P450 isoforms. Kinetic studies using HLM, CYP3A4, and CYP1A2 showed similar affinities (Km) for E2 with respect to 2-OHE2 production. Vmax and CLint values for HLM are 0.32 nmol/min/mg protein and 7.5 microl/min/mg protein; those for CYP3A4 are 6.9 nmol/min/nmol P450 and 291 microl/min/nmol P450; and those for CYP1A2 are 17.4 nmol/min/nmol P450 and 633 microl/min/nmol P450. Phenotyped HLM use showed that individuals with high levels of CYP1A2 and CYP3A4 have the greatest potential to metabolize E2. Preincubation of HLM with a variety of chemicals, including those used in military deployments, resulted in varying levels of inhibition of E2 metabolism. The greatest inhibition was observed with organophosphorus compounds, including chlorpyrifos and fonofos, with up to 80% inhibition for 2-OHE2 production. Carbaryl, a carbamate pesticide, and naphthalene, a jet fuel component, inhibited ca. 40% of E2 metabolism. Preincubation of CYP1A2 with chlorpyrifos, fonofos, carbaryl, or naphthalene resulted in 96, 59, 84, and 87% inhibition of E2 metabolism, respectively. Preincubation of CYP3A4 with chlorpyrifos, fonofos, deltamethrin, or permethrin resulted in 94, 87, 58, and 37% inhibition of E2 metabolism. Chlorpyrifos inhibition of E2 metabolism is shown to be irreversible.

Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation.

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Chen, Y K; Huang, Anderson H C; Chan, Anthony W S; Lin, L M

2016-06-01

Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen-stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well-differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic-like differentiation with morphological change from a spindle-shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver-specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation-medium culture. Positive immunofluorescence staining of low-density lipoprotein and albumin was observed from day 14 of differentiation-medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic-like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic-like cells. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

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Asuka Hirai-Yuki

2016-12-01

Full Text Available Hepatitis A virus (HAV is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99% progeny virions were released apically from Caco-2 cells, whereas basolateral (64% versus apical (36% release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1−/−Ifngr1−/− and Mavs−/− mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus.

Zebrafish have an ethanol-inducible hepatic 4-nitrophenol hydroxylase that is not CYP2E1-like.

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Hartman, Jessica H; Kozal, Jordan S; Di Giulio, Richard T; Meyer, Joel N

2017-09-01

Zebrafish are an attractive model organism for toxicology; however, an important consideration in translating between species is xenobiotic metabolism/bioactivation. CYP2E1 metabolizes small hydrophobic molecules, e.g. ethanol, cigarette smoke, and diesel exhaust components. CYP2E1 is thought to only be conserved in mammals, but recent reports identified homologous zebrafish cytochrome P450s. Herein, ex vivo biochemical measurements show that unlike mammals, zebrafish possess a low-affinity 4-nitrophenol hydroxylase (K m ∼0.6 mM) in hepatic microsomes and mitochondria that is inducible only 1.5- to 2-fold by ethanol and is insensitive to 4-methylpyrazole inhibition. In closing, we suggest creating improved models to study CYP2E1 in zebrafish. Copyright © 2017 Elsevier B.V. All rights reserved.

Studies on the metabolism of the α-pyrrolidinophenone designer drug methylenedioxy-pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-high-resolution MS and its detectability in urine by GC-MS.

Science.gov (United States)

Meyer, Markus R; Du, Peng; Schuster, Frank; Maurer, Hans H

2010-12-01

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples. Copyright © 2010 John Wiley & Sons, Ltd.

The Cytochrome P450-Mediated Metabolism Alternation of Four Effective Lignans From Schisandra chinensis in Carbon Tetrachloride-Intoxicated Rats and Patients With Advanced Hepatocellular Carcinoma.

Science.gov (United States)

Wu, Rongrong; Xiao, Zhiyong; Zhang, Xiaorui; Liu, Feng; Zhou, Wenxia; Zhang, Yongxiang

2018-01-01

It is highly valuable to study the pharmacokinetics of herbal components under the pathological condition of liver dysfunction for safe and rational use of herbal medicines. In this study, the pharmacokinetic profiles of four effective lignans from Schisandra chinensis (SC) , schisandrin, schisantherin A, deoxyshisandrin and γ-schisandrin, were investigated in carbon tetrachloride (CCl 4 )-intoxicated rats. The metabolism of the four lignans was also studied using microsomes from patients with advanced hepatocellular carcinoma. In situ intestinal and hepatic perfusions were conducted to clarify the contributions from impairments of gut and liver on the pharmacokinetics of the four schisandra lignans in CCl 4 -intoxicated rats. The metabolism in rat and human liver microsomes and transport in Caco-2 monolayer cell model were studied to reveal the key factors for the in vivo disposition of the four lignans. When SC alcoholic extract was orally administrated to CCl 4 -intoxicated rat for a short term (4 days), the pharmacokinetics of four active SC lignans was significantly changed while its hepatotherapeutic effect was not obviously observed. The plasma concentrations of the four schisandra lignans were dramatically elevated compared with the control. The Cmax, AUC and MRT were all increased or prolonged significantly while parameter CLz/F was obviously reduced in rat pretreated with CCl 4 . In hepatic perfusion study and liver microsomes incubation, it was found that the hepatic metabolism of the four lignans was markedly decreased mainly due to the activity reduction of multiple CYP450 isoenzymes involved the metabolism, which, eventually, might lead to the alternation of their pharmacokinetic profiles in CCl 4 -intoxicated rats or patients with advanced hepatocellular carcinoma. The pharmacokinetic studies of SC components in pathological situation of liver dysfunction are expected to provide useful data for rational and safe application of SC preparations in

Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

Science.gov (United States)

Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

1976-03-01

Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

Feature Hepatitis: Hepatitis Can Strike Anyone

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... Navigation Bar Home Current Issue Past Issues Feature Hepatitis Hepatitis Can Strike Anyone Past Issues / Spring 2009 Table ... from all walks of life are affected by hepatitis, especially hepatitis C, the most common form of ...

Molecular status of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus among transgender commercial sex workers in Surakarta, Indonesia

Science.gov (United States)

Prasetyo, Afiono Agung; Sari, Yulia; Dharmawan, Ruben; Marwoto

2017-02-01

Sexual contact and other risk behavior among transgender working as commercial sex workers are important factors for sexual and blood-borne virus (BBV) infections. However, there no data concerning the molecular status of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) circulated among transgender working as commercial sex workers. Blood samples obtained from transgender working as commercial sex workers in Surakarta were examined for HIV antibodies, HBsAg and HCV antibodies, respectively, by immunological assays. All blood samples were also subjected for viral nucleic acid extraction and molecular detection of HIV, HBV and HCV by nested RT-PCR. The PCR products were purified from agarose gels, and the nucleotide sequences were retrieved and molecular analyzed. HIV, HBV and HCV was detected in 26.9% (7/26), 19.2% (5/26) and 46.2% (12/26), respectively. HIV CRF01_AE and B were found to be circulating in the community. HBV genotype B3 predominated, followed by C1. HCV genotype 1a predominated among HCV-infected transgender working as commercial sex workers, followed by 1c, 3a, and 4a. HIV, HBV, and HCV were found circulating in the transgender working as commercial sex workers in Surakarta, Indonesia.

Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

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Arnaud Carpentier

2016-05-01

Full Text Available The establishment of protocols to differentiate human pluripotent stem cells (hPSCs including embryonic (ESC and induced pluripotent (iPSC stem cells into functional hepatocyte-like cells (HLCs creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.

[The effect of monensin, tiamulin and the simultaneous administration of both substances on the microsomal mixed function oxidases and on the peroxide formation in broilers].

Science.gov (United States)

Laczay, P; Simon, F; Lehel, J

1990-09-01

The influence of Monensin, Tiamulin and the simultaneous administration of the two substances on the microsomal, mixed function oxidases was studied on cockerels. Monensin was seen to cause a slight depression in the amount of cytochrome P-450 and cytochrome b5 as well as in the activities of aniline-p-hydroxylase, p-nitrophenol-hydroxylase and p-nitroanisole-O-demethylase. Tiamulin induced a moderate increase in the amount of cytochrome P-450 and in the activities of aniline-p-hydroxylase, p-nitrophenol-hydroxylase and aminopyrine-N-demethylase. The combined administration of monensin and tiamulin resulted in marked induction of the microsomal enzymes; the amount of cytochrome P-450 reduced by metyrapone or carbon monoxide increased 2.5 or 2-times, respectively, and the activities of the tested microsomal hydroxylases and demethylases showed also an expressed increase. At the same time the formation of lipid peroxides also markedly increased and the GSH concentration was reduced. In conclusion, the results of the investigations indicate that the simultaneous application of monensin and tiamulin cause a marked induction of the drug-metabolizing microsomal enzymes and a significant increase in the lipid peroxide formation.

Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry.

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Kachko, Alla; Costafreda, Maria Isabel; Zubkova, Iryna; Jacques, Jerome; Takeda, Kazuyo; Wells, Frances; Kaplan, Gerardo; Major, Marian E

2018-03-15

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81. IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the

Prevalence of Hepatitis A Virus Antibody in Portuguese Travelers: A New Paradigm.

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Rocha, Sónia; Tejo, Sandra; Ferreira, Eugénia; Trindade, Luís; Rabadão, Eduardo; Marques, Nuno; Saraiva da Cunha, José

2017-08-31

In Portugal, the prevalence of hepatitis A virus infection has decreased in the past decades, especially in young adults. The aim of this study was to detect the prevalence of antibody to hepatitis A virus in a population observed in our Travel Clinic. Antibodies against hepatitis A, hepatitis B, hepatitis C and human immunodeficiency virus were tested using standard enzyme immunoassay in patients older than 18. The exclusion criteria were: prior vaccination for hepatitis A virus, previous diagnosis of infection with hepatitis B virus, hepatitis C virus and/or human immunodeficiency virus, foreign travelers and long-term expatriates. We applied an epidemiological survey and data was statistically analyzed with SPSS® 18.0. In the 665 travelers studied, natural immunity to hepatitis A virus was present in 57.6% (n = 383). They were stratified into 8 age groups and for each one hepatitis A immunity was clarified: 5.0% (n = 1) in 18 - 25 years, 32.3% (n = 21) in 26 - 30 years, 40.9% (n = 47) in 31 - 35 years, 45.8% (n = 54) in 36 - 40 years, 68.7% (n = 79) in 41 - 45 years, 70.1% (n = 68) in 46 - 50 years, 80.8% (n = 63) in 51 - 55 years and 87.7% (n = 50) over 56 years old. In those who accepted further screening, positivity for hepatitis B core antibody was found in 0.6% (n = 3) travelers, hepatitis C virus infection in 1.1% (n = 6) and human immunodeficiency virus infection in 0.5% (n = 3) whose previous status was unknown. The most frequent travel destination was sub-Saharan Africa (72.6%; n = 483). We found 49.1% (n = 260) travelers under 50 years old susceptible to hepatitis A virus infection and for those between 40 and 50 years, 30.7% (n = 65) still need vaccine protection. Across age groups there is a trend towards lower prevalence of hepatitis A virus antibody, in particular among youngsters, when compared with older Portuguese studies.

Hepatic Encephalopathy

Medline Plus

Full Text Available ... Disease Type 1 (von Gierke) Hemochromatosis Hepatic Encephalopathy Hepatitis A Hepatitis B Hepatitis C Intrahepatic Cholestasis of Pregnancy ( ... Disease Type 1 (von Gierke) Hemochromatosis Hepatic Encephalopathy Hepatitis A Hepatitis B Hepatitis C Intrahepatic Cholestasis of Pregnancy ( ...

Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.

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Michiko Itoh

Full Text Available Although macrophages are thought to be crucial for the pathogenesis of chronic inflammatory diseases, how they are involved in disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH is poorly understood. Here we report the unique histological structure termed "hepatic crown-like structures (hCLS" in the mouse model of human NASH; melanocortin-4 receptor deficient mice fed a Western diet. In hCLS, CD11c-positive macrophages aggregate to surround hepatocytes with large lipid droplets, which is similar to those described in obese adipose tissue. Histological analysis revealed that hCLS is closely associated with activated fibroblasts and collagen deposition. When treatment with clodronate liposomes effectively depletes macrophages scattered in the liver, with those in hCLS intact, hepatic expression of inflammatory and fibrogenic genes is unaffected, suggesting that hCLS is an important source of inflammation and fibrosis during the progression of NASH. Notably, the number of hCLS is positively correlated with the extent of liver fibrosis. We also observed increased number of hCLS in the liver of non-alcoholic fatty liver disease/NASH patients. Collectively, our data provide evidence that hCLS is involved in the development of hepatic inflammation and fibrosis, thereby suggesting its pathophysiologic role in disease progression from simple steatosis to NASH.

Evaluation of the synergistic effect of Allium sativum, Eugenia jambolana, Momordica charantia, Ocimum sanctum and Psidium guajav on hepatic and intestinal drug metabolizing enzymes in rats

Directory of Open Access Journals (Sweden)

Devendra Kumar

2016-12-01

Full Text Available Aims/Background: Present study investigated the synergistic effect of polyherbal formulations (PHF of Allium sativum L Eugenia jambolana Lam., Momordica charantia L., Ocimum sanctum Linn and Psidium guajava L. in the inhibition/induction of hepatic and intestinal CYPs and Phase-II conjugated drug metabolizing enzymes. Consumption of these herbal remedy has been extensively documented for diabetes treatment in Auyureda. Methodology: PHF of these five herbs was prepared and different doses were orally administered to Sprague Dawley rats of different groups except control group. Expression of mRNA and activity of drug metabolizing enzymes were examined by RT-PCR and HPLC in isolated liver and intestine microsomes in PHF pretreated rats. Results: Activities of hepatic and intestinal Phase-II enzyme levels increased along with mRNA levels except CYP3A mRNA level. PHF administration increases the activity of hepatic and intestinal UDPGT and GST in response to dose and time; however, activity of hepatic SULT increased at higher doses. Conclusions: CYPs and Phase-II conjugated enzymes levels can be modulated in dose and time dependent manner. Observations suggest that poly herbal formulation might be a possible cause of herb-drug interaction, due to changes in pharmacokinetic of crucial CYPs and Phase-II substrate drug. [J Complement Med Res 2016; 5(4.000: 372-382

Prevalence of high-risk human papilloma virus among women with hepatitis C virus before liver transplantation.

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Tarallo, P A; Smolowitz, J; Carriero, D; Tarallo, J; Siegel, A; Jia, H; Emond, J C

2013-08-01

We sought to assess the prevalence and risk factors for high-risk human papillomavirus (HPV) infection among female liver transplant (LT) candidates. Traditional health screening before LT listing has included Pap smear and is typically carried out by the patient's local provider. The prevalence of high-risk HPV in this population has not been studied. With Institutional Review Board approval, 62 LT candidates received a liquid-based Pap smear with high-risk HPV testing as part of their pre-transplant evaluation by a single provider. Clinical variables included age, ethnicity, insurance status, prior Pap smear, and HPV results, HPV risk factors including age of first intercourse, number of lifetime partners, last sexual activity, smoking, birth control pill use, history of sexually transmitted infections, human immunodeficiency virus status, immunosuppressive medication, medical diagnoses, prescribed medications, and history of hepatitis A, B, C, or D. The 62 women had a median age of 56 years, and 39% had high-risk behavior known to be associated with HPV. Ten of 62 patients (16.1%) had high-risk HPV at baseline screening, 5 of whom had atypical cytology. All of the patients who were positive for high-risk HPV had an etiology of hepatitis C virus (HCV) as the underlying cause of liver disease, with the majority (90%) having no history of high-risk behavior for HPV. In contrast, all patients with high-risk behavior who were HCV negative were HPV negative. Fisher's exact test demonstrated a statistically significant relationship between HPV and HCV; odds ratio = 24.4, 95% confidence interval, 1.4, 438.7, P-value = 0.0013. None of the other potential risk factors were associated with HPV in this cohort. In this study, we provide evidence of a strong association between HCV and HPV in LT candidates, which has not been previously reported. HPV positivity was observed in non-sexually active women, suggesting a reactivation of dormant HPV. An association between

Changes induced by gamma radiation in microsomal membranes of storage of garlic; Cambios provocados por la radiacion gamma en membranas microsomales de hoja de reserva de ajo

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Perez, M B; Croci, C A [Universidad Nacional del Sur, Bahia Blanca (Argentina). Laboratorio de Radioisotopos; Aveldano, M I [Universidad Nacional del Sur, Bahia Blanca (Argentina). Instituto de Investigaciones Bioquimicas

2003-07-01

This study evaluates the effects of the radio inhibition process on garlic bulbs in terms of phase properties of microsomal membranes and their lipid and fatty acid composition. Garlic bulbs were irradiated with an average dose of 60 Gy of {sup 60}Co gamma rays 30-40 days after harvest. The treatment was carried out in the facilities of the National Atomic Energy Commission (CNEA). Rough and smooth microsomal membranes were isolated by ultracentrifugation from tissues of irradiated and non-irradiated storage leaves. Wide angle X-ray diffractograms of both fractions were recorded along 270 days of storage. Lipids were separated by thin layer chromatography. The fatty acid composition of major lipid fractions was studied by gas-liquid chromatography. The diffractograms featured peaks at Bragg spacing of 4.15 Armstrong and 3.75 Armstrong, revealing the presence of a gel (crystalline) phase, while the characteristic peak of the liquid-crystalline phase (4.6 Armstrong) was not observed in both sorts of membranes. Irradiation was found to bring about modifications in the intensity of 4.15 Armstrong and 3.75 Armstrong peaks from smooth microsomal membranes, but not in the behaviour along the studied period. Data from the rough microsomal fraction were erratic. Parallel to these changes, radiation induced significant modifications in the level of smooth microsomal membrane triacylglycerols in relation to phospholipids and their fatty acids. These findings indicate that the storage leaf tissues of garlic are radiosensitive both in terms of physical and chemical properties of their microsomal membranes. From the practical point of view, these results could be the basis for the development of techniques to be applied to storage garlic to evaluate if it was irradiated. (author)

Petroleum distillates suppress in vitro metabolic activation: higher [S-9] required in the Salmonella/microsome mutagenicity assay.

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Carver, J H; Machado, M L; MacGregor, J A

1985-01-01

To determine if standard conditions used in the Salmonella/mammalian microsome mutagenicity assay could reliably screen complex petroleum samples, two high-boiling (700-1,070 degrees F) distillates and their separated aromatic fractions were tested. The initial mutagenic activities were inconsistent with the samples' known polyaromatic hydrocarbon (PAH) contents and observed potencies in a dermal carcinogenesis bioassay. A significant mutagenic response was observed only at S-9 concentrations 5 to 10 times higher than those used in the standard assay, supporting the use of elevated levels of S-9 in the Salmonella/microsome assay to assess the carcinogenic potential of petroleum-derived materials. All four samples masked the expected mutagenic activity of added PAHs (benzo[a]pyrene and perylene). Data suggested that petroleum distillates suppress the functional efficacy of the S-9; possible mechanisms are discussed.

The effect of recombinant human growth hormone with or without rosiglitazone on hepatic fat content in HIV-1-infected individuals: a randomized clinical trial.

Science.gov (United States)

Kotler, Donald P; He, Qing; Engelson, Ellen S; Albu, Jeanine B; Glesby, Marshall J

2016-01-01

Hepatic fat is related to insulin resistance (IR) and visceral adipose tissue (VAT) in HIV+ and uninfected individuals. Growth hormone (GH) reduces VAT but increases IR. We evaluated the effects of recombinant human GH (rhGH) and rosiglitazone (Rosi) on hepatic fat in a substudy of a randomized controlled trial. HIV+ subjects with abdominal obesity and IR (QUICKI≤0.33) were randomized to rhGH 3 mg daily, Rosi 4 mg twice daily, the combination or double placebo. Hepatic fat was measured by magnetic resonance spectroscopy, visceral fat by MRI and IR by frequently sampled intravenous glucose tolerance tests at baseline and week 12. 31 subjects were studied at both time points. Significant correlations between hepatic fat and VAT (r=0.41; P=0.02) and QUICKI (r=0.39; P<0.05) were seen at baseline. IR rose with rhGH but not Rosi. When rhGH treatment groups were combined, hepatic fat expressed as percentage change decreased significantly (P<0.05) but did not change in Rosi (P=0.71). There were no correlations between changes in hepatic fat and VAT (P=0.4) or QUICKI (P=0.6). In a substudy of 21 subjects, a trend was noticed between changes in hepatic fat and serum insulin-like growth factor-1 (IGF-1; P=0.09). Hepatic fat correlates significantly with both VAT and IR, but changes in hepatic fat do not correlate with changes in VAT and glucose metabolism. Hepatic fat content is reduced by rhGH but Rosi has no effect. These results suggest an independent effect of GH or IGF-1 on hepatic fat. The study was registered at Clinicaltrials.gov (NCT00130286).