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Sample records for 2-acetylaminofluorene

  1. AM1 study of N-2-acetylaminofluorene bonded to deoxyguanosine at the minor adduct site

    Besson, Morgan

    2003-03-01

    We have computed the total energy as a function of six important torsion angles of the carcinogen N-2-acetylaminofluorene (AAF) bonded to the nitrogen N2 of deoxyguanosine using the semiempirical quantum mechanical method AM1. One global minimum and one local minimum are found separated by a modest barrier. We have computed the normal-mode frequencies of the relevant torsional motions and have determined the rate of conversion between the two minima.

  2. Effects of 2-acetylaminofluorene, dietary fats and antioxidants on nuclear envelope cytochrome P-450

    Carubelli, R.; Graham, S.A.; Griffin, M.J.; McCay, P.B.

    1986-05-01

    The authors reported a marked loss of cytochrome P-450 in hepatic nuclear envelope (NE) but not in microsomes of male Sprague-Dawley rats fed a semipurified diet containing 0.05% w/w 2-acetylaminofluorene (AAF) for 3 weeks. This may reflect loss of NE capacity to detoxify AAF metabolites generated by microsomal P-450. They are now investigating if dietary effects such as progressive decrease in the incidence of AAF-induced tumors in rats fed high polyunsaturated fat diet (HPUF) vs. high saturated fat diet (HSF) vs. low fat diet (LF), and the anticarcinogenic activity of butylated hydroxytoluene (BHT; 0.3% w/w) correlate with preservation of NE P-450. Rats fed AAF HSF (25.6% w/w corn oil) showed marked loss of NE P-450 after 3 weeks; BHT protected against this loss. Rats fed AAF in HSF (25.6% w/w; 18 parts beef tallow + 2 parts corn oil), on the other hand, experienced a marked drop in NE P-450 after 9 weeks; BHT protected against this loss. Comparison of NE P-450 levels in control rats fed HPUF or HSF for 3 weeks with those of rats fed a semipurified diet with 10% fat or Purina chow (ca. 5% fat), support the prediction of an inverse correlation between the levels of dietary fat and the NE P-450 content. Studies on AAF and BHT effects using LF (2% w/w corn oil) are in progress.

  3. Total energy of deoxyguanosine bonded to N-2-acetylaminofluorene by the semiempirical modified-neglect of differential diatomic overlap method

    Besson, Morgan

    2000-03-01

    We have computed the total energy surface as a function of two important torsion angles of the carcinogen N-2-acetylaminofluorene (AAF) bonded to the carbon C8 of deoxyguanosine using the semiempirical quantum mechanical method MNDO. One global minimum and one local minimum are found separated by an appreciable barrier. The equilibrium geometries show the rearrangement of AAF and the base consistent with experimental observations of DNA by previous investigators.

  4. Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene

    Fuchs, Robert P. P.; Schwartz, Nicole; Daune, Michel P.

    1983-01-01

    The covalent binding of an ultimate carcinogen to the DNA bases or phosphate groups creates a premutational lesion that in vivo is processed by the repair, replication and recombination enzymes, and eventually may be converted into a mutation. Being interested in the way that an initial premutational event is converted into a stable heritable mutation, we have sequenced stable mutations in a gene that has formed covalent adducts in vitro with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF, a mo...

  5. Stimulation of recombination between homologous sequences on plasmid DNA and chromosomal DNA in Escherichia coli by N-acetoxy-2-acetylaminofluorene.

    Luisi-DeLuca, C; Porter, R D; Taylor, W. D.

    1984-01-01

    A plasmid containing a wild-type lac operon and a tetracycline-resistance gene was covalently modified by N-acetoxy-2-acetylaminofluorene and used to transform two series of Lac- Escherichia coli cell types. Each set contained wild-type and repair-deficient mutants. One set of cells contained a lacY mutation and the other a deletion of the entire lac operon. Survival and mutagenesis of the plasmid were measured as a function of the N-acetoxy-2-acetylaminofluorene concentration. The results in...

  6. Formation and persistence of DNA adducts from the carcinogen N-hydroxy-2-acetylaminofluorene in rat mammary gland in vivo

    The rat mammary carcinogen, N-hydroxy-2-acetylaminofluorene (N-hydroxy-2-AAF), has been proposed to be metabolically activated by mammary cytosolic N,O-acetyltransferase to a DNA binding species. To test this hypothesis, adult female Sprague-Dawley derived CD rats were treated, i.p., with 4.0 mg/kg [ring-3H]N-hydroxy-2-AAF. After 4 h, 1, 3, 14, and 28 days, the animals were killed, the mammary epithelium DNA was isolated and the carcinogen-deoxyribonucleoside adducts present were analyzed by high pressure liquid chromatography. At each time, only one adduct was detected and it was chromatographically identical to N-(deoxyguanosin-8-yl)-2-aminofluorene. The level of the adduct was maximal at 4 h (1.5 adducts/10(6) nucleotides) and then decreased, following first order kinetics with a t1/2 of 14.2 days. The detection of a single non-acetylated aminofluorene adduct is consistent with N,O-acyltransferase being involved in the metabolic activation of N-hydroxy-2-AAF in the rat mammary gland

  7. Excision repair in ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome, and Bloom's syndrome after treatment with ultraviolet radiation and N-acetoxy-2-acetylaminofluorene

    Excision repair of damage due to ultraviolet radiation, N-acetoxy-2-acetylaminofluorene and a combination of both agents was studied in normal human fibroblasts and various cells from cancer prone patients (ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome and Bloom's syndrome). Three methods giving similar results were used: unscheduled DNA synthesis by radioautography, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and loss of sites sensitive to an ultraviolet endonuclease. All cell lines were proficient in repair of ultraviolet and acetoxy acetylaminofluorene damage and at saturation doses of both agents repair was additive. We interpret these data as indicating that the rate limiting step in excision repair of ultraviolet and acetoxy acetylaminofluorene is different and that there are different enzyme(s) working on incision of both types of damages. (Auth.)

  8. Detection of DNA adducts in N-acetoxy-2-acetylaminofluorene-treated human fibroblasts by means of immunofluorescence microscopy and quantitative immunoautoradiography

    An immunohistochemical procedure was developed for the detection of adducts in DNA of cultured cells exposed to N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) with the use of antibodies raised in rabbits against N-(guanosin-8-yl)-2-acetyl-aminofluorene (Guo-8-AAF) conjugated to bovine serum albumin. Binding of these antibodies to the nuclear DNA was visualized by means of immunofluorescence microscopy with fluorescein-conjugated anti-rabbit-Ig antibodies, or by means of immunoautoradiography with 125I-labeled protein A. The dose-response curve obtained when the number of autoradiographic grains developed over the nuclei was plotted as a function of the concentration of N-AcO-AAF used to treat the cells, indicated that the extent of specific antibody-binding is determined by the amount of adducts in the cells. DNA modification levels allowing for 20% survival of the cells could be detected with the immunofluorescence technique, while cells exposed to concentrations of N-AcO-AAF resulting in 60% survival were still positive with the immunoautoradiographic method

  9. Defective and enhanced postreplication repair in classical and variant xeroderma pigmentosum cells treated with N-acetoxy-2-acetylaminofluorene

    Xeroderma pigmentosum (XP) cells proficient in the excision repair of pyrimidine dimers (XP variants) were also found to be proficient in the excision repair of N-2-acetoxyacetylaminofluorene (AAAF)-induced lesions in their DNA, as assayed by the photolysis of 5-bromodeoxyuridine incorporated during repair. However, the time in which the small segments of newly synthesized DNA, made immediately after treatment of cells with AAAF, were joined together to form DNA of parental size by a process called postreplication repair was long in the XP variant and classical cells. Although increasing doses of AAAF increased the time for making daughter DNA of parental size for variant and classical XP cells, AAAF did not appear to affect this process in normal human cells. Treatment of variant and classical XP cells with a relatively small dose (2.5 μM) of AAAF or 2.5 J/sq m of uv radiation several hr before a 2- to 3-fold-larger dose decreased the time for the pulse-labeled DNA to appear as parental size

  10. The tumor-promoting activity of 2-acetylaminofluorene is associated with disruption of the p53 signaling pathway and the balance between apoptosis and cell proliferation

    The aromatic amine 2-acetylaminofluore (2-AAF) is a powerful complete genotoxic rat liver carcinogen that induces tumors without any additional interventions. While the tumor-initiating genotoxic activity of 2-AAF is well established, its tumor-promotion activity is far less understood. It is believed that the tumor-promoting property of 2-AAF is associated with selective enhancement of cell replication and sustained suppression of apoptosis in initiated cells. In the present study, we investigated the underlying mechanisms of tumor-promoting events induced by 2-AAF-exposure. Male Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 12 and 24 weeks, and the expression pattern of genes associated with the p53-signaling pathway and microRNA genes was determined in the livers of control and 2-AAF-fed rats. The results indicate that the tumor-promoting property of 2-AAF during hepatocarcinogenesis is associated predominantly with the up-regulation of anti-apoptotic growth-related genes and down-regulation of expression of pro-apoptotic genes. This disrupts the balance between cell proliferation and apoptosis, which leads to consequential unrestricted cell proliferation, especially of initiated cells. Also, the long-term-administration of 2-AAF resulted in disruption of regulatory miR-34a-p53 feed-back loop that mediates apoptosis. This was evidenced by an increased expression of miR-34a in response to genotoxic effects of 2-AAF in the absence of p53 up-regulation, and loss of regulatory control of mir-34a on SIRT1 function. Additionally, the livers of 2-AAF-exposed rats were characterized by the substantial deregulation of expression of miR-18, miR-21, miR-182, and miR-200 family, microRNAs involved in control of apoptosis/cell proliferation and cell-cell contact pathways, two major pathways disrupted during the promotion stage of hepatocarcinogenesis

  11. Hepatic Progenitor Cells Contribute to the Progression of 2-Acetylaminofluorene/Carbon Tetrachloride-Induced Cirrhosis via the Non-Canonical Wnt Pathway.

    Jiamei Chen

    Full Text Available Hepatic progenitor cells (HPCs appear to play an important role in chronic liver injury. In this study, cirrhosis was induced in F-344 rats (n = 32 via subcutaneous injection of 50% carbon tetrachloride (CCl4 twice a week for 8 weeks. Then, 30% CCl4 was administered in conjunction with intragastric 2-acetylaminofluorine (2-AAF for 4 weeks to induce activation of HPCs. WB-F344 cells were used to provide direct evidence for differentiation of HPCs to myofibroblasts. The results showed that after administration of 2-AAF, the hydroxyproline content and the expressions of α-SMA, Col I, Col IV, TGF-β1, CD68, TNF-α, CK19 and OV6 were significantly increased. OV6 and α-SMA were largely co-expressed in fibrous septum and the expressions of Wnt5b, frizzled2, frizzled3 and frizzled6 were markedly increased, while β-catenin expression was not statistically different among the different groups. Consistent with the above results, WB-F344 cells, treated with TGF-β1 in vitro, differentiated into myofibroblasts and α-SMA, Col I, Col IV, Wnt5b and frizzled2 expressions were significantly increased, while β-catenin expression was decreased. After blocking the non-canonical Wnt pathway via WIF-1, the Wnt5b level was down regulated, and α-SMA and F-actin expressions were significantly decreased in the WIF-1-treated cells. In conclusion, these results indicate that HPCs appear to differentiate into myofibroblasts and exhibit a profibrotic effect in progressive cirrhosis via activation of the non-canonical Wnt pathway. Blocking the non-canonical Wnt pathway can inhibit the differentiation of HPCs into myofibroblasts, suggesting that blocking this pathway and changing the fate of differentiated HPCs may be a potential treatment for cirrhosis.

  12. Hepatic Progenitor Cells Contribute to the Progression of 2-Acetylaminofluorene/Carbon Tetrachloride-Induced Cirrhosis via the Non-Canonical Wnt Pathway.

    Chen, Jiamei; Zhang, Xiao; Xu, Ying; Li, Xuewei; Ren, Shuang; Zhou, Yaning; Duan, Yuyou; Zern, Mark; Zhang, Hua; Chen, Gaofeng; Liu, Chenghai; Mu, Yongping; Liu, Ping

    2015-01-01

    Hepatic progenitor cells (HPCs) appear to play an important role in chronic liver injury. In this study, cirrhosis was induced in F-344 rats (n = 32) via subcutaneous injection of 50% carbon tetrachloride (CCl4) twice a week for 8 weeks. Then, 30% CCl4 was administered in conjunction with intragastric 2-acetylaminofluorine (2-AAF) for 4 weeks to induce activation of HPCs. WB-F344 cells were used to provide direct evidence for differentiation of HPCs to myofibroblasts. The results showed that after administration of 2-AAF, the hydroxyproline content and the expressions of α-SMA, Col I, Col IV, TGF-β1, CD68, TNF-α, CK19 and OV6 were significantly increased. OV6 and α-SMA were largely co-expressed in fibrous septum and the expressions of Wnt5b, frizzled2, frizzled3 and frizzled6 were markedly increased, while β-catenin expression was not statistically different among the different groups. Consistent with the above results, WB-F344 cells, treated with TGF-β1 in vitro, differentiated into myofibroblasts and α-SMA, Col I, Col IV, Wnt5b and frizzled2 expressions were significantly increased, while β-catenin expression was decreased. After blocking the non-canonical Wnt pathway via WIF-1, the Wnt5b level was down regulated, and α-SMA and F-actin expressions were significantly decreased in the WIF-1-treated cells. In conclusion, these results indicate that HPCs appear to differentiate into myofibroblasts and exhibit a profibrotic effect in progressive cirrhosis via activation of the non-canonical Wnt pathway. Blocking the non-canonical Wnt pathway can inhibit the differentiation of HPCs into myofibroblasts, suggesting that blocking this pathway and changing the fate of differentiated HPCs may be a potential treatment for cirrhosis. PMID:26087010

  13. Host-cell reactivation of uv-irradiated and chemically treated Herpes simplex virus type 1 strain MP in normal and xeroderma pigmentosum skin fibroblasts

    The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated herpes simplex virus type 1 strain mp was studied in normal human skin fibroblasts and xeroderma pigmentosum skin fibroblasts from XP genetic complementation groups A-D and in an XP variant. The increasing relative order for the host-cell reactivation of both types of damaged virus in the different complementation groups is A = D < B < C; XP variant = normal controls. XP complementation group D cells, which manifest the most severe inhibition of her ability for both UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus, can reactivate nitrogen mustard treated HSV-1 mp to the same extent as normal cells. Together, these results indicate that (1) Excision repair of UV and N-acetoxy-2-acetylaminofluorene DNA damaged viruses share a common rate limiting enzymatic step and (2) The repair defect in xeroderma pigmentosum cells plays little or no role in the recovery of nitrogen mustard treated virus. The results of studies on the effect of caffeine on the survival of both UV- and N-acetoxy-2-acetylaminofluorene-treated virus in normal and XP cells imply that the reactivation of HSV-1 mp is mediated by an excision repair process with little if any recovery contributed by post-replication repair mechanisms. The host-cell reactivation of N-acetoxy-2-acetylaminofluorene-treated HSV-1 mp was also correlated with the defective UV-induced unscheduled DNA synthesis in two skin fibroblast strains established from a skin biopsy obtained from each of two juvenile females who had been clinically diagnosed as xeroderma pigmentosum. These findings are discussed in relation to the further characterization of the xeroderma pigmentosum phenotype and their possible utilization for the selection and isolation of new mammalian cell DNA repair mutants

  14. Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen.

    Keler, T; Barker, C. S.; Sorof, S

    1992-01-01

    The hepatic carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) was shown previously to interact specifically with its target protein, liver fatty acid binding protein (L-FABP), early during hepatocarcinogenesis in rats. In search of the significance of the interaction, rat L-FABP cDNA in the sense and antisense orientations was transfected into a subline of the rat hepatoma HTC cell line that did not express L-FABP. After the transfections, the basal doubling times of the cells were no...

  15. Evaluation of Propineb, a dithiocarbamate pesticide, in the mouse-sperm morphology assay.

    Quinto, I; De Marinis, E

    1983-12-01

    Propineb, a dithiocarbamate fungicide, was studied by using the sperm morphology assay in (C57BL6 male X C3H female) F1 mice. At all dose levels, no statistically significant increase in the percentage of sperm abnormalities was observed. Methyl methanesulfonate (MMS) and 2-acetylaminofluorene (2-AAF), which were tested as positive controls, induced a dose-effect-related increase in teratospermia. PMID:6656825

  16. Arylamine-DNA adducts in vitro and in vivo: their role in bacterial mutagenesis and urinary bladder carcinogenesis

    Beland, Frederick A.; Beranek, David T.; Dooley, Kenneth L.; Heflich, Robert H; Kadlubar, Fred F.

    1983-01-01

    Hepatic N-oxidation, followed by N-glucuronidation, has been proposed as a route of metabolic activation for arylamine bladder carcinogens. It is postulated that the N-glucuronides are transported to the bladder lumen where they are hydrolyzed under slightly acidic conditions to release direct-acting carcinogenic and mutagenic N-hydroxyarylamines. In this study, 4-aminobiphenyl (ABP), 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), 2-acetylaminofluorene (AAF), 4-nitrobiphenyl (NBP), benzidine...

  17. Detection of strand breaks in phiX 174 RFI and PM2 DNA reacted with ultimate and proximate carcinogens.

    Thielmann, H W

    1977-10-01

    Supercoiled DNA duplexes of phages phiX 174 and PM2 were treated in aqueous solution at neutral pH with ultimate and proximate carcinogens. Subsequently, the carcinogen-treated phage DNAs were subjected to velocity sedimentation in neutral and alkaline sucrose to quantitative introduction of single strand breaks. Reaction of phage DNA with the ultimate carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr), 7-bromomethyl-benza[a]-anthracene, N-acetoxy-2-acetylaminofluorene [(Ac)2ONFln] and K-region oxides for short periods followed by sedimentation in neutral sucrose gradients led to very few breaks. Incubation with the proximate carcinogens N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene, 7-methyl-, and 7,12-dimethyl-benza[a]anthracene did not result in breaks. However, when the phage DNAs were reacted with the ultimate carcinogens under the same conditions but subsequently alkali-denatured and sedimented in alkaline sucrose gradients, single strand breaks were readily introduced. Incubation with the proximate carcinogens followed by alkali denaturation and sedimentation in alkaline sucrose showed that only 7,12-dimethyl-benz[a]anthracene and, to a minor extent, 7-methyl-benz[]anthracene caused alkali-inducible breaks. The ability of N-methyl-N'-nitro-N-nitrosoguanidine to effect breakdown of superhelical phage DNA in alkali was found enhanced in the presence of N-acetyl-cysteine. PMID:145749

  18. Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen.

    S H Khan; Sorof, S

    1990-01-01

    Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ2, and delta 12-PGJ2, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA2 and delta 12-PGJ2 in primary cul...

  19. Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, xp heterozygotes and normal skin fibroblasts

    The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus. (Auth.)

  20. Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease Pl treatment

    The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo(a)pyrene diol epoxide I, nuclease P1 treatment resulted in a significant reduction in detection of major adducts 1-amino-6-nitropyrene, 1-amino-8-nitropyrene, 2-aminofluorene, 2-naphthylamine and 4-aminobiphenyl modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene modified DNA. These results suggest that at least initially, both modications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitro-aromatics and aromatic amines

  1. Induction of active melanocytes in mouse skin by carcinogens: a new method for detection of skin carcinogens.

    Iwata, K; Inui, N; Takeuchi, T

    1981-01-01

    Application of potent skin carcinogens, such as 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene and 4-nitroquinoline-1-oxide, induced numerous dihydroxyphenylalanine (dopa)-positive cells in the interfollicular epidermis of C57BL/6 mice in a dose- and time-dependent fashion. Chrysene, a weak skin carcinogen, and croton oil, a tumor promoter, also induced 3--4 times more dopa-positive cells than acetone. Liver carcinogens, such as 3'-methyl-4-dimethylaminoazobenzene and N-2-acetylaminofluorene, and non-carcinogenic aromatic hydrocarbons, such as anthracene, fluoranthene, fluorene and pyrene, did not induce increase in these cells. These results indicate that increase in the number of dopa-positive cells after application of chemicals is well correlated with the abilities of these compounds to induce skin carcinogenesis and suppress sebaceous glands. PMID:7273337

  2. Expression and localization of regenerating gene I in a rat liver regeneration model

    Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.

  3. Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy

    Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions necessary to perform multiple sequence localization and signal amplification

  4. Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration

    Jelnes, Peter; Santoni-Rugiu, Eric; Rasmussen, Morten;

    2007-01-01

    The experimental protocols used in the investigation of stem cell-mediated liver regeneration in rodents are characterized by activation of the hepatic stem cell compartment in the canals of Hering followed by transit amplification of oval cells and their subsequent differentiation along hepatic...... the molecular phenotypes of oval cells in several of the most commonly used protocols of stem cell-mediated liver regeneration-namely, treatment with 2-acetylaminofluorene and partial (70%) hepatectomy (AAF/PHx); a choline-deficient, ethionine-supplemented (CDE) diet; a 3,5-diethoxycarbonyl-1...... results delineate remarkable phenotypic discrepancies exhibited by oval cells in stem cell-mediated liver regeneration between rats and mice and underline the importance of careful extrapolation between individual species....

  5. Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease P1 treatment.

    Gallagher, J E; Jackson, M A; George, M H; Lewtas, J; Robertson, I G

    1989-04-01

    The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo[a]pyrene diol epoxide I (BPDE-I), nuclease P1 treatment resulted in a significant reduction in detection of major adducts from 1-amino-6-nitropyrene (1-amino-6-NP), 1-amino-8-nitropyrene (1-amino-8-NP), 2-aminofluorene (2-AF), 2-naphthylamine (2-NA) and 4-aminobiphenyl (4-ABP) modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene (2-AAF) modified DNA. These results suggest that, at least initially, both modifications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitroaromatics and aromatic amines. PMID:2540901

  6. Clinical symptoms and DNA repair characteristics of xeroderma pigmentosum patients from Germany

    Sixty-one xeroderma pigmentosum (XP) patients living in the Federal Republic of Germany were investigated. Clinical symptoms were correlated with DNA repair parameters measured in fibroblasts grown from skin biopsies. Classification according to the international complementation groups revealed that of the 61 patients 3 belonged to group A, 26 to group C, 16 to group D, 3 to group E, and 2 to group F; 11 were of the XP variant type. A striking clinical aspect was the frequency of histogenetically different skin tumors varying from one XP complementation group to the other: squamous and basal cell carcinomas predominated in XP group C; lentigo maligna melanomas were most frequent in group D; basal cell carcinomas occurred preferentially in group E and XP variants. Three DNA repair parameters were determined for 46 fibroblast strains: colony-forming ability (D0); DNA repair synthesis (G0); and DNA-incising capacity (E0). Dose-response experiments with up to 13 dose levels were performed throughout to achieve sufficient experimental accuracy. DNA-damaging treatments included UV light, the 'UV-like' carcinogen N-acetoxy-2-acetylaminofluorene, and the alkylating carcinogens methyl methanesulfonate and N-methyl-N-nitrosourea. Comparison of clinical signs and repair data was made on the basis of D0, G0, and E0 values of both individual cell strains and weighted means of XP complementation groups. Despite considerable clinical and biochemical heterogeneity within complementation groups distinctive features emerged. In general, D0, G0, and E0 values of all XP strains investigated, including XP variants, were found to be reduced upon treatment with UV light or N-acetoxy-2-acetylaminofluorene

  7. Stabile Expression von Sulfotransferasen - allein oder in Kombination mit Cytochrom P450 - in Zelllinien für Mutagenitätsuntersuchungen

    Pabel, Ulrike

    2003-10-01

    Aromatische Amine und Amide (aAA) sind aufgrund ihrer starken Verbreitung in der menschlichen Umwelt und ihres kanzerogenen Potenzials von groer toxikologischer Bedeutung. Die Kanzerogenität der aAA wird durch die Mutagenität hochreaktiver Stoffwechselprodukte vermittelt, die in zwei sequenziellen katalytischen Reaktionen entstehen. Die erste ist meistens eine N-Hydroxylierung, die oft durch Cytochrom P450 1A2 (CYP1A2) katalysiert wird. Daran schliet sich eine O-Konjugation durch Sulfotransferasen (SULT) oder N-Acetyltransferasen (NAT) an. Die Bioaktivierung ist ein kritischer Parameter für die Übertragbarkeit von Ergebnissen aus Tiermodellen auf den Menschen. Rekombinante in vitro Systeme, die fremdstoffmetabolisierende Enzyme verschiedener Spezies exprimieren, ermöglichen die vergleichende Untersuchung der Bioaktivierung im Menschen und in Versuchstieren. Ziel des Projektes war die Aufklärung der Bioaktivierung der aAA durch humane Enzyme. Im Vordergrund stand die Untersuchung der Rolle humaner SULT in diesem Prozess. Es wurden rekombinante in vitro Systeme, konstruiert, die CYP1A2 und SULT des Menschen koexprimieren. SULT-cDNAs wurden in den Säugerzell Expressionsvektor pMPSV kloniert und in Standardindikatorzellen für Mutagenitätsuntersuchungen (V79 Zellen aus dem Chinesischen Hamster) transfiziert. Das Expressionsniveau von CYP1A2 und SULT wurde mittels Immunblotanalyse und radiometrischen Aktivitätsmessungen charakterisiert. In den rekombinanten Zellen wurden vier aAA als Modellsubstanzen (2-Acetylaminofluoren, 2-Aminoanthracen, 3′-Methyl-4-dimethylaminoazobenzol, 2,4-Diaminotoluol) auf ihre Mutagenität am hprt-Locus hin untersucht.Die aAA waren in Zellen, die keine rekombinanten Enzyme oder lediglich CYP1A2 exprimierten, nicht mutagen. In Zellen, die CYP1A2 und SULT der Subfamilie 1A koexprimierten, erzeugten sie bereits in geringen Konzentrationen klare mutagene Effekte (0,3 µM für 2-Acetylaminofluoren und 3′-Methyl-4-dimethylaminoazobenzol

  8. Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells

    Upon exposure to the carcinogens N-acetoxy-N-2-acetylaminofluorene and 7-bromomethyl-benz[a]anthracene, which bind covalently to DNA, ether-permeabilized (nucleotide-permeable) Escherichia coli wild-type cells responded with DNA excision repair. This repair was missing in mutants carrying defects in genes uvrA, uvrB and uvrC, whereas it was present in uvrD and several rec mutants. Enzymic activities involved were identified by measuring repair polymerization and size reduction of denatured DNA. An easily measurable effect in E. coli wild-type cells was carcinogen-induced repair polymerization. When initiated by N-acetoxy-N-2-acetylaminofluorene or 7-bromomethyl-benz[a]anthracene, it depended upton an ATP-requiring step; CTP, GTP or UTP did not substitute for ATP. DNA repair synthesis was inhibited by p-chloromercuribenzoate and quinacrine. In uvrA, uvrB and uvrC mutants no carcinogen-stimulated DNA synthesis could be detected, indicating that steps involved in pyrimidine dimer excision are also involved in chemorepair. In recA, recB and recC mutant cells, repair synthesis was stimulated by the carcinogens to a normal extent. This evidence excludes the ATP-dependent recB,C deoxyribonuclease and recA gene products as playing an important role in carcinogen-induced excision repair. polA1 cells showed drastically reduced levels of repair polymerization, indicating that DNA polymerase I is the main polymerizing enzyme. As determined by DNA size reduction in alkaline sucrose gradients, the arylalkylating carcinogens caused endonucleolytic cleavage of endogenous DNA in wild-type cells. This incision step was most effectively performed in the presence of ATP; UTP, CTP and GTP were only slightly effective. Incision was inhibited by p-chloromercuribenzoate and quinacrine. When exposed to the arylalkylating carcinogens, uvrA, uvrB and uvrC mutant cells did not perform the incision step in the presence of ATP, suggesting the involvement of the respective gene products in the

  9. Enhanced replication of UV-damaged Simian virus 40 DNA in carcinogen-treated mammalian cells

    The replication of UV-damaged Simian virus 40 (SV40) in carcinogen-treated monkey cells has been studied to elucidate the mechanism of carcinogen-enhanced reactivation. Carcinogen enhanced reactivation is the observed increase in UV-irradiated virus survival in host cells treated with low doses of carcinogen compared to UV-irradiated virus survival in untreated hosts. Carcinogen treatment of monkey kidney cells with either N-acetoxy-2-acetylaminofluorene (AAAF) or UV radiation leads to an enhanced capacity to replicate UV-damaged virus during the first round of infection. To further define the mechanism leading to enhanced replication, a detailed biochemical analysis of replication intermediates in carcinogen-treated cells was performed. Several conclusions can be drawn. First enhanced replication can be observed in the first four rounds of replication after UV irradiation of viral templates. The second major finding is that the relaxed circular intermediate model proposed for the replication of UV-damaged templates in untreated cells appears valid for replication of UV-damaged templates in carcinogen-treated cells. Possible mechanisms and the supporting evidence are discussed and future experiments outlined

  10. Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen

    Khan, S.H.; Sorof, S. (Fox Chase Cancer Center, Philadelphia, PA (United States))

    1990-12-01

    Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ{sub 2}, and {Delta}{sup 12}-PGJ{sub 2}, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA{sub 2} and {Delta}{sup 12}-PGJ{sub 2} in primary cultures of purified rat hepatocytes. As a model ligand, ({sup 3}H)PGA{sub 1} bound to L-FABP specifically, reversibly, rapidly, and with high affinity. Its dissociation constants were 134 nM (high affinity) and 3.6 {mu}M (low affinity). The high-affinity finding of ({sup 3}H)PGA{sup 1} correlated with their growth inhibitory activities reported previously and here. The in vitro actions of L-FABP are compatible with those of a specific and dissociable carrier of growth inhibitory prostaglandins in rat hepatocytes and suggest that the carcinogen may usurp the cellular machinery of the growth inhibitory prostaglandins.

  11. The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes

    The in vitro unscheduled DNA synthesis (UDS) assay was evaluated for inclusion in a battery of assays used at The Upjohn Company for evaluation of lead compounds in the development of new and existing drug entities. This evaluation process uncompassed aspects of the isolation of hepatocytes and tests of reference mutagens and genotoxins. The flow rate of perfusion solutions and their temperatures were critical in the isolation of high viability hepatocytes in good yield. The attachment of freshly isolated hepatocytes to coverslips was greatly enhanced by coating the coverslips with type III colagen. Results of testing 12 known genotoxic agents (UV light, cyclophosphamide, 7,12-dimethylbenzanthracene, dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, benzo[a]pyrene, methyl methanesulfonate, ethyl methanesulfonate, N-propyl-N'-nitro-N-nitrosoguanidine, benzidine and 4-aminobiphenyl) were in agreement with the literature. The use of X-ray did not induce unscheduled DNA synthesis in hepatocytes. This latter finding draws attention to the inability of this assay to detect agents which result in 'short-patch' repair of damage. (author). 35 refs.; 8 tabs

  12. Dracocephalum: Novel Anticancer Plant Acting on Liver Cancer Cell Mitochondria

    Mojtaba Talari

    2014-01-01

    Full Text Available Dracocephalum kotschyi Boiss. (Labiatae is a native Iranian medicinal plant which has been used in combination with Peganum harmala L. as a remedy for many forms of human cancer especially leukemia and gastrointestinal malignancies. Hepatocellular carcinoma (HCC is the third leading cause of cancer-related death worldwide. In this investigation HCC was induced by a single intraperitoneal injection of diethylnitrosamine (DEN in corn oil at 200 mg/kg body weight to rats. Two weeks after DEN administration, cancer development was promoted with dietary 2-acetylaminofluorene (2-AAF (0.02%, w/w for 2 weeks. Serum alpha-fetoprotein (AFP concentration, serum alanine transaminase (ALT, aspartate transaminase (AST, and alkaline phosphatase (ALP activities were also determined for confirmation of hepatocellular carcinoma induction. Then rat hepatocytes were isolated with collagen perfusion technique and tumoral hepatocytes were sorted by flow cytometry. Finally isolated mitochondria obtained from both tumoral and nontumoral hepatocytes were used for any probable toxic effect of Dracocephalum kotschyi ethanolic extract. Our results showed that D. kotschyi extract (250 µg/mL induced reactive oxygen species (ROS formation, mitochondrial membrane permeabilization (MMP, and mitochondrial swelling and cytochrome c release only in tumoral but not nontumoral hepatocyte. These findings propose Dracocephalum kotschyi as a promising candidate for future anticancer research.

  13. Differences in the sensitivity of children and adults to carciogenic substances - literature study

    A literature study was undertaken to investigate whether children are more sensitive to carcinogenic effects than adults. This question is especially important for regulatory decisions for situations with shorter than lifetime exposure. Adequate human data is scare except for ionizing radiation, where there is good evidence for a higher sensitivity of children for breast cancer, leukemia and thyroid cancer from epidemiological studies of japanese atom bomb survivors and tumor patients. For chemical substances main evidence comes from animal studies, which show for several substances (e.g. vinyl chloride, nitrosamines, polycyclic aromatic hydrocarbons, 2-acetylaminofluorene, benzidine, aflatoxin B1, cycasin, urethane) high incidences of tumors in the juvenile organisms whereas under comparable exposure conditions low numbers or no tumors were observed in adult animals. All of the mentioned substances are genotoxic carcinogens and mechanistic studies point towards the importance of high cell division rates in target organs of the juvenile organism which in combination with genotoxic activity leads to tumor development. Concerning nongenotoxic carcinogens there are data for saccharin which show that tumor incidence is higher when exposure periods include the period between birth and weaning. For other substances there is negative evidence under similar conditions. In conclusion there is ample evidence for a high sensitivity of the young towards some genotoxic carcinogens and therefore even less than lifetime exposures of children towards these substances may lead to a high carcinogenic risk. (orig.)

  14. Transcriptionally active and inactive genes are similarly modified by chemical carcinogens or X-ray in normal human fibroblasts

    Chemical carcinogens and ionizing radiation induce DNA modifications and strand breaks in cells. This damage is reported to be affected by chromatin proteins or chromatin of a higher structure order. To compare the sensitivity of transcriptionally active and inactive genes on chromatin toward DNA-damaging agents, we treated normal human fibroblasts (WI-38) cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), X-ray, 4-hydroxyaminoquinoline 1-oxide or N-acetoxy-2-acetylaminofluorene, and high molecular weight DNA was isolated. After digestion with EcoRI to completion, the DNA was electrophoresed on an alkaline agarose gel, blotted on a nitrocellulose filter and hybridized with a transcriptionally active gene probe (human type I(α2) procollagen gene) or an inactive gene probe (human β-globin gene). The results show that both genes are similarly modified by these agents. Repair of DNA damage caused by MNNG also occurred similarly in collagen and β-globin genes after removal of MNNG. (Auth.)

  15. UV stimulation of DNA-mediated transformation of human cells

    Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells, which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome

  16. Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen

    Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ2, and Δ12-PGJ2, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA2 and Δ12-PGJ2 in primary cultures of purified rat hepatocytes. As a model ligand, [3H]PGA1 bound to L-FABP specifically, reversibly, rapidly, and with high affinity. Its dissociation constants were 134 nM (high affinity) and 3.6 μM (low affinity). The high-affinity finding of [3H]PGA1 correlated with their growth inhibitory activities reported previously and here. The in vitro actions of L-FABP are compatible with those of a specific and dissociable carrier of growth inhibitory prostaglandins in rat hepatocytes and suggest that the carcinogen may usurp the cellular machinery of the growth inhibitory prostaglandins

  17. The multixenobiotic resistance mechanism in aquatic organisms

    Kurelec, B. (Center for Marine Research Zagreb, Ruder Boskovic Institute, Croatia (Yugoslavia))

    1992-01-01

    Many aquatic organisms thrive and reproduce in polluted waters. This fact indicates that they are well equipped with a defense system(s) against several toxic xenobiotics simultaneously because water pollution is typically caused by a mixture of a number of pollutants. We have found that the biochemical mechanism underlying such multixenobiotic' resistance in freshwater and marine mussel, in several marine sponges, and in freshwater fish is similar to the mechanism of multidrug resistance (MDR) found in tumor cells that became refractory to treatment with a variety of chemotherapeutic agents. All these organisms possess a verapamil-sensitive potential to bind 2-acetylaminofluorene and vincristine onto membrane vesicles. They all express mRNA for mdr1 gene, and mdr1 protein product, the glycoprotein P170. Finally, in in vivo experiments, the accumulation of xenobiotics is enhanced in all investigated organisms in the presence of verapamil, the inhibitor of the P170 extrusion pump. The knowledge that the presence of one xenobiotic may block the pumping out, and hence accelerating accumulation, of others, may help us to understand and interpret our present and past data on different environmental parameters obtained using indicator organisms.99 references.

  18. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    Wei Zhang; Xiao-Ping Chen; Wan-Guang Zhang; Feng Zhang; Shuai Xiang; Han-Hua Dong; Lei Zhang

    2009-01-01

    AIM: To elucidate the interaction between nonparenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover.CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

  19. In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

    Li, Z.; Chen, J. [Liaocheng People' s Hospital, Department of Hepatobiliary Surgery, Liaocheng, Shandong, China, Department of Hepatobiliary Surgery, Liaocheng People’s Hospital, Liaocheng, Shandong (China); Li, L.; Ran, J.H.; Liu, J. [The First People' s Hospital of Kunming, Kunming, Yunnan, China, The First People’s Hospital of Kunming, Kunming, Yunnan (China); Gao, T.X.; Guo, B.Y. [Dongchangfu Hospital of Women and Child Health Care, Liaocheng, Shandong (China); Li, X.H.; Liu, Z.H.; Liu, G.J.; Gao, Y.C.; Zhang, X.L. [Liaocheng People' s Hospital, Department of Hepatobiliary Surgery, Liaocheng, Shandong, China, Department of Hepatobiliary Surgery, Liaocheng People’s Hospital, Liaocheng, Shandong (China)

    2013-07-30

    Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

  20. In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

    Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation

  1. Evaluation of hydro-alcoholic extract of Eclipta alba for its multidrug resistance reversal potential: an in vitro study.

    Chaudhary, Harshita; Jena, Prasant Kumar; Seshadri, Sriram

    2013-01-01

    The development of multidrug resistance (MDR) causes problems in the chemotherapy of human cancer. The present study was designed to evaluate and establish the role of Eclipta alba as MDR reversal agent using multidrug resistant hepatocellular carcinoma cell line (DR-HepG2). To develop DR-HepG2, hepatocellular carcinoma cell line (HepG2) was transfected with 2-Acetylaminofluorene (AAF) and Aflatoxin B1 (AFB). Cytotoxic effects of the Eclipta alba hydroalcoholic extract (EAE) and standard anti-ancer drug Doxorubicin (DOX) were determined in DR-HepG2 and the parental cells HepG2 using MTT assay. The expression level of MDR1 gene and P-glycoprotein (P-gp) level was analyzed by RT-PCR and western blotting. From the present investigation, it was found that EAE (10 and 20 μg/ml) could significantly inhibit cell proliferation in DR-HepG2 whereas DOX (0.5 μg/ml) could not because of enhancement effect of MDR1/P-gp. This study demonstrated for the first time the antiproliferative activities of EAE in multidrug resistant DR-HepG2 cells. The findings revealed that Eclipta alba components are effective inhibitors of MDR1/P-gp. PMID:23859045

  2. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

  3. Hepatic microsomal mixed-function oxidase activity in ethanol-treated hamsters and its consequences on the bioactivation of aromatic amines to mutagens.

    Ioannides, C; Steele, C M

    1986-09-01

    Male golden Syrian hamsters were maintained on ethanol-containing liquid diets for 4 weeks, corresponding to an average daily intake of 17 g/kg body wt. The p-hydroxylation of aniline was markedly enhanced by this treatment while minimal effects were seen in benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities; there was no change in the microsomal levels of cytochromes P-450. Hepatic microsomal preparations from the ethanol-treated hamsters were more efficient than controls fed isocaloric diets in converting 2-aminofluorene, 4-aminobiphenyl, benzidine and 2-acetylaminofluorene into mutagens in the Salmonella mutagenicity test. The same treatment had no effect on the metabolic activation of 2-naphthylamine and even inhibited the mutagenicity of 2-aminoanthracene. No increase was seen in the activation of the two polycyclic aromatic hydrocarbons, benzo[a]pyrene and 3-methylcholanthrene to mutagens and an inhibitory effect was seen with the former. The ethanol-induced increase in the mutagenicity of 2-aminofluorene was inhibited by 2-butanol but not by the hydroxyl radical scavenger dimethylsulphoxide. It is concluded that chronic ethanol ingestion modulates the bioactivation of aromatic amines and amides to mutagens, the effect being substrate dependent. This effect of ethanol may be catalysed by unique form(s) of cytochrome P-450 whose synthesis is induced by such treatment. PMID:3021347

  4. Determination of site selectivity of different carcinogens for preferential mutational hot spots in oligonucleotide fragments by ion-pair reversed-phase nano liquid chromatography tandem mass spectrometry.

    Sharma, Vaneet K; Xiong, Wennan; Glick, James; Vouros, Paul

    2014-01-01

    Ion-pair reversed-phase nano liquid chromatography coupled with nanospray ion trap mass spectrometry was used to investigate site selectivity of the known carcinogens N-acetoxy-2-acetylaminofluorene, N-hydroxy-4-aminobiphenyl and (+/-)-anti-benzo[a]pyrene diol epoxide with the synthetic double-strand 14-mer long oligonucleotide fragment of the p53 gene containing two mutational hot-spot codons (5'-P-ACC155 CGC156 GTC157 CGC158 GC/5'-GCG CGG ACG CGG GT). The investigation was performed using a monolithic polystyrene divinylbenzene capillary column and triethylammonium bicarbonate as an ion-pair reagent. The exact location of the carcinogen on the modified oligonucleotide backbone was determined using characteristic collision-induced dissociation fragmentation patterns obtained under negative-ion mode ionization. In all these cases, the adducted, isomeric oligonucleotides formed were chromatographically resolved and structural identification was performed without any prior deoxyribonucleic acid cleavage or hydrolysis. The knowledge of the site specificity of a carcinogen, especially at purported mutational hot spots, is of paramount importance (1) in establishing the identity of biomarkers for an early risk assessment of the formed DNA adducts, (2) developing repair mechanisms for the formed carcinogen adducted DNA, and (3) understanding the nature of the covalent bond formed and mapping the frequency of the adduction process. PMID:24881456

  5. Differences in the sensitivity of children and adults to carciogenic substances - literature study; Unterschiede in der Empfindlichkeit von Kindern gegenueber krebserzeugenden Stoffen im Vergleich zu Erwachsenen - Literaturstudien

    Schneider, K. [Forschungs- und Beratungsinstitut Gefahrstoffe GmbH (FoBiG), Freiburg (Germany)

    1999-06-01

    A literature study was undertaken to investigate whether children are more sensitive to carcinogenic effects than adults. This question is especially important for regulatory decisions for situations with shorter than lifetime exposure. Adequate human data is scare except for ionizing radiation, where there is good evidence for a higher sensitivity of children for breast cancer, leukemia and thyroid cancer from epidemiological studies of japanese atom bomb survivors and tumor patients. For chemical substances main evidence comes from animal studies, which show for several substances (e.g. vinyl chloride, nitrosamines, polycyclic aromatic hydrocarbons, 2-acetylaminofluorene, benzidine, aflatoxin B1, cycasin, urethane) high incidences of tumors in the juvenile organisms whereas under comparable exposure conditions low numbers or no tumors were observed in adult animals. All of the mentioned substances are genotoxic carcinogens and mechanistic studies point towards the importance of high cell division rates in target organs of the juvenile organism which in combination with genotoxic activity leads to tumor development. Concerning nongenotoxic carcinogens there are data for saccharin which show that tumor incidence is higher when exposure periods include the period between birth and weaning. For other substances there is negative evidence under similar conditions. In conclusion there is ample evidence for a high sensitivity of the young towards some genotoxic carcinogens and therefore even less than lifetime exposures of children towards these substances may lead to a high carcinogenic risk. (orig.) [Deutsch] Im Rahmen einer Literaturstudie wurde der Frage nachgegangen, ob Kinder gegenueber Kanzerogenen im Vergleich zu Erwachsenen eine erhoehte Empfindlichkeit aufweisen. Insbesondere fuer die Bewertung von Situationen mit einer nicht lebenslangen Exposition von Kindern spielt dies eine Rolle. Beim Menschen sind die diesbezueglichen Informationen begrenzt. Nur fuer

  6. Chemically induced immunotoxicity in a medium-term multiorgan bioassay for carcinogenesis with Wistar rats

    A variety of chemicals can adversely affect the immune system and influence tumor development. The modifying potential of chemical carcinogens on the lymphoid organs and cytokine production of rats submitted to a medium-term initiation-promotion bioassay for carcinogenesis was investigated. Male Wistar rats were sequentially initiated with N-nitrosodiethylamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4hydroxybutyl)nitrosamine (BBN), dihydroxy-di-n-propylnitrosamine (DHPN), and 1,2-dimethylhydrazine (DMH) during 4 weeks. Two initiated groups received phenobarbital (PB) or 2-acetylaminofluorene (2-AAF) for 25 weeks and two noninitiated groups received only PB or 2-AAF. A nontreated group was used as control. Lymphohematopoietic organs, liver, kidneys, lung, intestines, and Zymbal's gland were removed for histological analysis. Interleukin (IL)-2, IL-12, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-10, and transforming growth factor beta1 (TGF-β1) levels were determined by ELISA in spleen cell culture supernatants. At the fourth week, exposure to the initiating carcinogens resulted in cell depletion of the thymus, spleen and bone marrow, and impairment of IL-2, IL-12, and IFN-γ production. However, at the 30th week, no important alterations were observed both in lymphoid organs and cytokine production in the different groups. The results indicate that the initiating carcinogens used in the present protocol exert toxic effects on the lymphoid organs and affect the production of cytokines at the initiation step of carcinogenesis. This early and reversible depression of the immune surveillance may contribute to the survival of initiated cells facilitating the development of future neoplasia

  7. Roles of human sulfotransferases in genotoxicity of carcinogens using genetically engineered umu test strains.

    Oda, Yoshimitsu; Zhang, Yu; Buchinger, Sebastian; Reifferscheid, Georg; Yang, Min

    2012-03-01

    Human sulfotransferase (SULT) 1A1, 1A2, and 1A3 cDNA genes were subcloned separately into the pTrc99A(KM) vector. The generated plasmids were introduced into the Salmonella typhimurium O-acetyltransferase-deficient strain NM6000 (TA1538/1,8-DNP/pSK1002), resulting in the new strains NM7001, NM7002, and NM7003. We compared the sensitivities of these three strains with the parental strain NM7000 against 51 chemicals including aromatic amines, nitroarenes, alkenylbenzenes, estrogens-like chemicals, and other compounds with and without S9 mix by making use of the umu test system that is based on the bacterial SOS induction. 2-Amino-6-methyl-dipyrido[1,2-α:3',2'-d]imidazole, 3-methoxy-4-aminoazobenzene, 3-nitrobenzanthrone, 5-nitroacenaphthene, and 3,9-dinitrofluoranthene caused high genotoxicity in the NM7001 strain. The genotoxic effects of 2-aminofluorene, 2-acetylaminofluorene, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-nitrofluorene, 1-nitropyrene, and 2-nitropropane were stronger in the NM7002 strain compared with the NM7001 and NM7003 strains. Among the tested benzylic and allylic compounds, 1-hydroxymethylpyrene was detected in the NM7001 strain with the highest sensitivity. Estragole and 1'-hydroxysafrole exhibited strong genotoxicity in the NM7003 strain. The estrogen-like chemicals such as bisphenol A, genistein, p,n-nonylphenol, and 4-hydroxytamoxifen were not detected as genotoxins in any strain used. Collectively, the present results suggest that the generated test strains are valuable tools in order to elucidate the role of SULT enzymes in the bioactivation of chemicals to environmental carcinogens. PMID:22072630

  8. Effects of dietary pantethine levels on drug-metabolizing system in the liver of rats orally administered varying amounts of autoxidized linoleate.

    Hiramatsu, N; Kishida, T; Natake, M

    1989-08-01

    The effects of dietary pantethine levels on the drug-metabolizing system were investigated under administration of varying amounts of autoxidized linoleate (AL) with rat liver microsomes and S-9 fractions. AL having 800 meq/kg of peroxide value and 1,700 meq/kg of carbonyl value was dosed to the rats of each group given drinking water containing 0 mg% (deficient), 6.25 mg% (normal), and 125 mg% pantethine (sufficient). The contents and activities of the enzymes in the drug-metabolizing system in the rat liver of each pantethine-level group changed essentially in a similar manner, that is, they were induced at an AL daily dose of 0.2 ml/100 g body weight (i.e., small dose) for 5 successive days and lowered at a daily dose of 0.4 ml/100 g body weight (i.e., large dose) by the same administration period, compared with respective non-AL groups in each of the three pantethine levels. In both non-AL and the small-dose AL, enzyme activities of the electron transfer system in rat liver microsomes, aminopyrine-N-demethylase activity, and metabolic activation of 2-acetylaminofluorene in S-9 fractions were significantly higher in the pantethine-deficient group than in the pantethine-normal and -sufficient groups. In the large-dose AL, the enzyme activities in the drug-metabolizing system decreased significantly in any pantethine levels, though the survival rate of the rats was higher in the pantethine-sufficient group than in the pantethine-normal groups. The results suggest that the pantethine relieves the effect of dosed AL on the drug-metabolizing system in rat liver. PMID:2585150

  9. Quantitative mammalian cell mutagenesis and mutagen screening: study with CHO cells

    The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. In all direct-acting chemical mutagens studied, mutation induction increases linearly as a function of the concentration, with no apparent threshold. Some chemicals induce mutation at non-cytotoxic concentrations. The mutagenicity of ethyl methanesulfonate has been quantified as a function of exposure concentration x treatment time. The sensitive and quantitative nature of the system enables studies of the structure-activity (mutagenicity) relationships of various classes of chemicals, including alkylating agents, heterocyclic nitrogen mustards, and platinum compounds. When rat liver S9-mediated metabolic activation is present, procarcinogens such as benzo(a)pyrene, 2-acetylaminofluorene, and dimethylnitrosamine are mutagenic, whereas their noncarcinogenic structural analogues pyrene, fluorene, and dimethylamine are not. The system has been shown to be useful in determining the interactive effects between physical and chemical agents, and in screening for mutagenicity of fractionated organic mixtures and industrial chemicals in both liquid and gaseous state. For the system to be used successfully in routine screening, further studies should be directed toward the development of a metabolic activation system suitable for a broad spectrum of chemicals, a sensitive and reliable statistical method, and an experimental design to determine compounds with low mutagenicity. The system has been expanded for determination of mutagen-induced chromosome aberration, sister-chromatid exchange, and micronucleus formation in addition to gene mutation and cytotoxicity; it can also be used to study inhibition of DNA synthesis

  10. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of [3H]thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity

  11. Crystal structures of human sulfotransferases SULT1B1 and SULT1C1 complexed with the cofactor product adenosine-3'- 5'-diphosphate (PAP)

    Dombrovski, Luidmila; Dong, Aiping; Bochkarev, Alexey; Plotnikov, Alexander N. (Toronto)

    2008-09-17

    Cytosolic sulfotransferases (SULTs), often referred as Phase II enzymes of chemical defense, are a superfamily of enzymes that catalyze the transfer of a sulfonate group from 3{prime}-phosphoadenosine 5{prime}-phosphosulfate (PAPS) to an acceptor group of substrates. This reaction modulates the activities of a large array of small endogenous and foreign chemicals including drugs, toxic compounds, steroid hormones, and neurotransmitters. In some cases, however, SULTs activate certain food and environmental compounds to mutagenenic and carcinogenic metabolites. Twelve human SULTs have been identified, which are partitioned into three families: SULT1, SULT2 and SULT4. The SULT1 family is further divided in four subfamilies, A, B, C, and E, and comprises eight members (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, and 1E1). Despite sequence and structural similarity among the SULTs, the family and subfamily members appear to have different biological function. SULT1 family shows substrate-binding specificity for simple phenols, estradiol, and thyroid hormones, as well as environmental xenobiotics and drugs. Human SULT1B1 is expressed in liver, colon, small intestine, and blood leukocytes, and shows substrate-binding specificity to thyroid hormones and benzylic alcohols. Human SULT1C1 is expressed in the adult stomach, kidney, and thyroid, as well as in fetal kidney and liver. SULT1C1 catalyzes the sulfonation of p-nitrophenol and N-hydroxy-2-acetylaminofluorene in vitro. However, the in vivo function of the enzyme remains unknown. We intend to solve the structures for all of the SULTs for which structural information is not yet available, and compare the structural and functional features of the entire SULT superfamily. Here we report the structures of two members of SULT1 family, SULT1B1 and SULT1C1, both in complex with the product of the PAPS cofactor, adenosine-3{prime}-5{prime}-diphosphate (PAP).

  12. Stellate cells from rat pancreas are stem cells and can contribute to liver regeneration.

    Claus Kordes

    Full Text Available The identity of pancreatic stem/progenitor cells is still under discussion. They were suggested to derive from the pancreatic ductal epithelium and/or islets. Here we report that rat pancreatic stellate cells (PSC, which are thought to contribute to pancreatic fibrosis, have stem cell characteristics. PSC reside in islets and between acini and display a gene expression pattern similar to umbilical cord blood stem cells and mesenchymal stem cells. Cytokine treatment of isolated PSC induced the expression of typical hepatocyte markers. The PSC-derived hepatocyte-like cells expressed endodermal proteins such as bile salt export pump along with the mesodermal protein vimentin. The transplantation of culture-activated PSC from enhanced green fluorescent protein-expressing rats into wild type rats after partial hepatectomy in the presence of 2-acetylaminofluorene revealed that PSC were able to reconstitute large areas of the host liver through differentiation into hepatocytes and cholangiocytes. This developmental fate of transplanted PSC was confirmed by fluorescence in situ hybridization of chromosome Y after gender-mismatched transplantation of male PSC into female rats. Transplanted PSC displayed long-lasting survival, whereas muscle fibroblasts were unable to integrate into the host liver. The differentiation potential of PSC was further verified by the transplantation of clonally expanded PSC. PSC clones maintained the expression of stellate cell and stem cell markers and preserved their differentiation potential, which indicated self-renewal potential of PSC. These findings demonstrate that PSC have stem cell characteristics and can contribute to the regeneration of injured organs through differentiation across tissue boundaries.

  13. Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

    Perrone, Carmen E.; Duan, Jian Dong; Jeffrey, Alan M.; Williams, Gary M. [New York Medical College, Department of Pathology, Valhalla (United States); Ahr, Hans-Juergen; Schmidt, Ulrich [Bayer AG, Institute of Toxicology, Wuppertal (Germany); Enzmann, Harald H. [Federal Institute for Drugs and Medical Devices, Bonn (Germany)

    2004-10-01

    Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovoturkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using {sup 32}P-postlabeling for DNA adducts. In ovoexposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had {sup 32}P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity. (orig.)

  14. mei-9/sup a/ mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair

    The mei-9/sup a/ mutant of Drosophila melanogaster, which reduces meiotic recombination in females, is deficient in the excision of uv-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus, which is specific for pyrimidine dimers, was employed to monitor uv-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10-20 times faster than that from mei-9/sup a/ cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9/sup a/ cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos

  15. Lack of effect of furfural on unscheduled DNA synthesis in the in vivo rat and mouse hepatocyte DNA repair assays and in precision-cut human liver slices.

    Lake, B G; Edwards, A J; Price, R J; Phillips, B J; Renwick, A B; Beamand, J A; Adams, T B

    2001-10-01

    The ability of furfural to induce unscheduled DNA synthesis (UDS) in hepatocytes of male and female B6C3F(1) mice and male F344 rats after in vivo administration and in vitro in precision-cut human liver slices has been studied. Preliminary toxicity studies established the maximum tolerated dose (MTD) of furfural to be 320 and 50 mg/kg in the mouse and rat, respectively. Furfural was dosed by gavage at levels of 0 (control), 50, 175 and 320 mg/kg to male and female mice and 0, 5, 16.7 and 50 mg/kg to male rats. Hepatocytes were isolated by liver perfusion either 2-4 h or 12-16 h after treatment, cultured in medium containing [3H]thymidine for 4 h and assessed for UDS by grain counting of autoradiographs. Furfural treatment did not produce any statistically significant increase or any dose-related effects on UDS in mouse and rat hepatocytes either 2-4 h or 12-16 h after dosing. In contrast, UDS was markedly induced in mice and rats 2-4 h after treatment with 20 mg/kg dimethylnitrosamine and 12-16 h after treatment of mice and rats with 200 mg/kg o-aminoazotoluene and 50 mg/kg 2-acetylaminofluorene (2-AAF), respectively. Precision-cut human liver slices from four donors were cultured for 24 h in medium containing [3H]thymidine and 0-10 mM furfural. Small increases in the net grain count (i.e. nuclear grain count less mean cytoplasmic grain count) observed with 2-10 mM furfural were not due to any increase in the nuclear grain count. Rather, it was the result of concentration-dependent decreases in the mean cytoplasmic grain counts and to a lesser extent in nuclear grain counts, due to furfural-induced cytotoxicity. In contrast, marked increases in UDS (both net grain and nuclear grain counts) were observed in human liver slices treated with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM aflatoxin B(1) and 0.005 and 0.05 mM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. This study demonstrates that furfural does not induce UDS in the hepatocytes of male and female B6C3F

  16. Differences in gene expression profiles in the liver between carcinogenic and non-carcinogenic isomers of compounds given to rats in a 28-day repeat-dose toxicity study.

    Nakayama, Koji; Kawano, Yukiko; Kawakami, Yuuki; Moriwaki, Norichika; Sekijima, Masaru; Otsuka, Masanori; Yakabe, Yoshikuni; Miyaura, Hideki; Saito, Koichi; Sumida, Kayo; Shirai, Tomoyuki

    2006-12-15

    Some compounds have structural isomers of which one is apparently carcinogenic, and the other not. Because of the similarity of their chemical structures, comparisons of their effects can allow gene expression elicited in response to the basic skeletons of the isomers to be disregarded. We compared the gene expression profiles of male Fischer 344 rats administered by daily oral gavage up to 28 days using an in-house oligo microarray. 2-Acetylaminofluorene (2-AAF), 2,4-diaminotoluene (2,4-DAT), 2-nitropropane (2-NP), and 2-nitro-p-phenylenediamine (2-NpP) are hepatocarcinogenic. However, their isomers, 4-acetylaminofluorene (4-AAF), 2,6-diaminotoluene (2,6-DAT), 1-nitropropane (1-NP), and 4-nitro-o-phenylenediamine (4-NoP), are non-hepatocarcinogenic. Because of the limited carcinogenicity of 2-NpP, we attempted to perform two-parametric comparison analyses with (1) a set of 4 isomers: 2-AAF, 2,4-DAT, 2-NP, and 2-NpP as "carcinogenic", and 4-AAF, 2,6-DAT, 1-NP, and 4-NoP as "non-carcinogenic"; and (2) a set of 3 isomers: 2-AAF, 2,4-DAT, and 2-NP, as "carcinogenic", and 4-AAF, 2,6-DAT, and 1-NP as "non-carcinogenic". After ratio filtering and Welch's approximate t-test analysis, 54 and 28 genes were selected from comparisons between the sets of 3 and 4 isomers, respectively, for day 28 data. Using hierarchical clustering analysis with the 54 or 28 genes, 2-AAF, 2,4-DAT, and 2-NP clustered into a "carcinogenic" branch. 2-NpP was in the same cluster as 4-NoP and 4-AAF. This clustering corresponded to the previous finding that 2-NpP is not carcinogenic in male Fischer 344 rats, which indicates that comparing the differences in gene expression elicited by different isomers is an effective method of developing a prediction system for carcinogenicity. PMID:17070881

  17. Insulin-like growth factor binding protein-3 is required for the regulation of rat oval cell proliferation and differentiation in the 2AAF/PHX model

    Nicole C Steiger-Luther

    2010-02-01

    RNA (siRNA treated animals determined that expression of TGFβ family members, including TGF-βRII and Smads 2–4, were significantly downregulated compared to animals at day 9 post-PHx alone or animals that received negative control siRNA. In conclusion, IGFBP-3 may function as a potent chemoattractant of oval cells during specific types of liver regeneration and may be involved in regulating oval cell proliferation and differentiation in vivo via the TGF-β pathway.Keywords: hepatic stem cells, transforming growth factor-beta, N-2-acetylaminofluorene (2AAF, partial hepatectomy (PHx

  18. Comparison of different initiation protocols in the resistant hepatocyte model

    Several models in rat liver have been developed to study multistage carcinogenesis, including the Solt-Farber resistant hepatocyte model. In this model, initiation consists of either a necrogenic dose of a hepatocarcinogen or a non-necrogenic dose in conjunction with partial hepatectomy (PH). As an alternative to PH, we investigated two different procedures: fasting for 96 h followed by refeeding, or the use of one-day-old neonates. Male Fisher 344 rats were injected p.o. with diethylnitrosamine (DEN) (0, 20, or 100 mg/kg) 24 h after refeeding or PH (controls received DEN alone with no proliferative stimulus). For the neonatal group, male and female Fisher 344 rats were treated with DEN (0 or 20 mg/kg, i.p.) at one day of age. All initiated animals were treated at the same age (11 weeks) with the following selection agents: three daily doses of 2-acetylaminofluorene (AAF) (30 mg/kg), followed by a single dose of carbon tetrachloride (2 ml/kg), followed by three additional daily treatments of AAF (30 mg/kg). Rats were euthanized 2 weeks after the last AAF injection. The PH, neonatal male, and neonatal female groups receiving DEN developed more γ-glutamyl transpeptidase (GGT)-positive foci per cubic centimeter and foci per liver as compared to untreated rats receiving the same proliferative stimulus, whereas the fasting/refeeding group and the group receiving no proliferative stimulus did not. All DEN-treated groups receiving one of the proliferative stimuli had more foci per cubic centimeter than the DEN-treated group receiving no proliferative stimulus. The volume fractions of GGT-positive foci in the PH/DEN and neonatal male/DEN groups were higher than those of both the DEN-treated group receiving no proliferative stimulus and the groups receiving the same proliferative stimulus without DEN. In neonatal females-receiving DEN, the volume fraction was not different from either neonatal females not receiving DEN or DEN-treated rats receiving no proliferative

  19. Selective Toxicity of Persian Gulf Sea Cucumber (Holothuria parva and Sponge (Haliclona oculata Methanolic Extracts on Liver Mitochondria Isolated from an Animal Model of Hepatocellular Carcinoma

    Seydi

    2015-12-01

    Full Text Available Background Natural products isolated from marine environments are well known for their pharmacodynamic potential in diverse disease treatments, such as for cancer or inflammatory conditions. Sea cucumbers are marine animals of the phylum Echinoderm and the class Holothuroidea, with leathery skin and gelatinous bodies. Sponges are important components of Persian Gulf animal communities, and the marine sponges of the genus Haliclona have been known to display broad-spectrum biological activity. Many studies have shown that sea cucumbers and sponges contain antioxidants and anti-cancer compounds. Objectives This study was designed to determine the selective toxicity of Persian Gulf sea cucumber (Holothuria parva and sponge (Haliclona oculata methanolic extracts on liver mitochondria isolated from an animal model of hepatocellular carcinoma, as part of a national project that hopes to identify novel potential anticancer candidates among Iranian Persian Gulf flora and fauna. Materials and Methods To induce hepatocarcinogenesis, rats were given diethylnitrosamine (DEN injections (200 mg/kg i.p. by a single dose, and then the cancer was promoted with 2-acetylaminofluorene (2-AAF (0.02 w/w for two weeks. Histopathological evaluations were performed, and levels of liver injury markers and a specific liver cancer marker (alpha-fetoprotein, were determined for confirmation of hepatocellular carcinoma induction. Finally, mitochondria were isolated from cancerous and non-cancerous hepatocytes. Results Our results showed that H. parva methanolic extracts (250, 500, and 1000 µg/mL and H. oculata methanolic extracts (200, 400, and 800 µg/mL increased reactive oxygen species (ROS formation, mitochondrial membrane potential (MMP, mitochondrial swelling, and cytochrome c release in the mitochondria obtained from cancerous hepatocytes, but not in mitochondria obtained from non-cancerous liver hepatocytes. These extracts also induced caspase-3 activation, which is

  20. Genotoxicity screening via the γH2AX by flow assay.

    Smart, D J; Ahmedi, K P; Harvey, J S; Lynch, A M

    2011-10-01

    The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on

  1. Performance of 181 chemicals in a Drosophila assay predominantly monitoring interchromosomal mitotic recombination.

    Vogel, E W; Nivard, M J

    1993-01-01

    An evaluation is presented of the effects of 181 chemicals in the (white/white+) (w/w+) eye mosaic assay, an in vivo short-term test measuring genetic damage in somatic cells of Drosophila after treatment of larvae. The genetic principle of this system is loss of heterozygosity for the wild-type reporter gene w+, an event predominantly resulting from homologous interchromosomal mitotic recombination between the two X chromosomes of female genotypes. The w/w+ eye mosaic test detects a broad spectrum of DNA modifications, since all distinct classes of genotoxins are monitored. Non-DNA-reactive chemicals are in principle not detected by this system. Occasional positive responses obtained for chemicals such as amitrole, ethionine and hexachloeroethane are probably not related to the mechanism responsible for their tumorigenicity. The principle outcome of this analysis is the necessity for classification of responses into three categories. (i) Positive, '++'. The 92 chemicals (Tables II and III) falling into this category were clearly recombinagenic in the assay, meaning that dose-response relations were obtained (or could have been established as was evident from the strong responses obtained at one or two exposure doses). Among the 92 chemicals were 49 promutagens including volatile chemicals such as vinyl bromide and vinyl chloride. (ii) Marginally positive, '+w'. The definition of a weakly positive response is the absence of a dose-response relationship due to the fact that a weak but reproducible effect, in most cases no more than a doubling of the spontaneous clone frequency, is inherently related to toxicity. The 40 chemicals (Tables IV and V) belonging to this category mainly represented four distinct types. (a) Procarcinogens, such as 2-acetylaminofluorene, dibenz[a,h]anthracene, p-dimethylaminoazobenzene, 2-naphthylamine and safrole, for which metabolic conversion was the apparent problem in the assay. (b) Electrophilic chemicals of high nucleophilic