Sample records for rat uterotrophic assay

  1. Comparative activities of p-nonylphenol and diethylstilbestrol in noble rat mammary gland and uterotrophic assays.

    Odum, J; Pyrah, I T; Foster, J R; Van Miller, J P; Joiner, R L; Ashby, J


    Colerangle and Roy (1996, Endocrine 4, 115-122) have described the apparent ability of both diethylstilbestrol (DES) and p-nonylphenol (NP) to cause extensive cell proliferation and lobular development in the mammary glands of young adult Noble rats. The chemicals were administered over 11 days via subcutaneously implanted minipumps. The dose level of DES used (0.076 mg/kg/day) was about 70 times higher than its minimum detection level in rodent uterotrophic and reproductive toxicology studies. In contrast, the lowest active dose level of NP (0.073 mg/kg/day) in the Noble rat mammary gland study was about 600 times lower than its minimum detection level in rat uterotrophic and multigeneration studies. The apparent enhanced sensitivity of the Noble rat mammary gland to the estrogenic activity of NP was considered worthy of further study. Ovariectomized Noble rat uterotrophic assays with NP (minimum detection level approximately 40 mg/kg/day, 3 or 11 days, oral gavage) revealed similar assay sensitivity to that observed for earlier immature and ovariectomized Alderley Park (AP) rat uterotrophic assays of this chemical. The response of the ovariectomized Noble rat uterotrophic assay to DES and estradiol was also as expected from earlier immature AP rat assays. It is concluded that the general sensitivity to estrogens of the Noble rat and the AP rat is similar. A repeat of the Noble rat mammary gland study with DES (11 x 0.076 mg/kg/day) and NP (11 x either 0.073 or 53.2 mg/kg/day), as originally reported by Colerangle and Roy (1996), revealed a strong positive response to DES and no response to NP. It is concluded that the minimum detection level of NP as a weakly estrogenic material in the rat should be based on the results of rat uterotrophic and multigeneration studies and therefore be set at approximately 40 mg/kg/day. It is also concluded that induced S-phase in the rodent mammary gland is best monitored using BRDU, as opposed to PCNA staining, and that use of

  2. The estrogenic effects of apigenin, phloretin and myricetin based on uterotrophic assay in immature Wistar albino rats.

    Barlas, Nurhayat; Özer, Saadet; Karabulut, Gözde


    Chemicals that occur in vegetal food and known as phytoestrogens, because of their structures similarity to estrogen, have benefits on chronic diseases. Despite this, when they are taken at high amounts, they can cause harmful effects on endocrine system of human and animals. In this study, it has been intended to determine the estrogenic potencies of phytoestrogens apigenin, phloretin and myricetin whose affinities for estrogen receptors in vitro. The female rats divided into 17 groups, each containing six rats. There was a negative control group and there were positive control dose groups which contains ethinyl estradiol, ethinyl estradiol+tamoxifen and genistein. The other dose groups which were tested for estrogenic activity contains apigenin, myricetin and phloretin All chemicals have been given to Wistar immature female rats with oral gavage for 3 consecutive days. By using uterotrophic analysis, uterus wet and blotted weights, vaginal opening, uterus length of female rats has been recorded at the end of the experiment. For detect of cell response, luminal epithelium height, gland number and lactoferrin intensity in luminal epithelium of uterus were evaluated. Biochemical analysises in blood were performed. Relative uterus weights of rats in 100 mg/kg/day dose group of myricetin were statistically increased according to vehicle control and positive control groups. In dose groups of apigenin and phloretin it was found that there were cell responses in uterus. All treatment groups had a significant difference in the high intensity of lactoferrin and uterine gland count compared to oil control group. There was no difference between phloretin and apigenin treatment groups in uterine weight statictically. Uterine heights were increased in positive control groups and 100 mg/kg/day dose group of myricetin. Epithelial cell heights were increased in treatment groups except apigenin and phloretin dose groups. There was no difference between all treatment groups in

  3. Usefulness of immature golden hamster (Mesocricetus auratus as a model for uterotrophic assay

    Radko Lidia


    Full Text Available The present study assessed the sensitivity of immature hamster uterotrophic assay to reference oestrogen agonists/antagonists in order to develop a sensitive model for evaluation of endocrine-active compounds in diets. After performing a baseline for control animals, the sensitivity of immature females (postnatal day 18 to reference compounds was evaluated in a three-day uterotrophic assay. The absolute and adjusted dry uterine weights, fold induction over control for absolute wet uterine weight, and wet uterine weight/body weight ratio (% were used as endpoints. The significantly active doses for reference oestrogens were as follows: 0.6 μg/kg for 17α-ethinyloestradiol (s.c.; 1 μg/kg/day (s.c. and 40 μg/kg (p.o. for diethylstilboestrol; 40 mg/kg (s.c. and 160 mg/kg (p.o. for bisphenol A. Co-treatment with tamoxifen at a dose of 1 mg/kg significantly antagonised the uterotrophic effect induced by 1 μg/kg 17α-ethinyloestradiol, and showed the attenuated proliferative effect in histopathological examination. We found immature hamster uterotrophic assay as a sensitive model that could be a good alternative to the rat assay.

  4. Uterotrophic and Hershberber Assays for Acrylonitrile in Rats%丙烯腈类性激素作用的整体动物筛检试验

    王宁; 荣顺兴; 朱菊一; 黄简抒; 周元陵; 金泰廙


    56-day-old ovariectomized rats for 3 days. In some rats, 17β-estradiol (EE) was also injected subcutaneously at a dose of 0.001 mg/kg as a positive control group. For the Hershberger assay, AN was administered orally at doses of 0, 10, 20, 30, 40 and 50 mg/kg to 56-day-old castrated rats for 10 days. 0.4 mg/kg testosterone propionate (TP) was also administered by subcutaneous injection after the administration of AN. In some rats, flutamide (FLU) was also administered orally at a dose of 25 mg/kg as the reference chemical for anti-vandrogenicity. [ Results ] For the uterotrophic assay, there were essentially no differences in the uterine wet or blotted weights between the control and any of the AN-treated groups. For the Hershberger assay,the weights of the accessory sex organs of the castrated rats showed no significant differences between the oil plus TP-treated group and any of the AN plus TP-treated groups. [ Conclusion ] These findings suggest that AN dose not have any endocrine-disrupting properties in in-vivo screening tests.

  5. Uterotrophic assay of two concentrations of migrates from each of 23 polystyrenes administered orally (by gavage) to immature female Wistar rats.

    Bachmann, S; Hellwig, J; Jäckh, R; Christian, M S


    The Styrene Steering Committee (SSC) of the European Chemical Industry Council (CEFIC) sponsored this work to address any concern that styrene dimers and trimers that might migrate from polystyrene containers into food could possess some estrogenic activity and thus possibly affect human health. All phases of the study were conducted in conformance with GLP regulations and without knowledge of the oligomer migrates tested. All activities were managed and audited under a third-party contract between the SSC and Argus International. Low and high doses of the styrene oligomer migrates of 23 polystyrene samples [i.e. 9 general purpose polystyrenes (GPPS), 8 high impact polystyrenes (HIPS) and 6 expandable polystyrenes (EPS)] were tested for estrogenicity in an in vivo uterotrophic assay (immature female rat model). This model is considered to be the "gold standard" for use in screening for estrogenic effects because it evaluates both direct and indirect potential effects. The two concentrations of migrates of each of the 23 polystyrenes tested were selected to simulate daily human consumption of a low and high amount of food. Representative dimer and trimer concentrations were obtained in conformance with EEC Council Directives and calculated to be at levels simulating human consumption of 0.5 or 5 kg of food for the GPPS and the HIPS samples and of 0.5 or 3.15 kg of food for the EPS samples, respectively. The study was conducted in a series of three blocks. Each block included concurrent untreated control (negative control), vehicle control (25% ethanol, 20 ml/kg/day) and positive control (diethylstilbestrol-dipropionate, DES-DP, 5 micrograms/kg/day) groups, and low and high doses of each of 7 (1 block) or 8 (2 blocks) polystyrene oligomer migrates. Each group in each block consisted of 10 immature Wistar (Chbb: THOM-SPF) female rats. Beginning when the rats were 22 +/- 1 days of age, each rat was appropriately handled (untreated control group) or administered twice

  6. Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

    Wang, S.; Aarts, J.M.M.J.G.; Evers, N.M.; Peijnenburg, A.A.C.M.; Rietjens, I.; Bovee, T.F.H.


    Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or

  7. Uterotrophic and Hershberger assays for endocrine disruption properties of plastic food contact materials polypropylene (PP) and polyethylene terephthalate (PET).

    Chung, Bu Young; Kyung, Minji; Lim, Seong Kwang; Choi, Seul Min; Lim, Duck Soo; Kwack, Seung Jun; Kim, Hyung Sik; Lee, Byung-Mu


    Plasticizers or plastic materials such as phthalates, bisphenol-A (BPA), and styrene are widely used in the plastic industry and are suspected endocrine-disrupting chemicals (EDC). Although plastic materials such as polypropylene (PP) and polyethylene terephthalate (PET) are not EDC and are considered to be safe, their potential properties as EDC have not been fully investigated. In this study, plastic samples eluted from plastic food containers (PP or PET) were investigated in Sprague-Dawley rats using Hershberger and uterotrophic assays. In the Hershberger assay, 6-wk-old castrated male rats were orally treated for 10 consecutive days with plastic effluent at 3 different doses (5 ml/kg) or vehicle control (corn oil, 1 ml/100 g) to determine the presence of both anti-androgenic and androgenic effects. Testosterone (0.4 mg/ml/kg) was subcutaneously administered for androgenic evaluation as a positive control, whereas testosterone (0.4 mg/ml/kg) and flutamide (3 mg/kg/day) were administered to a positive control group for anti-androgenic evaluation. The presence of any anti-androgenic or androgenic activities of plastic effluent was not detected. Sex accessory tissues such as ventral prostate or seminal vesicle showed no significant differences in weight between treated and control groups. For the uterotrophic assay, immature female rats were treated with plastic effluent at three different doses (5 ml/kg), with vehicle control (corn oil, 1 ml/100 g), or with ethinyl estradiol (3 μg/kg/d) for 3 d. There were no significant differences between test and control groups in vagina or uterine weight. Data suggest that effluents from plastic food containers do not appear to produce significant adverse effects according to Hershberger and uterotrophic assays.

  8. Lack of oestrogenic effects of food preservatives (parabens) in uterotrophic assays

    Hossaini, A.; Larsen, Jens-Jørgen; Larsen, John Christian


    The oestrogenic activity of the parabens, methyl-, ethyl- and propyl p-hydroxybenzoate, widely used as antimicrobials in food, and butyl p-hydroxybenzoate, which is used in cosmetic products, and their shared main metabolite p-hydroxybenzoic acid was investigated in a mouse uterotrophic assay...

  9. Raw drone milk of honeybees elicits uterotrophic effect in rats: evidence for estrogenic activity.

    Seres, Adrienn B; Ducza, Eszter; Báthori, Mária; Hunyadi, Attila; Béni, Zoltán; Dékány, Miklós; Gáspár, Róbert


    Numerous honeybee products are used in medicine, but the literature furnishes no information concerning the effects of the drone milk (DM), although drone brood, which is similar to DM, was reported to elicit a hormone-like strengthening effect. In certain countries, DM is traditionally used to treat infertility and to promote vitality in both men and women. The aim of this study was to determine the putative estrogen hormone-like effect of raw DM in rats and to identify the effective compounds. Uterotrophic assays revealed that DM increased the relative weight of the immature rat uterus. This effect was confirmed by reverse transcription polymerase chain-reaction and Western blot methods, in which the mRNA and protein expression of the estrogen-dependent peptide complement component C3 was determined. Column chromatography and uterotrophic assays were used to fractionate and check bioactivity, respectively. The active compound after the last fractionation was identified by the nuclear magnetic resonance and mass spectrometry techniques as E-dec-2-enedioic acid, which is very similar to the fatty acids with estrogenic activity that were previously isolated from royal jelly. These results lead us to suppose that E-dec-2-enedioic acid is responsible for the estrogen-like effect of DM. This appears to be the first report on the pharmacological effects of DM and E-dec-2-enedioic acid in mammals.

  10. Relationship between the results of in vitro receptor binding assay to human estrogen receptor alpha and in vivo uterotrophic assay: comparative study with 65 selected chemicals.

    Akahori, Yumi; Nakai, Makoto; Yamasaki, Kanji; Takatsuki, Mineo; Shimohigashi, Yasuyuki; Ohtaki, Masahiro


    For screening chemicals possessing endocrine disrupting potencies, the uterotrophic assay has been placed in a higher level in the OECD testing framework than the ER binding assay to detect ER-mediated activities. However, there are no studies that can demonstrate a clear relationship between these assays. In order to clarify the relationship between the in vitro ER binding and in vivo uterotrophic assays and to determine meaningful binding potency from the ER binding assay, we compared the results from these assays for 65 chemicals spanning a variety of chemicals classes. Under the quantitative comparison between logRBAs (relative binding affinities) and logLEDs (lowest effective doses), the log RBA was well correlated with both logLEDs of estrogenic and anti-estrogenic compounds at r(2)=0.67 (n=28) and 0.79 (n=23), respectively. The RBA of 0.00233% was found to be the lowest ER binding potency to elicit estrogenic or anti-estrogenic activities in the uterotrophic assay, accordingly this value is considered as the detection limit of estrogenic or anti-estrogenic activities in the uterotrophic assay. The usage of this value as cutoff provided the best concordance rate (82%). These findings are useful in a tiered approach for identifying chemicals that have potential to induce ER-mediated effects in vivo.

  11. The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1.

    Kanno, J; Onyon, L; Haseman, J; Fenner-Crisp, P; Ashby, J; Owens, W


    The Organisation for Economic Co-operation and Development has completed the first phase of an international validation program for the rodent uterotrophic bioassay. This uterotrophic bioassay is intended to identify the in vivo activity of compounds that are suspected agonists or antagonists of estrogen. This information could, for example, be used to help prioritize positive compounds for further testing. Using draft protocols, we tested and compared two model systems, the immature female rat and the adult ovariectomized rat. Data from 19 participating laboratories using a high-potency reference agonist, ethinyl estradiol (EE), and an antagonist, ZM 189,154, indicate no substantive performance differences between models. All laboratories and all protocols successfully detected increases in uterine weights using EE in phase 1. These significant uterine weight increases were achieved under a variety of experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). For each protocol, there was generally good agreement among laboratories with regard to the actual EE doses both in producing the first significant increase in uterine weights and achieving the maximum uterine response. Furthermore, the Hill equation appears to model the dose response satisfactorily and indicates general agreement based on calculated effective dose (ED)(10) and ED(50) within and among laboratories. The feasibility of an antagonist assay was also successfully demonstrated. Therefore, both models appear robust, reproducible, and transferable across laboratories for high-potency estrogen agonists such as EE. For the next phase of the OECD validation program, both models will be tested against a battery of weak, partial estrogen agonists. PMID:11564613

  12. A Demonstration of the Uncertainty in Predicting the Estrogenic Activity of Individual Chemicals and Mixtures From an In Vitro Estrogen Receptor Transcriptional Activation Assay (T47D-KBluc) to the In Vivo Uterotrophic Assay Using Oral Exposure.

    Conley, Justin M; Hannas, Bethany R; Furr, Johnathan R; Wilson, Vickie S; Gray, L Earl


    In vitro estrogen receptor assays are valuable tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently unable to fully account for absorption, distribution, metabolism, and excretion. To explore this issue, we calculated relative potency factors (RPF), using 17α-ethinyl estradiol (EE2) as the reference compound, for several chemicals and mixtures in the T47D-KBluc estrogen receptor transactivation assay. In vitro RPFs were used to predict rat oral uterotrophic assay responses for these chemicals and mixtures. EE2, 17β-estradiol (E2), benzyl-butyl phthalate (BBP), bisphenol-A (BPA), bisphenol-AF (BPAF), bisphenol-C (BPC), bisphenol-S (BPS), and methoxychlor (MET) were tested individually, while BPS + MET, BPAF + MET, and BPAF + BPC + BPS + EE2 + MET were tested as equipotent mixtures. In vivo ED50 values for BPA, BPAF, and BPC were accurately predicted using in vitro data; however, E2 was less potent than predicted, BBP was a false positive, and BPS and MET were 76.6 and 368.3-fold more active in vivo than predicted from the in vitro potency, respectively. Further, mixture ED50 values were more accurately predicted by the dose addition model using individual chemical in vivo uterotrophic data (0.7-1.5-fold difference from observed) than in vitro data (1.4-86.8-fold). Overall, these data illustrate the potential for both underestimating and overestimating in vivo potency from predictions made with in vitro data for compounds that undergo substantial disposition following oral administration. Accounting for aspects of toxicokinetics, notably metabolism, in in vitro models will be necessary for accurate in vitro-to-in vivo extrapolations.

  13. The effect of triclosan on the uterotrophic response to extended doses of ethinyl estradiol in the weanling rat

    Triclosan (TCS), a broad-spectrum antimicrobial agent found in many personal care products, has been detected in humans and has been shown to interact with endocrine systems in rats. We previously reported that TCS potentiated the estrogenic effect of ethinyl estradiol (EE) on u...

  14. Uterotrophic effects of benzophenone derivatives and a p-hydroxybenzoate used in ultraviolet screens.

    Koda, Tomoko; Umezu, Toyoshi; Kamata, Ryo; Morohoshi, Kaori; Ohta, Toshiko; Morita, Masatoshi


    Ultraviolet (UV) sunscreen products are popular because of concerns about UV radiation and skin cancer. Unfortunately, some of these products contain agents with estrogenic activity. We used an ovariectomized rat uterotrophic assay to measure the estrogenic activities of 2,4-dihydroxybenzophenone (2,4-DHBP), 2,2',4,4'-tetrahydroxybenzophenone (2,2',4,4'-THBP), and 4-hydroxybenzoic acid isobutyl ester (isobutyl-paraben), which are agents in UV sunscreens, and ethynyl estradiol (EE) and bisphenol A (BPA), which are positive controls. All chemicals increased rat uterine weights. The 10% effective doses (ED10, mg/kg/day) of EE, BPA, 2,4-DHBP, 2,2',4,4'-THBP, and isobutyl-paraben, as determined by Hill equation analysis, where 5E-5, 41.1, 544.6, 33.0, and 230.9, respectively, and their relative potencies against EE were about 1/800,000, 1/10,000,000, 1/600,000, and 1/4,000,000, respectively. Our findings indicated that UV screens contain weak estrogenic compounds.

  15. Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil.

    Maeda, Akihisa; Takahashi, Kei; Tsuchiyama, Hiromi; Oshida, Keiyu


    The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Long-term effects of in utero and lactational exposure to butyl paraben in female rats.

    Guerra, Marina Trevizan; Sanabria, Marciana; Cagliarani, Stephannie Vieira; Leite, Gabriel Adan Araújo; Borges, Cibele Dos Santos; De Grava Kempinas, Wilma


    Parabens are used as preservatives in cosmetic, pharmaceutical, and food industries, and are frequently detected as contaminants in human fluids and tissues. The endocrine disrupting effects of parabens in female rodents include uterotrophic response, steroidogenesis impairment, and ovarian disturbances. The objective of this study was to determine the effects of maternal butyl paraben (BP) exposure on female sexual development. Pregnant Wistar rats were treated subcutaneously with either corn oil or BP at doses of 10, 100, or 200 mg/kg, from gestational day (GD) 12 until GD 20 for female foetal gonad evaluation, and from GD 12 until the end of lactation to evaluate sexual parameters on the female offspring. Immature female rats were also used in the uterotrophic assay to evaluate the possible estrogenic action of parabens. Our results revealed that, in this experimental protocol, BP did not show estrogenic activity at the doses used and did not impair sexual development and fertility capacity in the female rats, but impaired sexual behavior. We conclude that brain sexual development may be more sensitive to BP effects and we speculate that doses higher than 100 mg/kg (the male lowest observed adverse effect level (LOAEL) for rodent reproductive parameters) would be necessary to promote damages in the female reproduction, regarding the same protocol of exposure. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 776-788, 2017.

  17. Calbindin-D9k mRNA expression in the rat uterus following exposure to methoxychlor: a comparison of oral and subcutaneous exposure.

    Shin, Jae-Ho; Moon, Hyun Ju; Kang, Il Hyun; Kim, Tae Sung; Lee, Su Jung; Oh, Ji Young; Lee, Young Joo; Hong, Eui-Ju; Jeung, Eui-Bae; Han, Soon-Young


    Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.

  18. Examining triclosan-induced potentiation of the estrogen uterotrophic effect

    Triclosan (TCS), a widely used antibacterial, has been shown to be an endocrine disruptor. We reported previously that TCS potentiated the estrogenic effect of ethinyl estradiol (EE) on uterine growth in rats orally administered 3 μg/kg EE and TCS (2 to 18 mg/kg) in the utero...

  19. The in vivo rat skin photomicronucleus assay: Phototoxicity and photogenotoxicity evaluation of six fluoroquinolones

    Reus, A.A.; Usta, M.; Kenny, J.D.; Clements, P.J.; Pruimboom-Brees, I.; Aylott, M.; Lynch, A.M.; Krul, C.A.M.


    An in vivo photomicronucleus test (MNT) using rat skin, the target organ for photoirritancy and carcinogenicity, was recently described. The assay was evaluated using fluoroquinolone (FQ) antibiotics with varying degrees of phototoxic potency (i.e. sparflocacin [SPFX], lomefloxacin [LOFX],

  20. Increased DNA damage in blood cells of rat treated with lead as assessed by comet assay

    Mohammad Arif


    Full Text Available A growing body of evidence suggests that oxidative stress is the key player in the pathogenesis of lead-induced toxicity. The present study investigated lead induced oxidative DNA damage, if any in rat blood cells by alkaline comet assay. Lead was administered intraperitoneally to rats at doses of 25, 50 and 100 mg/kg body weight for 5 days consecutively. Blood collected on day six from sacrificed lead-treated rats was used to assess the extent of DNA damage by comet assay which entailed measurement of comet length, olive tail moment, tail DNA (% and tail length. The results showed that treatment with lead significantly increased DNA damage in a dose-dependent manner. Therefore, our data suggests that lead treatment is associated with oxidative stress-induced DNA damage in rat blood cells which could be used as an early bio-marker of lead-toxicity.

  1. In vivo comet assay of acrylonitrile, 9-aminoacridine hydrochloride monohydrate and ethanol in rats.

    Nakagawa, Yuzuki; Toyoizumi, Tomoyasu; Sui, Hajime; Ohta, Ryo; Kumagai, Fumiaki; Usumi, Kenji; Saito, Yoshiaki; Yamakage, Kohji


    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, we examined the ability of acrylonitrile, 9-aminoacridine hydrochloride monohydrate (9-AA), and ethanol to induce DNA damage in the liver and glandular stomach of male rats. Acrylonitrile is a genotoxic carcinogen, 9-AA is a genotoxic non-carcinogen, and ethanol is a non-genotoxic carcinogen. Positive results were obtained in the liver cells of male rats treated with known genotoxic compounds, acrylonitrile and 9-AA.

  2. Prevalidation of the rat CFU-GM assay for in vitro toxicology applications.

    Pessina, Augusto; Bonomi, Arianna; Cavicchini, Loredana; Albella, Beatriz; Cerrato, Laura; Parent-Massin, Dominique; Sibiril, Yann; Parchment, Ralph; Behrsing, Holger; Verderio, Paolo; Pizzamiglio, Sara; Giangreco, Manuela; Baglio, Carolina; Coccè, Valentina; Sisto, Francesca; Gribaldo, Laura


    In vitro haematotoxicity assays are thought to have the potential to significantly reduce and refine the use of animals for haematotoxicity testing. These assays are used successfully in all types of studies - however, their use is not so common in human toxicology studies in the preclinical setting, as they are not required for regulatory testing in this case. Furthermore, these assays could play a key role in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. In previous studies, the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood) and for predicting the in vivo human maximal tolerated dose (MTD) by adjusting in vivo data on mouse toxicity. Recently, a Colony Forming Unit-Megakaryocyte (CFU-MK) assay has also been prevalidated for testing drug toxicity toward megakaryocytes. The rat CFU-GM assay has been used by many researchers for its ability to evaluate in vitro haematotoxicity. Although it is not yet available, a standardised procedure for data comparison could be very important, since the rat is the most widely-used species for the in vivo testing of toxicants. This report presents the results of the prevalidation study developed to analyse the intra-laboratory and inter-laboratory variability of a standardised operating procedure for this assay and its performance for the in vitro determination of the inhibitory concentration (IC) values of drugs on rat myeloid progenitors (CFU-GM). The results demonstrate that the CFU-GM assay can be performed with cryopreserved rat bone-marrow cells (rBMC). The assay represents a useful tool for evaluating the toxicity of a compound, in terms of both relative toxicity (when different molecules are compared) and the prediction of the degree of in vivo toxicity. The use of this assay could greatly reduce the number of rats used in experimental procedures, and

  3. The in vivo rat skin photomicronucleus assay: Phototoxicity and photogenotoxicity evaluation of six fluoroquinolones

    Reus, A.A.; Usta, M.; Kenny, J.D.; Clements, P.J.; Pruimboom-Brees, I.; Aylott, M.; Lynch, A.M.; Krul, C.A.M.


    An in vivo photomicronucleus test (MNT) using rat skin, the target organ for photoirritancy and carcinogenicity, was recently described. The assay was evaluated using fluoroquinolone (FQ) antibiotics with varying degrees of phototoxic potency (i.e. sparflocacin [SPFX], lomefloxacin [LOFX], ciproflox

  4. The Chernoff-Kavlock assay: its validation and application in rats.

    Wickramaratne, G A


    As the rat is the preferred species for teratogenicity studies in our laboratories, the Chernoff-Kavlock assay was modified and validated for use in this species. The Alpk:AP (Wistar-derived) strain, which is also used routinely for toxicology studies, was used, under standard SPF conditions. Twenty-six chemicals have been assayed to demonstrate the utility of the system as a predictor of rat teratogens and to develop a set of rules for interpretation of the results. Routes of chemical administration were largely gavage, with some dermal, subcutaneous, inhalation, and intraperitoneal applications. The use of the assay in five modes to assist the toxicological evaluation or understanding of chemicals is displayed. These modes are 1) as a pre-screen, 2) to compare analogues/homologues, 3) to provide a rapid hazard estimate, 4) to generate a dose-response, and 5) to test a hypothesis.

  5. Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde.

    Kraynak, A R; Barnum, J E; Cunningham, C L; Ng, A; Ykoruk, B A; Bennet, B; Stoffregen, D; Merschman, M; Freeland, E; Galloway, S M


    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery.

  6. Bioavailability of lysine for kittens in overheated casein is underestimated by the rat growth assay method.

    Larsen, J A; Fascetti, A J; Calvert, C C; Rogers, Q R


    Growth assays were performed to determine lysine bioavailability for kittens and rats in untreated and heated casein; these values were compared with estimates obtained with an in vitro method. Body weight, food intake, nitrogen and dry matter digestibility, and plasma lysine were determined during an 80-day growth trial using kittens (n = 16). Body weight and food intake were determined during a 21-day growth trial using weanling rats (n = 80). The growth data showed bioavailable lysine to be 102.4% and 100.2% (for untreated casein) and 66.1% and 51.7% (for heated casein) for kittens and rats, respectively. There was no relationship between plasma lysine and dietary lysine concentrations for kittens. There were no significant differences in nitrogen or dry matter digestibility among diets for kittens. The chemically reactive lysine content of untreated casein was 99.6%, and of heated casein was 67.1%. Heat treatment of casein resulted in significantly decreased lysine bioavailability as estimated by all methods. For untreated casein, both growth assays showed good agreement with the in vitro method for available lysine. For heated casein, the rat growth assay significantly underestimated bioavailable lysine as determined in kittens while the in vitro method closely approximated this value for the cat.

  7. Robust Array-Based Coregulator Binding Assay Predicting ERa-Agonist Potency and Generating Binding Profiles Reflecting Ligand Structure

    Aarts, J.M.M.J.G.; Wang, S.; Houtman, R.; Beuningen, van R.M.G.J.; Westerink, W.M.A.; Waart, van de B.J.; Rietjens, I.M.C.M.; Bovee, T.F.H.


    Testing chemicals for their endocrine-disrupting potential, including interference with estrogen receptor (ER) signaling, is an important aspect of chemical safety testing. Because of the practical drawbacks of animal testing, the development of in vitro alternatives for the uterotrophic assay and o

  8. Detection of gram-negative bacteremia by limulus amebocyte lysate assay: evaluation in a rat model of peritonitis.

    du Moulin, G C; Lynch, S E; Hedley-Whyte, J; Broitman, S A


    A spectrophotometric Limulus amebocyte lysate assay using lysis filtration and centrifugation has been developed for the detection of gram-negative bacteria in blood. The assay is directed at detection of endotoxin in viable and nonviable bacteria present in the blood-stream and not detection of free endotoxin in plasma. The assay was evaluated in a model of peritonitis in which rats were challenged with an inoculum consisting of sterilized human feces, barium sulfate, and one of eight species of bacteria. This assay was able to detect gram-negative bacteremia due to Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, and Klebsiella pneumoniae in the rat model when compared with sham-inoculated uninfected rats. The assay failed to detect bacteremia due to Bacteroides fragilis or Staphylococcus aureus, nor was there a significant rise in absorbance when a pellet containing sterilized feces was implanted in the rat.

  9. Jump-starting urban rat research: Conspecific pheromones recruit wild rats into a behavioral and pathogen-monitoring assay

    Michael H Parsons


    micro-chipped rats, irrespective of sex or age-class, where their individual identities, behaviors and pathogen loads could be consistently recorded. We discuss the potential for similar assays to help address several longstanding knowledge gaps in the literature regarding pathogen monitoring over time and space, rat dispersal patterns, population parameters and seasonal migrations through corridors.

  10. High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes.

    Tuttle, R H; Hall, J E; Tidwell, R R


    Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4'-amidinophenoxy)-3-pentanol and 1,5-di-(4'-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.

  11. Assay interference caused by antibodies reacting with rat kappa light-chain in human sera.

    Degn, Søren E; Andersen, Stig Henrik; Jensen, Lisbeth; Thiel, Steffen; Jensenius, Jens C


    The enzyme-linked immunosorbent assay (ELISA) and its derivatives are powerful tools used in research, in the clinic, and in many other analytical and quality control settings. In general, ELISAs are robust, reproducible and reliable. However, a number of pitfalls of ELISAs have been described over the years. The issue of rheumatoid factor (RF), autoantibodies against the Fc portion of IgG, is well recognized (yet often forgotten), as are problems arising from heterophilic antibodies induced by external antigens that cross-react with self-antigens. A few years ago focus was on human anti-mouse antibodies (HAMA) concomitant with the increased use of mouse monoclonal antibody therapy, a problem that is now diminishing due to development of humanized antibodies. Issues pertaining to food antigens or environmentally encountered antigens are less recognized. We report a recently encountered example of the latter resulting in interference in a solid-phase sandwich assay. Due to the set-up employing a monoclonal rat IgG for capture and a monoclonal rat IgM for development the interference had to be human antibodies reacting with rat light-chain. Out of 102 Danish Caucasian blood donors we found a prevalence of anti-rat kappa light chain antibodies of close to 40% (39/102, defined as at least 2-fold elevated measurements), with around 6% (6/102) having very high levels (defined as at least 4-fold elevated measurements), yielding significantly higher measurements in the assay designed to measure the complement component MAp19 in serum samples. The interference could be blocked by the addition of rat immunoglobulin to the sample buffer. An individual, who had been followed over time, demonstrated a periodic increase of interfering antibodies, highlighting that it is an independently varying parameter and thereby a variable interference in assays. Our results highlight a major pitfall of potential relevance to many sandwich-type assays, as well as an approach to rectify such

  12. Rapid one-step assays for on-site monitoring of mouse and rat urinary allergens.

    Koets, Marjo; Renström, Anne; Zahradnik, Eva; Bogdanovic, Jelena; Wouters, Inge M; van Amerongen, Aart


    Allergy to rodent proteins is common among laboratory animal workers. Sensitive methods to measure exposure to these allergens have been developed. These assays are, however, expensive, time-consuming, and require a laboratory facility and methodological expertise. A simple method to screen for allergen spread, or to test whether hygiene standards are maintained, would be useful. Lateral flow immunoassays (LFIAs) are especially suited for field settings; the tests are simple and results are visible within minutes. LFIAs were developed for detection of the rodent urinary allergens Mus m 1 and Rat n 1. Pilot studies were performed in animal facilities in three countries using both extracts from airborne dust samples and samples collected by wiping surfaces. For comparison and determination of sensitivity, the concentrations of rodent urinary allergens in the samples were also measured using enzyme immunoassays (EIAs). The LFIAs for rat and mouse urinary allergens had a detection limit of 31 pg allergen per mL in a buffer system with purified allergen standards. Results of environmental dust extracts tested in LFIAs correlated well with levels obtained using EIAs. Spread of rodent allergens, or non-adherence to hygiene around laboratory animal facilities, may aggravate rodent allergy. Using a simple, sensitive one-step assay, allergens can be detected to prevent allergen exposure. The results reveal that the rapid assays are suited for on-site demonstration of exposure to rodent allergens, and thus, useful in occupational hygiene practice.

  13. Therapeutic effects of Cassia angustifolia in a cadmium induced hepatotoxicity assay conducted in male albino rats

    Muhammad Tahir Haidry


    Full Text Available The present study aims to investigate the therapeutic effects of Senna plant (Cassia angustifolia L. in a cadmium induced hepatotoxicity assay by evaluating the activity of alanine transaminase (ALT, aspartate transaminase (AST, alkaline phosphatase (ALP and total protein (TP in the albino rats’ serum. A total of 30 white albino rats were taken and divided into three groups; each group comprising ten rats. The group A was taken as a control group; group B was given cadmium chloride concentration of 5 mg/kg (body weight for 42 days; and group C was given cadmium chloride 5 mg/kg body weight for first 21 days and then extract of C. angustifolia 100 mg/kg (body weight was given for remaining 21 days. The analysis were performed twice i.e., on 21st day and 42nd day. Results illustrated that the concentration of cadmium was significantly elevated (P<0.05 at the levels of serum biochemical markers namely ALT, AST, ALP which lowered the protein levels in albino rats. Moreover, treatment with the standard extracts of C. angustifolia observed to reverse the effects of the cadmium significantly (P<0.05. It is concluded that the C. angustifolia had hepatoprotective effects and therapeutic potential against the cadmium induced hepatotoxicity in albino rats.

  14. Therapeutic Effects of Cassia angustifolia in a Cadmium Induced Hepatotoxicity Assay Conducted in Male Albino Rats

    Muhammad Tahir Haidry


    Full Text Available The present study aims to investigate the therapeutic effects of Senna plant (Cassia angustifolia L. in a cadmium-induced hepatotoxicity assay by evaluating the activity of alanine transaminase (ALT, aspartate transaminase (AST, alkaline phosphatase (ALP and total protein (TP in the albino rats’ serum. A total of 30 white albino rats were taken and divided into three groups; each group comprising ten rats. The group A was taken as a control group; group B was given cadmium chloride concentration of 5 mg/kg (body weight for 42 days; and group C was given cadmium chloride 5 mg/kg body weight for first 21 days and then extract of C. angustifolia 100 mg/kg (body weight was given for remaining 21 days. The analysis were performed twice i.e., on 21stst day and 42nd day. Results illustrated that the concentration of cadmium was significantly elevated (P<0.05 at the levels of serum biochemical markers namely ALT, AST, ALP which lowered the protein levels in albino rats. Moreover, treatment with the standard extracts of C. angustifolia observed to reverse the effects of the cadmium significantly (P<0.05. It is concluded that the C. angustifolia had hepatoprotective effects and therapeutic potential against the cadmium-induced hepatotoxicity in albino rats.

  15. A novel operant-based behavioral assay of mechanical allodynia in the orofacial region of rats.

    Rohrs, Eric L; Kloefkorn, Heidi E; Lakes, Emily H; Jacobs, Brittany Y; Neubert, John K; Caudle, Robert M; Allen, Kyle D


    Detecting behaviors related to orofacial pain in rodent models often relies on subjective investigator grades or methods that place the animal in a stressful environment. In this study, an operant-based behavioral assay is presented for the assessment of orofacial tactile sensitivity in the rat. In the testing chamber, rats are provided access to a sweetened condensed milk bottle; however, a 360° array of stainless steel wire loops impedes access. To receive the reward, an animal must engage the wires across the orofacial region. Contact with the bottle triggers a motor, requiring the animal to accept increasing pressure on the face during the test. To evaluate this approach, tolerated bottle distance was measured for 10 hairless Sprague Dawley rats at baseline and 30 min after application of capsaicin cream (0.1%) to the face. The experiment was repeated to evaluate the ability of morphine to reverse this effect. The application of capsaicin cream reduced tolerated bottle distance measures relative to baseline (porofacial sensitivity is presented using an operant system. This operant device allows for consistent measurement of heightened tactile sensitivity in the orofacial regions of the rat. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. An in vitro clonogenic assay to assess radiation damage in rat CNS glial progenitor cells

    Maazen, R.W.M. van der; Verhagen, I.; Kogel, A.J. van der (Katholieke Univ., Nijmegen (Netherlands). Inst. of Radiotherapy)


    Normal glial progenitor cells can be isolated from the rat central nervous system (CNS) and cultured in vitro on a monolayer of type-1 astrocytes. These monolayers are able to support and stimulate explanted glial progenitor cells to proliferate. Employing these in vitro interactions of specific glial cell types, an in vivo-in vitro clonogenic assay has been developed. This method offers the possibility to study the intrinsic radiosensitivity, repair and regeneration of glial progenitor cells after in vitro or in vivo irradiation. (author).

  17. Binding of TH-iloprost to rat gastric mucosa: a pitfall in performing radioligand binding assays

    Beinborn, M.; Kromer, W.; Staar, U.; Sewing, K.F.


    Binding of TH-iloprost was studied in a 20,000 x g sediment of the rat gastric mucosa. When pH in both test tubes for total and non-specific binding was kept identical, no displaceable binding of iloprost could be detected. When no care was taken to keep the pH identical in corresponding test tubes of the binding assay, changes in pH simulated specific and displaceable binding of iloprost. Therefore it is concluded that - in contrast to earlier reports - it is not possible to demonstrate specific iloprost binding using the given method.

  18. Effects of seven chemicals on DNA damage in the rat urinary bladder: a comet assay study.

    Wada, Kunio; Yoshida, Toshinori; Takahashi, Naofumi; Matsumoto, Kyomu


    The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained.

  19. High-performance liquid chromatographic assay for metamizol metabolites in rat plasma: application to pharmacokinetic studies.

    Domínguez-Ramírez, Adriana Miriam; Calzadilla, Patricia Carrillo; Cortés-Arroyo, Alma Rosa; Hurtado Y de la Peña, Marcela; López, José Raúl Medina; Gómez-Hernández, Martín; López-Muñoz, Francisco Javier


    In order to evaluate the pharmacokinetics of metamizol in the presence of morphine in arthritic rats, after subcutaneous administration of the drugs, an easy, rapid, sensitive and selective analytical method was proposed and validated. The four main metamizol metabolites (4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine) were extracted from plasma samples (50-100μl) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography with diode-array detection. Standard calibration graphs for all metabolites were linear within a range of 1-100μg/ml (r(2)≥0.99). The intra-day coefficients of variation (CV) were in the range of 1.3-8.4% and the inter-day CV ranged from 1.5 to 8.4%. The intra-day assay accuracy was in the range of 0.6-9.6% and the inter-day assay accuracy ranged from 0.9 to 7.5% of relative error. The lower limit of quantification was 1μg/ml for all metabolites using a plasma sample of 100μl. Plasma samples were stable at least for 4 weeks at -20°C. This method was found to be suitable for studying metamizol metabolites pharmacokinetics in arthritic rats, after simultaneous administration of metamizol and morphine, in single dose. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Development of a streamlined rat whole embryo culture assay for classifying teratogenic potential of pharmaceutical compounds.

    Zhang, Cindy; Cao, June; Kenyon, James R; Panzica-Kelly, Julieta M; Gong, Lei; Augustine-Rauch, Karen


    This study describes a novel rat whole embryo culture (rWEC) teratogenicity assay that applies a simplified experimental design and statistical prediction model, resulting in reduced animal requirements and increased throughput with low prediction error rate for classifying teratogenic potential of compounds. A total of 70 compounds (38 teratogens and 32 nonteratogens) were evaluated, and the prediction model was generated from a dataset of 59 compounds. The rWEC assay requires only one test concentration (1μM) and three structural endpoints (group average morphological scores of spinal cord, heart, and number of somite pairs), which are used in a recursive partition model for classifying teratogenic liability. The model fitting concordance between the WEC assay and in vivo outcome was 83% with a standard deviation (SD) of 4.9%. The predictivity for future compounds was evaluated by using two statistical methods. Fivefold cross-validation estimated the predictivity of this model at 73% (SD 5.8%). A second estimation of predictivity was obtained from an independent test set of 11 compounds that were not used to build the prediction model and reached 82% (SD 11.6%). The overall estimate for prediction concordance is 74% (SD 5.2%). There is no statistically significant difference (p value > 0.50) in the predictivity between this model and the model supporting European Center for the Validation of Alternative Methods WEC assay with predictivity of 80% (SD 10.6%). Overall, the streamlined WEC assay is estimated to reduce animal use and operational costs by more than 50%. It substantially improves results turnaround with no loss of predictivity.

  1. Comet assay evaluation of six chemicals of known genotoxic potential in rats.

    Hobbs, Cheryl A; Recio, Leslie; Streicker, Michael; Boyle, Molly H; Tanaka, Jin; Shiga, Atsushi; Witt, Kristine L


    As a part of an international validation of the in vivo rat alkaline comet assay (comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomach of 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogens tested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidine produced an equivocal result in liver and negative results in stomach, and sodium arsenite was negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxic response. Sodium chloride, a non-genotoxic non-carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogens produced clearly positive results in the comet assay, the results obtained for o-anisidine and sodium arsenite in liver and stomach cells are consistent with the known mode of genotoxicity and tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxic mode-of-action and target organ carcinogenicity of 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assay validation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals.

  2. Effect of daily treatment with Thai herb, Kaempferia parviflora, in Hershberger assay using castrated immature rats.

    Trisomboon, Hataitip; Watanabe, Gen; Wetchasit, Phongphat; Taya, Kazuyoshi


    The aim of the present study was to investigate the testosterone-like effect of Kaempferia parviflora (KP). Castrated immature rats were randomized and divided into two groups (control and KP-treatment groups). The rats (n=7-8) were treated daily for 5 days by oral route with water in the control group and 1,000 mg/kg of KP in the treatment group. All rats were decapitated 24 h after their last dose and then blood samples were collected for assay of serum FSH, LH, testosterone, progesterone and corticosterone levels. The seminal vesicles plus coagulating glands, ventral prostate, levator ani muscle plus bulbocavernosus muscle, glans penis, kidneys and the adrenal glands were collected and weighed for organ wet weight. Body weight and weight of food intake were recorded throughout the study period. The results show that relative body weight gain in the KP-treatment group was significantly increased 24 and 48 h after the first dose (P<0.05) and then was indistinguishable from the control group. There were no significant differences in the relative reproductive and non-reproductive organ weights between the groups, although all organ weights, except for the glans penis, tended to increase in the KP-treatment group. The serum testosterone levels were significantly increased in the KP-treatment group. There were no significant differences in the serum FSH, LH, progesterone, or corticosterone levels between the groups, even though the serum progesterone level tended to increase and serum LH level tended to decrease in the KP-treatment group. The present study indicates that KP has no testosterone-like effect on reproduction in male rats.

  3. Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays.

    Bolle, P; Mastrangelo, S; Perrone, F; Evandri, M G


    Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity. This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays. In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor alpha, with a lower potency but comparable efficacy to that of 17 beta-estradiol. Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F(2alpha,) and bradykinin than tissues obtained from estradiol valerate treated rats. The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F(2alpha,) and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 beta-estradiol. Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.

  4. High Performance Liquid Chromatographic Assay for the Simultaneous Determination of Posaconazole and Vincristine in Rat Plasma

    Hadeel A. Khalil


    Full Text Available Purpose. Developing a validated HPLC-DAD method for simultaneous determination of posaconazole (PSZ and vincristine (VCR in rat plasma. Methods. PSZ, VCR, and itraconazole (ITZ were extracted from 200 μL plasma using diethyl ether in the presence of 0.1 M sodium hydroxide solution. The organic layer was evaporated in vacuo and dried residue was reconstituted and injected through HC-C18 (4.6 × 250 mm, 5 μm column. In the mobile phase, acetonitrile and 0.015 M potassium dihydrogen orthophosphate (30 : 70 to 80 : 20, linear gradient over 7 minutes pumped at 1.5 mL/min. VCR and PSZ were measured at 220 and 262 nm, respectively. Two Sprague Dawley rats were orally dosed PSZ followed by iv dosing of VCR and serial blood sampling was performed. Results. VCR, PSZ, and ITZ were successfully separated within 11 min. Calibration curves were linear over the range of 50–5000 ng/mL for both drugs. The CV% and % error of the mean were ≤18% and limit of quantitation was 50 ng/mL for both drugs. Rat plasma concentrations of PSZ and VCR were simultaneously measured up to 72 h and their calculated pharmacokinetics parameters were comparable to the literature. Conclusion. The assay was validated as per ICH guidelines and is appropriate for pharmacokinetics drug-drug interaction studies.

  5. Reference control data obtained from an in vivo comet-micronucleus combination assay using Sprague Dawley rats.

    Kasamoto, Sawako; Masumori, Shoji; Tanaka, Jin; Ueda, Maya; Fukumuro, Masahito; Nagai, Miho; Yamate, Jyoji; Hayashi, Makoto


    According to the International Conference on Harmonization Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (ICH S2(R1)), a positive response in any in vitro assay necessitates additional in vivo test(s) (other tissue/endpoint) in addition to the erythrocyte micronucleus test when Option 1 of the test battery is selected. When Option 2 of the test battery is selected, a bacterial gene mutation test and two in vivo tests with different tissues/endpoint are required. The in vivo alkaline comet assay is recommended as the second in vivo test because it can detect a broad spectrum of DNA damage in any tissue and can be combined with the erythrocyte micronucleus test. Considering animal welfare, a combination assay is preferable to an individual assay. Thus, we validated the protocol for the in vivo comet-micronucleus combination assay in rats with three daily administrations and determined the dose of the positive control (ethyl methanesulfonate; EMS, 200mg/kg/day). We also collected the negative control (vehicle) and positive control (EMS) data from the comet (liver, stomach, and kidney) and micronucleus (bone marrow) combination assay using male Sprague Dawley (SD) rats. The negative control data were comparable to our historical control data obtained from stand-alone assays. The positive control data showed clear and consistent positive responses in both endpoints.

  6. Ethanolic extract of dandelion (Taraxacum mongolicum) induces estrogenic activity in MCF-7 cells and immature rats.

    Oh, Seung Min; Kim, Ha Ryong; Park, Yong Joo; Lee, Yong Hwa; Chung, Kyu Hyuck


    Plants of the genus Taraxacum, commonly known as dandelions, are used to treat breast cancer in traditional folk medicine. However, their use has mainly been based on empirical findings without sufficient scientific evidence. Therefore, we hypothesized that dandelions would behave as a Selective estrogen receptor modulator (SERM) and be effective as hormone replacement therapy (HRT) in the postmenopausal women. In the present study, in vitro assay systems, including cell proliferation assay, reporter gene assay, and RT-PCR to evaluate the mRNA expression of estrogen-related genes (pS2 and progesterone receptor, PR), were performed in human breast cancer cells. Dandelion ethanol extract (DEE) significantly increased cell proliferation and estrogen response element (ERE)-driven luciferase activity. DEE significantly induced the expression of estrogen related genes such as pS2 and PR, which was inhibited by tamoxifen at 1 μmol·L(-1). These results indicated that DEE could induce estrogenic activities mediated by a classical estrogen receptor pathway. In addition, immature rat uterotrophic assay was carried out to identify estrogenic activity of DEE in vivo. The lowest concentration of DEE slightly increased the uterine wet weight, but there was no significant effect with the highest concentration of DEE. The results demonstrate the potential estrogenic activities of DEE, providing scientific evidence supporting their use in traditional medicine.

  7. Evaluation of 4,4'-diaminodiphenyl ether in the rat comet assay: Part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of in vivo rat alkaline comet assay.

    Priestley, Catherine C; Walker, Joanne S; O'Donovan, Michael R; Doherty, Ann T


    As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, 4,4'-diaminodiphenyl ether (DPE), a known rodent genotoxic carcinogen, was tested in this laboratory. Sprague Dawley rats (7-9 weeks of age) were given three oral doses of DPE, 24 and 21 h apart and liver or stomach sampled 3h after the final dose. Under the conditions of the test, no increases in DNA damage in liver and stomach were observed with DPE (up to 200 mg/kg/day). A dose-dependent decrease in DNA migration, compared to vehicle controls, was noted for DPE in rat stomach. Further analysis is required to elucidate fully whether this decrease is a consequence of the mode of action or due to the toxicity of DPE. What is perhaps surprising is the inability of the comet assay to detect a known rat genotoxic carcinogen in liver. Further investigation is needed to clarify whether this apparent lack of response results from limited tissue exposure or metabolic differences between species. This finding highlights a need for careful consideration of study design when evaluating assay performance as a measure of in vivo genotoxicity.

  8. Sleep Deprivation Alters Rat Ventral Prostate Morphology, Leading to Glandular Atrophy: A Microscopic Study Contrasted with the Hormonal Assays

    Daniel P. Venâncio


    Full Text Available We investigated the effect of 96 h paradoxical sleep deprivation (PSD and 21-day sleep restriction (SR on prostate morphology using stereological assays in male rats. After euthanasia, the rat ventral prostate was removed, weighed, and prepared for conventional light microscopy. Microscopic analysis of the prostate reveals that morphology of this gland was altered after 96 h of PSD and 21 days of SR, with the most important alterations occurring in the epithelium and stroma in the course of both procedures compared with the control group. Both 96 h PSD and 21-day SR rats showed lower serum testosterone and higher corticosterone levels than control rats. The significance of our result referring to the sleep deprivation was responsible for deep morphological alterations in ventral prostate tissue, like to castration microscopic modifications. This result is due to the marked alterations in hormonal status caused by PSD and SR.

  9. The Application of a Highly Purified Rat Leydig Cell Assay as a Complement to the H295R Steroidogenesis Assay for the Evaluation of Toxicant Induced Alterations in Testosterone Production

    U.S. Environmental Protection Agency — The greater dynamic range of testosterone production in a highly purified rat Leydig cell assay permitted the detection of chemical induced inhibition that was not...

  10. In vivo estrogenic potential of 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, an active metabolite of bisphenol A, in uterus of ovariectomized rat.

    Okuda, Katsuhiro; Takiguchi, Masufumi; Yoshihara, Shin'ichi


    4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an active metabolite of bisphenol A (BPA), has more potent estrogenic activity than BPA in vitro, but its activity in vivo is not established. Here, we examined in vivo estrogenic activity of MBP by means of uterotrophic assay in ovariectomized (OVX) female rats. MBP exhibited dose-dependent estrogenic activity, as evaluated in terms of effects on uterus weight, uterine luminal epithelial cell height and myometrium thickness. The highest concentration of MBP (10 mg/kg/day) completely reversed the changes caused by OVX, and its activity was equivalent to that of 5 microg/kg/day 17beta-estradiol (E2). We also investigated the effects of MBP on transcription of several estrogen-related genes. The changes of mRNA levels of estrogen receptors alpha and beta, c-fos and insulin-like growth factor 1 in MBP-treated OVX rats were qualitatively similar to those in E2-treated rats. BPA did not show any significant effect on OVX rat in these conditions. This study is the first to demonstrate that MBP, an active metabolite of BPA, has potent in vivo estrogenic activity, being about 500-fold more potent than BPA in OVX rats.

  11. Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test.

    Bowen, Damian E; Whitwell, James H; Lillford, Lucinda; Henderson, Debbie; Kidd, Darren; Mc Garry, Sarah; Pearce, Gareth; Beevers, Carol; Kirkland, David J


    With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(®) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these

  12. Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.

    Gealy, Robert; Wright-Bourque, Jennifer L; Kraynak, Andrew R; McKelvey, Troy W; Barnum, John E; Storer, Richard D


    In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

  13. DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay

    D. Zeljezic


    Full Text Available There are few studies of ochratoxin A (OTA genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay. Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg and positive control animals were treated with methyl methanesulfonate (40 mg/kg according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 ± 0.53, 7.52 ± 3.32, 7.85 ± 2.24 µg/mL, and 0.87 ± 0.09, 0.99 ± 0.06, 1.09 ± 0.15 µg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05. The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05, and still higher after 21 days (P < 0.05. The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05. OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

  14. Complex behavior of marine animal tissue extracts in the competitive binding assay of brevetoxins with rat brain synaptosomes.

    Whitney, P L; Delgado, J A; Baden, D G


    Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.

  15. Endocrine Activity of Bisphenol S (BPS) Using In Vitro Estrogenic/Anti-Androgenic Transcriptional Activation Assays and the In Vivo Uterotrophic Assay

    Bisphenol A (BPA) is gradually being phased out of many consumer products and processes leading to potential increases in human and environmental exposures to relatively understudied replacement compounds, including Bisphenol S (BPS). Research from our lab has shown that BPA and...

  16. Rat granulocyte colony-forming unit (CFU-G) assay for the assessment of drug-induced hematotoxicity.

    Matsumura-Takeda, K; Kotosai, K; Ozaki, A; Hara, H; Yamashita, S


    To assess the drug-induced hematotoxicity to granulocyte progenitors, we established a modified colony-forming assay using rat bone marrow cells (BMCs). In the presence of various colony-stimulating factors (CSFs), rat BMCs were disseminated on methylcellulose at a concentration of 1.3 x 10(4) cells/cm(2) (5 x 10(4) cells/0.5 ml/well in a 12-well plate). Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) stimulated the formation of almost all macrophage colonies. Human granulocyte colony-stimulating factor (hG-CSF) alone or in combination with mouse interleukin-3 (mIL-3) did not significantly effect on the number of rat colony-forming units in culture (CFU-C). When BMCs were seeded at 5.2 x 10(4) cells/cm(2) (5 x 10(5) cells/1 ml/dish in a 35-mm dish), hG-CSF increased the number of the colonies in a dose-dependent manner, and resulted in about 50 colonies at 50 ng/ml. The constituent cells of the colonies were identified as neutrophils. Under these conditions, the effects of 5-fluorouracil (5-FU) on granulocyte colony-forming units (CFU-G) were examined in rats and mice. The inhibitory effect of 5-FU on rat CFU-G was similar to the effect on mouse CFU-G. These results indicate that the rat CFU-G induced by hG-CSF is capable of being used for the evaluation of drug-induced hematotoxicity.

  17. Toxicologic Characterization of a Novel Explosive, Triaminoguanidinium-1-methyl-5-nitriminotetrazolate (TAG-MNT), in Female Rats and in vitro Assays


    exposures (that is, rat micronucleus assay). MATERIALS AND METHODS Test compound The TAG-MNT was synthesized and obtained from the Army Research...Jolla, CA). For the micronucleus assay, a one-way analysis of variance (ANOVA) was used to test for significant differences in %MN-RET and %RET... micronucleus assay of blood samples. Based on these data, the 14-day oral No observed adverse effect level (NOAEL) and the lowest observed adverse effect

  18. Activity of Antimicrobial Combinations against KPC-2-Producing Klebsiella pneumoniae in a Rat Model and Time-Kill Assay

    Aranha Junior, Ayrton Alves; Arend, Lavinia Nery; Ribeiro, Vanessa; Zavascki, Alexandre Prehn; Tuon, Felipe Francisco


    This study evaluated the efficacy of tigecycline (TIG), polymyxin B (PMB), and meropenem (MER) in 80 rats challenged with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae infection. A time-kill assay was performed with the same strain. Triple therapy and PMB+TIG were synergistic, promoted 100% survival, and produced negative peritoneal cultures, while MER+TIG showed lower survival and higher culture positivity than other regimens (P = 0.018) and was antagonistic. In vivo and in vitro studies showed that combined regimens, except MER+TIG, were more effective than monotherapies for this KPC-producing strain. PMID:25896686

  19. Evaluation of in vivo genotoxicity by thioacetamide in a 28-day repeated-dose liver micronucleus assay using male young adult rats.

    Sui, Hajime; Matsumoto, Hirotaka; Wako, Yumi; Kawasako, Kazufumi


    The repeated-dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens and can be integrated into general toxicological studies. In this study, thioacetamide (TAA) was tested in 14- and 28-day RDLMN assays to assess the performance of the assay. The test substance, TAA, was administered orally to 6-week-old male Crl:CD (SD) rats once daily for 14 or 28 days at a dosage of 5, 10 or 20mg/kg/day. Hepatocytes were collected approximately 24h after the last TAA administration, and the incidence of micronuclei was assessed. In this study, bone marrow micronucleus assays were also conducted in the same animals. The 14- and 28-day RDLMN assays indicated that none of the TAA dosages significantly increased the proportion of micronucleated hepatocytes. Bone marrow micronucleus assays with TAA also provided negative results. It is known that TAA is a liver carcinogen in mice and rats. In the previous genotoxic studies, the Ames test and the chromosomal aberration test using CHL/IU cells have yielded negative results [1-4]. The liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150mg/kg) also produced negative results [5]. TAA gave positive results only in the mouse bone marrow micronucleus assays [6,7].

  20. The rat whole embryo culture assay using the Dysmorphology Score system.

    Zhang, Cindy; Panzica-Kelly, Julie; Augustine-Rauch, Karen


    The rat whole embryo culture (WEC) system has been used extensively for characterizing teratogenic properties of test chemicals. In this chapter, we describe the methodology for culturing rat embryos as well as a new morphological score system, the Dysmorphology Score (DMS) system for assessing morphology of mid gestation (gestational day 11) rat embryos. In contrast to the developmental stage focused scoring associated with the Brown and Fabro score system, this new score system assesses the respective degree of severity of dysmorphology, which delineates normal from abnormal morphology of specific embryonic structures and organ systems. This score system generates an approach that allows rapid identification and quantification of adverse developmental findings, making it conducive for characterization of compounds for teratogenic properties and screening activities.

  1. A luminance-based heart chip assay for assessing the efficacy of graft preservation solutions in heart transplantation in rats.

    Maeda, Masashi; Kasahara, Naoya; Doi, Junshi; Iijima, Yuki; Kikuchi, Takeshi; Teratani, Takumi; Kobayashi, Eiji


    We developed a novel luciferase-based viability assay for assessing the viability of hearts preserved in different solutions. We examined whether this in vitro system could predict heart damage and survival after transplantation in rats. By our novel system, preserved heart viability evaluation and transplanted heart-graft functional research study. University basic science laboratory. Isolated Luciferase-transgenic Lewis (LEW) rat cardiac-tissue-chips were plated on 96-well tissue-culture plates and incubated in preservation solutions at 4°C. Viability was measured as photon intensity by using a bio-imaging system. Heart-grafts preserved in University of Wisconsin (UW), extracellular-trehalose-Kyoto (ETK), Euro-Collins (EC), histidin-tryptophan-ketoglutarat solution (HTK), lactated Ringer's (LR) or normal saline solution were transplanted cervically by using a cuff-technique or into the abdomens of syngeneic wild-type LEW rats by using conventional microsurgical suture techniques. Imaging an evaluation of preservation heart-graft and functional analysis. Cardiac-tissue-chips preserved with UW, HTK or ETK solution gave higher luminance than those preserved with EC, LR or normal saline (phearts in each solution at 4°C, the beating of the isolated hearts was evaluated. The success rate, evaluation of beating, of cervical heart transplants using UW and ETK solution exceeded 70%, but those using other preservation solutions were lower (UW: 100%, ETK: 75%, EC: 42.86%, HTK: 14.29%, normal saline: 0%). Histological analysis of cervical heart-grafts after 3 h preservation by myeloperoxidase (MPO), zona occludens-1(ZO-1), and caspase-3 immunostaining revealed different degrees of preservation damage in all grafts. Our novel assay system is simple and can test multiple solutions. It should therefore be a powerful tool for developing and improving new heart-graft preservation solutions.

  2. Development of a Highly Automated and Multiplexed Targeted Proteome Pipeline and Assay for 112 Rat Brain Synaptic Proteins

    Colangelo, Christopher M.; Ivosev, Gordana; Chung, Lisa; Abbott, Thomas; Shifman, Mark; Sakaue, Fumika; Cox, David; Kitchen, Rob R.; Burton, Lyle; Tate, Stephen A; Gulcicek, Erol; Bonner, Ron; Rinehart, Jesse; Nairn, Angus C.; Williams, Kenneth R.


    We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling (xMRM) that improves Signal/Noise by >2-fold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, Normalized Group Area Ratio (NGAR), MLR normalization, weighted regression analysis, and data dissemination through the Yale Protein Expression Database. As a proof of principle we developed a robust 90 minute LC-MRM assay for Mouse/Rat Post-Synaptic Density (PSD) fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our first method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay. PMID:25476245

  3. Triclosan Exposure enhances the Uterotrophic Response to Low Doses of Ethinyl Estradiol (EE) in the Female Wistar Rat

    Triclosan [TCS; (5-cbloro-2-(2, 4-dichlorophenoxy) phenol)] is an antimicrobial agent commonly found in personal care and sanitizing products, such as soaps, toothpaste and hair products. Recent studies have identified TCS in human breast milk, blood and urine. TCS has also been ...

  4. Development of an Assay Based on the Effects of PGBx on the Isolated Perfused Rat Heart and Rat Skeletal Muscle.


    and thyrotoxic rats. Eur. J. Pharac. 2,113-118. Aronson, C. E., Hess, M. E. and Shanfeld, J. (1972) Metabolic actions of tyramine and McNeil-A-343 in...that body weight and food intake of these diabetic mice also showed dose-dependency. As the dose of PGB administered to the animals was increased,x the...hypoglycemic effect became more pronounced, but food intake and body weight showed an inverse relationship to the quantity of drug which the mice

  5. Rapid one-step assays for on-site monitoring of mouse and rat urinary allergens

    Koets, M.; Renström, A.; Zahradnik, E.; Bogdanovic, J.; Wouters, I.M.; Amerongen, van A.


    llergy to rodent proteins is common among laboratory animal workers. Sensitive methods to measure exposure to these allergens have been developed. These assays are, however, expensive, time-consuming, and require a laboratory facility and methodological expertise. A simple method to screen for aller

  6. Refinement and optimisation of the rat CFU-GM assay to incorporate the use of cryopreserved bone-marrow cells for in vitro toxicology applications.

    Pessina, Augusto; Bonomi, Arianna; Baglio, Carolina; Cavicchini, Loredana; Gribaldo, Laura


    The colony-forming unit-granulocyte-macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons - not least because the rat is the most commonly-used species for the in vivo testing of toxicants. This report describes the refinement and optimisation of a standardised protocol for entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte-macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained.

  7. Effect of electromagnetic radiofrequency radiation on the rats' brain, liver and kidney cells measured by comet assay.

    Trosić, Ivancica; Pavicić, Ivan; Milković-Kraus, Sanja; Mladinić, Marin; Zeljezić, Davor


    The goal of study was to evaluate DNA damage in rat's renal, liver and brain cells after in vivo exposure to radiofrequency/microwave (Rf/Mw) radiation of cellular phone frequencies range. To determine DNA damage, a single cell gel electrophoresis/comet assay was used. Wistar rats (male, 12 week old, approximate body weight 350 g) (N = 9) were exposed to the carrier frequency of 915 MHz with Global System Mobile signal modulation (GSM), power density of 2.4 W/m2, whole body average specific absorption rate SAR of 0.6 W/kg. The animals were irradiated for one hour/day, seven days/week during two weeks period. The exposure set-up was Gigahertz Transversal Electromagnetic Mode Cell (GTEM--cell). Sham irradiated controls (N = 9) were apart of the study. The body temperature was measured before and after exposure. There were no differences in temperature in between control and treated animals. Comet assay parameters such as the tail length and tail intensity were evaluated. In comparison with tail length in controls (13.5 +/- 0.7 microm), the tail was slightly elongated in brain cells of irradiated animals (14.0 +/- 0.3 microm). The tail length obtained for liver (14.5 +/- 0.3 microm) and kidney (13.9 +/- 0.5 microm) homogenates notably differs in comparison with matched sham controls (13.6 +/- 0.3 microm) and (12.9 +/- 0.9 microm). Differences in tail intensity between control and exposed animals were not significant. The results of this study suggest that, under the experimental conditions applied, repeated 915 MHz irradiation could be a cause of DNA breaks in renal and liver cells, but not affect the cell genome at the higher extent compared to the basal damage.

  8. Protective effect of topical Cordia verbenacea in a rat periodontitis model: immune-inflammatory, antibacterial and morphometric assays

    Pimentel Suzana


    Full Text Available Abstract Background This study evaluated the effects of C. verbenacea essential oil topically administered in a rat periodontitis model. Methods Periodontitis was induced on rats in one of the mandibular first molars assigned to receive a ligature. Animals were randomly divided into two groups: a non-treatment group (NT (n = 18: animals received 1mL of vehicle; b C. verbenacea group (C.v. (n = 18: animals received 5mg/Kg of essential oils isolated from C. verbenacea. The therapies were administered topically 3 times daily for 11 days. Then, the specimens were processed for morphometric analysis of bone loss. The ligatures were used for microbiological assessment of the presence of Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Porphyromonas gingivalis using PCR. The gingival tissue was collected to Elisa assay of interleukin (IL-1α and IL-10 levels. Results Bone loss was inhibited by C. verbenacea when compared to the NT group (p p P. gingivalis was found in C.v. group (p  Conclusion C. verbenacea essential oil topically administered diminished alveolar bone resorption, promoting a positive local imbalance in the pro/anti-inflammatory system and reducing the frequency of detection of P. gingivalis.

  9. Evaluation of plasma antioxidant activity in rats given excess EGCg with reference to endogenous antioxidants concentrations and assay methods.

    Yokotani, Kaori; Umegaki, Keizo


    The contribution of (-)-epigallocatechin gallate (EGCg) intake to in vivo antioxidant activity is unclear, even with respect to plasma. In this study, we examined how administration of EGCg contributes to plasma antioxidant activity, relative to its concentration, endogenous antioxidants, and assay methods, namely oxygen radical absorbance capacity (ORAC) and ferric reducing/antioxidant power (FRAP). Administration of EGCg (500 mg/kg) to rats increased plasma EGCg (4μmol/L as free form) and ascorbic acid (1.7-fold), as well as ORAC (1.2-fold) and FRAP (3-fold) values. The increase in plasma ascorbic acid following EGCg administration was accompanied by its relocation from the adrenal glands and lymphocytes into plasma, and was related to the increase in FRAP. Plasma deproteinization and assays in plasma model solutions revealed that protein levels significantly contributed to ORAC values, where plasma ascorbic acid was not influenced by deproteinization, differences in FRAP values with and without deproteinization were estimated to determine the contribution of enhanced ascorbic acid attributable to EGCg administration. These results will help to understand the points that should be considered when evaluating EGCg antioxidant activity in plasma.

  10. Assay and properties of rat yolk sac 25-hydroxyvitamin D/sub 3/ 1. cap alpha. -hydroxylase

    Paulson, S.K.; Phelps, M.; DeLuca, H.F.


    An in vitro assay has been developed for the rat yolk sac 25-hydroxyvitamin D/sub 3/ 1..cap alpha..-hydroxylase (1..cap alpha..-hydroxylase). The subcellular location and some properties of the enzyme are described. 1,25-Dihydroxyvitamin D/sub 3/ produced from incubations of yolk sac homogenates was extracted, purified by Sephadex LH-20 chromatography and straight- and reverse-phase high-performance liquid chromatography (HPLC), and measured by a competitive binding assay using chick intestinal receptor. The reaction is linear with time for up to 45 min at a substrate concentration of 80 and 4-6 mg/mL microsomal protein. The enzyme, located in the microsomes, requires molecular oxygen and NADPH. Metyrapone (1 x 10/sup -3/ M) was found to inhibit 1-hydroxylation, but a 90% carbon monoxide-10% oxygen atmosphere did not, leaving open the question of involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, inhibited 1..cap alpha..-hydroxylation.

  11. Pharmaceutical evaluation of naftopidil enantiomers: Rat functional assays in vitro and estrogen/androgen induced rat benign prostatic hyperplasia model in vivo.

    Huang, Jun-Jun; Cai, Yi; Huang, Min-Yi; Zhu, Liu; He, Fei; Liu, Xia-Wen; Huang, Bi-Yun; Yi, Yan-Zhen; Yuan, Mu


    Naftopidil (NAF) is a α1D/1A adrenoceptor selective drug used for the treatment of both benign prostatic hyperplasia and lower urinary tract symptoms (BPH/LUTS). However, NAF is used as a racemate in clinic. To compare the differences and similarities among two enantiomers and racemate, pharmacological activities were evaluated through rat functional assays in vitro and estrogen/androgen (E/T) induced rat BPH model in vivo. NAF and the two enantiomers showed similar blocking activity on α1 receptor. S-NAF exhibited more α1D/1A adrenoceptor subtype selectivity than R-NAF and the racemate. The selectivity ratios pA2 (α1D)/pA2 (α1B) and pA2 (α1A)/pA2 (α1B) were 40.7- and 16.2-fold, respectively. NAF and its enantiomers effectively prevented the development of rat prostatic hyperplasia via suppressing the increase of the prostatic wet weight, visually. The quantitative analysis of the relative acinus volume, relative stroma volume, relative epithelial volume, epithelial height and expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were carried out. S-NAF showed an advantage on the effect of inhibiting prostate wet weight and stroma volume over R-NAF and racemate NAF (P<0.05). Nevertheless, no other significant difference was observed between these two enantiomers. In conclusion, both R-NAF and S-NAF not only relax prostate muscle but also inhibit the prostate growth, thus relieve BPH.

  12. Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP) - Assessment of corresponding epitopes

    Leeming, Diana Julie; Larsen, D.V.; Zhang, C.


    Objectives: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. Methods: Monoclonal antibodies were raised against...

  13. Two-generation reproduction toxicity study in rats with methoxychlor.

    Aoyama, Hiroaki; Hojo, Hitoshi; Takahashi, Ken L; Shimizu-Endo, Naoko; Araki, Masayuki; Takeuchi-Kashimoto, Yukiko; Saka, Machiko; Teramoto, Shoji


    A two-generation reproduction toxicity study was conducted in rats with a reference estrogenic pesticide, methoxychlor, to validate the sensitivity and competency of current guidelines recommended by the United States Environmental Protection Agency; Japanese Ministry of Agriculture, Forestry and Fisheries; and Organisation for Economic Co-operation and Development for predicting reproductive toxicity of the test compound based on estrogenic endocrine disrupting effects. Both sexes of SD rats were exposed to methoxychlor in the diet at concentrations of 0, 10, 500 and 1500 ppm for two successive generations. The present study has successfully detected estrogenic activities and reproductive toxicities of methoxychlor, as well as its systemic toxicity. Body weights, body weight gains and food consumption of both sexes of animals were suppressed significantly in the 500 and 1500 ppm groups. Typical reproductive toxicities observed in females of these groups included, but were not limited to, prolonged estrous cycle, reduced fertility, decreased numbers of implantation sites and newborns, decreased ovary weights and/or increased incidences of cystic ovary. Uterine weights of weanlings increased significantly in these groups, suggesting that the sensitivity of this parameter for predicting estrogenic ability of the test compound is comparable to that of the uterotrophic assay. Reproductive toxicities of methoxychlor seemed less potent in males than in females. Methoxychlor delayed preputial separation and significantly reduced sperm counts and reproductive organ weights of males of the 500 and/or 1500 ppm groups; however, most males that failed to impregnate females in the same group showed normal fertility when they were re-mated with untreated females. Neither systemic nor reproductive toxicities appeared in the 10 ppm group.

  14. Effects of nicotinic acetylcholine receptor agonists in assays of acute pain-stimulated and pain-depressed behaviors in rats.

    Freitas, Kelen C; Carroll, F Ivy; Negus, S Stevens


    Agonists at nicotinic acetylcholine receptors (nAChRs) constitute one drug class being evaluated as candidate analgesics. Previous preclinical studies have implicated α4β2 and α7 nAChRs as potential mediators of the antinociceptive effects of (–)-nicotine hydrogen tartrate (nicotine) and other nAChR agonists; however, these studies have relied exclusively on measures of pain-stimulated behavior, which can be defined as behaviors that increase in frequency, rate, or intensity after presentation of a noxious stimulus. Pain is also associated with depression of many behaviors, and drug effects can differ in assays of pain-stimulated versus pain-depressed behavior. Accordingly, this study compared the effects of nicotine, the selective α4/6β2 agonist 5-(123I)iodo-3-[2(S)-2-azetidinylmethoxy]pyridine (5-I-A-85380), and the selective α7 agonist N-(3R)-1-azabicyclo(2.2.2)oct-3-yl-4-chlorobenzamide in assays of pain-stimulated and pain-depressed behavior in male Sprague-Dawley rats. Intraperitoneal injection of dilute lactic acid served as an acute noxious stimulus to either stimulate a stretching response or depress the operant responding, which is maintained by electrical brain stimulation in an intracranial self-stimulation (ICSS) procedure. Nicotine produced a dose-dependent, time-dependent, and mecamylamine-reversible blockade of both acid-stimulated stretching and acid-induced depression of ICSS. 5-I-A-85380 also blocked both acid-stimulated stretching and acid-induced depression of ICSS, whereas N-(3R)-1-azabicyclo(2.2.2)oct-3-yl-4-chlorobenzamide produced no effect in either procedure. Both nicotine and 5-I-A-85380 were ≥10-fold more potent in blocking the acid-induced depression of ICSS than in blocking the acid-induced stimulation of stretching. These results suggest that stimulation of α4β2 and/or α6β2 nAChRs may be especially effective to alleviate the signs of pain-related behavioral depression in rats; however, nonselective behavioral effects

  15. In vivo genotoxicity assessment of aluminium oxide nanomaterials in rat peripheral blood cells using the comet assay and micronucleus test.

    Balasubramanyam, A; Sailaja, N; Mahboob, M; Rahman, M F; Hussain, Saber M; Grover, Paramjit


    Advances in nanotechnology and its usage in various fields have led to the exposure of humans to engineered nanomaterials (NMs) and there is a need to tackle the potential human health effects before these materials are fully exploited. The main purpose of the current study was to assess whether aluminium oxide NMs (Al(2)O(3)-30 nm and Al(2)O(3)-40 nm) could cause potential genotoxic effects in vivo. Characterization of Al(2)O(3)-30 nm and Al(2)O(3)-40 nm was done with transmission electron microscopy, dynamic light scattering and laser Doppler velocimetry prior to their use in this study. The genotoxicity end points considered in this study were the frequency of micronuclei (MN) and the percentage of tail DNA (% Tail DNA) migration in rat peripheral blood cells using the micronucleus test (MNT) and the comet assay, respectively. Genotoxic effects were evaluated in groups of female Wistar rats (five per group) after single doses of 500, 1000 and 2000 mg/kg body weight (bw) of Al(2)O(3)-30 nm, Al(2)O(3)-40 nm and Al(2)O(3)-bulk. Al(2)O(3)-30 nm and Al(2)O(3)-40 nm showed a statistically significant dose-related increase in % Tail DNA for Al(2)O(3)-30 nm and Al(2)O(3)-40 nm (P < 0.05). However, Al(2)O(3)-bulk did not induce statistically significant changes over control values. The MNT also revealed a statistically significant (P < 0.05) dose-dependent increase in the frequency of MN, whereas Al(2)O(3)-bulk did not show any significant increase in frequency of MN compared to control. Cyclophosphamide (40 mg/kg bw) used as a positive control showed statistically significant (P < 0.001) increase in % Tail DNA and frequency of MN. The biodistribution of Al(2)O(3)-30 nm and Al(2)O(3)-40 nm and Al(2)O(3)-bulk in different rat tissues, urine and feces was also studied 14 days after treatment using inductively coupled plasma mass spectrometry. The data indicated that tissue distribution of Al(2)O(3) was size dependent. Our findings suggest that Al(2)O(3) NMs were able to

  16. A 155-plex high-throughput in vitro coregulator binding assay for (anti-)estrogenicity testing evaluated with 23 reference compounds.

    Wang, Si; Houtman, René; Melchers, Diana; Aarts, Jac; Peijnenburg, Ad; van Beuningen, Rinie; Rietjens, Ivonne; Bovee, Toine F


    To further develop an integrated in vitro testing strategy for replacement of in vivo tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulators with estrogen receptor α was assessed using a PamChip® plate. The relative estrogenic potencies determined, based on ERα binding to coregulator peptides in the presence of ligands on the PamChip® plate, were compared to the relative estrogenic potencies as determined in the in vivo uterotrophic assay. The results show that the estrogenic potencies predicted by the 57 coactivators on the peptide microarray for 18 compounds that display a clear E2 dose-dependent response (goodness of fit of a logistic dose-response model of 0.90 or higher) correlated very well with their in vivo potencies in the uterotrophic assay, i.e., coefficient of determination values for 30 coactivators higher than or equal to 0.85. Moreover, this coregulator binding assay is able to distinguish ER agonists from ER antagonists: profiles of selective estrogen receptor modulators, such as tamoxifen, were distinct from those of pure ER agonists, such as dienestrol. Combination of this coregulator binding assay with other types of in vitro assays, e.g., reporter gene assays and the H295R steroidogenesis assay, will frame an in vitro test panel for screening and prioritization of chemicals, thereby contributing to the reduction and ultimately the replacement of animal testing for (anti-)estrogenic effects.

  17. Alkaline, Endo III and FPG modified comet assay as biomarkers for the detection of oxidative DNA damage in rats with experimentally induced diabetes.

    Kushwaha, S; Vikram, A; Trivedi, P P; Jena, G B


    Increased production of reactive oxygen species under diabetic condition underlines the higher oxidatively damaged DNA in different tissues. However, it is practically difficult to assess the oxidatively damaged DNA in different internal organs. Therefore, the present study was aimed to evaluate the extent of oxidative stress-induced DNA damage in different organs with the progression of diabetes. Diabetic and control Sprague Dawley rats were sacrificed in time-dependent manner and the lung, liver, heart, aorta, kidney, pancreas and peripheral blood lymphocytes (PBL) were analyzed for both alkaline and modified comet assay with endonuclease-III (Endo III) and formamidopyrimidine-DNA glycosylase (FPG) (hereafter called modified comet assay) for the detection of oxidative DNA damage. The statistically significant increase in olive tail moment (OTM) was found in all the tested tissues. The extent of DNA damage was increased with the progression of diabetes as revealed by the parameter of OTM in alkaline and modified comet assay. Further, the positive correlations were observed between OTM of the lung, liver, heart, aorta, kidney and pancreas with PBL of diabetic rat in the alkaline and modified comet assay. Moreover, significant increase in the 8-oxodG positive nuclei in the lung, liver, heart, aorta, kidney and pancreas was observed in 4th and 8th week diabetic rat as compared to control. Results of the present study clearly indicated the suitability of alkaline and modified comet assay for the detection of multi-organ oxidative DNA damage in streptozotocin (STZ)-induced diabetic rat and showed that damaged DNA of PBL can be used as a suitable biomarker to assess the internal organs response to DNA damage in diabetes.

  18. Inhibition of Human and Rat Sucrase and Maltase Activities To Assess Antiglycemic Potential: Optimization of the Assay Using Acarbose and Polyphenols.

    Pyner, Alison; Nyambe-Silavwe, Hilda; Williamson, Gary


    We optimized the assays used to measure inhibition of rat and human α-glucosidases (sucrase and maltase activities), intestinal enzymes which catalyze the final steps of carbohydrate digestion. Cell-free extracts from fully differentiated intestinal Caco-2/TC7 monolayers were shown to be a suitable source of sucrase-isomaltase, with the same sequence as human small intestine, and were compared to a rat intestinal extract. The kinetic conditions of the assay were optimized, including comparison of enzymatic and chromatographic methods to detect the monosaccharide products. Human sucrase activity was more susceptible than the rat enzyme to inhibition by acarbose (IC50 (concentration required for 50% inhibition) = 2.5 ± 0.5 and 12.3 ± 0.6 μM, respectively), by a polyphenol-rich green tea extract, and by pure (-)-epigallocatechin gallate (EGCG) (IC50 = 657 ± 150 and 950 ± 86 μM respectively). In contrast, the reverse was observed when assessing maltase activity (e.g. IC50 = 677 ± 241 and 14.0 ± 2.0 μM for human and rat maltase, respectively). 5-Caffeoylquinic acid did not significantly inhibit maltase and was only a very weak inhibitor of sucrase. The data show that for sucrase and maltase activities, inhibition patterns of rat and human enzymes are generally qualitatively similar but can be quantitatively different.

  19. 农药联苯菊酯对大鼠内分泌干扰作用的研究%Potential endocrine disrupting effects of bifenthrin in rats

    谭彦君; 王恒娟; 宋雁; 杨辉; 贾旭东; 李宁


    Objective To study the potential endocrine disrupting effects of bifenthrin (BIF)by using uterotrophic assay and Hershberger assay. Methods In uterotrophic assay ,60 female SD rats were randomly divided into 6 groups, 10 rats per group. Rats in bifenthrin-treated groups were given different doses of bifenthrin (1.47,4.41 and 13. 23mg/kg BW by gavage for 3 consecutive days). Rats in negative control groups were given com oil by gavage. Rats in ethinyl estradiol ( EE) oral positive control groups were given EE 1.0μg/kg BW by gavage. Rats in EE injected positive groups were given 0. 6μg/kg BW EE by subcutaneously injection while given corn oil by gavage. At necropsy, the wet and blotted uteri were weighed. The relative uteri weights were calculated,and the histology of uteri was observed. In Hershberger assay,60 castrated male SD rats were randomly divided into 6 groups, with 10 rats in each group. Rats in BIF-treated groups were given different doses of BIF(1.4,4. 2 and 12.6mg/kg BW) by gavage. Flutamide(3. 0mg/kg BW)were given to animals in the positive control group by gavage. Rats in the negative control group and testosterone propionate group were given corn oil by gavage for 10 consecutive days. Rats in all groups except the negative control group were also treated with testosterone propionate(TP,0.2mg/kg BW)by subcutaneous injection. At necropsy, ventral prostate (VP), seminal vesicle plus fluids and coagulating glands ( SVCG) , levator ani-bulbocavernosus muscle ( LABC ), paired Cowper's glands (COW) and the glands penis ( GP ) , liver, kidneys, adrenals were weighed. Serum triiodothyronine(T3)and thyroxine(T4)were determined. Results In uterotrophic assay, compared with the negative control group, the mean relative wet weight and relative blotted weight of uterine were increased significantly in the female rats given by BIF at 13. 23mg/kg BW for 3 days( P < 0. 05 ) . BIF resulted in a significant increase of epithelial cell heights of uteri at 4.41 and 13

  20. Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line

    Long, Manhai; Laier, Peter; Vinggaard, Anne Marie


    to be a rapid and sensitive assay for assessing the potency of AhR-activating compounds. We have used the AhR-CALUX assay to investigate the AhR-mediated activity of the persistent organochlorine insecticide dieldrin and twenty-two pesticides currently used in Denmark by employing the rat H4IIE and the human TV......101L hepatoma cell lines. In comparison the results indicated that the rat H4IIE cell line is more sensitive than the human TV101L for detection of TCDD inducing AhR-CALUX activity. The pesticides iprodione, chlorpyrifos and prochloraz showed dose-dependent AhR agonistic effects in both cell lines...... at concentrations above 10, 1 and 1 microM, respectively. However, some pesticides (methiocarb, chlorothalonil, tribenuron-methyl, paclobutrazol and tolchlofos-methyl) elicited differential responses in the two cell lines....

  1. Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

    Culman, J.; Torda, T.; Weise, V.K.


    A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-(methyl-/sup 3/H) adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.

  2. Selective Estrogen Receptor Modulator (SERM)-like Activities of Diarylheptanoid, a Phytoestrogen from Curcuma comosa, in Breast Cancer Cells, Pre-osteoblast Cells, and Rat Uterine Tissues.

    Thongon, Natthakan; Boonmuen, Nittaya; Suksen, Kanoknetr; Wichit, Patsorn; Chairoungdua, Arthit; Tuchinda, Patoomratana; Suksamrarn, Apichart; Winuthayanon, Wipawee; Piyachaturawat, Pawinee


    Diarylheptanoids from Curcuma comosa, of the Zingiberaceae family, exhibit diverse estrogenic activities. In this study we investigated the estrogenic activity of a major hydroxyl diarylheptanoid, 7-(3,4 -dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound 092) isolated from C. comosa. The compound elicited different transcriptional activities of estrogen agonist at low concentrations (0.1-1 μM) and antagonist at high concentrations (10-50 μM) using luciferase reporter gene assay in HEK-293T cells. In human breast cancer (MCF-7) cells, compound 092 showed an anti-estrogenic activity by down-regulating ERα-signaling and suppressing estrogen-responsive genes, whereas it attenuated the uterotrophic effect of estrogen in immature ovariectomized rats. Of note, compound 092 promoted mouse pre-osteoblastic (MC3T3-E1) cell differentiation and the related bone markers, indicating its positive osteogenic effect. Our findings highlight a new, nonsteroidal, estrogen agonist/antagonist of catechol diarylheptanoid from C. comosa, which is scientific evidence supporting its potential as a dietary supplement to prevent bone loss with low risk of breast and uterine cancers in postmenopausal women.

  3. Genotoxicity of doxorubicin in F344 rats by combining the comet assay, flow-cytometric peripheral blood micronucleus test, and pathway-focused gene expression profiling.

    Manjanatha, Mugimane G; Bishop, Michelle E; Pearce, Mason G; Kulkarni, Rohan; Lyn-Cook, Lascelles E; Ding, Wei


    Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7-week-old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species.

  4. Development of a liquid chromatography-mass spectrometry (LC/MS) assay method for the quantification of PSC 833 (Valspodar) in rat plasma.

    Binkhathlan, Ziyad; Somayaji, Vishwa; Brocks, Dion R; Lavasanifar, Afsaneh


    A liquid chromatography-mass spectrometry (LC/MS) assay method was developed for the quantification of PSC 833 in rat plasma, using amiodarone as internal standard (IS). Separation was achieved using a C(8) 3.5 microm (2.1 mm x 50 mm) column heated to 60 degrees C with a mobile phase consisting of acetonitrile-ammonium hydroxide 0.2% (90:10 v/v) pumped at a rate of 0.2 mL/min. Detection was accomplished by mass spectrometer using selected ion monitoring (SIM) in positive mode. An excellent linear relationship was present between peak height ratios and rat plasma concentrations of PSC 833 ranging from 10 to 5000 ng/mL (R(2)>0.99). Intra-day and inter-day coefficients of variation (CV%) were less than 15%, and mean error was less than 10% for the concentrations above the limit of quantification. The validated limit of quantification of the assay was 10 ng/mL based on 0.1 mL rat plasma. The method limit of detection, based on an average signal-to-noise (S/N) ratio of 3, was found to be 2.5 ng/mL. The assay was capable of measuring the plasma concentrations of PSC 833 in rats injected with a single dose of 5 mg/kg of the drug. PSC 833 and IS eluted within 4 min, free of interfering peaks. The method was found to be fast, sensitive, and specific for the quantification of PSC 833 in rat plasma.

  5. Effect of phytate reduction of sorghum, through genetic modification, on iron and zinc availability as assessed by an in vitro dialysability bioaccessibility assay, Caco-2 cell uptake assay, and suckling rat pup absorption model.

    Kruger, Johanita; Taylor, John R N; Du, Xiaogu; De Moura, Fabiana F; Lönnerdal, Bo; Oelofse, André


    Improved iron and zinc availability from sorghum, a commonly consumed staple, will benefit many malnourished communities in rural Africa burdened with high prevalence of iron and zinc deficiency. This research compared the effect of genetic phytate reduction in sorghum on iron and zinc bioaccessibility and uptake measured by in vitro dialysability and Caco-2 cell uptake assays to that of iron and zinc absorption measured by a suckling rat pup model. The phytate reduction (80-86%) in these sorghums significantly increased zinc availability. The Caco-2 cell method, but not the dialysability assay, proved useful in estimating zinc absorption. The measured increase in iron availability differed between the methods, possibly due to the effect of varying mineral (Ca, Fe, Zn, P) contents of the sorghums. This effect was most prominent in the iron uptake results. More research is needed to determine the effect of naturally occurring variations in mineral contents of sorghum on the iron uptake by Caco-2 cells.




    Full Text Available

    The absorption and bioavailability of the minerals Ca, Fe and Zn in balanced and mineral restricted diets with the addition of the food supplement multi-mixture (MM, was evaluated. A biological assay was carried out for 28 days with recently weaned Wistar rats, using 3 treatments and 8 animals in each group. The diets offered to each group were distributed as follows: CD – Control Diet (AIN-93G; CcD/MM – Control Diet + Supplement 5% MM; DRMIXc/MM – Control Diet with Mineral Restricted + Supplement 5% MM. The animals were monitored for weight; amount of diet consumed and amount of faeces excreted. They were sacrifi ced at the end of the experimental period and the pair of femurs, pancreas and liver removed for the determination of Ca, Fe and Zn. The CcD/MM diet presented absorption of Ca and Fe and bioavailability of Ca, Fe and Zn equal to that of CD, showing no statistical difference between the treatments. However the DRMIXcD/MM diet presented an increase in Ca absorption, equal absorption of Fe and an increase in the bioavailability of Ca, Fe and Zn when compared to CD. It was concluded that when fed on a balanced diet supplemented with 5% MM, there was no increase in the bioavailability of Ca, Fe or Zn, whereas when fed on a mineral defi cient diet, the group of animals that received supplementation with 5% MM, presented greater absorption and bioavailability of these minerals.

  7. Enzyme-linked immunosorbent assay for S100A9 in the stool of rats with dextran sulfate sodium-induced colitis.

    Sekiya, Shunsuke; Murata, Makoto; Arai, Satoshi; Murayama, Hiroshi; Kawasaki, Atushi; Ashida, Noriyuki; Okada, Kohki; Ikemoto, Masaki


    Calprotectin, a heterodimer of S100A8 and S100A9, has been reported to be a useful biomarker in inflammatory bowel disease (IBD); however, the relationship between the fecal level of S100A9 and the extent of inflammation in IBD remains unclear. Our aim was to develop a new enzyme-linked immunosorbent assay (ELISA) for rat S100A9, and to investigate whether changes in fecal S100A9 levels reflect the inflammatory conditions in the intestinal tracts of rats with dextran sulfate sodium (DSS)-induced colitis. Anti-rat S100A9 monoclonal antibodies were raised in mice and used for the development of a novel ELISA for rat S100A9. The performance of our ELISA was assessed by dilution and recovery tests, and the detection range was 3.75-240ng/mL. The dilution test showed good linearity. The recovery of fecal S100A9 was 95.1% (mean), with a range of 86.1%-108.8%. Colitis was induced in rats by oral administration of 3% DSS/drinking water (DW) for 11days (D group), while DW alone was provided to rats of the control group (C group) during the same period. The extent of inflammation was evaluated with the disease activity index (DAI), and the concentration of fecal S100A9 was determined by ELISA. Both the DAI scores and the fecal S100A9 levels were significantly higher in the D group than in the C group. Microscopic observation revealed that S100A9 was dominantly produced in many immune cells of myeloid origin in rat rectal tissues. These results indicate that the novel ELISA may be applied to clinically evaluate IBD in rats with high sensitivity. In conclusion, our ELISA is useful in toxicological and pharmacological evaluations. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Estrogen agonist/antagonist properties of dibenzyl phthalate (DBzP) based on in vitro and in vivo assays.

    Zhang, Zhaobin; Hu, Ying; Zhao, Liang; Li, Jun; Bai, Huicheng; Zhu, Desheng; Hu, Jianying


    The most commonly used phthalates have been banned or restricted for use as plasticizers in toys in some countries because of their endocrine-disrupting properties. Dibenzyl phthalate (DBzP) has been proposed as a possible alternative for the banned/restricted phthalates. In this study, the estrogen agonist/antagonist properties of DBzP were predicted by molecular docking and confirmed by yeast estrogen screen (YES) and immature mouse uterotrophic assays. The YES assay results showed a dose-dependent increase in DBzP estrogen agonist activity from 10⁻⁶ to 10⁻⁴ M, and at concentrations from 1.95×10⁻⁶ M to higher, DBzP significantly inhibited the agonist activity of 10⁻⁹ M 17β-estradiol (E₂), inhibiting 10⁻⁹ M E₂ by 74.5% at its maximum effectiveness. The in vivo estrogen agonist/antagonist activities of DBzP were demonstrated in immature mouse uterotrophic assays. The antagonist activity of DBzP inhibited E₂-induced uterine growth promoted at 40 and 400 μg/kg bw (body weight) (Pestrogen agonist/antagonist potentials of benzyl butyl phthalate (BBP) by YES, and found both were weaker than those of DBzP, suggesting DBzP would be more toxic than BBP and should not be used as an alternative plasticizer.

  9. Differential genotoxicity of acrylamide in the micronucleus and Pig-a gene mutation assays in F344 rats and B6C3F1 mice.

    Hobbs, Cheryl A; Davis, Jeffrey; Shepard, Kim; Chepelev, Nikolai; Friedman, Marvin; Marroni, Dennis; Recio, Leslie


    Acrylamide is used in many industrial processes and is present in a variety of fried and baked foods. In rodent carcinogenicity assays, acrylamide exposure leads to tumour formation at doses lower than those demonstrated to induce genotoxic damage. We evaluated the potential of acrylamide to induce structural DNA damage and gene mutations in rodents using highly sensitive flow cytometric analysis of micronucleus and Pig-a mutant frequencies, respectively. Male F344 rats and B6C3F1 mice were administered acrylamide in drinking water for 30 days at doses spanning and exceeding the range of acrylamide exposure tested in cancer bioassays-top dose of 12.0 and 24.0mg/kg/day in mice and in rats, respectively. A positive control, N-ethyl-N-nitrosourea, was administered at the beginning and end of the study to meet the expression time for the two DNA damage phenotypes. The results of the micronucleus and Pig-a assays were negative and equivocal, respectively, for male rats exposed to acrylamide at the concentrations tested. In contrast, acrylamide induced a dose-dependent increase in micronucleus formation but tested negative in the Pig-a assay in mice. Higher plasma concentrations of glycidamide in mice than rats are hypothesized to explain, at least in part, the differences in the response. Benchmark dose modelling indicates that structural DNA damage as opposed to point mutations is most relevant to the genotoxic mode of action of acrylamide-induced carcinogenicity. Moreover, the lack of genotoxicity detected at acrylamide-induced carcinogenicity in rodents. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail:

  10. Identification and Characterization of Novel Rat Polyomavirus 2 in a Colony of X-SCID Rats by P-PIT assay

    Rigatti, Lora H.; Toptan, Tuna; Newsome, Joseph T.


    ABSTRACT Polyomaviruses (PyVs) are known to infect a wide range of vertebrates and invertebrates and are associated with a broad spectrum of diseases, including cancers, particularly in immune-suppressed hosts. A novel polyomavirus, designated rat polyomavirus 2 (RatPyV2), was identified from a breeding colony of rats having X-linked severe combined immunodeficiency. Using a human panpolyomavirus immunohistochemistry test (P-PIT), RatPyV2 was initially detected in the parotid salivary gland of a colony member. Rolling circle amplification using DNA from harderian and parotid glands identified a novel 5.1-kb polyomavirus genome closely related to human Washington University (WU) and Karolinska Institute (KI) and vole polyomaviruses but notably divergent from Rattus norvegicus PyV1 (RnorPyV1; also designated RatPyV1). Further screening showed RatPyV2 inclusion body infection in the lung epithelium and variably in other respiratory, reproductive, and glandular tissues of 12/12 (100%) rats. IMPORTANCE Although P-PIT was developed to detect diseases associated with known human polyomaviruses, the identification of a new polyomavirus in rats suggests that it may have utility as a broad-based screen for new, as well as known polyomaviruses. Our findings suggest that RatPyV2 may be a commensal infection of laboratory rats that can lead to disseminated disease in T cell immune-deficient rats. Infection of the X-SCID rats with RatPyV2 and Pneumocystis carinii is a potential model for coinfection pathogenesis and treatment options during transplant preclinical studies. PMID:28028546

  11. Transgenic Animal Mutation Assays

    Tao Chen; Ph.D.D.A.B.T.


    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  12. In vivo assay for conversion of testosterone to dihydrotestosterone by rat prostatic steroid 5 alpha-reductase and comparison of two inhibitors

    Toomey, R.E.; Goode, R.L.; Petrow, V.; Neubauer, B.L. (Endocrine Research, Lilly Research Labs, Indianapolis, IN (USA))


    An in vivo assay for steroid 5 alpha-reductase in rat ventral prostate has been developed and used to compare the inhibitory activity of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and 6-methylene-4-pregnene-3,20-dione (LY207320). Immature rats (70-80 g) received test compounds 30 min prior to s.c. injection of (3H)-T. The rats were sacrificed 30 min later and the ventral prostates were analyzed for (3H)-T metabolites. Intraprostatic (3H)-T and (3H)-DHT reached peak levels within 5 min after injection of (3H)-T and declined to about 25% of peak levels after 2 hr. 4-MA was a very potent inhibitor of (3H)-DHT formation with an estimated IC50 of 0.2 mg/kg. LY207320, an inhibitor of 5 alpha-reductase in vitro, was weakly active in vivo and did not achieve greater than 45% inhibition at high doses (greater than 200 mg/kg, s.c.). Tissue uptake of (3H)-T was also inhibited by LY207320, which may contribute to its inhibitory activity on accessory sex organ growth in the rat.

  13. Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line

    Long, M.; Laier, Peter; Vinggaard, Anne;


    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biologic and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. The in vitro chemically activated luciferase expression (CALUX) assay has been proven...... TV101L hepatoma cell lines. In comparison the results indicated that the rat H4IIE cell line is more sensitive than the human TV101L for detection of TCDD inducing AhR-CALUX activity. The pesticides iprodione, chlorpyrifos and prochloraz showed dose-dependent AhR agonistic effects in both cell lines...

  14. DNA damage in isolated rat hepatocytes exposed to C.I. pigment orange 5 and C.I. pigment yellow 12 by the alkaline comet assay

    Møller, P; Wallin, Håkan; Grunnet, N


    The induction of DNA damage by commonly used printing ink pigments, C.I. pigment orange 5 (C.I. 12075) and C.I. pigment yellow 12 (C.I. 21090), was investigated in freshly isolated rat hepatocytes with the comet assay. C.I. pigment yellow 12 is a 3,3'-dichlorobenzidine-based diarylide pigment, an......]quinoxaline. Our data indicate that both C.I. pigment orange 5 and C.I. pigment yellow 12 are genotoxic in hepatocytes with metabolizing capacities. However, further investigation of the metabolism and disposition are required for the evaluation of the safety of these pigments....

  15. Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay.

    De Boeck, Marlies; van der Leede, Bas-jan; De Vlieger, Kathleen; Geys, Helena; Vynckier, An; Van Gompel, Jacky


    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague-Dawley rats (7-9 weeks of age) were given three oral doses of the test compounds, 24 and 21 h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000 mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells.

  16. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong


    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1–3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure.

  17. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong


    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1–3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure. PMID:27445126

  18. Optimisation of a 96-well electroporation assay for postnatal rat CNS neurons suitable for cost-effective medium-throughput screening of genes that promote neurite outgrowth

    Thomas eHutson


    Full Text Available Following an injury, central nervous system (CNS neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterised by their failure to express key regeneration-associated genes (RAGs and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimised a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth-inhibitory chondroitin sulphate proteoglycans. Optimal electroporation parameters resulted in 25% transfection efficiency and 50% viability for postnatal rat cerebellar granule neurons (CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates.

  19. Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

    Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H


    In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Optimal dose selection of N-methyl-N-nitrosourea for the rat comet assay to evaluate DNA damage in organs with different susceptibility to cytotoxicity.

    Kitamoto, Sachiko; Matsuyama, Ryoko; Uematsu, Yasuaki; Ogata, Keiko; Ota, Mika; Yamada, Toru; Miyata, Kaori; Funabashi, Hitoshi; Saito, Koichi


    The in vivo rodent alkaline comet assay (comet assay) is a promising technique to evaluate DNA damage in vivo. However, there is no agreement on a method to evaluate DNA damage in organs where cytotoxicity is observed. As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the comet assay, we examined DNA damage in the liver, stomach, and bone marrow of rats given three oral doses of N-methyl-N-nitrosourea (MNU) up to the maximum tolerated dose based on systemic toxicity. MNU significantly increased the % tail DNA in all the organs. Histopathological analysis showed no cytotoxic effect on the liver, indicating clearly that MNU has a genotoxic potential in the liver. In the stomach, however, the cytotoxic effects were very severe at systemically non-toxic doses. Low-dose MNU significantly increased the % tail DNA even at a non-cytotoxic dose, indicating that MNU has a genotoxic potential also in the stomach. Part of the DNA damage at cytotoxic doses was considered to be a secondary effect of severe cell damage. In the bone marrow, both the % tail DNA and incidence of micronucleated polychromatic erythrocytes significantly increased at non-hematotoxic doses, which were different from the non-cytotoxic doses for liver and stomach. These findings indicate that an optimal dose for detecting DNA damage may vary among organs and that careful attention is required to select an optimum dose for the comet assay based on systemic toxicity such as mortality and clinical observations. The present study shows that when serious cytotoxicity is suggested by increased % hedgehogs in the comet assay, histopathological examination should be included for the evaluation of a positive response.

  1. Estrogenic potencies of resorcylic acid lactones and 17 beta-estradiol in female rats.

    Everett, D J; Perry, C J; Scott, K A; Martin, B W; Terry, M K


    Uterotrophic response in sexually immature female rats has been used to rank the relative estrogenic potencies of six resorcylic acid lactones (RALs) and to compare their activities with that of 17 beta-estradiol. On oral administration, the estrogenic potency relative to 17 beta-estradiol is as follows: 7 alpha-zearalenol, 10 times less; zeranol, 150 times less; taleranol, 350 times less; zearalanone, 400 times less; zearalenone, 650 times less; 7 beta-zearalenol, 3500 times less. On subcutaneous administration, zeranol is 500 times less estrogenic than 17 beta-estradiol.

  2. An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

    Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Dillon, Debbie; Meredith, Clive


    Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies.

  3. Choline deficient diet enhances the initiating and promoting effects of methapyrilene hydrochloride in rat liver as assayed by the induction of gamma-glutamyltranspeptidase-positive hepatocyte foci.

    Perera, M. I.; Katyal, S. L.; Shinozuka, H.


    Earlier we demonstrated that short-term feeding of methapyrilene hydrochloride (MPH) and of a choline deficient (CD) diet to rats induced peroxidative damage of microsomal membrane lipids of liver cells. In the present study, we investigated whether a CD diet modifies the extent of MPH-induced lipid peroxidation and whether the modifications lead to changes in the initiating and promoting action of these agents using assays of the induction of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci. Addition of 0.1% MPH to a CD diet enhanced the extent of microsomal lipid peroxidation induced by a CD diet alone. Feeding a choline supplemented (CS) or a CD diet containing 0.1% MPH for 2 weeks followed by 7 weeks promotion by a CD diet plus phenobarbital was ineffective in inducing GGT-positive foci. Feeding MPH in a CS or a CD diet for 4 weeks, however, resulted in the development of substantial numbers of GGT-positive foci. There was a 3 fold increase in the number of foci in rats initiated with a CD + MPH diet over that in rats initiated with a CS + MPH diet. 0.1% MPH in a CS diet or a CD diet exerted significant promotional effects on the induction of GGT-positive foci in rats initiated with a single injection of diethylnitrosamine. Addition of MPH to a CD diet was additive in inducing GGT-positive foci. The results suggest that lipid peroxidation of the liver may be involved in the carcinogenic and/or promoting effects of MPH and a CD diet. PMID:2893639

  4. Angiogenesis Assays.

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P


    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.

  5. Evaluation of the antiandrogenic effects of flutamide, DDE, and linuron in the weanling rat assay using organ weight, histopathological, and proteomic approaches.

    Tinwell, Helen; Friry-Santini, Claire; Rouquié, David; Belluco, Sara; Elies, Laetitia; Pallen, Catherine; Bars, Remi


    The Organization for Economic Cooperation and Development (OECD) is currently funding the validation of the Hershberger assay as a rapid in vivo means of identifying (anti-) androgens. However, as the assay measures weight changes in the androgen-sensitive tissues of castrated rats, the evaluation of the androgen-stimulated intact weanling as a more ethical model to use in the assay has been requested. As part of the OECD validation exercise two weak antiandrogens, 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane (DDE) and linuron (LIN), were investigated in our laboratory at several dose levels in the testosterone propionate (TP)-stimulated weanling using flutamide (FM) as a positive control. In addition to weight measurements (sex accessory tissues [SATs], epididymides, and testes), histopathological assessment of the seminal vesicles, prostate, and testes was conducted for vehicle control, TP-stimulated, and TP-stimulated animals treated with FM or the top dose level of DDE or LIN. The modulation of a novel prostate protein associated with apoptosis, L-amino acid oxidase (LAO), was evaluated in these same treatment groups. Our gravimetric data (supported by the histopathology data) indicated that the weanling assay can detect SAT and epididymal weight changes induced by the antiandrogens evaluated. Inconsistent and variable data were recorded for the testicular weight and histopathological effects, suggesting that the testis is of little value in the identification of antiandrogens using this model. Three isoforms of LAO were identified, and all were regulated by TP. Modulation of LAO by the antiandrogens indicated that this protein could be a biomarker for endocrine disruption in male rodents.

  6. Ethanol-extracted Cameroonian propolis exerts estrogenic effects and alleviates hot flushes in ovariectomized Wistar rats.

    Zingue, Stéphane; Nde, Chantal Beatrice Magne; Michel, Thomas; Ndinteh, Derek Tantoh; Tchatchou, Jules; Adamou, Moïse; Fernandez, Xavier; Fohouo, Fernand-Nestor Tchuenguem; Clyne, Colin; Njamen, Dieudonné


    Since the biological properties of propolis depend to the plants that can be found in a specific region, propolis from unexplored regions attracts the attention of scientists. Ethanolic extract of Cameroonian propolis (EEP) is used to treat various ailments including gynecological problems and amenorrhea. Since there were no scientific data to support the above claims, the present study was therefore undertaken to assess estrogenic properties of Cameroonian propolis. To achieve our goal, the ability of EEP to induce MCF-7 cells proliferation in E-screen assay as well as to activate estrogen receptors α (ERα) and β (ERβ) in cell-based reporter gene assays using human embryonic kidney cells (HEK293T) transfected with ERs was tested. Further, a 3-day uterotrophic assay was performed and the ability of EEP to alleviate hot flushes in ovariectomized adult rats was evaluated. In vitro, EEP showed an antiestrogenic activity in both HEK293T ER-α and ER-β cells. In vivo, EEP induced a significant increase in a bell shape dose response manner of the uterine wet weight, the total protein levels in the uterus, the uterine and vaginal epithelium height and acini border cells of mammary gland with the presence of abundant eosinophil secretions. Moreover, EEP induced a significant decrease in the total number, average duration as well as frequency of hot flushes after 3 days of treatment in rat (equivalent to a month in woman). The dose of 150 mg/kg exhibited the most potent estrogenic effects among all the tested doses. The UPLC-HRMS analysis showed the presence of caffeic acid derivatives and trirtepernoids in EEP, which are well known endowed with estrogenic properties. These results suggest that Ethanolic extract of Cameroonian propolis has estrogen-like effects in vivo and may alleviate some menopausal problems such as vaginal dryness and hot flushes. Ethanol-extracted Cameroobian propolis exhibited in vitro and in vivo estrogen-like effects. This extract may contain

  7. Lack of marked cyto- and genotoxicity of cristobalite in devitrified (heated) alkaline earth silicate wools in short-term assays with cultured primary rat alveolar macrophages.

    Ziemann, Christina; Harrison, Paul T C; Bellmann, Bernd; Brown, Robert C; Zoitos, Bruce K; Class, Philippe


    Alkaline earth silicate (AES) wools are low-biopersistence high-temperature insulation wools. Following prolonged periods at high temperatures they may devitrify, producing crystalline silica (CS) polymorphs, including cristobalite, classified as carcinogenic to humans. Here we investigated the cytotoxic and genotoxic significance of cristobalite present in heated AES wools. Primary rat alveolar macrophages were incubated in vitro for 2 h with 200 µg/cm² unheated/heated calcium magnesium silicate wools (CMS1, CMS2, CMS3; heat-treated for 1 week at, or 4 weeks 150 °C below, their respective classification temperatures) or magnesium silicate wool (MS; heated for 24 h at 1260 °C). Types and quantities of CS formed, and fiber size distribution and shape were determined by X-ray diffraction and electron microscopy. Lactate dehydrogenase release and alkaline and hOGG1-modified comet assays were used, ± aluminum lactate (known to quench CS effects), for cytotoxicity/genotoxicity screening. Cristobalite content of wools increased with heating temperature and duration, paralleled by decreases in fiber length and changes in fiber shape. No marked cytotoxicity, and nearly no (CMS) or only slight (MS) DNA-strand break induction was observed, compared to the CS-negative control Al₂O₃, whereas DQ12 as CS-positive control was highly active. Some samples induced slight oxidative DNA damage, but no biological endpoint significantly correlated with free CS, quartz, or cristobalite. In conclusion, heating of AES wools mediates changes in CS content and fiber length/shape. While changes in fiber morphology can impact biological activity, cristobalite content appears minor or of no relevance to the intrinsic toxicity of heated AES wools in short-term assays with rat alveolar macrophages.

  8. Re-analysis results using medians of the data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay.

    Uno, Yoshifumi; Omori, Takashi


    The data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay were reported and analyzed statistically using the simple means of % tail DNA. However, OECD test guideline TG 489 recommends use of the median for data analysis due to the hierarchical nature of the data. Comparison between the simple mean approach and the median based approach for positive/negative/equivocal chemical calls was conducted using the % tail DNA data for the 40 chemicals tested in the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay, using liver and stomach as target organs. In the liver, two genotoxic chemicals, o-anisidine and 9-aminoacridine hydrochloride monohydrate, were positive using the median based approach but negative using the simple mean approach, and two genotoxic chemicals, 2-acetylaminofluorene and busulfan were equivocal using the median based approach but negative using the simple mean approach. In contrast, cadmium chloride (genotoxic carcinogen) was equivocal in both organs using the median based approach, while positive and equivocal in liver and stomach, respectively, using the simple mean approach. Two data sets of sodium arsenite showed equivocal and negative results for liver using the median based approach, although both data sets were equivocal using the simple mean approach. Overall, there are no large differences in terms of the genotoxic call between both approaches. However, the median based approach recommended in OECD TG 489 has an advantage toward higher precision within the groups treated with a test chemical, whereas the approach might show the lower values for the effect.

  9. Evaluation of a liver micronucleus assay in young rats (III): a study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

    Takasawa, Hironao; Suzuki, Hiroshi; Ogawa, Izumi; Shimada, Yasushi; Kobayashi, Kazuo; Terashima, Yukari; Matsumoto, Hirotaka; Aruga, Chinami; Oshida, Keiyu; Ohta, Ryo; Imamura, Tadashi; Miyazaki, Atsushi; Kawabata, Masayoshi; Minowa, Shigenori; Hayashi, Makoto


    We have been investigating a liver micronucleus assay to detect genotoxic chemicals using young rats for several years, and had established its advantages with respect to using autonomous proliferation of young rat hepatocytes. Nine chemicals known to induce hepatotoxic effects such as necrosis (2,6-dinitrotolune, bromobenzene, isoniazid, phenacetin, allyl alcohol and thioacetamide), cholestasis (chlorpromazine hydrochloride and alpha-naphthyl isothiocyanate) and oxidative stress (clofibrate) were selected for this study. A liver micronucleus assay was conducted in 4-week-old male F344 rats using two or three dose levels of test chemicals given orally by gavage to evaluate the compound's ability to induce micronucleated hepatocytes. Several of these test chemicals were additionally examined in a peripheral blood micronucleus assay conducted concurrently and in the same animals. The genotoxic rodent hepatocarcinogen, 2,6-dinitrotoluene showed a positive result in the liver micronucleus assay, but the nongenotoxic hepatocarcinogens, clofibrate and thioacetamide gave negative responses. Bromobenzene, known to produce DNA adducts but is noncarcinogenic in rodent liver, was judged equivocal in this assay. alpha-Naphthyl isothiocyanate is noncarcinogenic and showed negative response in the liver. The other four chemicals, known to be either noncarcinogenic or carcinogenic in other non-liver target organs, showed negative results in the liver micronucleus assay. Based on the results in the present study and previous report described above, it was concluded that this technique is able to effectively predict genotoxic rodent hepatocarcinogenicity, and does not give false positives due to hepatotoxicity.

  10. Toxic Effects of Titanium Dioxide Nanoparticles and Titanium Dioxide Bulk Salt in the Liver and Blood of Male Sprague-Dawley Rats Assessed by Different Assays.

    Shakeel, Muhammad; Jabeen, Farhat; Qureshi, Naureen Aziz; Fakhr-E-Alam, Muhammad


    This study evaluated the toxic effects of titanium dioxide (TiO2) bulk salt as well as its nanoparticles (NPs) in anatase phase with mean crystallite size of 36.15 nm in male Sprague-Dawley rats by subcutaneous injections at four different dose levels of either control (0), 50, 100 or 150 mg/kg of body weight (BW) of rat for 28 days on alternate days. Animal mortality, haematology, micronucleus assay, liver histology and activities of liver tissue damage markers like, alkaline phosphate (ALP), alanine transaminase (ALT), aspartate transaminase (AST), as well as oxidative stress indicators like superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) were investigated. The study revealed significant differences (P < 0.05) among control and experimental groups in all the haematological parameters at the end of experiment. Significantly elevated levels (P < 0.05) of ALT, AST and ALP were found for the group treated with TiO2 NPs at the dose of 150 mg/kg of body weight as compared to control. TiO2 and TiO2 NPs caused dose-dependent genotoxicity in the blood cells of the treated rat as revealed by micronuclei test. The highest frequency of micronuclei was observed in rats treated with NPs at the dose of 150 mg/kg BW which was significantly different (P < 0.001) from all other experimental groups after 28 days of exposure. Similarly, all the treatments showed dose-dependent oxidative stress in the treated rats. However, the significantly high decline in the activities of CAT, SOD, and GST as well as elevation in malondialdehyde and GSH was observed in the group receiving NPs at the rate of 150 mg/kg BW. TiO2 also caused histological alterations in the liver. The study revealed that higher dose of TiO2 NPs exerted significantly harmful effects on liver and blood as compared to its lower doses as well as from all other doses of their bulk counterparts.

  11. Data supporting the rat brain sample preparation and validation assays for simultaneous determination of 8 neurotransmitters and their metabolites using liquid chromatography–tandem mass spectrometry

    Aneta Wojnicz


    Full Text Available The data presented in this article supports the rat brain sample preparation procedure previous to its injection into the liquid chromatography–tandem mass spectrometry (LC–MS/MS system to monitor levels of adrenaline, noradrenaline, glutamic acid, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine, 5-hydroxyindole acetic acid, and 3-methoxy-4-hydroxyphenylglycol. In addition, we describe the method validation assays (such as calibration curve, lower limit of quantification, precision and accuracy intra- and inter-day, selectivity, extraction recovery and matrix effect, stability, and carry-over effect according to the United States Food and Drug Administration and European Medicine Agency to measure in one step different neurotransmitters and their metabolites. The data supplied in this article is related to the research study entitled: “Simultaneous determination of 8 neurotransmitters and their metabolite levels in rat brain using liquid chromatography in tandem with mass spectrometry: application to the murine Nrf2 model of depression” (Wojnicz et al. 2016 [1].

  12. Data supporting the rat brain sample preparation and validation assays for simultaneous determination of 8 neurotransmitters and their metabolites using liquid chromatography–tandem mass spectrometry

    Wojnicz, Aneta; Ortiz, José Avendaño; Casas, Ana I.; Freitas, Andiara E.; López, Manuela G.; Ruiz-Nuño, Ana


    The data presented in this article supports the rat brain sample preparation procedure previous to its injection into the liquid chromatography–tandem mass spectrometry (LC–MS/MS) system to monitor levels of adrenaline, noradrenaline, glutamic acid, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine, 5-hydroxyindole acetic acid, and 3-methoxy-4-hydroxyphenylglycol. In addition, we describe the method validation assays (such as calibration curve, lower limit of quantification, precision and accuracy intra- and inter-day, selectivity, extraction recovery and matrix effect, stability, and carry-over effect) according to the United States Food and Drug Administration and European Medicine Agency to measure in one step different neurotransmitters and their metabolites. The data supplied in this article is related to the research study entitled: “Simultaneous determination of 8 neurotransmitters and their metabolite levels in rat brain using liquid chromatography in tandem with mass spectrometry: application to the murine Nrf2 model of depression” (Wojnicz et al. 2016) [1]. PMID:27054183

  13. Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine.

    McNamee, J P; Bellier, P V


    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes.

  14. Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry.

    Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang, Leyu; Moore, Jennifer; Kuo, Ming-Shang T; LaMarr, William A; Ozbal, Can C; Bhat, B Ganesh


    Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K(m)=10.5 microM). The assay was highly reproducible with an average Z' value=0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC(50) values of 0.88 and 0.12 microM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 microM--14% conformation rate). Of the confirmed hits 172 had IC(50) values of <10 microM, including 111 <1 microM and 48 <100 nM. A large number of potent drug-like (MW<450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further

  15. Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

    Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang Leyu [Research Technology Center, Pfizer Global Research and Development, Cambridge, MA (United States); Moore, Jennifer; Kuo, Ming-Shang T. [Pfizer Global Research and Development, San Diego, CA (United States); LaMarr, William A.; Ozbal, Can C. [Biotrove, Inc., Woburn, MA (United States); Bhat, B. Ganesh [Pfizer Global Research and Development, San Diego, CA (United States)], E-mail:


    Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K{sub m} = 10.5 {mu}M). The assay was highly reproducible with an average Z' value = 0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC{sub 50} values of 0.88 and 0.12 {mu}M, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 {mu}M - 14% conformation rate). Of the confirmed hits 172 had IC{sub 50} values of <10 {mu}M, including 111 <1 {mu}M and 48 <100 nM. A large number of potent drug-like (MW < 450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable

  16. Neuroprotective effects of pretreatment with quercetin as assessed by acetylcholinesterase assay and behavioral testing in poloxamer-407 induced hyperlipidemic rats.

    Braun, Josiane B S; Ruchel, Jader B; Adefegha, Stephen A; Coelho, Ana Paula V; Trelles, Kelly B; Signor, Cristiane; Rubin, Maribel A; Oliveira, Juliana S; Dornelles, Guilherme L; de Andrade, Cinthia M; Castilhos, Lívia G; Leal, Daniela B R


    Hyperlipidemia is a group of disorders characterized by excessive lipids in the bloodstream. It is associated with the incidence of cardiovascular diseases and recognized as the most important factor underlying the occurrence of atherosclerosis. This study was conducted to investigate whether pretreatment with quercetin can protect against possible memory impairment and deterioration of the cholinergic system in hyperlipidemic rats. Animals were divided into ten groups (n=7): saline/control, saline/quercetin 5mg/kg, saline/quercetin 25mg/kg, saline/quercetin 50mg/kg, saline/simvastatin (0.04mg/kg), hyperlipidemia, hyperlipidemia/quercetin 5mg/kg, hyperlipidemia/quercetin 25mg/kg, hyperlipidemia/quercetin 50mg/kg and hyperlipidemia/simvastatin. The animals were pretreated with quercetin by oral gavage for a period of 30days and hyperlipidemia was subsequently induced by intraperitoneal administration of a single dose of 500mg/kg of poloxamer-407. Simvastatin was administered after the induction of hyperlipidemia. The results demonstrated that hyperlipidemic rats had memory impairment compared with the saline control group (P<0.001). However, pretreatment with quercetin and simvastatin treatment attenuated the damage caused by hyperlipidemia compared with the hyperlipidemic group (P<0.05). Acetylcholinesterase (AChE) activity in the cerebral hippocampus was significantly (P<0.001) reduced in the hyperlipidemic group compared with the control saline group. Pretreatment with quercetin and simvastatin treatment in the hyperlipidemic groups significantly (P<0.05) increased AChE activity compared with the hyperlipidemic group. Our results thus suggest that quercetin may prevent memory impairment, alter lipid metabolism, and modulate AChE activity in an experimental model of hyperlipidemia.

  17. A demonstration of the uncertainty in predicting the estrogenic activity of individual chemicals and mixtures from an in vitro estrogen receptor transcriptional activation assay (T47D-KBluc) to the in vivo uterotrophic assay using oral exposure

    U.S. Environmental Protection Agency — the data set contains the figures and tables from the publication in addition to the means, standard errors of the mean and the sample sizes used in each group for...

  18. Individual and mixture endocrine activity of BPS and BPC using in vitro estrogenic/anti-androgenic transcriptional activation assays and the in vivo uterotrophic assay- RAP 2.2-SSWR

    Bisphenol A (BPA) is gradually being phased out of many consumer products and processes leading to potential increases in human and environmental exposures to relatively understudied replacement compounds, including Bisphenol S (BPS) and Bisphenol C (BPC).Research from our lab ha...

  19. Radiosensitivity of glial progenitor cells of the perinatal and adult rat optic nerve studied by an in vitro clonogenic assay

    Maazen, R.W.M. van der; Verhagen, I.; Kleiboer, B.J.; Kogel, A.J. van der (Nijmegen University (Netherlands). Institute of Radiotherapy)


    The cellular basis of radiation-induced demyelination and white matter necrosis of the central nervous system (CNS), is poorly understood. Glial cells responsible for myelination in the CNS might be the target cells of this type of damage. Glial cells with stem cell properties derived from the perinatal and adult rat CNS can be cultured in vitro. These cells are able to differentiate into oligodendrocytes or type-2 astrocytes (O-2A) depending on the culture conditions. Growth factors produced by monolayers of type-1 astrocytes inhibit premature differentiation of O-2A progenitor cells and allow colony formation. A method which employs these monolayers of type-1 astrocytes to culture O-2A progenitor cells has been adapted to allow the analysis of colonies of surviving cells after X-irradiation. In vitro survival curves were obtained for glial progenitor cells derived from perinatal and adult optic nerves. The intrinsic radiosensitivity of perinatal and adult O-2A progenitor cells showed a large difference. Perinatal O-2A progenitor cells are quite radiosensitive, in contrast to adult O-2A progenitor cells. For both cell types an inverse relationship was found between the dose and the size of colonies derived from surviving cells. Surviving O-2A progenitor cells maintain their ability to differentiate into oligo-dendrocytes or type-2 astrocytes. This system to assess radiation-induced damage to glial progenitor cells in vitro systems to have a great potential in unraveling the cellular basis of radiation-induced demyelinating syndromes of the CNS. (author). 28 refs.; 4 figs.; 1 tab.

  20. Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera.

    Tang, W X; Bazaraa, H M; Magiera, H; Cooke, N E; Haddad, J G


    Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.

  1. Annexin V assay-proven anti-apoptotic effect of ascorbic acid 2-glucoside after cold ischemia/reperfusion injury in rat liver transplantation.

    Liu J


    Full Text Available Controversy exists over whether the predominant cell death of hepatocytes is due to apoptosis or necrosis after ischemia/reperfusion injury. In this study we investigated the predominant cell death of hepatocytes after cold ischemia/reperfusion injury using the Annexin V-based assay, and evaluated the anti-apoptotic effect of ascorbic acid 2-glucoside (AA-2G added to the University of Wisconsin solution (UW solution in rat liver transplantation. The retrieved liver was preserved in 4 UW solution for 24 h, and then transplanted orthotopically to the syngeneic Wistar recipient. The animals were divided into 2 groups, a control group (n=10, in which liver grafts were preserved in UW solution (4, and an AA-2G group (n=10, in which liver grafts were preserved in UW solution (4 with AA-2G (100 ug/ml. The serum AST level 4 h after reperfusion in the control group was significantly suppressed in the AA-2G group, and the bile production of the liver graft in the AA-2G group was well recovered. The mean survival time in the AA-2G group was significantly improved compared with that in the control group. Annexin-V and Propidium iodide staining 4 h after reperfusion showed a significantly higher percentage of viable hepatocytes in the AA-2G group compared with the control group (93.4 +/- 2.0 vs. 80.3 +- 2.1%, P<0.05. In the control group, the main cell death of hepatocytes was apoptosis (early apoptosis: 10.0 +- 4.7%, late apoptosis: 6.4 +/- 1.7%. The addition of AA-2G to the UW solution significantly inhibited both early and late apoptotic cell death 4 h after reperfusion (early apoptosis: 0.98 +/- 0.88%, late apoptosis: 2.2 +/- 1.1%. The expression of caspase 9 in the immunostaining of the liver graft was suppressed in the AA-2G group compared with in the control group. Our study using the Annexin V-based assay provided evidence that the predominant cell death of hepatocytes was apoptosis after 24 h cold ischemia/reperfusion injury in rat liver

  2. H2O2-mediated DNA damage and repair in the brain cells in the aging rats detected by comet assay

    Suming ZhangM.D., Ph.D; Zongchao Han, M.D.; Siyu Fang, M.D.; Ruan Yang, M.D; Wei Wang, M.D., Ph. D


    Objective: To identify the relation between DNA damage susceptibility/ DNA repair capability and aging process after insults, an observation of H2O2_induced DNA damage and the kinetics of DNA repair in senescent murine brain cells with the alkaline single cell gel electrophoresis (SCGE/Comet assay) was made. Methods: The dissociated brain cells harvested in the area of the cerebral cortex, hippocampus, basal gang]ion from 3-month (n=10), 8-month (n=8) and 26-month (n=5) old rats were respectively treated with H2O2 in gradient doses for 10 min, or without H2O2 as controls. The cells embedded in agarose were lysed, helix-untied, electrophoresed, stained with a fluorescence DNA binding stain, viewed under a fluorescence microscope. Individual image was optically recorded. The frequency of the tailed cells and the grade of tails wereused to analyze single strand breaks of DNA and injury intensity. Results: By the cell and DNA image like comets, a linear increase was noticed in vulnerability of DNA both to H2O2 doses and to the age. Regarding the damaged region of the brain, the cortex cells were more vulnerable to the insult than the hippocampus/basal ganglionic cells. Whatever aging or not the cells were, the maximum of ratio of DNA repair was only within 1 hour during the incubation for 0.5-4 hours after the insults. Furthermore, the more aging, the less ratio of DNA repair of sick cells. Conclusion: The DNA damagesusceptibility and the DNA repair capability of individual cells, whatever its age is, can be detected by this brain cell injury model. Comet assay is a sensitive way to find out DNA damage and repair of the cells. It should be more difficult for the cells to cope with an acute and excessive than with a persistent, chronic and mild DNA damage which is more related to an accumulating injury, the aging.

  3. In vivo and in vitro estrogenic activity of the antidepressant fluoxetine.

    Müller, Juliane C; Imazaki, Pedro H; Boareto, Ana C; Lourenço, Emerson L B; Golin, Munisa; Vechi, Marina F; Lombardi, Natália F; Minatovicz, Bruna C; Scippo, Marie-Louise; Martino-Andrade, Anderson J; Dalsenter, Paulo R


    Recent years have seen an increase in the use of antidepressant drugs, especially fluoxetine (FLX), in sensitive populations, such as pregnant and lactating women. Although some evidence suggests a possible endocrine action of FLX, no specific studies have been performed to investigate this hypothesis. In the present study, we investigated the possible (anti)androgenic and (anti)estrogenic actions of FLX using Hershberger, uterotrophic (0.4, 1.7, and 17mg/kg), and reporter gene (7.6-129μM) assays. In the Hershberger assay, no differences were observed in androgen-dependent organ weights. However, the uterotrophic and gene reporter assays indicated a possible estrogenic action of FLX. Uterine weight increased in the 1.7 and 17mg/kg/day groups in the 3-day uterotrophic assay in immature rats. Additionally, noncytotoxic concentrations of FLX induced estrogenic responses and increased the estrogenic response of estradiol in MCF-7 breast cancer cells transfected with luciferase.

  4. Repair capacity of adult rat glial progenitor cells determined by an in vitro clonogenic assay after in vitro or in vivo fractionated irradiation

    Maazen, R.W.M. van der; Kleiboer, B.J.; Verhagen, I.; Kogel, A.J. van der (Nijmegen Univ. (Netherlands). Inst. of Radiotherapy)


    Demyelination is one condition identified as a late response of the central nervous system (CNS) to irradiation. In the present study the repair capacity of glial stem cells was investigated and compared with the repair capacity of the CNS in vivo using functional endpoints. For this purpose, glial stem cells, derived from the adult rat optic nerve, were subjected to fractionated irradiation in vivo and in vitro and their survival was quantified with an in vitro clonogenic assay. The data were analysed by three different methods all based on the LQ-model (single dose survival curve; '[beta][sub RR]', 'F[sub e]-plot'). The resulting value of the [beta]-parameter of adult glial stem cells is consistent with values obtained for functional endpoints after irradiation of the CNS in vivo. The [alpha]/[beta]-ratio (4.9-7.3 Gy) of adult glial stem cells, however, is higher than the [alpha]/[beta]-ratio ([approx] 2 Gy) obtained for CNS in vivo and is closer to that of an acute responding tissue. (Author).

  5. HPLC assay for the determination of PD 117,302, a Kappa analgesic, and its application to disposition studies in rats

    Young, R.M.; Chang, T.


    PD 117,302-2 (I) trans-(+)-N-methyl-N-(2-(1-pyrrolidinyl) cyclohexyl)benzo(b)thiophene-4-acetamide, monohydrochloride is a new Kappa analgesic. To facilitate preclinical development, an HPLC assay was developed. (I) and an internal standard were extracted from alkalinized rat plasma with CH/sub 2/Cl/sub 2/. HPLC was performed on a Waters Nova-Pak C/sub 18/ column (3.9 mm x 15 cm) using 0.5% triethylamine in 50:50 CH/sub 3/CN/0.05M NH/sub 4/H/sub 2/PO/sub 4/ (pH 6.7) at a flow rate of 2 ml/min. UV absorbancy was monitored at 237 nm. Calibration curves were linear over a range of 80-4000 ng/ml. Within-day precision ranged 0.1-9.0% and between-day precision ranged 3.1-7.6%. /sup 14/C-(I) given orally (10 mg/kg) was completely absorbed and extensively metabolized. Elimination t 1/2 of (I) averaged 2 hr. Fecal and urinary /sup 14/C recoveries accounted for 54-76% and 9-12% of the dose, respectively. No intact drug was found in feces, indicating that the high fecal /sup 14/C recovery was due to the presence of metabolites(s) rather than poor absorption.

  6. Assay of 6-gingerol in CO2 supercritical fluid extracts of ginger and evaluation of its sustained release from a transdermal delivery system across rat skin.

    Chen, Yan; Zhang, Cuiping; Zhang, Mei; Fu, Xiaobing


    Ginger has been widely used as healthy food condiment as well as traditional Chinese medicine since antiquity. Multiple potentials of ginger for treatment of various ailments have been revealed. However, the biological half-life of 6-gingerol (a principal pungent ingredient of ginger) is only 7.23 minutes while taken orally. Delivery of ginger compositions by routes other than oral have scarcely been reported. Therefore, we studied a noninvasive transdermal drug delivery system (TDDS) of ginger to bypass hepatic first pass metabolism, avoid gastrointestinal degradation and achieve long persistent release of effective compositions. After establishment of a HPLC analysis method of 6-gingerol, assays of 6-gingerol were performed to compare two kinds of ginger extracts. Then, the characteristics of transdermal delivery of 6-gingerol in TDDS were exhibited. The results showed that the contents of 6-gingerol in two kinds of ginger extracts were significantly different. The maximal delivery percentage of 6-gingerol across rat skin at 20 h was more than 40% in different TDDS formulations. TDDS may provide long-lasting delivery of ginger compounds.

  7. In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test.

    Hayashi, Makoto; MacGregor, James T; Gatehouse, David G; Blakey, David H; Dertinger, Stephen D; Abramsson-Zetterberg, Lilianne; Krishna, Gopala; Morita, Takeshi; Russo, Antonella; Asano, Norihide; Suzuki, Hiroshi; Ohyama, Wakako; Gibson, Dave


    The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed

  8. In Vivo and In Vitro Effects of Bidens Pilosa L. (Asteraceae) Leaf Aqueous and Ethanol Extracts on Primed-Oestrogenized Rat Uterine Muscle

    Frida, Longo; Rakotonirina, Silvíre; Rakotonirina, Alice; Savineau, Jean-Pierre


    Bidens pilosa L. is an Asteraceae growing in tropical zones, and traditionally utilized worldwide in herbal medicine. The present work is based on its traditional use during child birth as a labour facilitator. In vivo tests of acute toxicity showed a weak toxic effect for both extracts but the toxicity of the ethanol extract (LD50=6.15g/kg) was upper than that of the aqueous extract (LD50=12.30g/kg). The three-days uterotrophic assay on immature mice showed body weight gain followed by a con...

  9. Scientific and Regulatory Policy Committee (SRPC) Points to Consider: Histopathology Evaluation of the Pubertal Development and Thyroid Function Assay (OPPTS 890.1450, OPPTS 890.1500) in Rats to Screen for Endocrine Disruptors.

    Keane, Kevin A; Parker, George A; Regan, Karen S; Picut, Catherine; Dixon, Darlene; Creasy, Dianne; Giri, Dipak; Hukkanen, Renee R


    The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a multitiered approach to determine the potential for environmental chemicals to alter the endocrine system. The Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female and Male Rats (OPPTS 890.1450, 890.1500) are 2 of the 9 EDSP tier 1 test Guidelines, which assess upstream mechanistic pathways along with downstream morphological end points including histological evaluation of the kidneys, thyroid, and select male/female reproductive tissues (ovaries, uterus, testes, and epididymides). These assays are part of a battery of in vivo and in vitro screens used for initial detection of test article endocrine activity. In this Points to Consider article, we describe tissue processing, evaluation, and nomenclature to aid in standardization of assay results across laboratories. Pubertal assay end points addressed include organ weights, estrous cyclicity, clinical pathology, hormonal assays, and histological evaluation. Potential treatment-related findings that may indicate endocrine disruption are reviewed. Additional tissues that may be useful in assessment of endocrine disruption (vagina, mammary glands, and liver) are discussed. This Points to Consider article is intended to provide information for evaluating peripubertal tissues within the context of individual assay end points, the overall pubertal assay, and tier I assays of the EDSP program.

  10. Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese center for the validation of alternative methods (JaCVAM) international validation study of the in vivo rat alkaline comet assay.

    Plappert-Helbig, Ulla; Junker-Walker, Ursula; Martus, Hans-Joerg


    As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship. 2,6-diaminotoluene induced significantly elevated DNA damage in the liver at each dose and a statistically significant dose-response relationship whereas no DNA damage was obtained in the stomach. 5-fluorouracil did not induce DNA damage in either liver or stomach.

  11. Rat Pig-a mutation assay responds to the genotoxic carcinogen ethyl carbamate but not the non-genotoxic carcinogen methyl carbamate.

    Bemis, Jeffrey C; Labash, Carson; Avlasevich, Svetlana L; Carlson, Kristine; Berg, Ariel; Torous, Dorothea K; Barragato, Matthew; MacGregor, James T; Dertinger, Stephen D


    Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500 mg/kg/day) or EC (250 mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9×10(-6) on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2×10(-6) on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail:

  12. Assays for calcitonin receptors

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.


    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  13. Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue™.

    Schoonen, Willem G E J; Stevenson, Joe C R; Westerink, Walter M A; Horbach, G Jean


    In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue™ (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72 h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5mM of glucose. The choice for the 109 reference compounds was based on the so-called Multicentre Evaluation for In vitro Cytotoxicity (MEIC) and on diverse drug categories. For 59 toxic reference compounds, an evaluation for both assays was carried up to 10(-3)M. Toxicity was demonstrated with MitoXpress for 23 (39%) and 36 (61%) compounds in H4IIE and HepG2 cells, respectively, and with AB for 44 (75%) and 40 (68%) compounds. For 50 more pharmaceutical drugs more physiological concentrations were used up to 3.16×10(-5)M, and only 19 (38%) of these compounds appeared to be toxic in both assays. In conclusion, overall 63 (58%) and 60 (55%) compounds showed toxic effects with the MitoXpress and AB assays on rat H4IIE and human HepG2 hepatocytes, respectively. AB assays were more sensitive with respect to H4IIE cells and MitoXpress assays with respect to HepG2 cells. At all tested time intervals, MitoXpress showed its sensitivity, while AB is more sensitive at 48 and 72 h. With AB more toxic compounds

  14. Predictive endocrine testing in the 21st century using in vitro assays of estrogen receptor signaling responses.

    Rotroff, Daniel M; Martin, Matt T; Dix, David J; Filer, Dayne L; Houck, Keith A; Knudsen, Thomas B; Sipes, Nisha S; Reif, David M; Xia, Menghang; Huang, Ruili; Judson, Richard S


    Thousands of environmental chemicals are subject to regulatory review for their potential to be endocrine disruptors (ED). In vitro high-throughput screening (HTS) assays have emerged as a potential tool for prioritizing chemicals for ED-related whole-animal tests. In this study, 1814 chemicals including pesticide active and inert ingredients, industrial chemicals, food additives, and pharmaceuticals were evaluated in a panel of 13 in vitro HTS assays. The panel of in vitro assays interrogated multiple end points related to estrogen receptor (ER) signaling, namely binding, agonist, antagonist, and cell growth responses. The results from the in vitro assays were used to create an ER Interaction Score. For 36 reference chemicals, an ER Interaction Score >0 showed 100% sensitivity and 87.5% specificity for classifying potential ER activity. The magnitude of the ER Interaction Score was significantly related to the potency classification of the reference chemicals (p vivo uterotrophic data, the ER Interaction Scores showed 91% sensitivity and 65% specificity. Overall, this study provides a novel method for combining in vitro concentration response data from multiple assays and, when applied to a large set of ER data, accurately predicted estrogenic responses and demonstrated its utility for chemical prioritization.

  15. Enzyme assays.

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie


    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  16. Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices

    Plazar, Janja; Hrejac, Irena; Pirih, Primoz; Filipic, Metka; Groothuis, Geny M. M.


    The comet assay is a simple and sensitive method for measuring DNA damage at the level of individual cells and is extensively used in genotoxicity studies. It is commonly applied to cultured cells. The aim of this study was to apply the comet assay for use in fresh liver tissue, where metabolic acti

  17. Characterization of degradation process of sucrase-isomaltase in rat jejunum with monoclonal-antibody-based enzyme-linked immunosorbent assay

    Goda, T; Quaroni, A; Koldovský, O


    ...]. To characterize further the process of sucrase-isomaltase degradation in jejunum, we determined the amounts of immunoreactive sucrase-isomaltase in rat jejunum by using a monoclonal-antibody-based...

  18. Evaluation of a repeated dose liver micronucleus assay in rats treated with two genotoxic hepatocarcinogens, dimethylnitrosamine and 2-acetylaminofluorene: the possibility of integrating micronucleus tests with multiple tissues into a repeated dose general toxicity study.

    Takashima, Rie; Takasawa, Hironao; Kawasako, Kazufumi; Ohyama, Wakako; Okada, Emiko; Narumi, Kazunori; Fujiishi, Yohei; Wako, Yumi; Yasunaga, Katsuaki; Hattori, Akiko; Kawabata, Masayoshi; Nakadate, Kiyoko; Nakagawa, Munehiro; Hamada, Shuichi


    As part of a collaborative study by the Collaborative Study Group for Micronucleus Test (CSGMT) of the Mammalian Mutagenicity Study Group (MMS) in the Japanese Environmental Mutagen Society (JEMS), the present study evaluated the effectiveness of the repeated dose liver micronucleus (RDLMN) assay. Two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF), were administered orally to male rats (6 weeks old at the initial dosing) once daily for 14 and 28 days to evaluate the micronucleus (MN) inducibility in the liver. In addition, these chemicals were evaluated for MN inducibility in the bone marrow (BM) and gastrointestinal (GI) tract, i.e. glandular stomach and colon of the same animals used in the RDLMN assay. As a result, both chemicals produced positive results in the liver, although a weak positive response was given by 2-AAF. DMN gave negative results in the tissues other than the liver. 2-AAF produced positive responses in the BM and glandular stomach, and a prominent response was particularly observed in the glandular stomach, which is directly exposed to the test chemicals by gavage. The present results suggest that the RDLMN assay is a useful method for detecting genotoxic hepatocarcinogens, and that it is especially effective for evaluating test chemicals, such as DMN, undetectable by the BM and GI tract MN assay. Moreover, the results in this investigation indicate that the use of multiple tissues in the study integrating the MN tests is more effective than using a single tissue, for detection of the MN induction produced by chemical exposure to rats, and helps to determine the characteristics of the test chemicals.

  19. Rats

    Alexey Kondrashov


    Full Text Available We aimed to perform a chemical analysis of both Alibernet red wine and an alcohol-free Alibernet red wine extract (AWE and to investigate the effects of AWE on nitric oxide and reactive oxygen species production as well as blood pressure development in normotensive Wistar Kyoto (WKY and spontaneously hypertensive rats (SHRs. Total antioxidant capacity together with total phenolic and selected mineral content was measured in wine and AWE. Young 6-week-old male WKY and SHR were treated with AWE (24,2 mg/kg/day for 3 weeks. Total NOS and SOD activities, eNOS and SOD1 protein expressions, and superoxide production were determined in the tissues. Both antioxidant capacity and phenolic content were significantly higher in AWE compared to wine. The AWE increased NOS activity in the left ventricle, aorta, and kidney of SHR, while it did not change NOS activity in WKY rats. Similarly, increased SOD activity in the plasma and left ventricle was observed in SHR only. There were no changes in eNOS and SOD1 expressions. In conclusion, phenolics and minerals included in AWE may contribute directly to increased NOS and SOD activities of SHR. Nevertheless, 3 weeks of AWE treatment failed to affect blood pressure of SHR.

  20. Comparison of culture and PCR assays for detection of bacteria in laboratory rats and mice%培养法和 PCR法用于实验大、小鼠细菌检测的比较分析

    冯洁; 谢建云; 冯丽萍; 魏晓锋; 高诚


    Objective To compare the efficiency of bacteria culture and PCR assays for detection of Staphylococcus aureus ( S.aureus) , Pseudomonas aeruginosa ( P.aeruginosa) and Klebsiella pneumoniae ( K.pneumoniae) in laboratory rats and mice.Methods Bacteria culture combined with biochemical identification and PCR assay were used to detect 78 SPF rats and 422 SPF mice and the results of the two methods were compared .Results All the 78 rats were negative .Of the 422 mice, the positive rate by culture was 7.11%(30/422), of which, 10 were S.aureus, 22 were P.aeruginosa, and 2 were K.pneumoniae.The positive rate by PCR was 7.58%(32/422), of which, 10 were S.aureus, 25 were P. aeruginosa, and 2 were K.pneumoniae.Conclusions The high sensitivity , rapid procedure and easy to operate of PCR assay makes it valuable for rapid bacteria diagnosis and large-scale screening in laboratory animals .%目的:比较培养法和PCR法对实验大、小鼠的细菌检测效果。方法分别采用传统细菌分离培养结合生化鉴定方法和PCR方法,对78只SPF级大鼠和422只SPF级小鼠进行检测,对两种方法的检测结果进行比较分析。结果78只SPF级大鼠均未检测出阳性。422只SPF级小鼠,培养法检测阳性率7.11%(30/422),其中金黄色葡萄球菌10只,绿脓杆菌22只,肺炎克雷伯杆菌2只。 PCR法检测阳性率7.58%(32/422),其中金黄色葡萄球菌10只,绿脓杆菌25只,肺炎克雷伯杆菌2只。结论 PCR法的敏感性高于培养法,操作简便、快捷,适用于实验动物细菌的快速诊断和大规模的质量筛查。

  1. 90-Day Inhalation Toxicity Study of Swedish Biofuel Alcohol-to-Jet (ATJ) Synthetic Kerosene with Aromatics (SPA) in Rats with Neurotoxicity Testing and Genotoxicity Assay


    effects . Numeric results for rears, stereotypical grooming bouts, hind limb splay, and forelimb and hindlimb grip strengths are shown in Table 6 (above... effects and evidence of genotoxicity and mutagenicity were both negative . Limited and minor neurological effects at 2000 mg/m3 were found. Some mild... effect to the respiratory by gender . All remaining lesions in the respiratory system are common background lesions found in research rats. The

  2. In vivo genotoxic effects of dietary heme iron on rat colon mucosa and ex vivo effects on colon cells monitored by an optimized alkaline comet assay.

    Océane, C Martin


    In conclusion, our results offer a suitable protocol to evaluate genotoxicity on in vivo cryopreserved colon mucosa and on in vitro murine colonic cells, with a middle throughput capacity. This protocol confirms the increase of genotoxicity in rat colon mucosa after an heme-iron diet. Moreover, this protocol enables the demonstration that aldehydes from heme-induced lipoperoxidation are responsible for this increase of genotoxicity.

  3. 药物类过敏反应的临床前评价方法研究(Ⅱ)——大鼠皮肤类过敏试验%Methodology for preclinical assay of pseudoallergy of injectable drugs ( Ⅱ )——rat model for assay of cutaneous pseudoallergy induced by injections

    梁爱华; 赵雍; 李春英; 易艳; 王云庭; 李桂琴; 叶祖光


    Objective: To develop animal models and methodologies for assay of pseudoallergy induced by injectable drugs. Method: Rats cutaneous anaphylactoid reaction model was developed by intravenous injection of 0. 6% Evans blue (EB) followed by intracutaneous injection of test substance solutions 50 μL Diameters of subcutaneous blue spots and EB exudation were assayed. Result: Rat anaphylactoid reaction was characterized as vascular hyperpermeability which was measured by diameters of blue spots inside the skin and the EB exudation of the blue spots. Compound 48/80 caused severe bluing and EB exudation in the skin by inducing obvious vascular hyperpermeability which indicated that it can induce rat skin pseudoallergy. Normal saline or 5% glucose injection showed no obvious reactions. The rat pseudoallergy model was validated by intracutaneous injections of western drug injections and Chinese medicine. Conclusion:Rats could be developed into skin pseudoallergy model for preclinical safety evaluation of injectable drugs. The pseudoallergy reaction in this model is of high clinic consistency, sensitivity, reproducibility, and maneuverability. The model is suitable for the evaluation for pseudoallergy induced by injectable products prepared from Chinese materia medica. This model can also be used for safety assay and quality control in manufacturing process, spot checking of marketed products, screening of allergen as well as studying of pseudoallergy mechanism.%目的:建立适用于注射剂类过敏评价的动物模型.方法:大鼠静脉注射含0.6%伊文思蓝(EB)的生理盐水溶液,10 min后,背部皮内注射受试物50μL,20 min后,测量皮内蓝斑直径和伊文思蓝渗出量,以评价类过敏反应程度.结果:生理盐水注射液(NS)、5%葡萄糖注射液造成很小的皮内蓝斑.阳性对照物Compound48/80可导致局部毛细血管通透性显著增高和水肿,从而引起较大的蓝斑和EB大量渗出.采用临床上可发生类过敏反

  4. Cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry for screening bioactive capilliposide C metabolites generated by rat intestinal microflora.

    Cheng, Zhongzhe; Huang, Meilin; Chen, Guiying; Yang, Guangjie; Zhou, Xin; Chen, Chang; Zhang, Yang; Xu, Yong; Feng, Yulin; Zhang, Lin; Jiang, Hongliang


    Many plant-derived glycosides are used as medications. It is common that these glycosides show poor intestinal absorption but their metabolites generated by intestinal microflora demonstrate strong bioactivity. Hence, it is crucial to develop a method for the identification and characterization of the metabolites, and consequently reveal the pathway in which the glycosides are processed in gut. In this study, cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) were developed for rapid discovery and evaluation of the metabolites of a glycoside compound, capilliposide C (LC-C). 92.7% of LC-C was biotransformed by rat intestinal microflora after 36-h incubation at 37°C. Human cancer cell lines HepG2, PC-3 and A549 was treated with metabolites pool, respectively, which was followed by cell viability assays and characterization of metabolites using UHPLC-QTOF-MS/MS. As a result, significant cytotoxicity was observed for the metabolites pool, from which six metabolites were identified. Based on the metabolites identified, deglycosylation and esterolysis were proposed as the major metabolic pathways of LC-C in rat intestinal microflora. In addition, M4, an esterolysis product of LC-C, was obtained and evaluated for its bioactivity in vitro. As a result, M4 exhibited a reduction in cell viability in HepG2 with an IC50 value of 17.46±1.55μg/mL.

  5. Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms at excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used

    Tiger, G. (Univ. of Umea (Sweden)); Fowler, C.J. (Astra Research Centre, Soedertalje (Sweden))


    The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 potentiated the response to carbachol at 6 mM K{sup +} when Ca{sup 2+}-free, but not when 2.52 mM Ca{sup 2+} assay buffer was used. In Ca{sup 2+}-free buffer, verapamil inhibited the response to carbachol at both 6 and 18 mM K{sup +} but higher concentrations were needed when 2.52 mM Ca{sup 2+} was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM ({sup 3}H)pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K{sup +} at 1.3 mM Ca{sup 2+}, but was without effect on the basal rate at other K{sup +} and Ca{sup 2+} concentrations. In the presence of NMDA or quisqualate, the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K{sup +} and Ca{sup 2+} concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.

  6. Effects of μ-opioid receptor agonists in assays of acute pain-stimulated and pain-depressed behavior in male rats: role of μ-agonist efficacy and noxious stimulus intensity.

    Altarifi, Ahmad A; Rice, Kenner C; Negus, S Stevens


    Pain is associated with stimulation of some behaviors and depression of others, and μ-opioid receptor agonists are among the most widely used analgesics. This study used parallel assays of pain-stimulated and pain-depressed behavior in male Sprague-Dawley rats to compare antinociception profiles for six μ-agonists that varied in efficacy at μ-opioid receptors (from highest to lowest: methadone, fentanyl, morphine, hydrocodone, buprenorphine, and nalbuphine). Intraperitoneal injection of diluted lactic acid served as an acute noxious stimulus to either stimulate stretching or depress operant responding maintained by electrical stimulation in an intracranial self-stimulation (ICSS). All μ-agonists blocked both stimulation of stretching and depression of ICSS produced by 1.8% lactic acid. The high-efficacy agonists methadone and fentanyl were more potent at blocking acid-induced depression of ICSS than acid-stimulated stretching, whereas lower-efficacy agonists displayed similar potency across assays. All μ-agonists except morphine also facilitated ICSS in the absence of the noxious stimulus at doses similar to those that blocked acid-induced depression of ICSS. The potency of the low-efficacy μ-agonist nalbuphine, but not the high-efficacy μ-agonist methadone, to block acid-induced depression of ICSS was significantly reduced by increasing the intensity of the noxious stimulus to 5.6% acid. These results demonstrate sensitivity of acid-induced depression of ICSS to a range of clinically effective μ-opioid analgesics and reveal distinctions between opioids based on efficacy at the μ-receptor. These results also support the use of parallel assays of pain-stimulated and -depressed behaviors to evaluate analgesic efficacy of candidate drugs.

  7. Effect of morin on pharmacokinetics of piracetam in rats, in vitro enzyme kinetics and metabolic stability assay using rapid UPLC method.

    Sahu, Kapendra; Shaharyar, Mohammad; Siddiqui, Anees A


    The aim of this study was to investigate the effect of Morin on the pharmacokinetics of Piracetam in rats, in vitro enzyme kinetics and metabolic stability (high throughput) studies using human liver microsomes in UPLC. For pharmacokinetics studies, male Wistar rats were pretreated with Morin (10 mg/kg) for one week and on the last day, a single dose of Piracetam (50 mg/kg) was given orally. In another group, both Morin and Piracetam were co-administered to evaluate the acute effect of Morin on Piracetam. The control group received oral distilled water for one week and administered with Piracetam on the last day. As Morin is an inhibitor of P- Glycoprotein (P-gp) and CYP 3A, it was anticipated to improve the bioavailability of Piracetam. Amazingly, relative to control, the areas under the concentration time curve and peak plasma concentration of Piracetam were 1.50- and 1.45-fold, respectively, greater in the Morin-pretreated group. However, co-administration of Morin had no significant effect on these parameters. Apart from the aforementioned merits, the results of this study are further confirmed by clinical trials; Piracetam dosages should be adjusted to avoid potential drug interaction when Piracetam is used clinically in combination with Morin and Morin-containing dietary supplements. The in vitro enzyme kinetics were performed to determined km, Vmax & CLins . The in vitro metabolic stability executed for the estimation of metabolic rate constant and half-life of Piracetam. These studies also extrapolate to in vivo intrinsic hepatic clearance (Clint, in vivo ) from in vitro intrinsic hepatic clearance (CLint, in vitro ). Copyright © 2012 John Wiley & Sons, Ltd.

  8. A reversed-phase HPLC-UV assay for simultaneous analysis of EGCG and ECG of tea polyphenols in rat plasma

    FU Ting; LIANG Jun; HAN Guo-zhu; L(U) Li; LI Nan


    Objective To develop a simple and specific reversed-phase HPLC-UV method for simultaneous determination of (-) Epigallocatechin-3-gallate (EGCG) and (-) Epicatechin-3-gallate (ECG), the main active ingredients of tea polyphenols (TP), in rat plasma. Methods EGCG and ECG were eluted on a Kromasil C18 analytical column (150 mm×4.6 mm, 5μm) protected by a C18 pre-column (4.6 mm×20 mm, 10μm) with a linear gradient mobile phase composed of CH3CN (A)-0.1% citric acid (B), which was run from initial 14 % A and 86 % B to 20 % A and 80 % B at a flow rate of 1.0 mL·min-1 in 14 min, then changed to 14% A and 86% B at a gradient flow rate of 1.0-1.5 mL·min-1 during 14-18 min, and then maintained until 22 min at a gradient flow rate of 1.5-1.0 mL·min-1. The UV detector was set at 280 nm. Plasma samples (200 μL each) were prepared by liquid-liquid extraction procedure with double volumes of EtoAc and then evaporation of organic phase under N2 stream to dry, followed by reconstitution with 100 μL of 20 % CH3CN aqueous solution. The peak area ratios of analytes to vanillin as internal standard vs concentration of analytes to construct calibration curves. Results The HPLC resulted in base-line separation of vanillin, EGCG, ECG and other components; there was no interference from blank plasma. The linear range was 0.5-300 μg·mL-1 for EC, CG (r=0.9999) and 0.1-60 μg·mL-1 for ECG (r = 0.9999). The intra-and inter-day precision (RSD) was better than 6.1% and 12.6%, respectively, and the average accuracy was between 86.25%-103.14%. The extraction recovery of EGCG and ECG was 79.80%-84.64% and 75.22 %-91.39 %, respectively. The plasma samples were stable for at least 30 days at -20 ℃ and 8 h at room temperature;EGCG, ECG and IS stock solutions 2 months at -20 ℃, and the EtoAc-extracted plasma samples 24 h at 4 ℃. Application of the method to the determination of EGCG and ECG in plasma of rats receiving iv 100 mg·kg-1 of TP showed that these 2 compounds

  9. Radioreceptor assay method for insulin

    Mori, K.F.; Wood, R.J. (Bureau of Drug Research, Health and Welfare Canada, Ottawa, Ontario. Health Protection Branch)


    A sensitive practical radioreceptor assay method for pharmaceutical insulin products has been developed with partially purified rat liver plasma membranes and the optimal conditions under which the best overall assay performance is obtainable have been defined. Intra- and inter-assay variations of the method averaged 7.3 and 12.2% of the man, respectively, when expressed as the coefficient of variation. Potency estimates of an insulin product obtained with the proposed method correlated well with those determined by the mouse convulsion bioassay method. Liver membranes prepared according to the method could be stored for up to ten weeks at 4/sup 0/C and for 6 months or more at -18/sup 0/C without losing insulin-binding ability.

  10. A rapid and sensitive UHPLC-MS/MS assay for the determination of trelagliptin in rat plasma and its application to a pharmacokinetic study.

    Hu, Xiao-Xia; Lan, Tian; Chen, Zhe; Yang, Cheng-Cheng; Tang, Peng-Fei; Yuan, Ling-Jing; Hu, Guo-Xin; Cai, Jian-Ping


    This study aims to develop and validate a simple, rapid and sensitive ultra-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for exploring pharmacokinetic characteristics of trelagliptin. Protein precipitation by acetonitrile was used to prepare plasma sample. A RRHD Eclipse Plus C18 (2.1×50mm, 1.8μ) column with gradient mobile phase (containing acetonitrile and 0.1% formic acid) help to achieve the separation of trelagliptin and carbamazepine (IS) with high selectivity. Detection of target fragment ions m/z 358.2→133.9 for trelagliptin, and m/z 237.1→194.0 for IS was performed in positive-ion electrospray ionization mass spectrometry by multiple reaction monitoring. Linear calibration plots were achieved in the range of 5-4000ng/mL for trelagliptin (R(2)=0.999) in rat plasma. The recovery of trelagliptin ranged from 87.8% to 93.7%. The method was showed to be accurate, precise and stable. No obvious matrix effect was found. It has been fully validated and successfully applied to pharmacokinetic study of trelagliptin.

  11. Assessment of the safety of hydrogenated resistant maltodextrin: reverse mutation assay, acute and 90-day subchronic repeated oral toxicity in rats, and acute no-effect level for diarrhea in humans.

    Yoshikawa, Yuko; Kishimoto, Yuka; Tagami, Hiroyuki; Kanahori, Sumiko


    A series of safety assessments were performed on hydrogenated resistant maltodextrin prepared by converting the reducing terminal glucose of resistant maltodextrin into sorbitol. The reverse mutation assay did not show mutagenicity. Acute and 90-day subchronic oral toxicity studies in rats showed no death was observed in any groups, including the group receiving the highest single dose of 10 g/kg body weight or the highest dose of 5 g/kg body weight per day for 90 days. Mucous or watery stools were observed in the hydrogenated resistant maltodextrin treatment group on the acute study, which were transient and were associated with the osmotic pressure caused by intake of the high concentrations. Subchronic study showed dose-dependent increases in the weights of cecum alone, cecal contents alone, and cecum with cecal contents as well as hypertrophy of the cecal mucosal epithelium, which are considered to be common physiological responses after intake of indigestible carbohydrates. These results indicated that the no observed adverse effect level (NOAEL) of hydrogenated resistant maltodextrin was 10 g/kg body weight or more on the acute oral toxicity study and 5.0 g/kg body weight/day or more on the 90-day subchronic repeated oral toxicity study in rats. Further study performed in healthy adult humans showed that the acute no-effect level of hydrogenated resistant maltodextrin for diarrhea was 0.8 g/kg body weight for men and more than 1.0 g/kg body weight for women. The results of the current safety assessment studies suggest that hydrogenated resistant maltodextrin is safe for human consumption.

  12. Development and application of assays for serotonin

    Gow, I.F.


    In this thesis, two assays for serotonin were developed, validated, and used to investigate the relationship between platelet aggregation, serotonin levels and sodium status and serotonin levels and platelet function in patients with cardiovascular disease. A radioimmunoassay (RIA) using an (/sup 125/I)-labelled tracer was developed and validated for the measurement of serotonin in human platelet-rich plasma (PRP) and rat serum. Antisera were raised against N-succinamylserotonin conjugated to bovine albumin and, to improve assay sensitivity, the analyte was made chemically similar to the immunogen by conversion to N-acetylserotonin prior to assay, using the specific amino reagent N-acetoxysuccinimide. An assay for serotonin using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) was developed, and used to validate the RIA. The RIA can be used to assay up to 100 samples/day compared with 10-20/day by the HPLC-ECD assay.

  13. Determination of newly synthesized lipoic acid-niacin dimer in rat plasma by UPLC/electrospray ionization tandem mass spectrometry: assay development, validation and application to a pharmacokinetic study.

    Chen, Xiao; Gao, Jingwen; Jiang, Yiming; Huang, Ping; Xie, Yuhui; Pi, Rongbiao; Zhu, Shuzhen; Yao, Meicun


    A simple, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed to determine the newly synthesized compound lipoic acid-niacin dimer (N2L) in plasma. Plasma samples were precipitated by methanol using tetrahydropalmatine as internal standard. Chromatographic separation was achieved on an Acquity BEH C18 (2.1 × 50 mm i.d., 1.7 µm) column; the mobile phase contains methanol and buffer solution (water with 0.5% formic acid and 10 mmol/L ammonium acetate). Multiple reaction monitoring (m/z 353.9 → 148.6 for N2L and m/z 356.0 → 192.0 for internal standard) was performed for detection and quantification. The method was validated to be rapid, specific, accurate and precise over the concentration range of 1-750 ng/mL; N2L was not stable on the bench-top or during freeze-freeze-thaw cycles in plasma, but was stable in the stock solution and after preparation in the autosampler for 24 h. The utility of the assay was confirmed by pharmacokinetic study of N2L in rats. Copyright © 2013 John Wiley & Sons, Ltd.

  14. [Treatment with isoflavones replaces estradiol effect on the tissue fat accumulation from ovariectomized rats].

    Torrezan, Rosana; Gomes, Rodrigo M; Ferrarese, Maria L; de Melo, Fernando Ben-Hur; Ramos, Aparecida M D; Mathias, Paulo C F; Scomparin, Dionizia X


    Isoflavones (ISO) present in soybean are named phytoestrogens because they show estrogen effect. The use of isoflavones has beneficial effect in disturbance of post-menopause, which is characterized by ovarian function suppression. Decreasing of estrogen secretion and consequent morphologic and metabolic disarrangements are observed in female hormonal decline. The aim of present work was to investigate the effect of ISO on the fat accretion of uterine endometric tissue, and HDL and glucose blood concentration from ovariectomized rats (OVX). Female Wistar rats with 60 days-old were submitted a surgery to remove bilaterally the ovarium. After 8-day recovery period the animals were distributed into three groups: sham operate (GC); OVX ISO untreated (GI) and OVX supplemented with ISO (G II). Total uterus mass, uterus fat and retroperitoneal fat pad, were removed, washed and weighted. Samples of uterus were histological processed to measure endometrium thickness. Blood samples were also collected to analyze the concentration of HDL and glucose. The OVX caused endometric atrophy, decrease of uterus weight and HDL reduction. The treatment with ISO provoked decrease of uterine and retroperitoneal fat pad. HDL increase and glycemia reduction were also observed. However, there was no uterotrophic effect. ISO treatment causes decrease in tissue fat accretion from ovariectomized rats.

  15. Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

    Hamada, Shuichi; Ohyama, Wakako; Takashima, Rie; Shimada, Keisuke; Matsumoto, Kazumi; Kawakami, Satoru; Uno, Fuyumi; Sui, Hajime; Shimada, Yasushi; Imamura, Tadashi; Matsumura, Shoji; Sanada, Hisakazu; Inoue, Kenji; Muto, Shigeharu; Ogawa, Izumi; Hayashi, Aya; Takayanagi, Tomomi; Ogiwara, Yosuke; Maeda, Akihisa; Okada, Emiko; Terashima, Yukari; Takasawa, Hironao; Narumi, Kazunori; Wako, Yumi; Kawasako, Kazufumi; Sano, Masaki; Ohashi, Nobuyuki; Morita, Takeshi; Kojima, Hajime; Honma, Masamitsu; Hayashi, Makoto


    The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

  16. Validation of a novel in vitro assay using ultra performance liquid chromatography-mass spectrometry (UPLC/MS) to detect and quantify hydroxylated metabolites of BDE-99 in rat liver microsomes.

    Erratico, Claudio A; Szeitz, András; Bandiera, Stelvio M


    The purpose of this study was to develop and validate an ultra performance liquid chromatography-mass spectrometry (UPLC/MS) method to investigate the hepatic oxidative metabolism of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), a widely used flame retardant and ubiquitous environmental contaminant. Hydroxylated metabolites were extracted using liquid-to-liquid extraction, resolved on a C18 column with gradient elution and detected by mass spectrometry in single ion recording mode using electrospray negative ionization. The assay was validated for linearity, accuracy, precision, limit of quantification, range and recovery. Calibration curves were linear (R2 > or = 0.98) over a concentration range of 0.010-1.0 microM for 4-OH-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90), 5'-OH-2,2',4,4',5-pentabromodiphenyl ether (5'-OH-BDE-99) and 6'-OH-2,2',4,4',5-pentabromodiphenyl ether (6'-OH-BDE-99), and a concentration range of 0.0625-12.5 microM for 2,4,5-tribromophenol (2,4,5-TBP). Inter- and intra-day accuracy values ranged from -2.0% to 6.0% and from -7.7% to 7.3%, respectively, and inter- and intra-day precision values ranged from 2.0% to 8.5% and from 2.2% to 8.6% (n=6), respectively. The limits of quantification were 0.010 microM for 4-OH-BDE-90, 5'-OH-BDE-99 and 6'-OH-BDE-99, and 0.0625 microM for 2,4,5-TBP. Recovery values ranged between 85 and 100% for the four analytes. The validated analytical method was applied to identify and quantify hydroxy BDE-99 metabolites formed in vitro. Incubation of BDE-99 with rat liver microsomes yielded 4-OH-BDE-90 and 6'-OH-BDE-99 as major metabolites and 5'-OH-BDE-99 and 2,4,5-TBP as minor metabolites. To our knowledge, this is the first validated UPLC/MS method to quantify hydroxylated metabolites of PBDEs without the need of derivatization.

  17. Evaluation of the repeated-dose liver micronucleus assay using N-nitrosomorpholine in young adult rats: report on collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study (MMS) Group.

    Hayashi, Aya; Kosaka, Mizuki; Kimura, Aoi; Wako, Yumi; Kawasako, Kazufumi; Hamada, Shuichi


    The present study was conducted to evaluate the suitability of a repeated-dose liver micronucleus (LMN) assay in young adult rats as a collaborative study by the Mammalian mutagenicity study (MMS) group. All procedures were performed in accordance with the standard protocols of the MMS Group. Six-week-old male Crl:CD(SD) rats (5 animals/group) received oral doses of the hepatocarcinogen N-nitrosomorpholine (NMOR) at 0 (control), 5, 10, and 30mg/kg/day (10mL/kg) for 14 days. Control animals received vehicle (water). Hepatocytes were collected from the liver 24h after the last dose, and the number of micronucleated hepatocytes (MNHEPs) was determined by microscopy. The number of micronucleated immature erythrocytes (MNIMEs) in the femoral bone marrow was also determined. The liver was examined using histopathologic methods after formalin fixation. The results showed statistically significant and dose-dependent increases in the number of MNHEPs in the liver at doses of 10mg/kg and greater when compared with the vehicle control. However, no significant increase was noted in the number of MNIMEs in the bone marrow at doses of up to 30mg/kg. Histopathology of the liver revealed hypertrophy and single cell necrosis of hepatocytes at doses of 5mg/kg and above. These results showed that the induction of micronuclei by NMOR was detected by the repeated-dose LMN assay, but not by the repeated-dose bone marrow micronucleus assay.

  18. The estrogenicity of methylparaben and ethylparaben at doses close to the acceptable daily intake in immature Sprague-Dawley rats

    Sun, Libei; Yu, Tong; Guo, Jilong; Zhang, Zhaobin; Hu, Ying; Xiao, Xuan; Sun, Yingli; Xiao, Han; Li, Junyu; Zhu, Desheng; Sai, Linlin; Li, Jun


    The estrogenicity of parabens at human exposure levels has become a focus of concern due to the debate over whether the estrogenicity of parabens is strong enough to play a role in the increased incidence of breast cancer. In this study, the uterotrophic activities of methylparaben (MP) and ethylparaben (EP) at doses close to the acceptable daily intake as allocated by JECFA were demonstrated in immature Sprague-Dawley rats by intragastric administration, and up-regulations of estrogen-responsive biomarker genes were found in uteri of the rats by quantitative real-time RT–PCR (Q-RT-PCR). At the same time, the urinary concentrations of MP and EP, as measured by gas chromatography–mass spectrometry (GC-MS) in rats that received the same doses of MP and EP, were found to be near the high urinary levels reported in human populations in recent years. These results show the in vivo estrogenicity of MP and EP at human exposure levels, and indicate that populations exposed to large amounts of MP and EP may have a high burden of estrogenicity-related diseases. In addition, a molecular docking simulation showed interaction between the parabens and the agonist-binding pocket of human estrogen receptor α (hERα). PMID:27121550

  19. Protein binding assay for hyaluronate

    Lacy, B.E.; Underhill, C.B.


    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 or less of hyaluronate, and is unaffected by the presence of proteins.

  20. Microbead agglutination based assays

    Kodzius, Rimantas


    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  1. Colorimetric protein assay techniques.

    Sapan, C V; Lundblad, R L; Price, N C


    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  2. Absolute nuclear material assay

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA


    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  3. Predictive Assay For Cancer Targets

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A


    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  4. GC-MS assay for hepatic DDAH activity in diabetic and non-diabetic rats by measuring dimethylamine (DMA) formed from asymmetric dimethylarginine (ADMA): evaluation of the importance of S-nitrosothiols as inhibitors of DDAH activity in vitro and in vivo in humans.

    Chobanyan, Kristine; Thum, Thomas; Suchy, Maria-Theresia; Zhu, Bijun; Mitschke, Anja; Gutzki, Frank-Mathias; Beckmann, Bibiana; Stichtenoth, Dirk O; Tsikas, Dimitrios


    Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, is hydrolyzed to dimethylamine (DMA) and L-citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). In the present article we report on a GC-MS assay for DDAH activity in rat liver homogenate in phosphate buffered saline. The method is based on the quantitative determination of ADMA-derived DMA by GC-MS as the pentafluorobenzamide derivative. Quantification was performed by selected-ion monitoring of the protonated molecules at m/z 240 for DMA and m/z 246 for the internal standard (CD3)2NH in the positive-ion chemical ionization mode. The assay was applied to determine the enzyme kinetics in rat liver, the hepatic DDAH activity in streptozotocin-induced (50 mg/kg) diabetes in rats, and to evaluate the importance of S-nitrosothiols as DDAH inhibitors. The KM and Vmax values were determined to be 60 microM ADMA and 12.5 pmol DMA/minmg liver corresponding to 166 pmol DMA/minmg protein. Typical DDAH activity values measured in rat liver homogenate were 8.7 pmol DMA/minmg liver at added ADMA concentration of 100 microM. DDAH activity was found to be 1.7-fold elevated in diabetic as compared to non-diabetic rats (P=0.01). The SH-specific agents HgCl2, S-nitrosocysteine ethyl ester (SNACET), a synthetic lipophilic S-nitrosothiol, S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO) and S-nitrosohomocysteine (HcysNO) were found to inhibit DDAH activity in rat liver homogenate. The IC50 values for HcysNO, SNACET, CysNO and GSNO were estimated to be 300, 500, 700 and 1000 microM, respectively. Oral administration of 15N-labelled SNACET to two healthy volunteers (1 micromol/kg) resulted in elevated urinary excretion of 15N-labelled nitrite and nitrate, but did not reduce creatinine-corrected excretion of DMA in the urine. Our results suggest that inhibition of DDAH activity on the basis of reversible nitros(yl)ation or irreversible N-thiosulfoximidation of the

  5. Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test.

    Hagio, Soichiro; Furukawa, Satoshi; Abe, Masayoshi; Kuroda, Yusuke; Hayashi, Seigo; Ogawa, Izumi


    Recently, the liver micronucleus (MN) assay using young adult rats with repeated administrations has been investigated by employing a new method without partial hepatectomy or in situcollagenase perfusion as the repeated dose liver MN (RDLMN) assay by Narumi et al. (2012). In our study, in order to investigate the possibility of the RDLMN assay using young adult mice instead of rats and the feasibility of employing some genotoxicity assays along with the RDLMN assay as a combination test, two genotoxic carcinogens (N,N-diethylnitrosoamine (DEN) and cisplatin (CIS)) and a nongenotoxic carcinogen (phenobarbital sodium (PHE)) were administered to mice for 15 or 29 days. Then, the liver MN assay, peripheral blood (PB) MN assay and comet assay using the liver and kidney were concurrently performed as a combination test. DEN showed positive responses to all endpoints except MN induction in PB after 15 days of repeat administration. A cross-linking agent, CIS, showed MN induction in liver after 29 days of repeat administration, and in PB after 15 and 29 days of repeat administration, although the comet assay yielded negative responses for both organs at both sampling times. PHE yielded negative responses for all endpoints. In conclusion, it is suggested that the RDLMN assay using mice is a feasible method to be integrated into the general repeated toxicity test along with the combination assays, i.e., comet assay or PB MN assay, which would help in risk assessment for carcinogenicity by comparing the results of combination assays with each other.

  6. Transport of puerarin in rat intestine in situ by modified gravimetry and phenol red assay%酚红法和改良重量法分别研究葛根素的大鼠在体肠吸收机制

    黄嗣航; 龙晓英; 袁飞; 陈莉; 蔡宝玲; 邱慧琳


    Objective To study the rat intestine absorption mechanism of puerarin by in situ single pass perfusion, and compare the difference of gravimetry and phenol red assay. Methods The rat single pass perfusion technique was applied. Gravimetry and phenol red assay were used to correct the perfusion volume. The concentration of puerarin was determined by HPLC. A series of studies were carried out including the absorption at different concentrations(100,200 and 400 (μg o mL-1 ) and at different intestinal regions. Results Phenol red assay's parameters were almost all of negative values. These results went against common sense. Gravimetry's results showed that puerarin could be absorbed in all rat intestines without special absorption sites and the differences were negligible. There was no highest saturated concentration raised with the increasing concentration of puerarin. Conclusion Gravimetry was more suitable than phenol red assay for studying the absorption mechanism of puerarin.%目的 通过在体单向肠灌流法研究葛根素的肠吸收机制,比较酚红法与改良重量法在研究葛根素吸收机制上的适用性.方法 采用酚红法、改良重量法校正灌流液体积,并用HPLC法对灌流前后葛根素的含量进行测定,研究不同质量浓度(100、200和400 μg· mL-1)的葛根素在大鼠不同肠段的吸收情况.结果 酚红法测得的葛根素的吸收参数基本为负值,其结果不符合常理.改良重量法测得葛根素在各肠段均有吸收,但各肠段之间差异无统计学意义,不存在明显吸收窗;葛根素浓度升高,肠吸收有所增加,且不存在饱和现象.结论 改良重量法相对酚红法更适用于葛根素的在体肠吸收研究.

  7. Cell viability assays: introduction.

    Stoddart, Martin J


    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  8. Tube-Forming Assays.

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy


    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  9. Assays for thrombopoietin

    McDonald, T.P.


    In summary, thrombopoietin levels have been determined indirectly by measuring thrombocytopoiesis in assay animals (platelet counting, measurement of isotope incorporation into newly formed platelets, changes in platelet sizes, or alterations in number and size of megakaryocytes) and by use of an immunoassay. Although much work remains, it seems clear at the present time that isotopic uptake into platelets of specially prepared assay mice (rebound-thrombocytosis) is superior to the other techniques now available for the measurement of thrombopoietin. However, the ideal assay for TSF which is specific, rapid, and inexpensive is yet to be developed. An immunoassay is in the development stage, but will require additional work before it can be utilized for the routine assay of TSF.

  10. New Rapid Spore Assay

    Kminek, Gerhard; Conley, Catharine


    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  11. Against vaccine assay secrecy.

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M


    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors.

  12. Against vaccine assay secrecy

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M


    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  13. Rover waste assay system

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)


    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  14. CTL ELISPOT assay.

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita


    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  15. New oligosaccharyltransferase assay method.

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi


    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  16. Hyaluronic Acid Assays

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H


    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  17. Instrument for assaying radiation

    Coleman, Jody Rustyn; Farfan, Eduardo B.


    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  18. Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM01183 (lurbinectedin), a novel antineoplastic agent, in mouse, rat, dog, Cynomolgus monkey and mini-pig plasma.

    Pernice, Tiziana; Bishop, Alan G; Guillen, Maria Jose; Cuevas, Carmen; Aviles, Pablo


    Lurbinectedin (PM01183) is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected sequences in the minor groove of DNA, inducing PM01183-DNA adducts that stall replication, DNA repair and transcription and gives rise to double-strand breaks and finally, caspase-dependent apoptotic cell death. PM01183 has demonstrated clinical antitumor activity in platinum resistant/refractory ovarian cancer patients. A rapid and sensitive liquid chromatography/tandem mass spectrometry assay was developed and validated to quantify PM01183 in plasma from nonclinical species. The bioanalysis consisted of a supported liquid extraction, followed by a gradient phase chromatography and, detection by positive ion electrospray tandem mass spectrometry. The calibration range for PM01183 was established using PM01183 standards from 0.1 to 100 ng/mL in blank plasma. The multiple reaction monitoring, based on the transition m/z 767.3→273.0, was specific for PM01183, and that based on the transition m/z 771.4→277.0 was specific for the internal standard (deuterated PM01183). No endogenous material interfered with the analysis of PM01183 and the internal standard from blank plasma. The limit of detection (LOD) of the assay was calculated as 0.025 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9937 to 0.9987. The mean inter-day accuracies for all calibration standards ranged from 92 to 108% (≤8% bias), and the mean inter-day precision for calibration standards was always less than 12%. The mean intra and inter-day assay accuracy for all quality control replicates remained between 91 and 109%. The mean intra and inter-day assay precision was less than 10% for all QC levels. The method was validated to demonstrate the specificity, recovery, limit of quantification, accuracy and precision of measurements. The assay has been used to support preclinical pharmacokinetic and toxicokinetic studies of PM01183 in nonclinical species. The main PK

  19. High-Dose Diosgenin Reduces Bone Loss in Ovariectomized Rats via Attenuation of the RANKL/OPG Ratio

    Zhiguo Zhang


    Full Text Available The aim of this study was to evaluate effect of diosgenin (DG on rats that had osteoporosis-like features induced by ovariectomy (OVX. Seventy-two six-month-old female Wistar rats were subjected to either ovariectomy (n = 60 or Sham operation (SHAM group, n = 12. Beginning at one week post-ovariectomy, the OVX rats were treated with vehicle (OVX group, n = 12, estradiol valerate (EV group, n = 12, or DG at three doses (DG-L, -M, -H group, n = 12, respectively. After a 12-week treatment, administration of EV or DG-H inhibited OVX-induced weight gain, and administration of EV or DG-H or DG-M had a significantly uterotrophic effect. Bone mineral density (BMD and indices of bone histomorphometry of tibia were measured. Levels of protein and mRNA expression of osteoprotegerin (OPG and receptor activator of nuclear factor kappa-B ligand (RANKL in tibia were evaluated by immunohistochemistry and in situ hybridization. Our results show that DG at a high dose (DG-H had a significant anti-osteoporotic effect compared to OVX control. DG-H treatment down-regulated expression of RANKL and up-regulated expression of OPG significantly in tibia from OVX rats compared to control, and thus lowered the RANKL/OPG ratio. This suggests that the anti-osteoporotic effect of DG might be associated with modulating the RANKL/OPG ratio and DG had potential to be developed as alternative therapeutic agents of osteoporosis induced by postmenopause.

  20. The corneal pocket assay.

    Ziche, Marina; Morbidelli, Lucia


    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  1. Kinetic Tetrazolium Microtiter Assay

    Pierson, Duane L.; Stowe, Raymond; Koenig, David


    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  2. B cell helper assays.

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo


    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  3. Detection of Over-Expressed Integrin-Linked Kinase in DNA Synthesis of Cardiomyocytes in Newborn Rats by EDU Immunofluorescence Assay%EDU免疫荧光法检测整合素连接激酶(ILK)高表达对新生大鼠心肌细胞DNA合成的影响

    王丙剑; 白剑; 孙静娴; 张娜; 徐标


    Objective: To investigate the effects of over-expressed integrin-linked kinase on rat neonatal DNA synthesis of cardiomyocytes by EDU immunofluorescence assay. Methods: Primary neonatal rat (1-3 days old) cardiomyocytes were cultured in vitro and randomly divided into two groups 72 h after isolation: infected by either adeno-GFP (control group) or adeno-GFP containing adeno-ILK (ILK group). 48h after infection, DNA synthesis of cardiomyecytes in two groups were determined by 5-ethynyl-2-deoxyuridinc(EdU) immunofiuorescence assay. Results: Compared with the control groups, DNA synthesis of cardiomyocytes increased dramatically in ILK groups (P <0.05). Conclusion: The over-expression of ILK could promote Rat Neonatal DNA synthesis of cardiomyocytes.%目的:通过EDU免疫荧光法观察整合素连接激酶(Integrin-Linked Kinase,ILK)在新生大鼠心肌细胞内高表达后对心肌细胞DNA合成的影响.方法:取新生(1-3天内)大鼠原代心肌细胞,培养72小时后随机分为正常对照组、ILK转染组.对照组转染重组腺病毒载体(adeno-GFP),ILK组转染重组腺病毒载体+ILK基因(adeno-ILK).转然成功后48小时将两组心肌细胞分别通过5-乙基-2'-脱氧尿嘧啶核苷(EDU)免疫荧光法测定心肌细胞DNA合成.结果:ILK转染组心肌细胞内DNA合成较对照组明显增加(P<0.05).结论:ILK高表达具有促进新生大鼠心肌细胞的DNA合成的能力.

  4. Comparación entre dos biomodelos murinos (ratones Balb/c y ratas Sprague Dawley en el ensayo de micronúcleos transplacentarios Comparison between two murine biomodles (Balb/c mice and Sprague Dawley rats in a transplacental micronucleus assay

    Daniel Francisco Arencibia Arrebola


    Full Text Available Objetivo: comparar fetos de ratones Balb/c y ratas Sprague Dawley como biomodelos en el ensayo de micronúcleos transplacentarios, determinando la frecuencia espontánea e inducida, y vincular el efecto genotóxico y reproductivo. Métodos: se formaron tres grupos experimentales/especie: control negativo (simulacro, control solvente NaCl (0,9 % y ciclofosfamida 50 mg/kg. Todos los grupos se administraron por vía intraperitoneal los días 14, 15 y 16 de la gestación, y 24 h después de la última inoculación en ratones y 48 h en ratas se procedió a realizar la eutanasia de las gestantes, para obtener las muestras de hígado fetal. Resultados: los fetos de ratas Sprague Dawley demostraron tener menores índices de citotoxicidad y de genotoxicidad, y se obtuvieron los resultados más bajos de micronúcleos endógenos. Los mejores resultados de inducción de citotoxicidad y genotoxicidad por la alta formación de micronúcleos con ciclofosfamida fueron en fetos de ratas Sprague Dawley, los que resultaron más susceptibles al daño genotóxico de este mutágeno. Se corroboró el poder clastogénico transplacentario de la ciclofosfamida, vinculando este ensayo de genotoxicidad a toxicología de la reproducción. Conclusiones: los resultados sugieren el mejor uso de fetos de ratas Sprague Dawley como biomodelo en este ensayo cuando es utilizada la ciclofosfamida como control positivo por la vía y dosis estudiada; además, se pudieran utilizar en la evaluación de nuevas drogas con carácter antigenotóxico por vía transplacentaria.Objectives: to compare fetuses from Balb/c mice and Sprague Dawley rats as biomodels in transplacental micronuclei assay and to determine the spontaneous and induced frequency to link the genotoxic and reproductive effect. Methods: three experimental groups by species were formed: a negative control (simulation, NaCl (0.9 % solvent control and 50 mg/kg cyclophosphamide. All the groups were intraperitoneally administered

  5. Ultra-performance liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma: application to a pharmacokinetic study.

    Gu, Wei; Wang, Dongyue; Pan, Zihao; Liu, Xiao; Cai, Baochang; Chen, Jun


    A rapid, simple and sensitive UHPLC-MS/MS method was developed and validated for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma using huperzine A as an internal standard (IS) after protein precipitation with methanol. The analytes were separated on a Purospher® STAR RP18 UHPLC column (2 µm, 2.1 × 100 mm) by gradient elution using a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Brucine, strychnine, brucine N-oxide and IS were detected in positive ion multiple reaction monitoring mode by means of an electrospray ionization interface (m/z 395.2 → 324.1, m/z 335.2 → 184.1, m/z 411.2 → 394.2, m/z 243.1 → 226.1). The calibration curve was linear over the range of 1-500 ng/mL for brucine and strychnine and 0.2-50 ng/mL for brucine N-oxide. The intra- and inter-day precisions of these analytes were all within 15% and the accuracy ranged from 85 to 115%. The stability experiment indicated that the plasma samples at three concentration levels were stable under different conditions. The developed method was successfully applied for the first time to pharmacokinetic studies of brucine, strychnine and brucine N-oxide following a single oral and intravenous administration of modified total alkaloid fraction in rats. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Growth cone collapse assay.

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J


    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.



    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  8. Radon assay for SNO+

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)


    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  9. RAS - Screens & Assays - Drug Discovery

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  10. Bacterial assays for recombinagens.

    Hoffmann, G R


    Two principal strategies have been used for studying recombinagenic effects of chemicals and radiation in bacteria: (1) measurement of homologous recombination involving defined alleles in a partially diploid strain, and (2) measurement of the formation and loss of genetic duplications in the bacterial chromosome. In the former category, most methods involve one allele in the bacterial chromosome and another in a plasmid, but it is also possible to detect recombination between two chromosomal alleles or between two extrachromosomal alleles. This review summarizes methods that use each of these approaches for detecting recombination and tabulates data on agents that have been found to be recombinagenic in bacteria. The assays are discussed with respect to their effectiveness in testing for recombinagens and their potential for elucidating mechanisms underlying recombinagenic effects.

  11. Efeitos do uso crônico do nelfinavir sobre a prenhez da rata albina: ensaio biológico Effects of chronic nelfinavir treatment on rat pregnancy: biological assay

    Camila Fernandes Venneri Mathias


    ratas prenhes e não mostrou efeitos deletérios sobre os conceptos.PURPOSE: to evaluate the chronic effects of nelfinavir on body weight gain of pregnant albino rats and their concepts, as well as on the number of implantations, reabsorptions, fetuses, placentae, and maternal and fetal mortality. METHODS: fifty pregnant EPM-1 Wistar albino rats were randomly divided into five groups: two controls, Contr1 (control of stress and Contr2 (drug vehicle control, and 3 experimental groups, Exp40, Exp120, Exp360, which received 40, 120 or 360 mg/kg per day of oral solution of nelfinavir, respectively. The drug and the vehicle (distilled water were administered twice a day (12/12 h by gavage from the first up to the 20th day of pregnancy. After sacrifice under deep anesthesia, the following parameters were evaluated: number of implantations and reabsorptions, the weight of fetuses and placentae, and the number of intrauterine deaths as well as inspection for major malformations. Data were evaluated by ANOVA followed by the Kruskal-Wallis multiple comparison test. RESULTS: body weight gain during pregnancy was normal for all the groups, and no significant differences were detected between them. ANOVA did not reveal any significant effect of nelfinavir on the studied parameters. The means of number of fetuses were: control = 9.7±0.50; nelfinavir-treated groups = 9.7±0.81. Regarding the means of number of placentae and implantations, controls = 9.7±0.50; nelfinavir-treated groups = 9.6±0.78. The mean fetal weights were as follows: controls = 4.04±0.50; nelfinavir-treated groups = 3.91±0.33 g. Finally, control placental weights averaged 0.64±0.02; nelfinavir-treated groups = 0.67±0.02 g. CONCLUSION: nelfinavir was well tolerated at all the administered doses; no damage was produced on the fetuses.

  12. Herbicide resistance screening assay.

    Peterson, Joan M


    Herbicide resistance screening is a method that can be used not only to determine presence of the enzyme, phosphinothricin acetyltransferase, encoded by either the Bar or the Pat gene in transgenic maize, but also to assess the inheritance ratio of those genes in a segregating population. Herbicide screening can also be used to study linkage of a transgene of interest that was cotransformed with the herbicide resistance marker gene. By combining the herbicide screen assay with a PCR-based screen of leaf tissue DNA for the presence of both the Bar or the Pat gene marker and a cotransformed transgene of interest from the same seedling tissue and maintaining that seedling identity, the researcher can identify linkage or the possible breakdown in linkage of the marker gene and the transgene of interest. Further, the occurrence of "DNA silencing" can be evaluated if an individual seedling that was susceptible to the applied herbicide nonetheless gave PCR data that indicated presence of the gene responsible for herbicide resistance. Similarly, "DNA silencing" of the gene of interest may be investigated if the seeds can be screened and scored for that phenotypic trait in a nondestructive manner prior to planting.

  13. Disagreement between Human Papillomavirus Assays

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller


    assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41......We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four......% tested positive on all four. Agreement was lower in women aged ≥ 30 years (30%, vs. 49% at samples (29%, vs. 38% in follow-up samples), and in women with concurrent normal cytology (22%, vs. 68% with abnormal cytology). Among primary screening samples from women aged 30...

  14. Evaluation of the Erythrocyte Malondialdehyde (MDA) Release Assay.


    Jolla, CA 92037) (95). Triglycerides, in this assay, undergo lipase hydrolysis , phosphorylation, and oxidation/reduction reactions. The final product...tocopherol in the guinea pig . J Nutr 109, 105-109(1979). 82. Mino M, Kasugai 0, Nagita A. Relationship between red blood cell and liver tocopherol...blood cell tocopherol and liver tocopherol in hyperlipemic rats as compared with plasma tocopherol. Lipids 1985;20:488-491. 6. Moudgil KD, Narang BS

  15. Repeated-dose liver micronucleus assay: an investigation with 2-nitropropane, a hepatocarcinogen.

    Kawakami, Satoru; Araki, Tetsuro; Nakajima, Mikio; Kusuoka, Osamu; Uchida, Keisuke; Sato, Norihiro; Tanabe, Yoko; Takahashi, Kaori; Wako, Yumi; Kawasako, Kazufumi; Tsurui, Kazuyuki


    The utility of the repeated-dose liver micronucleus (RDLMN) assay in the detection of a genotoxic hepatocarcinogen was evaluated. In this paper, a rat hepatocarcinogen, 2-nitropropane (2-NP), was administered orally to young adult rats for 14 and 28 days without a partial hepatectomy or a mitogen, and the micronucleus induction in liver was examined using a simple method to isolate hepatocytes. In addition, a bone marrow micronucleus assay was conducted concomitantly. The frequency of micronucleated hepatocytes induced by 2-NP increased significantly in both the 14- and 28-day repeated-dose studies, while the bone marrow micronucleus assays were negative in each study. These results indicate that the RDLMN assay is useful for detecting a genotoxic hepatocarcinogen that is negative in bone marrow micronucleus assays and is a suitable in vivo genotoxicity test method for integration into a repeated-dose general toxicity study.

  16. Roxatidine, an H(2) receptor blocker, is an estrogenic compound--experimental evidence.

    Agrawal, Shyam Sundar; Alvin Jose, Manonmani


    Roxatidine is an H(2) receptor blocker frequently used in the treatment of peptic ulcers. H(2) receptor blockers are reported to show antifertility activity. To examine the mechanism of antifertility, estrogenic and antiestrogenic activity was studied using an in vitro rat and rabbit uterine receptor binding assay and in vivo using the uterotrophic assay in immature Wistar rats. The results revealed that roxatidine showed mild receptor binding affinity to both rat and rabbit uterine receptors when compared to estradiol. Interstingly, in vivo roxatidine increases the wet uterine weight of immature Wistar rats significantly (Proxatidine treated group was somewhat similar to that of the estradiol treated group. Histopathological results and the structure of the roxatidine support that H(2) receptor blocker roxatidine is an estrogenic compound.

  17. In vivo and in vitro estrogen bioactivities of alkyl parabens.

    Lemini, Cristina; Jaimez, Ruth; Avila, María Estela; Franco, Yanira; Larrea, Fernando; Lemus, Ana Elena


    The alkyl esters of p-hydroxybenzoic acid known as parabens (Pbens) are used as preservatives in food, pharmaceutical and cosmetic formulations. They have been reported as estrogenic. Here, we present evidence for the in vivo and in vitro bioactivities and receptor binding affinities of methylparaben (MePben), ethylparaben (EtPben), propylparaben (PrPben), and butylparaben (BuPben) compared with those of estradiol (E2). Estrogenicity was studied using the uterotrophic assay in immature (Im) and adult ovariectomized (Ovx) CD1 mice, and in immature female Wistar rats (IW). Animals were subcutaneously (sc) treated for three consecutive days with different molar equivalent doses ranging from 3.62 to 1086 micromol/kg body weight of Pbens, E2 (0.036 micromol/kg), or vehicle. Pbens increased uterine weight in Im and Ovx animals and their relative uterotrophic effect to E2 (100) (RUEE2) were from 34 to 91. The relative uterotrophic potencies related to E2 (100) (RUPE2) of these compounds were from 0.003 to 0.007. The E2 ED50 for CD1 animals able to increase the uterine weight was 7 microg/kg (0.9-55 confidence limits); and that of Pbens ranged from 18 to 74 mg/kg. In IW rats, the ED50 were from 33 to 338 mg/kg. All Pbens, except MePb, competed with [3H]E2 for the estrogen receptor binding sites. The uterotrophic effects of Pbens in Im mice have a positive correlation with the side-chain length of the ester group of these compounds. The E2 and Pbens relative binding affinities (RBA) and Ki values correlated to their estrogenic activity. The NOELs values for Pbens uterotrophic activity in Im were from 0.6 to 6.5 mg/kg per day; and Ovx from 6 to 55 mg/kg. The NOELs IW ranged from 16.5 to 70 mg/kg indicating that Im were more susceptible than Ovx and IW to these effects. The data shown here confirm the estrogenicity of Pbens.

  18. Practical assay issues with the PERT/PBRT assay: a highly sensitive reverse transcriptase assay.

    Chang, A; Dusing, S


    Product safety testing for retroviruses can be achieved by a panel of screening assays, including electron microscopy, viral gene specific PCRs, virus propagation, and detection of reverse transciptase activity. The application of PCR-based reverse transcriptase assays (PERT) that are approximately a million-fold more sensitive than conventional nucleotide incorporation assays in the testing of biologicals is described. Use of PERT assays can be applied to three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load and (iii) monitoring levels of infectious retrovirus generation in cell lines that contain endogenous retroviruses.

  19. From Antenna to Assay

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.


    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  20. Transporter assays and assay ontologies: useful tools for drug discovery.

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P


    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  1. Experiences with the in vivo and in vitro comet assay in regulatory testing.

    Frötschl, Roland


    The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages.

  2. Comparative evaluation of alkylphenolic compounds on estrogenic activity in vitro and in vivo.

    Kwack, Seung Jun; Kwon, Oran; Kim, Hyung Sik; Kim, Soon Sun; Kim, So Hee; Sohn, Kyung Hee; Lee, Rhee Da; Park, Chul Hoon; Jeung, Eui Bae; An, Beum-Soo; Park, Kui Lea


    This study was undertaken to compare the sensitivity of screening test methods and to investigate the structure-activity relationships of the estrogenic activity of alkylphenolic compounds (APs) using in vitro and in vivo assays. Two in vitro systems, MCF-7 cell proliferation (E-screen assay) and competitive binding assay to estrogen receptor (ER), were selected to evaluate the estrogenic effects. Uterotrophic assay and Calbindin-D9K (CaBP9K) mRNA expression were also examined in ovariectomized Sprague-Dawley female rats. A series of APs with various alkyl groups were examined, namely, 4-propylphenol, 4-butylphenol, 4-t-butylphenol, 4-pentylphenol, 4-nonylphenol, 4-octylphenol, 4-t-octylphenol, and 4-phenylphenol, and 17beta-estradiol (E2) was used as a positive control. In the E-screen assay, E2 was found to induce maximum proliferation of MCF-7 cells at 1 nM. Among the APs, 4-t-octylphenol and 4-nonylphenol were found to be considerably more potent than any other compound and estrogenic effects were detectable at 1 and 10 microM, respectively. 4-t-Octylphenol and 4-nonylphenol inhibited the binding of E2 to the ER of MCF-7 cells in a competitive ER binding assay. The uterotrophic effects to APs (10, 50, 200, and 400 mg/kg/d) were compared to E2 (1 microg/kg) in ovariectomized rats after treatment for 3 d. 4-Nonylphenol, 4-t-octylphenol, and 4-phenylphenol produced dose-dependent increases in the uterine weights of ovariectomized rats. In the CaBP-9K mRNA expression test, CaBP-9K mRNA levels were detected in the uteri of ovariectomized rats treated with 4-pentylphenol (400 mg/kg), 4-nonylphenol, 4-phenylphenol (200 and 400 mg/kg), and 4-t-octylphenol (50 mg/kg and above), respectively. In the dot blot assay, CaBP-9K mRNA levels were significantly increased in rats exposed to 4-t-octylphenol (200 and 400 mg/kg), 4-pentylphenol, 4-nonylphenol, and 4-phenylphenol (400 mg/kg), respectively. Among the APs, compounds with bulky alkyl groups or higher carbon numbers

  3. Development of a thyroperoxidase inhibition assay for high-throughput screening.

    Paul, Katie B; Hedge, Joan M; Rotroff, Daniel M; Hornung, Michael W; Crofton, Kevin M; Simmons, Steven O


    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach.


    Bishnoi Kapil*, , ,; Kataria Mahesh; Singhal Vipin; Gupta Deepika


    Micronutrients added to foods are analyzed using various procedures depending on their nature and properties. The microbiological assays are better than chemical method because any suitable change in vitamin molecule which may not be detected by chemical method will be revealed by change in microbial activity. The microbiological assay of vitamins is based upon the comparison of the stimulation of growth of bacteria by measured concentration of vitamin with that produced by known concentratio...

  5. Development of a hydrophilic interaction chromatography-UPLC assay to determine trigonelline in rat plasma and its application in a pharmacokinetic study%建立HILIC-UPLC联用分析法测定血浆中葫芦巴碱及在药动学研究中的应用

    程再兴; 吴锦俊; 刘中秋; 林娜


    目的:葫芦巴碱是咖啡豆中含量较多的生物碱之一,为了研究其药代动力学特征,本研究建立了一种简便、灵敏、准确的亲水相互作用色谱-超高效液相联用分析方法,并研究了葫芦巴碱和咖啡豆甲醇提取物的药代动力学.方法:大鼠灌胃和尾静脉注射给予葫芦巴碱和咖啡豆甲醇提取物后,眼眶静脉丛取血,血液样本采用亲水相互作用色谱-超高效液相联用分析法进行分析,药代动力学参数按非房室模型,通过药动学软件87/97进行计算.结果:对所建立的方法进行了方法学的考察,线性范围为0.12-1 00 μg·mL-1,最低定量下限为0.12 μg·mL-1,日内日间精密度、准确度、提取回收率和稳定性等均达到体内药物分析要求.葫芦巴碱口服和尾静脉注射给药后,AUC(0-∞)分别为(4 066.83±l 244.41)和(3 544.29±908.80) min·μg·mL-1,生物利用度达到57.37%.咖啡豆甲醇提取物口服后,AUC(0-∞)为(4 566.75±1 435.64) min· μg·mL-1,绝对生物利用度为64.42%,相对生物利用度为112.29%.结论:所建立的亲水作用色谱-超高效液相联用分析法简单、准确、重现性好;葫芦巴碱无论是纯品还是在咖啡豆甲醇提取物中都有较好的生物利用度,而且没有明显的差异.%AIM:Trigonelline (Tr) is the second most abundant alkaloid in coffee beans.This study developed an assay combining hydrophilic interaction chromatography with ultra performance liquid chromatography (HILIC-UPLC) for the quantification of Tr in rat plasma to determine its pharmacokinetic behavior.METHODS:After the administration of Tr by gavage as well as intravenous injection and that of methanol extract of coffee beans (MECB) orally,blood samples from the experimental rats were analyzed using the HILIC-UPLC assay.Pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using Practical Pharmacokinetic Program Version 87/97.RESULTS

  6. Analysis of Hexanitrostilbene (HNS) and Dipicryethane (DPE) for Mutagenicity by the Ames/Salmonella Assay

    Wu, R; Felton, J


    The Ames/Salmonella assay, developed by Professor Bruce Ames at the University of California, Berkeley, is a rapid and sensitive assay for detecting mutagenicity of various chemical compounds (Maron and Ames, 1983). It is a widely accepted short-term assay for detecting chemicals that induce mutations in the histidine (his) gene of Salmonella typhimurium. This is a reverse mutation assay that detects the mutational reversion of his-dependent Salmonella to the his-independent counterpart. Thereby, mutagenic compounds will increase the frequency of occurrence of his-independent bacterial colonies. The assay utilizes the specific genetically constructed strains of bacteria either with or without mammalian metabolic activation enzymes (S9), Aroclor induced rat liver homogenate to assess the mutagenicity of different compounds. In this study, we will use the Ames/Salmonella assay to investigate the mutagenicity of Hexanitrostilbene (HNS) from both Bofors and Pantex, and Dipicryethane (DPE).

  7. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Carina Ladeira


    The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

  8. Effect of combining in vitro estrogenicity data with kinetic characteristics of estrogenic compounds on the in vivo predictive value.

    Punt, Ans; Brand, Walter; Murk, Albertinka J; van Wezel, Annemarie P; Schriks, Merijn; Heringa, Minne B


    With the ultimate aim of increasing the utility of in vitro assays for toxicological risk assessment, a method was developed to calculate in vivo estrogenic potencies from in vitro estrogenic potencies of compounds by taking into account systemic availability. In vitro estrogenic potencies of three model compounds (bisphenol A, genistein, and 4-nonylphenol) relative to ethinylestradiol (EE2), determined with the estrogen receptor alpha (ERα) transcriptional activation assay using hER-HeLa-9903 cells, were taken from literature and used to calculate the EE2 equivalent (EE2EQ) effect doses in the predominantly ERα-dependent rat uterotrophic assay. Compound-specific differences in hepatic clearance relative to the reference compound EE2 were determined in vitro to examine whether in vivo estrogenic potencies reported in literature could be more accurately estimated. The EE2EQ doses allowed to predict in vivo uterotrophic responses within a factor of 6-25 and the inclusion of the hepatic clearance further improved the prediction with a factor 1.6-2.1 for especially genistein and bisphenol A. Yet, the model compounds still were less potent in vivo than predicted based on their EE2 equivalent estrogenic potency and hepatic clearance. For further improvement of the in vitro to in vivo predictive value of in vitro assays, the relevance of other kinetic characteristics should be studied, including binding to carrier proteins, oral bioavailability and the formation of estrogenic metabolites.

  9. Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins


    streptavidin-phycoerythrin (PE) similar to sandwich enzyme-linked immunosorbent assays (ELISAs). The 3 fluorescent markers (2 beads plus PE) allow for...least expensive platform. It uses a magnetic plate to create a monolayer of beads that can be imaged with a light-emitting-diode-based imager capable... Magnetic Luminex Screening Assay Rat Premixed Multi-Analyte Kit, a kit was purchased that included all of the 17 analytes included in company’s catalog

  10. New competition assay for the solubilized hepatic galactosyl receptor

    Ray, D.A.; Weigel, P.H.


    The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total /sup 125/I-asialo-orosomucoid (/sup 125/I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both /sup 125/I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the /sup 125/I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described.

  11. Barcoded microchips for biomolecular assays.

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu


    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  12. A standardized Humulus lupulus (L.) ethanol extract partially prevents ovariectomy-induced bone loss in the rat without induction of adverse effects in the uterus.

    Keiler, Annekathrin M; Helle, Janina; Bader, Manuela I; Ehrhardt, Tino; Nestler, Kristin; Kretzschmar, Georg; Bernhardt, Ricardo; Vollmer, Günter; Nikolić, Dejan; Bolton, Judy L; Pauli, Guido F; Chen, Shao-Nong; Dietz, Birgit M; van Breemen, Richard B; Zierau, Oliver


    Hops (Humulus lupulus (L.)) dietary supplements are of interest as herbal remedies to alleviate menopausal symptoms, such as hot flushes, depression and anxiety. So far, the evidence regarding estrogenic and related properties of hops preparations has been considered insufficient for a market authorization for menopausal indications. The study aims to investigate a chemically standardized hops extract regarding its safety in the uterus, as wells as its efficacy to prevent bone loss in the ovariectomized rat model. Female Wistar rats were ovariectomized and divided into a control group receiving phytoestrogen-free diet, a group treated with E2benzoate (0.93 mg/kg body weight/d) and a group treated with the standardized hops extract (60 mg/kg body weight/d) for 8 weeks. Micro-computed tomography of the tibiae and vertebrae, as wells as histological changes in the uterus and tibia were analyzed. Neither uterotrophic nor proliferative effects were observed in the endometrium in response to the oral 8-week administration of the hops extract. However, site-dependent skeletal effects were observed. The hops extract significantly decreased the number of osteoclasts in the tibial metaphysis and prevented reduction of the trabecular thickness that resulted from estradiol depletion. In contrast, the hops extract did not prevent the ovariectomy-induced micro-architectural changes in the lumbar vertebra. Certain parameters (e.g. thickness and number of trabeculae) were even found to be below the values determined in the ovariectomized control group. Taken together, the results provide evidence for the safety of the standardized hops extract and point to a weak bone type-specific, protective effect on bone loss following estradiol depletion. Copyright © 2017. Published by Elsevier GmbH.

  13. In vivo genotoxicity assessment of sertraline by using alkaline comet assay and the cytokinesis-block micronucleus assay.

    Battal, Dilek; Aktas, Ayca; Sungur, Mehmet Ali; Kadioglu, Ela; Eker, Ebru Derici; Sahin, Nefise Ozlen; Saygi, Sahan


    Sertraline, a leading antidepressant in the selective serotonin reuptake inhibitor (SSRI) group of medicine, is the most frequently prescribed drug. In this study, the alkaline comet assay and the cytokinesis-block micronucleus (CBMN) assay were used to investigate genotoxicity potential of sertraline in the peripheral blood lymphocytes (PBLs) of acute and chronic sertraline-treated Wistar albino rats. Male Wistar albino rats (n = 48) were administered low, medium and high doses of sertraline (10, 40, 80 mg/kg) for acute and chronic treatment by employing the gavage method to investigate genotoxicity of the administered drug. The data (tail length, tail intensity and tail moment) were analysed and indicated that there was no statistically significant difference between sertraline-treated groups and the negative control group with respect to DNA damage (p > 0.05). However, it was observed that acute sertraline administration had caused much more DNA damage in comparison with chronic treatment (p sertraline treatment. Based on the outcome of comet assay, detection of statistically insignificant DNA damage may be due to the fact that sertraline did not cause damage on DNA. Also, increase in frequency of MN in chronic sertraline treatment suggests that chronic sertraline administration might influence some mechanisms of cell division. Therefore, dose adjustment in depressed patients seems significant as it may help prevent further prognosis of the diseases.

  14. Chromosome aberration assays in Allium

    Grant, W.F.


    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  15. On-line radiochemical assay for monoamine oxidase utilizing high-performance liquid chromatography

    Nissinen, E.; Linko-Loeppoenen SMae; Maennistoe P4


    A fast and sensitive assay for the determination of monoamine oxidase activity was developed. The method is based on the separation and quantitation of /sup 14/C-labeled assay products by high-performance liquid chromatography, which is interfaced directly into a flow-through radioactivity detector. This allows on-line quantitation of the radioactive compounds with picomole sensitivity. The method makes possible the complete separation and detection of the deaminated products of monoamine oxidase A and B substrates benzylamine and 5-hydroxytryptamine, respectively. This assay has been applied to the measurement of monoamine oxidase A and B activities in rat brain.

  16. A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds.

    Augustine-Rauch, Karen; Zhang, Cindy X; Panzica-Kelly, Julieta M


    Increasing need for proactive safety optimization of pharmaceutical compounds has led to generation and/or refinement of in vitro developmental toxicology assays. Our laboratory has developed three in vitro developmental toxicology assays to assess teratogenic liability of pharmaceutical compounds. These assays included a mouse molecular embryonic stem cell assay (MESCA), a dechorionated zebrafish embryo culture (ZEC) assay, and a streamlined rat whole embryo culture (rWEC) assay. Individually, the assays presented good (73-82%) predictivity. However, it remains to be determined whether combining or tiering the assays could enhance performance. Seventy-three compounds representing a broad spectrum of pharmaceutical targets and chemistry were evaluated across the assays to generate testing strategies that optimized performance. The MESCA and ZEC assays were found to have two limitations: compound solubility and frequent misclassification of compounds with H1 receptor or GABAnergic activity. The streamlined rWEC assay was found to be a cost-effective stand-alone assay for supporting poorly soluble compounds and/or ones with H1 or GABAnergic activity. For all other compounds, a tiering strategy using the MESCA and ZEC assays additionally optimized throughput, cost, and minimized animal use. The tiered strategy resulted in improved performance achieving 88% overall predictivity and was comparable with 89% overall predictivity achieved with frequency analysis (final teratogenic classification made from most frequent teratogenic classification from each individual assay). Furthermore there were 21 compounds in the test set characterized as definitive or suspect human teratogens and the multiassay approach achieved 95 and 91% correct classification using the tiered or frequency screening approach, respectively.

  17. A system for performing high throughput assays of synaptic function.

    Chris M Hempel

    Full Text Available Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.

  18. An enzyme-linked immunosorbent assay for autoantibodies against the nuclear protein Scl-70

    Geisler, C; Høier-Madsen, M


    This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established...

  19. 21 CFR 225.158 - Laboratory assays.


    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  20. Comet Assay in Cancer Chemoprevention.

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina


    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  1. The skin-blanching assay.

    Smit, P; Neumann, H A M; Thio, H B


    The skin-blanching assay is used for the determination and bioequivalence of dermatologic glucocorticoids (GCs). The exact mechanism of the production of blanching is not fully understood, but it is considered that local vasoconstriction of the skin microvasculature and the consequent blood-flow reduction cause this phenomenon. Several factors influence skin blanching, including drug concentration, duration of application, nature of vehicle, occlusion, posture and location. The intensity of vasoconstriction can be measured in several ways: visual or quantitative methods, such as reflectance spectroscopy, thermography, laser Doppler velocimetry and chromametry. In literature, contradicting results in the correlation of the skin-blanching assay with different tests to determine GC sensitivity have been reported, limiting its clinical usefulness.

  2. Bioluminescence assay for cell viability.

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N


    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  3. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Carina Ladeira; Susana Viegas; Manuel C. Gomes


    The cytokinesis-block micronucleus cytome (CBMN) assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells. The endpoints possible to be measured are micronuclei (MN), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/...

  4. Calibrated user-friendly reverse transcriptase-PCR assay

    Bor, M V; Sørensen, B S; Rammer, P


    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...... and to accept or reject the results according to existing rules for quality assurance....... and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17......). We conclude that the CURT-PCR method developed is suitable for routine applications such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run...


    Paniagua-Pérez, Rogelio; Flores-Mondragón, Gabriela; Reyes-Legorreta, Celia; Herrera-López, Brígida; Cervantes-Hernández, Isabel; Madrigal-Santillán, Osiris; Morales-González, José Antonio; Álvarez-González, Isela; Madrigal-Bujaidar, Eduardo


    Background: Beta-sitosterol (BS) is a compound discovered to be present in numerous plants. A number of interesting biomedical properties have been attributed to BS, including immuno-modulating and anti-inflammatory activities. Therefore, the aim of this report was to evaluate its anti-inflammatory capacity by applying various rodent experimental tests. Methods: To carry out the objective of the study we applied the methods indicated here. Two of the adopted methods were based on the passive reverse Arthus reaction: the rat paw edema test and the rat pleurisy assay. We also applied two methods related with the non-specific acute inflammation: the mouse ear edema test, and the mouse mieloperoxidase activity assay. Results: The results obtained in all tests established a significant anti-inflammatory potential of BS. In the rat paw edema test we found an inhibitory effect which goes from 50-70%; in the rat pleurisy assay our findings with respect to the volume of pleural exuded showed a reduction of 46%, as well as a 20% low amount of neutrophils in comparison with the level of the control group. In the mouse ear edema test we found a mean inflammatory inhibition of 75%, and with respect to mieloproxidase activity the results showed a significant inhibition induced by the three doses of BS. Conclusions: In the present study we determined a potent anti-inflammatory capacity of BS in specific and non-specific types of acute inflammation in rodents. PMID:28480389

  6. Potential neoplastic effects of parathion-methyl on rat liver



    The mutagenic and carcinogenic effects of parathion-methyl were examined by bacterial reverse assay and a long term experiment with Wistar rats. The potential mutagenic effect of parathion-methyl in Salmonella typhimurium TA100 bacterial cells was observed without rat liver S9 metabolic activation. Parathion-methyl was further investigated for pathological changes in rat pancreas and liver. The long-term rat experiments showed that parathion-methyl exposure for 3 months can cause pathological changes in rat pancreases acinar cells and pancreatic hepatocytes. Atypical acinar cell focuses (AACF) were determined in the liver and pancreas of the rats. The results from short-term Ames test and long-term rat experiments suggest that parathion-methyl would be potential carcinogenic.

  7. Quantification assays for total and polyglutamine-expanded huntingtin proteins.

    Douglas Macdonald

    Full Text Available The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD. Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

  8. 21 CFR 864.7525 - Heparin assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and... HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A heparin assay is a device used to determine the level of the anticoagulant heparin in the...

  9. A colorimetric assay for cytokinin oxidase.

    Libreros-Minotta, C A; Tipton, P A


    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  10. Multicentre comparison of a diagnostic assay

    Waters, Patrick; Reindl, Markus; Saiz, Albert;


    ) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

  11. 21 CFR 866.3210 - Endotoxin assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a) Identification. An endotoxin assay is a device that uses serological techniques in whole blood. The device...

  12. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida;


    of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...

  13. Steroid assays in paediatric endocrinology.

    Honour, John W


    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation.

  14. Genotoxicity assessment of melamine in the in vivo Pig-a mutation assay and in a standard battery of assays.

    Tu, Honggang; Zhang, Ming; Zhou, Changhui; Wang, Zheng; Huang, Pengcheng; Ou, Hongmei; Chang, Yan


    The genotoxicity of melamine was evaluated with the combined Pig-a mutation/micronucleus assay, the bacterial reverse mutation assay, and the in vitro cytokinesis-block micronucleus assay (CBMN). Five groups of six- to eight-week-old male Sprague-Dawley (SD) rats were given three daily doses of vehicle control (100% pure sesame oil), melamine (500, 1000, and 2000 mg/kg) or positive control (N-ethyl-N-nitrosourea, ENU, 20 mg/kg) by oral gavage. Peripheral blood was sampled pre-dose (day -1) and at time points up to day 60. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies, on days -1, 15, 29 and 60, and micronucleus frequencies were measured in RETs on day 4. No significant increases in RBC(CD59-) or RET(CD59-) frequencies were observed for the melamine-treated group at any of the time points studied, but the positive control, ENU, induced statistically significant increases compared with the vehicle control. Similar results were obtained in the micronucleus assay. Melamine did not induce statistically significant increases in %MN-RET. In the bacterial reverse mutation assay, melamine was tested from 62.5 to 1000 μg/plate in tester strains TA97a, TA98, TA100, TA102, and TA1535, with and without metabolic activation, and no evidence of toxicity or mutagenicity was observed at any dose tested. In the in vitro CBMN assay, in Chinese hamster ovary (CHO) cells, melamine was tested (75, 150, and 300 μg/mL) in the presence and absence of S9 mix, and no positive increases in the number of cells containing micronuclei were seen. These results suggest that melamine does not exhibit significant genotoxic potential. These data could be valuable for risk assessment purposes and also for further characterizing the new in vivoPig-a gene mutation assay.

  15. Activity assay of membrane transport proteins

    Hao Xie


    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  16. treated rats



    Jan 8, 2014 ... Our results show, for the first time, that oral administration of C. edulis ... the exact mechanisms of these hematological changes produced by .... Hematological analysis .... rats are subjected to the additional stress of hypoxia to.

  17. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

    Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine Lydia


    different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after...... tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts...

  18. Effects of currently used pesticides in the AhR-CALUX assay

    Long, Manhai; Laier, Peter; Vinggaard, Anne Marie


    to be a rapid and sensitive assay for assessing the potency of AhR-activating compounds. We have used the AhR-CALUX assay to investigate the AhR-mediated activity of the persistent organochlorine insecticide dieldrin and twenty-two pesticides currently used in Denmark by employing the rat H4IIE and the human TV......101L hepatoma cell lines. In comparison the results indicated that the rat H4IIE cell line is more sensitive than the human TV101L for detection of TCDD inducing AhR-CALUX activity. The pesticides iprodione, chlorpyrifos and prochloraz showed dose-dependent AhR agonistic effects in both cell lines...... at concentrations above 10, 1 and 1 microM, respectively. However, some pesticides (methiocarb, chlorothalonil, tribenuron-methyl, paclobutrazol and tolchlofos-methyl) elicited differential responses in the two cell lines....

  19. Simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay.

    Demir, Ihsan Ekin; Tieftrunk, Elke; Schäfer, Karl-Herbert; Friess, Helmut; Ceyhan, Güralp O


    Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

  20. Neural plasticity occurs in the adrenal medulla of asthmatic rats

    FENG Jun-tao; LI Xiao-zhao; HU Cheng-ping; WANG Jun; NIE Hua-ping


    Background Airway symptoms in asthma are related to decrease of epinephrine secretion, which may be ascribed to elevated nerve growth factor (NGF) in the organism.The aim of this study was to monitor the neuroendocrine alteration in the adrenal medulla of asthmatic rats.Methods Sixteen rats were randomly divided into two groups (n=8), control group and asthma group, and the asthmatic rats were sensitized and challenged with ovalbumin (OVA).The levels of NGF, epinephrine and norepinephrine in serum were detected by enzyme linked immunosorbent assay (ELISA), the NGF expression in adrenal medulla was detected by immunohistochemistry, and the changes in the ultrastructure of the adrenal medulla was observed by electron microscopy.Results The NGF expression was increased in asthmatic rats compared with control rats.Compared with control rats,the results indicated that the epinephrine level was decreased in asthmatic rats, but no significant difference was found in norepinephrine levels.We found more ganglion cells in the adrenal medulla of asthmatic rats than in control rats, with NGF immunostaining mainly located in these ganglion cells.Electron microscopic images showed the density of chromaffin granula decreased and there was shrunken nucleolemma in the adrenal medullary cells of asthmatic rats.Conclusion The innervation of the adrenal medulla is changed in asthmatic rats, and it may contribute to the epinephrine decrease in asthma.

  1. Direct Spectrophotometric Assay for Benzaldehyde Lyase Activity

    Dessy Natalia


    Full Text Available Benzaldehyde lyase from Pseudomonas fluorescens Biovar I. (BAL, EC is a versatile catalyst for the organic synthesis of chiral α-hydroxy ketones. To allow fast assessment of enzyme activity, a direct spectrophotometric assay is desirable. Here, a new robust and easy-to-handle assay based on UV absorption is presented. The assay developed is based on the ligation of the α-hydroxy ketone (R-2,2′-furoin from 2-furaldehyde. A robust assay with direct monitoring of the product is facilitated with a convenient concentration working range minimising experimental associated with low concentrations.

  2. The comet assay – how to recognise “good data”

    William Barfield


    Full Text Available Testing of potentially genotoxic materials currently involves use of in vitro assays such as the Ames test (gene mutations, the chromosome aberration assay and in vitro micronucleus assay (predominantly using human peripheral lymphocytes to detect clastogenicity and/or aueuploidy and the mouse lymphoma assay (gene mutations. In addition, one in vivo assay, predominately the in vivo micronucleus assay, is “normally” required to satisfy regulatory testing requirements and on occasion a second in vivo assay is required to assist in interpretation of results. Following release of the OECD 489 guideline “In vivo mammalian alkaline comet assay” in September 2014 the in vivo comet assay is now considered to be the second in vivo genetic toxicology assay of choice, effectively replacing the use of the UDS assay. The comet assay can be used to detect single strand and double strand breaks by measuring the median %tail intensity under alkaline conditions (pH>13. Following the JaCVAM validation trial, subsequent publication of the OECD 489 guideline recommended using the liver (site of metabolism and glandular stomach (site of contact of rats. However, any tissue can be examined if experimental competency with the tissue of interest has been proven. A number of key areas need careful consideration when performing the assay and interpreting the data. Both duration and temperature of tissue preparation have been shown to be critical parameters for obtaining “good data” along with the correct electrophoresis conditions to detect ‘weak’ effects. Study design requires careful thought in terms of animals per group, slides analysed per animal and cells analysed per slide and to facilitate this a special interest group within industry has published recommendations on statistical analysis of the comet assay. The sensitivity of the comet assay within the laboratory can be determined with the use of ‘power curves’ and identifying a high

  3. Identification multiplex assay of 19 terrestrial mammal species present in New Zealand.

    Ramón-Laca, Ana; Linacre, Adrian M T; Gleeson, Dianne M; Tobe, Shanan S


    An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species-specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species-specific fragments, making the assay self-confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost-effective manner.

  4. Pathogenic assays of acanthamoeba belonging to the t4 genotype.

    Hamed Mirjalali


    Full Text Available Acanthamoeba genus is introduced as opportunistic and cosmopolitan parasite. Monkey and wistar rat are appropriate models for experimental study on Acanthamoeba infection. In this study Acanthamoeba spp. were isolated from hot spring (HS, windows dust (WD and a corneal sample of keratitis patient (KP and their pathogenicity surveyed by in vitro and in vivo tests.Isolates of Acanthamoeba were cultivated axenically for 12 months in PYG medium. Overall, 30 wistar rats, in 6 equal groups were used for developing experimental Acanthamoeba keratitis (AK and Granulomatous Amoebic Encephalitis (GAE. The Keratitis and Granulomatous Encephalitis experiments were performed by intrastromal and intranasal inoculation of Acanthamoeba cysts, respectively. Pathogenicity of the three isolates was also evaluated by in vitro test using osmotolerance and temperature tolerance assays. Identification of genotypes were performed by PCR technique and sequencing.None of the isolates could perform AK and GAE in wistar rats, although all isolates were described as T4 genotype. Isolates obtained from KP and WD could grow only in 30 °C, but not in 37 °C and 40 °C. On the other hand, HS isolate grew in 30 °C and 37 °C but not in 40 °C. Moreover, all of isolate grew in 0.5 M mannitol but not in 1 M and 1.5 M.T4 isolates with a long-term axenic culture and different factors related to host and parasite may play role in pathogenicity of these free-living amoebae.

  5. Assay-dependent variability of serum insulin concentrations: a comparison of eight assays.

    Tohidi, Maryam; Arbab, Parvaneh; Ghasemi, Asghar


    Although insulin measurement is essential for both clinical and research purposes, there is currently no reference method for insulin assays. The aim of this study was to compare results of serum insulin determined by a number of commercially available assays. We compared eight insulin assays by analyzing 165 serum samples. Assays included two chemiluminescence (Roche and DiaSorin), four ELISA (Tosoh, Mercodia, Monobind, and Diametra), and two IRMA (Izotop and BioSource) methods. Each assay was compared with the mean of all assay methods and Bland-Altman difference plots were used to measure agreement between each assay and overall mean. Least squared perpendicular distance regression analysis (Deming's method) was used to calculate slope and intercept for bias and also for each assay vs. mean of eight assays. Findings showed that the lowest and highest median insulin concentrations varied by a factor of 1.8. Maximum and minimum correlations with mean of assays were observed for Roche (0.992) and BioSource (0.844), respectively. Significant bias was observed in six assays. In pairwise comparisons of different assays, the highest and least mean differences were 7.78 μU/mL and -0.14 μU/mL, respectively. In conclusion, serum insulin measurement with different assays showed a maximum of 1.8-fold difference, a point that should be taken into consideration in the interpretation of circulating insulin levels in both clinical and research fields.

  6. Surface modification of poly(dimethylsiloxane) for microfluidic assay applications

    Seguin, Christine; McLachlan, Jessica M.; Norton, Peter R. [Department of Chemistry, University of Western Ontario, 1151 Richmond St., London, ON, N6A 5B7 (Canada); Lagugne-Labarthet, Francois, E-mail: [Department of Chemistry, University of Western Ontario, 1151 Richmond St., London, ON, N6A 5B7 (Canada)


    The surface of a poly(dimethylsiloxane) (PDMS) film was imparted with patterned functionalities at the micron-scale level. Arrays of circles with diameters of 180 and 230 {mu}m were functionalized using plasma oxidation coupled with aluminum deposition, followed by silanization with solutions of 3-aminopropyltrimethoxy silane (3-APTMS) and 3-mercaptopropyltrimethoxy silane (3-MPTMS), to obtain patterned amine and thiol functionalities, respectively. The modification of the samples was confirmed using X-ray photoelectron spectroscopy (XPS), gold nanoparticle adhesion coupled with optical microscopy, as well as by derivatization with fluorescent dyes. To further exploit the novel surface chemistry of the modified PDMS, samples with surface amine functionalities were used to develop a protein assay as well as an array capable of cellular capture and patterning. The modified substrate was shown to successfully selectively immobilize fluorescently labeled immunoglobulin G (IgG) by tethering Protein A to the surface, and, for the cellular arrays, C2C12 rat endothelial cells were captured. Finally, this novel method of patterning chemical functionalities onto PDMS has been incorporated into microfluidic channels. Finally, we demonstrate the in situ chemical modification of the protected PDMS oxidized surface within a microfluidic device. This emphasizes the potential of our method for applications involving micron-scale assays since the aluminum protective layer permits to functionalize the oxidized PDMS surface several weeks after plasma treatment simply after etching away the metallic thin film.




    Receptor assays occupy a particular position in the methods used in bioanalysis, as they do not exploit the physico-chemical properties of the analyte. These assays make use of the property of the analyte to bind to the specific binding site (receptor) and to competitively replace a labelled ligand

  8. Assessing sediment contamination using six toxicity assays

    Allen G. BURTON Jr.


    Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

  9. Radioreceptor assay: theory and applications to pharmacology

    Perret, G. (U.E.R. de Medecine, Sante et Biologie Humaine, 93 - Bobigny (France)); Simon, P. (Faculte de Medecine Pitie-Salpetriere, 75 - Paris (France))

    The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, ..beta..-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising.

  10. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay

    Galleano, Iacopo; Schiedel, Matthias; Jung, Manfred


    and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2...

  11. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana


    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  12. Assays for Determination of Protein Concentration.

    Olson, Bradley J S C


    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc.

  13. A combination assay for simultaneous assessment of multiple signaling pathways.

    Goetz, A S; Liacos, J; Yingling, J; Ignar, D M


    We have developed an assay in which modulation of two or more signaling pathways can be assessed concurrently by combining reporter gene systems with fluorescent probe technology. The validation of this method was achieved by indirect analysis of adenylyl cyclase activation with the use of a cyclic AMP response element (CRE)-luciferase reporter system in combination with the measurement of calcium mobilization by Calcium Green-1 AM fluorescence on a fluorescent imaging plate reader. To demonstrate the utility of the method in studying the pharmacology of receptors that couple to more than one G protein, Chinese hamster ovary (CHO) cells, which stably expressed both the CRE-luciferase reporter gene and the human pituitary adenylyl cyclase-activating peptide (PACAP) receptor, were treated with PACAP 1-27 and 1-38. Calcium mobilization and the induction of adenylyl cyclase activity in response to each concentration of peptide were assessed in individuals wells. This assay may also be used to screen for ligands of two or more unrelated receptors simultaneously without compromising the assessment of either signaling pathway. To illustrate this point, Rat-1 fibroblasts, which expressed human alpha1A receptors, were cocultured with CRE-luciferase CHO cells, which expressed human GLP-1 receptors. Calcium mobilization elicited by phenylephrine agonism of the alpha1A receptor was assessed in the same assay as GLP-1-induced activation of adenylyl cyclase. The pEC(50) for each agonist was similar to that observed when the cell lines were not cocultured. The number of different receptors that can be screened per well is limited only by the ability to distinguish different reporter gene signals and fluorescent indicators.

  14. A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

    Gentry-Nielsen Martha J


    Full Text Available Abstract Background Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS to recruit PMNs to their lungs. They are then infected with live 5(-and 6 carboxyfluorescein diacetate succinimidyl ester (CFDA/SE labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. Results The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. Conclusion This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

  15. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C. [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States); Miller, R.T., E-mail: [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States)


    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  16. In vitro screening assay for teratogens using growth inhibition of human embryonic cells.

    Pratt, R M; Willis, W D


    We have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. We suggest that this assay, along with the complementary tumor cell-attachment assay of Braun et al. [Braun, A. G., Emerson, D. J. & Nichinson, B. B. (1979) Nature (London) 282, 507-509] may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo. Images PMID:3862095

  17. In vitro screening assay for teratogens using growth inhibition of human embryonic cells

    Pratt, R.M.; Willis, W.D.


    The authors have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. The authors suggest that this assay, along with the complementary tumor cell-attachment assay of Braun may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.

  18. Improved benzodiazepine radioreceptor assay using the MultiScreen (R) Assay System

    Janssen, MJ; Ensing, K; de Zeeuw, RA


    In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time, The filtration in this method was performed by using the MultiScreen(R) Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom,

  19. Nano-immunosafety: issues in assay validation

    Boraschi, Diana; Italiani, Paola [Institute of Biomedical Technologies, National Research Council, Via G. Moruzzi 1, 56124 Pisa (Italy); Oostingh, Gertie J; Duschl, Albert [Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Casals, Eudald; Puntes, Victor F [Institut Catala de Nanotecnologia, Campus de la UAB - Facultat de Ciencies, Edifici CM7, 08193 Bellaterra (Spain); Nelissen, Inge, E-mail: [VITO NV, Boeretang 200, BE-2400 Mol (Belgium)


    Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

  20. Mobile non-destructive assay system

    Colarusso, A.P.; Audas, J.H.; Bieri, J.M.; Herrera, G.C.; Hastings, R.D.; Horton, W.S.; Kuckertz, T.H.; Kunz, W.E.; Medvick, P.A.; Vogel, P.A.


    A mobile system for non-destructive assay (NDA), developed at the Los Alamos National Laboratory, provides accurate and sensitive measurements for transuranic (TRU) isotopes contained in 208-iota drums of miscellaneous nuclear wastes. The NDA unit consists of four major subsystems: an assay chamber, counting and digital electronics, data acquisition, and a neutron generator. It performs both active and passive neutron waste measurements. The former determines the amount of fissile isotopes at a sensitivity level of 1 mg plutonium. The latter determines spontaneous fission and ..cap alpha..,n) isotopes at a comparable level. A complete assay consists of sequential active and passive measurements. The assay measurement and other supporting data are incorporated in a commercial spreadsheet program (Lotus 1,2,3) for further analysis, which includes various matrix corrections and a determination of whether or not the drum exceeds the 100-nCi/g threshold for TRU wastes. Field tests have been performed on three separate occasions, accomplishing more than 1800 waste drum assays. These waste drum assays are discussed, especially those comparing passive and active neutron measurements with independent segmented gamma scan assays. Results obtained with a set of 15 drums containing plutonium prepared from standards and actual hot waste matrices are also reviewed.

  1. Analytical characterization of the APTIMA HPV Assay.

    Dockter, Janel; Schroder, Astrid; Eaton, Barbara; Wang, Ann; Sikhamsay, Nathan; Morales, Liezel; Giachetti, Cristina


    Human papillomavirus (HPV) testing has improved the sensitivity for the detection of cervical pre-cancer and cancer as compared to Pap testing. Several HPV tests are commercially available and most target the DNA from 13 or 14 high-risk HPV types. The APTIMA HPV Assay however, detects HPV E6/E7 mRNA from 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. To determine the analytical performance characteristics of the APTIMA HPV Assay. Analytical sensitivity, analytical specificity, reproducibility, and the effect of potentially interfering substances was determined for the APTIMA HPV Assay on both the DTS (semi-automated) and TIGRIS DTS (fully automated) systems. The 95% detection limit for both systems was between 17 and 488 copies/reaction, depending on the HPV type. The assay did not cross-react with normal flora and opportunistic organisms that may be found in cervical samples, or low-risk HPV types. Spermicides, anti-fungal and anti-itch medications, whole blood, glacial acetic acid, and most lubricants did not interfere with assay performance. Those lubricants containing polyquaternium 15 did interfere with assay performance. Inter-instrument, inter-operator, inter-lot, and inter-run signal variability were 99% of the data. Intra-run variability was HPV Assay showed excellent performance and robustness.

  2. Oxymatrine reduces neuroinflammation in rat brain A signaling pathway

    Jiahui Mao; Yae Hu; Ailing Zhou; Bing Zheng; Yi Liu; Yueming Du; Jia Li; Jinyang Lu; Pengcheng Zhou


    Cerebral neuroinflammation models were established by injecting 10 μg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats.The rats were treated with an intraperitoneal injection of 120,90,or 60 mg/kg oxymatrine daily for three days prior to the lipopolysaccharide injection.Twenty-four hours after model induction,the hippocampus was analyzed by real-time quantitative PCR,and the cerebral cortex was analyzed by enzyme-linked immunosorbent assay and western blot assay.The results of the enzyme-linked immunosorbent assay and the real-time quantitative PCR showed that the secretion and mRNA expression of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α were significantly decreased in the hippocampus and cerebral cortex of model rats treated with oxymatrine.Western blot assay and real-time quantitative PCR analysis indicated that toll-like receptor 4 mRNA and protein expression were significantly decreased in the groups receiving different doses of oxymatrine.Additionally,120 and 90 mg/kg oxymatrine were shown to reduce protein levels of nuclear factor-kB p65 in the nucleus and of phosphorylated IkBα in the cytoplasm of brain cells,as detected by western blot assay.Experimental findings indicate that oxymatrine may inhibit neuroinflammation in rat brain via downregulating the expression of molecules in the toll-like receptor 4/nuclear factor-kB signaling pathway.

  3. No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay Ausência de mutações em éxons dos genes TP53, H-RAS e K-RAS em fígado de ratos wistar submetidos a ensaio de carcinogênese química de média duração

    Erick da Cruz Castelli


    Full Text Available The standard protocol to evaluate the carcinogenic potential of chemicals is the long-term bioassay in rodents, not performed in developing countries due to its high cost and complex operational procedures. Our laboratory has established an alternative medium-term bioassay in Wistar rats, also called DMBDD assay, based on the paradigm initiation/promotion of chemical carcinogenesis. This method was accepted by the Brazilian Environment Agency (IBAMA as an official source of evidence of carcinogenicity. The aim of this study was to evaluate alterations in exons 5 to 8 of the tumor suppressor gene TP53 and exons 1 and 2 of oncogenes K-RAS and H-RAS in neoplastic and preneoplastic hepatic lesions observed in DMBDD assay. The characterization of these alterations may contribute to the recognition of patterns of damage in critical genes, as well as to suggest mechanisms of action of the compounds tested in the protocol. Sixty male Wistar rats were separated into 3 groups: the first was treated with no chemicals; the second received five initiating agents and the third received initiation followed by phenobarbital. Liver DNA samples (obtained from formalin-fixed and paraffin-embedded tissues after histological analysis were evaluated by the non-isotopic PCR-SSCP technique. No changes in any analyzed exons were detected by the PCR-SSCP banding pattern in all experimental groups. This result suggests that liver mutations in exons 5 to 8 of TP53 and exons 1 and 2 of H-RAS and K-RAS are not among the early molecular alterations occurring in the hepatic carcinogenesis process induced by the DMBDD protocol in male Wistar rats.O teste padrão para identificar o potencial cancerígeno de compostos químicos é o estudo de longa duração em roedores, não realizado no Brasil. Nosso laboratório estabeleceu um teste alternativo de média duração (ensaio DMBDD, baseado no paradigma iniciação-promoção da carcinogênese química, adotado pelo Instituto

  4. Tissue distribution and subcellular localization of phosphatidylcholine transfer protein in rats as determined by radioimmunoassay

    Teerlink, T.; Krift, T.P. van der; Post, M.; Wirtz, K.W.A.


    A radioimmunoassay for the phosphatidylcholine-transfer protein from rat liver was used to measure levels of PC-transfer protein in rat tissues. The assay as described before (Teerlink T., Poorthuis B.J.H.M., Van der Krift T.P. and Wirtz K.W.A., Biochim. Biophys. Acta 665 (1981) 74–80) was modified

  5. Hepatic oxidative stress, genotoxicity and vascular dysfunction in lean or obese zucker rats

    Løhr, Mille; Folkmann, Janne Kjærsgaard; Sheykhzade, Majid;


    ) sensitive sites as measured by the comet assay. There were decreasing levels of oxidatively damaged DNA with age in the liver of lean rats, which occurred concurrently with increased expression of Ogg1. The 37 week old lean rats also had higher expression level of Hmox1 and elevated levels of DNA strand...

  6. Effects of Extended Exposure to the Antibacterial Triclosan in the the Adult Female Rat

    Triclosan (TCS), an antibacterial, has been shown to have endocrine disrupting activity in the rat. We reported previously that TCS advanced puberty in the female rat in the female pubertal assay and potentiated the estrogenic effect of ethinyl estradiol (EE) on uterine growth i...

  7. Differential regulation of renal prostaglandin receptor mRNAs by dietary salt intake in the rat

    Jensen, B L; Mann, Birgitte; Skøtt, O


    BACKGROUND: In this study, we tested the hypothesis that prostaglandin (PG) receptor expression in the rat kidney is subject to physiological regulation by dietary salt intake. METHODS: Rats were fed diets with 0.02 or 4% NaCl for two weeks. PG receptor expression was assayed in kidney regions...

  8. Polymerase chain reaction assay for avian polyomavirus.

    Phalen, D.N.; Wilson, V G; Graham, D L


    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By us...

  9. Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: historical database and influence of technical aspects.

    Pant, Kamala; Springer, S; Bruce, S; Lawlor, T; Hewitt, N; Aardema, M J


    There is increased interest in the in vivo comet assay in rodents as a follow-up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin-treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays.

  10. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    Buch Karl


    Full Text Available Abstract For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98 were included in the experiment to study its principal and general applicability.

  11. Isolation, production, purification, assay and characterization of ...

    Isolation, production, purification, assay and characterization of fibrinolytic ... are isolated from Bacillus subtilis, β-haemolytic Streptococci and urine sample. ... recombinant E.coli containing short fragment genomic DNA of Pseudomonas sp.

  12. 21 CFR 864.7250 - Erythropoietin assay.


    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass)...

  13. The comet assay: a heavenly method!

    Collins, Andrew R


    The contributions to this special issue of Mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of DNA damage and repair. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the comet assay in a historical context is illustrated by a bibliometric survey.

  14. Electrochemical Assay of Gold-Plating Solutions

    Chiodo, R.


    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  15. Variables Affecting Two Electron Transport System Assays

    Burton, G. Allen; Lanza, Guy R.


    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methan...

  16. Ovariectomized rats as a model of postmenopausal osteoarthritis

    Høegh-Andersen, Pernille; Tankó, László B; Andersen, Thomas L


    inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide......We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague-Dawley rats...... for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0...

  17. Radioimmune assay of ganglioside GM/sub 1/ synthase using cholera toxin

    Honke, K.; Taniguchi, N.; Makita, A.


    A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM/sub 2/ galactosyltransferase, which synthesizes GM/sub 1/, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM/sub 2/ were incubated with an enzyme sample, and a radiolabeled product, GM/sub 1/, was reacted with cholera toxin. The GM/sub 1/-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reaction product, GM/sub 1/. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn/sup 2 +/. The K/sub m/ values for UDP-Gal and GM/sub 2/ were 0.12 mM and 6, respectively.

  18. Validation of assays to monitor immune responses in the Syrian golden hamster (Mesocricetus auratus).

    Zivcec, Marko; Safronetz, David; Haddock, Elaine; Feldmann, Heinz; Ebihara, Hideki


    The Syrian golden hamster (Mesocricetus auratus) is a valuable but under-utilized animal model for studies of human viral pathogens such as bunyaviruses, arenaviruses, flaviviruses, henipaviruses, and SARS-coronavirus. A lack of suitable reagents and specific assays for monitoring host responses has limited the use of this animal model to clinical observations, pathology and humoral immune responses. The objective of this study was to establish and validate assays to monitor host immune responses in the hamster including important pro-inflammatory, anti-inflammatory and innate immune responses, as well as markers of apoptosis, cell proliferation, cell junction integrity and coagulation. Commercially available mouse and rat ELISA and luminex panels were screened for potential cross-reactivity, but were found to be of limited value for studying host responses in hamsters. Subsequently, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays for the detection of 51 immune-related and four internal reference genes were developed. To validate the immune-related assays, hamsters were infected with vesicular stomatitis virus (VSV), Indiana species, or treated with lipopolysaccharide (LPS) and host immune responses were monitored in selected organs. Ribosomal protein L18 was identified as the most stable internal reference gene. In conclusion, these new assays will greatly improve the use of the hamster as an important small animal model in infectious disease research.

  19. Development of an albumin copper binding (ACuB) assay to detect ischemia modified albumin.

    Eom, Ji-Eun; Lee, Eunyoung; Jeon, Kyung-Hwa; Sim, Jeongeun; Suh, Minah; Jhon, Gil-Ja; Kwon, Youngjoo


    Myocardial ischemia (MI) induces many changes in the body, including pH decrease and electrolyte imbalance. No obvious symptoms of MI appear until irreversible cellular injuries occur. Since early treatment is critical for recovery from ischemia, the development of reliable diagnostic tool is demanded to detect the early ischemic status. Ischemia modified albumin (IMA), formed by cleavage of the last two amino acids of the human serum albumin (HSA) N-terminus, has been considered so far as the most trustworthy and accurate marker for the investigation of ischemia. IMA levels are elevated in plasma within a few minutes of ischemic onset, and may last for up to 6 h. In the present study, we developed a novel assay for the examination of IMA levels to ameliorate the known albumin cobalt binding (ACB) test established previously. We observed a stronger copper ion bound to the HSA N-terminal peptide than cobalt ion by HPLC and ESI-TOF mass spectrometric analyses. The copper ion was employed with lucifer yellow (LY), a copper-specific reagent to develop a new albumin copper binding (ACuB) assay. The parameters capable of affecting the assay results were optimized, and the finally-optimized ACuB assay was validated. The result of the IMA level measurement in normal versus stroke rat serum suggests that the ACuB assay is likely to be a reliable and sensitive method for the detection of ischemic states.

  20. Effect of adrenalectomy and hydrocortisone on ventral prostate of rats

    Neena Nair; R.S. Bedwal; R.S. Mathur


    Aim: To study the effects of adrenalectomy and hydrocortisone on the ventral prostate of SD rats. Methods: In adrenalectomised (ADX) and ADX + hydrocortisone (1, 2, or 4 mg) treated rats, the prostatic histology and the cholesterol, protein, zinc, and copper levels and the enzymic profile (acid phosphatase, alkaline phosphatase, aryl sulphatase, lactic dehydrogenase, and leucine aminopeptidase) in the prostatic tissue were determined; the serum hormonal profile (testosterone, FSH and LH) was also assayed. Results: Adrenalectomy caused a progressive degeneration in prostatic structure that was not reversed by hydrocortisone treatment. The serum testosterone were significantly lower in ADX than in sham operated rats and lower in ADX + hydrocortisone than in ADX-C rats ( P < 0.01) The serum FSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham operated rats and higher in ADX + hydrocortisone than in ADX-C rats ( P <0.05-0.01). The prostatic zinc levels were significantly higher in sham operated than in ADX, and higher in ADX-C than in ADX + hydrocortisone rats ( P < 0.05 -0.01). The prostatic copper level was significantly lower in sham operated than in ADX, and lower in ADX-C than in the ADX + hydrocortisone rats ( P < 0.01). Conclusion: In rats, adrenalectomy leads to pathological and functional changes of the prostate. Hydrocortisone treatment at the doses employed did not reverse these changes.

  1. Controlling variation in the comet assay

    Andrew Richard Collins


    Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

  2. Cell Culture Assay for Human Noroviruses [response

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.


    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  3. The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

    Bas-jan Van Der Leede


    Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.


    Dwiyati Pujimulyani


    Full Text Available A study on the antioxidative properties of white saffron extract (in vivo has been conducted. The purpose of this study was to determine the antioxidative effects of white saffron extract in in vivo assay. Fresh white saffrons were peeled, washed, blanched at 100˚C in 0.5% citric acid solution for 5 minutes and grated. The ratio between grated white saffron and distilled water was 1:1; 1:2; 1:3 and 1:4. Then it was filtered in order to obtain white saffron extract. The extract was evaluated in terms of its antioxidant activity by using in vivo. Five-week old male Wistar rats were purchased from Experimental Animal Development Unit, Gadjah Mada University. After one week of adaptation, the rats were divided into six groups, feed and drinking water were provided ad libitum. White saffron extract was orally administrated using a syringe at 09.00 a.m and 14.00 p.m daily, for 14 days. The livers and serum were removed for analysis of thiobarbituric acid reactive subtances (TBARS, α-tocopherols and superoxide dismutase (SOD. The results of this study showed that white saffron extract has an antioxidative activity in the in vivo assay. The higher concentration of white saffron extract, the higher α-tocopherols and superoxide dismutase, but the TBARS value was lower. Keywords: Curcuma mangga Val., rat, antioxidant activity, in vivo

  5. Random assay in radioimmunoassay: Feasibility and application compared with batch assay

    Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)


    The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

  6. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J; Meyer, Matthew R.; Mesri, Mehdi; Abbatiello, Susan E; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J. C.; Fenyö, David; Hiltke, Tara; Ketchum, Karen A.; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C.; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D.; Thomas, Stefani; Townsend, R. Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G


    Summary The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal ( to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories. PMID:26867747

  7. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G.


    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal ( to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  8. Comet assay on mice testicular cells

    Anoop Kumar Sharma


    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  9. Research highlights: digital assays on chip.

    Kim, Donghyuk; Wei, Qingshan; Kong, Janay Elise; Ozcan, Aydogan; Di Carlo, Dino


    The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

  10. Biochemical and behavioral effects of long-term citalopram administration and discontinuation in rats Role of serotonin synthesis

    Bosker, Fokko J.; Tanke, Marit A. C.; Jongsma, Minke E.; Cremers, Thomas I. F. H.; Jagtman, Evelien; Pietersen, Charmaine Y.; van der Hart, Marieke G. C.; Gladkevich, Anatoliy V.; Kema, Ido P.; Westerink, Ben H. C.; Korf, Jakob; den Boer, Johan A.


    We have investigated effects of continuous SSRI administration and abrupt discontinuation on biochemical and behavioral indices of rat brain serotonin function and attempted to identify underlying mechanisms Biochemistry of serotonin was assessed with brain tissue assays and microdialysis behavior w

  11. Quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis in an experimental rat model

    M.J. Becker (Martin); S. de Marie (Siem); D. Willemse; H.A. Verbrugh (Henri); I.A.J.M. Bakker-Woudenberg (Irma)


    textabstractTwo diagnostic tests, an Aspergillus-specific PCR and an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of galactomannan, were compared for diagnosing and monitoring invasive pulmonary aspergillosis. Persistently neutropenic rat

  12. EDTA interference in electrochemiluminescence ACTH assay.

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer


    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  13. Comet assay on tetraploid yeast cells

    Rank, Jette; Syberg, Kristian; Jensen, Klara


    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  14. Fungicide resistance assays for fungal plant pathogens.

    Secor, Gary A; Rivera, Viviana V


    Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the field. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular procedures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol demethylation inhibitor; DMI) fungicides, and inhibition of spore germination for quinone outside inhibitor (QoI) fungicides.

  15. Nonsulfated cholecystokinins in the small intestine of pigs and rats

    Agersnap, Mikkel; Rehfeld, Jens F


    -step assay specific for nonsulfated CCK. For further characterization, the intestinal extracts were subjected to size- and ion exchange-chromatography. The intestinal concentrations of sulfated and nonsulfated CCK were highest in the duodenum and the proximal part of jejunum both in the pig and the rat...

  16. Isomers of broparoestrol and antiestrogen action: comparison with tamoxifen.

    Edery, M; Barnova, A; Drosdowsky, M; Guggiari, M; Vives, C; Rudali, G


    This study compares the relative biological potencies of a known antiestrogen tamoxifen to two triarylethylene compounds which have been shown previously to be potent inhibitors of rodent mammary tumorigenesis. Based on a) uterotrophic and anti-uterotrophic tests, b) indexes of cellularity, and c) protein content, these studies indicate that the trans, as well as the cis, isomers of bromotriphenylethylene are partial estrogen antagonists with no estrogenic effects in rat uteri and partial agonists in mouse uteri, whereas tamoxifen shows partial antiestrogenic/estrogenic effects in rats and is fully estrogenic in mice.

  17. Detection of Eperythrozoon wenyoni by PCR assay

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO


    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  18. A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats.

    Chang, Yen-Jui; Huang, Wei-Ju; Lin, Han-Wei; Chang, Ling-Ling; Chang, Full-Young; Wang, Paulus S


    Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.

  19. Chuanxiongzine-astragaloside V decreases IL-1β and Caspase-3 gene expressions in rat brain damaged by cerebral ischemia/reperfusion: A study of real-time quantitgtive PCR assay%川芎嗪-黄芪甲苷降低缺血/再灌注损伤的大鼠脑组织中IL-1β和Caspase-3基因的表达:实时定量PCR的研究

    朱振洪; 万海同; 李金辉


    -time PCR. Rats were randomized into the following groups: sham operation group, model group (cerebral I/R group), astragaloside V (AST V) group, chuanxiongzine-AST V group and nimodipine group (n = 10 in each group). The rats in all the groups except sham operation group were subjected to cerebral I/R treatment. Sham operation and model groups were treated by normal saline (5 mL/kg). AST V, chuanxiongzine-AST V and nimodipine groups received 20 mg/kg AST V, 10 mg/kg chuanxiongzine plus 20 mg/kg AST V, and 10 mg/kg nimodipine treatments, respectively. The administrations of drugs were performed with intraperitoneal injections at 0 and 12 h, 1 d, 2 d, 3 d, till to 7 d after I/R. A real-time quantitative PCR assay was developed for absolute quantification of the expressions of IL-1β and Caspase-3 genes. The absolute quantification approach relies on the construction of an accurate standard curve. Thus, two plasmids which contained rat IL-1β and Caspase-3 genes respectively were constructed. The cloned circular plasmids were then quantified using a spectrophotometer and used as standards. Standard curves were generated, and the copy numbers of IL-1 β and Caspase-3 mRNA isolated from I/R-damaged brain tissue were also calculated by SYBR Green I dye method using specific primers. The results showed that melting curves exhibited sharp peaks, and PCR product also generated prominent band with expected size in agarose gel electrophoresis, which validated the optimization of the selected primer sets of IL-1β and Caspase-3 genes. The optimal annealing temperatures of IL-1β and Caspase-3 genes were 59 °C and 61.2 °C, respectively. Real-time PCR results showed that the expression of IL- 1 β and Caspase-3 genes in the model group was significantly elevated compared to that in the sham operation group. However, compared to those in the model group, IL-1β and Caspase-3 gene expressions were obviously decreased in AST V, chuanxiongzine-AST V and nimodipine groups.Especially in

  20. Instructions for Uploading Data to the Assay Portal - Instructions for Uploading Data to the Assay Portal

    This document provides instructions for configuring and uploading data files to the CPTAC Assay Portal. It is divided into sections, with an overview checklist provided at the end. If help is needed at any stage of the process, please use the support page:

  1. Protection of GBE50 on brain mitochondria in rats with hyperlipidemia

    KaiSUN; GangLIU; Yun-xuanZHANG; Ghen-liangYIN; Gaj-junTIAN; Jia-huPAN


    AIM To study the protective effects of the new standardized preparation of Ginkgo biloba extracts(GBE50) anainst brain mitochondrial damages induced by hyperlipemia in rats. METHODS Rat model with hyperlipemia was established by feeding the young male SD rats (3 weeks after born) with high lipid food for 3 months. Then the rats were treated with different dosage of GBE50(ig) for 4 weeks. The prophylaxis rat group were fed with the mixture of high lipid food and GBE50 (50mg/kg/d). The brain mitochondria was separated for determination of the R3, R4 and RCR of the respiration function with the method of Clark's electrode. The mitochondrial transmembrane potential(Δψm) was derected by the Rho123 assay and the cytochrome c release was checked by spectrography. In addition, the parameters of oxidative stress (the activities of SOD, GSH-Px, and the MDA leve) and the activities of ATPase were assayed.

  2. Quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters

    Varghese, S.; Christakos, S.


    A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and /sup 125/I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and /sup 125/I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous /sup 125/I immunobinding assays which were not quantitative but were used as antigen spot tests or which required iodination of the antibody.

  3. Assay of mitochondrial functions by resazurin in vitro

    Hai-xia ZHANG; Guan-hua DU; Jun-tian ZHANG


    AIM: To study the mechanism of resazurin as indicator of mitochondrial function and to develop a rapid and sensitive assay for measuring metabolic activity of isolated mitochondria from rat liver in vitro. METHODS: The screening was carried out on 96-well microtitre plates by monitoring fluorescence intensity of resazurin reduced by mitochondria. Experimental conditions were optimized and influences of several inhibitors on mitochondrial function were observed. RESULTS: Fluorescence intensity increased in a linear manner when the mitochondrial protein concentration from 5 to 50 μg protein per well was incubated with resazurin (5 μmol/L) during 230 min period at 37 ℃. Edetic acid could promote the reduction of resazurin in mitochondria. The fluorescence intensity decreased greatly after pretreatment with NaN3, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),and oligomycin compared with the control. However, the typical complex I inhibitor, rotenone enhanced the fluorescence intensity without mitochondria. CONCLUSION: Using resazurin to determine mitochondrial function is sensitive, inexpensive and could be easily automated for high throughput screening.

  4. Drugs and brain death: drug assay perspectives.

    Morris, R G


    The ability to make any meaningful interpretation of a drug assay result is very dependent upon a knowledge of the limitations of the method(s) used (sensitivity, specificity etc.), and the concentration that may be measured in plasma and its relationship to CNS effects. We need more information about 'critical' concentrations for each drug and sedation in the setting of the brain-injured patient before meaningful interpretation can be applied to such data. While the above discussion is critical of screen-type assays, the alternative specific assays are not easily provided for, as obviously the resourcing of laboratories to be able to deliver such specialized services for a range of therapeutic drugs, in addition to 'social' drugs or other toxins (e.g. glues, pesticides, solvents, environmental substances etc), becomes an increasingly complex issue in the current economic climate. Hence, the analytical laboratory can offer valuable support to the clinical team however, the interpretation of such results must be assessed in the light of many limitations of such assay methods and not seen as the 'gold standard' for assessment of brain function.

  5. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W


    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  6. Endoproteolytic activity assay in malting barley

    Blanca Gómez Guerrero


    Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.

  7. Comet assay on mice testicular cells

    Sharma, Anoop Kumar


    for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity...

  8. In vitro solubility assays in drug discovery.

    Kerns, Edward H; Di, Li; Carter, Guy T


    The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies.

  9. Benzodiazepine Synthesis and Rapid Toxicity Assay

    Fletcher, James T.; Boriraj, Grit


    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  10. Assessment of the potential genotoxic risk of medicinal Tamarindus indica fruit pulp extract using in vivo assays.

    Silva, F M V; Leite, M F; Spadaro, A C C; Uyemura, S A; Maistro, E L


    Tamarindus indica has been used in folk medicine as an antidiabetic, a digestive aid, and a carminative, among other uses. Currently, there is no information in the toxicology literature concerning the safety of T. indica extract. We evaluated the clastogenic and/or genotoxic potential of fruit pulp extract of this plant in vivo in peripheral blood and liver cells of Wistar rats, using the comet assay, and in bone marrow cells of Swiss mice, using the micronucleus test. The extract was administered by gavage at doses of 1000, 1500 and 2000 mg/kg body weight. Peripheral blood and liver cells from Wistar rats were collected 24 h after treatment, for the comet assay. The micronucleus test was carried out in bone marrow cells from Swiss mice collected 24 h after treatment. The extract made with T. indica was devoid of clastogenic and genotoxic activities in the cells of the rodents, when administered orally at these three acute doses.

  11. Estrogenic effects of Sedum sarmentosum Bunge in ovariectomized rats.

    Kim, Won-Hee; Park, Yun-Ja; Park, Mi-Ra; Ha, Tae-Yeul; Lee, Sang-Hyeon; Bae, Song-Ja; Kim, Mihyang


    The aim of this study was to evaluate the effects of Sedum sarmentosun Bunge (SS) on the lipid on serum and the collagen content of the connective tissues in ovariectomized estrogen-deficient rats. Three groups were surgically ovariectomized. The fourth group was sham operated. From day 2 until day 37 after the ovariectomy, Sprague-Dawley female rats were randomly assigned to the following groups: sham-operated rats (sham), ovariectomized control rats (OVX-control), ovariectomized rats supplemented with an ethyl ether fraction of SS at 10 mg/kg bw/d (OVX-EE), ovariectomized rats supplemented an ethyl acetate fraction of SS at 10 mg/kg bw/d (OVX-EA). The SS fractions were orally administrated at 1 mL per day. The estrogenic effects of the ethyl ether and ethyl acetate fractions of SS, were investigated using one in vitro assay and two in vivo assays. The treatment of the partition of the ethyl ether and ethyl acetate layers of SS increased the transcriptional activity 0.7-fold and 0.5-fold compared to those that were given 17beta-estradiol treatment, respectively. The OVX rats were significantly heavier than the sham-operated rats at all times, but supplementation with the SS extracts tended to result in less weight gain than OVX-control. The serum triglyceride levels were significantly decreased after supplementation with the SS portion EE and EA layers. Supplementation with the SS extracts prevented a decrease in the collagen level in bone and cartilage tissues. This result indicates that the SS affects the collagen synthesis in ovariectomized rats. These results are consistent with the conclusions based on the estrogenic activities of SS. Therefore, it may be used to possibly improve the quality of life in menopausal women.

  12. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats



    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function...

  13. Production and assay of forskolin antibodies

    Ho, L.T.; Ho, R.J.


    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  14. Fluorimetric assay for ornithine decarboxylase by high-performance liquid chromatography.

    Haraguchi, K; Kai, M; Kohashi, K; Ohkura, Y


    A highly sensitive method for the assay of ornithine decarboxylase in sample solutions prepared from rat tissue homogenate is described which employs high-performance liquid chromatography with fluorescence detection. Putrescine formed from ornithine under the optimal conditions for the enzyme reaction is treated by Cellex P column chromatography for clean-up and converted into the fluorescamine derivative in the presence of cupric ion which inhibits the reaction of interfering amines with fluorescamine. The derivative is separated by reversed-phase chromatography on LiChrosorb RP-18 with linear gradient elution. The lower limit of detection for putrescine formed enzymatically is 5 pmol.

  15. Ceftriaxone attenuates hypoxic-ischemic brain injury in neonatal rats

    Huang Yen


    Full Text Available Abstract Background Perinatal brain injury is the leading cause of subsequent neurological disability in both term and preterm baby. Glutamate excitotoxicity is one of the major factors involved in perinatal hypoxic-ischemic encephalopathy (HIE. Glutamate transporter GLT1, expressed mainly in mature astrocytes, is the major glutamate transporter in the brain. HIE induced excessive glutamate release which is not reuptaked by immature astrocytes may induce neuronal damage. Compounds, such as ceftriaxone, that enhance the expression of GLT1 may exert neuroprotective effect in HIE. Methods We used a neonatal rat model of HIE by unilateral ligation of carotid artery and subsequent exposure to 8% oxygen for 2 hrs on postnatal day 7 (P7 rats. Neonatal rats were administered three dosages of an antibiotic, ceftriaxone, 48 hrs prior to experimental HIE. Neurobehavioral tests of treated rats were assessed. Brain sections from P14 rats were examined with Nissl and immunohistochemical stain, and TUNEL assay. GLT1 protein expression was evaluated by Western blot and immunohistochemistry. Results Pre-treatment with 200 mg/kg ceftriaxone significantly reduced the brain injury scores and apoptotic cells in the hippocampus, restored myelination in the external capsule of P14 rats, and improved the hypoxia-ischemia induced learning and memory deficit of P23-24 rats. GLT1 expression was observed in the cortical neurons of ceftriaxone treated rats. Conclusion These results suggest that pre-treatment of infants at risk for HIE with ceftriaxone may reduce subsequent brain injury.

  16. Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

    Edwin C. Ouyang; Catherine H. Wu; Cherie Walton; Kittichai Promrat; George Y. Wu


    AIM To determine whether normal geneticallyirnmunocornpetent rodent hosts could bemanipulated to accept human hepatocytetransplants with long term survival withoutirnrnunosuppression.METHODS Tolerance towards humanhepatocytes was established by injection ofprimary human hepatocytes or Huh7 humanhepatoma cells into the peritoneal cavities offetal rats. Corresponding cells weresubsequently transplanted into newborn rats viaintrasplenic injection within 24 h after birth.RESULTS Mixed lymphocyte assays showedthat spleen cells from non-tolerized rats werestimulated to proliferate when exposed to humanhepatocytes, while cells from tolerized ratswere not. Injections made between 15 d and 17 dof gestation produced optimal tolerizaton.Transplanted human hepatocytes in rat liverswere visualized by immunohistochemicalstaining of human albumin. By dot blotting ofgenomic DNA in livers of tolerized rats 16 weeksafter hepatocyte transplantation, it was foundthat approximately 2.5 × 105 human hepatocytessurvived per rat liver. Human albumin mRNA wasdetected in rat livers by RT-PCR for 15 wk, andhuman albumin protein was also detectable in ratserum.CONCLUSION Tolerization of an immuno-competent rat can permit transplantation, andsurvival of functional human hepatocytes.

  17. Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature

    Dawson, R.M.


    Benzodiazepine receptors of rat cerebellum were assayed with (/sup 3/H)-labeled flunitrazepam at 37/sup 0/C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37/sup 0/C until just prior to filtration.

  18. Pavlovian fear conditioning as a behavioral assay for hippocampus and amygdala function: cautions and caveats.

    Maren, Stephen


    Pavlovian fear conditioning has become an important model for investigating the neural substrates of learning and memory in rats, mice and humans. The hippocampus and amygdala are widely believed to be essential for fear conditioning to contexts and discrete cues, respectively. Indeed, this parsing of function within the fear circuit has been used to leverage fear conditioning as a behavioral assay of hippocampal and amygdala function, particularly in transgenic mouse models. Recent work, however, blurs the anatomical segregation of cue and context conditioning and challenges the necessity for the hippocampus and amygdala in fear learning. Moreover, nonassociative factors may influence the performance of fear responses under a variety of conditions. Caution must therefore be exercised when using fear conditioning as a behavioral assay for hippocampal- and amygdala-dependent learning.

  19. Acute toxicity of the aqueous-methanolic Moringa oleifera (Lam leaf extract on female Wistar albino rats

    Mitchel O. Okumu


    Conclusions: Based on these results, the LD50 of the AQ-ME MO leaf extract was found to be >2000 mg/kg in female wistar albino rats. Keywords: Moringa oleifera, Aqueous-methanol, Wistar rats, OECD 425, Biochemical assays, Liver [Int J Basic Clin Pharmacol 2016; 5(5.000: 1856-1861

  20. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  1. Development of a central nervous system axonal myelination assay for high throughput screening.

    Lariosa-Willingham, Karen D; Rosler, Elen S; Tung, Jay S; Dugas, Jason C; Collins, Tassie L; Leonoudakis, Dmitri


    Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

  2. Detection of Hypoxia in Human Brain Tumor Xenografts Using a Modified Comet Assay

    Jingli Wang


    Full Text Available We used the standard comet assay successfully to generate in vitro dose-response curves under oxic and hypoxic conditions. We then made mixtures of cells that had been irradiated with 3 and 9 Gy of X-rays to simulate two subpopulations in a tumor, but efforts to accurately detect and quantify the subpopulations using the standard comet assay were unsuccessful. Therefore, we investigated a modified comet assay to determine whether it could be used for measuring hypoxia in our model systems. U251 MG cells were grown as subcutaneous tumors in athymic mice; U251 MG and U87 MG cells were grown as intracerebral (i.c. tumors in athymic rats. Animals were injected with RSU 1069, irradiated, and euthanized. Tumors and normal brains were removed, and the cells were analyzed using a modified comet assay. Differences in comet tail moment distributions between tumor and contralateral normal brain, using tail moments at either the 25th or 50th percentile in each distribution, were taken as measures of the degree of tumor hypoxia. For U251 MG tumors, there was a positive relationship between tumor size and the degree of hypoxia, whereas preliminary data from U87 MG i.c. tumors showed less hypoxia and no apparent relationship between tumor size and hypoxia.

  3. A highly sensitive cell assay for validation of purification regimes of alginates.

    Leinfelder, U; Brunnenmeier, F; Cramer, H; Schiller, J; Arnold, K; Vásquez, J A; Zimmermann, U


    Among the hydrogels used for microencapsulation of cells and tissues, alginate has been and will continue to be one of the most important biomaterials. A mandatory requirement for clinical immunoisolated transplantations is the reproducible production of biocompatible alginate. As shown here for alginates extracted from freshly collected algal stipes, the current assays used for validation of the quality of the alginate are not sufficient to screen for impurities arising from spores of gram-positive bacteria (and related contaminants). To assess the quality of alginate, we have developed a cell assay based on the induction of apoptosis in Jurkat cells. This assay allows in combination with the "modified mixed lymphocyte" assay a rapid and sensitive screening for any fibrosis-inducing impurities in alginate samples (even during the purification regime) as demonstrated by transplantation experiments performed in parallel with BB rats (exhibiting an elevated macrophage activity). The results clearly demonstrate that the quality of the input algal material is of key relevance for the production of transplantation-grade alginate.

  4. Radioreceptor assay for analysis of fentanyl and its analogs in biological samples

    Alburges, M.E.


    The assay is based on the competition of these drugs with ({sup 3}H) fentanyl for opioid receptors in membrane preparations of rat forebrain in vitro. The binding in stereospecific, reversible and saturable. Scatchard plots of saturation suggest the presence of high and low affinity binding sites. Morphine and hydromorphone complete with ({sup 3}H)fentanyl for the opioid receptor, but other morphine-like compounds were relatively weak displacers of ({sup 3}H)fentanyl. Many other commonly abused drugs do not compete with ({sup 3}H)fentanyl for the opioid receptors. Urine samples from animals injected with fentanyl, ({plus minus})-cis-3-methylfentanyl, alpha-methylfentanyl, butyrylfentanyl and benzylfentanyl were analyzed by radioreceptor assay, radioimmunoassay, and gas chromatography/mass spectrometry. Urinary analysis of fentanyl showed a good correlation with these three methods; however, discrepancies were observed in the analysis of fentanyl analogs. This radioreceptor assay is well-suited as an initial assay for the detection of active analogs of fentanyl in urine with good correlation with other techniques in the analysis of fentanyl; however, there is substantial disagreement between techniques in the quantitation of fentanyl analogs. The implications of these discrepancies are discussed.

  5. Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

    Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AK (USA) Univ. of Arkansas for Medical Sciences, Little Rock (USA))


    A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3{prime},4,4{prime}-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4{prime}-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4{prime}-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.

  6. Comparison of prostate gene expression and tissue weight changes as monitors of antiandrogen activity in GNRH-inhibited rats

    Nellemann, Christine Lydia; Lefevre, P. A.; Ashby, J.


    BACKGROUND: The Hershberger assay for antiandrogens and modifiers of steroid biosynthesis uses surgically-castrated rats. We described an adaptation of the assay using the GnRH inhibitor Antarelix in place of surgical castration [Ashby J, Lefevre PA, Deghenghi R, Wallis N. Regulatory Toxicology...

  7. Indigestible material attenuated changes in apoptosis in the fasted rat jejunal mucosa.

    Kakimoto, Takashi; Fujise, Takehiro; Shiraishi, Ryosuke; Kuroki, Tsukasa; Park, Jae Myung; Ootani, Akifumi; Sakata, Yasuhisa; Tsunada, Seiji; Iwakiri, Ryuichi; Fujimoto, Kazuma


    We have previously demonstrated that fasting induced apoptosis and decreased cell proliferation in the rat intestinal mucosa. The aim was to investigate the effect of expanded polystyrene as indigestible material on apoptosis and cell proliferation in rat small intestinal mucosa during fasting. Male SD rats were divided into 3 groups. The first group was fed with chow and water ad libitum. The second group fasted for 72 hrs. The third group was fasted for 24 hrs and was fed expanded polystyrene. Intestinal apoptosis was evaluated by percent fragmented DNA assay, terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end-labeling (TUNEL) staining, and caspase-3 assay. Cell proliferation was analyzed by 5-bromo-2'-deoxyuridine (5-BrdU) uptake. Truncal vagotomy was performed to evaluate a role of the central nervous system. In the 72-hr fasted rat, mucosal height of the rat jejunum was decreased to 73% of that in rats fed ad libitum, and this decrease was partly restored to 90% in rats fed expanded polystyrene. The fragmented DNA was increased in fasted rats (28.0%) when compared with that in rats fed ad libitum (2.6%). The increase in fragmented DNA in fasted rats was recovered by feeding them expanded polystyrene (8.3%). TUNEL staining confirmed this result. The effect of polystyrene on apoptosis was decreased by truncal vagotomy. Expression of cleaved caspase-3 was increased in fasted rats, which was then decreased by feeding of expanded polystyrene. In contrast to apoptosis, feeding of expanded polystyrene had no reconstructive effect on 5-BrdU uptake in the intestinal epithelium, which was decreased by fasting to 60% of that in rats fed ad libitum. In conclusion, feeding of indigestible material partly restored the decrease in intestinal mucosal length in the fasted rats through the apoptotic pathway without any influence on BrdU uptake. Further exploration focused on the mechanism of this effect of indigestible material is required.

  8. Lump corrections for radioactive waste assay.

    Miller, T J


    Previous studies have shown that automated radioactive waste assay techniques, such as segmented gamma scanner (SGS) and automated qualitative and quantitative (AQ2), have severely underestimated fissile material due to either the malfunction or absence of appropriate lump correction routines. This paper examines the application of manual techniques, such as Monte Carlo N particle (MCNP) and spectral non-destructive assay platform (SNAP) software, to lump corrections in plutonium (Pu), enriched uranium (EU) and depleted uranium (DU) waste streams. Excellent results have been obtained when comparing MCNP with SNAP and applying the SNAP lump correction routine to a range of simulated and typical wastes containing various Pu and EU lump sizes. It has been concluded that the need for lump corrections was relatively rare and usually apparent from abnormal gamma ray peak area ratios, since most AWE waste streams are only lightly shielded.

  9. Transient expression assays in tobacco protoplasts.

    Vanden Bossche, Robin; Demedts, Brecht; Vanderhaeghen, Rudy; Goossens, Alain


    The sequence information generated through genome and transcriptome analysis from plant tissues has reached unprecedented sizes. Sequence homology-based annotations may provide hints for the possible function and roles of particular plant genes, but the functional annotation remains nonexistent or incomplete for many of them. To discover gene functions, transient expression assays are a valuable tool because they can be done more rapidly and at a higher scale than generating stably transformed tissues. Here, we describe a transient expression assay in protoplasts derived from suspension cells of tobacco (Nicotiana tabacum) for the study of the transactivation capacities of transcription factors. To enhance throughput and reproducibility, this method can be automated, allowing medium-throughput screening of interactions between large compendia of potential transcription factors and gene promoters.

  10. Identification of irradiated pepper with comet assay

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra [Centro de Aplicaciones Tecnologicas y Desarrollo Nuclear. (CEADEN), Ciudad de La Habana (Cuba)]. E-mail:;; Iglesia Enriquez, Isora [Instituto de Investigacion para la Industria Alimenticia (IIIA), Ciudad de La Habana (Cuba)


    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  11. Posttranslational Modification Assays on Functional Protein Microarrays.

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng


    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  12. Comparative assay of Vipera ammodytes antivenom potency.

    Capitanescu, Cristian; Macovei Oprescu, Anca Monica; Supeanu, Alexandru; Coculescu, Bogdan Ioan; Strambu, Victor; Macovei, Radu Alexandru; Manole, Gheorghe


    The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A2 antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A2 activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.

  13. Assay of adenosine 3',5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

    Handa, A.K.; Bressan, R.A.


    In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many /sup 32/P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of (..gamma..-/sup 32/P)ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of (..gamma..-/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: (ATP) = (ATP)/sub 0/ e/sup -(cAMP)kt/. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the potein kinase stimulation assay based on transfer of (/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.

  14. Kinetic viability assays using DRAQ7 probe.

    Wlodkowic, Donald; Akagi, Jin; Dobrucki, Jurek; Errington, Rachel; Smith, Paul J; Takeda, Kazuo; Darzynkiewicz, Zbigniew


    Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal. There is a need therefore for rapid and high-throughput bioassays capable of continuously tracking viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative, DRAQ7, is gaining increasing interest as an easy-to-use marker capable of long-term monitoring of cell death in real-time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect the cells' susceptibility to the death-inducing agents. However, when the membrane integrity is compromised, DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single-color DRAQ7 real-time assay to dynamically track cell viability. The second protocol outlines a simplified end-point DRAQ7 staining approach. The final protocol highlights the real-time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans-membrane electrochemical potential (ΔΨm) sensing probe.

  15. A new fluorescent assay for sialyltransferase.

    Kajihara, Y; Kamitani, T; Sakakibara, T


    A new fluorescent assay for the sialyltransferase reaction was established. After incubation of the sialyltransferase reaction, the sialyloligosaccharide obtained was treated by acid hydrolysis, and then the NeuAc that was released was labeled with 1,2-diamino-4,5-methylenedioxibenzene. The fluorescent-labeled NeuAc could be estimated by HPLC (excitation: 373 nm; emission: 448 nm) and a Lineweaver-Burk plot could be plotted with the data from this analysis.

  16. Delivery of High-Quality Biomarker Assays

    Brian N. Swanson


    Full Text Available Biomarker measurements now support key decisions throughout the drug development process, from lead optimization to regulatory approvals. They are essential for documenting exposure-response relationships, specificity and potency toward the molecular target, untoward effects, and therapeutic applications. In a broader sense, biomarkers constitute the basis of clinical pathology and laboratory medicine. The utility of biomarkers is limited by their specificity and sensitivity toward the drug or disease process and by their overall variability. Understanding and controlling sources of variability is not only imperative for delivering high-quality assay results, but ultimately for controlling the size and expense of research studies. Variability in biomarker measurements is affected by: biological and environmental factors (e.g., gender, age, posture, diet and biorhythms, sample collection factors (e.g., preservatives, transport and storage conditions, and collection technique, and analytical factors (e.g., purity of reference material, pipetting precision, and antibody specificity. The quality standards for biomarker assays used in support of nonclinical safety studies fall under GLP (FDA regulations, whereas, those assays used to support human diagnostics and healthcare are established by CLIA (CMS regulations and accrediting organizations such as the College of American Pathologists. While most research applications of biomarkers are not regulated, biomarker laboratories in all settings are adopting similar laboratory practices in order to deliver high-quality data. Because of the escalation in demand for biomarker measurements, the highly-parallel (multi-plexed assay platforms that have fueled the rise of genomics will likely evolve into the analytical engines that drive the biomarker laboratories of tomorrow.

  17. Methods and devices for protein assays

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee


    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  18. Immunoreagents and competitive assays to fludioxonil

    Abad Fuentes, Antonio; Agulló, Consuelo; Esteve Turrillas, Francesc Albert; Abad Somovilla, Antonio; Mercader Badia, Josep Vicent


    Fludioxonil is a new-generation fungicide widely used for postharvest fruit protection. The aim of this study was to produce hitherto unreported immunoreagents for Fludioxonil analysis by immunoassay. Derivatives of this agrochemical were synthesized with different linker tethering sites. Those functionalized haptens were activated, and the purified active esters were efficiently conjugated to different carrier proteins for immunogen and assay antigen preparation. Antibodies to Fludioxonil we...

  19. Cell based assay for hypoglycemic drugs screening

    LiZHANG; Juan-juanHU; Guan-huaDU


    OBJECTIVE: To establish a cell based assay for hypoglyc emicdrugs. METHODS: The five cell lines, BALB/c3T3, HepG2, NIH3T3, Be17402, and L929 were incubated with insulin (0-125n mol/L) for 48 h. Their sensitivities to insulin were studied by detecting glucose consumption. The dose-response and time-response relationship between the sensitive cell line (BALB/c 3T3)

  20. FY 1998 results of the intellectual basement project using functions of private companies (venture promotion type basement creation R and D project). Development of endocrine disrupter testing method and development of environmental assessment method; 1998 nendo naibunpi kakuran busshitsu ni taisuru shiken hoho kaihatsu oyobi eikyo hyoka shuho kaihatsu hokokusho



    For the purpose of assessing the risk in relation to endocrine disrupting chemicals, the following were conducted: development of testing/assessment method for endocrine disrupting chemicals, survey of the actual exposure assessment, development of measuring method for the concentration in the environment. In the development of the testing method, the following were carried out: development of a high-throughput screening method for evaluating endocrine disrupting chemicals; as screening testing method using mammals, uterotrophic assay, Hershberger assay using castrated male rats, thyroid hormone assay in pubertal rats, enhanced OECD 407 test guideline for 28-day toxicity test; study on yeast two-hybrid assay for endocrine disrupter; sex-reversal assay for suspected endocrine disrupting chemicals using S-rR strain medaka. In the development of exposure assessment method, estrogenic potency of individual nonylphenol congeners isolated from technical mixtures; determination of endocrine disrupters and related chemicals from industry and nature origin in river water and sediment; research for the flow of industrial origin chemicals; reconstruction of pollution history of chemicals using sediment cores. (NEDO)

  1. Adolescent social isolation influences cognitive function in adult rats

    Feng Shao; Xiao Han; Shuang Shao; Weiwen Wang


    Adolescence is a critical period for neurodevelopment. Evidence from animal studies suggests that isolated rearing can exert negative effects on behavioral and brain development. The present study aimed to investigate the effects of adolescent social isolation on latent inhibition and brain-derived neurotrophic factor levels in the forebrain of adult rats. Male Wistar rats were randomly divided into adolescent isolation (isolated housing, 38–51 days of age) and social groups. Latent inhibition was tested at adulthood. Brain-derived neurotrophic factor levels were measured in the medial prefrontal cortex and nucleus accumbens by an enzyme-linked immunosorbent assay. Adolescent social isolation impaired latent inhibition and increased brain-derived neurotrophic factor levels in the medial prefrontal cortex of young adult rats. These data suggest that adolescent social isolation has a profound effect on cognitive function and neurotrophin levels in adult rats and may be used as an animal model of neurodevelopmental disorders.

  2. Polymerase chain reaction assay for avian polyomavirus.

    Phalen, D N; Wilson, V G; Graham, D L


    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

  3. Diagnostic Certified Assay: Neuromuscular and Cardiac Assessments

    Rea Valaperta


    Full Text Available The expansion of the specific trinucleotide sequence, [CTG], is the molecular pathological mechanism responsible for the clinical manifestations of DM1. Many studies have described different molecular genetic techniques to detect DM1, but as yet there is no data on the analytical performances of techniques used so far in this disease. We therefore developed and validated a molecular method, “Myotonic Dystrophy SB kit,” to better characterize our DM1 population. 113 patients were examined: 20 DM1-positive, 11 DM1/DM2-negative, and13 DM1-negative/DM2-positive, who had a previous molecular diagnosis, while 69 were new cases. This assay correctly identified 113/113 patients, and all were confirmed by different homemade assays. Comparative analysis revealed that the sensitivity and the specificity of the new kit were very high (>99%. Same results were obtained using several extraction procedures and different concentrations of DNA. The distribution of pathologic alleles showed a prevalence of the “classical” form, while of the 96 nonexpanded alleles 19 different allelic types were observed. Cardiac and neuromuscular parameters were used to clinically characterize our patients and support the new genetic analysis. Our findings suggest that this assay appears to be a very robust and reliable molecular test, showing high reproducibility and giving an unambiguous interpretation of results.

  4. Assessment of plaque assay methods for alphaviruses.

    Juarez, Diana; Long, Kanya C; Aguilar, Patricia; Kochel, Tadeusz J; Halsey, Eric S


    Viruses from the Alphavirus genus are responsible for numerous arboviral diseases impacting human health throughout the world. Confirmation of acute alphavirus infection is based on viral isolation, identification of viral RNA, or a fourfold or greater increase in antibody titers between acute and convalescent samples. In convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque reduction neutralization test. To identify the best method for alphavirus and neutralizing antibody recognition, the standard solid method using a cell monolayer overlay with 0.4% agarose and the semisolid method using a cell suspension overlay with 0.6% carboxymethyl cellulose (CMC) overlay were evaluated. Mayaro virus, Una virus, Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV) were selected to be tested by both methods. The results indicate that the solid method showed consistently greater sensitivity than the semisolid method. Also, a "semisolid-variant method" using a 0.6% CMC overlay on a cell monolayer was assayed for virus titration. This method provided the same sensitivity as the solid method for VEEV and also had greater sensitivity for WEEV titration. Modifications in plaque assay conditions affect significantly results and therefore evaluation of the performance of each new assay is needed.

  5. Hyperpolarized NMR Probes for Biological Assays

    Sebastian Meier


    Full Text Available During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

  6. A single step protein assay that is both detergent and reducer compatible: The cydex blue assay.

    Rabilloud, Thierry


    Determination of protein concentration is often an absolute prerequisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. It was found that inclusion of cyclodextrins in a Coomassie blue-based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, a single-step assay that is both detergent and reducer compatible was developed. Depending on the type and concentration of detergents present in the sample buffer, either beta-cyclodextrin or alpha-cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2-10 mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1 mg/mL protein, in the presence of up to 2% detergent and reducers such as 5% mercaptoethanol or 50 mM DTT in a single step with a simple spectrophotometric assay.

  7. Assessment of the in vivo genotoxicity of cadmium chloride, chloroform, and D,L-menthol as coded test chemicals using the alkaline comet assay.

    Wada, Kunio; Fukuyama, Tomoki; Nakashima, Nobuaki; Matsumoto, Kyomu


    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) international validation study of in vivo rat alkaline comet assays, we examined cadmium chloride, chloroform, and D,L-menthol under blind conditions as coded chemicals in the liver and stomach of Sprague-Dawley rats after 3 days of administration. Cadmium chloride showed equivocal responses in the liver and stomach, supporting previous reports of its poor mutagenic potential and non-carcinogenic effects in these organs. Treatment with chloroform, which is a non-genotoxic carcinogen, did not induce DNA damage in the liver or stomach. Some histopathological changes, such as necrosis and degeneration, were observed in the liver; however, they did not affect the comet assay results. D,L-Menthol, a non-genotoxic non-carcinogen, did not induce liver or stomach DNA damage. These results indicate that the comet assay can reflect genotoxic properties under blind conditions.

  8. The JaCVAM international validation study on the in vivo comet assay: Selection of test chemicals.

    Morita, Takeshi; Uno, Yoshifumi; Honma, Masamitsu; Kojima, Hajime; Hayashi, Makoto; Tice, Raymond R; Corvi, Raffaella; Schechtman, Leonard


    The Japanese Center for the Validation of Alternative Methods (JaCVAM) sponsored an international prevalidation and validation study of the in vivo rat alkaline pH comet assay. The main objective of the study was to assess the sensitivity and specificity of the assay for correctly identifying genotoxic carcinogens, as compared with the traditional rat liver unscheduled DNA synthesis assay. Based on existing carcinogenicity and genotoxicity data and chemical class information, 90 chemicals were identified as primary candidates for use in the validation study. From these 90 chemicals, 46 secondary candidates and then 40 final chemicals were selected based on a sufficiency of carcinogenic and genotoxic data, differences in chemical class or genotoxic or carcinogenic mode of action (MOA), availability, price, and ease of handling. These 40 chemicals included 19 genotoxic carcinogens, 6 genotoxic non-carcinogens, 7 non-genotoxic carcinogens and 8 non-genotoxic non-carcinogens. "Genotoxicity" was defined as positive in the Ames mutagenicity test or in one of the standard in vivo genotoxicity tests (primarily the erythrocyte micronucleus assay). These chemicals covered various chemicals classes, MOAs, and genotoxicity profiles and were considered to be suitable for the purpose of the validation study. General principles of chemical selection for validation studies are discussed.

  9. Rat Genome Database (RGD)

    U.S. Department of Health & Human Services — The Rat Genome Database (RGD) is a collaborative effort between leading research institutions involved in rat genetic and genomic research to collect, consolidate,...

  10. A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.

    Yamamoto, N; Lindsay, D D; Naraparaju, V R; Ireland, R A; Popoff, S N


    Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages.

  11. Development of a Rapid Real-Time PCR Assay for Quantitation of Pneumocystis carinii f. sp. Carinii

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida


    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD...... 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro...... axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer...

  12. Citrus-derived flavonoid naringenin exerts uterotrophic effects in female mice at human relevant doses

    Breinholt, Vibeke Miller; Svendsen, Gitte Winkel; Dragsted, Lars Ove


    ingestion of 400-760 ml of orange juice (Erlund et al. 2001). This could be taken to suggests that ingestion of orange juice and other citrus fruits and juices may give rise to sufficiently high tissue levels of naringenin in man to exert a biological effect....

  13. Evaluation of the estrogenic activity of the wild Pueraria mirifica by vaginal cornification assay.

    Cherdshewasart, Wichai; Kitsamai, Yosaporn; Malaivijitnond, Suchinda


    The aim of this study was to evaluate the estrogenic activity of tuberous samples of phytoestrogen-rich Pueraria mirifica collected from 25 of 76 provinces in Thailand by vaginal cornification assay. Tuberous powders were prepared and administered to ovariectomized rats for 14 consecutive days at dosages of 10, 100 and 1,000 mg/kg BW respectively, and were compared with a daily treatment with 2 mg/kg BW 17beta-estradiol (E(2)). Rats treated with 10 mg/kg BW Pueraria mirifica showed no vaginal cornification. Treatment with 100 mg/kg BW Pueraria mirifica from 13 out of 25 plant samples resulted in development of vaginal cornification. The cell count percentages of the vaginal smeared cells for the treatment with the 2 plant samples that exhibited the fastest vaginal cornification revealed large variation in their estrogenic activities. Treatment with 1,000 mg/kg BW Pueraria mirifica from all plant samples produced vaginal cornification with the mean value for the period (day) of first appearance of cornified cells being 4.08 days compared to 2 days with 2 mg/kg BW E(2). The overall appearance period (day) of cornified cells during the treatment and post-treatment period with 1,000 mg/kg BW per day Pueraria mirifica was shorter than treatment with 2 mg/kg BW E(2). The results demonstrate that the plant population shows differential estrogenic activity as evaluated by vaginal cornification assay.

  14. Probing regenerative potential of Moringa oleifera aqueous extracts using In vitro cellular assays

    Evangeline E Fernandes


    Full Text Available Background: Molecules stimulating regeneration and proliferation of cells are of significance in combating ailments caused due to tissue injury, inflammation, and degenerative disorders. Moringa oleifera is one of the most valued food plants having the profile of important nutrients and impressive range of medicinal uses. Objective: To evaluate the potential of M. oleifera aqueous leaf and flower extracts to promote the proliferation of cells and explore their effect on cancer cell lines for assessment of safety. Materials and Methods: Aqueous leaf and flower extracts of M. oleifera were investigated for effect on rat-derived primary fibroblast, mesenchymal stem cells (MSCs, and cancer cell lines using cell proliferation assay. They were also tested and compared for wound healing, angiogenesis, and hepatoprotective effect using in vitro assays. Results: Statistically significant increase in the proliferation of primary rat fibroblast, MSCs, and angiogenesis was observed after treatment with aqueous flower extract. The aqueous leaf extract determined a comparatively moderate increment in the proliferation of MSCs and angiogenesis. It however showed prominent cytotoxicity to cancer cell lines and a significant hepatoprotective effect. Conclusion: A very clear difference in response of the two extracts to different types of cells was detected in this study. The aqueous flower extract exhibited a higher potential to stimulate cell proliferation while not exerting the same effect on cancer cell lines. The leaf extract on the other hand, had a prominent antitumor and hepatoptotective effects.

  15. A novel assay detecting recall response to Mycobacterium tuberculosis: Comparison with existing assays.

    Hsu, Denise C; Zaunders, John J; Plit, Marshall; Leeman, Craig; Ip, Susanna; Iampornsin, Thatri; Pett, Sarah L; Bailey, Michelle; Amin, Janaki; Ubolyam, Sasiwimol; Avihingsanon, Anchalee; Ananworanich, Jintanat; Ruxrungtham, Kiat; Cooper, David A; Kelleher, Anthony D


    A strategy to reduce the burden of active TB is isoniazid preventive therapy for latent TB infection (LTBI). However, current assays used to diagnose LTBI all have limitations. In these proof of concept studies, we compared the agreement of a novel flow cytometry assay detecting CD25/CD134 co-expression with QuantiFERON-TB Gold In-Tube (QFN-GIT) and Tuberculin skin test (TST) in the detection of recall immune response to TB. The CD25/CD134 assay, QFN-GIT and TST were performed on 74 participants referred for TB screening in Sydney and on 50 participants with advanced HIV infection (CD4 ≤ 350 × 10(6) cells/L) in Bangkok. The agreement between CD25/CD134 assay and QFN-GIT was 93.2% (Kappa 0.631 95% CI 0.336-0.926) in Sydney and 90% (Kappa 0.747 95% CI 0.541-0.954) in Bangkok. Discordant results occurred around the cut off of both tests. The agreement between CD25/CD134 assay and TST was 73.6% (Kappa 0.206 95% CI 0.004-0.409) in Sydney and 84% (Kappa 0.551 95% CI 0.296-0.806) in Bangkok. The CD25/CD134 assay showed good agreement with QFN-GIT in detecting recall response to TB both in well and less resourced setting as well as in persons with advanced HIV infection. Further study into the performance of this assay is thus warranted.

  16. The course of Schistosoma mansoni infection in thymectomized rats.

    Cioli, D; Dennert, G


    Inbred rats were thymectomized, irradiated, and reconstituted with T cell-free bone marrow cells. Thymectomized-reconstituted (B rats) and control rats were infected with Schistosoma mansoni cercariae and the number of worms recovered was determined at various times after infection. The extent of immunosuppression was assessed by two criteria: 1) response to an injection of sheep erythrocytes (plaque assay, hemagglutination, hemolysis); 2) response to schistosome antigens (passive hemagglutination). Humoral responses to worm antigens were completely suppressed in almost all instances and anti-sheep erythrocyte responses showed a more variable but always very definite depression in B rats. The number of worms in B rats was about 4 times higher than in control animals at 5 weeks and about 3 times higher at 6 weeks. In a different experiment, rats were perfused at 4, 6, and 9 weeks after infection and the number of worms was found to be consistently higher in B rats, by a factor of about 2 at 4 weeks to a factor of about 4 or 6 at subsequent times. Although B rats had more worms than controls even at 9 weeks, a slow drop in their worm burden was noticeable with time in both experiments. Moreover, the size of worms in B rats was smaller than in controls and even 9-week-old worms failed to develop to normal size and appearance and could not be shown to produce fertile eggs. These experiments show a definite involvement of the immune system in the "self-cure" phenomenon, but may at the same time suggest that other non-immune mechanisms are involved in determining the pattern of S. mansoni infection in the rat.

  17. Olive Oil effectively mitigates ovariectomy-induced osteoporosis in rats

    Saleh Hanan A


    Full Text Available Abstract Background Osteoporosis, a reduction in bone mineral density, represents the most common metabolic bone disease. Postmenopausal women are particularly susceptible to osteoporosis when their production of estrogen declines. For these women, fracture is a leading cause of morbidity and mortality. This study was conducted to evaluate the protective effects of olive oil supplementation against osteoporosis in ovariectomized (OVX rats. Methods We studied adult female Wistar rats aged 12-14 months, divided into three groups: sham-operated control (SHAM, ovariectomized (OVX, and ovariectomized rats supplemented with extravirgin olive oil (Olive-OVX orally for 12 weeks; 4 weeks before ovariectomy and 8 weeks after. At the end of the experiment, blood samples were collected. Plasma levels of calcium, phosphorus, alkaline phosphatase (ALP, malondialdehyde (MDA, and nitrates were assayed. Specimens from both the tibia and the liver were processed for light microscopic examination. Histomorphometric analysis of the tibia was also performed. Results The OVX-rats showed a significant decrease in plasma calcium levels, and a significant increase in plasma ALP, MDA, and nitrates levels. These changes were attenuated by olive oil supplementation in the Olive-OVX rats. Light microscopic examination of the tibia of the OVX rats revealed a significant decrease in the cortical bone thickness (CBT and the trabecular bone thickness (TBT. In addition, there was a significant increase in the osteoclast number denoting bone resorption. In the Olive-OVX rats these parameters were markedly improved as compared to the OVX group. Examination of the liver specimens revealed mononuclear cellular infiltration in the portal areas in the OVX-rats which was not detected in the Olive-OVX rats. Conclusions Olive oil effectively mitigated ovariectomy-induced osteoporosis in rats, and is a promising candidate for the treatment of postmenopausal osteoporosis.

  18. Pulmonary Immune Responses to Aspergillus Fumigatus in Rats



    Objective To evaluate immunologic mechanisms underlying Aspergillus fumigatus pulmonary infections in immunocompetent Dark Agouti (DA) and Albino Oxford (AO) rats recognized as being susceptible to some inflammatory diseases in different manners. Methods Lung fungal burden (quantitative colony forming units, CFU, assay), leukocyte infiltration (histology, cell composition) and their function (phagocytosis, oxidative activity, CD11b adhesion molecule expression) and cytokine interferon-γ(IFN-γ) and interleukin-17 and-4 (IL-17 and IL-4) lung content were evaluated following infection (intratracheally, 1x107 conidia). Results Slower reduction of fungal burden was observed in AO rats in comparison with that in DA rats, which was coincided with less intense histologically evident lung cell infiltration and leukocyte recovery as well as lower level of most of the their activities including intracellular myeloperoxidase activity, the capacity of nitroblue tetrazolium salt reduction and CD11b adhesion molecule expression (except for phagocytosis of conidia) in these rats. Differential patterns of changes in proinflammatory cytokine levels (unchanged levels of IFN-γand transient increase of IL-17 in AO rats vs continuous increase of both cytokines in DA rats) and unchanged levels of IL-4 were observed. Conclusion Genetically-based differences in the pattern of antifungal lung leukocyte activities and cytokine milieu, associated with differential efficiency of fungal elimination might be useful in the future use of rat models in studies of pulmonary aspergillosis.

  19. DNA damage after intracerebroventricular injection of ouabain in rats.

    Jornada, Luciano K; Valvassori, Samira S; Arent, Camila O; Leffa, Daniela; Damiani, Adriani A; Hainzenreder, Giana; Ferreira, Camila L; Moretti, Morgana; Andrade, Vanessa M; Quevedo, João


    There is an emerging body of data suggesting that bipolar disorder is associated with DNA damage. Intracerebroventricular (i.c.v.) administration of ouabain in rats results in manic-like alterations. We evaluated DNA damage of peripheral blood, cerebrospinal fluid and hippocampus of rats after i.c.v. ouabain injection. Ouabain-induced hyperlocomotion was examined in an open field. Additionally, we used single cell gel electrophoresis (comet assay) to measure early transient damage in cerebrospinal fluid (CSF), hippocampus and blood; and the micronucleus test to measure persistent damage in total blood samples of rats after ouabain administration. Our findings demonstrated that ouabain induced hyperlocomotion in rats, and this response remained up to 7 days following a single i.c.v. injection. In addition, we observed that the persistent increase in the rat spontaneous locomotion is associated with increased hippocampal and peripheral index of early DNA damage in rats. No significant alterations were observed in the micronucleus frequency in total blood samples of the rats after the ouabain i.c.v. injection. These results suggest that ouabain may induce peripheral and central early DNA damage, but this early damage may be repaired.

  20. Detection of genotoxic carcinogens in the in vivo-in vitro hepatocyte DNA repair assay.

    Mirsalis, J C; Tyson, C K; Butterworth, B E


    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful in the evaluation of the carcinogenic potential of chemicals. The purpose of this study was to apply this assay to determining the genotoxicity of the compounds from a wide variety of structural classes. Male Fischer-334 rats were treated by gavage or ip injection with compounds dissolved in either corn oil, water, or dimethyl sulfoxide (DMSO). At selected times after treatment, hepatocytes were isolated by liver perfusion and cultured with 3H-thymidine, Unscheduled DNA synthesis (UDS) was measured by quantitative autoradiography as net grains/nucleus (NG); greater than or equal to 5 NG was considered positive. Water, corn oil, or DMSO controls produced -3 to -6 NG with less than or equal to 6% of the cells in repair. All genotoxic hepatocarcinogens tested produced strong positive responses of greater than 15 NG including dimethylnitrosamine, 2-acetylaminofluorene (2-AAF), azoxymethane, 1,2-dimethylhydrazine, benzidine, aflatoxin B1, 2,6-dinitrotoluene, and 2,4-diaminotoluene. The noncarcinogen, 2,6-diaminotoluene, was negative. The mutagen and rat brain carcinogen methyl methanesulfonate (MMS) and the rat pancreatic carcinogen azaserine were also positive. The carcinogens benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene yielded from -2 to -4 NG. This negative response is consistent with their lack of carcinogenic activity in rat liver. MMS produced the greatest amount of UDS 2 hr after treatment whereas 2-AAF did not induce its maximum response until 12 hr post-treatment. The potent hepatotoxin carbon tetrachloride induced a 40-fold elevation in DNA replication 48 hr after a 400 mg/kg dose, but no UDS was observed at 2, 12, 24, or 48 hr post-treatment. The weak hepatocarcinogen safrole induced no UDS suggesting that it is either nongenotoxic or is metabolized to an active form at an extremely slow rate following a single administration. These results demonstrate that this assay is

  1. Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules

    Joosten, I; Wauben, M H; Holewijn, M C


    must be able to assess peptide-MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptides. So far no information has been available on the peptide binding...... characteristics of the Lewis rat MHC class II RT1.B1 molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptides can be assessed while employing detection of biotinylated marker peptides...... by chemiluminescence. The assay is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues in the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found...

  2. Preventive effect of lipid-free soy powder on bone loss in ovariectomized rats%脱脂豆粉对去卵巢大鼠骨质量的保护作用

    张彤; 李万根


    Objective  To investigate the effect of lipid-free soy powder (SP) in preventing bone loss in ovariectomized(OVX) rats. Methods Thirty SD rats were randomly divided into three groups. In Sham group, sham-operated rats were fed with a casein-based diet; In SP group, OVX rats were with fed with a SP-based diet and in Casein group, OVX rats were fed with a casein-based diet. The experiments lasted for 8 weeks. Results ①The lumbar vertebral bone mineral density (BMD) and compressive strength in SP group were significantly lower than in Sham group (P<0.05, P<0.01, respectively). The femoral BMD and bending strength showed no difference between the two groups. BMD and strength of both bones were both significantly higher in SP group than in Casein group (P<0.05 for BMD and P<0.01 for strength of both bones). ②The uterine weight of rats in both SP group and Casein group were significantly lower than in Sham group (P<0.05 for both). Conclusion SP can entirely prevent bone loss of the femour and can partially prevent bone loss of lumbar vertebra in OVX rats without uterotrophic activity.%目的探讨脱脂豆粉对去卵巢大鼠骨质量减低的预防作用。方法 30只3月龄SD大鼠随机分为3组:假手术(sham,S)组、脱脂豆粉(lipid-free soy powder,SP)组和酪蛋白(casein,C)组。8周后测骨密度(BMD)等指标。结果①SP组的腰椎BMD低于S组( P<0.05),股骨BMD与S组无差异,两者均高于C组(P均<0.05)。②SP组腰椎的抗压强度低于S组(P<0.01),股骨的抗弯强度与S组无差异,两者均高于C组(P均<0.01)。③SP组和C 组的子宫重量均低于S组(P均<0.05)。结论脱脂豆粉可以预防去卵巢大鼠股骨及部分预防其腰椎骨质量的降低,对子宫无不良影响。

  3. Effects of Diosgenin on Myometrial Matrix Metalloproteinase-2 and -9 Activity and Expression in Ovariectomized Rats

    Chi-Chen Chang, Tang-Ching Kuan, Yao-Yuan Hsieh, Ying-Jui Ho, Yu-Ling Sun, Chih-Sheng Lin


    Full Text Available Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day for 8 weeks. Serum was then sampled for progesterone (P4 and estradiol (E2 assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.

  4. Bacterial assay of contact lens wearers.

    Hart, D E; Hosmer, M; Georgescu, M; Farris, R L


    The goal of the project was to determine the quantity of bacteria on the contact lens and adjacent areas of the eye. This paper is a quantitative study of the contact lens and ocular aerobic microbiota in a mixed group of daily and extended wear disposable contact lens users. The contact lens, the lower fornix, tears collecting at the lower fornix, and edge of the lower lid at the Meibomian gland margin were assayed for the quantity of bacterial colony forming units (CFU). Eighteen patients wearing 49 disposable high water content hydrogel contact lenses were assayed and the mean lens age was 8.8 +/- 4.6 days. Three patients wore their lenses on a daily wear basis and 15 on an extended wear schedule. Tear samples were obtained with sterile microbial loops and the lens was macerated into small particles with a tissue grinder. The samples were poured onto the surface of chocolate agar plates and incubated at 35 degrees C for 48 h in 5% Co2. The lid margin revealed the greatest bacterial presence (mean = 9.7 CFU; median = 2 CFU; mode = 0 CFU). The lens showed the next greatest presence of CFU (mean = 4.5 CFU; median = 1 CFU; mode = 0). The fornix and tears revealed the least bacterial presence (fornix: mean = 2.6 CFU; median = 0 CFU; mode = 0 CFU). The bacteria were coagulase-negative staphylococci. The bacterial assay of disposable lens wearing contact lens subjects indicates that the lid margins are the greatest source of bacteria with the tears being the lowest. These studies support the concept that in the eye, the lens typically does not possess a large number of bacteria under normal conditions.

  5. Nondestructive boxed transuranic (TRU) waste assay systems

    Caldwell, John T.; Jones, Stephanie A.; Lucero, Randy F.


    A brief history of boxed waste assay systems (primarily those developed at Los Alamos National Laboratory) is presented. The characteristics and design process involved with current generation systems--as practiced by BII--are also discussed in some detail. Finally, a specific boxed waste assay system and acceptance test results are presented. This system was developed by BII and installed at the Waste Receiving and Packaging (WRAP) facility in Hanford, Washington in early 1997. The WRAP system combines imaging passive/active neutron (IPAN) techniques with gamma- ray energy analysis (GEA) to assay crates up to 2.5 m X 2.5 m X 6.5 m in size. (Systems that incorporate both these methodologies are usually denoted IPAN/GEA types.) Two separate gamma-ray measurements are accomplished utilizing 16 arrayed NaI detectors and a moveable HPGe detector, while 3He detectors acquire both active and passive neutron data. These neutron measurements use BII's proprietary imaging methodology. Acceptance testing of the system was conducted at Hanford in January 1998. The system's operating performance was evaluated based on accuracy and sensitivity requirements for three different matrix types. Test results indicate an average 13% active mode accuracy for 10 nCi/g loadings of Pu waste and 5% passive mode accuracy for 10 g loadings of Pu waste. Sensitivity testing demonstrated an active mode lower limit of detection of less than 5 nCi/g of 239Pu for the medium matrix and less than 20 pCi/g of fission and activation products at 3(sigma) above background.

  6. A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants.

    Németi, Balázs; Poór, Miklós; Gregus, Zoltán


    The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.

  7. The comet assay in eight mouse organs: results with 24 azo compounds.

    Tsuda, S; Matsusaka, N; Madarame, H; Ueno, S; Susa, N; Ishida, K; Kawamura, N; Sekihashi, K; Sasaki, Y F


    The genotoxicity of 24 azo compounds selected from IARC (International Agency for Research on Cancer) groups 2A, 2B, and 3 were determined by the comet (alkaline single cell gel electrophoresis, SCG) assay in eight mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8, and 24 h after treatment. For the 17 azo compounds, the assay was positive in at least one organ; (1) 14 and 12 azo compounds induced DNA damage in the colon and liver, respectively, (2) the genotoxic effect of most of them was greatest in the colon, and (3) there were high positive responses in the gastrointestinal organs, but those organs are not targets for carcinogenesis. One possible explanation for this discrepancy is that the assay detects DNA damage induced shortly after administration of a relatively high dose, while carcinogenicity is detected after long treatment with relatively low doses. The metabolic enzymes may become saturated following high doses and the rates and pathways of metabolic activation and detoxification may differ following high single doses vs. low long-term doses. Furthermore, considering that spontaneous colon tumors are very rare in rats and mice, the ability to detect tumorigenic effects in the colon of those animals might be lower than the ability to detect genotoxic events in the comet assay. The in vivo comet assay, which has advantage of reflecting test chemical absorption, distribution, and excretion as well as metabolism, should be effective for estimating the risk posed by azo dyes to humans in spite of the difference in dosage regimen.

  8. Antibacterial effect of protamine assayed by impedimetry

    Johansen, Charlotte; Gill, T.; Gram, Lone


    Impedimetric measurements were used to assay the antibacterial effect of protamine. A good linear correlation between the impedance detection time and the initial cell counts was obtained (r = 0 . 99, n = 2). As basic peptides may cause clumping of cells, this correlation curve was used when esti...... A suspended in buffer was not lethal as was the effect on growing cells; however, protamine (50-500 mu g ml(-1)) killed the Gram-negative fish spoilage bacteria Shewanella putrefaciens when the live cells were suspended in buffer....

  9. Test procedure for boxed waste assay system

    Wachter, J. [Los Alamos National Lab., NM (United States)


    This document, prepared by Los Alamos National Laboratory`s NMT-4 group, details the test methodology and requirements for Acceptance/Qualification testing of a Boxed Waste Assay System (BWAS) designed and constructed by Pajarito Scientific Corporation. Testing of the BWAS at the Plutonium Facility (TA55) at Los Alamos National Laboratory will be performed to ascertain system adherence to procurement specification requirements. The test program shall include demonstration of conveyor handling capabilities, gamma ray energy analysis, and imaging passive/active neutron accuracy and sensitivity. Integral to these functions is the system`s embedded operating and data reduction software.

  10. Purification and kinase assay of PKN.

    Mukai, Hideyuki; Ono, Yoshitaka


    PKN is a serine/threonine protein kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC) in the carboxyl-terminal region and three repeats of the antiparallel coiled coil (ACC) domain in the amino-terminal region. Mammalian PKN has three isoforms each derived from different genes, PKN1 (PKNalpha/PRK1/PAK1), PKN2 (PRK2/PAK2/PKNgamma), and PKN3 (PKNbeta). PKN isoforms show different enzymatic properties and tissue distributions and have been implicated in various distinct cellular processes (reviewed in Mukai [2003]). This chapter discusses methods to prepare purified enzymes and to assay substrate phosphorylation activities.

  11. Bicarbonate alters cellular responses in respiration assays.

    Krycer, James R; Fisher-Wellman, Kelsey H; Fazakerley, Daniel J; Muoio, Deborah M; James, David E


    Metabolic assay buffers often omit bicarbonate, which is susceptible to alkalinisation in an open environment. Here, we assessed the effect of including bicarbonate in respirometry experiments. By supplementing HEPES-buffered media with low concentrations of bicarbonate, we found increased respiration in adipocytes and hepatocytes, but not myotubes. This was observed across multiple respirometry platforms and was independent of effects on enhanced insulin sensitivity, pH drift, or mitochondrial function. Permeabilised cell experiments suggest that bicarbonate increases substrate availability, likely by acting as a cofactor for carboxylase enzymes. This emphasises the importance of buffer choice in experimental biology. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Mass-based readout for agglutination assays

    Chunara, Rumi; Godin, Michel; Knudsen, Scott M.; Manalis, Scott R.


    We present a mass-based readout for agglutination assays. The suspended microchannel resonator (SMR) is used to classify monomers and dimers that are formed during early stage aggregation, and to relate the total count to the analyte concentration. Using a model system of streptavidin functionalized microspheres and biotinylated antibody as the analyte, we obtain a dose-response curve over a concentration range of 0.63-630nM and show that the results are comparable to what has been previously achieved by image analysis and conventional flow cytometry.

  13. [Human angiogenin: expression, purification, biological assay].

    Yang, H; Zhang, Y Q; Yan, Z; Han, W; Yao, L B; Su, C Z


    Angiogenin cDNA was obtained by RT-PCR, and cloned into the fusion expression vector pRSETB. The recombinant Angiogenin protein was fused with His6 at its N-terminal and expressed as inclusion body. The expression level was about 10% of the total bacteria protein. After dissolved in 8 mol/L urea, the recombinant protein was purified by Ni2(+)-NTA chelating resin, according to the high affinity of His6 with Ni2+. The biological assay indicated that purified rhANG could induced the new blood vessel formation of CAM and degraded tRNA in vitro.

  14. Standardization of cytokine flow cytometry assays

    Cox Josephine


    Full Text Available Abstract Background Cytokine flow cytometry (CFC or intracellular cytokine staining (ICS can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online. Results Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC were shipped to various sites, where ICS assays using cytomegalovirus (CMV pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V. ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24% for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82% for samples with a mean of Conclusion ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more

  15. Synthesis and biological evaluation of some novel 4H-benzopyran-4-one derivatives as nonsteroidal antiestrogens.

    Ismail, K A; Abd El Aziem, T


    The preparation and characterization of some novel 2- and 3-substituted-7-methoxy-4H-1-benzopyran-4-one are presented. The synthesized compounds were evaluated for their uterotrophic, antiuterotrophic and antiimplantation activities in mature female albino rats. 3-Benzyl-7-methoxy-4H-1-benzopyran-4-one (14) showed the highest uterotrophic activity (87%) based on dry uterine weight gain. The antifertility activity, as assessed by the post-coital antiimplantation activity test, was of weak potency for most compounds (14-29%). Among the products, the 2-(4'-methoxyphenyl)-7-methoxy-4H-1-benzopyran-4-one (19) exhibited the highest antiestrogenic activity of 65%. It also elicited 31% of the uterotrophic activity of estradiol.

  16. Autoradiographic studies of the rat renotropic system.

    Castillo, O; Robertson, D; Goldin, H; Preuss, H G


    Rat sera, 10-30 h after unilateral nephrectomy (UNI), enhance 3H-thymidine ("3H-Tdr) incorporation into DNA of incubating renal tissue from control rats. Stimulation is even greater when extracts from remaining growing kidneys 20 h after UNI are combined with sera from rats after UNI. UNI extracts, i.e., extracts from the kidney remaining after uninephrectomy, are nonstimulatory alone. UNI sera and UNI sera plus UNI extracts could theoretically augment 3H-Tdr incorporation into renal DNA via dilutional means rather than enhanced DNA synthesis. To determine if our results were secondary to enhanced DNA synthesis, we performed our in vitro assay using the labelling of nuclei via autoradiography as another index. The addition of UNI sera compared to sera from sham-operated rats (SHAM) in seven paired experiments enhanced incorporation of 3H-Tdr into DNA by 30% (p less than 0.02) and the addition of both UNI sera and UNI extracts compared to SHAM sera and SHAM extracts enhanced incorporation by 48% (p less than 0.001). Unlike a dilutional effect, nuclear labelling also increased in these same seven experiments: UNI sera versus SHAM sera increased 25% (p less than 0.05) and UNI sera + UNI extracts versus SHAM sera + SHAM extracts increased 37% (p less than 0.01). We conclude that UNI sera and UNI sera + UNI extracts enhance 3H-Tdr incorporation into DNA by augmenting DNA synthesis, driving cells into the "S" phase. The use of 3H-Tdr incorporation into DNA in our assay does estimate DNA synthesis.

  17. Assaying environmental nickel toxicity using model nematodes.

    Rudel, David; Douglas, Chandler D; Huffnagle, Ian M; Besser, John M; Ingersoll, Christopher G


    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  18. Assaying environmental nickel toxicity using model nematodes.

    David Rudel

    Full Text Available Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water, we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  19. Response definition criteria for ELISPOT assays revisited.

    Moodie, Z; Price, L; Gouttefangeas, C; Mander, A; Janetzki, S; Löwer, M; Welters, M J P; Ottensmeier, C; van der Burg, S H; Britten, Cedrik M


    No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses.

  20. Standardization of anti-DNA antibody assays.

    Pisetsky, David S


    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  1. Assaying environmental nickel toxicity using model nematodes

    Rudel, David; Douglas, Chandler; Huffnagle, Ian; Besser, John M.; Ingersoll, Christopher G.


    Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegansand P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

  2. Cryptosporidium cell culture infectivity assay design.

    King, B J; Keegan, A R; Robinson, B S; Monis, P T


    Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

  3. Evidence of Absence: Estrogenicity Assessment of a New Food-Contact Coating and the Bisphenol Used in Its Synthesis.

    Soto, Ana M; Schaeberle, Cheryl; Maier, Mark S; Sonnenschein, Carlos; Maffini, Maricel V


    Consumer concerns about exposure to substances found in food contact materials with estrogenic activity (EA) have created substantial demand for alternatives. We assessed the potential EA of both a new bisphenol monomer used to synthesize polymeric coatings for metal food-contact applications and the nonintentionally added substances (NIAS) that may migrate into food. We evaluated tetramethyl bisphenol F (TMBPF) using in vitro and in vivo assays. We extracted the polymeric coating using food simulants ethanol (50% v/v) and acetic acid (3% w/v) and measured migration using tandem liquid chromatography (LC)/mass spectrometry (MS) and LC time-of-flight MS for TMBPF and NIAS, respectively. We also tested migrants for EA using the E-SCREEN assay. TMBPF did not show estrogenic activity in the uterotrophic assay and did not alter puberty in male and female rats or mammary gland development in female rats. Neither TMBPF nor the migrants from the final polymeric coating increased proliferation of estrogen-sensitive MCF7 cells. TMBPF did not show estrogen-agonist or antagonist activity in the estrogen receptor-transactivation assay. TMBPF migration was below the 0.2 parts per billion detection limit. Our findings provide compelling evidence for the absence of EA by TMBPF and the polymeric coating derived from it and that human exposure to TMBPF would be negligible.

  4. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    Harding, Ethelynda E.; Kimsey, R. Scott


    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  5. Assay Method for 235U in Low-Density Waste


    <正>235U assay method will provide a semi-quantitative assay for any uranium lumps that might exist in low-density, low-Z material waste boxes within a short count time. These materials will consist of

  6. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    Satomi Kawaguchi


    Full Text Available Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test. WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU, ethyl nitrosourea (ENU, methyl methanesulfonate (MMS, ethyl methanesulfonate (EMS, bleomycin (BLM, or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC and without (Comet assay DNA repair inhibitors (araC and hydroxyurea. Furthermore, acellular Comet assay (acellular assay was performed to determine how single-strand breaks (SSBs as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD, which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1 for all mutagens studied, LGDs were MN test ≦ Comet assay; (2 except for BLM, LGDs were Comet assay/araC ≦ MN test; (3 except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1 LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2 for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were

  7. Assay optimization: a statistical design of experiments approach.

    Altekar, Maneesha; Homon, Carol A; Kashem, Mohammed A; Mason, Steven W; Nelson, Richard M; Patnaude, Lori A; Yingling, Jeffrey; Taylor, Paul B


    With the transition from manual to robotic HTS in the last several years, assay optimization has become a significant bottleneck. Recent advances in robotic liquid handling have made it feasible to reduce assay optimization timelines with the application of statistically designed experiments. When implemented, they can efficiently optimize assays by rapidly identifying significant factors, complex interactions, and nonlinear responses. This article focuses on the use of statistically designed experiments in assay optimization.

  8. Performance Characteristics of Xpert Flu/RSV XC Assay.

    Popowitch, Elena B; Miller, Melissa B


    The Xpert Flu/RSV XC assay was compared to laboratory-developed tests (LDTs) (n = 207) and the Xpert Flu assay (n = 147) using archived nasopharyngeal swabs. The percentages of positive agreements with LDTs were 97.8% for influenza A, 97.2% for influenza B, and 89.3% for RSV. The sensitivity of influenza detection was improved with the Xpert Flu/RSV XC assay compared to the Xpert Flu assay.

  9. Effects of Brain-Derived Neurotrophic Factor on Local Inflammation in Experimental Stroke of Rat

    Yongjun Jiang; Ning Wei; Juehua Zhu; Tingting Lu; Zhaoyao Chen; Gelin Xu; Xinfeng Liu


    This study was aimed to investigate whether brain-derived neurotrophic factor (BDNF) can modulate local cerebral inflammation in ischemic stroke. Rats were subjected to ischemia by occluding the right middle cerebral artery (MCAO) for 2 hours. Rats were randomized as control, BDNF, and antibody groups. The local inflammation was evaluated on cellular, cytokine, and transcription factor levels with immunofluorescence, enzyme-linked immunosorbent assay, real-time qPCR, and electrophoretic mobil...

  10. The Potential Benefits and Adverse Effects of Phytic Acid Supplement in Streptozotocin-Induced Diabetic Rats

    Omoruyi, F. O.; Budiaman, A.; Eng, Y.; F. E. Olumese; Hoesel, J. L.; Ejilemele, A.; Okorodudu, A. O.


    In this study, the effect of phytic acid supplement on streptozotocin-induced diabetic rats was investigated. Diabetic rats were fed rodent chow with or without phytic acid supplementation for thirty days. Blood and organ samples were collected for assays. The average food intake was the highest and the body weight gain was the lowest in the group fed phytic acid supplement compared to the diabetic and normal control groups. There was a downward trend in intestinal amylase activity in the gro...

  11. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C


    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  12. Thyroid endocrine system disruption by pentachlorophenol: an in vitro and in vivo assay.

    Guo, Yongyong; Zhou, Bingsheng


    The present study aimed to evaluate the disruption caused to the thyroid endocrine system by pentachlorophenol (PCP) using in vitro and in vivo assays. In the in vitro assay, rat pituitary GH3 cells were exposed to 0, 0.1, 0.3, and 1.0 μM PCP. PCP exposure significantly downregulated basal and triiodothyronine (T3)-induced Dio 1 transcription, indicating the antagonistic activity of PCP in vitro. In the in vivo assay, zebrafish embryos were exposed to 0, 1, 3, and 10 μg/L of PCP until 14 days post-fertilization. PCP exposure resulted in decreased thyroxine (T4) levels, but elevated contents of whole-body T3. PCP exposure significantly upregulated the mRNA expression of genes along hypothalamic-pituitary-thyroid (HPT) axis, including those encoding thyroid-stimulating hormone, sodium/iodide symporter, thyroglobulin, Dio 1 and Dio 2, alpha and beta thyroid hormone receptor, and uridinediphosphate-glucuronosyl-transferase. PCP exposure did not influence the transcription of the transthyretin (TTR) gene. The results indicate that PCP potentially disrupts the thyroid endocrine system both in vitro and in vivo.

  13. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan


    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Systems, devices, and methods for agglutination assays using sedimentation

    Schaff, Ulrich Y.; Sommer, Gregory J.; Singh, Anup K.


    Embodiments of the present invention include methods for conducting agglutination assays using sedimentation. Aggregates may be exposed to sedimentation forces and travel through a density medium to a detection area. Microfluidic devices, such as microfluidic disks, are described for conducting the agglutination assays, as are systems for conducting the assays.

  15. 21 CFR 866.2350 - Microbiological assay culture medium.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... organism in the innoculated medium. Test results aid in the diagnosis of disease resulting from either...

  16. 21 CFR 864.7470 - Glycosylated hemoglobin assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section... Glycosylated hemoglobin assay. (a) Identification. A glycosylated hemoglobin assay is a device used to measure the glycosylated hemoglobins (A1a, A1b, and A1c) in a patient's blood by a column...

  17. 21 CFR 864.7400 - Hemoglobin A2 assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hemoglobin A2 assay. 864.7400 Section 864.7400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7400 Hemoglobin A2 assay. (a) Identification. A hemoglobin A2 assay is a device used to determine the hemoglobin A2...

  18. 21 CFR 864.7500 - Whole blood hemoglobin assays.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  19. 21 CFR 864.7415 - Abnormal hemoglobin assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Abnormal hemoglobin assay. 864.7415 Section 864... hemoglobin assay. (a) Identification. An abnormal hemoglobin assay is a device consisting of the reagents... hemoglobin types. (b) Classification. Class II (performance standards)....

  20. 21 CFR 866.3402 - Plasmodium species antigen detection assays.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plasmodium species antigen detection assays. 866... Plasmodium species antigen detection assays. (a) Identification. A Plasmodium species antigen detection assay... malaria caused by the four malaria species capable of infecting humans: Plasmodium falciparum, Plasmodium...

  1. 40 CFR 79.64 - In vivo micronucleus assay.


    ... micronucleus assay. (a) Purpose. The micronucleus assay is an in vivo cytogenetic test which uses erythrocytes...) Test evaluation. (i) Positive results in the micronucleus test provide information on the ability of a..., Mammalian Bone Marrow Cytogenetics Tests: Micronucleus Assay. (2) Cihak, R. “Evaluation of Benzidine by...

  2. 21 CFR 866.3305 - Herpes simplex virus serological assays.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Herpes simplex virus serological assays. 866.3305... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3305 Herpes simplex virus serological assays. (a) Identification. Herpes simplex virus serological assays are devices...

  3. 21 CFR 864.7100 - Red blood cell enzyme assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  4. The sensitivity of male rat reproductive organs to monosodium glutamate

    Sitthichai Iamsaard


    Full Text Available Objective. This study aimed to investigate the sensitivity of the testis, epididymis, seminal vesicle, and sperm acrosome reaction (AR to monosodium L- glutamate (MSG in rats. Materials and methods. Rats were divided into four groups and fed with non-acidic MSG at 0.25, 3 or 6 g/kg body weight for 30 days or without MSG. The morphological changes in the reproductive organs were studied. The plasma testosterone level, epididymal sperm concentration, and sperm AR status were assayed. Results. Compared to the control, no significant changes were discerned in the morphology and weight of the testes, or the histological structures of epididymis, vas deferens and seminal vesicle. In contrast, significant decreases were detected in the weight of the epididymis, testosterone levels, and sperm concentration of rats treated with 6 g/kg body weight of MSG. The weight loss was evident in the seminal vesicle in MSG-administered rats. Moreover, rats treated with MSG 3 and 6 g/kg exhibited partial testicular damage, characterized by sloughing of spermatogenic cells into the seminiferous tubular lumen, and their plasma testosterone levels were significantly decreased. In the 6 g/kg MSG group, the sperm concentration was significantly decreased compared with the control or two lower dose MSG groups. In AR assays, there was no statistically significant difference between MSG-rats and normal rats. Conclusion. Testicular morphological changes, testosterone level, and sperm concentration were sensitive to high doses of MSG while the rate of AR was not affected. Therefore, the consumption of high dose MSG must be avoided because it may cause partial infertility in male.

  5. Estrogenic effects in vitro and in vivo of the fungicide fenarimol

    Andersen, Helle Raun; Bonefeld-Jørgensen, Eva C.; Nielsen, Flemming


    in fenarimol-treated animals. To our knowledge, only two other pesticides (o.p-DDT and methoxychlor) have previously been reported to induce an estrogenic response in the rodent uterotrophic bioassay. A pronounced xenoestrogenicity in serum samples from rats treated with fenarimol and estradiol benzoate (E2B...

  6. Toxicogenomics of resveratrol in rat liver.

    Hebbar, Vidya; Shen, Guoxiang; Hu, Rong; Kim, Bok-Ryang; Chen, Chi; Korytko, Peter J; Crowell, James A; Levine, Barry S; Kong, A-N Tony


    Resveratrol, a polyphenolic compound found in grape skin and peanuts has been shown to prevent many diseases including cardiovascular diseases and cancer. To better understand resveratrol's potential in vivo toxicity, we studied the dose response using cDNA stress arrays coupled with drug metabolizing enzymatic (DME) assays to investigate the expression of stress-responsive genes and Phase I and II detoxifying enzymes in rat livers. Male and female CD rats were treated with high doses of resveratrol (0.3, 1.0 and 3.0 gm/kg/day) for a period of 28 days. Total RNA from rat liver was reverse-transcribed using gene-specific primers and hybridized to stress-related cDNA arrays. Among female rats, Phase I DME genes were repressed at 0.3 and 1.0 gm/kg/day doses, while genes such as manganese superoxide dismutase, cytochrome P450 reductase, quinone oxidoreductase and thiosulfate sulfurtransferase demonstrated a dose-dependent increase in gene expression. The modulation of these liver genes may implicate the potential toxicity as observed among the rats at the highest dose level of resveratrol. Real-Time PCR was conducted on some of the Phase II DME genes and anti-oxidant genes to validate the cDNA array data. The gene expression from real-time PCR demonstrated good correlation with the cDNA array data. UGT1A genes were amongst the most robustly induced especially at the high doses of resveratrol. We next performed Phase I and Phase II enzymatic assays on cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Induction of Phase II detoxifying enzymes was most pronounced at the highest dose of resveratrol. CYP1A1 activity demonstrated a decreasing trend among the 3 dose groups and CYP2E1 activity increased marginally among female rats over controls. In summary, at lower doses of resveratrol there are few significant changes in gene expression whereas the


    黄颖秋; 萧树东; 莫剑忠; 张德中


    Objective To investigate the effects of hemoglobin (Hb) on serum nitric oxide (NO) concentration and hemodynamics pattern changes in rats with cirrhosis. Methods Cirrhosis model was induced in male SD rats by injection of 60% CCl4 oily solution subcutaneously. Cirrhotic rats were treated with erythropoietin (l00U/kg) injected subcutaneously for 2 weeks. Mean arterial pressure (MAP), cardiac output (CO), cardiac index (CI), splanchnic vascular resistance (SVR), splanchnic blood flow (SBF) and serum NO concentration were determined in erythropoietin - treated, erythropoietin - untreated cirrhotic rats and controls by using 57Co- labelled microsphere technique and a fluorometric assay, respectively. In addition, blood Hb levels were also measured in the 3 groups. Results Untreated cirrhotic rats had significantly lower MAP, SVR, Hb and higher CO, CI, SBF and NO concentration than those of the controls (P<0.01). In treated cirrhotic rats, erythropoietin significantly attenuated the increase of CO, CI, SBF, NO concentration and the decrease of MAP and SVR. In cirrhotic rats,epoetin beta in subcutaneous dose of 100U· kg-1· d-1 induced a markedly increment of blood Hb levels and decrement of NO concentration in comparison with untreated cirrhotic rats (181±11g/L vs 120±15g/L;1.14±0.62μmol/L vs 4.20±1.25μmol/L). Conclusion The endogenous NO may play an important role in the changes of hemodynamics pattern in cirrhosis, and hyperdynamic circulatory status in rats with cirrhosis might be ameliorated by inactivation of overproduced NO by increasing hemoglobin with erythropoietin.

  8. Assays for investigating deSUMOylation enzymes.

    Madu, Ikenna G; Chen, Yuan


    Post-translational modifications by the SUMO (Small Ubiquitin-like MOdifier) family of proteins are recently discovered essential regulatory mechanisms. All SUMO proteins are synthesized as larger precursors that are matured by SUMO-specific proteases, known as SENPs, which remove several C-terminal amino acids of SUMO to expose the Gly-Gly motif. SENPs also remove SUMO modifications from target proteins, making this modification highly dynamic. At least six deSUMOylation enzymes, all of which are encoded by essential genes, have been identified in mammals. SENP1 has been shown to play an important role in the development of prostate cancer and in angiogenesis. This unit describes and discusses methods for characterizing the deSUMOylation enzymes. These assays enable the identification of inhibitors of these enzymes and investigation of their mechanism of inhibition in order to develop research tools and future therapeutics.

  9. Nondestructive Assay Options for Spent Fuel Encapsulation

    Tobin, Stephen J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Jansson, Peter [Uppsala Univ. (Sweden)


    This report describes the role that nondestructive assay (NDA) techniques and systems of NDA techniques may have in the context of an encapsulation and deep geological repository. The potential NDA needs of an encapsulation and repository facility include safeguards, heat content, and criticality. Some discussion of the facility needs is given, with the majority of the report concentrating on the capability and characteristics of individual NDA instruments and techniques currently available or under development. Particular emphasis is given to how the NDA techniques can be used to determine the heat production of an assembly, as well as meet the dual safeguards needs of 1) determining the declared parameters of initial enrichment, burn-up, and cooling time and 2) detecting defects (total, partial, and bias). The report concludes with the recommendation of three integrated systems that might meet the combined NDA needs of the encapsulation/repository facility.

  10. Assay of Endocannabinoid Oxidation by Cyclooxygenase-2

    Kudalkar, Shalley N.; Kingsley, Philip J.; Marnett, Lawrence J.


    Summary The endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide (AEA) are endogenous ligands for the cannabinoid receptors (CB1 and CB2) and are implicated in a wide array of physiological processes. These neutral arachidonic acid (AA) derivatives have been identified as efficient substrates for the second isoform of the cyclooxygenase enzyme (COX-2). A diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) is generated by the action of COX-2 (and downstream prostaglandin synthases) on 2-AG and AEA. As the biological importance of the endocannabinoid system becomes more apparent, there is a tremendous need for robust, sensitive and efficient analytical methodology for the endocannabinoids and their metabolites. In this chapter we describe methodology suitable for carrying out oxygenation of endocannabinoids by COX-2, and analysis of products of endocannabinoid oxygenation by COX-2 and of endocannabinoids themselves from in-vitro and cell assays. PMID:27245906

  11. Stable isotope dilution assays in mycotoxin analysis

    Rychlik, Michael; Asam, Stefan [Universitaet Muenchen, Lehrstuhl fuer Lebensmittelchemie der Technischen, Garching (Germany)


    The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis. (orig.)

  12. Lab-on-a-Chip Multiplex Assays.

    Peter, Harald; Wienke, Julia; Bier, Frank F


    Lab-on-a-chip multiplex assays allow a rapid identification of multiple parameters in an automated manner. Here we describe a lab-based preparation followed by a rapid and fully automated DNA microarray hybridization and readout in less than 10 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable rapid identification of bacterial species and detection of antibiotic resistance. The use of DNA microarrays allows a fast adaptation of new biomarkers enabling the identification of different genes as well as single-nucleotide-polymorphisms (SNPs) within these genes. In this protocol we describe a DNA microarray developed for identification of Staphylococcus aureus and the mecA resistance gene.

  13. Development of a Radioactive Waste Assay System

    Kang, Duck Won; Song, Myung Jae; Shin, Sang Woon; Sung, Kee Bang; Ko, Dae Hach [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Kil Jeong; Park, Jong Mook; Jee, Kwang Yoong [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)


    Nuclear Act of Korea requires the manifest of low and intermediate level radioactive waste generated at nuclear power plants prior to disposal sites.Individual history records of the radioactive waste should be contained the information about the activity of nuclides in the drum, total activity, weight, the type of waste. A fully automated nuclide analysis assay system, non-destructive analysis and evaluation system of the radioactive waste, was developed through this research project. For the nuclides that could not be analysis directly by MCA, the activities of the representative {gamma}-emitters(Cs-137, Co-60) contained in the drum were measured by using that system. Then scaling factors were used to calculate the activities of {alpha}, {beta}-emitters. Furthermore, this system can automatically mark the analysis results onto the drum surface. An automated drum handling system developed through this research project can reduce the radiation exposure to workers. (author). 41 refs., figs.

  14. Stable isotope dilution assays in mycotoxin analysis.

    Rychlik, Michael; Asam, Stefan


    The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis.

  15. Universal fieldable assay with unassisted visual detection

    Chelyapov, Nicolas (Inventor)


    A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.




    Full Text Available Casein, the main milk protein was determined by different assay methods: the gravimetric method, the method based on the neutralization of the NaOH excess used for the casein precipitate solving and the method based on the titration of the acetic acid used for the casein precipitation. The last method is the simplest one, with the fewer steps, and also with the lowest error degree. The results of the experiment revealed that the percentage of casein from the whole milk protein represents between 72.6–81.3% in experiment 1, between 73.6–81.3% in experiment 2 and between 74.3–81% in experiment 3.

  17. Enzymatic assay for methotrexate in erythrocytes

    Schrøder, H; Heinsvig, E M


    Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection...... limit was 3 nmol/l. Within run and between-run precision was 7.4% and 13.5% for control 10 nmol/l and 1.2% and 3.2% for control 50 nmol/l. Recovery was 85-115% of MTX added to haemolysed erythrocytes. We found the method useful for pharmacokinetic studies of MTX in erythrocytes in MTX-treated patients...

  18. Advancement in biochemical assays in andrology

    Wolf-BernhardSchill; RaftHenkel


    Determination of maikers of sperm function, accessory sex gland secretion and silent male genital tract inflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemical tests into the analysis of male factor has the advantage that standardized assays with a coefficient of variafion characteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability .Biochemical parameters may be used in clinical practice to evaluate the sperm fertitizing capacity (acrosin, aniline blue,ROS), to characterize male accessory sex gland secretinns (fructose, a-glucosidase, PSA), and to identify men with silent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS). (As/an J Androl 1999 Jun; 1: 45-51)

  19. Direct measurement of human plasma corticotropin-releasing hormone by two-site immunoradiometric assay

    Linton, E.A.; McLean, C.; Nieuwenhuyzen Kruseman, A.C.; Tilders, F.J.; Van der Veen, E.A.; Lowry, P.J.


    A ''two-site'' immunoradiometric assay (IRMA) which allows the direct estimation of human CRH (hCRH) in plasma is described. Using this IRMA, basal levels of CRH in normal subjects ranged from 2-28 pg/mL (mean, 15 +/- 7 (+/- SD) pg/mL; n = 58). Values in men and women were similar. Plasma CRH values within this range were also found in patients with Cushing's syndrome, Addison's disease, and Nelson's syndrome, with no correlation between plasma CRH and ACTH levels in these patients. Elevated plasma CRH levels were found in pregnant women near term (1462 +/- 752 (+/- SD) pg/mL; n = 55), and the dilution curve of this CRH-like immunoreactivity paralleled the IRMA standard curve. After its immunoadsorption from maternal plasma, this CRH-like material eluted on reverse phase high performance liquid chromatography with a retention time identical to that of synthetic CRH and had equipotent bioactivity with the synthetic peptide in the perfused anterior pituitary cell bioassay. Circulating CRH was not detected in Wistar rats, even after adrenalectomy and subsequent ether stress. Synthetic hCRH was degraded by fresh human plasma relatively slowly; 65% of added CRH remained after 1 h of incubation at 37 C. Degradation was inhibited by heat treatment (54 C; 1 h), cold treatment (4 C; 4 h), or freezing and thawing. Loss of synthetic rat CRH occurred more rapidly when fresh rat plasma was used; only 20% of added CRH remained under the same conditions. The inability to measure CRH in peripheral rat plasma may be due to the presence of active CRH-degrading enzymes which fragment the CRH molecule into forms not recognized by the CRH IRMA.

  20. Medical Devices; Immunology and Microbiology Devices; Classification of the Assayed Quality Control Material for Clinical Microbiology Assays. Final order.


    The Food and Drug Administration (FDA, Agency, or we) is classifying the assayed quality control material for clinical microbiology assays into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the assayed quality control material for clinical microbiology assays' classification. The Agency is classifying the device into class II (special controls) to provide a reasonable assurance of safety and effectiveness of the device.

  1. Improved internal control for molecular diagnosis assays.

    Vinayagamoorthy, T; Maryanski, Danielle; Vinayagamoorthy, Dilanthi; Hay, Katie S L; Yo, Jacob; Carter, Mark; Wiegel, Joseph


    The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1-4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step. •The analyte and internal control have the same PCR and sequencing annealing sequences.•This method assures for little or no false negatives and false positives due to the method's design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively.•This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay.

  2. An ethanolic extract of black cohosh causes hematological changes but not estrogenic effects in female rodents

    Mercado-Feliciano, Minerva; Cora, Michelle C.; Witt, Kristine L. [National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Research Triangle Park, NC (United States); Granville, Courtney A.; Hejtmancik, Milton R.; Fomby, Laurene; Knostman, Katherine A.; Ryan, Michael J. [Battelle Memorial Institute, Columbus, OH (United States); Newbold, Retha; Smith, Cynthia; Foster, Paul M.; Vallant, Molly K. [National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Research Triangle Park, NC (United States); Stout, Matthew D., E-mail: [National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Research Triangle Park, NC (United States)


    Black cohosh rhizome (Actaea racemosa) is used as a remedy for pain and gynecological ailments; modern preparations are commonly sold as ethanolic extracts available as dietary supplements. Black cohosh was nominated to the National Toxicology Program (NTP) for toxicity testing due to its widespread use and lack of safety data. Several commercially available black cohosh extracts (BCE) were characterized by the NTP, and one with chemical composition closest to formulations available to consumers was used for all studies. Female B6C3F1/N mice and Wistar Han rats were given 0, 15 (rats only), 62.5 (mice only), 125, 250, 500, or 1000 mg/kg/day BCE by gavage for 90 days starting at weaning. BCE induced dose-dependent hematological changes consistent with a non-regenerative macrocytic anemia and increased frequencies of peripheral micronucleated red blood cells (RBC) in both species. Effects were more severe in mice, which had decreased RBC counts in all treatment groups and increased micronucleated RBC at doses above 125 mg/kg. Dose-dependent thymus and liver toxicity was observed in rats but not mice. No biologically significant effects were observed in other organs. Puberty was delayed 2.9 days at the highest treatment dose in rats; a similar magnitude delay in mice occurred in the 125 and 250 mg/kg groups but not at the higher doses. An additional uterotrophic assay conducted in mice exposed for 3 days to 0.001, 0.01, 0.1, 1, 10, 100 and 500 mg/kg found no estrogenic or anti-estrogenic activity. These are the first studies to observe adverse effects of BCE in rodents. -- Highlights: ► Mice and rats were dosed with black cohosh extract for 90 days starting at weaning. ► Hematological changes were consistent with a non-regenerative macrocytic anemia. ► Peripheral micronucleated red blood cell frequencies increased. ► Puberty was delayed 2.9 days in rats. ► No estrogenic/anti-estrogenic activity was seen in the uterotrophic assay.

  3. Effects of Acute Oral 5-aminotetrazole (5-AT) Exposure to Rats (Rattus norvegicus)


    Micronucleus Assay (MNA) Male rats from the 5-AT study (three highest dose groups and the vehicle control) were tested for DNA damage in their peripheral...Dose Test (Sub-acute Study) - Sperm Collection and Analysis 09/25/2013 10/07/2013 14-Day Repeated Dose Test (Sub-acute Study) - Micronucleus Assay...2015 Prepared by: Valerie H Adams, Ph.D. Approved for public release; distribution unlimited. Specialty: 500C Toxicity Test Toxicity Report No. S

  4. Antioxidant assay using genetically engineered bioluminescent Escherichia coli

    Bartolome, Amelita; Macalino, Bernadette; Pastoral, Ian Lemuel; Sevilla, Fortunato, III


    A new antioxidant activity assay based on the reactive oxygen species (ROS)-inducible bacterial strain (E. coli DPD2511) is described. The strain harbors the plasmid pKatG::luxCDABE and responds to hydrogen peroxide treatment by increasing light emission at 490 nm. Antioxidant capacity is evaluated through the ability of an agent to inhibit the hydrogen peroxide-induced bioluminescence of E. coli DPD2511. Applicability of the developed assay in detecting levels of antioxidants in various aqueous plant extracts is demonstrated. The assay was validated against 2,2-diphenylpicrylhydrazyl (DPPH) assay, a known antioxidant assay.

  5. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Jean-Louis Reymond


    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  6. Performance of MycAssay Aspergillus DNA real-time PCR assay compared with the galactomannan detection assay for the diagnosis of invasive aspergillosis from serum samples.

    Danylo, Alexis; Courtemanche, Chantal; Pelletier, René; Boudreault, Alexandre A


    Invasive aspergillosis (IA) is a major problem in the immunocompromised population, and its diagnosis is difficult due to the low sensitivity of available tests. Detection of Aspergillus nucleic acid by polymerase chain reaction (PCR) in serum samples is a promising diagnostic tool; however, use of multiple "in-house" methods precludes standardization. The first commercial PCR assay, MycAssay Aspergillus (Myconostica, Ltd), became available recently, and its performance in the diagnosis of IA was evaluated and compared with the galactomannan (GM) assay. Serum samples obtained from patients with hematological cancer were tested retrospectively with MycAssay Aspergillus PCR. Per-episode and per-test analyses were undertaken with 146 sera from 35 hematological patients. Sixteen patients had proven or probable IA and 19 had possible or no IA. In per-episode analysis, MycAssay Aspergillus had a sensitivity of 43.8% (95% confidence interval [CI], 19.8%-70.1%) and a specificity of 63.2% (95% CI, 38.4%-83.7%) for IA diagnosis. In per-test analyses, MycAssay Aspergillus had a lower specificity than the GM assay (83.3% vs. 93.1%, P = 0.04). The addition of PCR to routine clinical practice would have permitted the diagnosis of one additional probable IA in our cohort. Use of PCR instead of GM assay would have delayed the diagnosis in two cases. Aspergillus DNA detection by PCR with serum specimens using MycAssay showed a lower specificity than the GM assay and was associated with a low sensitivity for IA diagnosis. More studies are needed to determine the exact role of MycAssay in IA diagnosis in patients with hematological malignancy.

  7. Effect of Vagotomy on Acute Pancreatitis in Rats


    One hundred and eighty-two SD rats were randomly divided into the normal control group, fast operating group and food-intake operating group. The experimental model of acute pancreatitis (AP) in rats was established by injecting 5 % sodium taurocholate into the pancreatic duct of rat according to Aho's method. The sandostatin was used for positive contrast. The concentrations of serum amylase, calcium, C reaction protein (CRP) and interleukin-6 (IL-6) were assayed respectively at different time points. The pathological sections were observed. Each operating group contained 10 rats. The mortality of the operating groups was observed during the 24 h. The serum amylase level in the AP rats was reduced after receiving vagotomy (VG, P<0.05). Although the serum calcium level in most groups was decreased, the reduction in the group with VG plus sandostatin was not obvious (P>0.05). The increase of CRP and IL-6 was not obvious after VG (P>0.05). The change of mortality was not significant (P>0.05). The pathological sections showed that the AP pathological change was mild after VG. The disease condition of food-intake operating group was more serious than that of fast operating group. It was suggested that VG had some influence on the prognosis of AP in rats.

  8. Reproductive toxicity of Roundup herbicide exposure in male albino rat.

    Owagboriaye, Folarin O; Dedeke, Gabriel A; Ademolu, Kehinde O; Olujimi, Olarenwaju O; Ashidi, Joseph S; Adeyinka, Aladesida A


    The incidence of infertility in human is on the increase and the use of Roundup herbicide and presence of its residues in foodstuff is a major concern. This study therefore aim to assess the effect of Roundup on the reproductive capacity of 32 adult male albino rats randomized into 4 groups of 8 rats per group orally exposed to Roundup at 3.6mg/kg body weight(bw), 50.4mg/kgbw and 248.4mg/kgbw of glyphosate concentrations for 12 weeks while the control group was given distilled water. Serum level of reproductive hormone (testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin), oxidative stress indices in the testicular tissue, epididymal sperm morphology assessment and testicular histopathology of the rats were used as a diagnostic marker of reproductive dysfunction. Significant (p<0.05) alterations in the level of all the reproductive hormones and oxidative stress markers assayed were observed in rats exposed to Roundup. Significant reductions (p<0.05) in sperm count, percentage motility and significant (p<0.05) increased in abnormal sperm cells were observed in the exposed rats. Histopathologically, severe degenerative testicular architectural lesions were seen in the Roundup exposed rats. Roundup may interfere with spermatogenesis and impair fertility in male gonad. Copyright © 2017 Elsevier GmbH. All rights reserved.

  9. Detection of Genotoxicity of Cinnabaris by Using Micronucleus Assay and Comet Assay%微核试验和彗星试验检测朱砂的遗传毒性

    张超超; 吴文斌; 汤家铭


    Objective:To study the effect of Cinnabaris on mouse/rat chromosome damage,and to compare the detection of genotoxicity by short-term and long-term dose administration, exploring the feasibility of integrating micronucleus assay and comet assay into reproductive toxicity test I period ( male fertility and early embryonic development). Method; Cinnabaris suspension was orally administrated to male mice by ig at doses of 10,5. 0,2. 5 g ·kg-1 respectively (equal to 100,50, and 25 times of the human highest clinical equivalent doses) , after 2 days mice were sacrificed. Cinnabaris suspension was orally administrated to rats by ig at doses of 1. 0, 0. 3,0. 1 g· kg-1 respectively ( equal to 20,6. 4, and 2. 0 times of the human highest clinical equivalent doses) , according to the protocol of rat fertility and early embryo development toxicity by ig administration of Cinnabaris, male rats were sacrificed after 42 day ' 8 continuous administration and mating, and female rats after 20 day' s continuous administration and on D15 of pregnancy. Then the bone marrows were taken to do micronucleus assay and comet assay. Result:The micronucleus rates of mice administrated were 0. 175% ,0. 108% and 0. 092% respectively,and had statistical significance when compared with the negative control. But in comet assay, the results werenegative. The micronucleus rates of rats, which were integrated into the protocol of rat fertility and early embryo development toxicity test, had a tendency of increase as the doses increase, but no statistical significance. But in comet assay,the results showed that both tailed cell numbers and tailed lengths in high and middle dose groups statistically increased in both male and female rats when compared with the negative control. Conclusion; ① Both high dose, short-term and low dose, long-term administrations of Cinnabaris may cause chromosome damage; ② it is feasible that both micronucleus assay and comet assay be integrated into the protocol of

  10. Expression of AT2 receptors in the developing rat fetus.

    Grady, E F; Sechi, L. A.; Griffin, C A; Schambelan, M.; Kalinyak, J E


    Angiotensin II is known primarily for its effects on blood pressure and electrolyte homeostasis, but recent studies suggest that angiotensin II may play a role in the regulation of cellular growth. This study was undertaken to identify the angiotensin II receptor subtypes expressed during fetal and neonatal development and to characterize their cellular localization. Using an in situ receptor binding assay on sagittal frozen sections of fetal and neonatal rats, bound 125I-[Sar1,Ile8]-angioten...

  11. Insulin regulation of rat growth hormone gene transcription.


    We have previously shown that insulin suppresses growth hormone (GH) messenger (m) RNA levels in rat pituitary cells. To further delineate the molecular mechanism of insulin action, the effect of insulin treatment on GH gene transcription rates was examined in GH3 pituitary cells grown in serum-free defined medium. A transcriptional run-off assay was performed when intact isolated nuclei were allowed to continue RNA synthesis in an in vitro reaction. Specific incorporation of [32P]GTP into RN...

  12. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    Simon, R H; DeHart, P D; Todd, R F


    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neut...

  13. Calcium-independent phospholipase A2 in rat tissue cytosols

    Pierik, A.J.; Nijssen, J.G.; Aarsman, A.J.; Bosch, H. van den


    Cytosols (105000 X g supernatant) from seven rat tissues were assayed for Ca²⁺-independent phospholipase A₂ activity with either 1-acyl-2-[1-¹⁴C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[l-¹⁴C]linoleoyl-snglycero- 3-phosphoethanohunine or 1-0-hexadecyl-2-[9,10-³H₂]oleoyl-sn-glycero-3-phosphoc


    Pontari, M.A.; LUTHIN, G. R.; Braverman, A. S.; Ruggieri, M. R.


    The purpose of this study was to characterize the muscarinic receptor subtypes in the individual lobes of the rat prostate. Immunoprecipitation was performed on homogenates of these 3 lobes using antibodies to the m1-m4 muscarinic receptor subtypes. Reverse transcriptase polymerase chain reaction assays (RT-PCR) were also performed using primers specific for each of the five muscarinic receptor subtypes (m1-m5). The susceptibility of the receptors to degradation by endogenous prostate proteas...

  15. Micronuclei rate and hypoxanthine phosphoribosyl transferase mutation in radon-exposed rats

    Fengmei Cui; Saijun Fan; Mingjiang Hu; Jihua Nie; Hongmei Li; Jian Tong


    The genetic changes in rats with radon exposure were studied by the micronucleus technology and detection of hypoxanthine phosphoribosyl transferase (hprt) mutations.The rate of the micronuclei in peripheral blood lymphocytes and tracheal-bronchial epithelial cells in the radon-inhaled rats was higher than that of the controls (P < 0.05).A similar result was obtained from the hprt assay,which showed a higher mutation frequency in radon-exposed rats.Our results suggested that micronuclei rate and hprt deficiency could be used as biomarkers for the genetic changes with radon exposure.

  16. SWEEP Project RAT

    Ibsen, Lars Bo; Madsen, Søren; Petersen, L. B.

    This report presents the results from the design analyses made for the clustered suction caisson used as foundation for a Riser Access Tower (RAT). The RAT is intended built next to the K15-FA-1 Platform in the Dutch Sector of the North Sea.......This report presents the results from the design analyses made for the clustered suction caisson used as foundation for a Riser Access Tower (RAT). The RAT is intended built next to the K15-FA-1 Platform in the Dutch Sector of the North Sea....

  17. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay.

    Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine


    Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

  18. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J


    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

  19. Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke.

    Roemer, Ewald; Lammerich, Hans-Peter; Conroy, Lynda L; Weisensee, Dirk


    Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment.

    Gaucher, Sonia; Jarraya, Mohamed


    MTT assay is the gold standard for assessing skin sample viability but it is time-consuming. Here we compared the MTT test with two other assays for the assessment of skin viability. The MTT, PrestoBlue (colorimetric method) and LDH release assays were applied to fresh and cryopreserved skin. Skin viability was considered proportional to the optical density values of the relevant analytes. PrestoBlue did not reliably distinguish between fresh and cryopreserved skin. The LDH release assay did not allow us to establish a viability index. We recommend the MTT assay for assessing skin viability.

  1. Development and characterization of an in vivo skin photomicronucleus assay in rats

    Reus, A.A.; Usta, M.; Meeuwen, R.N.C. van; Maas, W.J.M.; Robinson, S.A.; Kenny, J.D.; Pruimboom-Brees, I.; Clements, P.J.; Lynch, A.M.; Krul, C.A.M.


    For pharmaceuticals, current regulatory guidance for photosafety testing states that studies are warranted for drug candidates that both absorb light in the range of 290-700 nm and that are either applied topically or reach the skin or eyes by systemic exposure. In contrast to standard genotoxicity

  2. Development and characterization of an in vivo skin photomicronucleus assay in rats

    Reus, A.A.; Usta, M.; Meeuwen, R.N.C. van; Maas, W.J.M.; Robinson, S.A.; Kenny, J.D.; Pruimboom-Brees, I.; Clements, P.J.; Lynch, A.M.; Krul, C.A.M.


    For pharmaceuticals, current regulatory guidance for photosafety testing states that studies are warranted for drug candidates that both absorb light in the range of 290-700 nm and that are either applied topically or reach the skin or eyes by systemic exposure. In contrast to standard genotoxicity

  3. Tube leukocyte adherence inhibition assay : the assessment of tumor immunity in cancer patients and in rats

    B. Tank (Bhupendra)


    textabstractFor the past two decades, intensive search has been made for the existence of tumor-specific antigens of human cancer. The recent succesful development of monoclonal antibodies against TAA on human cell membrane has not yet resulted in the identification of any tumor-specific determinant

  4. Conditioned stress prevents cue-primed cocaine reinstatement only in stress-responsive rats.

    Hadad, Natalie A; Wu, Lizhen; Hiller, Helmut; Krause, Eric G; Schwendt, Marek; Knackstedt, Lori A


    Neurobiological mechanisms underlying comorbid posttraumatic stress disorder (PTSD) and cocaine use disorder (CUD) are unknown. We aimed to develop an animal model of PTSD + CUD to examine the neurobiology underlying cocaine-seeking in the presence of PTSD comorbidity. Rats were exposed to cat urine once for 10-minutes and tested for anxiety-like behaviors one week later. Subsequently, rats underwent long-access (LgA) cocaine self-administration and extinction training. Rats were re-exposed to the trauma context and then immediately tested for cue-primed reinstatement of cocaine-seeking. Plasma and brains were collected afterwards for corticosterone assays and real-time qPCR analysis. Urine-exposed (UE; n = 23) and controls not exposed to urine (Ctrl; n = 11) did not differ in elevated plus maze behavior, but UE rats displayed significantly reduced habituation of the acoustic startle response (ASR) relative to Ctrl rats. A median split of ASR habituation scores was used to classify stress-responsive rats. UE rats (n = 10) self-administered more cocaine on Day 1 of LgA than control rats (Ctrl + Coc; n = 8). Re-exposure to the trauma context prevented cocaine reinstatement only in stress-responsive rats. Ctrl + Coc rats had lower plasma corticosterone concentrations than Ctrls, and decreased gene expression of corticotropin releasing hormone (CRH) and Glcci1 in the hippocampus. Rats that self-administered cocaine displayed greater CRH expression in the amygdala that was independent of urine exposure. While we did not find that cat urine exposure induced a PTSD-like phenotype in our rats, the present study underscores the need to separate stressed rats into cohorts based on anxiety-like behavior in order to study individual vulnerability to PTSD + CUD.

  5. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia


    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.

  6. No sign of decreased burrowing behavior in the genetically depressive flinders rats

    Baastrup, C. S.; Wegener, Gregers; Finnerup, N. B.


    Background: Burrowing is a natural behavior in rats and has been observed in several different strains. Furthermore decreased burrowing behavior has been shown in different rodent disease models like Multiple sclerosis, peripheral nerve injury and knee inflammation when compared with control...... animals. Burrowing has thus been suggested as a behavioral outcome of the tendency to engage in natural behaviors -a simplified surrogate measure of 'rat quality of life' (rQoL). Most of the disease models used to develop the assay, also concomitantly result in different levels of motor dysfunction...... of each drug. Results: The genetically depressive FSL rats did not burrow less than FRL control rats. Treatment with imipramin or citalopram-S did not change the burrowing behavior of the FSL rats. In contrast treatment with imipramin and citalopram-S unexpectedly decreased the burrowing behavior...

  7. Piwil 2 gene transfection changes the autophagy status in a rat model of diabetic nephropathy.

    Wu, Weihua; Zhang, Maoping; Liu, Qi; Xue, Ling; Li, Ying; Ou, Santao


    This study aims to investigate effects of Piwil2 on autohpagy in a DN rat model. Sixty health SD rats were selected and divided into four group, including normal group, control, DN and Piwil2 therapy group. DN model (DN group) was established by injecting the streptozotocin (50 mg/kg) into rats. Piwil2 therapy group was injected with viral plasmid carrying Piwil2 mRNA to DN rats. The urinary protein concentrations were determined by placing the animals in individual metabolic cages for a timed urine collection every 8 weeks. Blood and soleus muscle samples were collected after animals were sacrificed. Blood glucose was examined by using commercial detection kits. Western blot assay was employed to examine expression of Beclin 1 and LC3 (LC3 I and LC3 II) protein. Results indicated that urinary protein levels were remarkably higher in DN group compared to Normal and Control group (Pdiabetic nephropathy rats.

  8. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M


    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  9. Comparison of five assays for detection of Clostridium difficile toxin.

    Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B


    Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  10. Fluoride assay methodology for carbonated beverages.

    Heilman, Judith R; Levy, Steven M; Wefel, James S; Patterson, Kristine Y; Cutrufelli, Rena; Pehrsson, Pamela R; Holden, Joanne M


    The purpose of this paper was to review different methodological techniques used for the assessment of fluoride in carbonated beverages, and compare results using a fluoride ion electrode direct read method with and without a prior decarbonation treatment. The carbonated beverages in this study were either purchased locally at grocery stores in Iowa City, Iowa, or purchased as part of a national representative sampling approach included in the National Fluoride Database and Intake Assessment Study (NFDIAS). The samples were compared with and without a decarbonating process. Soda pop and beer samples were analyzed by removing a 1-ml sample and adding a 1-ml buffer solution. The fluoride concentration of the sample and buffer combination was then determined using a fluoride ion specific electrode. There was no significant difference in the fluoride concentration of the samples with or without prior decarbonation. The mean absolute difference between the soda pop group with and without decarbonation was 0.01 ppm F, while results from the beer samples showed variation of 0.00 to 0.02 parts per million fluoride (ppm F). These differences were not statistically significant for the soda pop or beer groups (P=.50 and P=.74, respectively). Whether or not decarbonation was conducted prior to analysis, the fluoride assay results were the same. Therefore, decarbonation of soda pop and beer was deemed unnecessary prior to fluoride analysis.

  11. The synchronous active neutron detection assay system

    Pickrell, M.M.; Kendall, P.K.


    We have begun to develop a novel technique for active neutron assay of fissile material in spent nuclear fuel. This approach will exploit a 14-MeV neutron generator developed by Schlumberger. The technique, termed synchronous active neutron detection (SAND), follows a method used routinely in other branches of physics to detect very small signals in presence of large backgrounds. Synchronous detection instruments are widely available commercially and are termed ``lock-in`` amplifiers. We have implemented a digital lock-in amplifier in conjunction with the Schlumberger neutron generator to explore the possibility of synchronous detection with active neutrons. The Schlumberger system can operate at up to a 50% duty factor, in effect, a square wave of neutron yield. Results are preliminary but promising. The system is capable of resolving the fissile material contained in a small fraction of the fuel rods in a cold fuel assembly; it also appears resilient to background neutron interference. The interrogating neutrons appear to be non-thermal and penetrating. Work remains to fully explore relevant physics and optimize instrument design.

  12. Prandiology of Drosophila and the CAFE assay

    Ja, William W.; Carvalho, Gil B.; Mak, Elizabeth M.; de la Rosa, Noelle N.; Fang, Annie Y.; Liong, Jonathan C.; Brummel, Ted; Benzer, Seymour


    Studies of feeding behavior in genetically tractable invertebrate model systems have been limited by the lack of proper methodology. We introduce the Capillary Feeder (CAFE), a method allowing precise, real-time measurement of ingestion by individual or grouped fruit flies on the scale of minutes to days. Using this technique, we conducted the first quantitative analysis of prandial behavior in Drosophila melanogaster. Our results allow the dissection of feeding into discrete bouts of ingestion, defining two separate parameters, meal volume and frequency, that can be uncoupled and thus are likely to be independently regulated. In addition, our long-term measurements show that flies can ingest as much as 1.7× their body mass over 24 h. Besides the study of appetite, the CAFE can be used to monitor oral drug delivery. As an illustration, we used the CAFE to test the effects of dietary supplementation with two compounds, paraquat and ethanol, on food ingestion and preference. Paraquat, a prooxidant widely used in stress tests, had a strong anorexigenic effect. In contrast, in a feeding preference assay, ethanol-laced food, but not ethanol by itself, acted as an attractant. PMID:17494737

  13. Assaying Visual Memory in the Desert Locust

    Senne Dillen


    Full Text Available The involvement of associative learning cues has been demonstrated in several stages of feeding and food selection. Short neuropeptide F (sNPF, an insect neuropeptide whose effects on feeding behavior have previously been well established, may be one of the factors bridging feeding and learning behavior. Recently, it was shown in Drosophila melanogaster that the targeted reduction of Drome-sNPF transcript levels significantly reduced sugar-rewarded olfactory memory. While Drosophila mainly relies on olfactory perception in its food searching behavior, locust foraging behavior is likely to be more visually orientated. Furthermore, a feeding-dependent regulation of Schgr-sNPF transcript levels has previously been observed in the optic lobes of the locust brain, suggesting a possible involvement in visual perception of food and visual associative memory in this insect species. In this study, we describe the development of a robust and reproducible assay allowing visual associative memory to be studied in the desert locust, Schistocerca gregaria. Furthermore, we performed an exploratory series of experiments, studying the role of Schgr-sNPF in this complex process.

  14. Liquid chromatographic assay for dicloxacillin in plasma.

    Alderete, Oscar; González-Esquivel, Dinora F; Del Rivero, L Misael; Castro Torres, Nelly


    A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.

  15. Fluorometric enzymatic assay of L-arginine

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo


    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.


    Lilly, Laura E.; Blinebry, Sara K.; Viscardi, Chelsea M.; Perez, Luis; Bonaventura, Joe; McMahon, Tim J.


    Methods to systematically analyze in parallel the function of multiple protein or cell samples in vivo or ex vivo (i.e. functional proteomics) in a controlled gaseous environment have thus far been limited. Here we describe an apparatus and procedure that enables, for the first time, parallel assay of oxygen equilibria in multiple samples. Using this apparatus, numerous simultaneous oxygen equilibrium curves (OECs) can be obtained under truly identical conditions from blood cell samples or purified hemoglobins (Hbs). We suggest that the ability to obtain these parallel datasets under identical conditions can be of immense value, both to biomedical researchers and clinicians who wish to monitor blood health, and to physiologists studying non-human organisms and the effects of climate change on these organisms. Parallel monitoring techniques are essential in order to better understand the functions of critical cellular proteins. The procedure can be applied to human studies, wherein an OEC can be analyzed in light of an individual’s entire genome. Here, we analyzed intraerythrocytic Hb, a protein that operates at the organism’s environmental interface and then comes into close contact with virtually all of the organism’s cells. The apparatus is theoretically scalable, and establishes a functional proteomic screen that can be correlated with genomic information on the same individuals. This new method is expected to accelerate our general understanding of protein function, an increasingly challenging objective as advances in proteomic and genomic throughput outpace the ability to study proteins’ functional properties. PMID:23827235

  17. Microrheological Coagulation Assay Exploiting Micromechanical Resonators.

    Padovani, Francesco; Duffy, James; Hegner, Martin


    Rheological measurements in biological liquids yield insights into homeostasis and provide information on important molecular processes that affect fluidity. We present a fully automated cantilever-based method for highly precise and sensitive measurements of microliter sample volumes of human blood plasma coagulation (0.009 cP for viscosity range 0.5-3 cP and 0.0012 g/cm(3) for density range 0.9-1.1 g/cm(3)). Microcantilever arrays are driven by a piezoelectric element, and resonance frequencies and quality factors of sensors that change over time are evaluated. A highly accurate approximation of the hydrodynamic function is introduced that correlates resonance frequency and quality factor of cantilever beams immersed in a fluid to the viscosity and density of that fluid. The theoretical model was validated using glycerol reference solutions. We present a surface functionalization protocol that allows minimization of unspecific protein adsorption onto cantilevers. Adsorption leads to measurement distortions and incorrect estimation of the fluid parameters (viscosity and density). Two hydrophilic terminated self-assembled monolayers (SAMs) sensor surfaces are compared to a hydrophobic terminated SAM coating. As expected, the hydrophobic modified surfaces induced the highest mass adsorption and could promote conformational changes of the proteins and subsequent abnormal biological activity. Finally, the activated partial thromboplastin time (aPTT) coagulation assay was performed, and the viscosity, density, and coagulation rate of human blood plasma were measured along with the standard coagulation time. The method could extend and improve current coagulation testing.

  18. Fluorescence Polarization Assays in Small Molecule Screening

    Lea, Wendy A.; Simeonov, Anton


    Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies. PMID:22328899

  19. Background Assay and Rejection in DRIFT

    Brack, Jeff; Dorofeev, Alexei; Ezeribe, Anthony; Gauvreau, Jean-Luc; Gold, Michael; Harton, John; Lafler, Randy; Lauer, Robert; Lee, Eric R; Loomba, Dinesh; Matthews, John; Miller, Eric H; Monte, Alissa; Murphy, Alex; Paling, Sean; Phan, Nguyen; Sadler, Steve; Scarff, Andrew; Snowden-Ifft, Daniel; Spooner, Neil; Telfer, Sam; Walker, Daniel; Williams, Matt; Yuriev, Leonid


    The DRIFT-IId dark matter detector is a m$^3$-scale low-pressure TPC with directional sensitivity to WIMP-induced nuclear recoils. Its primary backgrounds were due to alpha decays from contamination on the central cathode. Efforts to reduce these backgrounds led to replacing the 20 \\mu m wire central cathode with one constructed from 0.9 \\mu m aluminized mylar, which is almost totally transparent to alpha particles. Detailed modeling of the nature and origin of the remaining backgrounds led to an in-situ, ppt-sensitive assay of alpha decay backgrounds from the central cathode. This led to further improvements in the thin-film cathode resulting in over 2 orders of magnitude reduction in backgrounds compared to the wire cathode. Finally, the addition of O$_2$ to CS$_2$ gas was found to produce multiple species of electronegative charge carriers, providing a method to determine the absolute position of nuclear recoils and reject all known remaining backgrounds while retaining a high efficiency for nuclear recoil...

  20. Mouse lung adhesion assay for Bordetella pertussis

    Burns, K.A.; Freer, J.H. (Department of Microbiology, Alexander Stone Building, Bearsden, Glasgow, Scotland)


    The ability of Bordetella pertussis to adhere to cell surfaces has been demonstrated by adhesion to tissue culture cells and adhesion to chicken, hamster or rabbit trachea in organ culture. In this report a mouse lung assay for adhesion is described and the results obtained using two virulent strains of B. pertussis and their avirulent counterparts. These were a C modulation of one of the original virulent strains and a phase IV variant of the other virulent strain. Organisms were radiolabelled by adding 1 (37 K Bq) of (/sup 14/C)glutamic acid per 10 ml of culture medium before inoculation and incubation for 5 days. The lungs were washed by perfusion in situ with at least two volumes (1 ml) of sterile 1% (w/v) casamino acids. The percentage of the inoculated organisms retained in the lungs was determined, after removal of the lungs, by one of the following two methods: viable count or radioactive count. Results for both methods were expressed as the percentage of the inoculum retained in the lungs plus or minus one standard deviation.

  1. Rho family and Rap GTPase activation assays.

    Jennings, Richard T; Knaus, Ulla G


    The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione S-transferase (GST). Such domains bind preferentially to the GTP-bound form of the upstream Rho or Ras GTPase. Coupling these probes to beads enables extraction of the complex and subsequent quantification of the active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for every small GTPase, analysis of certain members of the Rho and Ras GTPase family is now routinely performed. Here, we describe affinity-based pulldown assays for detection of Rho GTPase (Rac1/2, Cdc42, RhoA/B) and Rap1/2 activity in stimulated neutrophils or macrophages.

  2. Establishment of indirect immunofluorescence assay for rotavirus.

    Tao, J; Zhang, J; Liu, X; Jin, H; Jiang, C; Yin, Y


    Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a significant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most effective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofluorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. The activating concentration of trypsin was 1 µg/ml for healthy monolayers of MA104 cells at 100% confluence. After incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fluorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID50-ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certification of rotavirus vaccine production.

  3. Effect of vanadium on insulin and leptin in Zucker diabetic fatty rats.

    Wang, J; Yuen, V G; McNeill, J H


    Vanadium exhibits a variety of insulin-mimetic actions in vitro and in vivo. The mechanism(s) of the effect of vanadium on leptin in Zucker diabetic fatty (ZDF) rats, a model of Type 2 diabetes, is unclear. Since insulin is a stimulator of leptin production and secretion and vanadium is an insulin-mimetic or insulin-enhancing agent, we studied how vanadium affected plasma leptin levels in vivo and the relationship between plasma insulin, leptin and body fat in ZDF rats. Zucker lean and ZDF rats at 9-week old were chronically treated with bis(ethylmaltolato)oxovanadium(IV) (BEOV), an organic vanadium compound, by oral gavage daily for 3 weeks. At termination, the total body fat was weighed and blood was collected for insulin, leptin and glucose assay. BEOV treatment (0.1 mmol/kg/day) significantly decreased plasma glucose levels in ZDF rats and did not change food intake and body fat content either in lean or ZDF rats. Following 3-week treatment, plasma insulin and leptin levels in BEOV treated ZDF rats were significantly higher, 1.5 and 0.5 fold than untreated rats, respectively. The correlation coefficients in ZDF rats showed that plasma leptin levels were correlated to plasma insulin levels, but not to body fat. These data indicate that plasma leptin levels parallel plasma insulin levels, and the effects of vanadium on leptin appear to be mediated by insulin in ZDF rats.

  4. Effect of diabetes and insulin treatment on nitric oxide synthase content in rat corpus cavemosum

    Zhi-Shun XU; Qiang FU; Sheng-Tian ZHAO; Hai-Nan LIU


    Aim: To study the effect of diabetes mellitus and insulin treatment on rat penile nitric oxide synthase content.Methods: Male Wistar rats were divided at random into two groups: the Control ( n = 8) and the Diabetic ( n =17). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. The diabetic animals were then ran domly divided into two subgroups: diabetic rats without insulin treatment ( n = 7) and diabetic rats with insulin treat ment ( n = 10). The neuronal nitric oxide synthase (nNOS) in the penile corpus cavemosum were assayed by immrmo histochemical staining with specific antibody to nNOS and the nNOS-positive nerve fibers were counted semiquantita tively under a high power microscope. Results: The nNOS- positive nerve fibres in diabetic rats with treatment was higher than that in diabetic rats without treatment ( P < 0.05) and lower than that in the controls ( P < 0.01 ). The nNOS-positive nerve fibres in diabetic rat without treatment were also lower than that in the controls ( P < 0.01). Con clusion: In streptozotocin-induced diabetic rats, the nNOS content in the penile corpus cavernosum was significantly decre~ed. Insulin treatment at the dose level employed partially restores the penile nNOS content in these rats.

  5. Analgesic Activity of Tramadol and Buprenorphine after Voluntary Ingestion by Rats (Rattus norvegicus).

    Taylor, Bryan F; Ramirez, Harvey E; Battles, August H; Andrutis, Karl A; Neubert, John K


    Effective pain management for rats and mice is crucial due to the continuing increase in the use of these species in biomedical research. Here we used a recently validated operant orofacial pain assay to determine dose-response curves for buprenorphine and tramadol when mixed in nut paste and administered to male and female rats. Statistically significant analgesic doses of tramadol in nut paste included doses of 20, 30, and 40 mg/kg for female rats but only 40 mg/kg for male rats. For male rats receiving buprenorphine mixed in nut paste, a significant analgesic response was observed at 0.5 and 0.6 mg/kg. None of the doses tested produced a significant analgesic response in female rats. Our results indicate that at the doses tested, tramadol and buprenorphine produced an analgesic response in male rats. In female rats, tramadol shows a higher analgesic effect than buprenorphine. The analgesic effects observed 60 min after administration of the statistically significant oral doses of both drugs were similar to the analgesic effects of 0.03 mg/kg subcutaneous buprenorphine 30 min after administration. The method of voluntary ingestion could be effective, is easy to use, and would minimize stress to the rats during the immediate postoperative period.

  6. Evaluation of the immunotoxicity of low-level PCB (polychlorinated biphenyl) exposure in the rat

    Smialowicz, R.J.; Andrews, J.E.; Riddle, M.M.; Rogers, R.R.; Luebke, R.W.


    Weanling male Fischer 344 rats were exposed daily by gastric intubation for up to 15 weeks to the polychlorinated biphenyl (PCB) Aroclor 1254 at 0.1, 1, 10, or 25 mg/kg body weight. At 5, 10 and 15 weeks groups of rats were killed and immune functions were evaluated. The immune parameters examined included the following: body and lymphoid organ weights, mitogen stimulated lymphoproliferative (LP) responses, natural killer (NK) cell activity, mixed lymphocyte reaction (MLR), and cytotoxic T lymphocyte (CTL) response. After 10 and 15 weeks of dosing body weights were reduced in rats receiving 25 mg/kg PCB while thymus weights were decreased in rats receiving 10 and 25 mg/kg. NK cell activity was reduced in rats dosed for 15 weeks at 10 and 25 mg/kg. The LP response to phytohemagglutinin was enhanced in rats dosed for 15 weeks at 25 mg/kg PCB. Exposure of rats to PCB did not affect the MLR or CTL responses. Other groups of rats were exposed to cyclophosphamide (CY) and served as positive controls for the immune assays employed. CY induced alterations in all of the immune parameters measured, indicating that this is a sensitive battery of immune function tests which is capable of detecting immune alterations in the rat.

  7. Antenatal taurine reduces cerebral cell apoptosis in fetal rats with intrauterine growth restriction*

    Jing Liu; Xiaofeng Wang; Ying Liu; Na Yang; Jing Xu; Xiaotun Ren


    From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12th day of pregnancy, 300 mg/kg rine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neo-natal rats at 6 hours after delivery, neonatal rats with intrauterine growth restriction, and neonatal rats with intrauterine growth restriction undergoing taurine supplement were obtained for further experiments. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling assay revealed that the number of apoptotic cel s in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cel apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. nohistochemical staining revealed that taurine supplement increased glial cel line-derived neuro-trophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cel apoptosis through the glial cel line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.

  8. Location and Quantification of iNOS in Testis of Dahl Hypertensive Rats

    Jing LIU; Rui-li LI; Huan-Ying ZHAO; Xiu-Ling YIN


    Objective To study the effect of hypertension on inducible isoform of nitrogen oxide syntheses (iNOS) expression and reproductive function in testes of Dahl hypertensive rats Method The iNOS expression in Dahl rat testes was localized and assayed semi-quan titatively by immunohistochemistry.Results iNOS was expressed and localized predominantly in the cytoplasm of Sertoli and Leydig cells in both normal and hypertensive rats. However, in the early stage of hypertension, the expression of iNOS was stronger in testes than that of the normal rats (P<0. 05). With the development, the staining intensity of iNOS decreased gradually in the late stage. Moreover, the level of testosterone decreased with the increase of blood pressure. But in vitro, there was no difference in the expression of iNOS between cultured Sertoli cells from normal rats and hypertensive rats.Conclusion High-salt food induced hypertension in Dahl rats, which was characterized by the high expression of iNOS in rat Sertoli and Leydig cells; excessive NO produced by iNOS reduced the level of testosterone in testicle artery, and may thus affect the reproductive function of rats testis.

  9. Pavlovian Conditioning of Rat Mucosal Mast Cells to Secrete Rat Mast Cell Protease II

    MacQueen, Glenda; Marshall, Jean; Perdue, Mary; Siegel, Shepard; Bienenstock, John


    Antigen (egg albumin) injections, which stimulate mucosal mast cells to secrete mediators, were paired with an audiovisual cue. After reexposure to the audiovisual cue, a mediator (rat mast cell protease II) was measured with a sensitive and specific assay. Animals reexposed to only the audiovisual cue released a quantity of protease not significantly different from animals reexposed to both the cue and the antigen; these groups released significantly more protease than animals that had received the cue and antigen in a noncontingent manner. The results support a role for the central nervous system as a functional effector of mast cell function in the allergic state.

  10. Molecular cloning and pharmacological characterization of rat melatonin MT1 and MT2 receptors.

    Audinot, Valérie; Bonnaud, Anne; Grandcolas, Line; Rodriguez, Marianne; Nagel, Nadine; Galizzi, Jean-Pierre; Balik, Ales; Messager, Sophie; Hazlerigg, David G; Barrett, Perry; Delagrange, Philippe; Boutin, Jean A


    In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT(1) receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT(2) receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT(2) receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT(1) and rMT(2). A 55% homology was observed between both rat isoforms. The entire contiguous rat MT(1) and MT(2) receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[(125)I]-Iodomelatonin. The dissociation constants (K(d)) for rMT(1) and rMT(2) were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT(1) and MT(2) receptors were evaluated at rMT(1) and rMT(2) receptors, for their binding affinity and functionality in [(35)S]-GTPgammaS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT(1) subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models.

  11. Increased angiogenesis in portal hypertensive rats: role of nitric oxide.

    Sumanovski, L T; Battegay, E; Stumm, M; van der Kooij, M; Sieber, C C


    Systemic and especially splanchnic arterial vasodilation accompany chronic portal hypertension. Different soluble mediators causing this vasodilation have been proposed, the strongest evidence being for nitric oxide (NO). No data exist if structural vascular changes may partly account for this vasodilatory state. Here, we developed a new in vivo quantitative angiogenesis assay in the abdominal cavity and determined if: 1) portal hypertensive rats show increased angiogenesis; and 2) angiogenesis is altered by inhibiting NO formation. Portal hypertension was induced by partial portal vein ligation (PVL). Sham-operated rats served as controls (CON). During the index operation (day 0), a teflon ring filled with collagen I (Vitrogen 100) was sutured in the mesenteric cavity. After 16 days, rings were explanted, embedded in paraffin, and ingrown vessels counted using a morphometry system. The role of NO was tested by adding an antagonist of NO formation (Nomega-nitro-L-arginine [NNA], 3.3 mg/kg/d) into the drinking water. The mean number of ingrown vessels per implant was significantly higher in PVL rats compared with CON rats, i.e., 1,453 +/- 187 versus 888 +/- 116, respectively (P <.05; N = 5 per group). NNA significantly (P <.01) inhibited angiogenesis in PVL (202 +/- 124; N = 5) and in CON (174 +/- 25; N = 6) rats, respectively. In contrast, the beta-adrenergic blocker, propranolol, did not prevent angiogenesis either in PVL or CON rats in a separate set of experiments (data not shown). The conclusions drawn from this study are that: 1) rats with portal hypertension show increased angiogenesis; and 2) inhibition of NO formation significantly prevents angiogenesis in both PVL and CON rats. Therefore, splanchnic vasodilation in chronic portal hypertension may also be a result of structural changes.

  12. Piracetam and vinpocetine ameliorate rotenone-induced Parkinsonism in rats.

    Zaitone, Sawsan A; Abo-Elmatty, Dina M; Elshazly, Shimaa M


    To evaluate the neuroprotective effect of the nootropic drugs, piracetam (PIR) and vinpocetine (VIN), in rotenone-induced Parkinsonism in rats. Sixty male rats were divided into 6 groups of 10 rats each. The groups were administered vehicle, control (rotenone, 1.5 mg/kg/48 h/6 doses, s.c.), PIR (100 and 200 mg/kg/day, p.o.) and VIN (3 and 6 mg/kg/day, p.o.). The motor performance of the rats was evaluated by the open field and pole test. Striatal dopamine level, malondialdehyde (MDA), reduced glutathione (GSH) and tumor necrosis factor-α (TNF-α) were assayed. Histopathological study of the substantia nigra was also done. Results showed that rotenone-treated rats exhibited bradykinesia and motor impairment in the open-field test. In addition, GSH level was decreased whereas MDA and TNF-α increased in striata of rotenone-treated rats as compared to vehicle-treated rats. Marked degeneration of the substantia nigra pars compacta (SNpc) neurons and depletion of striatal dopamine was also observed in the rotenone-treated rats. Treatment with PIR or VIN significantly reversed the locomotor deficits and increased striatal dopamine level. Treatment with VIN significantly (P<0.05) reduced the striatal level of MDA and GSH in comparison to rotenone group whereas TNF-α production was found to be significantly decreased in PIR group (P<0.05). VIN and PIR exhibit neuroprotective activity in rotenone-induced Parkinsonism. Hence, these nootropic agents may be considered as possible candidates in the treatment of Parkinson's disease.

  13. Enzyme-linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S)

    Leeming, Diana J; Nielsen, Mette J; Dai, Yueqin


    Aim:  The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100...... were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P collagen...... expression in BDL rats (r = 0.49, P collagen in CCL4 treated livers (P 

  14. Rat Bite Fever

    ... Español Text Size Email Print Share Rat Bite Fever Page Content Article Body Rat-bite fever is a disease that occurs in humans who ... ingestion of contaminated food or milk products (Haverhill fever). Most cases in the United States are caused ...

  15. SWEEP Project RAT

    Ibsen, Lars Bo; Madsen, Søren; Petersen, L. B.

    This report presents the results from the design analyses made for the clustered suction caisson used as foundation for a Riser Access Tower (RAT). The RAT is intended built next to the K15-FA-1 Platform in the Dutch Sector of the North Sea....

  16. A comprehensive company database analysis of biological assay variability.

    Kramer, Christian; Dahl, Göran; Tyrchan, Christian; Ulander, Johan


    Analysis of data from various compounds measured in diverse biological assays is a central part of drug discovery research projects. However, no systematic overview of the variability in biological assays has been published and judgments on assay quality and robustness of data are often based on personal belief and experience within the drug discovery community. To address this we performed a reproducibility analysis of all biological assays at AstraZeneca between 2005 and 2014. We found an average experimental uncertainty of less than a twofold difference and no technologies or assay types had higher variability than others. This work suggests that robust data can be obtained from the most commonly applied biological assays.

  17. Optimization of cell motility evaluation in scratch assay

    Gotsulyak N. Ya.


    Full Text Available A scratch test is one of the most popular methods of classical cell migration assay in a monolayer culture. At the same time, the scratch assay has some disadvantages that can be easily corrected. Aim. Optimization the existent scratch assay on the base of detection of scratch wound surface area and the length of the field of observation which is more objective and less time consuming. Methods. Scratch assay. Results. The modification of scratch assay enables to perform measurement more accurately and rapidly. This approach is more simple and eliminates the main disadvantages of the classical method. Conclusions. The procedure of scratch wound width measurement calculated on the base of detection of cell free area and the length of the field of observation is more effective than the classical wound healing assay. It will be useful for the estimation of cell migration dynamics in monolayer culture for a wide range of live cell based experiments.

  18. Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay



    Full Text Available Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The applications of this assay suggest that it is a useful assay to assess aquatic genotoxicants. However, there are some factors, which should be taken into account when using this assay as aquatic ecotoxicological assessment device such as inter-animal and cell variability.

  19. Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis

    Masson Véronique


    Full Text Available Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix. To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or screen "pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer.

  20. Liposomal butamben gel formulations: toxicity assays and topical anesthesia in an animal model.

    Cereda, Cintia Maria Saia; Guilherme, Viviane Aparecida; Alkschbirs, Melissa Inger; de Brito Junior, Rui Barbosa; Tofoli, Giovana Radomille; Franz-Montan, Michelle; de Araujo, Daniele Ribeiro; de Paula, Eneida


    The aim of this study was to evaluate the in vitro cytotoxicity and the in vivo analgesic effect and local toxicity of the local anesthetic butamben (BTB) encapsulated in conventional or elastic liposomes incorporated in gel formulations. The results showed that both gel formulations of liposomal BTB reduced the cytotoxicity (p < 0.001; one-way ANOVA/Tukey's test) and increased the topical analgesic effect (p < 0.05; one-way ANOVA/Tukey's test) of butamben, compared to plain BTB gel. The gel formulations presented good rheological properties, and stability assays detected no differences in physicochemical stability up to 30 d after preparation. Moreover, histological assessment revealed no morphological changes in rat skin after application of any of the gel formulations tested.

  1. Evaluation of beta-naphthoxyacetic acid for mutagenic activity in the Salmonella/mammalian microsome assay.

    Rashid, K A; Mumma, R O


    Beta-naphthoxyacetic acid (BNOA) is used as a plant growth regulator on tomatoes and strawberries. It is the active ingredient in Blossom-Set and Berry-Set, two plant hormone sprays for fruit-set. The mutagenic activity of BNOA was evaluated in four strains of Salmonella typhimurium (TA97, TA98, TA100 and TA1535) in the presence and absence of liver microsomal and cytosolic enzymes derived from Aroclor induced rats. BNOA did not produce any significant increase (p less than 0.05) in the reversion of any of the four tester strains in the standard plate incorporation assay. Results of the agar overlay toxicity tests indicates that the chemical shows toxic effects at concentrations above 500 micrograms/plate. It was concluded that under the conditions of these tests, BNOA did not exhibit any mutagenic activity.

  2. In vitro and in vivo evaluation of the estrogenic, androgenic, and progestagenic potential of two cyclic siloxanes.

    Quinn, Anne L; Regan, Jane M; Tobin, Joseph M; Marinik, Brian J; McMahon, Joan M; McNett, Debra A; Sushynski, Christopher M; Crofoot, Steven D; Jean, Paul A; Plotzke, Kathleen P


    The purpose of these experiments was to determine the potential estrogenic, androgenic, and progestagenic activity of two cyclic siloxanes, octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5). Receptor-binding experiments and a luciferase reporter gene assay were used to determine if the materials were able to bind and activate either the estrogen receptors (ERs) or progesterone receptors (PRs)-alpha or beta. The rat uterotrophic assay (RUA) for estrogenic activity and the Hershberger assay for androgenic activity were utilized as the in vivo assays. For the ER-binding studies, D4 was shown to bind to ERalpha but not to ERbeta. D5 did not bind to either of the two receptors. D4 activated the reporter gene at 10 microM, while D5 was considered negative in the estrogen reporter gene assay. Neither material was a ligand for the PRs. Both the RUA and Hershberger assays were conducted using whole-body inhalation of the two materials for 16 h/day. D4 resulted in a small but significant increase in both wet and blotted uterine weight as well as increases in both luminal and glandular epithelial cell height in both Sprague Dawley and Fischer 344 rats. D5 was negative in both rat strains, indicating that D5 does not possess estrogenic activity. Neither material possessed any significant antiestrogenic activity. Both materials were negative in the Hershberger assay indicating that neither material possesses any significant androgenic activity. Our studies have shown that D4 exhibits a low affinity for ERalpha in vitro and a weakly estrogenic response in vivo.

  3. Establishment of an Enzyme Linked Immunosorbent Assay for Neonatal Thyrotropin


    A sensitive and specific ELISA for neonatal thyrotropin(Neonatal TSH) is established with using twoanti-TSH monoclonal antibody. One of them is coated on the microtiter plate, the other is conjugate ofbiotin. The label is horseradish peroxidase(HRP) conjugate of streptavidin. TMB-H2O2 solution is used asthe substrate of HRP.The sensitivity of the assay is 0.5 mIU/L, the intra-assay CVs and the intre-assay

  4. Validation of Laboratory-Developed Molecular Assays for Infectious Diseases

    Burd, Eileen M.


    Summary: Molecular technology has changed the way that clinical laboratories diagnose and manage many infectious diseases. Excellent sensitivity, specificity, and speed have made molecular assays an attractive alternative to culture or enzyme immunoassay methods. Many molecular assays are commercially available and FDA approved. Others, especially those that test for less common analytes, are often laboratory developed. Laboratories also often modify FDA-approved assays to include different e...

  5. An ultrasensitive rapid immunocytotoxicity assay for detecting Clostridium difficile toxins

    He, Xiangyun; Wang, Jufang; Steele, Jennifer; Sun, Xingmin; Nie, Weijia; Tzipori, Saul; Feng, Hanping


    We describe a novel ultrasensitive cell-based immunocytotoxicity assay for detecting less then 1 pg/ml of Clostridium difficile toxins in porcine clinical samples. The assay is simple to perform with a turnaround time of approximately 3 hours and capable of detecting less then 1 pg/ml of toxin A. Using this assay, we were able to detect the presence of C. difficile toxins in the fecal and serum specimens of experimentally infected piglets. PMID:19393695

  6. The Spheroplast Lysis Assay for Yeast in Microtiter Plate Format

    Ovalle, Rafael; Spencer, Moyah; Thiwanont, Monthiwa; Lipke, Peter N.


    A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes. PMID:10427014




    Full Text Available Three new, simple, and cost-effective visible spectrophotometric methods are proposed for determination of gatifloxacin (GTF using bromate-bromide mixture, and three dyes, methyl orange, indigocarmine and thymol blue, as reagents.The methods engross the addition of a known excess of bromate-bromide mixture to GTF in hydrochloric acid medium followed by determination of residual bromine by reacting with a fixed amount of either methyl orange andmeasuring the absorbance at 520 nm (method A or indigo carmine and measuring the absorbance at 610 nm (method B or thymol blue and measuring the absorbance at 550 nm (method C. In all the methods, the amount of brominereacted corresponds to the amount of GTF, and the absorbance is found to increase linearly with the concentration of GTF. Under the optimum conditions, GTF could be assayed in the concentration range 0.25-1.5, 0.5-6.0, and 0.5-10μg/mL by method A, method B and method C, respectively. The apparent molar absorptivities are calculated to be 1.6x105, 4.0x104 and 3.2x104 L mol-1 cm-1 for the method A, method B and method C, respectively, and the corresponding Sandell sensitivity values are 0.0025, 0.010 and 0.012 μg/cm2. The intra-day and inter-day precision, and the accuracy of the methods were evaluated as per the current ICH guidelines. The methods were successfully applied to the determination of GTF in pharmaceutical preparations without the interference from any of the pharmaceutical adjuvants.

  8. Inferring Protein Associations Using Protein Pulldown Assays

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.


    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  9. Comparative investigation of various cellulase assay procedures

    Canevascini, G.; Gattlen, C.


    The cellulolytic activity of crude enzyme preparations from different cellulolytic fungi (namely Trichoderma viride, Trichoderma koningii, Fusarium solani, Sporotrichum pulverulentum, Sporotrichum thermophile) was assayed comparatively with several common analytical procedures described in the literature. The investigation was carried out with the objective of evaluating, with raw culture filtrates, the different cellulase tests in relation to their specificity for endo- and exo-cellulase action as well as to allow comparisons to be made between results from different research groups using different methods. 1) Cellulase activity was tested viscometrically as well as chemically (determination of reducing end groups) with different carboxymethylcelluloses as substrates. Essentially constant ratios between both kinds of activities were obtained, indicating that they are directly related. By estimating cellulase activity with insoluble cellulosic substrates no direct relationship could be established with the above-described activities except in the case where the cellulose was amorphous. The ratio profile between activities thus obtained and endo-cellulase activities determined viscometrically shows that some enzyme preparations (such as those from both Trichoderma sp.) are clearly more active than others against crystalline cellulose reflecting quantitative differences in enzyme composition. Nevertheless, for a biological understanding of cellulolysis, analytical procedures using crystalline celluloses are not adequate for specifically monitoring exo-cellulase activity in crude enzyme solutions for essentially two reasons: a) they are not sufficiently sensitive to detect small changes in enzyme activity during the early phase of growth, and b) exo-cellulase activity in crude enzyme solutions also depends on the endo-cellulase activity present. (Refs. 39).

  10. Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

    Takasawa, Hironao; Takashima, Rie; Narumi, Kazunori; Kawasako, Kazufumi; Hattori, Akiko; Kawabata, Masayoshi; Hamada, Shuichi


    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone.

  11. Robust versatile tyrosine kinase assay for HTS in drug discovery

    Deshpande, Sudhir S.; Mineyev, I.; Owicki, John C.


    A fluorescence polarization assay was developed as an alternative to the radiolabeled SPA assays currently used to monitor the activity of tyrosine kinases in drug discovery. The assay can be used with enzymes having substrate specificity similar to that of the insulin receptor, the EGF receptor and the Src kinase receptor enzymes. The assay is easy to configure in 96, 384 and 1536-well microplates in assay volumes ranging from (mu) L with minimal efforts. The reconstituted reagents are stable for up to 24 hr at ambient temperatures, thereby minimizing the need for replenishing the stock solutions during the course of a high-throughput screen. Because of the stability and equilibrium kinetics, the assay allows the user the luxury of scheduling the reading of plates any time up to 24 hr after the completion of the assay without substantial deterioration in the assay signal. The antibody and the tracer solutions can also be premixed and added as a preformed complex in a single step. The performance of the assay with the insulin receptor kinase is described. In addition, given the diversity of the substrates used in measuring the activity of different tyrosine kinases, LJL's on-going efforts to provide different antibodies of wide ranging specificity and sensitivity are described.

  12. Click Chemistry-Mediated Nanosensors for Biochemical Assays

    Chen, Yiping; Xianyu, Yunlei; Wu, Jing; Yin, Binfeng; Jiang, Xingyu


    Click chemistry combined with functional nanoparticles have drawn increasing attention in biochemical assays because they are promising in developing biosensors with effective signal transformation...

  13. A Multi-detection Assay for Malaria Transmitting Mosquitoes

    Lee, Yoosook; Weakley, Allison M.; Nieman, Catelyn C.; Malvick, Julia; Lanzaro, Gregory C.


    The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays. PMID:25867057

  14. Intradermal injection of Botulinum toxin type A alleviates infraorbital nerve constriction-induced thermal hyperalgesia in an operant assay.

    Kumada, A; Matsuka, Y; Spigelman, I; Maruhama, K; Yamamoto, Y; Neubert, J K; Nolan, T A; Watanabe, K; Maekawa, K; Kamioka, H; Yamashiro, T; Kuboki, T; Oguma, K


    Recent studies have shown that infraorbital nerve constriction (IoNC)-induced mechanical allodynia has been attenuated by administration of highly purified 150-kDa Botulinum neurotoxin type A (BoNT/A). Here, we extend these studies to determine whether BoNT/A could attenuate IoNC-induced symptoms of thermal hyperalgesia. Instead of testing head withdrawal thresholds, a thermal operant assay was used to evaluate cortical processing of sensory input following IoNC. In this assay, a fasted rat's desire to obtain a food reward (sweetened condensed milk) is coupled to its ability to tolerate facial contact with a warm (45 °C) thermode. Bilateral IoNC decreased the ratio of thermode contact duration/event, which is an indicative of thermal hyperalgesia. BoNT/A injection intradermally in the area of infraorbital nerve (IoN) innervation 7 days after IoNC resulted in decreased number of facial contacts and increased the ratio of contact duration/event (measured at 14 days after IoNC). The BoNT/A (2-200 pg) effects were dose dependent and statistically significant at 100 and 200 pg (P thermal hyperalgesia symptoms was obtained with a 200-pg dose, without affecting sham rat behaviour. Off-site (neck) injection of BoNT/A did not relieve thermal hyperalgesia, while co-injection of BoNT/A with a neutralising antibody in the area of IoN innervation prevented relief of thermal hyperalgesia. Neither IoNC nor BoNT/A injection affected operant assay parameters with a 24 °C thermode, indicating selectivity of thermal hyperalgesia measurements. These results strongly suggest that intradermal injection of BoNT/A in the area of IoN innervation alleviates IoNC-induced thermal hyperalgesia in an operant assay.

  15. A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

    M.F. Alves


    Full Text Available A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(DnpP-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl was optimized for the measurement of angiotensin I-converting enzyme (ACE in human plasma and rat tissues. Abz-FRK(DnpP-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(DnpP-OH correlated closely (r = 0.90, P < 0.001. The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(DnpP-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

  16. A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.

    Kubota, Kazuyuki; Mizukoshi, Toshimi; Miyano, Hiroshi


    Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 μM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.

  17. Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in Wistar rats

    Ven, van der L.T.M.; Kuil, van de T.; Leonards, P.E.G.; Slob, W.; Lilienthal, H.; Litens, S.; Herlin, M.; Hakansson, H.; Canton, R.F.; Berg, M.; Visser, T.J.; Loveren, van H.; Vos, J.G.; Piersma, A.H.


    The brominated flame retardant (BFR) hexabromocyclododecane was tested in a one-generation reproduction assay in Wistar rats, enhanced for endocrine parameters. A solution of the compound in corn oil was mixed in the feed, targeting at dietary exposure of 0-0.1-0.3-1-3-10-30-100 mg/kg body weight/da

  18. Endocrine effects of hexabromocyclododecane (HBCD) in a one-generation reproduction study in Wistar rats.

    van der Ven, L.T.; van de Kuil, T.; Leonards, P.E.; Slob, W.; Lilienthal, H.; Litens, S.; Herlin, M.; Hakansson, H.; Canton, R.F.; van den Berg, M.; Visser, T.J.; van Loveren, H.; Vos, J.G.; Piersma, A.H.


    The brominated flame retardant (BFR) hexabromocyclododecane was tested in a one-generation reproduction assay in Wistar rats, enhanced for endocrine parameters. A solution of the compound in corn oil was mixed in the feed, targeting at dietary exposure of 0-0.1-0.3-1-3-10-30-100 mg/kg body weight/da

  19. Occurrence and partition of the β-carboline norharman in rat organs

    D. Fekkes (Durk); W.T. Bode (Willem)


    textabstractThe β-carboline norharman was determined in plasma, brain, liver, kidney, spleen, heart and lung of the rat using HPLC with fluorescence detection. In order to improve the speed and sensitivity of this assay an earlier published sample clean-up extraction procedure and HPLC method were a




    The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on-line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluorimetric detection, was set up. W