共查询到20条相似文献,搜索用时 46 毫秒
1.
Katsuyoshi Horibata Akiko Ukai Takafumi Kimoto Tetsuya Suzuki Nagisa Kamoshita Kenichi Masumura Takehiko Nohmi Masamitsu Honma 《Environmental and molecular mutagenesis》2013,54(9):747-754
The recently developed Pig‐a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor‐deficient red blood cells caused by a forward mutation in the Pig‐a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig‐a assay could be integrated into repeat‐dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig‐a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N‐ethyl‐N‐nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4‐nitroquinoline‐1‐oxide (4NQO, 50 mg/kg) in the Pig‐a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig‐a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7‐week terminal sacrifice. ENU increased both Pig‐a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig‐a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two‐ or threefold above control) were detected in the bone marrow in both the Pig‐a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig‐a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
2.
Shin Ohtani Aiko Unno Akira Ushiyama Takafumi Kimoto Daishiro Miura Naoki Kunugita 《Environmental and molecular mutagenesis》2012,53(8):579-588
The in vivo Pig‐a mutation assay has been adapted for measuring mutation in rats, mice, monkeys, and humans. To date, the assay has been used mainly to assess the mutagenicity of chemicals that are known to be powerful point mutagens. The assay has not been used to measure the biological effects associated with ionizing radiation. In this study, we modified the Pig‐a gene mutation assay (Kimoto et al. [2011b]: Mutat Res 723:36‐42) and used 3‐color staining with fluorescently labeled anti‐CD24, anti‐TER‐119, and anti‐CD71 to detect the Pig‐a mutant frequencies in total red blood cells (RBCs) and in reticulocytes (RETs) from X‐irradiated mice. Single exposures to X‐irradiation resulted in dose‐ and time‐dependent increases in Pig‐a mutant frequencies, and these subsequently declined over time returning to background frequencies. The same total amount of radiation, delivered either as a single dose or as four repeat doses at weekly intervals, increased Pig‐a mutant frequencies to comparable levels, reaching maxima 2–3 weeks after the single dose or 2–3 weeks after the last of the repeat doses. These increased frequencies subsequently returned to background levels. Our results indicated that the 3‐color Pig‐a assay was useful for evaluating the in vivo genotoxicity of radiation. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc. 相似文献
3.
Yiying Wang Javier Revollo Page McKinzie Mason G. Pearce Azra Dad Berran Yucesoy Hans Rosenfeldt Robert H. Heflich Vasily N. Dobrovolsky 《Environmental and molecular mutagenesis》2018,59(1):4-17
The X‐linked Pig‐a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig‐a mutant cells fail to synthesize GPI and to express GPI‐anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig‐a mutation in various in vivo assays. Here, we describe an in vitro Pig‐a mutation assay in L5178YTk+/– mouse lymphoma cells, in which mutant‐phenotype cells are measured by flow cytometry using a fluorescent anti‐CD90 antibody. Increased frequencies of CD90‐deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N‐ethyl‐N‐nitrosourea (ENU), ethyl methanesulphonate, and 7,12‐dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting‐dilution cloning assay using proaerolysin selection. Individual CD90‐deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig‐a gene. Pig‐a mutations with agent‐specific signatures were found in nearly all clones that developed from sorted CD90‐deficient cells. These results indicate that a Pig‐a mutation assay can be successfully conducted in L5178YTk+/? cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig‐a assays. Environ. Mol. Mutagen. 59:4–17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA. 相似文献
4.
Applying the erythrocyte Pig‐a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The Pig‐a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)‐anchored cell surface markers. We hypothesized that the erythrocyte Pig‐a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI‐anchored CD59 as the Pig‐a mutation marker and examined the frequency of CD59‐negative sperm using flow cytometry. A reconstruction experiment that spiked un‐labeled sperm (mutant–mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant–mimic sperm, demonstrating the analytical ability for CD59‐negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N‐ethyl‐N‐nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid‐lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose‐dependent increase in the frequency of CD59‐negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59‐negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment‐related changes were detected in the frequency of CD59‐negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59‐negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig‐a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485–493, 2017. © 2017 Wiley Periodicals, Inc. 相似文献
5.
Vasily N. Dobrovolsky Javier Revollo Mason G. Pearce M. Monserrat Pacheco‐Martinez Haixia Lin 《Environmental and molecular mutagenesis》2015,56(8):674-683
A major question concerning the scientific and regulatory acceptance of the rodent red blood cell‐based Pig‐a gene mutation assay is the extent to which mutants identified by their phenotype in the assay are caused by mutations in the Pig‐a gene. In this study, we identified T‐lymphocytes deficient for the glycosylphosphatidylinositol‐anchored surface marker, CD48, in control and 7,12‐dimethylbenz[a]anthracene (DMBA)‐treated rats using a flow cytometric assay and determined the spectra of mutations in the endogenous Pig‐a gene in these cells. CD48‐deficient T‐cells were seeded by sorting at one cell per well into 96‐well plates, expanded into clones, and exons of their genomic Pig‐a were sequenced. The majority (78%) of CD48‐deficient T‐cell clones from DMBA‐treated rats had mutations in the Pig‐a gene. The spectrum of DMBA‐induced Pig‐a mutations was dominated by mutations at A:T, with the mutated A being on the nontranscribed strand and A→T transversion being the most frequent change. The spectrum of Pig‐a mutations in DMBA‐treated rats was different from the spectrum of Pig‐a mutations in N‐ethyl‐N‐nitrosourea (ENU)‐treated rats, but similar to the spectrum of DMBA mutations for another endogenous X‐linked gene, Hprt. Only 15% of CD48‐deficient mutants from control animals contained Pig‐a mutations; T‐cell biology may be responsible for a relatively large fraction of false Pig‐a mutant lymphocytes in control animals. Among the verified mutants from control rats, the most common were frameshifts and deletions. The differences in the spectra of spontaneous, DMBA‐, and ENU‐induced Pig‐a mutations suggest that the flow cytometric Pig‐a assay detects de novo mutation in the endogenous Pig‐a gene. Environ. Mol. Mutagen. 56:674–683, 2015. © 2015 Wiley Periodicals, Inc. 相似文献
6.
John Nicolette Joel Murray Paul Sonders Bruce Leroy 《Environmental and molecular mutagenesis》2018,59(1):91-95
The in vivo rodent Pig‐a mutation assay is a sensitive test to identify exposure to mutagenic substances, and has been proposed as an assay for the identification of impurities for pharmaceuticals. Red blood cells (RBCs) and reticulocytes (RETs) are analyzed by flow cytometry after exposure to potentially mutagenic chemicals for cells deficient in the cell surface anchored protein CD59, representing mutation in the X‐linked Pig‐a gene. The full potential of the assay as well as its limitations are currently being explored. The current study investigated the effects of regenerative erythropoietic bone marrow responses on the frequency of Pig‐a mutated reticulocytes (RETCD59‐) and erythrocytes (RBCCD59‐). We hypothesized that a robust regenerative erythropoietic response would not increase the basal frequency of RETCD59‐ or RBCCD59‐ cells. Two groups of six male Sprague‐Dawley rats either had 2 mL of blood sampled each day via an indwelling catheter over a period of 5 days or were minimally sampled for hematology and used as controls. Blood was also then collected and evaluated 5, 18, and 49 days after the initial bleed period for the number of Pig‐a mutant cells in either the RET or RBC population. Despite the expected decrease in hematocrit and the correlative increase in reticulocytes after bleeding, no increase in the number of Pig‐a mutant cells was observed in male Sprague‐Dawley rats that were bled for five consecutive days. These results indicate that changes in erythropoiesis and hematology parameters in rats appear to have no effect on the background levels of Pig‐a mutated RETs and RBCs. Environ. Mol. Mutagen. 59:91–95, 2018. © 2017 Wiley Periodicals, Inc. 相似文献
7.
Comparison of integrated genotoxicity endpoints in rats after acute and subchronic oral doses of 4‐nitroquinoline‐1‐oxide
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Daniel J. Roberts Marie McKeon Yong Xu Leon F. Stankowski 《Environmental and molecular mutagenesis》2016,57(1):17-27
During interlaboratory validation trials for the Pig‐a gene mutation assay we assessed the genotoxicity of 4‐nitroquinoline‐1‐oxide (4NQO) across endpoints in multiple tissues: induction of Pig‐a mutant red blood cells (RBCs) and reticulocytes (RETs); micronucleated RETs (MN RETs); and DNA damage in blood and liver via the alkaline Comet assay (%tail intensity [TI]). In a previous subchronic toxicity study with 28 daily doses, biologically meaningful increases were observed only for Pig‐a mutant RBCs/RETs while marginal increases in the frequency of MN RET were observed, and other clastogenic endpoints were negative. Follow up acute studies were performed using the same cumulative doses (0, 35, 70, 105, and 140 mg/kg) administered in a bolus, or split over three equal daily doses, with samples collected up to 1 month after the last dose. Both of the acute dosing regimens produced similar results, in that endpoints were either positive or negative, regardless of 1 or 3 daily doses, but the three consecutive daily dose regimen yielded more potent responses in TI (in liver and blood) and Pig‐a mutant frequencies. In these acute studies the same cumulative doses of 4NQO induced positive responses in clastogenic endpoints that were negative or inconclusive using a subchronic study design. Additionally, a positive control group using combination doses of cyclophosphamide and ethyl methanesulfonate was employed to assess assay validity and potentially identify a future positive control treatment for integrated genetic toxicity studies. Environ. Mol. Mutagen. 57:17–27, 2016. © 2015 Wiley Periodicals, Inc. 相似文献
8.
Sublinear response in lacZ mutant frequency of Muta™Mouse spermatogonial stem cells after low dose subchronic exposure to N‐ethyl‐N‐nitrosourea
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Jason M. O'Brien Mike Walker Ahalya Sivathayalan George R. Douglas Carole L. Yauk Francesco Marchetti 《Environmental and molecular mutagenesis》2015,56(4):347-355
The transgenic rodent mutation assay was used to compare the dose–response relationship of lacZ mutant frequency (MF) in spermatogonial stem cells exposed acutely or subchronically to N‐ethyl‐N‐nitrosourea (ENU). Muta?Mouse males were exposed orally to 0, 25, 50, or 100 mg/kg ENU for acute exposures and 0, 1, 2, or 5 mg/(kg day) for 28‐day subchronic exposures. LacZ MF was measured in sperm collected 70 days post‐exposure to target spermatogonial stem cells. Dose–response data were fit to linear, quadratic, exponential, or power models. Acute exposure resulted in a dose‐dependent increase in MF that was significant (P < 0.05) at all doses tested and was best described by a quadratic dose–response model that was linear in the low dose range. In contrast, similar total doses fragmented over a 28‐day subchronic exposure only resulted in a significant increase in lacZ MF at the highest dose tested. Therefore, the subchronic no observable genotoxic effect level (NOGEL) was 2 mg/(kg day) (or 56 mg/kg total dose). The subchronic dose–response was best described by the exponential and power models, which were sublinear in the low dose range. Benchmark dose lower confidence limits (BMDLs) for acute and subchronic exposure were 3.0 and 1.0 mg/(kg day) (or 27.4 mg/kg total dose), respectively. These findings are supportive of a saturable DNA repair mechanism as the mutagenic mode of action for ENU in spermatogonia and imply that sufficiently low exposures would not cause appreciable genotoxic effects over background. This may have important implications for the quantitative risk assessment of germ cell mutagens. Environ. Mol. Mutagen. 56:347–355, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. 相似文献
9.
Marian Raschke Bernd‐W. Igl Julia Kenny Joanne Collins Stephen D. Dertinger Carson Labash Javed A. Bhalli Cameron C.M. Tebbe Kylie M. McNeil Andreas Sutter 《Environmental and molecular mutagenesis》2016,57(9):678-686
The rodent Pig‐a assay is an in vivo method for the detection of gene mutation, where lack of glycosylphosphatidylinositol‐anchored proteins on the surface of circulating red blood cells (RBCs) serves as a reporter for Pig‐a gene mutation. In the case of rats, the frequency of mutant phenotype RBCs is measured via fluorescent anti‐CD59 antibodies and flow cytometry. The Pig‐a assay meets the growing expectations for novel approaches in animal experimentation not only focusing on the scientific value of the assay but also on animal welfare aspects (3Rs principles), for example, amenable to integration into pivotal rodent 28‐day general toxicology studies. However, as recommended in the Organisation for Economic Co‐operation and Development Test Guidelines for genotoxicity testing, laboratories are expected to demonstrate their proficiency. While this has historically involved the extensive use of animals, here we describe an alternative approach based on a series of blood dilutions covering a range of mutant frequencies. The experiments described herein utilized either non‐fluorescent anti‐CD59 antibodies to provide elevated numbers of mutant‐like cells, or a low volume blood sample from a single N‐ethyl‐N‐nitrosourea treated animal. Results from these so‐called reconstruction experiments from four independent laboratories showed good overall precision (correlation coefficients: 0.9979–0.9999) and accuracy (estimated slope: 0.71–1.09) of mutant cell scoring, which was further confirmed by Bland–Altman analysis. These data strongly support the use of reconstruction experiments for training purposes and demonstrating laboratory proficiency with very few animals, an ideal situation given the typically conflicting goals of demonstrating laboratory proficiency and reducing the use of animals. Environ. Mol. Mutagen. 57:678–686, 2016. © 2016 Wiley Periodicals, Inc. 相似文献
10.
Mouse Pig‐a and micronucleus assays respond to N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and ethyl carbamate,but not pyrene or methyl carbamate
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Kristine Carlson Ariel Berg Dorothea K. Torous Steven M. Bryce Jeffrey C. Bemis James T. MacGregor Stephen D. Dertinger 《Environmental and molecular mutagenesis》2016,57(1):28-40
This laboratory previously described a method for scoring the incidence of peripheral blood Pig‐a mutant phenotype rat erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends the method to mouse blood, using the frequency of CD24‐negative reticulocytes (RETCD24−) and erythrocytes (RBCCD24−) as phenotypic reporters of Pig‐a gene mutation. Following assay optimization, reconstruction experiments demonstrated the ability of the methodology to return expected values. Subsequently, the responsiveness of the assay to the genotoxic carcinogens N‐ethyl‐N‐nitrosourea, benzo[a]pyrene, and ethyl carbamate was studied in male CD‐1 mice exposed for 3 days to several dose levels via oral gavage. Blood samples were collected on Day 4 for micronucleated reticulocyte analyses, and on Days 15 and 30 for determination of RETCD24− and RBCCD24− frequencies. The same design was used to study pyrene, with benzo[a]pyrene as a concurrent positive control, and methyl carbamate, with ethyl carbamate as a concurrent positive control. The three genotoxicants produced marked dose‐related increases in the frequencies of Pig‐a mutant phenotype cells and micronucleated reticulocytes. Ethyl carbamate exposure resulted in moderately higher micronucleated reticulocyte frequencies relative to N‐ethyl‐N‐nitrosourea or benzo[a]pyrene (mean ± SEM = 3.0 ± 0.36, 2.3 ± 0.17, and 2.3 ± 0.49%, respectively, vs. an aggregate vehicle control frequency of 0.18 ± 0.01%). However, it was considerably less effective at inducing Pig‐a mutant cells (e.g., Day 15 mean no. RETCD24− per 1 million reticulocytes = 7.6 ± 3, 150 ± 9, and 152 ± 43 × 10−6, respectively, vs. an aggregate vehicle control frequency of 0.6 ± 0.13 × 10−6). Pyrene and methyl carbamate, tested to maximum tolerated dose or limit dose levels, had no effect on mutant cell or micronucleated reticulocyte frequencies. Collectively, these results demonstrate the utility of the cross‐species Pig‐a and micronucleated reticulocyte assays, and add further support to the value of studying both endpoints in order to cover two distinct genotoxic modes of action. Environ. Mol. Mutagen. 57:28–40, 2016. © 2015 Wiley Periodicals, Inc. 相似文献
11.
Diet‐induced obesity increases the frequency of Pig‐a mutant erythrocytes in male C57BL/6J mice
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Jeffrey K. Wickliffe Stephen D. Dertinger Dorothea K. Torous Svetlana L. Avlasevich Bridget R. Simon‐Friedt Mark J. Wilson 《Environmental and molecular mutagenesis》2016,57(9):668-677
Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig‐a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30‐week old male mice reared on either a high‐fat diet (60% calories from fat) that exhibit an obese phenotype or a normal‐fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N‐ethyl‐N‐nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig‐a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non‐obese mice with respect to Pig‐a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig‐a mutant frequencies (increased 2.5‐3.7‐fold, p < 0.02) in erythrocytes as compared to non‐obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668–677, 2016. © 2016 Wiley Periodicals, Inc. 相似文献
12.
Dorothea K. Torous Souk Phonethepswath Svetlana L. Avlasevich Jared Mereness Steven M. Bryce Jeffrey C. Bemis Pamela Weller Sara Bell Carol Gleason Laura L. Custer James T. MacGregor Stephen D. Dertinger 《Environmental and molecular mutagenesis》2012,53(6):420-428
Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood‐based genotoxicity endpoints—micronucleated reticulocyte frequency and gene mutation at the Pig‐a locus—thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short‐term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague‐Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3‐day and 28‐day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig‐a mutation with a dual labeling method (SYTO 13 in combination with anti‐CD59‐PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3‐day study (43‐fold increase) and on Day 42 of the 28‐day study (171‐fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28‐day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate that Pig‐a and micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that the Pig‐a endpoint is suitable for both repeated‐dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on‐going toxicology studies, and use as a short‐term assay that provides efficient screening and mechanistic information in vivo. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc. 相似文献
13.
Evaluation of the Pig‐a,micronucleus, and comet assay endpoints in a 28‐day study with ethyl methanesulfonate
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
William C. Gunther Stephanie L. Coffing Donna A. Dickinson Maria E. Engel Ronald D. Fiedler Susan D. O'Lone Kelley E. Sanok Catherine J. Thiffeault Thomas J. Shutsky Maik J. Schuler Krista L. Dobo 《Environmental and molecular mutagenesis》2014,55(6):492-499
Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig‐a mutation assay and compared with other well‐established in vivo genotoxicity endpoints. Male Sprague‐Dawley (SD) rats were given a daily dose of 0, 6.25, 12.5, 25, 50, or 100 mg/kg/day EMS for 28 days, and evaluated for a variety of genotoxicity endpoints in peripheral blood, liver, and colon. Blood was sampled pre‐dose (Day 1) and at various time points up to Day 105. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBCCD59? and RETCD59? frequencies. The first statistically significant increases in mutant frequencies were seen in RETs on Day 15 and in RBCs on Day 29 with the maximum RETCD59? on Day 29 and of RBCCD59? on Day 55. The lowest dose producing a statistically significant increase of RETCD59? was 12.5 mg/kg on Day 55 and 25 mg/kg for RBCCD59? on Day 55. EMS also induced significant increases in % micronucleated RETs (MN‐RETs) in peripheral blood on Days 3, 15, and 28. No statistically significant increases in micronuclei were seen in liver or colon. Results from the in vivo Comet assay on Day 29 showed generally weak increases in DNA damage in all tissues evaluated with little evidence for accumulation of damage seen over time. The results with EMS indicate that the assessment of RBCCD59? and/or RETCD59? in the Pig‐a assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints. Environ. Mol. Mutagen. 55:492–499, 2014. © 2014 Wiley Periodicals, Inc. 相似文献
14.
Svetlana L. Avlasevich Carson Labash Dorothea K. Torous Jeffrey C. Bemis James T. MacGregor Stephen D. Dertinger 《Environmental and molecular mutagenesis》2018,59(1):30-37
The Pig‐a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig‐a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty‐eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days ?4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well‐established aneugenic effect on hematopoietic cells, the lack of a Pig‐a response indicates that this tubulin‐binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30–37, 2018. © 2017 Wiley Periodicals, Inc. 相似文献
15.
Assessment of 5‐fluorouracil and 4‐nitroquinoline‐1‐oxide in vivo genotoxicity with Pig‐a mutation and micronucleus endpoints
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Changhui Zhou Min Zhang Pengcheng Huang Honggang Tu Zheng Wang Stephen D. Dertinger Dorothea K. Torous Yan Chang 《Environmental and molecular mutagenesis》2014,55(9):735-740
Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5‐fluorouracil (5‐FU) and 4‐nitroquinoline‐1–oxide (4NQO) as part of an international validation trial of the Pig‐a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5‐FU for three consecutive days (Days 1–3); blood was sampled on Days ?1, 4, 15, 29, and 45. Pig‐a mutant phenotype reticulocyte (RETCD59?) and mutant phenotype erythrocyte (RBCCD59?) frequencies were determined on Days ?1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN‐RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBCCD59? and RETCD59? frequencies were determined on Days ?1, 15, and 29, and MN‐RET were quantified on Day 29. Whereas 5‐FU was found to increase %MN‐RET, no significant increases were observed for RBCCD59? or RETCD59? at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBCCD59? and RETCD59? frequencies, and this same dose level caused a weak but significantly elevated increase in MN‐RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig‐a mutation and MN‐RET into acute and 28‐day repeat‐dose studies. Environ. Mol. Mutagen. 55:735–740, 2014. © 2014 Wiley Periodicals, Inc. 相似文献
16.
《Environmental and molecular mutagenesis》2018,59(5):366-374
Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N‐ethyl‐N‐nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA‐deficient diet had a 1.3‐fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control‐fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig‐a MF, but there was no effect of FA intake on ENU‐induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU‐induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366–374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018. 相似文献
17.
Souk Phonethepswath Svetlana L. Avlasevich Dorothea K. Torous Jared Mereness Jeffrey C. Bemis James T. MacGregor Stephen D. Dertinger 《Environmental and molecular mutagenesis》2013,54(4):294-298
Procarbazine is a genotoxic carcinogen whose DNA‐damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD‐1 mice using a multi‐endpoint study design that scored micronucleated reticulocyte (MN‐RET) frequency and gene mutation at the Pig‐a locus. CD‐1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN‐RETs, with the high dose group exhibiting a mean 29‐fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig‐a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Environ. Mol. Mutagen. 54:294–298, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
18.
Lemieux CL Douglas GR Gingerich J Phonethepswath S Torous DK Dertinger SD Phillips DH Arlt VM White PA 《Environmental and molecular mutagenesis》2011,52(9):756-765
In this study we compared the response of the Pig‐a gene mutation assay to that of the lacZ transgenic rodent mutation assay, and demonstrated that multiple endpoints can be measured in a 28‐day repeat dose study. Muta?Mouse were dosed daily for 28 days with benzo[a]pyrene (BaP; 0, 25, 50 and 75 mg/kg body weight/day) by oral gavage. Micronucleus (MN) frequency was determined in reticulocytes (RETs) 48 hr following the last dose. 72 h following the last dose, mice were euthanized, and tissues (glandular stomach, small intestine, bone marrow and liver) were collected for lacZ mutation and DNA adduct analysis, and blood was evaluated for Pig‐a mutants. BaP‐derived DNA adducts were detected in all tissues examined and significant dose‐dependent increases in mutant Pig‐a phenotypes (i.e., RETCD24‐ and RBC CD24‐) and lacZ mutants were observed. We estimate that mutagenic efficiency (i.e., rate of conversion of adducts into mutations) was much lower for Pig‐a compared to lacZ, and speculate that this difference is likely explained by differences in repair capacity between the gene targets, and differences in the cell populations sampled for Pig‐a versus lacZ. The BaP doubling doses for both gene targets, however, were comparable, suggesting that similar mechanisms are involved in the accumulation of gene mutations. Significant dose‐related increases in % MN were also observed; however, the doubling dose was considerably higher for this endpoint. The similarity in dose response kinetics of Pig‐a and lacZ provides further evidence for the mutational origin of glycosylphosphatidylinositol (GPI)‐anchor deficiencies detected in the Pig‐a assay. Environ. Mol. Mutagen. 2011. © 2011 Wiley‐Liss, Inc. 相似文献
19.
Stephanie L. Coffing Michelle O. Kenyon Joel I. Ackerman Thomas J. Shutsky Krista L. Dobo 《Environmental and molecular mutagenesis》2015,56(3):322-332
Understanding the mutagenic dose response could prove beneficial in the management of pharmaceutically relevant impurities. For most alkyl ester impurities, such as isopropyl methanesulfonate (IPMS), little in vivo mutagenicity data exist for dose analysis. The likelihood of a sublinear dose response for IPMS was assessed by comparing the Swain Scott constant, the SN1/SN2 reaction mechanism and the O6:N7 guanine adduct ratio to that of more well‐known alkyl esters. Based on available information, IPMS was predicted to have a mutagenic profile most like ethyl nitrosourea. To test this hypothesis, mature male Wistar Han rats were administered IPMS using acute (single administration at 3.5 to 56 mg/kg) or subchronic (28 days at 0.125 to 2 mg/kg/day) exposures. The in vivo Pig‐a mutation assay was used to identify mutant phenotype reticulocyte (Ret) and red blood cell (RBC) populations. The maximum mutant response occurred approximately 15 and 28 days after the last dose administration in the mutant Ret and RBC populations respectively in the acute study and on Day 29 and 56 in the mutant Ret and RBC populations, respectively, in the subchronic study. A comparison of RBC mutant frequencies from acute and subchronic protocols suggests a sublinear response; however, this was not substantiated by statistical analysis. A No Observed Effect Level (NOEL) of 0.25 mg/kg/day resulted in a Permitted Daily Exposure equivalent to the Threshold of Toxicological Concern. An estimate of the NOEL based on the previously mentioned factors, in practice, would have pre‐empted further investigation of the potent mutagen IPMS. Environ. Mol. Mutagen. 56:322–332, 2015. © 2014 Wiley Periodicals, Inc. 相似文献
20.
Quantitative dose–response analysis of ethyl methanesulfonate genotoxicity in adult gpt‐delta transgenic mice
下载免费PDF全文
![点击此处可从《Environmental and molecular mutagenesis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Xuefei Cao Roberta A. Mittelstaedt Mason G. Pearce Bruce C. Allen Lya G. Soeteman‐Hernández George E. Johnson C. Anita H. Bigger Robert H. Heflich 《Environmental and molecular mutagenesis》2014,55(5):385-399
The assumption that mutagens have linear dose–responses recently has been challenged. In particular, ethyl methanesulfonate (EMS), a DNA‐reactive mutagen and carcinogen, exhibited sublinear or thresholded dose‐responses for LacZ mutation in transgenic Muta?Mouse and for micronucleus (MN) frequency in CD1 mice (Gocke E and Müller L [2009]: Mutat Res 678:101–107). In order to explore variables in establishing genotoxicity dose–responses, we characterized the genotoxicity of EMS using gene mutation assays anticipated to have lower spontaneous mutant frequencies (MFs) than Muta?Mouse. Male gpt‐delta transgenic mice were treated daily for 28 days with 5 to 100 mg/kg EMS, and measurements were made on: (i) gpt MFs in liver, lung, bone marrow, kidney, small intestine, and spleen; and (ii) Pig‐a MFs in peripheral blood reticulocytes (RETs) and total red blood cells. MN induction also was measured in peripheral blood RETs. These data were used to calculate Points of Departure (PoDs) for the dose responses, i.e., no‐observed‐genotoxic‐effect‐levels (NOGELs), lower confidence limits of threshold effect levels (Td‐LCIs), and lower confidence limits of 10% benchmark response rates (BMDL10s). Similar PoDs were calculated from the published EMS dose–responses for LacZ mutation and CD1 MN induction. Vehicle control gpt and Pig‐a MFs were 13–40‐fold lower than published vehicle control LacZ MFs. In general, the EMS genotoxicity dose–responses in gpt‐delta mice had lower PoDs than those calculated from the Muta?Mouse and CD1 mouse data. Our results indicate that the magnitude and possibly the shape of mutagenicity dose responses differ between in vivo models, with lower PoDs generally detected by gene mutation assays with lower backgrounds. Environ. Mol. Mutagen. 55:385–399, 2014. © 2014 Wiley Periodicals, Inc. 相似文献