Glycosylphosphatidylinositol (GPI) anchored protein deficiency serves as a reliable reporter of Pig‐a gene Mutation: Support from an in vitro assay based on L5178Y/Tk+/− cells and the CD90.2 antigen

Lack of cell surface glycosylphosphatidylinositol (GPI)‐anchored protein(s) has been used as a reporter of Pig‐a gene mutation in several model systems. As an extension of this work, our laboratory initiated development of an in vitro mutation assay based on the flow cytometric assessment of CD90.2 expression on the cell surface of the mouse lymphoma cell line L5178Y/Tk^+/?. Cells were exposed to mutagenic and nonmutagenic compounds for 24 hr followed by washout and incubation for an additional 7 days. Following this mutant manifestation time, cells were labeled with fluorescent antibodies against CD90.2 and CD45 antigens. These reagents indicated the presence of GPI‐anchored proteins and general cell surface membrane receptor integrity, respectively. Instrument set‐up was aided by parallel processing of a GPI anchor‐deficient subclone. Results show that the mutagens reproducibly caused increased frequencies of mutant phenotype cells, while the nonmutagens did not. Further modifications to the method, including application of a viability dye and an isotype control for instrument set‐up, were investigated. As a means to verify that the GPI‐anchored protein‐negative phenotype reflects bona fide Pig‐a gene mutation, sequencing was performed on 38 CD90.2‐negative L5178Y/Tk^+/? clones derived from cultures treated with ethyl methanesulfonate. All clones were found to have mutation(s) within the Pig‐a gene. The continued investigation of L5178Y/Tk^+/? cells, CD90.2 labeling, and flow cytometric analysis as the basis of an in vitro mutation assay is clearly supported by this work. These data also provide evidence of the reliability of using GPI anchor‐deficiency as a valid reporter of Pig‐a gene mutation. Environ. Mol. Mutagen. 59:18–29, 2018. © 2017 Wiley Periodicals, Inc.     

CD48‐deficient T‐lymphocytes from DMBA‐treated rats have de novo mutations in the endogenous Pig‐a gene

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.     

Evaluation of in vivo genotoxicity induced by N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and 4‐nitroquinoline‐1‐oxide in the Pig‐a and gpt assays

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.     

Applying the erythrocyte Pig‐a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation

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.     

The frequency of Pig‐a mutant red blood cells in rats exposed in utero to N‐ethyl‐N‐nitrosourea

The Pig‐a assay has been developed as a rapid sensitive measure of gene mutation in adult rats; however, no data exist on its ability to detect mutation following in utero exposures or in neonatal animals. Pregnant Sprague‐Dawley rats were treated daily on gestational days 12–18 with oral doses of 0, 6, or 12 mg/kg/day N‐ethyl‐N‐nitrosourea (ENU); following parturition, the offspring and dams were monitored over a period of 5 months for the frequency of CD59‐deficient erythrocytes as a marker of Pig‐a mutation. Significant dose‐related increases in Pig‐a mutant red blood cells (RBCs) were observed in ENU‐treated dams. However, only very weak increases in RBC Pig‐a mutant frequency (MF) were noted in offspring treated in utero with the lower ENU dose. The higher ENU dose produced extremely variable responses in the offspring as a function of age, even among littermates, ranging from a steady low or moderately high Pig‐a MF to a rapidly increasing or decreasing Pig‐a MF. The manifestation kinetics of Pig‐a mutant RBCs in the offspring suggest that the change from predominantly hepatic to predominantly bone marrow erythropoiesis that occurs during early development may have contributed to this variability. Our results indicate that using the RBC Pig‐a model for mutation detection in animals treated in utero may require analysis of multiple offspring from the same litter to account for potential “jack pot” effects, and that detection of the earliest treatment effect (i.e., in neonates using the hepatic RBC fraction) may require optimization of blood processing. Environ. Mol. Mutagen. 2012. Published 2012 Wiley Periodicals, Inc.     

A regenerative erythropoietic response does not increase the frequency of Pig‐a mutant reticulocytes and erythrocytes in Sprague‐Dawley rats

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 (RET^CD59‐) and erythrocytes (RBC^CD59‐). We hypothesized that a robust regenerative erythropoietic response would not increase the basal frequency of RET^CD59‐ or RBC^CD59‐ 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.     

Mouse Pig‐a and micronucleus assays respond to N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and ethyl carbamate,but not pyrene or methyl carbamate

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 (RET^CD24−) and erythrocytes (RBC^CD24−) 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 RET^CD24− and RBC^CD24− 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. RET^CD24− 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.     

Simultaneous measurement of benzo[a]pyrene-induced Pig-a and lacZ mutations, micronuclei and DNA adducts in Muta™ Mouse

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., RET^CD24‐ 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.     

The in vivo Pig‐a gene mutation assay is useful for evaluating the genotoxicity of ionizing radiation in mice

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.     

Evaluation of the Pig‐a,micronucleus, and comet assay endpoints in a 28‐day study with ethyl methanesulfonate

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 RBC^CD59? and RET^CD59? 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 RET^CD59? on Day 29 and of RBC^CD59? on Day 55. The lowest dose producing a statistically significant increase of RET^CD59? was 12.5 mg/kg on Day 55 and 25 mg/kg for RBC^CD59? 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 RBC^CD59? and/or RET^CD59? 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.     

In Vivo Pig‐a gene mutation assay: Guidance for 3Rs‐friendly implementation

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.     

Assessment of 5‐fluorouracil and 4‐nitroquinoline‐1‐oxide in vivo genotoxicity with Pig‐a mutation and micronucleus endpoints

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 (RET^CD59?) and mutant phenotype erythrocyte (RBC^CD59?) 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. RBC^CD59? and RET^CD59? 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 RBC^CD59? or RET^CD59? at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC^CD59? and RET^CD59? 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.     

Diet‐induced obesity increases the frequency of Pig‐a mutant erythrocytes in male C57BL/6J mice

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.     

Comparison of integrated genotoxicity endpoints in rats after acute and subchronic oral doses of 4‐nitroquinoline‐1‐oxide

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.     

Diethylnitrosamine genotoxicity evaluated in sprague dawley rats using Pig‐a mutation and reticulocyte micronucleus assays

Diethylnitrosamine (DEN) is a genotoxic carcinogen, but in vivo DNA‐damaging activities are not usually evident in hematopoietic cells because the short‐lived active metabolite is formed mainly in the liver. DEN therefore represented an interesting case for evaluating the performance characteristics of blood‐based endpoints of genotoxicity that have been automated using flow cytometric analysis—frequency of micronucleated reticulocytes and Pig‐a mutant phenotype reticulocytes (RET^CD59?) and erythrocytes (RBC^CD59?). Male Sprague Dawley rats were treated for 28 consecutive days with DEN at levels up to 12.5 mg/kg/day. Serial blood samples were collected and micronucleus frequencies were determined on Days 4 and 29, while RET^CD59? and RBC^CD59? frequencies were determined on Days 15, 29, and 42. The Pig‐a analyses were conducted with an enrichment step based on immunomagnetic column separation to increase the statistical power of the assay. Modest but significant reductions to reticulocyte frequencies demonstrated that bone marrow was exposed to reactive intermediates. Even so, DEN did not affect micronucleus frequencies at any dose level tested. However, RET^CD59? frequencies were significantly elevated in the high dose group on Day 29, and RBC^CD59? were increased at this same dose level on Days 29 and 42. These results demonstrate that the Pig‐a assay is sufficiently sensitive to evaluate chemicals for genotoxic potential, even in the case of a promutagen that has traditionally required direct assessment(s) of liver tissue for detection of DNA‐damage. Environ. Mol. Mutagen. 55:400–406, 2014. © 2014 Wiley Periodicals, Inc.     

Mutation analysis with random DNA identifiers (MARDI) catalogs Pig‐a mutations in heterogeneous pools of CD48‐deficient T cells derived from DMBA‐treated rats

Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a “clone‐by‐clone” approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high‐fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS‐ and PCR‐induced sequencing artifacts. When applied to the Pig‐a cDNA analysis of heterogeneous pools of CD48‐deficient T cells derived from DMBA‐treated rats, MARDI detected nearly all Pig‐a mutations that were previously identified by conventional clone‐by‐clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non‐transcribed DNA strand. Environ. Mol. Mutagen. 57:114–124, 2016. © 2015 Wiley Periodicals, Inc.     

Flow cytometric analysis of Pig‐a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD‐1 mice

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.     

Human erythrocyte PIG‐A assay: An easily monitored index of gene mutation requiring low volume blood samples

This laboratory has previously described a method for scoring the incidence of rodent blood Pig‐a mutant phenotype erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends this approach to human blood. The frequencies of CD59‐ and CD55‐negative reticulocytes (RET^CD59?/CD55?) and erythrocytes (RBC^CD59?/CD55?) serve as phenotypic reporters of PIG‐A gene mutation. Immunomagnetic separation was found to provide an effective means of increasing the number of reticulocytes and erythrocytes evaluated. Technical replicates were utilized to provide a sufficient number of cells for precise scoring while at the same time controlling for procedural accuracy by allowing comparison of replicate values. Cold whole blood samples could be held for at least one week without affecting reticulocyte, RET^CD59?/CD55? or RBC^CD59?/CD55? frequencies. Specimens from a total of 52 nonsmoking, self‐reported healthy adult subjects were evaluated. The mean frequency of RET^CD59?/CD55? and RBC^CD59?/CD55? were 6.0 × 10^?6 and 2.9 × 10^?6, respectively. The difference is consistent with a modest selective pressure against mutant phenotype erythrocytes in the circulation, and suggests advantages of studying both populations of erythrocytes. Whereas intra‐subject variability was low, inter‐subject variability was relatively high, with RET^CD59?/CD55? frequencies differing by more than 30‐fold. There was an apparent correlation between age and mutant cell frequencies. Taken together, the results indicate that the frequency of human PIG‐A mutant phenotype cells can be efficiently and reliably estimated using a labeling and analysis protocol that is well established for rodent‐based studies. The applicability of the assay across species, its simplicity and statistical power, and the relatively non‐invasive nature of the assay should benefit myriad research areas involving DNA damage, including studies of environmental factors that modify “spontaneous” mutation frequencies. Environ. Mol. Mutagen. 56:366–377, 2015. © 2014 Wiley Periodicals, Inc.     

In vivo flow cytometric Pig‐a and micronucleus assays: Highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated‐dose designs

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.     

Folate deficiency increases chromosomal damage and mutations in hematopoietic cells in the transgenic mutamouse model

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.