Comparison of male versus female responses in the Pig-a mutation assay

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

doi:10.1093/mutage/geu055

Cited by 17 publications

(14 citation statements)

References 25 publications

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“…It is therefore important to verify that the lyonization process in females does not introduce any unexpected problems with the conduct of the Pig-a assay. Our present data and that of previous reports (9-10) demonstrate that female rats are similarly responsive to genotoxic agents and show the same kinetic behavior as male rats. Thus, we believe that integration of the Pig-a assay with routine toxicology testing has the major advantages of eliminating the need for any additional animals for genotoxicity evaluation while at the same time providing sensitive genotoxicity information in both sexes, and in the context of relevant information on toxicity, pharmacokinetics, and metabolism that is routinely assessed as part of toxicity studies.…”

Section: Discussionsupporting

confidence: 88%

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Induction of Pig-a mutant erythrocytes in male and female rats exposed to 1,3-propane sultone, ethyl carbamate, or thiotepa

Labash

Carlson

Avlasevich

et al. 2015

Mutation Research/Genetic Toxicology and Environmental Mutagene

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Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, groups of five male and female Sprague Dawley rats were exposed to the mutagens 1,3-propane sultone (80 mg/kg/day), ethyl carbamate (600 mg/kg/day), or thiotepa (7.5 mg/kg/day) for three consecutive days (study Days 1-3). Pig-a mutant phenotype reticulocyte (RETCD59-) and mutant phenotype erythrocyte (RBCCD59-) frequencies were determined on study Days -4, 15, 30 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). While the percentage of reticulocytes (%RET) was markedly higher for pre-treatment blood samples from males compared to females (6.6% vs. 3.5%), this sex effect was slight or nonexistent at later time points. Treatment-related effects to %RET were generally modest owing to the 12-day interval between last exposure and blood sampling. Mean RETCD59-and RBCCD59- frequencies were consistently low in vehicle control animals of both sexes, with 77% of samples exhibiting mutant cell frequencies ≤ 1 × 10-6 over study Days 15-46. Treatment with each mutagen caused significant increases to mean RETCD59- and RBCCD59- frequencies. Whereas genotoxicant-induced RETCD59- values were maximal on Day 15, induced RBCCD59- frequencies were highest at the last sampling time. Sex did not affect 1,3-propane sultone- or thiotepa-induced mutant cell frequencies. While ethyl carbamate-exposed females exhibited higher mean mutant cell frequencies compared to like-treated males, statistical significance was achieved only for RBCCD59- at one time point (7.6 ± 1.0 × 10-6 compared to 4.7 ± 0.6 × 10-6 on Day 30). Thus, while some quantitative differences were evident, there were no qualitative differences in how males and females responded to three diverse mutagens. These data support the use of both sexes for Pig-a gene mutation studies.

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Section: Discussionsupporting

confidence: 88%

“…Labash et al . reported that while spontaneous frequencies were similar between the sexes, three consecutive days of exposure at 25 mg ENU/kg/day caused modest quantitative differences in young male versus female Sprague Dawley rats, possibly related to differences in hematopoietic activity (10). …”

Section: Introductionmentioning

confidence: 99%

Induction of Pig-a mutant erythrocytes in male and female rats exposed to 1,3-propane sultone, ethyl carbamate, or thiotepa

Labash

Carlson

Avlasevich

et al. 2015

Mutation Research/Genetic Toxicology and Environmental Mutagene

Self Cite

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“…No clear sex bias has been found for Pig‐a mutation in rodent studies (Labash et al ; Labash et al ), while studies in different ethnic human populations have produced inconsistent results (Dobrovolsky et al ; Dertinger et al ; Cao et al ). The Pig‐a gene is on the X chromosome, suggesting that X‐chromosome inactivation in females may affect spontaneous MFs.…”

Section: Discussionmentioning

confidence: 99%

The potential application of human PIG‐A assay on azathioprine‐treated inflammatory bowel disease patients

Cao

Wang²,

Liu

et al. 2019

Environ and Mol Mutagen

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The rodent Pig-a assay has been used extensively as a potential regulatory assay for evaluating the in vivo mutagenicity of test substances. Although the assay can be conducted in different mammalian species, there have been only a few reports describing its use in humans, and rarely in genotoxicant-exposed human populations. In this study, PIG-A mutation frequencies (MFs) were evaluated in 36 azathioprine (AZA; human carcinogen)-treated inflammatory bowel disease (IBD) patients and 36 healthy volunteers. IBD patients exhibited a slight but statistically higher MF (6.10 AE 4.44 × 10 −6 ) than healthy volunteers (4.97 AE 2.74 × 10 −6 ) (P = 0.0489). The estimated relative risk for the exposed patients was 1.22 which indicated that AZA is a risk factor for inducing PIG-A mutation. However, the PIG-A MF showed no associations with AZA treatment duration or total AZA exposure. In addition, we performed the cytokinesis-block micronucleus test on the same samples. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in IBD patients (MN: 4.70 AE 2.86‰; NBUD: 1.89 AE 0.95‰) were significantly higher than in healthy volunteers (MN: 1.47 AE 0.77‰, P < 0.001; NBUD: 0.90 AE 0.58‰, P = 0.004). MN frequency also had significant correlations with AZA treatment duration (P = 0.011) and total AZA exposure (P = 0.018).Our findings indicate that AZA-treated IBD patients have only a marginally significant increase in PIG-A MF; in contrast, a much stronger AZA-associated increase in genotoxicity was detected with the lymphocyte MN assay. Environ. Mol. Mutagen. 61:456-464, 2020.

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“…Le séquençage du gène PIG-A réalisé sur des cellules issues de patients HPN et de sujets sains a ainsi identifié plus de 100 mutations inactivantes (parmi lesquelles des mutations non-sens, faux-sens, des décalages du cadre de lecture, des substitutions, et de larges délétions) aboutissant toutes au même phénotype de déficience en GPI, suggérant que le gène PIG-A puisse être sensible à un grand nombre de modes d'actions génotoxiques [15,34]. Deux lacunes identifiées par l'IWGT en 2015 ont été comblées par la publication de deux études récentes : les performances du test ne dépendent en fait pas du nombre de copies du chromosome X portées par l'individu, les fréquences spontanées et induites de cellules mutées pour le gène PIG-A n'étant pas différentes entre mâles et femelles [23,35,36] ; l'érythropoïèse compensatoire observée après exposition à un agent hémolysant non génotoxique (comme le 2-butoxyéthanol) n'entraîne pas d'augmentation de la fréquence des cellules GPI-déficientes [37]. Ces qualités ont initié l'évaluation du test PIG-A in vitro : le séquen-çage du gène PIG-A réalisé sur des lymphoblastes humains cultivés in vitro et soumis à divers génotoxiques connus, a révélé des résultats comparables à ceux obtenus in vivo chez la souris après exposition aux mêmes agents [38].…”

Section: Cytométrie De Fluxunclassified