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

Abstract

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 dosedependent 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.

Genotoxicity assessment: opportunities, challenges and perspectives for quantitative evaluations of dose–response data

“…The mammalian erythrocyte Pig-a gene mutation assay, for which an OECD guideline (OECD TG 470) has recently been adopted (OECD 2022 ), is limited to the phenotypic detection of mutations in the endogenous phosphatidylinositol glycan, class A ( Pig-a ) gene. Up to now, the Pig-a gene mutation assay is only fully validated for erythrocytes sampled from peripheral blood, but it has also been adapted to germ cells and blood cells other than erythrocytes (Dobrovolsky et al 2017 ; Ji and LeBaron 2017 ; Kirkland et al 2019 ). The in vivo mammalian alkaline comet assay (OECD TG 489) can be applied to cells or nuclei isolated from many target tissues (OECD 2016b ).…”