Effective use of the <i>Pig-a</i> gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals

Abstract

-The Pig-a gene mutation assay using perpherial blood erythrocytes is being investigated as a screening tool for assessing mutagenicity in vivo. In this study, we evaluated two distinct approaches for performing the Pig-a assay in rats. We used antibodies to CD45 or the erythroid marker HIS49 to identify red blood cells (RBCs), and then monitored the kinetics of Pig-a mutant frequency, as measured by the frequency of CD59-deficient RBCs, in rats treated with the genotoxic chemicals, N-ethyl-N-nitrosourea, cyclophosphamide, 4-nitroquinoline-1-oxide, and ethylmethanesulfonate. In some instances, micronucleus frequency also was measured in the same animals. Time-and dose-related increases in Pig-a mutant frequency were found in all the chemical-treated groups, except for the groups treated with cyclophosphamide, which was a potent inducer of micronuclei. The two different approaches we employed were comparable for measuring induced mutant frequencies, but our historical data showed that the mean background frequencies for the CD45/CD59 method and the HIS49/CD59 method were 12.7 × 10 -6 and 5.5 ×10 -6 , respectively. The relatively low, stable background mutant frequency associated with the HIS49/CD59 method indicates that it may have greater power for discriminating weak induced responses. These results suggest that the HIS49/CD59 method is a promising tool for measuring Pig-a mutant RBCs. In addition, differences in their manifestation kinetics and in their relative sensitivity for detecting different test compounds suggest that the combination of the Pig-a assay and the micronucleus assay may be effective in identifying in vivo genotoxicity.

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

“…This is in agreement with a report in rats using the higher powered version of the Pig‐a gene mutation assay, where 3 mkd was negative after 1 month of daily dose administrations [Zhou et al, ]. In an equally powered assay to the format used herein, where 1 million RBC from five animals are evaluated for mutant phenotype, higher single doses of 50 and 100 mg/kg 4NQO induced near linear MFs that were ∼2‐fold apart in magnitude [Kimoto et al, ]. A similar response was observed here after single doses of 70 and 140 mg/kg, even when using different antibodies and gating logic to identify and enumerate mutant phenotype cells (Fig.…”

Pilot studies evaluating the nongenotoxic rodent carcinogens phenobarbital and clofibrate in the rat Pig‐a assay

“…Comparison of the preand post-exposure data of the %RET values indicated a substantial decrease in the F344 rats utilized in this study. This was consistent with the reports in Sprague Dawley rats from other laboratories (Cammerer et al, 2011;Dertinger et al, 2011b;Lynch et al, 2011a;Stankowski et al, 2011;Kimoto et al, 2012;Torous et al, 2012;Gunther et al, 2014), indicating that this is a biological aging phenomenon.…”

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

“…In Japan, many studies have been conducted using a fluorescent antibody to detect erythroid cells (HIS49 for rats [55,56]), plus fluorescent antibodies to detect a GPI-anchored marker (CD59 in rats) and transferrin receptor (CD71, which is used as a marker for RETs). This method has also begun incorporating a magnetic separation step to increase the number of cells scored in the assay, in this case, CD71-positive RETs [57].…”