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

Gunther, William C.; Coffing, Stephanie L.; Dickinson, Donna; Engel, Maria; Fiedler, Ronald D.; O'Lone, Susan D.; Sanok, Kelley E.; Thiffeault, Catherine J.; Shutsky, Thomas; Schuler, Maik; Dobo, Krista L.

doi:10.1002/em.21863

Cited by 8 publications

(7 citation statements)

References 39 publications

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“…In comparison to the pre‐exposure data, the postexposure %RET in both studies (study day 63 in the ENU study and study day 29 in the clofibrate study) was substantially decreased. This finding is consistent with the reports in rats from other labs, which indicate a biological aging phenomenon [Cammerer et al, ; Dertinger et al, ,c; Lynch et al, ; Stankowski et al, ; Dertinger et al, ; Kimoto et al, ; Torous et al, ; Dertinger et al, ; Gunther et al, ].…”

Section: Resultssupporting

confidence: 91%

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

LeBaron

2017

Environ and Mol Mutagen

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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.

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Section: Resultssupporting

confidence: 91%

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

LeBaron

2017

Environ and Mol Mutagen

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“…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.…”

Section: Pig-a Assaysupporting

confidence: 93%

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

Settivari

LeBaron

2018

Environ and Mol Mutagen

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The Pig‐a assay is an emerging and promising in vivo method to determine mutagenic potential of chemicals. Since its development in 2008, remarkable progress has been made in harmonizing and characterizing the test procedures, primarily using known mutagenic chemicals. The purpose of the present study was to evaluate specificity of the Pig‐a assay using two nongenotoxic and well‐characterized rodent liver carcinogens, phenobarbital and clofibrate, in male F344/DuCrl rats. Daily oral administration of phenobarbital or clofibrate at established hepatotoxic doses for 28 days resulted in substantial hepatic alterations, however, did not increase the frequency of Pig‐a mutation markers (RETCD59‐ and RBCCD59‐) compared to vehicle control or pre‐exposure (Day −5) mutant frequencies. These results are consistent with the existing literature on the nonmutagenic mode of action (MoA) of phenobarbital and clofibrate liver tumors. The present study contributes to the limited, but expanding evidence on the specificity of the Pig‐a assay and further for the investigations of carcinogenic MoAs, i.e., mutagenic or nonmutagenic potential of chemicals. Environ. Mol. Mutagen. 60:42–46, 2019. © 2018 Wiley Periodicals, Inc.

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“…The maximal mean RET Pig-a MF was observed on Day 29, suggesting that there was an accumulation of mutations in stem cells with repeated dosing, consistent with the observed results for other genotoxic chemicals (6). This accumulative feature might increase the value of Pig-a mutation as a genotoxic endpoint that is integrated in a repeat dose toxicity study, because the highest dose in a repeat dose study is generally lower than an acute study, and the sensitivity of other non-accumulative endpoints such as micronuclei in reticulocytes would be doubtful (10). There are no effects on RBC Pig-a MF after MMS treatment.…”

Section: Discussionsupporting

confidence: 64%

Pig-a Gene Mutation and Micronucleus Induction in Rat Peripheral Blood by Methyl Methanesulfonate

Muto¹,

Yamada²,

Kato³

et al. 2014

Genes and Environment

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A recent report on an interlaboratory trial on the Pig-a mutation assays using an antibody against the erythroid marker (HIS49 Pig-a assays) has shown transferability and reproducibility of the assays. In this report, methyl methanesulfonate (MMS) was used to further evaluate the ability of HIS49 Pig-a assays in different study designs (28-day repeated dose and single dose). MMS was administered to male Sprague Dawley (SD) rats orally for 28 days at a dose of 7.5, 15, or 30 mg/kg/day or by single oral gavage at 50, 100 and 200 mg/kg in 28-day study and single dose study, respectively. Pig-a assays for total red blood cells (RBC Pig-a assay), for reticulocytes (PIGRET assay) and micronucleated reticulocytes (MN-RET) measurement were performed. In the 28-day study, there were significant increases in the Pig-a mutant frequency (Pig-a MF) in PIGRET assay at 30 mg/kg/day on Days 8, 15 and 29 with the maximum Pig-a MF on Day 29. On the contrary, there was no change in the Pig-a MF in RBC Pig-a assay. In the single dose study, MMS resulted in a steady increase in Pig-a MF in PIGRET assay from Day 8 through to Day 29 and a delayed increase in Pig-a MF in RBC Pig-a assay at Day 15 and 29 with maximam Pig-a MF on Day 29. MMS also induced significant increases in the MN-RET frequency on Day 4 of the 28-day study and on Day 3 of the single dose study. These results suggest that the PIGRET assay has the following advantages. The accumulated properties with repeated doses would be useful when the assay is integrated with a repeated dose general toxicity study, and the rapid detection of mutagenesis using a single dose protocol would be useful for early assessment of the in vivo mutagenicity of chemicals.

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

Cited by 8 publications

References 39 publications

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

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

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

Pig-a Gene Mutation and Micronucleus Induction in Rat Peripheral Blood by Methyl Methanesulfonate

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