Single cell gel electrophoresis (SCGE) and Pig-a mutation assay in vivo-tools for genotoxicity testing from a regulatory perspective: a study of benzo[a]pyrene in Ogg1(-/-) mice

Abstract

The OECD has developed test guidelines (TG) to identify agents with genotoxic effects. The in vivo alkaline single cell gel electrophoresis (SCGE) assay is currently being prepared to become such a TG. The performance of a combined SCGE/Pig-a gene mutation study was evaluated with the prototypical genotoxicant benzo[a]pyrene (BaP) at an exposure level known to induce germ cell mutation. We aimed to better understand (i) the strengths and weaknesses of the two methods applied in blood and their potential to predict germ cell mutagenicity, and (ii) the involvement of reactive oxygen species (ROS) following in vivo BaP-exposure. To explore the involvement of ROS on BaP genotoxicity, we utilised a mouse model deficient in a DNA glycosylase. Specifically, C57BL/6 mice (Ogg1(+/+) and Ogg1(-/-)) were treated for three consecutive days with 50 mg BaP/kg/day. DNA damage in nucleated blood cells was measured four hours after the last treatment with the SCGE assay, with and without formamidopyrimidine DNA glycosylase (Fpg). Pig-a mutant phenotype blood erythrocytes were analysed two and four weeks after treatment. BaP-induced DNA lesions were not significantly increased in either version of the SCGE assay. The phenotypic mutation frequencies for immature and mature erythrocytes were significantly increased after two weeks. These effects were not affected by genotype, suggesting oxidative damage may have a minor role in BaP genotoxicity, at least in the acute exposure situation studied here. While both assays are promising tools for risk assessment, these results highlight the necessity of understanding the limitations regarding each assay's ability to detect chemicals' genotoxic potential.

Keywords: Alkaline single cell gel electrophoresis (SCGE); Benzo[a]pyrene (BaP); Genotoxicity; Hazard identification; Pig-a mutation assay; Risk assessment.

Figure 1. Metabolism of Benzo[a]pyrene (BaP)

The metabolism primarily occurs via cytochrome P450s (CYP1A1 and CYP1B1), peroxidases (epoxide hydrolase) and aldo-keto reductase. The major metabolite which is thought to cause carcinogenesis is BaP-7,8-diol-9,10-epoxide (BPDE) which is metabolised via BaP-7,8-epoxide and BaP-7,8-diol. BPDE forms DNA-adducts which can induce G→T transversions. In addition, many metabolites (such as catechol and dione within a futile cycle) form reactive oxygen species (ROS) thus adding to oxidative stress and contributing to the formation of oxidative DNA lesions such as 8-oxoguanine, a pre-mutagenic DNA lesion. Radical cations are formed in a minor quantity.

Figure 2. Study design

Eight C57BL/6 mice of two different genotypes (Ogg1^+/+ and Ogg1^−/−) were treated with BaP on three consecutive days (total of 150 mg/kg bw, ip). In addition, three mice of each genotype were injected with corn oil as vehicel control. Blood for the Pig-a mutation assay was drawn one week prior BaP-treatment (day −5), and two weeks (day 16) and four weeks (day 34) after the last chemical treatment. Blood for the SCGE was drawn four hours after the last injections of BaP or vehicle control.

Figure 3. DNA lesions in mouse blood cells measured by the alkaline SCGE without (A) and with Fpg (B) four hours after treatment with BaP

Each circle represents data for one mouse as mean (n = 8 for BaP, n = 3 for corn oil), based on three technical replicates per mouse. Mouse genotypes and treatments are indicated on the horizontal axis. The box plot shows the median and the first and third quartile.

Figure 4. Pig-a mutant frequencies and percent reticulocytes in blood cells of mice treated with BaP

Data is given as median (SD) for the percentage of reticulocytes (A), and the mutation frequencies of red blood cells (B) and reticulocytes (C). Data was compared to each other by Dunnett’s multiple comparison within a one way ANOVA. Significant differences to the vehicle control (corn oil) of the same genotype and sampling day are indicated by asterisks: p ≤ 0.05 (*) and p ≤ 0.001 (**). Significant differences between day 16 and day 34, respectively, to day −5 are marked with crosses: p ≤ 0.05 (⁺) and p ≤ 0.001 (⁺⁺).