title: 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.
authors.affiliation: Department of Chemicals and Radiation, Norwegian Institute of Public Health, Oslo 0403, Norway.
authors.forename: Anne
authors.initials: A
authors.lastname: Graupner
authors.affiliation: Department of Chemicals and Radiation, Norwegian Institute of Public Health, Oslo 0403, Norway.
authors.forename: Christine
authors.initials: C
authors.lastname: Instanes
authors.affiliation: Litron Laboratories, Rochester, NY 14623, United States.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Dertinger
authors.affiliation: Department of Chemicals and Radiation, Norwegian Institute of Public Health, Oslo 0403, Norway.
authors.forename: Jill Mari
authors.initials: JM
authors.lastname: Andersen
authors.affiliation: Department of Chemicals and Radiation, Norwegian Institute of Public Health, Oslo 0403, Norway.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Lindeman
authors.affiliation: Department of Chemicals and Radiation, Norwegian Institute of Public Health, Oslo 0403, Norway.
authors.forename: Tonje Danielsen
authors.initials: TD
authors.lastname: Rongved
authors.affiliation: Department of Chemicals and Radiation, Norwegian Institute of Public Health, Oslo 0403, Norway.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Brunborg
authors.affiliation: Department of Chemicals and Radiation, Norwegian Institute of Public Health, Oslo 0403, Norway. Electronic address: ann.karin.olsen@fhi.no.
authors.forename: Ann-Karin
authors.initials: AK
authors.lastname: Olsen
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000009:adverse effects* / Q000494:pharmacology; D004249:DNA Damage; D045647:DNA Glycosylases; D004586:Electrophoresis / Q000379:methods; D004913:Erythrocytes, Abnormal / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009153:Mutagens / Q000009:adverse effects* / Q000494:pharmacology; D009154:Mutation; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D009153:Mutagens; D017382:Reactive Oxygen Species; C080086:phosphatidylinositol glycan-class A protein; D001564:Benzo(a)pyrene; D045647:DNA Glycosylases; C485990:Ogg1 protein, mouse
keywords: Alkaline single cell gel electrophoresis (SCGE); Benzo[a]pyrene (BaP); Genotoxicity; Hazard identification; Pig-a mutation assay; Risk assessment