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  • Evaluation of in vivo genotoxicity induced by N-ethyl-N-nitrosourea, benzo[a]pyrene, and 4-nitroquinoline-1-oxide in the Pig-a and gpt assays.

Evaluation of in vivo genotoxicity induced by N-ethyl-N-nitrosourea, benzo[a]pyrene, and 4-nitroquinoline-1-oxide in the Pig-a and gpt assays.

Environmental and molecular mutagenesis (2013-10-10)
Katsuyoshi Horibata, Akiko Ukai, Takafumi Kimoto, Tetsuya Suzuki, Nagisa Kamoshita, Kenichi Masumura, Takehiko Nohmi, Masamitsu Honma
ABSTRACT

The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N-ethyl-N-nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4-nitroquinoline-1-oxide (4NQO, 50 mg/kg) in the Pig-a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig-a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7-week terminal sacrifice. ENU increased both Pig-a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig-a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two- or threefold above control) were detected in the bone marrow in both the Pig-a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig-a mutation assay in order to use it as an alternative to the TGR mutation assay.

MATERIALS
Product Number
Brand
Product Description

Supelco
Benzo[a]pyrene solution, 100 μg/mL in cyclohexane, analytical standard
Supelco
Benzo[a]pyrene solution, certified reference material, TraceCERT®, 1000 μg/mL in acetone
Supelco
Benzo[a]pyrene solution, certified reference material, TraceCERT®, 200 μg/mL in methylene chloride
Sigma-Aldrich
Benzo[a]pyrene, ≥96% (HPLC)
Sigma-Aldrich
4-Nitroquinoline N-oxide, ≥98%
Supelco
Benzo[a]pyrene, analytical standard, for environmental analysis
Supelco
Benzo[a]pyrene, certified reference material, TraceCERT®, Manufactured by: Sigma-Aldrich Production GmbH, Switzerland