Duplex sequencing identifies unique characteristics of ENU-induced mutations in male mouse germ cells.

Abstract

Germ cell mutagenicity testing is increasingly required for chemical risk assessment. Duplex Sequencing (DS) is rapidly gaining acceptance as a method to assess in vivo mutagenesis, and as a valid alternative to transgenic rodent (TGR) mutation models such as the MutaMouse. We used a DS panel of 20 genomic targets and the TGR assay to measure mutations in the germ cells of MutaMouse males exposed to 0, 1, 2, or 5 mg/kg N-ethyl-N-nitrosourea (ENU) for 28 days. Germ cells from the seminiferous tubules were collected 28 days post-exposure. The TGR assay showed a significant increase in mutant frequencies at the high (p < 0.001) and medium (p = 0.01) ENU doses relative to controls, while DS revealed a significant increase (p < 0.001) in ENU-induced mutations only at the high-dose. DS mutation frequencies were lower in genic than in intergenic targets, suggesting a protective role for transcription-coupled repair. Interestingly, we observed several unique germ cell characteristics with respect to DS data from rodent somatic tissues: 1) larger inter-animal variability in clonally expanded mutations that affects the ability to detect significant increases in mutation frequency; 2) a target on chromosome 2 showing much higher susceptibility to spontaneous and chemical-induced mutagenesis than other targets; and 3) a mutation spectrum consistent with that observed in the offspring of ENU-treated males but not with the spectrum in bone marrow of directly-exposed males. These results suggest that DS is a promising approach for characterizing germ cell mutagenesis and that mutagenic mechanisms operating in germ cells differ from those in somatic tissues.