Duplex Sequencing Provides Detailed Characterization of Mutation Frequencies and Spectra in the Bone Marrow of MutaMouse Males Exposed to Procarbazine Hydrochloride

Abstract

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with >80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Adopting Duplex Sequencing™ Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with N-Ethyl-N-Nitrosourea (ENU)-Exposed Rats

“…How this technology will perform for various types of mutagens still needs to be determined, and a general understanding of the background spontaneous mutation rate and mutation spectra in untreated animals will be necessary to distinguish the DNA mutations associated with chemical exposure via MF or spectra. These findings and those reported by others [15][16][17] support research to explore the adoption of DuplexSeq for the purpose of creating standardized rodent genetic toxicity tests to meet regulatory needs for the detection of point mutations [13], similar to efforts made to develop OECD test guidelines for the TGR and Pig-a gene mutation assays. Compared to currently used assays, direct detection of mutations and identification of mutational spectra in animal models has greater applicability to understanding human exposures to mutagenic substances and may provide more informative data for human cancer risk assessment.…”

“…Recently, MF and spectrum obtained by the cII plaque assay using DNA from BigBlueÒ mice exposed to either N-ethyl-N-nitrosourea (ENU) or benzo[a]pyrene (B[a]P) were replicated by DuplexSeq [15]. A high level of concordance was also observed between DuplexSeq and mutagenesis of the lacZ transgene in the bone marrow of MutaMouse animals exposed to B[a]P [16] or when exposed to procarbazine [17]. An analogous human version of the DuplexSeq mutagenesis assay panel was also used successfully to identify ENU-induced mutations in human lymphoblastoid TK6 cells, an OECDrecommended cell line for genetic toxicity testing [18] and ethyl methanesulfonate (EMS)-induced mutations an in vitro air-liquid-interface airway tissue model used to evaluate the toxicity of inhaled substances [19].…”