Application of PECC-Seq for genome-wide mutation analysis with rat tissues from a 28-day general toxicity study

In vivo mutagenicity tests are essential for predicting cancer risks. However, each conventional assay presents distinct challenges. For example, transgenic rodent tests are reliable but have difficulty in incorporating into other regulatory tests and the Pig-a assay can be integrated into other tests but has limitations in applicable tissues. Therefore, alternative methods that overcome such limitations are highly required. Error-corrected sequencing (ECS) technology can detect low-frequency somatic mutations in any tissues of model rodents, making it a promising approach to overcome those limitations at once. To incorporate ECS into general toxicity studies, however, it remains to be determined whether ECS can detect mutations under conditions relevant to such studies. In this study, we applied paired-end and complementary consensus sequencing (PECC-Seq), one of the ECS technologies, to samples from Sprague-Dawley (SD) rats treated with N-ethyl-N-nitrosourea (ENU) as a mutagen. The strain used and study schedule were based on a 28-day general toxicity test and the lower dose level was set to tumorigenic dose 50 (TD₅₀). PECC-Seq successfully detected a dose-dependent increase in mutation frequency and ENU specific mutation spectra in liver and brain. Our results demonstrated that ECS can directly detect mutations in SD rats in conditions of repeated-dose toxicity tests, indicating that ECS can be incorporated into general toxicity tests. Additionally, the detection of mutations even in a TD₅₀-related dose level within a short-term might imply the possibility of assessing carcinogenicity using ECS technology. Collectively, this study provides valuable information for incorporating ECS technology into toxicological workflows.