Detecting N-ethyl-N-nitrosourea-induced mutation in the tissues of mice
using whole-genome HiFi Sequencing
Abstract
Direct sequencing can be used for characterizing mutagenicity in complex
biological models, e.g., in various tissues of mammalian species. We
employed whole-genome High-Fidelity Sequencing (HiFi Sequencing) for
detecting mutations induced in male CD-1 mice by N-ethyl-N-nitrosourea
(ENU). Mice were treated by gavage with a single 40 mg/kg dose of ENU at
12 weeks of age; negative control mice were untreated. Peripheral blood
and solid tissues (liver, spleen, and kidney) were harvested 4 weeks
post-exposure; the erythrocyte Pig-a assay was used to measure mutant
frequencies in peripheral blood, while mutations were evaluated in the
solid tissues by HiFi Sequencing. The frequency of Pig-a phenotypically
mutant total red blood cells and reticulocytes in ENU-treated mice
increased 7- and 30-fold, while the frequency of mutations in the
genomic DNA of solid tissues increased up to 7-fold, with the greatest
increase observed in the spleen and the smallest increase in the liver.
The most common mutations detected by HiFi Sequencing in ENU-treated
mice were T>A transitions and T>C
transversions. The data suggest that HiFi Sequencing complements the
Pig-a reporter gene mutation assay, detecting mutagenicity in tissues
where performing the Pig-a assay is impossible and providing information
on the spectra of mutations which potentially may be useful for
characterizing the genotoxicity of novel compounds.