Adopting Duplex Sequencing Technology for Genetic Toxicity Testing: A Proof-of-concept Mutagenesis Experiment with N-ethyl-N-nitrosourea (ENU)-exposed Rats

Overview

Journal Mutat Res Genet Toxicol Environ Mutagen

Publisher Elsevier

Specialties Environmental Health
Genetics
Toxicology

Date 2023 Sep 28

PMID 37770135

Authors

Stephanie L Smith-Roe

Cheryl A Hobbs

Victoria Hull

J Todd Auman

Leslie Recio

Michael A Streicker

Miriam V Rivas

Gabriel A Pratt

Fang Yin Lo

Jacob E Higgins

Elizabeth K Schmidt

Lindsey N Williams

Daniela Nachmanson

Charles C Valentine Iii

Jesse J Salk

Kristine L Witt

Affiliations

Soon will be listed here.

Abstract

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 10 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays.

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