ArticleGenes and environment : the official journal of the Japanese Environmental Mutagen Society2026
Development of a DNA damage assay system using stable human hepatocytes.
Article in Genes and environment : the official journal of the Japanese Environmental Mutagen Society, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 2 papers.
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Who cites it
2 citing papers in PubMed.
- Correction: Development of a DNA damage assay system using stable human hepatocytes.Genes and environment : the official journal of the Japanese Environmental Mutagen Society · 2026Article
- A simple suspension culture method for generating human iPSC-derived liver organoids.Biology methods & protocols · 2026Article
Corrections and comments
- Erratum issued
Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundOvercoming species differences in metabolism between humans and animals remains a critical challenge in toxicological studies. Rat liver S9 fraction has long been the gold standard for exogenous metabolic activation in in vitro genotoxicity tests. Experiences with human S9 or human primary hepatocytes have suggested that the human materials are unsuitable for standardized testing due to high variability. Nevertheless, there is growing interest in genotoxicity evaluation using metabolic systems that more closely mimic human physiology.
resultsWe developed an in-cell ELISA system to measure γH2AX as a DNA damage marker in stable human hepatocytes (γH2AX-SHE). HepaSH cells are consistently available human hepatocytes that stably express a range of metabolic enzymes and drug transporters in vitro. Due to their highly differentiated and non-proliferative nature, conventional genotoxicity endpoints such as micronuclei formation, chromosomal aberrations, or mutant colony assays are not applicable. We used γH2AX, a sensitive DNA damage marker, in this assay system. Indirect mutagens including benzo(a)pyrene, aristolochic acid, and 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine induced dose-dependent increases in γH2AX across all three HepaSH strains. Time-course analysis following benzo(a)pyrene exposure indicated that a treatment duration of 16 hours or longer was necessary to detect genotoxic responses. Prolonged exposure for 48 hours resulted in extensive cell death, which may interfere with γH2AX quantification.
conclusionsWe demonstrated that γH2AX-SHE can serve as a valuable tool for detecting DNA damage under conditions that mimic human metabolic activity. Based on the findings in this study, we recommend the following assay conditions for γH2AX-SHE: a 24-hour treatment period, a DMSO concentration not exceeding 1%, and careful interpretation of positive responses observed at highly cytotoxic doses - defined as approximately less than 60% cell survival - as these may lack biological relevance.
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