One of the stubborn problems in toxicology is that laboratory animals and humans do not metabolize chemicals in the same way. For decades, the workhorse solution has been rat liver S9 fraction, a preparation of liver enzymes from rats treated with inducing chemicals such as aroclor 1254, which is added to standard in vitro genotoxicity tests like the Ames test and mammalian cell assays. But tests of certain compounds using alternative S9 sources have produced divergent results, and interest is growing in systems that mimic human metabolism more closely. A new study published in the journal Genes and Environment describes a step toward that goal: an assay that measures DNA damage directly in stable human hepatocytes that carry a broad repertoire of human metabolic enzymes.
The research team, led by Masayuki Mishima and Kei-Ichi Sugiyama, built what they call γH2AX-SHE, an in-cell ELISA system that quantifies γH2AX, a sensitive marker of DNA damage, in HepaSH cells. HepaSH cells are commercially available human hepatocytes produced by incubating and expanding cryopreserved primary human hepatocytes in immunodeficient mice before harvesting. They stably express a range of metabolic enzymes and drug transporters for up to one month in vitro, and previous work showed that a marker of inflammation and tissue injury, C-reactive protein, drops to low levels in these cells, suggesting recovery from the initial damage of isolation.
The choice of γH2AX was dictated by the biology of the cells themselves. HepaSH cells are highly differentiated and non-proliferative, which rules out the conventional genotoxicity endpoints that depend on cell division, including micronucleus formation, chromosomal aberration analysis, and mutant colony assays. γH2AX, by contrast, was originally identified as a marker of DNA double strand breaks after ionizing radiation and is later induced by single strand breaks and base or backbone modifications, largely through replication fork collapse or DNA repair processes. Because the HepaSH cells do not proliferate, the authors suggest the γH2AX induction they observed likely reflects transcription-coupled repair mechanisms.
To establish a reliable baseline, the team compiled data from 246 untreated control wells collected over nine months, spanning eleven production lots of the three HepaSH strains, which derive from three human donors. The mean γH2AX index was 1.00 with a standard deviation of 0.09, and values ranged from 0.78 to 1.57. After excluding the upper and lower 2.5 percent of the distribution, the 95th percentile range fell between 0.85 and 1.22. On that basis, the researchers defined a γH2AX index greater than 1.2 as the threshold for a positive result, while acknowledging that strain-specific thresholds could not yet be established because control data for some strains were insufficient.
The assay then faced its key test: could it detect damage from indirect mutagens, compounds that only become genotoxic after metabolic activation? The answer was yes. Benzo(a)pyrene, aristolochic acid, and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, known as PhIP, all produced dose-dependent increases in γH2AX across all three HepaSH strains after 24-hour exposure. The key human enzymes that generate genotoxic metabolites of these compounds are CYP1A1 for benzo(a)pyrene, NQO1 for aristolochic acid, and CYP1A2 for PhIP, and the positive responses indicate these enzymes effectively activated the test chemicals inside the cells. Notably, PhIP, which is activated by CYP1A2, induced γH2AX at lower doses in HepaSH-L cells, consistent with a reported higher CYP1A2 activity in that strain.
Direct-acting mutagens worked too. Mitomycin C raised the γH2AX index at concentrations of 1 micromolar and above, with the highest index at 63 micromolar where minimal cytotoxicity was detected, and oxaliplatin exceeded the positive threshold at 25 micromolar and higher after 24 hours. Timing mattered, however. In a time-course with benzo(a)pyrene, no positive response appeared at 8 hours or shorter; dose-dependent induction became evident only after 16 hours or longer, with the lowest positive concentrations at 8 micromolar at 16 and 24 hours and 4 micromolar at 48 hours. The authors attribute the longer exposure requirement to the fact that compounds must cross the cell membrane to reach intracellular metabolic enzymes, unlike standard S9-based tests where compounds interact directly with enzymes in the well and treatment typically lasts 3 to 6 hours.
The 48-hour exposure came with a warning. Although γH2AX values were slightly higher at 48 hours than at 24, microscopic examination revealed cells with shrunken nuclei and condensed green fluorescence, a pattern absent at 24 hours. Nuclear pyknosis is a morphological hallmark of apoptosis triggered by genotoxic stress, and since dying cells are not considered a direct cause of carcinogenesis, the authors recommend excluding apoptotic cells when applying γH2AX as a biomarker for genotoxic hazard. They therefore propose 24 hours as the appropriate treatment period for the assay.
Two other conditions proved critical. First, the solvent DMSO suppressed the response: with 200 micromolar benzo(a)pyrene, mean γH2AX indices stayed between 1.50 and 1.80 at DMSO concentrations of 0.2 to 2.0 percent, but fell into the negative range at 3.0 percent or higher, even though cell viability remained at 85 percent or above at every concentration tested. The team recommends keeping DMSO at or below 1 percent, consistent with standard genotoxicity tests, because its effects on enzymatic activity in HepaSH cells remain unclear. Second, cytotoxicity itself can generate false positives. The non-mutagenic compounds sodium dodecyl sulfate and sodium chloride produced hockey-stick dose-response curves, with γH2AX indices exceeding the threshold only at the highest concentrations where cell viability dropped below 60 percent. Such positives at highly cytotoxic doses are considered biologically irrelevant in standard in vitro cytogenetic assays, and the authors adopt the same rule here, while noting that DAPI-based cell counts may underestimate cytotoxicity because damaged cells remain attached to the plate.
Reproducibility across donors was broadly encouraging. Benzo(a)pyrene produced dose-dependent γH2AX induction in all three strains across multiple test days, with minimum positive concentrations of 8 or 16 micromolar for HepaSH-E, 16 micromolar for HepaSH-AD, and 4 micromolar for HepaSH-L, and peak mean indices of 2.52 to 3.69, 2.30, and 2.84 respectively. Strain-specific differences also emerged with aristolochic acid and PhIP, which the authors suggest may reflect individual donor responses, genetic polymorphisms, or enzyme inducibility, though they caution that further confirmation is needed to clarify the extent of strain variability.
Compared with widely used liver-derived cell lines such as HepG2 and HepaRG, HepaSH cells occupy a distinct niche. HepaRG cells, derived from a liver tumor, express various Phase I and Phase II enzymes and have shown good sensitivity to indirect mutagens in comet, micronucleus, and γH2AX assays, but HepaSH cells are non-proliferative, making comet and micronucleus assays inapplicable. What HepaSH offers instead is donor diversity: strains come from different donors and new ones can be readily added, an advantage for studying inter-individual variability and for safety assessments that need to cover a genetically varied human population. The authors conclude that γH2AX-SHE, with its recommended conditions of 24-hour treatment, DMSO at or below 1 percent, and careful interpretation of positives at roughly less than 60 percent cell survival, can serve as a valuable tool for detecting DNA damage under conditions that mimic human metabolic activity. A correction to the article was published on 2 March 2026.
Subject of Research: A γH2AX in-cell ELISA genotoxicity assay developed in stable human hepatocytes expressing human metabolic enzymes.
Article Title: Development of a DNA damage assay system using stable human hepatocytes
Article References: Mishima, M., Izawa, K., Tsuda, M., Higuchi, Y., Uehara, S., Suemizu, H., & Sugiyama, K.-I. (2026). Development of a DNA damage assay system using stable human hepatocytes. Genes and Environment, 48(1), Article 3. https://doi.org/10.1186/s41021-025-00347-7
Image Credits: AI Generated
DOI: 10.1186/s41021-025-00347-7
Keywords: genotoxicity, γH2AX, HepaSH cells, human hepatocytes, DNA damage, in-cell ELISA, metabolic activation, benzo(a)pyrene, aristolochic acid, PhIP, cytochrome P450, in vitro toxicology
News Source: Daisy Hatcher. (October 29, 2026). New DNA Damage Assay Uses Stable Human Hepatocytes to Mimic Human Metabolism. Scienmag.
