Development of a DNA damage assay system using stable human hepatocytes

Abstract

Background: Overcoming 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.

Results: We 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.

Conclusions: We 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.

Keywords: Genotoxicity; HepaSH cells; Human hepatocytes; Liver s9; Metabolic activation; γh2AX.

Fig. 1

Histogram of γH2AX indices of 246 control wells. The data included indices of different production lots of three strains of HepaSH cells. The range of 95 percentile was from 0.85 to 1.22

Fig. 2

γH2AX index of HepaSH-E cells treated with non-genotoxic test compounds after 24 hours exposure. Blue bars (left vertical axis) indicate the mean gH2AX index from duplicate wells. Circles represent the index of each individual culture well: red indicates values exceeding the positive criteria, black indicates values within the background negative range. Gray lines (right vertical axis) represent the mean relative cell survival

Fig. 3

Dose dependent increase of γH2AX index of HepaSH-E cells treated with direct mutagens after 24 hours exposure. Blue bars (left vertical axis) indicate the mean gH2AX index from duplicate wells. Circles represent the index of each individual culture well: red indicates values exceeding the positive criteria, black indicates values within the background negative range. Gray lines (right vertical axis) represent the mean relative cell survival

Fig. 4

Time course of induction of γH2AX in HepaSH-E cells exposed to BaP. Blue bars (left vertical axis) indicate the mean gH2AX index from duplicate wells. Circles represent the index of each individual culture well: red indicates values exceeding the positive criteria, black indicates values within the background negative range. Gray lines (right vertical axis) represent the mean relative cell survival. Slight precipitation of the test compound was seen at 250 μM

Fig. 5

Microscopic observation of Hepa SH-E cells in in cell ELISA plates after treatment with Bap for 24 or 48 hours. HepaSH cells stained with DAPI on the left column and Alexa488 labeled anti-γH2AX antibody on the right. There were shrunk nuclei with condensed green fluorescent (arrows) after 48 hours treatment with 125 μM BaP

Fig. 6

Reduction of γH2AX index with 3% or higher DMSO concentrations in culture wells. HepaSH-E cells were exposed to 200 μM BaP together with DMSO. Blue bars (left vertical axis) indicate the mean gH2AX index from duplicate wells. Circles represent the index of each individual culture well: red indicates values exceeding the positive criteria, black indicates values within the background negative range. Gray lines (right vertical axis) represent the mean relative cell survival

Fig. 7

Reproducible dose related increase of γH2AX index of HepaSH cells from three donors after treatment with Bap for 24 hours. Tests with HepaSH-E were repeated on different days. Blue bars (left vertical axis) indicate the mean gH2AX index from duplicate wells. Circles represent the index of each individual culture well: red indicates values exceeding the positive criteria, black indicates values within the background negative range. Gray lines (right vertical axis) represent the mean relative cell survival. Slight precipitation of test compound was seen at 250 μM

Fig. 8

Dose dependent increase of γH2AX index of HepaSH cells from three donors after treatment with aristolochic acid for 24 hours. Blue bars (left vertical axis) indicate the mean gH2AX index from duplicate wells. Circles represent the index of each individual culture well: red indicates values exceeding the positive criteria, black indicates values within the background negative range. Gray lines (right vertical axis) represent the mean relative cell survival

Fig. 9

Dose dependent increase of γH2AX index of HepaSH cells from three donors treated with PhIP after 24 hours exposure. Blue bars (left vertical axis) indicate the mean gH2AX index from duplicate wells. Circles represent the index of each individual culture well: red indicates values exceeding the positive criteria, black indicates values within the background negative range. Gray lines (right vertical axis) represent the mean relative cell survival