Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

Abstract

In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96-well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In-Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90-100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high-throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics.

Genotoxicity of 11 heavy metals detected as food contaminants in two human cell lines

“…The In‐Cell Western technique was performed as previously described [Graillot et al, ; Khoury et al, ,b; Chevereau et al, ]. Briefly, after the treatment, cells were fixed with 4% paraformaldehyde (Electron Microscopy Science) in phosphate buffered saline (PBS) and permeabilized with 0.2% Triton X‐100.…”

“…The γH2AX assay was chosen in the present study to simultaneously measure cell viability (relative cell count) and overall genotoxicity [Graillot et al, ; Khoury et al, ]. The phosphorylation of histone H2AX (γH2AX) is an early and sensitive marker of genotoxicity because this biomarker can be induced by different type of DNA lesions including notably DNA adducts, DNA abasic sites, DNA strand breaks and DNA replication or transcription blocking lesions [Jeggo and Lobrich, ; Khoury et al, ], and including those hypothesized to be induced by heavy metals.…”

A Central Role for Heme Iron in Colon Carcinogenesis Associated with Red Meat Intake

“…H2AX phosphorylation (gH2AX) is a rapid and sensitive cellular response to genotoxicity (25,26). Genotoxicity and cytotoxicity of aldehydes were measured after 24 hours of treatment of Apc þ/þ and Apc À/þ cells using a gH2AX in-cell Western blot assay according to Audebert and colleagues (27,28).…”

Are effects of common ragwort in the Ames test caused by pyrrolizidine alkaloids?

“…Generally, a combination of the Ames test and the micronucleus test is regarded as a powerful in vitro genotoxicity battery [19]. Another promising genotoxicity assay is the test based on the quantification of the phosphorylation of the histone H2AX in HepG2 cells [18]. The combined outcomes of a set of tests might improve the risk assessment of compounds, complex mixtures and products like plants that can be part of our food chain [12].…”