Direct quantification of gamma H2AX by cell‐based high throughput screening for evaluation of genotoxicity of pesticides in a human thyroid cell lines

Abstract

Genotoxicity is thought to be the cause of many cancers. Genotoxicity due to environmental toxins may be partly responsible for the dramatic increase in the incidence of papillary thyroid cancer over the past two decades. Here, we present a fully automatable assay platform that directly quantifies the phosphorylation of nuclear histone gamma H2AX (γH2AX), a specific cellular marker for DNA double strand breaks (DSBs) via immunohistochemistry and laser scanning cytometry. It multiplexes γH2AX with total cell number measured as propidium iodide and calculates the percentage of cells with DSBs. Validation of this assay using NTHY-ori-3–1 human thyroid cells and etoposide showed that it was an excellent choice for high throughput applications. We used the assay to test the genotoxic effects of the EPA Toxcast Phase 1 pesticide library of 309 compounds. Compounds were evaluated in dose response and the DSB was quantified. We found that 19 pesticides induce DSB in vitro, highlighting a need to further assess these pesticides for their long-term oncogenic effects on the thyroid gland.

A High-Throughput, Time-Resolved Fluorescence Approach Increases γ-H2AX Detection

“…This approach is tedious and has led to the development of high-throughput automated foci detection to quantify DNA DSBs within cell nuclei 6,7,33,34,37,38,42 . Some of these methods include laser-scanning cytometry 7 , infrared imaging scanners 36 , imaging flow cytometry 37 , and automated γ-H2AX immunocytochemistry 33 . Most of these methods have been proven robust and exhibit different degrees of resolution.…”

“…Many studies have shown that the number of γ-H2AX foci directly correlates with the number of DNA DSBs in cells 11,20,21 . In recent years, many high-and low-throughput methods have been developed to detect and quantify γ-H2AX expression as a function of DNA DSBs in different cells derived from various tissues 6,7,[33][34][35][36][37][38] . Immunofluorescence techniques and epifluorescent microscopy have been commonly used to count γ-H2AX foci with the naked eye 3,13,31,[39][40][41] .…”

“…They are difficult to repair and can lead to genotoxicity, cell death, and misrepaired DNA DSBs with potential for neoplastic progression. Exogenous factors inducing DNA DSBs include cytotoxic chemical agents and environmental and physical factors such as radiation and heat [2][3][4][5][6][7][8][9][10] . The ability of different forms of irradiation to induce DNA DSBs has been harnessed in radiotherapy treatments to kill cancer cells.…”

“…These methods are often less sensitive, tedious, and require counting γ-H2AX foci using an epifluorescent microscope or other expensive equipment such as a flow cytometer or confocal microscope. A variety of robust high-throughput methods have been established to quantify γ-H2AX in cells 6,7,[33][34][35][36][37][38] . Most of these systems are automated and designed to detect γ-H2AX in large-scale studies and are therefore suitable for laboratories specialized in handling large amounts of samples.…”

Lead Induces Genotoxicity via Oxidative Stress and Promoter Methylation of DNA Repair Genes in Human Lymphoblastoid TK6 Cells

“…There is a positive correlation between DSBs and γ-H2AX and γ-H2AX is an acknowledged marker for DSBs. The quantification of γ-H2AX has been widely used for evaluation of genotoxicity in many studies [ 28 – 30 ]. In addition, γ-H2AX is a DSB biomarker that reveals different types of DNA damage, notably DNA adducts and oxidative DNA lesions [ 31 , 32 ].…”