Comparative potency approach based on H2AX assay for estimating the genotoxicity of polycyclic aromatic hydrocarbons

Abstract

a b s t r a c t a r t i c l e i n f oPolycyclic Aromatic Hydrocarbons (PAHs) constitute a family of over one hundred compounds and can generally be found in complex mixtures. PAHs metabolites cause DNA damage which can lead to the development of carcinogenesis. Toxicity assessment of PAH complex mixtures is currently expressed in terms of toxic equivalents, based on Toxicity Equivalent Factors (TEFs). However, the definition of new TEFs for a large number of PAH could overcome some limitations of the current method and improve cancer risk assessment. The current investigation aimed at deriving the relative potency factors of PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a new genotoxic assay (γH2AX) with two human cell lines (HepG2 and LS-174T) to analyze the genotoxic properties of 13 selected PAHs at low doses after 24 h treatment. The dose-response for genotoxic effects was modeled with a Hill model; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. In the two cell lines tested, we observed a clear dose-response for genotoxic effects for 11 tested compounds. LS-174T was on average ten times more sensitive than HepG2 towards PAHs regarding genotoxicity. We developed new TEFs, which we named Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were generally higher than the TEF usually used. Our study proposed a new in vitro based method for the establishment of relevant TEFs for PAHs to improve cancer risk assessment.

Evidence of the in vitro genotoxicity of methyl-pyrazole pesticides in human cells

“…It was also reported that micronucleus formation is correlated with H2AX phosphorylation [7] and that ␥H2AX is a reliable biomarker of pre-cancerous cells in vivo [8,9]. These data support the assumption that H2AX phosphorylation could be an appropriate biomarker of genotoxicity, as suggested in recent in vitro and in vivo studies [10][11][12][13][14][15][16][17][18][19].…”

“…The In Cell Western technique was previously reported to allow the determination of cell viability in parallel to genotoxicity for adherent cells (ACHN, HepG2, LS-174T and SH-SY5Y) [10,11,18,19]. Briefly, after treatment with pesticides, cells were fixed with 4% paraformaldehyde (Electron Microscopy Science) for 20 min for adherent cells.…”

“…Then, from these 21 pesticides, we retained at least 14 compounds: ten pesticides registered in the Annex I of the 2008 European directive 91/414 (benalaxyl, esfenvalerate, penconazole, pirimicarb, propyzamide, pyriproxyfen, tebufenpyrad), three for which the use was still authorized in 2008 (acrinathrin, bupirimate, myclobutanil), and four pesticides that were considered to be persistent in the environment [23] (dieldrin, chlordane, lindane, heptachlor) ( Table 1). The aim of this study was to screen the cytotoxicity and genotoxicity of the 14 selected pesticides with the ␥H2AX In Cell Western (ICW) assay [10,11,18,19]. The advantage of the ICW methodology is the simultaneous determination of cytotoxicity and genotoxicity of xenobiotics on cells cultured in a 96-well plate format [10,11].…”

Novel approaches to improving the chemical safety of the meat chain towards toxicants

“…More than 30 PAHs have been identified; among them 13 are genotoxic (Audebert, Zeman, Beaudoin, Pery, & Cravedi, 2012;Audebert et al, 2010) or carcinogenic (EFSA, 2008; IARC, 2010b) and they are most probably neurotoxic (Crépeaux et al, 2013;Schroeder, 2010). It should be emphasized that processing procedures, such as smoking, drying, and cooking of food are commonly thought to be the main sources of contamination by PAHs (Scientific Committee on Food, 2002).…”

Absolute quantification of γH2AX using liquid chromatography–triple quadrupole tandem mass spectrometry

“…The findings that one γH2AX focus corresponds to one double strand break [3,5] and that a broad spectrum of mutagens can induce γH2AX (reviewed in [6]) ensures that γH2AX is a sensitive indicator of DNA damage. In regulatory science, γH2AX is expected to be a useful biomarker of genotoxicity of chemicals [7][8][9][10][11][12] and is also used for detection of genotoxic environmental pollutants such as polyaromatic hydrocarbons and cigarette smoke [13][14][15]. In pharmacology, γH2AX is a good indicator of efficacy of cancer drugs which damage DNA and also may be a potential target of cancer drugs [16,17].…”