Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines

Audebert, Marc; Riu, Anne; Jacques, Carine; Hillenweck, Anne; Jamin, Emilien L.; Zalko, Daniel; Cravedi, Jean Pierre

doi:10.1016/j.toxlet.2010.08.022

Cited by 100 publications

(92 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The In Cell Western technique was performed essentially as previously described (Audebert et al, 2010;Audebert et al, 2011;Graillot et al, 2012). Briefly, cells were dispensed in 96-well cell culture plate (40 × 10 3 cells/200 μL/well) and were treated in duplicate, 16 h later, with compounds or vehicle (DMSO) in serum free medium.…”

Section: In Cell Western (Icw) Assaymentioning

confidence: 99%

“…Raw absorbance data was averaged for the duplicate, corrected for background; the relative fluorescence units from the scanning allowed a quantitative analysis. The ICW technique allows the determination of cytotoxicity and genotoxicity in a single experiment (O'Brien et al, 2006;Audebert et al, 2010;Audebert et al, 2011). For determination of genotoxicity, relative fluorescent units for γH2AX per cell (as determined by γH2AX divided by DNA content) were divided by the respective controls (vehicle only), in order to determine the change in phosphorylation of H2AX levels relative to control.…”

Section: In Cell Western (Icw) Assaymentioning

confidence: 99%

“…A new sensitive genotoxic assay based on histone H2AX phosphorylation detection (Audebert et al, 2010;Audebert et al, 2011) was used to determine the genotoxicity of 13 different PAHs on HepG2 cell line. All PAHs were analyzed for cytotoxicity and genotoxicity after 24 h treatment at different concentrations covering a four order of magnitude range (Fig.…”

Section: Genotoxic Data In Human Cell Linesmentioning

confidence: 99%

“…In a previous study, we examined three different cell Toxicology and Applied Pharmacology 260 (2012) 58-64 lines for their ability to biotransform PAHs (Audebert et al, 2010). We concluded that liver cells (HepG2) and colon cells (Caco-2) were able to metabolize PAHs.…”

Section: Introductionmentioning

confidence: 99%

“…We concluded that liver cells (HepG2) and colon cells (Caco-2) were able to metabolize PAHs. Nevertheless, the Caco-2 cell line was not suited to our genotoxic assay (Audebert et al, 2010). This new genotoxic assay was based on the detection of the phosphorylation of the histone H2AX (named γH2AX) that reflects a global genotoxic insult resulting from diverse type of DNA damage, notably DNA adducts and oxidative lesions (Zhou et al, 2006;Watters et al, 2009;Audebert et al, 2011;Graillot et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Audebert

Zeman

Beaudoin

et al. 2012

Toxicology and Applied Pharmacology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: In Cell Western (Icw) Assaymentioning

confidence: 99%

Section: In Cell Western (Icw) Assaymentioning

confidence: 99%

Section: Genotoxic Data In Human Cell Linesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Audebert

Zeman

Beaudoin

et al. 2012

Toxicology and Applied Pharmacology

Self Cite

View full text Add to dashboard Cite

show abstract

Genotoxicity of pesticide mixtures present in the diet of the French population

Graillot

Takakura

Hégarat

et al. 2012

Environ and Mol Mutagen

View full text Add to dashboard Cite

Consumers may be simultaneously exposed to several pesticide residues in their diet. A previous study identified the seven most common pesticide mixtures to which the French population was exposed through food consumption in 2006. The aim of this study was to investigate if the seven mixtures are potentially cytotoxic and genotoxic and if so, whether compounds in a same mixture have a combined effect. The cytotoxicity and genotoxicity of the seven mixtures were investigated with a new assay (γ-H2AX) using four human cell lines (ACHN, SH-SY5Y, LS-174T, and HepG2). Mixtures were tested at equimolar concentrations and also at concentrations reflecting their actual proportion in the diet. Irrespective of the cell line tested, parallel cytotoxicity of the seven mixtures was observed. Only one mixture was genotoxic for the HepG2 cells at concentrations = 3 μM in equimolar proportion and at 30 μM in actual proportion. Caspase 3/7 activity, the comet assay, and reactive oxygen species production were also investigated using the same mixture and HepG2 cells. Our results suggest that pesticide metabolites from the mixture generated by HepG2 cells were responsible for the observed damage to DNA. Among the five compounds in the genotoxic mixture, only fludioxonil and cyprodinil were genotoxic for HepG2 cells alone at concentrations = 4 and 20 μM, respectively. Our data suggest a combined genotoxic effect of the mixture at low concentrations with a significantly higher effect of the mixture of pesticides than would be expected from the response to the individual compounds. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.

show abstract

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

Khoury

Zalko

Audebert

2013

Environ and Mol Mutagen

View full text Add to dashboard Cite

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.

show abstract

Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines

Cited by 100 publications

References 73 publications

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

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

Genotoxicity of pesticide mixtures present in the diet of the French population

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

Contact Info

Product

Resources

About