Currently, the only carcinogenicity test to detect the carcinogenic potential of a compound is carried out on rodents. Due to inter-species differences (metabolism, DNA repair,...), the validity of animal tests in predicting human effects can be questioned. Since 2007, the European Union REACH regulation calls for the development of alternative in vitro methods to minimize the use of animals. These regulations make it possible to quickly and efficiently characterize the genotoxic and carcinogenic potential of the thousands of chemical compounds placed on the market annually.
The γH2AX/pH3 assay that we have developed is a genotoxicity in vitro assay, realized with the In-Cell Western technique (ICW) using human cell lines (INRA license know-how). This innovative test permits to study the genotoxicity and the cytotoxicity of a compound, directly in cellulo. Furthermore, the γH2AX/pH3 method is the only one to allowed determining the genotoxic mode of action of the tested compounds precisely, by differentiating aneugenic and clastogenic compounds (see references, Khoury et al 2015). This test can also make it possible to visualize a protective effect (antioxidant property) of the tested compound on DNA.
A valid innovative and predictive assay
γH2AX assay (INRA license know-how): Our test is based on the phosphorylation of histone H2AX (named γH2AX) that can be considered as a global indicator of genotoxicity. The γH2AX assay thus allows to determined compounds with different mode of genotoxic action, such as DNA adducts, oxidative stress, alkylating agents, aneugenic agents, topoisomerase inhibitors, modification of the dNTP pool... 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.
Differenciate aneugenic and clastogenic compoundsIt is now required to be able to differentiate efficiently genotoxic compounds with aneugenic (modifications in the number of chromosomes) from clastogenic (inducing DNA breaks) mode of action.
In order to meet this purpose, we have implemented our γH2AX assay by the quantification of the phosphorylation of Ser10 of histone H3 (named pH3) which represents a biomarker of aneugenic compounds. Thus, we demonstrated that combined use of these two biomarkers (γH2AX and pH3) allows to efficiently differentiated the aneugenic and clastogenic from cytotoxic compounds (see references, Khoury et al 2015).
Principles: Aneugenic compounds induced either an increase or a decrease in pH3 depending on their mode of action. Clastogens induced γH2AX, and cytotoxic compounds generated a marked decrease in these two biomarkers. Moreover, the use of different cell lines permits to discriminate direct from bioactivated genotoxins without the need of an exogenous metabolic activation system.
Also a cytotoxicity assay
The γH2AX/pH3 test is realized with the In-Cell Western technique, which makes possible to study the cytotoxicity of a compound at the same time as his genotoxic effect. This viability measure is more sensitive than the classical MTT assay (see references, Graillot EMM 2012).
Examples of the application of the γH2AX/pH3 assay
Fig. 1 : Image of a 96-well plate obtained from a scanner after treatment with different compounds. DNA is shown in red, the biomarker of interest is in green.
|Compounds||Metabolic activation required||Mode of actiona||Ames assayb||MN assayc||γH2AX/pH3 assayd`|
Fig. 2 : Results obtained by different genotoxicity tests. a Mode of action: C = Clastogen; A = Aneugen; C/A = both, b Ames assay: in vitro assay realized on bacteria, c Micronucleus assay: in vitro assay on HepG2 cells, d γH2AX/pH3 assay: realized with the In-Cell Western technique on HepG2 cells.