Genotoxicity biomarkers ex vivo et in vivo. – GENOTOXTRACK

Genotoxic substances induce DNA damage. DNA double-dtrand breaks (DSBs) are considered the most lethal form of DNA damage as they can result in cell death and, if misrepaired, they have the potential to result in chromosomal translocations and genomic instability.
The H2AX protein phosphorylation at its C-terminal part, gH2AX, is a sensitive and a quantitative way to study DSB and to measure the DNA repair. However, the detection of gH2AX depends on antibodies used on fixed cells. Here, we propose to develop biomarkers to detect and follow DNA damage and their repair mechanisms ex vivo, within living cells, and in alternative animal models.
As a proof-of concept, nanobodies directed against gH2AX will be developed but we will also develop other DSBs and/or repair biomarkers. In addition to the conventional antibodies, llamas produce antibodies only composed of heavy chains and their antigen-binding site is formed by a single domain (VHH or nanobody). After immunization, a llama VHH library will be made and screened by phage display. Nanobodies binding to the gH2AX target will be isolated and their sequence subcloned in fusion with a fluorescent tag in a mammalian vector. Each fluobody will be tested in human cells and different parameters will be assayed (foci formation, signal amplification/disappearance, fluorescent background) to validate the biomarkers.
At this step of the program, we will develop the FRET technology to enhance the signal and follow, at the same time, two markers. Particularly, new markers of repair or apoptosis, on H2AX histone, have been recently described in human cells. We will test different markers association to differenciate specifically among these processes and to follow the cell output of a genotoxic exposure. Biomarkers allowing to follow, real-time, the occurrence of DSB and to determine genotoxicity in vivo, will be patented.
A direct application of our tools will be to study genotoxins, produced by E. coli bacteria -pathogenic or not. Genotoxins induce cell cycle defect and DSBs in host cells. We will study the mechanisms leading to DSBs in order to define new targets for biomarkers and we will use our tools to screen bacteria strains and assay their potential genotoxicity. We will particularly focus on intra-S checkpoint as it bridges together replication, repair and cell cycle in a coordinated network. We will also particularly study which repair mechanism is set up in different context (toxic dose, time exposure, mutations/knocked-down) as a deregulation of these processes may lead to genomic instability.
Finally, we plan to transfert our tools in alternative animal models -zebrafish or nematod- to track genotoxic pollutants in rivers and/or soils.
Overall, this project will help to develop exclusive tools to determine the toxicity of known or new substances, and to follow genotoxic stress ex vivo or in vivo. It will help to analyse the cell output after a genotoxic exposure and o predict processes leading to genetic instability.
In conclusion, our tools will help to gain insights on these mechanisms and be used in the prevention and/or diagnosis of exposure to genotoxic, improving environmental protection and human health.