Genetic Toxicology
 

       The group of "Genetic Toxicology" is involved in a preventive approach consisting in screening environmental compounds (genotoxic agents) able to damage the genome of our cells (genotoxic effects) and  to generate mutations at the origin of human cancers .

       We have used the genotoxic response of the bacterial cell to devise a screening test for potential mutagenic and/or carcinogenic agents: the SOS chromotest. The test allows to rapidly evaluate, by a simple colorimetric assay, the capacity of an agent to dammage DNA. It has been evaluated with a large number of compounds (see data base).

       We are also involved in studying the mechanism of action of genotoxic agents. Particularly, we have studied in details the mechanism of action of a very powerful bacterial mutagen, R7000, a nitrofuran. Nitrofuran derivatives are widely used as antibacterial agent in medecine.

      Our objective is to explain, in terms of molecular interactions, why R7000 reaches such a powerful mutagenic potency. We have located the R7000 induced DNA damages and the resulting mutations in Escherichia coli. We have found that R7000 induced DNA adducts with a very high efficiency. These R7000 induced DNA adducts are essentially on modified guanines and the resulting mutations are mainly G:C>T:A base pair substitutions and G:C base pair deletions. The genotoxic activity of nitrofurans requires their metabolic activation catalyzed by nitroreductases. During this metabolism a soxRS-dependant oxidative stress is also generated. Although it is likely a side effect it could contribute to the nitrofuran genotoxicity.

      The genotoxic effects of R7000 has also been studied in an animal model in order to try to evaluate the possible consequences of nitrofurans exposition in human. Transgenic mice, so called "Big-Blue" have been used. These mice present inserted in their genomic DNA a transgenic vector containing the E.coli lacI or phage lambda genes used as reporters genes for the detection of mutations in any mice organs. We found that, when administrated by intraperitoneal route, R7000 induces mutations in small intestine, caecum and colon, organs belonging to the digestive apparatus, the target of the therapeutic action of most nitrofurans. However, when administered orally, this compound is mutagenic in stomach. The mutation spectrum induced by R7000 in mice is very similar to what had been found in E.coli suggesting that the mechanism of its genotoxic action is similar in both organisms.

      Nitrofurantoin and nifuroxazide are two nitrofuran derivatives widely used in human medecine for therapy of urinary tract infections and acute diarrhoea from bacterial origin, respectively. The two compounds are mutagenic in bacteria and consequently they could have long term adverse effects on human health. The mutagenic action of these  two compounds have been evaluated in "Big-Blue" transgenic mice after oral administration. We found that nitrofurantoin induced a mutagenic effect in kidney. No mutagenic effect in any of the organs tested was induced by nifuroxazide.

      In order to study the functions induced by a broader spectrum of toxic agents (potentially carcinogenic genotoxic agents, non-genotoxic carcinogens, various pollutants of the natural environment), we use high density DNA arrays containing all PCR-amplified open reading frame (ORFs) from E.coli K12. The objective is to define classes of toxic agents on the basis of genes for which the expression is modified, and to define genes, or groups of genes, diagnostic for each classe of agents. One considers the spectrum of genes for which the expression is modified by a chemical or physical agent as a "signature"  of the action of the agent on the cell.

      The knowledge of diagnostic genes for classes of toxic agents may bring informations on various aspects of their action (metabolism, activation, stress response) and may reveal the properties of ORF unidentified yet. On an other hand, we hope that this work will lead to the construction of bacterial strains carrying fusions between these diagnostic genes and a reporter gene to allow the screening of these agents in the environment (P.Quillardet).

      The aim of another study in our laboratory is to analyze why infection from bacterial origin are susceptible to induce genotoxic events and lead to cancer development in the population. The same transgenic mice cited above constitute an appropriate tool to analyze if the infection results in an increase of the mutation frequency at the infected site, which could be responsible for events at the origin of precancerous lesions. The model choosen is the infection of the gastric mucosa by Helicobacter pylori  (work in collaboration with Dr A. Labigne's and Dr M. Huerre's teams at the Institut Pasteur). There is now considerable evidence that infection with Helicobacter pylori is an important aetiological factor in the development of gastric carcinoma. Bacterial and host factors as well as diet and environmental conditions are likely to play an important role in this process. The main goals of our study are : i) to evaluate the mutagenic effects susceptible to be induced at the gastric level, during chronic infection by Helicobacter pylori in mice ; ii) to identify host, bacterial or environmental factors which could lead to precancerous lesions upon this infection. Big Blue mice were inoculated intragastrically with a suspension of H.pylori SS1 or H.felis CS1. Animals were sacrificed 6 months later and their stomachs were isolated. A severe gastritis was observed in 100% of H.felis-infected mice and a moderate gastritis in only 25% of the H.pylori-infected mice. However, analyses of the gastric mutant frequency showed an increase of 4.5-fold and 1.7-fold in H.pylori and H.felis-infected mice respectively, as compared to the non-infected groups. This work constitues an animal model for the study of this infectious disease (E.Touati, V.Michel).

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