Fraunhofer Institute for Toxicology and Experimental Medicine
In recent years, drugs have been recalled from the market, due to detection of low levels of impurities consisting of N-nitrosamines (NAs). NAs are considered potent carcinogens with a mutagenic mode of action. The regulatory authorities, therefore, recently established a very low threshold of 18 ng/d for all NAs for which no specific carcinogenicity studies are available. But is this threshold justified for all types of NAs? This question has been addressed in the MutaMind project, funded by the European Medicines Agency (EMA) and led by Fraunhofer ITEM. In this project, eight partners – from academia, a regulatory authority, and a contract research organization – aim to better understand the processes contributing to the mutagenicity of NAs and to develop custom-tailored in-vitro tools for sensitive genotoxicity screening. A special focus is on NAs resulting from drugs.
To this end, the consortium is evaluating about 40 NAs and their capacity to form different types of DNA adducts (i.e., chemical modifications of DNA bases) upon metabolic activation. Differences in the corresponding DNA repair processes are investigated by using repair-proficient as well as repair-deficient human and mouse cell models.
To define NA-adapted, sensitive, and predictive genotoxicity screening tools, both DNA base mutations and DNA repair-dependent DNA strand breaks serve as endpoints. The standard Ames test is evaluated regarding the impact of the type and amount of solvent and the metabolic activation system (rat versus hamster) on the detection of mutagenic NAs. In addition, the researchers are evaluating the usefulness and feasibility of the Ames fluctuation assay for NA testing. As an alternative tool, they investigate the sensitivity and predictivity of the in-vitro alkaline comet assay with different liver cell models (HepG2 cells, primary rat and human hepatocytes). First promising results have already been obtained with primary hepatocytes.
To complete the picture, the scientists are exploring the endogenous formation of NAs by bacteria from the human stomach and gut microbiome. To this end, they culture bacteria under anaerobic conditions and expose them individually or in combination to different drugs under physiological conditions. Fist result have already demonstrated differences in the kinetics of NA formation.
The obtained data will eventually be used to better distinguish groups of potent from less potent mutagenic NAs based on their structural properties and biological effects.
