Research at the Department of Genetic and Molecular Toxicology is focused on the fundamental molecular mechanisms involved in the cascade of genotoxic/carcinogenic effects of xenobiotics as well as on the factors of individual susceptibility modulating genotoxic and/or carcinogenic effects.
Current research focuses on the following main topics:
- The molecular events involved in the mechanisms of the genotoxic effects induced by environmental and occupational xenobiotics.
- The determination of specific DNA damage and its repair in the light of individual susceptibility.

      Numerous environmental and/or occupational xenobiotics to which humans are exposed cause various adverse effects (general toxicity, disturbances in the immune system) and may tend to accumulate in the body if the enzymatic metabolizing system is less efficient. On the other hand, metabolic conversion may result in the formation of highly reactive intermediates, which are usually responsible for genotoxic effects due to a direct attack on the nucleophillic centers of biological macromolecules.
      Current projects in our laboratory are focused on the determination of various biomarkers (defined as indicators of exposure, effect and individual susceptibility) that are intended to cast some light on possible causality both in carcinogenesis and teratogenesis. Particular attention is being paid to the specific detection and quantitation of DNA damage (DNA adducts, single-strand breaks in DNA) and DNA repair processes. In general, in our studies we employ in vitro systems (e.g. mammalian cell lines) and in vivo models (mice, rats). Finally, the results obtained serve as a basis for comprehensive studies in the human population.
      In order to clearly understand the molecular events involved in the mechanisms of the genotoxic effects induced by environmental and occupational xenobiotics, the environmental and occupational contaminants styrene and 1,3-butadiene have been selected as model compounds.

Fig. 1: Events involved in the cascade of genotoxic effects following exposure to xenobiotics.

      Styrene, one of the most frequently used monomers in the plastics industry, serves as an excellent model compound. The mechanisms of styrene genotoxicity are not fully understood. The quantitative and qualitative determination of various styrene-induced DNA adducts at N7- and N2- guanine, N3-, N1- and N6-adenine, N3-uracil, N1-hypoxanthine and N4-cytidine represents an useful, very sensitive tool for studying primary DNA damage. Special effort is devoted to the relationships among different types of styrene-specific DNA adducts, single-strand breaks in DNA and HPRT (or APRT) mutant frequencies, and to the understanding of DNA repair processes in cell cultures and experimental animals. Similarly, the relationship between occupational exposure to styrene, DNA and haemoglobin adducts, SSB, HPRT mutant frequencies and cytogenetic endpoints has been studied in a group of styrene-exposed workers. Very recently, a new urinary metabolite of styrene, 4-vinyl phenol, has been identified in humans. This finding suggests biotransformation via arene oxidation, resulting in highly reactive 3,4-arene oxide, which may contribute to the genotoxic burden of styrene.

Fig. 2: An illustration of the specific N1-adenine DNA adducts in human (HPLC/radioisotope detection).

      1,3-Butadiene (BD), an important industrial and environmental xenobiotic, was shown to be genotoxic, but the mechanisms of BD genotoxicity are not clear. BD has been classified by IARC as a possible human carcinogen, group 2A. An investigation of the relationships between various biomarkers, DNA repair as well as adaptive response in vitro and in animal experiments would be of particular interest. The studied biomarkers will be applied in a population study of BD-exposed workers in a tyre plant. Within this project, new aspects will be implemented:
1. Quantitative and qualitative detection of primary DNA lesions (specific DNA adducts),
2. An emphasis on individual susceptibility at the metabolic level (genotyping the whole set of main metabolizing enzymes) on on the level of DNA repair (individual repair capacity) and the level of adaptive responses. An additional novel contribution is the validation of individual biomarkers as a tool for predicting individual risk and for implementing preventive measures in humans exposed to BD. Current projects, employing a wide range of different biomarkers (markers of exposure, effect and individual susceptibility) in different systems (in vitro on various cell cultures, in vivo on animals and in population studies), may contribute to our understanding of the complex mechanisms of genotoxic effects.

      DNA repair processes play a key role in the removal of tentative DNA damage, thus preventing mutagenicity and/or carcinogenicity. Our effort has been aimed at understanding the DNA repair processes of the damage induced by various xenobiotics in cell cultures and experimental animals. Apparently, DNA repair seems to remove DNA damage induced by simple alkyl epoxides by different mechanisms (BER, short-patch repair) in comparison to methylating agents (BER, long-patch repair) and polycyclic aromatic hydrocarbons (NER). The mechanisms of DNA repair of the damage induced by styrene and 1,3-butadiene are currently studied on human xeroderma pigmentosum cell lines (XPA and XPC cells) with deficient nucleotide excision repair.

Fig. 3: A simplified schema of the most important DNA repair pathways

      Human subjects vary widely in their susceptibility to genotoxic chemicals. The genotyping of metabolizing (CYP 2E1) and detoxifying enzymes (GST, EPHX), along with the determination of individual DNA repair capacity, may represent a valid approach to individual risk assessment in styrene-exposed humans. Additionally, variant alleles were discovered for various DNA repair genes, such as XPD, XPG and XPC involved in NER, XRCC1 involved in BER and XRCC3 associated with double-strand break/recombination repair. DNA repair polymorphisms and their associations with the levels of biomarkers in humans are at present under investigation. The project aims to identify the role of individual occupational risk factors and to develop relevant markers that could be used in the identification of individuals at risk in workplaces.