A Functional Toxicogenomics Approach to Characterize Genotoxicity Mechanisms of the Environmental Contaminant Trichloroethylene

Trichloroethylene (TCE) is an environmental contaminant and human carcinogen that remains an environmental health hazard decades after its introduction. While evidence from rodent and epidemiological studies suggests a mutagenic mode of action mediating TCE kidney carcinogenesis, there remains a lack of molecular evidence to support the association between TCE exposure, mutagenesis and cancer. A clearer understanding of the molecular mechanisms mediating TCE exposure and cancer will strengthen risk assessment analyses, exposure standards and policies regarding TCE cleanup. Advances in genomic technologies make a functional genomics approach in Saccharomyces cerevisiae an appealing platform for elucidating toxicity mechanisms for a range of environmental contaminants. The studies of this dissertaion aim to utilize functional profiling platforms in model organisms to (1) identify novel insights into heavy metal and TCE toxicity; (2) assess and characterize the genotoxicity of TCE; and (3) identify candidate human toxicant susceptibility genes./nHeavy metals are widely distributed environmental contaminants increasingly associated with a range of adverse health effects, including neurological disease, developmental abnormalities and cancer. We used functional profiling in yeast to identify ion specific and common molecular pathways that mediate heavy metal toxicity. Our studies with the metals cadmium, lead and zinc revealed that a common subset of pathways and processes, including intracellular trafficking, vacuolar function and protein catabolism are required in response to heavy metal exposure. In the presence of Pb, Cd, and Zn, mutants deficient in components of iron and copper metabolic pathways were hypersensitive, suggesting that metal toxicity is mediated by alterations in iron metabolism. Copper is required for iron uptake, suggesting iron deficiency may be a secondary effect of copper deficiency. Thus, some of the cytotoxic effects associated with these metals could result from disruption of metal homeostasis. These studies revealed the importance of metal homeostasis and identified additional mechanisms important in heavy metal toxicity./nA similar functional genomics approach in yeast was employed to profile trichloroethylene metabolites, including the implicated penultimate metabolite in the kidney, dichlorovinyl cysteine (DCVC). Specific DNA repair pathways such as 1) error prone translesion synthesis repair 2) nucleotide excision repair and 3) homologous recombination were required in response to DCVC exposure. The phenotypic profile generated by DCVC showed high similarity to those of known DNA interstrand crosslinking agents, implicating direct DNA damage and mutagenic repair as a potential mechanism of renal toxicity. A combination of functional studies in the avian DT40 system and human cell lines deficient in DNA repair were conducted to confirm and further characterize the DNA damage repair response to DCVC. While DNA repair in eukaryotes is highly conserved, these platforms allowed for additional analysis of DNA repair systems not present in yeast. DT40 translesion synthesis mutants were hypersensitive to DCVC, as observed in yeast and supported a role for mutagenic DNA repair in TCE toxicity. This hypothesis was further supported by an increased frequency of point mutations, insertions and deletions at low DCVC exposure levels. Interestingly, strains defective in the Fanconi anemia (FA) repair pathway and homologous recombination were unaffected and hyper-resistant, respectively, to DCVC exposure. This finding is in contrast to prior studies describing the repair of ICL agents and suggests a recombination independent mechanism for repairing DCVC induced DNA damage. Taken together these in vitro studies provide mechanistic evidence supporting a mutagenic mode of action for TCE that is mediated by the metabolite DCVC.

Advisor: Vulpe, Christopher