The overall goal of the proposed research is to determine the mechanistic basis for the preferential effects of carcinogenic metals on inducible gene expression. Four of the eight heavy metals on the OSWER list of twenty-two agents of concern, namely, chromium, nickel, arsenic and cadmium, are considered human carcinogens. However, the mechanism(s) by which these metals exert their carcinogenic effects is poorly understood. Previous studies have demonstrated that a variety of genotoxic chemical carcinogens, including chromium and nickel, have strong preferential effects on the expression of several inducible genes in both in vivo and cell culture systems, and that these effects are closely correlated with carcinogen-induced DNA damage. These effects appear to be a result of a unique response of the cell to low overall levels of DNA damage. Other studies have suggested that the carcinogenic but non-genotoxic metals, arsenic and cadmium, can also have profound effects on inducible gene expression, but through different and poorly understood mechanisms. The specific objectives of this research are to determine the molecular basis for the various effects of individual metals on expression of the model hormone-inducible phosphoenolpyruvate carboxykinase (PEPCK) gene, and to investigate the interactions of these pathways when exposure to combinations of carcinogenic metals occurs.
Our specific aims will be to: 1. determine whether specific DNA regulatory regions within the PEPCK promoter are responsible for the cis effects of metals on PEPCK gene expression in the chick embryo in vivo, in H4IIE cells in culture, and in stably transfected H4IIE cell lines containing integrated PEPCK promoter/CAT reporter gene constructs; 2. determine whether functional alterations in specific transcriptional factors are responsible for the trans effects of metals on PEPCK gene expression using DNA footprinting, gel mobility shift and in vitro transcription assays in stably transfected H4IIE cell lines containing integrated PEPCK promoter/CAT reporter gene constructs and/or portions of the PEPCK gene in vitro; and 3. examine the effects of combinations of metals on PEPCK gene expression, and determine whether these effects are a result of interactions between the pathways delineated in Specific Aims 1 and 2 above. We will compare pure samples with samples obtained from toxic waste sites containing known amounts of these metals, to determine whether environmental sources of these agents have the same effects on biological systems when present in complex mixtures. Determining the mechanisms by which these carcinogenic metals selectively alter gene expression would have important implications for understanding the molecular basis for the impact of these agents on the cancer process. While each of these agents may have specific effects and act through independent mechanisms, they may have profound, but different effects when present in different combinations in the environment. Understanding these interactions at the molecular level is critical for an accurate assessment of the overall health effects of these substances on the human population.
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