Metabolism and Detoxification of Base Propenals

University Of Louisville, Louisville KY

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Abstract

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the metabolic pathways that regulate the cytotoxic and mutagenic potential of base propenals (BP) generated from oxidant-induced DNA damage. BP are derived from selective hydrogen abstraction from C4' of the deoxyribose ring, and their formation leads to strand scission. High concentrations of BPs are generated by anticancer drugs such as bleomycin and environmental agents such as Cr (VI). BPs have also been detected in normal, untreated, human cells, suggesting that they are produced from background DNA damage. The BPs are highly reactive unsaturated aldehydes which readily attack cellular nucleophiles such as glutathione and guanine. At high concentrations BPs are cytotoxic and cytostatic, whereas at low concentrations they form potentially mutagenic DNA adducts. Propenal-derived DNA adducts have been detected in high abundance in normal and cancerous human tissues and it has been suggested that they are derived mainly from BPs originating from oxidant-mediated DNA damage. Nonetheless, the metabolic processes that regulate the cytotoxic and mutagenic potential of BPs are not well understood. Based on our preliminary data and literature evidence, we propose that glutathione S-transferase (GSTP1-1) and aldose reductase (AR) are the major enzymes that participate in the metabolism of base propenals and that the sequential biotransformation by these enzymes prevents the toxicity and mutagenicity of base propenals. To test this hypothesis we will identify the major cellular and urinary metabolites of base propenals generated by COS-7 and HepG2 cells or mice exposed to radiolabeled adenine and thymine propenals (Aim 1). Next we will delineate the contribution of AR and GSTP1-1 to the overall metabolism of base propenals using pharmacological and molecular strategies, and elucidate the metabolism of propenal in AR-inhibitor treated or GSTP1-1 null mice (Aim 2). To test the catalytic efficiency of AR and GSTP1-1, we will examine their steady-state kinetic properties and determine whether product or substrate inhibition limits propenal metabolism via this pathway (Aim 3). Finally, to delineate the role of GSTP1-1 and AR in preventing the acute toxicity of base propenals and their ability to form DNA adducts (Aim 4), we will test whether inhibition or overexpression of these enzymes prevents base propenal cytotoxicity and adduct formation, and whether the mutagenic burden due to base propenal exposure is altered in GSTP1-1-null or AR inhibitor-treated mice. The results of these studies will provide a better understanding of the cytotoxic effects of DNA degradation products and the mechanism by which they indirectly induce potentially mutagenic DNA lesions. Our results may also help in designing more effective and targeted anticancer interventions and could lead to the identification of organ systems and individuals more sensitive to endogenous and environmental oxidants.