In the past we have observed that AZQ undergoes bioreductive activation by 1e or 2e reducing the enzymes, and that this activity results in redox cycling producing oxyradicals, the semiquinone, and the hydroquinone. The 2e reduced species of AZQ has also been shown to alkylate more readily than the oxidized parent compound. These results suggest that the mechanism of AZQ cytotoxicity consists of: a) redox cycling with the production of oxyradicals and b) aziridine alkylation enhanced by the reduction of the quinone. This proposal studies the fee radical and alkylation aspects of AZQ activity by focusing first on the glutathione redox cycle, on the specificity of DNA damage and repair and second on the alkylation of AZQ to DNA and to glutathione (GSH). The assessment of relative importance of oxidative stress over alkylation or vice versa will be carried out in MCF-7 human breast cancer cells. Finally, a practical application to pharmacology is achieved by exploring the use of AZQ-GSH adducts as possible markers for toxicity and clinical response. Thus AZQ-GSH adducts will be extracted from human plasma of patients treated with AZQ-treated cells. The GSH redox cycle will be studies by assessing the response of the hexose monophosphate shunt to oxidative stress. This study will be complemented by quantifying intracellular H2O2, by identifying and quantifying oxygen and quinone free radicals in GSH depleted cells, and by determining GSH and GSSG pools. Studies of DNA damage by AZQ involve demarcating the types and specificity of this damage as well as fidelity of this repair. The chemical structure of AZQ-nucleoside/nucleotide adducts will be elucidate. the reaction of AZQ-GSH adducts and establishing the chemical reactions that lead to them. This will identify another pathway for depleting GSH and will provide standards in developing the methodology to extract and quantify these adducts in biological systems. We will also investigate the possibility that the AZQ-GSH adducts redox cycle themselves thus contributing to oxidative stress rather than serving as a way of detoxify AZQ.
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