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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA053491-02
Application #
2095358
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Project Start
1994-01-01
Project End
1996-12-31
Budget Start
1995-01-01
Budget End
1995-12-31
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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Gu, Y; Desai, T; Gutierrez, P L et al. (2001) Alteration of DNA base excision repair enzymes hMYH and hOGG1 in hydrogen peroxide resistant transformed human breast cells. Med Sci Monit 7:861-8
Gutierrez, P L (2000) The metabolism of quinone-containing alkylating agents: free radical production and measurement. Front Biosci 5:D629-38
Li, B; Blough, N V; Gutierrez, P L (2000) Trace detection of hydroxyl radicals during the redox cycling of low concentrations of diaziquone: a new approach. Free Radic Biol Med 29:548-56
Li, B; Gutierrez, P L; Amstad, P et al. (1999) Hydroxyl radical production by mouse epidermal cell lines in the presence of quinone anti-cancer compounds. Chem Res Toxicol 12:1042-9
Li, B; Gutierrez, P L; Blough, N V (1999) Trace determination of hydroxyl radical using fluorescence detection. Methods Enzymol 300:202-16
Ngo, E O; Nutter, L M; Sura, T et al. (1998) Induction of p53 by the concerted actions of aziridine and quinone moieties of diaziquone. Chem Res Toxicol 11:360-8
Li, B; Gutierrez, P L; Blough, N V (1997) Trace determination of hydroxyl radical in biological systems. Anal Chem 69:4295-302
Lenehan, P F; Gutierrez, P L; Wagner, J L et al. (1995) Resistance to oxidants associated with elevated catalase activity in HL-60 leukemia cells that overexpress multidrug-resistance protein does not contribute to the resistance to daunorubicin manifested by these cells. Cancer Chemother Pharmacol 35:377-86
Gutierrez, P L; Siva, S (1995) Diaziquone-glutathione conjugates: characterization and mechanisms of formation. Chem Res Toxicol 8:455-64

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