Journal Article

Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: evidence for reductive activation by human NAD(P)H:quinone oxidoreductase

Marie Stiborová, Eva Frei, Bruno Sopko, Klára Sopková, Vladimíra Marková, Martina Laňková, Tereza Kumstýřová, Manfred Wiessler and Heinz H. Schmeiser

in Carcinogenesis

Volume 24, issue 10, pages 1695-1703
Published in print October 2003 | ISSN: 0143-3334
Published online October 2003 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgg119
Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: evidence for reductive activation by human NAD(P)H:quinone oxidoreductase

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Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA metabolism is important in the assessment of an individual's susceptibility to this carcinogen. Using the 32P-postlabeling assay we examined the ability of enzymes of cytosolic samples from 10 different human livers and from one human kidney to activate the major component of the plant extract AA, 8-methoxy- 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI), to metabolites forming adducts in DNA. Cytosolic fractions of both organs generated AAI–DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II, indicating a possible demethoxylation reaction of AAI, were identified as AA–DNA adducts formed from AAI by all human hepatic and renal cytosols. To define the role of human cytosolic reductases in the activation of AAI, we investigated the modulation of AAI–DNA adduct formation by cofactors or selective inhibitors of the NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO) and aldehyde oxidase. We also determined whether the activities of NQO1 and XO in different human hepatic cytosolic samples correlated with the levels of AAI–DNA adducts formed by the same cytosolic samples. Based on these studies, we attribute most of the activation of AA in human cytosols to NQO1, although a role of cytosolic XO cannot be ruled out. With purified NQO1 from rat liver and kidney and XO from buttermilk, the major role of NQO1 in the formation of AAI–DNA adducts was confirmed. The orientation of AAI in the active site of human NQO1 was predicted from molecular modeling based on published X-ray structures. The results demonstrate for the first time the potential of human NQO1 to activate AAI by nitroreduction.

Keywords: AA, aristolochic acid; AAN, aristolochic acid nephropathy; AAI, 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid; AAII, 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid; dA–AAI, 7-(deoxyadenosin-N6-yl)aristolactam I; dA–AAII, 7-(deoxyadenosin-N6-yl)aristolactam II; dG–AAI, 7-(deoxyguanosin-N2-yl) aristolactam I; dG–AAII, 7-(deoxyguanosin-N2-yl) aristolactam II; NQO1, NAD(P)H:quinone oxidoreductase; polyethylenimine; XO, xanthine oxidase

Journal Article.  7328 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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