Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase - P-32-postlabeling analysis of DNA adduct formation

Aristolochic acid (All), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding w hich enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinog en. We examined the ability of prostaglandin H synthase (PHS) to activate A A to metabolites forming DNA adducts with the nuclease P1 and 1-butanol ext raction enrichment procedure of the P-32-postlabeling assay. PHS is a promi nent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patt erns reproducing those found in renal tissues in CHN patients. 7-(Deoxyaden osin-N-6-yl)aristolactam I, 7-(deoxyguanosin-N-2-yl)aristolactam I and 7-(d eoxyadenosin-N-6-yl)aristolactam II were identified as AA-DNA adducts forme d by AAI. Two adducts, 7-(deoxyguanosin-N2-yl)aristolactam II and 7-(deoxya denosin-N6-yl)aristolactam II, were generated from AAII. According to the s tructures of the DNA adducts identified, nitroreduction is the crucial path way in the metabolic activation of AA. The identity of PHS as the activatin g enzyme in RSV microsomes was proven with different cofactors and inhibito rs. Only indomethacin, a selective inhibitor of PHS, significantly decrease d the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CW reductase (cr-lipoic acid) and some selective inhibitors of cytochromes P4 50 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic a cid and hydrogen peroxide, supported the DNA adduct formation of AAI and AA II, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1. (C) 2001 Elsevier Science B .V. All rights reserved.