Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:Cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid‐DNA adducts found in patients with urothelial cancer

Abstract

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 activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. Using the 32 P postlabeling assay, we examined the ability of microsomal samples from 8 human livers and from 1 human kidney to activate AAI, the major component of the plant extract AA, to metabolites forming adducts in DNA. Microsomes of both organs generated DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(deoxyadenosin-N 6 -yl)aristolactam I, 7-(deoxyguanosin-N 2 -yl)aristolactam I and 7-(deoxyadenosin-N 6 -yl)aristolactam II were identified as AA-DNA adducts formed from AAI by all human hepatic and renal microsomes. To define the role of human microsomal enzymes in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors and selective inhibitors of microsomal reductases, cytochrome P450 (CYP) enzymes, NADPH:CYP reductase and NADH:cytochrome b 5 reductase. We also determined whether the activities of CYP and NADPH:CYP reductase in different human hepatic microsomal samples correlated with the levels of AAI-DNA adducts formed by the same microsomal samples. On the basis of these studies, we attribute most of the activation of AAI in human hepatic microsomes to CYP1A2. In contrast to human hepatic microsomes, in human renal microsomes NADPH:CYP reductase is more effective in AAI activation. In addition, prostaglandin H synthase is another enzyme activating AAI in renal microsomes. The results demonstrate for the first time the potential of microsomal enzymes in human liver and kidney to activate AAI by nitroreduction.

The mechanism of cytotoxicity and DNA adduct formation by the anticancer drug ellipticine in human neuroblastoma cells

“…Incubations were also carried out in the presence of a peroxidase cofactor, hydrogen peroxide (0.1 mM) [33,34]. Incubations A c c e p t e d M a n u s c r i p t 10 were carried out at 37ºC for 30 minutes; ellipticine-DNA adduct formation was found to be linear up to 30 min of incubation [3].…”

“…The following chemicals were used to inhibit the activation of ellipticine to form DNA adducts in the presence of S9 subcellular fraction of neuroblastoma cells: -NF, which inhibits CYP1A1 and 1A2 [34][35][36], and activates oxidation of some substrates by CYP3A4 [36], and ketoconazole, an inhibitor of CYP3A4 [36]. Inhibitors were dissolved in 7.5 l of methanol, to yield final concentrations of 0.1 mM in the incubation mixtures.…”

The Role of Biotransformation Enzymes in the Development of Renal Injury and Urothelial Cancer Caused by Aristolochic Acid: Urgent Questions and Difficult Answers

“…This leads to dA-AAI, the most persistent adduct in initiating car- 25 . Several in-vitro studies report that the most important human and rat enzyme to activate AAI in vitro in hepatic or renal cytosolic subcellular fractions, is NAD(P)H:quinone oxidoreductase (NQO1) [34][35][36] followed by cytochrome P450 (CYP) 1A1/2 in hepatic microsomes 37,38 and NADPH:CYP reductase (POR) in renal microsomes 36,39 . In addition, prostaglandin H synthase (cyclooxygenase, COX) is able to bioactivate AAI 36,40 , which is highly expressed in urothelial tissue.…”

“…An increase in AAIa excretion due to its conjugation with glucuronide, caused by induction of UDP-glucuronosyltransferase with MC, could occur. However as CYP1A enzymes also activate AAI to species forming DNA adducts 25,[36][37][38] , the decrease in AAI in liver and kidney might also result from this reaction. Also, NQO1 which is also efficiently induced by MC, could contribute to decreased AAI levels in MC-treated mice.…”

β-Naphthoflavone protects mice from aristolochic acid-I-induced acute kidney injury in a CYP1A dependent mechanism

“…Among the P450 superfamily, CYP1A is known to be involved in activa- tion/detoxification of a variety of procarcinogens such as 3-methylcholanthrene (3-MC) [12] . It has been reported that in hepatic microsomes, AA is metabolized to aristolochic acid Ia (AAIa) under aerobic conditions in vitro [13,14] . An in vivo study suggests that aristolactams (AL) are the major metabolites in kidney [15] .…”