The human carcinogen aristolochic acid i is activated to form DNA adducts by human NAD(P)H:quinone oxidoreductase without the contribution of acetyltransferases or sulfotransferases

Abstract

Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with AA nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. We investigated the efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) to activate aristolochic acid I (AAI) and used in silico docking, using soft-soft (flexible) docking procedure, to study the interactions of AAI with the active site of human NQO1. AAI binds to the active site of NQO1 indicating that the binding orientation allows for direct hydride transfer (i.e., two electron reductions) to the nitro group of AAI. NQO1 activated AAI, generating DNA adduct patterns reproducing those found in urothelial tissues from humans exposed to AA. Because reduced aromatic nitro-compounds are often further activated by sulfotransferases (SULTs) or N,O-acetlytransferases (NATs), their roles in AAI activation were investigated. Our results indicate that phase II reactions do not play a major role in AAI bioactivation; neither native enzymes present in human hepatic or renal cytosols nor human SULT1A1, -1A2, -1A3, -1E, or -2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAI. Instead under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAI through the formation of a cyclic hydroxamic acid (N-hydroxyaristolactam I) favored by the carboxy group in peri position to the nitro group without additional conjugation. These results emphasize the major importance of NQO1 in the metabolic activation of AAI and provide the first evidence that initial nitroreduction is the rate limiting step in AAI activation.

Mechanisms of Enzyme-Catalyzed Reduction of Two Carcinogenic Nitro-Aromatics, 3-Nitrobenzanthrone and Aristolochic Acid I: Experimental and Theoretical Approaches

“…In contrast, the contribution of these conjugation enzymes to the activation of 3-NBA, NAT1, NAT2, SULT1A1 and SULT1A2 was ineffective to influence AAI-DNA adduct formation mediated by NQO1 (Table 1) [69,73]. Further, similar results were found in human hepatic and renal cytosols.…”

“…Similarly, a major role of NQO1 in the activation of both nitro-aromatics was found in human hepatic and renal cytosolic fractions. The formation of 3-NBA- and AAI-DNA adducts in these subcellular fractions was dependent on the presence of the cofactor of NQO1, NADPH, and again dicoumarol was shown to be a potent inhibitor [48,52,68,69]. Recently, this NQO1 inhibitor was also utilized to study the role of NQO1 in AAI activation in vivo [62,70].…”

“…Amenability of 3-NBA and AAI to chemical and enzymatic reduction might be one of the reasons. Ab initio calculations were employed to test this hypothesis [52,69,73,94,95,96]. …”

Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity

“…Studies on the involvement of phase II metabolism in further bioactivation of AL-I-NOH via a sulfonation step in vivo have revealed conflicting results in different model systems (6,7,(14)(15)(16). To assess which metabolite of AA-I is the toxic form transported from liver to kidney, human kidney MPS were treated with synthetic sulfate-conjugated AL-I metabolite (AL-I-NOSO 3 ) or AL-I-NOH.…”

In vitro genotoxicity tests point to an unexpected and harmful effect of a Magnolia and Aristolochia association

“…When improperly repaired, these DNA lesions lead to a characteristic A:T to T:A transversion mutation (Grollman et al 2007). NAD(P)H:quinone oxidoreductase (NQO1) is the main enzyme responsible for metabolic activation of AAs in ALs by reduction of the nitro group (Stiborová et al 2003;Stiborová et al 2011); acetyl-and sulfotransferases may be involved in the bioactivation of AAs but their precise roles are still unclear (Stiborová et al 2011;Sidorenko et al 2014). Interestingly NQO1 is up-regulated by broccoli sprout extracts (Afaq 2011) and β-lapachone, a quinone obtained from the bark of the lapacho tree (Handroanthus impetiginosus (Mart.…”