Role of Cytochromes P450 1A1/2 in Detoxication and Activation of Carcinogenic Aristolochic Acid I: Studies with the Hepatic NADPH:Cytochrome P450 Reductase Null (HRN) Mouse Model

Abstract

Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

Effect of Hepatic Cytochrome P450 (P450) Oxidoreductase Deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 Reductase Conditional Null Mice

“…We have previously used Hepatic P450 Reductase Null (HRN) mice to investigate hepatic versus extrahepatic P450-mediated carcinogen metabolism (Arlt et al, 2005(Arlt et al, , 2006Stiborová et al, 2008;Levová et al, 2011). In HRN mice, hepatic POR expression has been deleted, resulting in almost complete ablation of hepatic P450 function (Henderson et al, 2003).…”

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

“…However, its expression levels are low (<0.005 pmol CYP1A1/mg protein) [77,88], indicating that the overall impact of this enzyme on AAI activation in this organ seems to be low. CYP3A5 which is also expressed in human kidney has recently been found to be capable of activating AAI, but less efficiently than CYP1A1 and 1A2 [89]. Hence, a contribution of CYP3A5 to AAI activation might partially explain the efficacy of microsomes isolated from a human kidney to activate AAI used in a previous study [77].…”

“…, oxidative) conditions both CYP enzymes are able to O -demethylate AAI to a detoxification metabolite, 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa; Figure 2). Under these (aerobic) conditions, AAI behaves as a classical substrate of human CYP1A1 and 1A2, where one oxygen atom is utilized for O -demethylation of AAI to form AAIa [46,47,62,86,87,89,91,92,93]. These experimental results strongly suggest that, besides CYP1A1 and 1A2 expression levels, the oxygen level in tissues affects the balance between AAI nitroreductive activation and oxidative detoxication by these CYPs in vivo [47,62,86,87,89].…”

Impact of genetic modulation of SULT1A enzymes on DNA adduct formation by aristolochic acids and 3-nitrobenzanthrone

“…It is well known that conjugation reactions catalysed by phase II enzymes are often involved in the activating metabolism of carcinogenic nitroaromatics (Arlt et al 2005; Glatt and Meinl 2004; Glatt et al 2016; Rendic and Guengerich 2012). Phase II metabolites of AA have been found in the urine and faeces of AA-treated rodents and include the N - and O -glucuronide of aristolactam Ia and the O 8 -glucuronide, the O 8 -acetate and O 8 -sulfonated ester of AAIa (Chan et al 2007); AAIa is considered to be a detoxification product (Levova et al 2011; Stiborova et al 2012). As shown for other aromatic hydroxylamines (Glatt and Meinl 2004; Glatt et al 2016), like those generated during the metabolism of 3-nitrobenzanthrone (3-NBA) (Arlt et al 2002b, 2003, 2005), the N -hydroxyaristolactams may be further activated by O -acetylation or O -sulfonation to reactive esters capable of generating the same electrophilic species (aristolactam-nitrenium ions) as formed by direct cleavage of the hydroxyl group.…”