František Bárta

Bioactivation versus Detoxication of the Urothelial Carcinogen Aristolochic Acid I by Human Cytochrome P450 1A1 and 1A2

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

Role of P450 1A1 and P450 1A2 in Bioactivation versus Detoxication of the Renal Carcinogen Aristolochic Acid I: Studies in Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1a1/1a2(-/-) Mice

Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

The influence of ochratoxin A on DNA adduct formation by the carcinogen aristolochic acid in rats

Abstract Exposure to the plant nephrotoxin and carcinogen aristolochic acid (AA) leads to the development of AA nephropathy, Balkan endemic nephropathy (BEN) and upper urothelial carcinoma (UUC) in humans. Beside AA, exposure to ochratoxin A (OTA) was linked to BEN. Although OTA was rejected as a factor for BEN/UUC, there is still no information whether the development of AA-induced BEN/UUC is influenced by OTA exposure. Therefore, we studied the influence of OTA on the genotoxicity of AA (AA–DNA adduct formation) in vivo. AA–DNA adducts were formed in liver and kidney of rats treated with AA or AA combined with OTA, but no OTA-related DNA adducts were detectable in rats treated with OTA alone or OTA combined with AA. Compared to rats treated with AA alone, AA–DNA adduct levels were 5.4- and 1.6-fold higher in liver and kidney, respectively, of rats treated with AA combined with OTA. Although AA and OTA induced NAD(P)H:quinone oxidoreductase (NQO1) activating AA to DNA adducts, their combined treatment did not lead to either higher NQO1 enzyme activity or higher AA–DNA adduct levels in ex vivo incubations. Oxidation of AA I (8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid) to its detoxification metabolite, 8-hydroxyaristolochic acid, was lower in microsomes from rats treated with AA and OTA, and this was paralleled by lower activities of cytochromes P450 1A1/2 and/or 2C11 in these microsomes. Our results indicate that a decrease in AA detoxification after combined exposure to AA and OTA leads to an increase in AA–DNA adduct formation in liver and kidney of rats.

The influence of dicoumarol on the bioactivation of the carcinogen aristolochic acid I in rats

UNLABELLED Aristolochic acid I (AAI) is the major toxic component of the plant extract AA, which leads to the development of nephropathy and urothelial cancer in human. Individual susceptibility to AAI-induced disease might reflect variability in enzymes that metabolise AAI. In vitro NAD(P)H quinone oxidoreductase (NQO1) is the most potent enzyme that activates AAI by catalyzing formation of AAI-DNA adducts, which are found in kidneys of patients exposed to AAI. Inhibition of renal NQO1 activity by dicoumarol has been shown in mice. Here, we studied the influence of dicoumarol on metabolic activation of AAI in Wistar rats in vivo. In contrast to previous in vitro findings, dicoumarol did not inhibit AAI-DNA adduct formation in rats. Compared with rats treated with AAI alone, 11- and 5.4-fold higher AAI-DNA adduct levels were detected in liver and kidney, respectively, of rats pretreated with dicoumarol prior to exposure to AAI. Cytosols and microsomes isolated from liver and kidney of these rats were analysed for activity and protein levels of enzymes known to be involved in AAI metabolism. The combination of dicoumarol with AAI induced NQO1 protein level and activity in both organs. This was paralleled by an increase in AAI-DNA adduct levels found in ex vivo incubations with cytosols from rats pretreated with dicoumarol compared to cytosols from untreated rats. Microsomal ex vivo incubations showed a lower AAI detoxication to its oxidative metabolite, 8-hydroxyaristolochic acid (AAIa), although cytochrome P450 (CYP) 1A was practically unchanged. Because of these unexpected results, we examined CYP2C activity in microsomes and found that treatment of rats with dicoumarol alone and in combination with AAI inhibited CYP2C6/11 in liver. Therefore, these results indicate that CYP2C enzymes might contribute to AAI detoxication.

The effect of aristolochic acid I on expression of NAD(P)H:quinone oxidoreductase in mice and rats--a comparative study.

UNLABELLED Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined. We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver. NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Induction of cytochromes P450 1A1 and 1A2 suppresses formation of DNA adducts by carcinogenic aristolochic acid I in rats in vivo.

Highlights • Oxidation and reduction of aristolochic acid I (AAI) dictate its (geno)toxicity in vivo.• Cytochrome P450 (CYP) 1A1 and 1A2 are induced in rats treated with Sudan I and AAI.• Induced CYP1A enzyme activity resulted in decreased AAI-DNA adduct levels in vivo.• CYP1A1 and 1A2 mainly detoxify AAI and attenuate its genotoxicity in vivo.

Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using 32P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.

Comparison of the oxidation of carcinogenic aristolochic acid I and II by microsomal cytochromes P450 in vitro: experimental and theoretical approaches

The herbal drug aristolochic acid, a natural mixture of 8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid (AAI) and 6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid (AAII), is derived from Aristolochia species and is the cause of two nephropathies. Ingestion of aristolochic acid is associated with the development of urothelial tumors linked with aristolochic acid nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. The O-demethylated metabolite of AAI, 8-hydroxyaristolochic acid (AAIa), is the detoxification product of AAI generated by its oxidative metabolism. Whereas the formation of AAIa from AAI by cytochrome P450 (CYP) enzymes has been found in vitro and in vivo, this metabolite has not been found from AAII as yet. Therefore, the present study has been designed to compare the amenability of AAI and AAII to oxidation; experimental and theoretical approaches were used for such a study. In the case of experimental approaches, the enzyme (CYP)-mediated formation of AAIa from both carcinogens was investigated using CYP enzymes present in subcellular microsomal fractions and recombinant CYP enzymes. We found that in contrast to AAI, AAII is oxidized only by several CYP enzymatic systems and their efficiency is much lower for oxidation of AAII than AAI. Using the theoretical approaches, such as flexible in silico docking methods and ab initio calculations, contribution to explanation of these differences was established. Indeed, the results found by both used approaches determined the reasons why AAI is better oxidized than AAII; the key factor causing the differences in AAI and AAII oxidation is their different amenability to chemical oxidation.Graphical abstract