Sudan I (1-phenylazo-2-hydroxynaphthol) is a suspected human carcinogen causing tumors in the livers and urinary bladders of rats, mice, and rabbits. Here, we investigated for the first time the influence of Sudan I exposure on the expression of several biotransformation enzymes in the livers, kidneys, and lungs of rats concomitantly at the mRNA and protein levels and assayed their enzymatic activities. We also studied its effect on the formation of Sudan I-derived DNA adducts in vitro. Sudan I increased the total amounts of cytochrome P450 (P450) in all organs tested. Western blots using antibodies raised against various P450s, NADPH:P450 reductase, and NAD(P)H:quinone oxidoreductase 1 (NQO1) showed that the expression of P450 1A1 and NQO1 was induced in the liver, kidney, and lung of rats treated with Sudan I. The higher protein levels correlated with increased enzyme activities of P450 1A1/2 and NQO1. Furthermore, 9.9-, 5.9-, and 2.8-fold increases in the formation of Sudan I oxidative metabolites catalyzed by microsomes isolated from the liver, kidney, and lung, respectively, of rats treated with Sudan I were found. The relative amounts of P450 1A and NQO1 mRNA, measured by real-time polymerase chain reaction (RT-PCR) analysis, demonstrated that Sudan I induced the expression of P450 1A1 and NQO1 mRNA in the liver, kidney, and lung, and of P450 1A2 mRNA in kidney and lung. Finally, microsomes isolated from livers, kidneys, and lungs of Sudan I exposed rats more effectively catalyzed the formation of Sudan I-DNA adducts than microsomes from organs of control rats. This was attributable to the higher P450 1A1 expression. Because P450 1A1 is playing a major role in the bioactivation of Sudan I in rat and human systems, its induction by Sudan I may have a profound effect on cancer risk by this azo dye. In addition, the induction of P450 1A1/2 and NQO1 enzymes can influence individual human susceptibility to other environmental carcinogens and have an effect on cancer risk.

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.

Cytochrome P450 (CYP) 2S1 is ''orphan'' CYP that is overexpressed in several epithelial tissues and many human tumors. The pure enzyme is required for better understanding of its biological functions. Therefore, human CYP2S1 was considered to be prepared by the gene manipulations and heterologous expression in Escherichia coli. Here, the conditions suitable for efficient expression of human CYP2S1 protein from plasmid pCW containing the human CYP2S1 gene were optimized and the enzyme purified to homogeneity. The identity of CYP2S1 as the product of heterologous expression was confirmed by dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. To confirm the presence of the enzymatically active CYP2S1, the CO spectrum of purified CYP2S1 was recorded. Since CYP2S1 was shown to catalyze oxidation of compounds having polycyclic aromatic structures, the prepared enzyme has been tested to metabolize the compounds having this structural character; namely, the human carcinogen benzo[a]pyrene (BaP), its 7,8-dihydrodiol derivative, and an anticancer drug ellipticine. Reaction mixtures contained besides the test compounds the CYP2S1 enzyme reconstituted with NADPH:CYP reductase (POR) in liposomes, and/or this CYP in the presence of cumene hydroperoxide or hydrogen peroxide. High performance liquid chromatography was employed for separation of BaP, BaP-7,8-dihydrodiol, and ellipticine metabolites. The results found in this study demonstrate that CYP2S1 in the presence of cumene hydroperoxide or hydrogen peroxide catalyzes oxidation of two of the test xenobiotics, a metabolite of BaP, BaP-7,8-dihydrodiol, and ellipticine. Whereas BaP-7,8,9,10-tetrahydrotetrol was formed as a product of BaP-7,8-dihydrodiol oxidation, ellipticine was oxidized to 12-hydroxyellipticine, 13-hydroxyellipticine, and the ellipticine N 2 -oxide.

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