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Resveratrol is a polyphenolic compound present in grapes and wine with anticancer activities that undergoes pronounced metabolism in humans. In order to determine whether metabolism of resveratrol also occurs in tumor cells and whether biotransformation has any impact on cytotoxicity, metabolism experiments were conducted with hormone-dependent ZR-75-1 and hormone-independent MB-MDA-231 human breast cancer cells. Along with resveratrol, it was possible to identify one metabolite, namely, resveratrol-3-O-sulfate in both cell lines. Its concentration in the cytoplasm and culture medium was 5.4- to 9-fold higher in ZR-75-1 cells than in MDA-MB-231 cells, concomitant with a 3.1-fold higher IC(50) value in the ZR-75-1 cell line (74 microM compared to 38 microM). By using RT-PCR, expression of sulfotransferase (SULT)1A1 mRNA, but not of other SULTs investigated, showed a close correlation with resveratrol 3-O-sulfate formation which was particularly high in ZR-75-1 and very low in MDA-MD-231 cells. In conclusion, we demonstrate that SULT1A1-based biotransformation reduces the anticancer activity of resveratrol in breast cancer cells, which must be considered in humans following oral uptake of dietary resveratrol as a chemopreventive agent.

Human CYP2B6 and CYP2E1 were used to investigate the extent to which differential substrate selectivities between cytochrome P450 subfamilies reflect differences in active-site residues as opposed to distinct arrangement of the backbone of the enzymes. Reciprocal CYP2B6 and CYP2E1 mutants at active-site positions 103, 209, 294, 363, 367, and 477 (numbering according to CYP2B6) were characterized using the CYP2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin, the CYP2E1-selective substrate p-nitrophenol, and the common substrates 7-ethoxycoumarin, 7-butoxycoumarin, and arachidonic acid. This report is the first to study the active site of CYP2E1 by systematic site-directed mutagenesis. One of the most intriguing findings was that substitution of CYP2E1 Phe-477 with valine from CYP2B6 resulted in significant 7-ethoxy-4-trifluoromethylcoumarin deethylation. Use of three-dimensional models of CYP2B6 and CYP2E1 based on the crystal structure of CYP2C5 suggested that deethylation of 7-ethoxy-4-trifluoromethylcoumarin by CYP2E1 is impeded by van der Waals overlaps with the side chain of Phe-477. Interestingly, none of the CYP2B6 mutants acquired enhanced ability to hydroxylate p-nitrophenol. Substitution of residue 363 in CYP2E1 and CYP2B6 resulted in significant alterations of the metabolite profile for the side chain hydroxylation of 7-butoxycoumarin. Probing of CYP2E1 mutants with arachidonic acid indicated that residues Leu-209 and Phe-477 are critical for substrate orientation in the active site. Overall, the study revealed that differences in the side chains of active-site residues are partially responsible for differential substrate selectivities across cytochrome P450 subfamilies. However, the relative importance of active-site residues appears to be dependent on the structural similarity of the compound to other substrates of the enzyme.

The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis., (Copyright 2001 Academic Press.)

The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC(50) value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC(50) values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.

Differences in expression of CYP1A isoforms (CYP1A1 and CYP1A2) in liver and small intestine of male Wistar rats and their inducibility by 3-methylcholanthrene as well as the effect of different CYP1A1/1A2 expression on caffeine metabolism were investigated. In rat liver, CYP1A2 is the predominant isoform and CYP1A1 protein expression in liver is significantly increased after treatment by 3-methylcholanthrene. In contrast, only CYP1A1 was detected in control and 3-methylcholanthrene induced small intestine microsomes. Treatment with 3-methylcholanthrene (40 mg/kg intraperitoneally daily during 1, 2, 3 or 4 days) demonstrated that liver CYP1A1 is more sensitive for the induction effects than CYP1A2 and also that significant induction of CYP1A1 in rat small intestine only occurred after 3 to 4 days pretreatment. Caffeine metabolism and inhibition studies by furafylline, CYP1A1 antiserum and ketoconazole revealed that the differences in the expression of CYP1A1 and CYP1A2 in the two tissues led to significant changes in the contribution of the various isoenzymes involved in the biotransformation of caffeine. Whereas in liver paraxanthine formation was almost exclusively catalyzed by CYP1A2, in rat proximal intestine it was formed by CYP1A1. In addition, other CYP enzymes (most probably CYP3A) play a significant role in theobromine and theophylline formation from caffeine in rat intestine. Overall, this study shows different expression and inducibility of CYP1A1/1A2 by 3-methylcholanthrene in rat liver and small intestine. Furthermore in rat intestine cytochrome P450 isozymes such as CYP1A1 and CYP3A replace CYP1A2 in the caffeine metabolism.

A new HPLC assay was developed to study dextromethorphan O-demethylation to dextrorphan in vitro using human liver microsomes to investigate the activity of the polymorphic monooxygenase cytochrome P450 2D6 (CYP 2D6). The separation of dextromethorphan and its main metabolite dextrorphan was performed on a polymeric C18 reversed-phase column with UV-detection using levallorphan as an internal standard. Liver samples from ten subjects were screened for dextrorphan formation whereby three groups with different abilities to metabolize dextromethorphan could be found. Seven microsomal preparations from extensive metabolizers showed an average dextrorphan formation rate of 298 +/- 68 pmol/mg protein.min, one sample was classified to belong to an intermediate dextromethorphan metabolizer (79 pmol/mg protein.min), whereas two samples of poor metabolizers exhibited significantly lower rates of dextromethorphan metabolism with values of 11 and 27 pmol/mg protein.min, respectively. This assay permits not only a fast in vitro screening for cytochrome P450 2D6 monooxygenase activity but is also an excellent tool to determine potential drug-drug interactions with this important metabolizing enzyme.

A specific and sensitive method for the determination of PFP 6, a new compound for reverting multidrug resistance, and its two main metabolites in human liver microsomes has been developed. Samples were almost quantitatively isolated by solid phase extraction and further analysed by HPLC. Separation was achieved on an endcapped reversed phase C-18 column with triethylamine as an organic modifier in the mobile phase. The method exhibits high recovery and a good sensitivity for analyses of small sample volumes. The application of the method for screening the metabolism of this new drug in human liver microsomes is presented.