Effect of natural analogues of trans-resveratrol on cytochromes P4501A2 and 2E1 catalytic activities.

The aim was to assess the inhibitory effect of a series of naturally occurring trans-resveratrol analogues on cytochromes P450, namely CYP1A2 and CYP2E1, in vitro in order to analyse any structure-activity relationships. 3,5-Dimethoxy-4'-hydroxy-trans-stilbene (pterostilbene), 3,4',5-trimethoxy-trans-stilbene (TMS), 3,4'-dihydroxy-5-methoxy-trans-stilbene (3,4'-DH-5-MS) and 3,5-dihydroxy-4'-methoxy-trans-stilbene (3,5-DH-4'-MS) inhibited the activity of CYP1A2, with K(i) = 0.39, 0.79, 0.94 and 1.04 microM, respectively. Piceatannol (3,3',4,5'-tetrahydroxy-trans-stilbene) was the least potent inhibitor of CYP1A2 with a K(i) = 9.67 microM. Piceatannol and TMS in the concentration range 1-100 microM did not inhibit CYP2E1 activity. The activity of this enzyme likewise was not significantly influenced by pterostilbene and 3,5-DH-4'-MS with IC(50) > 100 microM, whereas 3,4'-DH-5-MS appeared to be a moderately potent, competitive inhibitor of CYP2E1 (K(i) = 42.6 microM). Structure-activity relationship analysis leads to the conclusion that the substitution of hydroxy groups of resveratrol with methoxy groups increases the inhibition of CYP1A2, yet the number and position of methylation are not essential. However, the 4'-hydroxy group in trans-resveratrol and its analogues may play an important role in the interaction with a binding site of CYP2E1.

Formation and persistence of benzo[a]pyrene--DNA adducts in different tissues of C57BL/10 and DBA/2 mice.

Synchronous fluorescence spectrophotometry (SFS) developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)--DNA adducts was used to measure the formation and disappearance of DNA adducts in the lung, liver, kidney, spleen and small intestine of genetically responsive C57BL/10 (B10) and nonresponsive DBA/2 (D2) mice. After single stomach intubation of 100 mg/kg of benzo[a]pyrene (B[a]P) in both strains, binding of BPDE to DNA reached a peak 48 h after treatment. However, the levels of binding in the lung, liver, kidney and spleen were higher in D2 than in B10 mice. In contrast to this, in the small intestine the higher level of BPDE binding was found in B10 mice and reached its maximum 24 h earlier. Thereafter a very rapid drop in the level of BPDE--DNA adducts to a value of approximately 50% after 48 h was observed in this tissue. In the other tissues of the B10 mice the rate of adducts removal was slower, but by 14 days after treatment 90-100% of adducts were removed. In the D2 mice up to the 4th day after treatment the rates of removal of the BPDE--DNA adducts were similar to that of the B10 mice. Thereafter the level of bound hydrocarbon decreased at a slower rate. During the whole period after B[a]P treatment distinct differences between organs in the amount of BPDE--DNA adducts were observed. In D2 mice the highest level of binding was found in the spleen followed by the lung, kidney, liver and small intestine. In B10 mice the highest level of binding was observed in the DNA of small intestine. The data suggest that the decreased rate of B[a]P metabolism in D2 mice may be at least in some tissues the reason of higher binding of BPDE--DNA adducts in comparison with B10 mice.

Monoclonal antibody characterization of NADH- and NADPH-dependent hydroxylation of benzo(a)pyrene in liver microsomes from 5,6-benzoflavone-induced C57Bl/6 mice.

Monoclonal antibodies (MAb 1-7-1) directed against the isoenzymes of rat liver cytochrome P-450 induced by methylcholanthrene, inhibited benzo(a)pyrene hydroxylase less strongly at low (0.04 mM) than at high (2 mM) NADH concentration. Inhibition of NADPH dependent hydroxylation was the same irrespective of NADPH concentrations and corresponded to that observed at high NADH concentration. The same was also the h.p.l.c. pattern of the reaction products of benzo(a)pyrene hydroxylation supported by high concentration of both coenzymes. It is postulated that different cytochrome P-450 isoenzymes participate in benzo(a)pyrene hydroxylation, whereas the second one acts at high concentration of both NADH and NADPH.

[Benzo(a)pyrene metabolism in human fibroblasts--effect of 7,8-benzoflavone]. / Metabolizm benzo(a)pirenu w fibroblastach ludzkich--wplyw 7,8-benzoflawonu.

Benzo(a)pyrene (BP) metabolism was studied in cultured fibroblasts from healthy donor. Cells were cultured in Eagle medium supplemented with 10% fetal calf serum, 2 mM 1-glutamine, 100 U/ml penicillin and 100 micrograms/ml streptomycin. Cultures were grown at 37 degrees C to confluence in 45 cm2 culture flasks containing 15 ml medium and refed with fresh medium 24 h prior to treatment with BP. Cells were treated for 24 h with [G-3H]BP diluted with BP to give a final concentration 10 microM and a specific activity of 0.3 Ci/mmol. The metabolites were extracted by ethyl acetate, dried with Na2SO4, evaporated with nitrogen and injected into high pressure liquid chromatograph. The column (LiChrosorb RP 18) was eluted with a linear gradient of 60% methanol in water to 100% methanol in 45 min at a flow rate 0.8 ml/min. Radioactivity of fractions was measured. Following metabolites were identified: 3-hydroxy-BP (23.7%), 9-hydroxy-BP (17.0%), quinones (22.9%), 7,8-dihydroxy-BP (6.1%), 9,10-dihydroxy-BP and other derivatives (30.3%). 7,8-benzoflavone--an inhibitor of BP hydroxylase and epoxide hydrase, strongly inhibited the metabolism of BP in human fibroblasts, changing the proportions in the amounts of the metabolites. The ratio of phenols to the other metabolites increased twice under the influence of 7,8-benzoflavone. This suggests that 7,8-benzoflavone has the stronger inhibitory effect on epoxide and diol formation in comparison with BP hydroxylation.

[Effect of monoclonal antibody against methylcholanthrene-induced cytochrome P-450 forms on benzo(a)pyrene metabolism in hepatic microsomes of C57BL/10 mice]. / Wplyw przeciwciala monoklonalnego skierowanego przeciwko formom cytochromu P-450, indukowanym przez metylocholantren, na metabolizm benzo(a)pirenu w mikrosomach watroby myszy C57BL/10.

In this study, hepatic microsomes from 5,6-benzoflavone induced C57BL/10 mice were used. To inhibit monooxygenase activities, the monoclonal antibody MAb 1-7-1 recognizing two isoenzymes of methylcholanthrene-induced cytochrome P-450 was applied. Microsomes were incubated with tritium labeled benzo(a)pyrene [G-3H]BP for 10 min at 37 degrees C. The incubation mixture contained: 50 mM potassium phosphate buffer, pH 7.25; 30 mM KCl; 3 mM MgCl2; 2 mM NADPH; 80 microM [G-3H]BP (specific activity 50 mCi/mmol); and monoclonal antibody MAb 1-7-1 or ascites fluid (NBS) containing nonspecific IgG as a control. The ratio of antibody protein/microsomal protein was 2:5. BP metabolites were extracted from incubation mixtures by ethyl acetate. The organic layer was dried over sodium sulfate, and evaporated under a stream of nitrogen. To separate BP metabolites HPLC technology was used. The column was eluted with methanol gradient (60-100%) for 45 minutes. The radio-activity of collected samples was determined using liquid scintillation counter. Differential inhibitory effects of MAb 1-7-1 on BP-metabolites formation were found, e.g. 7,8-diol was inhibited by 86.1% and quinones by 62.5%. The predominant metabolite, 3-OH-BP, was inhibited by 80.4%. Moreover, it was found that MAb 1-7-1 inhibition of benzo(a)pyrene hydroxylase activity (by 75.8%, as measured by fluorescent technique) was very similar to the inhibition of 3-OH-BP along with 9-OH-BP formation (as measured by HPLC).