[Benzo(a)pyrene metabolism in human fibroblasts--effect of 7,8-benzoflavone]

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.