Piperine impairs cytochrome P4501A1 activity by direct interaction with the enzyme and not by down regulation of CYP1A1 gene expression in the rat hepatoma 5L cell line.

Modulation of benzo(a)pyrene (BP) metabolism and regulation of CYP1A1 gene expression by piperine in 5L cells in culture was studied. Treatment of cultures with 60 microM piperine for different time periods inhibited metabolism of BP by 50% within 4-8 h. Piperine uptake in 5L cells attained saturation plateau at 8 h and this related with the maximal impairment of BP metabolism. Exposure of cell cultures to piperine for 24 h indicated activation of CYP1A1 gene transcription. There was a 10-fold increase in CYP1A1mRNA and an approximately 7-fold increase in arylhydrocarbon hydroxylase (AHH) activity, while treatment with benzanthacene (BA) increased CYP1A1mRNA by 86- and AHH by 56-fold. Combined treatment with BA plus piperine further increased CYP1A1mRNA contents by about 25%. The BA-inducible AHH activity, however, registered a decrease of about 45%. Piperine neither destroyed CYP1A1-protein nor affected the total cellular protein contents. Exposure of cultures to 0.01 to 3.0 microM trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene for 24 h reduced maximum cellular growth by about 50%. Piperine at 60 microM offered full protection against the diol cytotoxicity. The results, suggest that piperine mediated inhibition of the AHH activity and consequent suppression of the procarcinogen activation is the result of direct interaction of piperine with CYP1A1-protein and not because of down regulation of the CYP1A1 gene expression.

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on cytochrome P-450-dependent monooxygenase activities in rat hepatoma cells: induction of P-450I and suppression of P-450II.

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytochrome P-450-dependent monooxygenase activities in several differentiated and dedifferentiated Reuber rat hepatoma cell lines using aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and aldrin epoxidase (AE) as test systems. The following results were obtained: (1) Exposure of cultures to 400 nM TPA for 18-24 h increased AHH activities in the differentiated lines 2sFou, H41IEC3/G- and Fao as well as in the dedifferentiated line 5L, 1.5-2.5-fold. The phorbol ester did not affect AHH activity in the dedifferentiated line H5. (2) EROD, a marker for P-450I, was induced by the phorbol ester to a similar degree as AHH. (3) A monoclonal antibody directed against P-450I strongly inhibited the AHH activity induced by TPA. (4) The onset of AHH or EROD induction by TPA was much later than that elicited by benz[a]anthracene. (6) In contrast to the induction of AHH and EROD, TPA decreased AE activity, a marker for P-450II, by about 50% in all the cell lines containing this monooxygenase activity. (7) The half-maximum-effect concentration of TPA for inducing or suppressing AHH and AE, respectively, was approximately 20 nM. (8) TPA did not interfere with AHH induction by benz[a]anthracene. However, the phorbol ester moderately decreased AHH induction and markedly suppressed AE induction by dexamethasone. The results indicate that TPA simultaneously induces P-450I and suppresses P-450II forms in rat hepatoma cells. P-450I induction by TPA in these cells did not appear to depend on their status of differentiation. Furthermore, the results suggest that the mechanism of P-450I induction by TPA differs from that elicited by polycyclic aromatic hydrocarbons or glucocorticoids.