EXPOSURE OF MAMMALIAN CELLS TO DIOL EPOXIDES FROM BENZO[A]PYRENE AND DIBENZO[A,L]PYRENE AND EFFECTS ON Mdm2 AND P53

Abstract

Dibenzo[a,l]pyrene (DBP) is considerably more carcinogenic than benzo[a]pyrene (BP). This fact is most probably related to differences in DNA binding efficiency of their ultimate carcinogenic diol epoxide (DE) intermediates, (+)-anti-BPDE in the case of BP and (−)-anti-DBPDE with DBP; differences in structural features of the DNA adducts and differences in DNA adduct recognition; and the subsequent lesion removal by nucleotide excision repair (NER). The complex signaling network involved in adduct recognition, cell cycle check points, and adduct removal is rapidly emerging. In this study we have focused on the effects of (+)-anti-BPDE and (±)-anti-DBPDE on Mdm2, a protein that regulates p53 stabilization/accumulation in an autoregulatory negative feedback loop. The data available so far indicate that both DEs induce accumulation of Mdm2 in HepG2 cells in concentrations without detectable effects on p53 stabilization. However, BPDE was found to be several orders of magnitude more powerful as inducer than the more complex DBPDE. BPDE caused Mdm2 accumulation at low pM concentrations whereas nM concentrations of DBPDE were required for a similar effect. The Mdm2 accumulation induced by each DE was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. This suggests that phosphorylation of Mdm2 is an essential step in accumulation of the protein. Furthermore, available RT-PCR results indicate that the DEs at higher concentrations (1 μM BPDE and 0.1 μM DBPDE) actually lowered the levels of Mdm2 mRNA indicating transcriptional downregulation of Mdm2. As expected, the effect was associated with p53 phosphorylation on serine 15 and, accordingly, stabilization of the protein.