The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are consider ed as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibe nzo[a,1]pyrene (DB[a,1]P), the most potent carcinogenic PAH ever tested, an d benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (H EL) using multiple endpoints. DNA adduct levels were measured by P-32-postl abelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA a dduct formation was proportional to the time of exposure and dependent on t he stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24 h exposure, 0.01 muM for both PAHs). The high est DNA adduct levels were observed after 24 h of exposure in near-confluen t cells (>90% of cells at GO/G1 phase), but DNA damage induced by DB[a,l]P was similar to 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 muM and for DB[a,1]P at 0.1 mu M: 237 +/- 107 and 2360 +/- 798 adducts/10(8) nucleotides, respectively). T he induction of p53 and p21(WAF1) protein occurred subsequent to the induct ion of DNA adducts. The DNA adduct levels correlated with both p53 (R = 0.8 32, P < 0.001 and R = 0.859, P < 0.001, for DB[a,1]P and B[a]P, respectivel y) and p21(WAF1) levels (R = 0.808, P < 0.001 and R = 0.797, P = 0.001, for DB[a,1]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (<greater than or equal to>1.5-fold as compared with controls) requires a minimal DNA adduct level of similar to 200-250 adduct s/10(8) nucleotides for both PAHs tested and suggest that the level of addu cts rather than their structure triggers the p53 and p21(WAF1) responses. T he cell cycle was altered after 12-16 h of treatment, and after 24 h of exp osure to 0.1 muM DB[a,1]P in growing cells, there was similar to 24% increa se in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) c ells. Cell treatment with 1.0 muM B[a]P resulted in more subtle alterations . We conclude that DB[a,1]P, and to a lesser degree B[a]P, are able to indu ce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M ar rests but rather an S phase delay/arrest. Whether the S phase delay observe d in our study is beneficial for the survival of the cells remains to be fu rther established. (C) 2000 Elsevier Science B.V. All rights reserved.