The effect of dibenzo[a,l]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

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Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered 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 dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32 P -postlabelling, the expression of p53 and p21 WAF1 proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24 h exposure, 0.01 μM for both PAHs). The highest DNA adduct levels were observed after 24 h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was ∼ 8–10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 μM and for DB[a,l]P at 0.1 μM: 237±107 and 2360±798 adducts/10 8 nucleotides, respectively). The induction of p53 and p21 WAF1 protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 ( R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21 WAF1 levels ( R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]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 (≥1.5-fold as compared with controls) requires a minimal DNA adduct level of ∼ 200–250 adducts/10 8 nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21 WAF1 responses. The cell cycle was altered after 12–16 h of treatment, and after 24 h of exposure to 0.1 μM DB[a,l]P in growing cells, there was ∼ 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 μM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21 WAF1 without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.