Carcinogenic polycyclic aromatic hydrocarbons : theoretical, molecular, in vitro and cellular characterization of biotransformation and DNA damage

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are widespread mutagenic and carcinogenic environmental pollutants, which require metabolic activation to electrophilic intermediates and subsequent covalent binding to critical targets in DNA to elicit their biological activity. Bayand fjord-region diol epoxides (DEs) have been identified as the ultimate mutagenic and carcinogenic metabolites of PAHs. The balance between metabolic activation and detoxification routes influences the extent of DE-DNA adduct-formation. The most important detoxification pathway of DEs is glutathione transferase (GST) catalyzed conjugation with glutathione (GSH).

Human GSTs of Alpha class have been assayed with the ultimate carcinogenic ( )-anti- and (+)-syn-DEs derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-DE of the planar benzo[a]pyrene [(+)-anti-BPDE]. In general, the activities were in the order: (+)-syn- DBPDE > ( )-anti-DBPDE > (+)-anti-BPDE. GSTA1-1 was found to be the most efficient enzyme and demonstrated a remarkable catalytic efficiency (kcat/Km) of 464 mM-1s-1 with (+)- syn-DBPDE. The higher activity of GSTA1-1 with (+)-syn-DBPDE relative to ( )-anti-DBPDE was explained by molecular modeling showing the formation of more favorable interactions between the substrate and the enzyme-GSH complex. The results showed that the spatial orientation of the hydroxyl groups are important determinants for the catalytic efficiency and thus responsible for the observed difference in catalytic activity. Investigating preferences in structure of (+)-syn- and ( )-anti-DBPDE using DFT showed several levels of flexibility. Because of the distorted structure, the molecule can readily flip its DE moiety relative to the aromatic ring system ( in and out ). Furthermore, the hydroxyl groups on the saturated DE ring were found to be either in a diequatorial or in a diaxial conformation. Our results showed a lower energy profile and thus a preference for the in-diequatorial conformation for both DEs. The possibility of transversions on different levels might have biological consequences, both in detoxication and in DNA adduct formation, adapting to DNA and thus escape recognition/repair by the DNA repair machinery.

To study individual GSTs in a more biologically relevant system, V79 cells stably overexpressing different human GSTs were constructed and characterized. Factors governing the accessibility of lipophilic DE substrates for GSTs in the cell were investigated. With the highly reactive (+)-anti-BPDE, 1-2 % of the expected activity was observed, whereas the corresponding values for the less reactive ( )-anti-DBPDE were up to 13 %. Furthermore, the protective effect of individual GSTs against DE induced DNA adduct formation was determined. In general, an increase in GST activity was concomitant with a decrease in DNA adduct formation. DBPDE showed the highest DNA binding capacity among the DEs tested. GSTA1-1 showed the highest GSH conjugating capacity and offered best protection against DBPDE induced DNA adduct formation. With BPDE, GSTP1-1 was most active in GSH conjugate formation whereas GSTM1-1 displayed the most effective protection against formation of DNA adducts. Overall, this demonstrates the difficulty in extrapolating data obtained with pure enzymes to the complex situation in the intact cell.

We have compared the formation and removal of adducts as a function of time formed by ( )- anti-DBPDE and (+)-anti-BPDE in A549 human epithelial lung carcinoma cells. The treatment with DBPDE resulted in an initial increase of adducts to a maximal level of adducts after 1 hr of incubation. This was followed by an apparent, although not statistically significant, slow removal of adducts. In cells treated with BPDE the maximal level adducts was reached within 20 min of exposure. The formation was followed by an initial rapid decline in the adduct level and a later statistically significant 10-fold slower rate of adduct removal. Comparing the rate of removal of adducts derived from BPDE with those of DBPDE, the latter are obviously more refractory to the NER coupled repair than the former. Moreover, we observe a significant increase in ratio of dA/dG adducts for DBPDE, indicating that dA adducts are especially refractory to repair. The apparent resistance of adducts from DBPDE to be eliminated may reflect the ability of such adducts to escape recognition and/or the subsequent removal by the NER machinery.