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The polycyclic aromatic hydrocarbons (PAHs) dibenzo[a,l]pyrene (DBP) and benzo[a]pyrene (BP) are widespread environmental contaminants and potent carcinogens. The fjord-region DBP is considerably more carcinogenic than the bay-region BP. This fact can be ascribed to differences in DNA binding efficiency of their ultimate carcinogenic diol epoxide (DE) intermediates, differences in structural features of the DNA adducts, and differences in DNA adduct recognition and the subsequent lesion removal by nucleotide excision repair (NER). We have compared the formation and removal of adducts as a function of time formed by the carcinogenic metabolites (−)-anti-DBPDE and (+)-anti-BPDE in A549 human epithelial lung carcinoma cells. Cells were exposed to 0.1 or 1.0 μM (−)-anti-DBPDE and (+)-anti-BPDE, respectively. Adducts were measured at various post-treatment times (up to 6 h) by enzymatic DNA hydrolysis and a HPLC procedure that allows monitoring of all cis- and trans-nucleoside adducts of dA and dG. Treatment with 0.1 μM (−)-anti-DBPDE resulted in an initial increase of adducts to a maximal level of 144 pmol adducts/mg of DNA after 1 h of incubation. This was followed by an apparent, although not statistically significant, slow removal of adducts. After 6 h of incubation, at least 80% seems to remain. In cells treated with 1.0 μM (+)-anti-BPDE, the maximal level of 140 pmol adducts/mg of DNA was reached within 20 min of exposure. The formation was followed by an initial rapid decline in the adduct level (1.54 pmol adducts/mg of DNA/min) and a later statistically significant slower rate (0.14 pmol adducts/mg of DNA/min) of adduct removal. After 1 h of incubation, about 45% of the adducts are removed followed by 75% at 6 h. The biphasic pattern of BPDE removal has been observed previously in mammalian cells and, at least in part, may reflect the action of transcription-coupled repair (TCR) and the subsequent global genomic repair (GGR). 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. Furthermore, 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. Further analysis of DNA adduct distribution as a function of incubation time reveals that the dA/dG adduct ratio for BPDE was independent of time (4% dA, 96% dG), whereas the corresponding ratio for DBPDE was significantly increased from 2.9 (74% dA, 26% dG) at 20 min to 4.0 (80% dA, 20% dG) after 6 h of incubation. The results presented here on DNA adduct removal in mammalian cells are in part consistent with recent results on NER-coupled activity on bay- and fjord-region DE-modified oligonucleotides in vitro and further substantiate the hypothesis that the high carcinogenicity of the nonplanar PAHs arise from the ability of the preferentially formed dA adducts to escape recognition by surveillance systems and the subsequent NER-coupled lesion removal.

The (−)-anti- and (+)-syn-diol epoxides of dibenzo[a,l]pyrene (DBPDE, 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene) and the stereochemically corresponding benzene diol epoxides (BDE, 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrobenzene) have been studied by density functional theory (DFT) to determine the structures, energies, dynamics, thermal properties, and solvent effects on the different conformers. The smaller BDE is used as a model compound for studies of transitions between diequatorial and diaxial conformations of the hydroxyl groups. It was found that DBPDE is distorted due to overcrowding in the fjord region and that the arene oxide prefers to be on the same side of the saturated ring as the distal ring (“in”) in most stereoisomeric states. For the anti-diastereomer, a diequatorial orientation of the hydroxyl groups is preferred, while the orientation preference in the syn-diastereomer seems to depend on the solvent and the in/out conformation. Transition states for the interconversions between in and out conformations of DBPDE as well as between diequatorial and diaxial conformations on BDE have been found, and transition rates have been estimated by transition state theory. The barriers are found to be moderate, the highest being 9.6 kcal/mol. Solvent effects as well as zero-point vibrational energy and thermal effects were included and found to be significant in some cases. The results presented here are in agreement with previous experimental studies.

In this study, human glutathione transferases (GSTs) of alpha class have been assayed with the ultimate carcinogenic (−)-anti- and (+)-syn-diol epoxides (DEs) derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-diol epoxide of the planar benzo[a]pyrene [(+)-anti-BPDE] in the presence of glutathione (GSH). In all DEs, the benzylic oxirane carbon reacting with GSH, possess R-absolute configuration. GSTA1-1 demonstrated activity with all DEs tested whereas A2-2 and A3-3 only were active with the DBPDE enantiomers. With GSTA4-4, no detectable activity was observed. GSTA1-1 was found to be the most efficient enzyme and demonstrated a catalytic efficiency (kcat/Km) of 464 mM^-1 s^-1 with (+)-syn-DBPDE. This activity was about 7-fold higher than that observed with (−)-anti-DBPDE and more than 65-fold higher than previously observed with less complex fjord-region DEs. GSTA3-3 also demonstrated high kcat/Km with the DEs of DBP and a high preference for the (+)-syn-DBPDE enantiomer [190 vs 16.2 mM^-1 s^-1 for (−)-anti-DBPDE]. Lowest kcat/Km value of the active enzymes was observed with GSTA2-2. In this case, 30.4 mM^-1 s^-1 was estimated for (+)-syn-DBPDE and 3.4 mM^-1 s^-1 with (−)-anti-DBPDE. Comparing the activity of the alpha class GSTs with (−)-anti-DBPDE and (+)-anti-BPDE revealed that GSTA1-1 was considerable more active with the former substrate (about 25-fold). Molecular modeling studies showed that the H-site of GSTA1-1 is deeper and wider than that of GSTA4-4. This is mainly due to the changes of Ser212→Tyr212 and Ala216→Val216, which cause a shallower active site, which cannot accommodate large substrates such as DBPDE. The higher activity of GSTA1-1 with (+)-syn-DBPDE relative to (−)-anti-DBPDE is explained by the formation of more favorable interactions between the substrate and the enzyme−GSH complex. The presence of GSTA1-1 in significant amounts in human lung, a primary target tissue for PAH carcinogenesis, may be an important factor for the protection against the harmful action of this type of potent carcinogenic intermediates.

Mammalian V79 cells stably expressing human glutathione transferase (GST) A1-1, M1-1, and P1-1 (the allelic variant with Val105 and Ala114) have been constructed and characterized. The cells have been used to study the capacity of individual GST isoenzymes in conjunction with GSH to detoxify diol epoxides from dibenzo[a,l]pyrene (DBPDE), the most carcinogenic polycyclic aromatic hydrocarbon (PAH) identified so far, and diol epoxides from benzo[a]pyrene (BPDE). The relationship between GSH-conjugation and DNA adduct-formation has been investigated as well as factors governing the accessibility of lipophilic diol epoxide substrates for the soluble GSTs in the cells. Relative to control cells, those expressing GSTA1-1 showed the highest rate (about 50-fold increase) to perform GSH-conjugation of (−)-anti-DBPDE (R-absolute configuration at the benzylic oxirane carbon in the fjord-region) followed by GSTM1-1 (25-fold increase) and GSTP1-1 (10-fold increase). GSTA1-1 was found to be strongly inhibited when expressed in cells (10% of fully functional protein). Taking this factor into account, the rates of conjugation found in the cells fairly well reflected the order of catalytic efficiencies (kcat/Km) obtained with the pure enzymes. Increased GSH conjugation of (−)-anti-DBPDE was associated with a reduction in DNA adduct formation. GSTA1-1 inhibited the formation of adducts more than 6-fold and GSTM1-1 and GSTP1-1 about 2-fold. With (+)-anti-BPDE, GSTP1-1-expressing cells demonstrated a substantially higher rate of GSH-conjugate formation than cells with GSTA1-1 and GSTM1-1 cells (33- and 10-fold increase, respectively). Relative to control cells, GSTM1-1 was found to inhibit DNA adduct formation of (+)-anti-BPDE most effectively followed by GSTP1-1 and GSTA1-1 (12-, 4-, and 3-fold, respectively). Values of kcat/Km and estimated oil/water partition coefficients of DBPDE and BPDE were used to calculate the concentration of free diol epoxides in solution and expected rates of GSH conjugate formation in cells, and these theoretical results were compared with the observed ones. 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%. The most obvious explanations for the low observed rate with (+)-anti-BPDE are rapid and competing reactions such as hydrolysis and/or more unspecific chemical and physical reactions with cellular constituents (proteins, lipids, nucleic acids, etc.). In addition, the difference between the theoretical and observed rates may also reflect participation of factors such as macromolecular crowding and reduced rates of diffusion, factors expected to further restrict the accessibility of GST and the diol epoxides in the intact cell.