37571-88-3

Reactions of the benzo[a]pyrene (BP) and benz[a]anthracene (BA) metabolites, (±)-trans-7, 8-dihydroxy-anti-9, 10-epoxy-7, 8, 9, 10- tetrahydro-BP (BPDE), (±)-trans-3, 4-dihydroxy-anti-1, 2-epoxy-1, 2, 3, 4- tetrahydro-BA (BADE), (±)-BP-4, 5-oxide (BPO), and (±)-BA-5, 6-oxide (BAO), were examined under pseudo-first-order conditions at varying Na+ (2.0-100 mM) and native, calf thymus DNA (ctDNA) concentrations. In 0.2 mM ctDNA and 2.0 mM Na+, at a pH of 7.3, most BPDE, BADE, BPO, and BAO (87-95%) undergo DNA catalyzed hydrolysis or rearrangement. For BPDE and BPO, overall, pseudo- first-order rate constants, k, in 2.0 mM Na+ and 0.2 mM ctDNA are 21-72 times larger than values obtained without DNA. For BADE and BAO, the rate constants are less strongly influenced by DNA; k values in 0.2 mM ctDNA are only 9-12 times larger than values obtained without DNA. Kinetic data for BPDE, BPO, BADE, and BAO and DNA intercalation association constants (K(A)) for BP and BA diols which are model compounds indicate that K(A) values for BPDE and BPO in 2.0 mM Na+ are 6.6-59 times larger than those of BADE and BAO. The greater DNA enhancement of rate constants for BPDE and BPO, versus BADE and BAO, correlates with the larger K(A) values of the BP metabolites. DNA adducts, which account for less than 10% of the yields, also form. For BPDE in 0.20 mM ctDNA, k decreases 5.1 times as the Na+ concentration increases from 2.0 to 100 mM. Nevertheless, the DNA adduct level remains constant over the range of Na+ concentrations examined. These results provide evidence that, for BPDE in 0.20 mM DNA and 2.0 mM Na+, ctDNA adduct formation follows a mechanism which is similar to that for DNA catalyzed hydrolysis. The pseudo-first-order rate constant for adduct formation, k(Ad), given approximately by k(Ad) ? (k(cat, Ad)K(A)[DNA])/(1 + K(A)[DNA]), where k(cat, Ad) is a catalytic rate constant. For BADE, BPO, and BAO, the influence of varying DNA and Na+ concentrations on k values is similar to that for BPDE, and provides evidence that the formation of adducts follows the same rate law.

Fluorescence intensity measurements of overall, pseudo-first-order rate constants for two epoxide-containing metabolites of benzo[a]pyrene (BP) were carried out in Tris, EDTA buffer (pH 7.3) without DNA, and in buffer with double-stranded calf thymus DNA (DS ctDNA) and with closed-circular, single-stranded viral M13mp19 DNA (SS M13 DNA). Highly purified SS M13 DNA was employed in order to avoid polymeric contamination which is present in DNA samples obtained using a standard preparation method relying on phenol extraction and which influences results from measurements of DNA-ligand interactions. The BP metabolites examined were highly carcinogenic (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[e]pyrene (BPDE) and less genotoxic benzo[a]pyrene 4,5-oxide (BPO). Without DNA, BPDE hydrolyzes to 7,8,9,10-tetrahydroxytetrahydro-BP, while BPO hydrolyzes to trans-4,5-dihydroxy-4,5-dihydro-BP (BP45D) and rearranges to 4-hydroxy-BP. With DNA, BPDE and BPO hydrolysis and rearrangement are catalyzed, and DNA modification occurs. In DS ctDNA, previous kinetic and binding measurements indicate that catalysis occurs primarily at intercalation sites. In SS M13 DNA (0.20 mM), BPDE has overall, pseudo-first-order rate constants (k) of (12 ± 1) × 10-3 and (2.8 ± 0.5) × 10-3 s-1, at Na+ concentrations of 2.0 and 100 mM, respectively. At these Na+ concentrations, values of k measured in SS M13 DNA are 3-16 times larger than values measured without DNA, but smaller than values measured in DS ctDNA. For BPO, the ordering of k values without DNA, with SS M13 DNA, and with DS ctDNA is the same as for BPDE. At 2.0 mM Na+, the nonreactive diols, trans-7,8-dihydroxy-7,8-dihydro-BP (BP78D) and BP45D, which are model compounds for BPDE and BPO, respectively, have SS M13 DNA association constants [(7.2 ± 0.5) × 103 and (2.7 ± 0.5) × 103 M-1] that are 2.3 times smaller than DS ctDNA association constants. In contrast, at 100 mM Na+, association constants for SS M13 DNA are 2.9-3.1 times larger than for DS ctDNA. Fluorescence lifetime measurements indicate that, in SS M13 DNA, reversible binding involves intercalation into local duplex regions. Estimated catalytic rate constants (kcat) for BPDE hydrolysis in SS M13 DNA, obtained from BP78D association constants and from k values measured with and without DNA, are (22.8 ± 2.5) × 10-3 and (3.5 ± 0.7) × 10-3 s-1, at 2.0 and 100 mM Na+, respectively. For this Na+ concentration range, the ratio of kcat values for DS ctDNA versus SS M13 DNA is almost constant (1.7 ± 0.6) even though the absolute kcat values vary by more than a factor of 5. The similar magnitudes of kcat values for SS M13 DNA and DS ctDNA provide evidence that catalytic sites in SS M13 DNA are similar to intercalated catalytic sites in DS ctDNA.

Time-resolved fluorescence and UV photoelectron measurements have been employed to examine the influence of calf thymus DNA on the reactivities of epoxides derived from benzo[a]pyrene (BP) and benz[a]anthracene (BA). By monitoring the increase in fluorescence intensity, which accompanies reaction at 23°C, overall, pseudo-first-order rate constants have been measured for reactions of the highly carcinogenic bay region epoxide (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and of two less carcinogenic K region epoxides benzo[a]pyrene 4,5-oxide (BPO) and benz[a]anthracene 5,6-oxide (BAO). Overall rate constants for hydrolysis and rearrangement reactions have been measured for BPDE, BPO, and BAO in buffer alone (1.0 mM sodium cacodylate, pH 7.1). The rate constants increase in the order BPO ((3.8 ± 0.1) × 10-6 s-1) -5 s-1) -4 s-1). These results have been compared with overall rate constants for reactions, carried out in calf thymus DNA, which result in catalyzed hydrolysis and rearrangement, as well as DNA adduct formation. In DNA, the ordering of the rate constants for BPO and BAO changes from that observed in buffer alone. The rate constants increase in the order BAO ((2.8 ± 0.1) × 10-3 s-1) -2 s-1) -1 s-1). This ordering is the same as the ordering of association constants for the reversible binding to DNA of the fluorescent diols trans-7,8-dihydroxy-7,8-dihydro-BP (BP78D), trans-4,5-dihydroxy-4,5-dihydro-BP (BP45D) and cis-5,6-dihydroxy-5,6-dihydro-BA (BAD), which are model compounds of BPDE, BPO, and BAO, respectively. For the model compounds, the association constants for intercalation increase in the order BAD ((3.6 ± 0.9(× 102 M-1) 3 M-1) 4 M-1). This ordering is consistent with the ordering of the association constants of BPDE ((2.5 ± 0.3) × 104 M-1) and of BPO ((6.0 ± 1.0) × 103 M-1). The temperature dependence of the association constants of the model compounds demonstrates that, for the intercalation of the BP diols into DNA, differences in the enthalpy of binding contribute significantly to differences in the free energy of binding. UV photoelectron data and results from ab initio molecular orbital calculations on BPDE, BPO, and BAO indicate that, for these three epoxides, the association constants increase as the ionization potentials decrease and the polarizabilities increase. The percentage of epoxide reaction that yields DNA adducts has been compared under varying conditions. For long reaction times (>1 h) in systems containing native, calf thymus DNA at low salt concentrations, the ordering of adduct yields is BPO (14.9 ± 1.1%) > BPDE (10.1 ± 3.0%) > BAO (3.6 ± 0.4%). For short reaction times (10 min) in systems containing native DNA stabilized with 0.10 mM Mg2+, the ordering of adduct yields is BPDE (7.3 ± 1.9%) > BPO (1.3 ± 0.1%) > BAO (0.1 ± 0.1%). These results suggest that the ability of an epoxide to form adducts with exposed DNA during long reaction times is less indicative of the genotoxic potency of the epoxide than its ability to form adducts with stabilized DNA during short reaction times.

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