The reactivities of polycyclic aromatic hydrocarbon epoxides in DNA complexes

摘要

$(pm)$-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo (a) pyrene (BPDE) reacts faster than benz (a) anthracene 5,6-oxide (BAO) which reacts faster than benzo (a) pyrene 4,5-oxide (BPO) when the epoxides are reacted in 1.0 mM sodium cacodylate at pH 7.1. When 0.2 mM double-stranded calf thymus DNA (DS CT DNA) was added BPO became more reactive than BAO. Separate UV absorption measurements of BPDE and BPO binding constants for reversible intercalation into DNA and in fluorescence quenching measurements of BPDE, BPO, and BAO model compounds indicate that in DNA the ordering of epoxide reactivities is the same as that of the associations constants. The correlation between association constants provides evidence that reversible intercalation into DS CT DNA is important to catalysis of the benzo (a) pyrene (BP) and benz (a) anthracene epoxide reactions examined. Results from fluorescence lifetime measurements and from measurements of the dependance of associations constants of the BP diols trans-7,8-dihydroxy-7,8-dihydrobenzo (a) pyrene (BP78D) and trans-4,5-dihydroxy-4,5-dihydrobenzo (a) pyrene (BP45D) as a function of Na$sp+$ concentration are consistent with the conclusion that in single-stranded M13mp19 DNA (SS M13 DNA) reversible binding occurs primarily via intercalation into regions where local duplex formation occurs. A comparison of approximate values of catalytic rate constants for BPDE hydrolysis in DS CT DNA and SS M13 DNA at varying Na$sp+$ concentrations indicates that the absolute values of the catalytic rate constants decrease approximately 5.1 and 6.5 fold, respectively, as the Na$sp+$ concentration increases from 2 to 100 mM. However, for this range of Na$sp+$ concentrations the ratios of the catalytic rate constants for reaction in DS CT DNA to the rate constant in SS M13 DNA is almost constant with values (1.4 to 1.8) nearly equal to 1. The similarity of the magnitudes of the catalytic rate constants provides evidence that catalytic sites in SS M13 DNA are similar to catalytic intercalation sites in DS CT DNA. Finally, the percentage of BPDE which forms adducts with DS CT DNA in 10 mM Tris, 1 mM EDTA (pH 7.3) was measured as a function of Na$sp+$ concentration. The results indicate that while the overall, pseudo-first-order rate constant varies 6.5 fold over this range of Na$sp+$ concentrations the percentage of BPDE reaction leading to adduct formation (6.5 $pm$ 0.5) remains nearly constant. This observation demonstrates that DNA not only catalyzes the BPDE hydrolysis reaction but also catalyzes DNA modification reactions.