Modulation of microsomal benzo[a]pyrene metabolism by DNA

Inclusion of calf thymus DNA during microsomal benzo[a]pyrene (BP) metabolism increases product formation by decreasing the accessibility of microsomal enzymes to inhibitory BP quinones. The relief of product inhibition of BP metabolism, the stimulation of the formation of BP 7,8-dihydrodiol-9,10-oxides (DE), and the corresponding DNA adducts were all dependent to varying extents on DNA concentration. The role of BP quinones was evidenced by effects of DNA on all aspects of quinone reactivity: (a) inhibition of microsomal reduction of quinones, (b) inhibition of quinone glucuronidation, (c) inhibition of quinone monooxygenation, (d) a substantial reduction of the inhibition of BP metabolism and diol epoxide (DE) formation by added BP 6,12-quinone, and (e) a stimulation of BP metabolism even though quinine levels were also increased. DNA inhibited the reduction of 1,6- and 3,6-quinone to a similar degree under both oxygen-depleted and aerobic conditions. Other effects of DNA were very selective; glucuronidation of added 1,6- and 6,12-quinone was inhibited less than glucuronidation of 3,6-quinone (35% and 50% versus over 80%). However, monooxygenation of 3,6-quinone was not inhibited, whereas monooxygenation of 1,6-quinone was reduced by 60-70%. There was no measurable monooxygenation of 6,12-quinone. This specificity may indicate that DNA exerts its effect not simply by sequestering BP quinones. The interaction with DNA produced a distinct 25-nm red shift in the visible spectra of 1,6- and 3,6-quinone, while the change in the 6,12-quinone spectrum was less pronounced. RNA induced a similar red shift in the 1,6-quinone spectrum. Spectral measurements indicated binding of one molecule of 1,6-quinone per 50 DNA base pairs, while binding to RNA was 10-fold less extensive. The binding of 1,6-quinone to DNA was decreased by Mg2+, suggesting that 1,6-quinone binds by intercalation. DNA perturbs BP metabolism in a second way by increasing the ratio of 9-phenol to 9,10-dihydrodiol 4-fold. This is probably due to a DNA-catalyzed rearrangement of 9,10-oxide to 9-phenol. This effect contributes substantially to the greater sensitivity of the formation of 9-phenol 4,5-oxide-DNA adducts to the DNA concentration. It is evident from these data that, in addition to binding carcinogens covalently, DNA can affect the kinetics and product distribution of carcinogen metabolism. The high capacity of DNA to sequester BP quinones from cellular membranes is likely to be associated with additional DNA damage which may contribute to carcinogenesis.