DNA repair of trans-(+)-anti-, ($-$)-anti-, (+)-syn, and ($-$)-syn-benzo(a);pyrene diol epoxides using human cell extracts: Effects of sequence context and adduct stereochemistry

Lung cancer is the leading cause of cancer deaths in the U.S. Benzo (a) pyrene, a component of cigarette smoke, produces DNA adducts in lung tissue. Mutation and epidemiologic data suggest that persistent unrepaired benzo (a) pyrene diol epoxide (BPDE) adducts are responsible for some mutation hot spots and lung cancers. The +2 BPDE isomer has been shown to be far more mutagenic, and tumorigenic than the other isomers. Different repair rates between BPDE stereoisomers has been proposed as a contributing factor to the mutagenic and tumorigenic activity of BP. In this work the effect of adduct stereochemistry and sequence context on the extent and rate of DNA repair were investigated. Covalently-closed-plasmids containing a site-specific single trans (+)-anti-, $({-})$-anti-, (+)-syn-, or $({-})$-syn-benzo (a) pyrene diol epoxide were constructed. The adduct was attached to $\rm G\sb1$ or $\rm G\sb2$ of the K-ras codon 12 sequence, $5\sp\prime$-$\rm GCTG\sb1G\sb2TGGC$-$3\sp\prime .$ Extent and rate of repair were determined using human whole cell extracts in an in vitro repair synthesis assay. Both repair rate and extent were dependent upon adduct stereochemistry. Overall repair efficiency decreased in the following order: ${+}2\ge{-}1\ge{+}1\gg{-}2.$ Repair efficiency was ${\sim}6$-fold higher for the +2 isomer compared to the ${-}2$ isomer. Although others have found sequence context differences in repair, for the $\rm TG\sb1G\sb2T$ sequence extent and rate of repair was independent of adduct position. To determine if inefficient repair of the ${-}2$ adduct was due to difficulty in making the $3\sp\prime$ incision, single lesion plasmids were prepared that contained a nick at the 6th phosphodiester bond $3\sp\prime$ to the adducted base. The overall repair signal increased for nicked constructs compared to the single lesion closed-circular-constructs and repair for the ${-}2$ adduct increased to +2 levels. Although this study does not support inefficient repair as a basis for increased +2 mutagenicity, it does suggest that BPDE adducts persist in human cells because they are poorly repaired.