Inhibition of Werner Syndrome Helicase Activity by Benzo[ c ]phenanthrene Diol Epoxide dA Adducts in DNA Is Both Strand-and Stereoisomer-dependent

Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis- and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (-)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (+)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3' or the 5' side of the adducted deoxyadenosine for the 1S- and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts

Hepatotoxicity of precocene I in rats: role of metabolic activation in vivo

The mechanism of the hepatotoxicity of precocene I has been investigated in male, Sprague-Dawley rats. Administration of a single dose of precocene I caused a large depletion of liver glutathione (GSH) levels that was both time and dose dependent. Concomitant with the decrease of liver GSH, there was an increase in serum glutamic pyruvic transaminase (GPT) levels which was also time and dose dependent. Administration of a single dose of [4-3H]precocene I resulted in extensive covalent binding of the radiolabel to liver proteins and DNA in the liver; the extent of binding increased with increasing dose. Treatment of the rats with the mixed-function oxidase inhibitor piperonyl butoxide, before administration of precocene I, significantly decreased the proportion of the radiolabel bound covalently to proteins and DNA, although the total radioactivity (bound and unbound) in the liver remained the same. Piperonyl butoxide pretreatment limited both the liver GSH depletion and the hepatic necrosis normally caused by precocene I. These results are consistent with the view that the hepatotoxicity of precocene I is due to reactive metabolites formed through cytochrome P-450 mediated metabolism of precocene I