Benzo[c]phenanthrene−DNA Adducts in Mouse Epidermis in Relation to the Tumorigenicities of Four Configurationally Isomeric 3,4-Dihydrodiol 1,2-Epoxides

Abstract

P-Postlabeling assays were used to monitor the binding to epidermal DNA that resulted from the application of each of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide to mouse skin in vivo. For three of these configurational isomers, there was a reasonable correlation between the relative level of binding to epidermal DNA and the known tumorigenic effects of these compounds. However, for the 4(S),3(R)-dihydrodiol 2(S),1(R)-epoxide, the tumorigenic response was considerably greater in relation to the level of DNA modification than was the case for the other isomers. This greater tumorigenic response was consistent with previous observations indicating that this isomer was more mutagenic, at equivalent levels of DNA modification, than the other two tumorigenic dihydrodiol epoxides. Additionally, the 4(S),3(R)-dihydrodiol 2(S),1(R)-epoxide reacts with DNA to generate predominantly (approximately 80%) adducts on the amino group of adenine residues. These findings might imply a greater intrinsic biological effect of such adenine adducts with respect to the other major adduct formed on the amino group of guanine residues.

DNA adduct levels associated with p53 induction and delay of MCF-7 cells in S phase after exposure to benzo[g]chrysene dihydrodiol epoxide enantiomers

“…To obtain an independent assessment of the levels of DNA modification by the two enantiomeric BgChDEs in these cells, DNA was isolated from cells exposed to various concentrations of each isomer for 21 h, and DNA adduct levels were determined by postlabeling [13,20,21]. Typical chromatograms showing the labeled adducted nucleoside bis-phosphates obtained from MCF-7 cells are shown in Figure 2, along with adducts obtained from reaction of the optically active BgChDEs with calf-thymus DNA.…”

“…Quantitation in postlabeling assays is difficult for a variety of reasons, and adduct detection is never completely efficient. In previous work in our laboratory, the efficiencies of detection of benzo[c]phenanthrene-DNA adducts were approximately 25-50% [21], and differences in the efficiency of detection for adducts derived from enantiomeric dihydrodiol epoxides have been noted [13,21]. Therefore, the postlabeling data provide only an estimate of DNA binding lev-els.…”

“…Five-microliter aliquots (2 µg) of calf-thymus DNA that had been reacted with optically active dihydrodiol epoxide were digested to deoxyribonucleoside-3′phosphates by using 2 µg each of calf-spleen phosphodiesterase and micrococcal nuclease at 37°C for 3 h [20,21]. This was followed by digestion for 1 h with nuclease P1 (2 µg) [22].…”

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

“…BcPh DEs react extensively with dA residues in DNA (37,38), and the DEs and their resultant adducts induce a variety of mutations at dA sites in vivo (39,(63)(64)(65). These BcPh DE-dA adducts are of particular significance since they are known to elude cellular repair processes (66) and thus are highly likely to be encountered by other DNA-processing enzymes.…”

“…Notably, human liver microsomes metabolize BcPh via the carcinogenic diol epoxide pathway to a greater extent than do microsomes from rat liver (36). The metabolically derived BcPh DEs react extensively with dA residues in DNA in vitro (37) and in vivo (38), and induce mutations at dA in mammalian cells (39). Such mutations provide an attractive mechanism for the induction of cell transformation leading to cancer.…”