Bay region dihydrodiol epoxide

Figure 2. 7-Methylbenz[a]anthracene and benzo[a]pyrene indicating those regions defined as bay regions and the structures of the corresponding bay region dihydrodiol epoxides.

Figure 4. The bay region dihydrodiol epoxide route of metabolism of benzo[a]pyrene.

Structures of 5-MeC bay region dihydrodiol epoxides and their precursor dihydrodiols. 

Identification of DNA-Reactive Metabolites Generated in a Target Tissue, Mouse Skin, In Vivo. Our initial studies focused on activation of DMBA in mouse embryo cells in culture because of the ease of isolation of sufficient DNA for adduct characterization. The cells were exposed to DMBA and the isolated DNA enzymatically hydrolyzed to deoxyribonucleosides. DMBA-deoxyribonucleoside adducts were characterized by fluorescence measurements (11,22), by photosensitivity studies (12) and by column chromatography (23,24). These studies provided evidence that the DNA-reactive metabolite generated in these cells is a bay region dihydrodiol epoxide. The enzymatic steps in this activation pathway (Figure 1) involve oxidation of DMBA by mixed function oxidases to a 3,4-epoxide which is converted by epoxide hydrase to a 3,4-dihydro-diol. This is, in turn, oxidized by mixed function oxidases to the dihydrodiol epoxide. 

In contrast, we did not find these concentration-dependent qualitative changes when activation occurred in intact cellular systems (16). We examined the adducts formed in mouse embryo cells in culture and in mouse skin ijri vivo over 40- and 100-fold DMBA concentration ranges, respectively, and found quantitative, but no qualitative, changes in binding (16). At all concentrations, activation appeared to be through the bay region dihydrodiol epoxide pathway. The cellular systems are physically very different from the homogenate systems and it is difficult to... 

The relative amounts of syn and anti adducts produced in mouse embryo cells did not vary substantially with DMBA concentration (20). However, we found a dramatic difference in the relative amounts of these adducts when the dose of DMBA applied to mouse skin was varied (jl). Figure 9 shows the HPLC elution profiles for adducts formed at a low dose of 14 nmol [ HJ-DMBA. Peaks A,C and D are present in approximately equal amounts, i.e. 29, 21 and 22% of total radioactivity, respectively. However, at a 100-fold higher dose of 1400 nmol, peak C has increased to 39% while A and D have decreased to 13% and 9%. These results indicate that the formation of syn-bay region dihydrodiol epoxide adducts is favored at high doses. Due to this, the total binding to deoxyadenosine (peaks C and D) also increases with dose and ranges from 27% to 48% of the total DNA binding. 

In studies on the metabolic activation of 5-MeC (Fig. 9), in vitro metabolism was coupled with mutagenicity and carcinogenicity assays. This work illustrates some techniques which may be used to elucidate structure and determine active metabolites. The metabolic activation of 5-MeC was of interest because of its high carcinogenicity and mutagenicity compared with the other methylchrysene isomers (183). In addition, 5-MeC has two dissimilar bay regions available for formation of bay region dihydrodiol epoxides. 

In accordance with the above results, a bioassay of 1,2-dihydro-1,2-dihydroxy-5-MeC as a tumor initiator on mouse skin indicated high tumorigenic activity. The 1,2-dihydrodiol was more tumorigenic than 5-MeC or than 7,8-dihydro-7,8-dihydroxy-5-MeC 9,10-dihydro-9,10-dihydroxy-5-MeC was inactive (795). These results are in agreement with the bay region hypothesis discussed earlier. However, they are of further interest since both the 1,2-dihydrodiol and the 7,8-dihydrodiol could form bay region dihydrodiol epoxides, but the former was more tumorigenic than the latter. 

---