Mouse embryo cell adducts

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. 

Figure 2. Sephadex LH-20 column chromatography of DMBA-deoxyribo-nucleoside adducts formed by enzymatic digestion of DNA from mouse embryo cells exposed to [l Cj-DMBA (0.2 yg/ml) for 24 h ( - ) and of DNA from the skin of female NIH Swiss mice treated with [3h]-DMBA (10 yg/mouse) for 24 h (0-0). The arrow denotes the position of elution of an added uv-absorbing marker 4-(p-nitrobenzyl)pyridine. "Reproduced with permission from Ref. 15. Copyright 1980, IRL Press".

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. 

Secondary cultures of hamster embryo cells metabolized 90% of the added DMBA in 48 hours. The major organic-soluble metabolite was the 8,9-dihydrodiol (19, 147). Hydroxymethyl derivatives were only minor metabolites. Glucuronides of DMBA phenols were also major metabolites. The results of this study suggest that there may be important differences between microsomal and whole cell metabolism of DMBA (19). Such differences were also evident when the products of binding of DMBA to DNA in mouse embryo cell cultures and mouse skin or catalyzed by Aroclor induced rat liver microsomes were compared. The major adducts observed in mouse skin or mouse embryo cell cultures resulted from metabolism in the 1-4 ring, presumably through the 3,4-dihydrodiol-1,2-epoxide, whereas the main adduct formed in the microsomal system resulted from reaction with the 4,5-epoxide of DMBA (35). 

The presence in cellular systems of conjugating enzymes may partially explain some of the differences which have been observed in the nature of BaP-DNA adducts when microsomal versus cellular systems were used for activation. While the major adducts formed in rodent embryo cells or mouse skin resulted from reaction of BaP-7,8-diol-9,10-epoxide, isomer I, with DNA, another adduct was formed from BaP in the presence of rat liver microsomes 238, 271), This adduct appears to result from reaction of DNA with the 4,5-epoxide of 9-hydroxy-BaP. The factors controlling formation of this adduct are not completely understood, since it has not always been observed when microsomal systems were used for BaP activation 444). 

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