Teresa A. Smolarek

Temperature-induced alterations in the metabolic activation of benzo[a]pyrene to DNA-binding metabolites in the Bluegill fry cell line BF-2

Abstract Many carcinogenic chemicals such as the environmental pollutant benzo[ a ]pyrene (B[ a ]P) require metabolic activation to reactive DNA-binding intermediates in order to induce cancer formation. To determine whether the temperature of incubation affects the proportion of B[ a ]P metabolized to DNA-binding metabolites as well as the amount of B[ a ]P metabolized, cultures of the Bluegill ( Lepomis macrochirus ) fry cell line BF-2 maintained at 23°C and 35°C were exposed to [ 3 H]B[ a ]P. Exposure at 35°C resulted in an increase in the amount of B[ a ]P metabolized by 61% at 24 h compared with cultures incubated at 23°C. The amount of B[ a ]P-9,10-diol present in the medium was similar at both temperatures, but the amount of water-soluble metabolites was greater in the cultures maintained at 35°C. The amount of B[ a ]P bound to DNA in cultures exposed to B[ a ]P at 35°C was 97% greater at 24 h and 61% greater at 48 h than in cultures exposed at 23°C. The increase in the major B[ a ]P-deoxyribonucleoside adduct, (+)- anti -B[ a ]P-7,8-diol-9,10-epoxide (B[ a ]PDE; the isomer with the epoxide and benzylic hydroxyl on opposite faces of the molecule)-deoxyguanosine at 35°C, was 314% and 100% at 24 h and 48 h, respectively. There was no significant change in the amount of syn -B[ a ]PDE (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule)-deoxyguanosine. These results indicate that although the amount of B[ a ]P metabolized by BF-2 cell cultures is 60% greater at 35°C than at 23°C, the amount of B[ a ]P bound to DNA through the (+)- anti -B[ a ]PDE increased by more than 300%. Thus increased temperature can increase the proportion of B[ a ]P metabolized to an ultimate carcinogenic metabolite in BF-2 cells in culture.

Benz[a]anthracene-induced alterations in the metabolic activation of benzo[a]pyrene by hamster embryo cell cultures

Co-administration of benz[a]anthracene (BA) with benzo[a]pyrene (B[a]P) to hamster embryo cell cultures for 24 h resulted in a decrease in the metabolism of benzo[a]pyrene by 40%, a decrease in the level of binding of B[a]P to DNA by 70% and a 10-fold reduction in mutation induction in a hamster embryo cell-mediated V79 cell mutation assay. This data indicates that the biological effects of co-administration of BA with B[a]P result from inhibition of the metabolic activation of B[a]P rather than induction of enzymes that detoxify the B[a]P.

Effects of pretreatment with benzo[a]pyrene on the stereochemical selectivity of metabolic activation of benzo[a]pyrene to DNA-binding metabolites in hamster embryo cell cultures

The proportions of individual benzo[a]pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600). To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing [3H]BaP. After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group. Cells pretreated for 24 h with BaP and then exposed to [3H]BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to [3H]BaP for 5 h. In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to [3H]BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct. Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced [3H]uridine incorporation into RNA by more than 65% at all times. These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells. The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE.

Species-Dependent Differences in the Metabolic Activation of Polycyclic Aromatic Hydrocarbons in Cells in Culture

Polycyclic aromatic hydrocarbons are widespread environmental contaminants that require metabolic activation in order to induce biological effects1. One of the most widely studied carcinogenic hydrocarbons is benzo-(a)pyrene (BaP). Most pathways of BaP metabolism result in the production of metabolites that are detoxification products. However, a small proportion of the BaP metabolites are reactive derivatives that bind to DNA in cells and these DNA interactions are involved in the initiation of the cancer induction process1. Although these reactive metabolites cannot be isolated from cells so that their production can be quantitated, it is possible to measure their formation through detection of the DNA adducts they produce. The DNA serves as both a critical target for the “ultimate carcinogenic metabolites” and as a nucleophilic trapping agent for detection and measurement of these reactive electrophiles.

7,12-Dimethylbenz[a]anthracene-DNA adduct formation in Wistar rat and Syrian hamster embryo cell cultures

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA was examined in Syrian hamster and Wistar rat embryo cell cultures exposed to DMBA for 5, 24, 48 and 72 h. The level of binding of DMBA to DNA was about twice as great in the hamster embryo cells as in the rat embryo cells at all times. Analysis of the DMBA-deoxyribonucleoside adducts by immobilized boronate chromatography demonstrated that the ratio of adducts with no cis vicinal hydroxyl groups to those containing cis vicinal hydroxyl groups was much greater in the rat embryo cells (from 2.2:1 to 2.9:1) than in the hamster embryo cells (from 1.3:1 to 1.6:1). The hamster embryo cells contained three major DMBADE-DNA adducts: based upon their chromatographic behavior and comparison with the three major DMBA-DNA adducts described by Dipple et al. in mouse embryo cell cultures (Biochemistry, 24 (1985) 2291), two were tentatively identified as resulting from the reaction of anti-DMBADE (the isomer of 1,2-epoxy-3,4-dihydroxy-1,2,3,4-tetrahydro-DMBA with the epoxide and benzylic hydroxyl on the opposite faces of the molecule) with deoxyguanosine and deoxyadenosine and one adduct resulted from reaction of syn-DMBADE (epoxide and benzylic hydroxyl on the same face of the molecule) with deoxyadenosine. The anti-DMBADE-deoxyguanosine, syn-DMBADE-deoxyadenosine, and anti-DMBADE-deoxyadenosine adducts were present in hamster embryo cell DNA in a ratio of 1.2:2:1. The Wistar rat embryo cell DNA contained a much larger proportion of the syn-DMBADE-deoxyadenosine adduct. The relative proportions of the three major DMBA-DNA adducts in Syrian hamster embryo cells were similar at all times, but the proportion of syn-DMBADE-deoxyadenosine adduct decreased slightly with time in the rat embryo cells. These results indicate that there are species specific differences in the stereospecificity of activation of DMBA to DNA-binding diol epoxides which parallel those observed for benzo[a]pyrene (BaP). The high proportion of deoxyadenosine adducts suggests that they may have an important role in the induction of biological effects by DMBA.