In Vivo Mutagenicity and Carcinogenicity of Heterocyclic

点击翻译按钮获取中文摘要

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a food-borne mutagen and mammary gland carcinogen in female rats. A high fat diet has been shown to increase the incidence of PhIP-induced mammary gland tumors. We used Big Blue rats harboring the lambda lacI mutational reporter transgene, to address whether the promotional effect of a high fat diet is mediated via modulation in mammary gland mutagenesis. Big Blue rats were given 10 doses of PhIP (75 mg/kg, p.o.) and place on defined low fat (5% corn oil) or high fat (23.5% corn oil) diet for 6 weeks prior to collecting mammary glands. The lacI mutant frequency (mean + standard error, n=3 rats) was 231 + 15 (x10-6) and 193 + 12 (x10-6) in the low and high fat group, respectively. Values were increased 12-fold over control but were not significantly different between the two diets. In a parallel study, diet did not alter the mutant frequency induced by 7,12-dimethylbenz[a]anthracene (DMBA) (125 mg/kg, p.o.) in the mammary gland. The findings suggest that the promotion by the high fat diet is not mediated via an increase in mutations. Consistent with the high potency of DMBA as a mammary carcinogen, the mutant frequency was 20-30% higher with DMBA than with PhIP. Sixty-nine and 56 PhIP-induced lacI mutants were sequenced from the low and high fat diet groups, respectively. While the percentage of various types of mutations were identical between the diet groups, some difference in the distribution of mutations along the lacI gene was observed. The mutation spectrum in the mammary gland from rats on both diets was consistent with the formation of PhIP-guanine adducts which were detected by 32P-postlabeling assay. Guanine base substitutions accounted for about 85% of all mutations irrespective of diet. Single base pair deletions at guanine occurred in 11-17% of mutants. G:C to T:A transversions were the predominant base substitution mutation accounting for 35-43% of all mutations. The majority of all guanine mutations (74%) occurred at guanine bases adjacent to another G:C pair. Five out of 125 (4%) mutations involved a G deletion in the 5'-GGGA-3' sequence. Twelve out of 125 (10%) mutations involved the guanine base in the sequence 5'-CAG(Pu)-3'. The findings from these studies suggest that 5'-CAG(Pu)-3' is an characteristic target site for PhIP guanine adduct-induced mutations in vivo in the mammary gland. N-Hydroxy-PhIP is the proximate reactive metabolite of PhIP associated with PhIP-DNA adduct formation and mutagenesis. Whole mammary glands obtained from transgenic C57Bl/6 mice carrying the plasmid-lacZ mutational reporter gene were cultured in defined medium and exposed to various concentrations of N-hydroxy-PhIP for 24 h. At various times after N-hydroxy-PhIP exposure, PhIP-DNA adduct levels were determined by the 32P-postlabeling assay and the lacZ-mutant frequency determined by the positive selection system. Glands were cultured in either medium containing insulin (I medium), necessary for maintenance of the gland, or I medium containing prolactin, aldosterone, and hydrocortisone (IPAH medium) to induce lobuloalveolar development. At 3 and 7 days after exposure to 10 mM N-hydroxy-PhIP, mutant frequency was upwards of 9-fold higher in glands incubated in IPAH medium than in I medium (15.2 + 1.9 and 1.6 + 0.7, [(mean + standard error) x 10-3], IPAH and I medium, respectively, 3-day time point). PhIP-DNA adduct levels were 1.7-fold higher in glands cultivated in IPAH medium than in I medium immediately after exposure to 10 mM N-hydroxy-PhIP. A statistically significant reduction in PhIP-DNA adduct levels occurred with time in glands cultivated in IPAH medium but not I medium (one-way analysis of variance, p<0.05). By 7 days after exposure, PhIP-DNA adduct levels were similar in glands cultured in I and IPAH media (3.2 + 0.2 and 2.8 + 0.29 adducts per 107 nucleotides, respectively). DNA synthesis as measured by [3H]thymidine labeling was approximately 2-fold higher in glands culture in IPAH medium than in I medium. The higher mutant frequency in glands cultivated in IPAH medium versus I medium appeared to be due to a combination of higher initial PhIP-DNA adduct levels and a greater fixation of mutations that occurred at higher proliferation rates. The findings indicate that mammotrophic hormones influence the mutagenicity of PhIP in the mammary gland in vitro and emphasize the importance of hormonal milieu on carcinogen-DNA adduct-induced mutations in this organ.