In Vivo Mutagenicity and Carcinogenicity of Heterocyclic

2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶是一种食源性诱变剂和雌性大鼠乳腺致癌物。高脂肪饮食已被证明会增加phip诱导的乳腺肿瘤的发生率。我们使用携带lambda lacI突变报告基因的大鼠来研究高脂肪饮食的促进作用是否通过调节乳腺诱变来介导。在采集乳腺前，给大鼠10剂PhIP (75 mg/kg, p.o.)，并给予规定的低脂（5%玉米油）或高脂（23.5%玉米油）饮食6周。低脂组和高脂组lacI突变频率（平均值+标准误差，n=3只大鼠）分别为231 + 15 （x10-6）和193 + 12 （x10-6）。数值比对照组增加了12倍，但两种饮食之间没有显著差异。在一项平行研究中，饮食并没有改变7,12-二甲基苯[a]蒽（DMBA） （125 mg/kg, p.o）在乳腺中引起的突变频率。研究结果表明，高脂肪饮食的促进作用并不是通过增加突变来介导的。与DMBA作为乳腺癌致癌物的高效力一致，DMBA的突变频率比PhIP高20-30%。从低脂和高脂饮食组分别测序了69和56个phip诱导的lacI突变体。虽然不同饮食组之间的各种类型突变的百分比相同，但观察到lacI基因突变的分布存在一些差异。两种饮食的大鼠乳腺突变谱与phip -鸟嘌呤加合物的形成一致，这是通过32p后标记法检测到的。鸟嘌呤碱基置换占所有突变的85%，与饮食无关。11-17%的突变体出现鸟嘌呤单碱基对缺失。G:C到T:A的转换是主要的碱基取代突变，占所有突变的35-43%。大多数鸟嘌呤突变（74%）发生在相邻G:C对的鸟嘌呤碱基上。125个突变中有5个（4%）涉及5‘-GGGA-3’序列的G缺失。125个突变中有12个（10%）涉及5‘-CAG(Pu)-3’序列中的鸟嘌呤碱基。这些研究结果表明，5‘-CAG(Pu)-3’是PhIP鸟嘌呤加合物诱导的乳腺体内突变的特征性靶点。

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.