Mechanisms of Mammary Gland Carcinogenesis of Heterocycl

在CYP1A2基因缺失(KO)和野生型(WT)小鼠中，研究了CYP1A2在熟肉衍生杂环胺(HCAs)2-氨基-3-甲基咪唑并[4，5-f]喹啉(IQ)和2-amino-1-methyl-6-phenylimidazo[4，5-b]pyridine(PhIP)DNA加合物形成中的作用。给小鼠灌胃IQ(25 mg和75 mg/kg)和PhIP(150 mg/kg)，用~(32)P-后标记法检测肝、肾、乳腺和结肠的DNA加合物水平。无论服用哪种剂量的智商，3小时后，KO组小鼠肝脏和肾脏中的加合物水平是WT组小鼠的20-30%，这一差异具有统计学意义(学生t检验，p&lt；0.05)。在结肠中，仅在最低IQ剂量时，KO小鼠的加合物水平显著低于WT小鼠(分别为1.6±0.6和4.6±0.7，相对加合物标记(AL)×108，平均值+扫描电子显微镜，n=3-5)。然而，在乳腺，无论哪种剂量的IQ，KO和WT小鼠的IQ-DNA加合物水平没有差异。服用PhIP 3小时后，在所有被检测的组织中，KO小鼠的PhIP-DNA加合物水平显著低于WT小鼠。WT小鼠肝脏和肾脏的PhIP-DNA加合物分别为9.9±1.1和22.5±6.9，而KO小鼠的肝脏和肾脏均未检测到PhIP-DNA加合物(检测下限为1.4-2.8×109)。WT小鼠乳腺和结肠的PhIP-DNA加合物水平分别为47.1±9.5和58.0±21.7，而KO小鼠仅为3.8±0.7和5.4±0.9。结果表明，代谢作用的第一步，负责IQ和PhIP N-羟化的CYP1A2显著影响体内DNA加合物的形成。然而，这些数据提出了这样一种可能性，即其他细胞色素P450以及其他激活途径可能有助于特定器官中DNA加合物的形成，这取决于HCA底物。 利用HC11小鼠乳腺上皮细胞系，一种激素介导的分化模型，我们检测了PhIP是否改变了由促乳素激素地塞米松、胰岛素和催乳素(DIP)调控的基因的表达。将稳定表达β-酪蛋白启动子荧光素酶的HC-11-Lux细胞培养在含DIP的培养液中，100 mM的PhIP可使荧光素酶活性提高11倍。只有在含有乳源激素的情况下，才能观察到PhIP对荧光素酶活性的增强作用。在激素浓度较高的培养液中，PhIP处理的HC11细胞内源性b-酪蛋白基因的表达也较高。随着b型酪蛋白基因表达的增加，调节b型酪蛋白基因表达的转录因子磷酸化STAT5A在PhIP作用下的表达水平也随之升高。JAK2特异性抑制剂AG490可阻断PhIP对b-酪蛋白基因表达的影响。PhIP处理的细胞也表现出较高的Bcl2表达和较低的Bax表达，这与PhIP可能的抗细胞凋亡作用一致。研究结果表明，PhIP通过增强STAT5A的磷酸化来调节乳腺上皮细胞产乳激素介导的基因表达。这一发现对乳腺癌致癌物PhIP的新作用机制具有重要意义。

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20-30% the levels in WT mice, a difference that was statistically significant (Student's t-test, p<0.05). In colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6 + 0.6 versus 4.6 + 0.7, respectively, relative adduct labeling (RAL) x 108, mean + SEM, n=3-5 mice). In mammary gland, however, there was no difference in IQ-DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP-DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP-DNA adducts in liver and kidney of WT mice were 9.9 + 1.1 and 22.5 + 6.9, respectively, whereas no PhIP-DNA adducts were detected in either organ of KO mice (limit of detection, 1.4-2.8 x 109). PhIP-DNA adduct levels in mammary gland and colon of WT mice were 47.1 + 9.5 and 58.0 + 21.7, respectively, but accordingly only 3.8 + 0.7 and 5.4 + 0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate. Using the HC11 mouse mammary epithelial cell line, a well-characterized model for hormone-mediated differentiation, we examined whether PhIP altered the expression of genes regulated by lactogenic hormones dexamethasone, insulin, and prolactin (DIP). When HC-11-Lux cells (stably transfected with a b-casein promoter luciferase construct) were cultured in DIP-containing medium, PhIP (100 mM) enhanced luciferase activity 11-fold over that observed in DIP medium alone. The effect of PhIP on augmenting luciferase activity was observed only when lactogenic hormones were included in the medium. Expression of the endogenous b-casein gene was also higher in HC11 cells treated with PhIP in hormone-enriched medium. With the increased expression of b-casein gene, the level of phospho-STAT5A, the transcription factor regulating b-casein gene expression, was elevated in PhIP-exposed HC11 cells. AG490, a JAK2-specific inhibitor, blocked the effect of PhIP on b-casein gene expression. PhIP-treated cells also showed higher expression of Bcl-2 and lower expression of Bax, consistent with a possible anti-apoptotic action of PhIP. The findings indicate that PhIP modulates lactogenic hormone-mediated gene expression in mammary epithelial cells, apparently via enhanced phosphorylation of STAT5A. The findings have implications for a novel mechanism of action of the mammary gland carcinogen PhIP.