Carcinogenic and Toxicologic Effects of PhIP on the Mammary Gland

苯丙氨酸(2-Amino-1-methyl-6-phenylimidazo[4，5-b]pyridine，PhIP)是一种在熟肉中发现的芳香胺，是一种已确定的雌性大鼠乳腺致癌物质。在本项目中，我们使用大鼠模型来研究PhIP对乳腺的致癌靶向作用的机制和涉及的因素。在最近的一项研究中，四种剂量的PhIP(150 mg/kg，P.O.，每天一次)被给予与其10日龄幼鼠分开的哺乳SD大鼠，以启动腺体的退化。末次给药24小时后，TUNEL法检测对照组大鼠乳腺细胞凋亡指数显著高于苯丙酮组(4.757%1.066 vs 1.905%0.248，p&lt0.05)。与对照组相比，PhIP处理组大鼠乳腺中的肺泡也明显较大，并含有更多的分泌上皮细胞。Western blotting检测细胞凋亡刺激因子Bax和细胞凋亡抑制因子Bcl2的表达。相应地，对照组大鼠Bax的表达是对照组的2.7倍，而PhIP处理组的Bcl2的表达是对照组的3.1倍，两者的变化在统计学上是不同的(学生t检验，p&lt；0.05)。免疫组织化学进一步证实，经PhIP处理的乳腺分泌腺泡上皮细胞中Bax表达降低，而Bcl2表达增强。这一发现与PhIP暴露通过部分抑制程序性细胞死亡而延缓退化的观点一致。为了探讨PhIP抑制乳腺退缩的可能机制，测定了正常发情周期的处女大鼠给予PhIP(150 mg/kg，P.O.)后血清催乳素的水平。在发情的早晨。经过一个发情周期后，发情当天清晨，PHIP组的血清催乳素水平是对照组的1.3倍(单因素方差分析，Fisher LSD多重比较检验，p&lt；0.05)。在退缩期间暴露于PhIP与良性乳腺肿瘤的诱发有关。12只大鼠中有7只发生纤维腺瘤，1只在退化期给予PhIP(150 mg/kg，每天一次，连续5天)和高脂饮食(23.5%玉米油)后一年内发展为管乳头状癌。血清催乳素水平的升高和PhIP对乳腺细胞凋亡的影响可能与该化合物的致癌机制有关。进一步研究了PhIP对不同细胞系信号转导通路的影响，包括人乳腺细胞系MCF10A。结果表明，PhIP通过增加ERK-2的磷酸化和增加Bcl2的表达来延缓细胞的凋亡。结果表明，PhIP对乳腺上皮细胞具有促增殖作用，这可能与其致癌机制有关。

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is an arylamine found in cooked meat which is an established mammary gland carcinogen in female rats. In this project, we use the rat model to investigate the mechanisms and factors involved in the carcinogenic targeting of PhIP to the mammary gland. In a recent study, four doses of PhIP (150 mg/kg, p.o., once per day) were given to lactating Sprague-Dawley rats separated from their 10-day old pups to initiate involution of the gland. Twenty-four hours after the last dose, apoptotic index in the mammary gland, as measured by the TUNEL assay, was significantly higher in the gland from control rats than in the PhIP-treated rats (4.757% 1.066 vs 1.905% 0.248, p<0.05). In comparison to controls, alveoli in the mammary gland of PhIP-treated rats were also visibly larger and contained more secretory epithelial cells. The expression of Bax, a stimulator of apoptosis, and Bcl-2, an inhibitor of apoptosis, were quantitated by Western blotting. Accordingly, Bax expression was 2.7-fold higher in control rats while Bcl-2 expression was 3.1-fold higher in PhIP-treated rats, both changes being statistically different (Student's t-test, p<0.05). Immunohistochemistry further confirmed a lower expression of Bax and higher expression of Bcl-2 in secretory alveolar epithelial cells of the PhIP-treated mammary gland. The findings are consistent with the notion that exposure to PhIP retarded involution via partial inhibition of programmed cell death. To investigate possible mechanisms for the inhibitory effects of PhIP on mammary gland involution, serum levels of prolactin were measured in virgin rats with regular estrous cycles given PhIP (150 mg/kg, p.o.) on the morning of diestrous. After one estrous cycle, on the morning of proestrous, serum prolactin levels were 1.3-fold higher after PhIP than after control vehicle (one way ANOVA, Fisher LSD multiple comparison test, p<0.05). PhIP exposure during involution was associated with the induction of benign mammary tumors. Seven out of 12 rats developed fibroadenomas, and one developed a tubulopapillary carcinoma within one year after receiving PhIP administration during involution (150 mg/kg, p.o.once per day for five days) and a high fat diet (23.5% corn oil). An increase in serum prolactin level and the effects on mammary gland apoptosis seen with PhIP may have implications for the mechanisms of mammary gland carcinogenesis of this compound. Further studies were carried out to address the effects of PhIP on signal transduction pathways in various cell lines, including human breast cell line, MCF10A. The results showed that PhIP delayed apoptosis via a mechanism involving an increase in Erk-2 phosphorylation and an increase in Bcl-2 expression. The findings show that PhIP has pleotropic effects on mammary epithelial cells that may be pertinent to understanding its mechanism of mammary carcinogenesis.