Carcinogenic and Toxicologic Effects of PhIP on the Mammary Gland

2-氨基-1-甲基-6-苯基咪唑并[4，5-B]吡啶（PhIP）是一种在熟肉中发现的芳胺，是一种已确定的雌性大鼠乳腺致癌物。 在这个项目中，我们使用大鼠模型来研究PhIP对乳腺的致癌靶向机制和相关因素。在最近的一项研究中，四个剂量的PhIP（150 mg/kg，p.o.，每天一次）给予与其10日龄幼鼠分离的哺乳期Sprague-Dawley大鼠以引发腺体退化。 在最后一次给药后24小时，通过TUNEL分析测量的乳腺中的凋亡指数在来自对照大鼠的腺体中显著高于PhIP处理的大鼠（4.757% ± 1.066 vs 1.905% ± 0.248，p<0.05）。 与对照组相比，PhIP处理的大鼠乳腺中的肺泡也明显更大，含有更多的分泌上皮细胞。Western blotting法检测Bax和Bcl-2的表达。因此，Bax表达在对照大鼠中高2.7倍，而Bcl-2表达在PhIP处理的大鼠中高3.1倍，两种变化都是统计学上不同的（Student’s t检验，p<0.05）。 免疫组化进一步证实了较低的表达Bax和较高的表达Bcl-2的分泌腺泡上皮细胞的PhIP处理的乳腺。 这些发现与以下观点一致：暴露于PhIP通过部分抑制程序性细胞死亡延缓退化。 为了研究PhIP对乳腺退化的抑制作用的可能机制，在具有规律发情周期的未交配大鼠中测量了给予PhIP（150 mg/kg，p.o.）在diestrous的早晨。在一个发情周期后，在发情前期的早晨，PhIP后的血清催乳素水平比对照载体后高1.3倍（单因素ANOVA，Fisher LSD多重比较检验，p<0.05）。 在退化过程中暴露于PhIP与良性乳腺肿瘤的诱导有关。 在退化期间接受PhIP给药（150 mg/kg，p.o.每天一次，持续5天）和高脂肪饮食（23.5%玉米油）后，12只大鼠中有7只发生了纤维腺瘤，1只在1年内发生了小管乳头状癌。 血清催乳素水平的增加和对乳腺细胞凋亡的影响，看到与PhIP可能有意义的乳腺癌的这种化合物的机制。 进一步研究了PhIP对各种细胞系（包括人乳腺细胞系MCF 10A）中信号转导途径的影响。 结果表明，PhIP通过增加Erk-2磷酸化和增加Bcl-2表达来延迟凋亡。研究结果表明，PhIP对乳腺上皮细胞具有多效性效应，这可能有助于了解其乳腺癌发生机制。

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.