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. These studies are being extended to investigate mechanisms of apoptosis inhibition in greater detail and the effects of PhIP on cell signaling in mammary epithelial cells. The purpose of these studies is to better understand the mechanisms of mammary carcinogenesis of PhIP especially factors in addition to DNA adduct formation and DNA-adduct-induced mutagenesis.
