Mechanisms of Mammary Gland Carcinogenesis of Heterocycl

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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.