Elizabeth G. Snyderwine

Some perspectives on the nutritional aspects of breast cancer research. Food‐derived heterocyclic amines as etiologic agents in human mammary cancer

Epidemiologic and experimental evidence indicates that dietary factors influence the incidence of mammary gland cancer. The dietary causes of this cancer, however, remain largely unknown. This paper reviews the experimental studies implicating the food‐derived heterocyclic amines (HAs) in human breast cancer. Heterocyclic amines are formed at the parts‐per‐billion levels in meats, such as beef, chicken, pork, and fish, cooked by ordinary methods. 2‐Amino‐1‐methyl‐6‐phenylimidazo [4, 5‐b]pyridine (PhIP) is among the most prevalent of the HAs in fried and barbecued beef, a staple of the American diet. Chronic administration of PhIP in the diet has been shown to cause mammary gland cancer in rats. Two other food‐derived HAs, 2‐amino‐3‐methylimidazo[4, 5‐flquinoline and 2‐amino‐3, 4‐dimethylimidazo[4, 5‐flquinoline, also have been shown to be mammary carcinogens in rodent models. In rats, heterocyclic amines produce DNA adducts in the mammary gland after metabolic activation. Studies examining human urine for HAs and metabolites confirm that humans who consume cooked meats are exposed to HAs. Studies also reveal that humans can activate HAs metabolically. Therefore, the experimental evidence suggests that the food‐derived HAs may be eliologic agents in human breast cancer. Humans, however, are exposed to a complex mixture of carcinogenic and anticarcinogenic agents through their diets. Experimental studies examining the interaction between HAs and other dietary factors with respect to mammary carcinugenesis are warranted.

Metabolism of food-derived heterocyclic amines in nonhuman primates.

During the cooking of meats, several highly mutagenic heterocyclic amines (HCAs) are produced. Three HCAs, IQ, MeIQx, and PhIP have been under study for carcinogenicity in cynomolgus monkeys, and to date, IQ has been shown to be a potent hepatocarcinogen. Concomitantly, the metabolic processing of these HCAs has been examined. Metabolism studies show that the potent hepatocarcinogenicity of IQ is associated with the in vivo metabolic activation of IQ via N-hydroxylation and the formation of DNA adducts. In monkeys undergoing carcinogen bioassay with IQ, N-hydroxylation was confirmed by the presence of the N-hydroxy-N-glucuronide conjugate of IQ in urine. The N-hydroxylation of IQ appears to be carried out largely by hepatic CYP3A4 and/or CYP2C9/10, and not by CYP1A2, an isoform not expressed in liver of this species. Notably MeIQx is poorly activated in cynomolgus monkeys and lacks the potency of IQ to induce hepatocellular carcinoma after a 5-year dosing period. The poor activation of MeIQx appears to be due to the lack of constitutive expression of CYP1A2 and an inability of other cytochromes P450, such as CYP3A4 and CYP2C9/10, to N-hydroxylate the quinoxalines. MeIQx is detoxified in monkeys largely by conjugation with glucuronide at the N-1 position. Although the carcinogenicity of PhIP is not yet known, the metabolic data suggest that PhIP will be carcinogenic in this species. PhIP is metabolically activated in vivo in monkeys by N-hydroxylation, as discerned by the presence of the N-hydroxy-N-glucuronide conjugate in urine, bile, and plasma. PhIP also produces DNA adducts that are widely distributed in tissues. The results from these studies support the importance of N-hydroxylation in the carcinogenicity of HCAs in nonhuman primates and by analogy, the importance of this metabolic activation step in the possible carcinogenicity of dietary HCAs in humans.

Mammary gland carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Sprague-Dawley rats on high- and low-fat diets

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic amine derived from cooked meat. Mammary gland tumors were induced in female Sprague-Dawley rats given 10 doses of PhIP (75 mg/kg po) once per day from 43 days of age and then placed on a defined high-fat (23.5% corn oil) or low-fat (5% corn oil) diet for 25 weeks. Mammary tumor incidence was 49% (44 of 90 rats) and 31% (27 of 88 rats) in the high- and low-fat groups, respectively. No tumors were found in vehicle control rats on the high-or the low-fat diet (n = 44 and 43, respectively). The higher tumor incidence in the high-fat group was due to an increase specifically in carcinomas (classified as tubulopapillary carcinomas) rather than benign tumors (tubular adenomas and fibroadenomas). The incidence of carcinomas was 45% and 24% in PhIP-treated rats on the high- and low-fat diets, respectively. In addition, the percentage of carcinomas showing stromal invasion was highest in the high-fat diet group (22% vs. 8%, high- vs. low-fat diet). Proliferating cell nuclear antigen immunostaining (PCNA) index revealed six times more proliferation in carcinomas from rats on the high-fat diet than in rats on the low-fat diet. Adenomas from rats on different diets had similar PCNA indexes. The tumor apoptotic index, quantitated by immunohistochemical detection (terminal deoxynucleotidyl transferase nick end labeling), was twice as high in carcinomas from rats on the high-fat diet as in carcinomas from rats on the low-fat diet but was similar between the two groups of adenomas. The PCNA-to-apoptosis ratio was 43 and 17 in carcinomas from rats on the high- and low-fat diets, respectively, indicating that the growth rate of carcinomas was greater in rats on the high-fat diet. The results from this study show that the high-fat diet increases the incidence, invasiveness, and growth of PhIP-induced mammary gland carcinomas.

Mammary gland carcinogenesis by food-derived heterocyclic amines and studies on the mechanisms of carcinogenesis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

The heterocyclic amines (HCAs) comprise a family of mutagenic/carcinogenic compounds found in cooked meat. Several HCAs including 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are mammary gland carcinogens in rats. One mammary gland carcinogen, PhIP, is the most prevalent in the human diet. This article reviews the mechanisms of mammary gland carcinogenesis of PhIP including metabolic processing, DNA adduct formation, effects on mammary gland development, cell signaling, and the genomic alterations found in PhIP-induced rat mammary gland carcinomas.

Id4 Regulates Mammary Epithelial Cell Growth and Differentiation and Is Overexpressed in Rat Mammary Gland Carcinomas

Id4 belongs to a family of helix-loop-helix (HLH) proteins that impact cellular growth and differentiation via regulation of basic HLH transcription factors. Herein the rat Id4 gene was cloned (GenBank Accession No. AF468681). The expression of rat Id4 was examined in rat mammary gland tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in the human diet. By real-time polymerase chain reaction analysis, relative expression of Id4 mRNA in carcinomas, adenomas, and normal tissue was 27, 6, and 1, respectively. Immunohistochemical analysis indicated statistically elevated nuclear expression for Id4 protein in carcinomas in comparison to adenomas and normal mammary gland. In carcinomas, Id4 nuclear expression was positively correlated with proliferation, invasiveness, and tumor weight (Fisher Exact Test or Spearman Correlation, P < 0.05). The consequence of enforced expression of Id4 on mammary epithelial cell proliferation, differentiation, and growth in soft agar was examined in HC11 cells, a well-characterized model for studying various aspects of mammary epithelial cell biology. After transient and stable transfection of HC11 cells, Id4 overexpression increased cell proliferation and inhibited lactogenic hormone-mediated differentiation as revealed by inhibition of beta-casein promoter activity and beta-casein expression. In addition, enforced expression of Id4 in HC11 cells induced a statistically significant increase in colony growth in soft agar. The results implicate Id4 in rat mammary gland carcinogenesis and suggest that Id4 may contribute to carcinogenesis by inhibiting mammary epithelial cell differentiation and stimulating mammary epithelial cell growth.

Transgenic mouse models in carcinogenesis research and testing

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c-myc and TGF-alpha transgenes during the neoplastic process. We show experimental evidence that co-expression of TGF-alpha and c-myc transgenes in mouse liver promotes overproduction of ROS and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-alpha/c-myc mouse model. Also, the role of mutagenesis in hepatocarcinogenesis induced by 2-amino-3,8-dimethylimidazo(4,5-f)-quinoxaline (MeIQx) was demonstrated in C57BL/lacZ (Muta Mice) and double transgenic c-myc/lacZ mice that carry the lacZ mutation reporter gene. The MeLQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.

In vivo mutagenicity and DNA adduct levels of heterocyclic amines in Muta™Mice and c-myclacZ double transgenic mice

The cooked meat derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) are established mutagens in the Salmonella assay and hepatocarcinogens in mice. The current study uses transgenic mice to examine hepatic HCA-DNA adduct formation and mutagenesis in vivo and the impact of hepatic overexpression of the c-myc oncogene on HCA-induced mutagenesis. C57B1/lacZ and c-myc/lacZ mice strains, produced by crossbreeding Muta Mice (carrying the lacZ mutation target gene) with either C57B1 control or c-myc transgenic mice, respectively, were treated with 10 daily doses of IQ, MelQx or A alpha C (20 micrograms/g, p.o.). Four weeks after dosing, the frequency of mutations in the lacZ gene in liver of either C57B1/lacZ or c-myc/lacZ mice was significantly higher in mice treated with any one of the three HCAs than in mice given vehicle only. In addition, all three HCAs formed hepatic DNA adducts, as measured by the 32P-postlabeling analysis 24 h after dosing. In both strains of mice, hepatic DNA adduct levels were 2-3-fold higher with A alpha C than with either IQ or MeIQx, although the mutant frequencies in the lacZ gene were 30-40% lower in mice dosed with A alpha C. These results suggest that A alpha C-DNA adducts may be less mutagenic in vivo than either IQ- or MeIQx-DNA adducts. The lacZ mutant frequencies observed with all three HCAs appeared to be influenced by c-myc transgene expression: after HCA treatment, transgenic mice carrying the c-myc gene showed a 30-40% higher lacZ mutant frequency than mice not carrying this transgene. Notably, lacZ mutant frequencies were not different among C57B1/lacZ and c-myc/lacZ mice that received vehicle control. DNA adduct studies showed that the levels of IQ- and MeIQx-DNA adducts were 2-3-fold higher in c-myc/lacZ mice than in C57B1/lacZ mice; however, A alpha C DNA adducts were not statistically different between the two strains. In addition, phase 1 metabolic activation of these HCAs, as assessed by hepatic microsomal mutagenic activation, was also similar in both strains of mice. These results support the notion that overexpression of the c-myc oncogene cooperates with the HCAs to enhance in vivo mutagenicity. Further studies are needed to assess the mechanisms of this cooperative effect.

Mammary gland carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rats: possible mechanisms

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine derived from cooked meat. Mammary gland cancer can be induced in female Sprague-Dawley rats by administration of several oral doses of PhIP. The mechanism of mammary gland carcinogenesis by PhIP in this rat model is not fully understood but appears to involve several factors. One factor is the formation of PhIP-DNA adducts in the mammary gland after metabolic activation of PhIP. Possible target cell populations include the epithelial cells of the mammary gland terminal end buds (TEBs), putative sites of origin of carcinomas. Another factor involved in the mammary carcinogenicity of PhIP may be an increased proliferation in epithelial cells of the TEBs which occurs after a carcinogenic dose of PhIP is administered. This proliferation would be likely to enhance the fixation of mutations from PhIP-DNA adducts in target cells and facilitate the initiation of carcinogenesis. PhIP exposure also transiently inhibits the development of the mammary gland by retarding the differentiation of TEBs to alveolar buds and lobules. As a consequence, more TEBs are available for neoplastic transformation. Recent studies in rats have also shown that PhIP increases the levels of serum prolactin, a well-recognized promoter of mammary gland cancer, which may further explain the targeting of PhIP to the mammary gland. The results to date indicate that PhIP has multiple effects on the mammary gland and hormone status in rats that could potentially play a role in its ability to induce mammary gland cancer.

Effect of dietary fat on codon 12 and 13 Ha-ras gene mutations in 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine–induced rat mammary gland tumors

Activating mutations in and expression of the Ha‐ras gene were examined in benign and malignant female Sprague‐Dawley rat mammary gland tumors induced by the heterocyclic amine 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) and promoted by a diet high in polyunsaturated fat. Ha‐ras mutations were detected in codons 12 and 13 by selective polymerase chain reaction amplification of mutated sequences and nucleotide sequencing. The percentage of Ha‐ras mutations in carcinomas from PhIP‐treated rats was significantly higher in rats on a low‐fat diet than in rats on a high‐fat diet (82% (nine of 11) vs 26% (seven of 27), respectively, P < 0.01). In addition, whereas 56% of the carcinomas with Ha‐ras mutations from rats on a low‐fat diet carried double Ha‐ras mutations, none of the carcinomas from rats on a high‐fat diet had double mutations. Ha‐ras mutations were also detected in benign tumors (largely adenomas) induced by PhIP in rats on different diets; two of eight and three of four benign tumors examined from rats on low‐fat and high‐fat diets, respectively, had Ha‐ras mutations, suggesting that activating Ha‐ras mutations alone are not sufficient for PhIP‐induced tumors to become malignant. No differences were observed in the level of Ha‐ras mRNA expression in the different groups. In our animal model, a high‐fat diet increased the incidence and percentage of malignant PhIP‐induced mammary gland tumors yet decreased the percentage of carcinomas showing Ha‐ras mutations. Thus, the complement of genetic alterations associated with PhIP‐induced mammary gland carcinogenesis is probably altered by the level of dietary fat. Mol. Carcinog. 20:348–354, 1997.

Possible role of Stat5a in rat mammary gland carcinogenesis

Signal transducer and activator of transcription (Stat) 5a is a transcription factor mediating the action of specific cytokines, growth factors and hormones on gene expression. In the mammary gland, Stat5a is well recognized for its function in prolactin signaling, lobuloalveolar development, and milk protein expression during pregnancy and lactation. Latent cytoplasmic Stat5a is activated by tyrosine phosphorylation and following dimerization undergoes nuclear import. In the current study, Stat5a expression was examined immunohistochemically in carcinomas induced by the chemical carcinogens 7,12-dimethylbenz[a]anthracene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. A high percentage of carcinomas showed nuclear labeling of Stat5a [44 of 68 (65%)] with Stat5a nuclear labeling index ranging from 18 to 77%. In contrast, control normal mammary gland tissue displayed cytosolic expression. Carcinomas with different Stat5a staining patterns (cytoplasmic or nuclear) showed a statistical difference for the proliferating cell nuclear antigen (PCNA) labeling, tumor differentiation, nuclear grade, mitotic activity, and tumor size. High Stat5a nuclear expression was closely correlated with the higher-grade carcinomas. Stat5a nuclear expression was also detected in intraductal proliferations (10 of 21 lesions) and in ductal carcinomas in situ (13 of 15 lesions). Immunohistochemical analysis was further carried out in human breast cancers. Stat5a nuclear expression was detected in ductal and lobular carcinomas and DCIS at a frequency of 48% (15/31), 33% (2/6), and 40% (2/5), respectively. Nuclear expression of Stat5a in human breast cancers also correlated with the PCNA nuclear labeling index. The findings implicate activated Stat5a in mammary gland cancer development in the rat and human.