Heterocyclic amines (HCAs) are formed when meat products such as beef, chicken, pork and fish are cooked at high temperatures. The most abundant HCA found in the human diet is 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP). PhIP causes mammary carcinomas in female rats and mice, and is associated with an increased risk of developing colon, breast, and prostate cancer in humans. PhIP is metabolized by cytochrome P-450s producing N-OH-PhIP. The N-OH-PhIP can be esterified by phase II enzymes forming an arylnitrenium ion that binds to DNA causing adducts. Furthermore, N-OH-PhIP may be reduced by cytochrome b5 reductase producing superoxide anions and hydroxyl radicals causing DNA strand breaks. Diallyl sulfide (DAS) has been shown to prevent cancer in several animal models, presumably by metabolic modulation. We hypothesize that PhIP produces reactive oxygen species causing DNA strand breaks and that DAS will inhibit the formation of PhIP induced DNA strand breaks. To test this hypothesis we treated normal breast epithelial (MCF-10A) cells with PhIP, DAS and a combination of PhIP and DAS. The detection of lipid peroxides was used as a surrogate for ROS. Lipid peroxides were detected using a PeroxiDetect kit (Sigma). PhIP increased the production of lipid peroxides and DAS decreased the PhIP-induced peroxidation by 47%. To determine if PhIP causes DNA strand breaks in MCF-10A cells, cells were treated for 3, 6, 9, and 24 h with PhIP (100 μM), DAS (100 μM) and a combination of PhIP (100 μM) and DAS (100 μM). DNA strand breaks were evaluated using the Comet assay. PhIP produced DNA strand breaks in a dose-and time-dependent fashion. We have shown that DAS inhibits PhIP-induced DNA strand breaks by inhibiting the production of reactive oxygen species. Therefore, we propose that DAS can prevent PhIP-induced breast cancer.
Previous studies in this lab have shown that diallyl sulfide (DAS) inhibits DNA adduct formation, lipid peroxidation and modulate detoxification and DNA repair genes and enzymes in breast tissue of diethylstilbesterol (DES) treated female ACI rats. These findings are important since estrogen and estrogenic compounds such as DES have been shown to induce breast cancer in humans and animals. In addition, scientists have postulated that DAS, a constituent of garlic, is an effective cancer prevention agent. In this study, we hypothesize that the release of inteleukin 6 by DES induced cancerous cells leads to activation of the JAK‐ Stat pathway which can increase cell proliferation and inhibit apoptosis. DAS may modulate these changes. To prove this hypothesis, 6 week old female ACI rats were implanted with Control (silicon implant alone) and DES (50mg/kg) silicon pellets were implanted s.c. In addition, these animals were fed normal chow or DAS (as co‐treatment) containing chow ad libitum for six months. At sacrifice, representative mammary tissueand all palpable mammary tumors were removed for genetic and histopathologicalanalysis. Genetic analysis was done by microarray analysis and validated using PCR array. Our data show significant increase in interleukin 6 gene expression in cancerous cells (DES group) and a significant decrease in the DAS co‐treatment group. Furthermore, a significant modulation of genes associated with the JAK/STAT pathway was observed. Animals co‐treated with DAS were free of tumor. For the first time our lab was able to show that DAS prevents DES induced breast tumors in female ACI rats.Funded by RCMI Grant #2G12RR03020‐24
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