PROJECT SUMMARY Benzo(a)pyrene [B(a)P], an ubiquitous environmental toxicant, is a member of the polycyclic aromatic hydrocarbon (PAH) family of compounds and has been implicated in the causation of colorectal cancer (CRC). Additionally, consumption of red meat and saturated fats, toxicants such as PAHs has also been implicated as one of the risk factors for sporadic colon cancer. Our preliminary studies not only show that B(a)P causes colon cancer, but that administration of Western diet to B(a)P-treated Polyposis in Rat Coli (PIRC) rats increases the development of adenomas in the colon. Our proposed studies will test the hypothesis that Western diet exacerbates B(a)P-initiated colon carcinogenesis through altered biotransformation and DNA damage. We will test our hypothesis in adult male transgenic PIRC rats with the following specific aims: 1. Characterize and validate the PIRC rat as a model to study how Western diet potentiates B(a)P-induced colon carcinogenesis; 2. Determine diet-induced alteration of B(a)P biotransformation enzyme activities, expression, and metabolite disposition in the PIRC rat; and 3. Assess the contribution of regular and Western diet in the formation and persistence of B(a)P-DNA adducts and oxidative DNA damage in the PIRC rat. This project will provide new and interesting information with regard to the contribution of Western diet towards increasing the development of colorectal polyps. The goals of this application are also to provide the intellectual environment and resources for the applicant to enrich herself in various approaches in her chosen area of research via a training plan that incorporates: 1) Mentored research activity; 2) Active participation & presentation in meetings, seminars and journal clubs; 3) Interaction with visiting scientists; 4) Periodic evaluation of applicant's research progress through individual development plan; 5) Publishing research findings in peer-reviewed journals; and 6) Participation in career development activities. It is expected that by the end of the training period, the applicant will be successful in her chosen research endeavor and contribute to the diversity of the research workforce, a goal of the Ruth L. Kirschstein predoctoral fellowship. Relevance of this project to human health Colorectal cancer contributes to significant mortalities in the United States, with 50,000 deaths per year. Consumption of fatty foods contaminated with B(a)P was implicated as a causative factor. One-third of adults and 17% of adolescents in the United State are obese. Obese people have a higher risk for colon cancer due to the excess energy intake from Western diet. We plan to study the progression of CRC, using a rat model that mirrors the human CRC scenario. We also propose to use Western diet, (>58% fat), as an accelerator of CRC. Our findings will be of use to determining the mechanistic effects of diet on B(a)P-caused initiation, promotion and progression of CRC.

PROJECT NARRATIVE This project explores how sporadic colorectal cancer caused by environmental toxicants can be influenced by dietary habits. Specifically, this project focuses on how consumption of Western diet, a diet composed of 58% fat, accelerates the development of sporadic colorectal cancer induced by the environmental toxicant, benzo(a)pyrene [B(a)P]. __SpecificAimsTextDelimiter__ SPECIFIC AIMS Epidemiological studies show that both environmental factors and diet play an important role in sporadic colon cancer susceptibility (Martinez, 2005). Diet is estimated to contribute to 80% of known colorectal cancer (CRC) cases (Bingham, 2000) and although several environmental chemicals are implicated as the contributing factors to CRC in humans, polycyclic aromatic hydrocarbons (PAHs) have evoked a great deal of interest as they are not only formed in red meat cooked at high temperatures (Butler and Pearson, 2005), but also contributed from several environmental sources (Diggs et al., 2011). Studies also document that exposure of people to PAHs is associated with obesity (Rundle et al., 2012). With increasing epidemiological evidence demonstrating obesity being associated with increasing risk of colon cancer (Bianchini et al., 2002), a more in- depth understanding of benzo(a)pyrene [B(a)P] metabolic activation in target tissues (colon, liver, and adipose) of individuals with poor dietary habits (frequent Western diet, and fast food, but not fruit & vegetable eaters) is essential to formulate effective detoxification or intervention strategies. In this way, inflammation arising out of signaling pathways involving B(a)P reactive metabolites could be curtailed. The long range goal of this proposal is to determine the role of Western diet in the etiology of B(a)P (a PAH compound)-induced colon carcinogenesis. The rationale underlying this initiative is that the formation and progression of colon tumors depends on altered B(a)P biotransformation by Western diet. The central hypothesis is that Western diet exacerbates B(a)P-initiated colon carcinogenesis through altered biotransformation and DNA damage. Specific Aim 1: To characterize and validate the polyposis in rat coli (PIRC) rat as a model to study how Western diet potentiates B(a)P-induced colon carcinogenesis. The underlying rationale for this study is that obesity is documented to increase the risk for colon cancer and studies involving animal models demonstrate that ingested exposure to B(a)P in saturated fats caused increase in adenoma formation in the colon in comparison to animals exposed to B(a)P in unsaturated fats (Calle and Kaaks, 2004; Harris et al., 2009). The objective is to identify the relationships that may exist between B(a)P-exposure in regular, and Western diets and adenoma development. Information gained will allow for further understanding of the potential pathways through which Western diet contaminated with B(a)P exert its harmful effects. Our strategy is to use seven-week-old male PIRC rats (Taconic Labs, Hudson, NY) which are genetically altered to mimic sporadic cancer as seen in humans. Animals will receive B(a)P via oral gavage while being fed a regular (5% fat) or Western diet (58% fat) for 60 days. Also, weekly food consumption and body weight gains of rats will be measured. Blood will be drawn to assay the production of metabolic regulatory proteins [C-reactive protein (CRP)] and, inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-¿ (TNF-¿)] and markers of oxidative stress [8-iso-PGF2¿ (F2-isoprostanes)]. Colon tissues will be harvested and scored for number and size of polyps. Additionally, histopathological evaluation will be performed to categorize the polyps as adenomas or invasive carcinomas. Specific Aim 2: To determine diet-induced alteration of B(a)P and biotransformation enzyme activities, expression and metabolite disposition in the PIRC rat. The rationale for this aim is that the biotransformation of a carcinogen and the accumulation of metabolic products is an important event prior to cellular damage in target tissues (Arakaki et. al., 2008). To forge a link between biotransformation and colon carcinogenesis, the key enzymes involved in B(a)P biotransformation will be assayed and B(a)P metabolites will be measured from target tissues (colon and liver) of rats that received the B(a)P for the above mentioned- treatment groups. Further, in this specific aim, we will determine the activities and expression of the CYP family enzymes (CYP1A1 and 1B1), and glutathione-S-transferase. The experiments in this specific aim will provide information on the balance between the toxification and detoxification processes in B(a)P-treated rats and how Western diet alters this key event. Specific Aim 3: To assess the contribution of regular and Western diet in the formation and persistence of B(a)P-DNA adducts and oxidative DNA damage in the PIRC rat. The rationale for this research is that DNA adducts are markers of tumorigenesis; examining the relationship among B(a)P alone, B(a)P+ regular diet, B(a)P+ Western diet treatment groups, DNA adducts and adenomas may provide an insight into the extent to which dietary type increases the formation of adducts and the length of the time the affected DNA resides in the tissues (adduct persistence). DNA will be isolated, digested, and labeled with 32P from colon and liver of rats from the above-mentioned treatment groups that received 25-100 ¿g B(a)P /kg BW. The B(a)P-DNA adducts will be resolved through thin layer chromatography. The adduct concentrations will be measured at various time points after B(a)P exposure. Further, this aim will also determine the role of Western diet exposure, during various time points, as a modulator of B(a)P-induced oxidative DNA damage by measuring aldehyde lesions.