Source: UTAH STATE UNIVERSITY submitted to NRP
IMPACT OF TYPICAL WESTERN DIET ON CANCER PREVENTION BY GREEN TEA USING A MULTI-GENERATION MODEL OF COLORECTAL CANCER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1001602
Grant No.
2014-67017-21755
Cumulative Award Amt.
$496,654.00
Proposal No.
2013-03494
Multistate No.
(N/A)
Project Start Date
Jan 15, 2014
Project End Date
Jan 14, 2018
Grant Year
2014
Program Code
[A1341]- Food Safety, Nutrition, and Health: Function and Efficacy of Nutrients
Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322
Performing Department
School of Veterinary Medicine
Non Technical Summary
Diet is widely recognized as an important factor in lifetime cancer risk, yet Americans routinely consume foods that are energy-dense and nutrient-poor. Animal model studies to identify functional foods for cancer prevention generally do not account for typical Western dietary patterns with respect to macro- and micronutrient content. The long-term goals of our research program are to identify functional foods and to optimize dietary intervention strategies for cancer prevention. For this study, our primary objective is to determine the efficacy of green tea in a murine model of inflammation-associated To this end, our research team has developed the new Total Western Diet (TWD) for rodents which recapitulates typical human macro- (sugars, fats, proteins) and micronutrient (vitamins and minerals) intakes on an energy-density basis. Secondarily, we also seek to determine whether multi-generation or ancestral exposure to the typical U.S. diet increases risk of colorectal cancer in offspring. The hypotheses to be tested in this study are 1) that green tea will have a substantially greater health benefit in mice exposed to a Westernized diet and 2) that ancestral exposure to this new Western diet will increase risk of colon cancer in offspring. Our proposal is highly responsive to the Function and Efficacy of Foods priority area in that we propose to determine whether green tea, the most popular beverage in the world (besides water), prevents inflammation-associated colorectal cancer in mice consuming a diet representative of typical American nutrition.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70238401010100%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
The long-term goals of our research program are to identify functional foods and/or bioactive food chemicals with anticancer properties and to optimize dietary intervention strategies for cancer prevention. For this study, our primary objective is to determine the efficacy of green tea for cancer prevention in a murine model of inflammation-associated To this end, our research team has developed the new Total Western Diet (TWD) for rodents which recapitulates typical human macro- and micronutrient intakes on an energy-density basis [31]. Secondarily, we also seek to determine whether multi-generation or ancestral exposure to the typical U.S. diet increases risk of A. Employ the well-established azoxymethane + dextran sodium sulfate (AOM+DSS) model of inflammation-promoted B. Compare the efficacy of green tea extract for suppression of colon tumorigenesis in offspring exposed to TWD, DIO or AIN93G, either directly or via prior generations. 2) Gut inflammation: Determine the impact of all three test diets on health endpoints associated with gut inflammation and metabolic syndrome, including alterations of inflammatory cytokines, insulin resistance, glucose tolerance, body weight and composition. 3) Mechanism of colon carcinogenesis: A. Assess the impact of green tea supplementation in mice exposed to TWD, DIO or AIN93 diets on mRNA expression and methylation status of genes critical to colon carcinogenesis. B. Additionally, compare effects of green tea supplementation on expression of molecular biomarkers of colorectal cancer.
Project Methods
Mice will be bred for 3 generations prior to carcinogen exposure in order to test the impact of experimental diet on cancer risk in offspring. In 3rd generation mice, we will employ the well-established azoxymethane + dextran sodium sulfate (AOM+DSS) model of inflammation-promoted Additionally, at the 3rd generation, offspring will be divided into two subgroups, one receiving regular drinking water, the other receiving water with a green tea extract. Generation and experimental diet groups are as follows: Generations: F0 = great-grand parents F1 = first generation F2 = second generation F3 = third generation Diets Control diet, AIN93G Total Western Diet (TWD) 45% fat diet-induced obesity diet (DIO) Diet exposure protocol (according to generation): F0 F1 F2 F3 (cancer study) Group 1AIN93G AIN93GAIN93GAIN93G with water Group 2AIN93G AIN93GAIN93GAIN93G with green tea Group 3AIN93G AIN93GTWD AIN93G with water Group 4AIN93G AIN93GTWD AIN93G with green tea Group 5TWD TWD TWD AIN93G with water Group 6TWD TWD TWD AIN93G with green tea Group 7TWD AIN93GAIN93GAIN93G with water Group 8TWD AIN93GAIN93GAIN93G with green tea Group 9AIN93G AIN93GDIO AIN93G with water Group 10AIN93G AIN93GDIO AIN93G with green tea Group 11DIO DIO DIO AIN93G with water Group 12DIO DIO DIO AIN93G with green tea Group 13DIO AIN93GAIN93GAIN93G with water Group 14DIO AIN93GAIN93GAIN93G with green tea Following exposure to the carcinogen in F3 offspring at 7 weeks of age, mice will be monitored for 16 weeks (body weight, food consumption, blood glucose levels). At the end of the study, mice will be euthanized and tumor outcome will be assessed (incidence, number of tumors per tumor-bearing animal and tumor size). Colon tissues will be retained for analysis of protein markers of colon carcinogenesis by antibody staining. Also, colon tissues will be preserved for analysis of gene expression and DNA methylation by established methods. The experiment design above requires the use of multivariate analyses of variance (one- or two-way ANOVA) for determination of significant differences in cancer outcome, and for determination of potential litter effects. For binary data (tumor incidence, methylation incidence), Fisher's exact test or the Chi-square test will be used. For all statistical tests, a p value <0.05 (two-sided test) will be considered significant. Statistical analyses of gene expression and DNA methylation data will involve a per-gene ANOVA with multiple testing adjustments to control the false discovery rate. For the methylation data, we will calculate the frequency of methylation at each CpG site, the percentage of methylated sites for the amplicon and the number of biological replicates with methylated DNA detected.

Progress 01/15/14 to 01/14/18

Outputs
Target Audience:Scientists Changes/Problems:Though grant project period is over, some further data analysis will be needed to complete the last objective work regarding assessment of gene expression and DNA methylation. Per a change in the research plan approved with NIFA and the program officer, we shifted from a focus on only a handful of gene targets for methylation analysis to a genome-wide approach. The original plan to was to employ reduced Reduced Representation Bisulfite Sequencing (RRBS), which captures the vast majority of promoter CpG islands but only sequences about 10% of the overall genome. This strategy would have enabled us to examine methylation for all samples (all treatments, all diets) with the available funding support. Moreover, the experiment design and collection of samples required analysis of RNA expression and DNA methylation in the same samples, which were preserved in TRI-Reagent to avoid degradation of RNA. RNA isolation and sequencing proceeded as expected. However, as the student optimized procedures for DNA isolation, she determined that about 86% of the DNA product was single stranded, which would be incompatible with restriction endonuclease enzymes used in RRBS, as those enzymes recognize only a double-stranded substrate. (This was a new discovery, as neither our genomics core experts nor the supplier of TRI-Reagent was unaware of this effect of the protocol materials on DNA structure.) After careful evaluation of other options, we decided to perform methylated DNA immunoprecipitation (MeDIP) followed by Illumina sequencing. Another advantage to MeDIP-Seq is that mC antibodies are highly specific to methylated cytosine and will not also bind to hydroxymethylated cytosine, as compared to RRBS where hydroxymethylated cytosine behaves as methylated cytosine and is resistant to bisulfite treatment. Hydroxymethylated cytosine is believed to be present in the demethylation transition, a very different role than that played by methylated cytosine. In collaboration with the Director of the USU CORE Sequencing Facility, Ms. Phatak developed an MeDIP-Seq protocol suitable for these samples. Sequencing using the Illumina NextSeq500 platform has been completed as of this final report. Given this change, and the very large genome-wide methylation sequencing data sets generated by this approach, we identified a new collaborator, Dr. Rakesh Kaundal, to assist in further bioinformatics analysis. What opportunities for training and professional development has the project provided?This project constituted the dissertation research of Sumira Phatak. As such, Sumira has had extensive one-on-one interaction with the project PI, as well as the Co-I's, and gained extensive experience in animal husbandry, experiment management, nutrition, animal necropsy, and genome-wide assessment of gene expression and DNA methylation. Sumira also received professional development training through various workshops on campus, including technical writing and scientific presentation. Additionally, two other graduate students and 10 undergraduate students have provided technical assistance, and have gained extensive experience in animal husbandry. A new opportunity realized in 2015 was the added focus on reproductive endpoints, in alignment with the research interests of another two other students, on graduate and one undergraduate. With no changes to the experiment design, these students collected data on reproductive outcomes (successful matings, litter size, litter growth, subsequent reproductive performance, estrus cycling in F3 and collected ovarian samples for possible future immunohistochemistry work) to evaluate the impact of these diets on reproductive competence. Although not specifically designed for this purpose, we hope to identify some preliminary data that may support further investigation supported through other funding opportunities. Two highly significant outcomes of the training opportunities for students working on this project include the following: In 2017, Ashli Hunter, an undergraduate student who worked on this USDA project, received the Joyce Kinkead Outstanding Honors Capstone Project Award for Utah State University. She also published related work, which was made possible by her training on this project, as an Honors Thesis. Second, Sumira Phatak successfully competed for a NIFA predoctoral fellowship award (Grant #2018-67011-28043), which started March 2018. This new fellowship project "Basal diet, green tea extract and gut microbiome interactions in a mouse multi-generation study" is an extension of this USDA NIFA project that focuses on effects of diet and green tea on the composition of the gut microbiome. How have the results been disseminated to communities of interest?Dissemination of this research has thus far principally been in the form of conference presentations at international conferences (Experimental Biology, Society of Toxicology, Environmental Mutagenesis and Genomics Society, American College of Toxicology), invited lectures (Tsukuba, Japan; University of Illinois), regional meetings (Western Region of Society of Toxicology, USDA W-3122 multi-state group) and local conferences (University students research symposia, Hansen Life Science Retreat, etc.) and publication in conference proceedings. These presentations were delivered principally by graduate and undergraduate student participants and project personnel (Benninghoff, Hintze, Ward). During this grant period, several associated publications were also produced, including manuscripts in Nutrition Research, Molecular Food and Nutrition Research, Journal of Developments in Sustainable Agriculture, Functional Foods in Health and Disease, and Advances in Nutrition. Additional publication of research results is expected, with future papers detailing cancer outcomes tied to changes in DNA methylation and gene expression as that data analysis is completed in collaboration with bioinformatics specialist. What do you plan to do during the next reporting period to accomplish the goals?Manuscript preparation is in progress, with the first paper outlining cancer outcomes planned for submission later this year. Additionally, analysis of RNAseq and MeDIP-Seq data is ongoing with our new collaborator, Dr. Rakesh Kaundal.

Impacts
What was accomplished under these goals? 1) Major activities completed: By the second year of this program, all mouse breeding had been completed to generate all needed mice for the colorectal cancer study that was performed on the F3 generation offpsring. This cancer study was also completed, with more than 500 necropsies performed, by November 2015. As of this final report, all in vivo aspects of this project have been completed. Also, we developed and optimized procedures for paired extraction of nucleic acids (RNA and DNA) from colon tissue samples for use in RNAseq to assess changes in the transcriptome and methylated DNA immunoprecipitation sequencing (MeDIP-seq) to assess differential methylation on a genome-wide scale. 2) Specific objectives met: All animal breeding, the F3 generation cancer study, data collection from the in vivo study has been completed. During 2016, data analysis was completed for parameters including (in part) food intake, energy intake, water intake, mating success, rate of weight gain during pregnancy, liter size at birth, weight of offspring at weaning, rate of weight gain during grow out of offspring for each generation, final body weight at each generation, body composition of offspring, fasting glucose level, glucose tolerance, estrus cycling (F3 generation). Also, we completed analysis for F3 generation endpoints related to cancer outcome (incidence, multiplicity, size and burden) and endpoints related to metabolic syndrome (body weight, body composition, glucose tolerance), including mice that received or did not receive green tea supplementation. Though we encountered substantial difficulty in optimizing procedures for genome-wide assessment of DNA methylation in our colon mucosa samples (reduced representation bisulfite sequencing was not possible), we were able to optimize a suitable protocol and have completed all sequencing for DNA methylation and gene expression by RNAseq and MeDIP-Seq, respectively. As of this final report, these data are with a new colleague that specializes in bioinformatics, Dr. Rakesh Kaundal, to enable more sophisticated statistics and bioinformatics analyses. 3) Significant results achieved, including major findings, developments, or conclusions (both positive and negative): A significant observation was that the TWD did not overtly negatively impact breeding success, as assessed by frequency of successful pregnancies, weight gain during pregnancy, litter size, weight gain during nursing or offspring weight at weaning. As mentioned in the proposal, this was a potential pitfall that may have caused problems in ramping up the breeding program to generate sufficient numbers of animals. This pitfall was, in fact, not realized and breeding proceeded normally. Cancer endpoints in F3 generation offspring - Notably, incidence of CAC was significantly higher in F3 offspring exposed ancestrally to the TWD (92%) as compared to their F3 counterparts fed AIN93G (56%). Moreover, tumor burden was markedly higher (> 3-fold increase) in F3 offspring exposed to TWD over multiple generations when compared to F3 offspring provided TWD directly. Alternatively, neither ancestral or multi-generation exposure to the DIO diet altered CAC incidence, whereas a small, significant increase in tumor burden in F3 offspring ancestrally-exposed to the DIO was observed as compared to direct-fed mice. GTE consumption decreased tumor multiplicity and burden in both sexes after multi-generation exposure to TWD. GTE increased relative cecum mass in females after trans-generational TWD exposure. These analyses suggest that GTE reduced colorectal cancer severity only after cumulative TWD exposure over multiple generations Cancer endpoints in F3 generation offspring (with green tea extract (GTE) supplementation): Fasting glucose and glucose tolerance - Glucose tolerance (assessed as AUC) in F3 offspring was not significantly affected by diet regimen for the TWD or the DIO groups nor by supplementation with green tea. A significant main effect of green tea on fasting glucose was apparent for the TWD set of diets, though not for the DIO groups. Also, a significant main effect of diet on difference from baseline (an indicator of persistent glucose elevation) was evident for the TWD groups. Strong cage effects were identified, which reduced the statistical power of the analysis. Interestingly, this phenomenon has been previously observed by our research group in a prior study testing the DIO diet. Overall, these observations point to a complex relationship between basal diet and the green tea supplement, even though neither treatment resulted in severe glucose abnormalities. Reproductive endpoints - In F3 offspring, neither gender ratio or litter size were impacted by either the TWD or DIO diets as compared to the control AIN93G diet. With respect to estrus cycling (an added focus described in 2015's report), no significant differences were observed among any of the diet treatment groups for any of the estrous cycle stages (p>0.18). These data disagree with prior observations that a high fat diet prolonged time in diestrus, although the current study used diets with fewer calories from fat (35% for TWD and 45% for DIO) compared to the study by Brothers, et al (2010), which employed a 60% fat diet. 4) Key impacts or other accomplishments realized: As part of this breeding program, we have measured a number of additional parameters beyond the scope of the primary hypothesis to enhance the impact of this work, including continuous assessment of food and energy intakes, frequency of successful matings, litter size, weight gain of dams and pups, etc. to assess impacts of diet on fertility and health of offspring. Over multiple generations, these additional parameters will provide insight into the effect of a western style diet on early life parameters. A likely key impact of this study is the novel observation that ancestral exposure to a Western type diet markedly increased colon cancer incidence and disease severity in F3 offspring that were not fed this diet directly. Also, exposure to TWD over multiple generations markedly exacerbated the disease in F3 offspring as compared to those fed TWD directly. These observations point to the important influence of (grand)parental lifestyle factors on health outcomes in offspring. Put simply, the poor diet of the great-grandparents' appeared to have influenced cancer development in their great-grandchildren, who otherwise ate a healthy diet. Abbreviations: total Western diet, TWD; diet-induced obesity diet, DIO; GTE, green tea extract, CAC, colitis associated colorectal cancer

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Phatak, S. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Neal, C. (Author Only), Contreras, K., Pontsler, K. (Author Only), Armbrust, T. (Author Only), Rodriguez, D. (Author Only), Abercrombie, B. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), Experimental Biology 2017, "Ancestral and multi-generational consumption of the total Western diet in mice promotes colitis-associated colorectal cancer in third-generation offspring," American Society of Nutrition, Chicago, IL. (April 22, 2017 - April 26, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Benninghoff, A. (Presenter & Author), Hunter, A. (Author Only), Monsanto, S. (Author Only), Phatak, S. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Lefevre, M. (Author Only), Experimental Biology 2017, "Consumption of the total Western diet (TWD) enhanced and sustained colonic inflammation and promoted colon tumorigenesis in mice, which led to marked changes in the composition of the gut microbiome in mice," American Society of Nutrition, Chicago, IL. (April 22, 2017 - April 26, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Hintze, K. J. (Presenter & Author), Hisatome, T. (Author Only), Kellen, S. (Author Only), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), Lefevre, M. (Author Only), Experimental Biology 2017, "Effects of the Food Matrix and Western Diet on the Mouse Microbiome," American Society of Nutrition, Chicago, IL. (April 22, 2017 - April 26, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Hintze, K. J. (Presenter & Author), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), Lefevre, M. (Author Only), Experimental Biology 2017, "The Total Western Diet: Effects on Metabolism, Colon Cancer, and the Microbiome.," American Society for Nutrition, Chicago, IL. (April 23, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Hunter, A. (Presenter & Author), Neal, C. (Presenter & Author), Armbrust, T. (Presenter & Author), Eddy, F. (Presenter & Author), Phatak, S. (Author Only), Hintze, K. J. (Author Only), Benninghoff, A. (Author Only), Student Research Symposium, Research Week, "TNF as a biomarker of gut inflammation in a multi-intervention study examining functional foods, micronutrients and thermally abused oil as modulators of inflammation-associated colorectal cancer," Office of Research and Graduate Studies, Utah State University, Logan, UT. (April 13, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Hunter, A. (Presenter & Author), Larson, D. (Author Only), Phatak, S. (Author Only), Hintze, K. J. (Author Only), Benninghoff, A. (Author Only), 31st Annual National Conference on Undergraduate Research, "Dietary supplementation with tart cherries for prevention of inflammation-associated colorectal cancer in mice," Council on Undergraduate Research, Memphis, TN. (April 6, 2017 - April 8, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Hintze, K. J. (Presenter & Author), Ward, R. E., Benninghoff, A., Lefevre, M., 21st Annual Conference of the Functional Foods Center, "Modifying Animal Diets to Increase Relevance to Human Populations," San Diego, CA. (March 25, 2017 - March 26, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Hunter, A. (Author Only), Larson, D. (Author Only), Phatak, S. (Author Only), Hintze, K. J. (Author Only), Benninghoff, A. (Author Only), 34th Annual Meeting Mountain West Regional Chapter of the Society of Toxicology, "Dietary supplementation with tart cherries for prevention of inflammation-associated colorectal cancer in mice," Society of Toxicology, Salt Lake City, Utah. (September 21, 2016 - September 23, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Rodriguez, D. (Presenter & Author), Neal, C. (Presenter & Author), Phatak, S. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Esquer, H. (Author Only), Contreras, K. (Author Only), Pontsler, K. (Author Only), Armbrust, T. (Author Only), Abercrombie, B. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Lefevre, M. (Author Only), Benninghoff, A. (Author Only), 34th Annual Meeting Mountain West Regional Chapter of the Society of Toxicology, "Impact of the total Western diet fed over multiple generations on fasting glucose and glucose tolerance in F3 generation offspring.," Society of Toxicology, Salt Lake City, Utah. (September 21, 2016 - September 23, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Phatak, S. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Esquer, H. (Author Only), Neal, C. (Presenter & Author), Contreras, K. (Author Only), Pontsler, K. (Author Only), Armbrust, T. (Presenter & Author), Rodriguez, D. (Author Only), Abercrombie, B. (Author Only), Hintze, K. J. (Author Only), Lefevre, M. (Author Only), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), 34th Annual Meeting Mountain West Regional Chapter of the Society of Toxicology, "Impact of ancestral or multi-generation exposure to a typical Western diet on colorectal carcinogenesis in mice," Society of Toxicology, Salt Lake City, Utah. (September 21, 2016 - September 23, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Armbrust, T. (Presenter & Author), Phatak, S. (Author Only), Neal, C. (Presenter & Author), Contreras, K. (Author Only), Rodriguez, D. (Author Only), Abercrombie, B. (Author Only), Pontsler, K. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Esquer, H. (Author Only), Cuthbert, J. (Author Only), Lefevre, M. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), Hansen Life Science Retreat 2016, "Impact of the total Western diet fed over multiple generations on breeding success and offspring growth in mice," College of Science, Utah State University, Logan, Utah. (September 17, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Rodriguez, D. (Presenter & Author), Neal, C. (Presenter & Author), Phatak, S. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Esquer, H. (Author Only), Contreras, K. (Author Only), Pontsler, K. (Author Only), Armbrust, T. (Author Only), Abercrombie, B. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Lefevre, M. (Author Only), Benninghoff, A. (Author Only), Hansen Life Science Retreat 2016, "Impact of the total Western diet fed over multiple generations on fasting glucose and glucose tolerance in F3 generation offspring.," College of Science, Utah State University, Logan, Utah. (September 17, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Contreras, K. (Author Only), Cuthbert, J. (Author Only), Phatak, S. (Author Only), Larson, D. (Author Only), Benninghoff, A. (Author Only), 2016 Student Research Symposium, "Effect of the total Western diet via direct or ancestral exposure on estrous cycling in third generation offspring in mice," Department of Animal, Dairy and Veterinary Sciences. (August 10, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Phatak, S. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Esquer, H. (Author Only), Neal, C. (Presenter & Author), Contreras, K. (Author Only), Pontsler, K. (Author Only), Armbrust, T. (Presenter & Author), Rodriguez, D. (Author Only), Abercrombie, B. (Author Only), Hintze, K. J. (Author Only), Lefevre, M. (Author Only), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), 2016 Student Research Symposium, "Impact of ancestral or multi-generation exposure to a typical Western diet on colorectal carcinogenesis in mice," Department of Animal, Dairy and Veterinary Sciences. (August 10, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Bellgrau, A. (Presenter & Author), Hunter, A. (Author Only), Van Wettere, A. (Author Only), Larson, D. (Author Only), Phatak, S. (Author Only), Hintze, K. J. (Author Only), Benninghoff, A. (Author Only), 2016 Student Research Symposium, "Impact of the total Western diet and supplementation with tart cherries on colitis: histopathology and biomarkers of inflammation in C57BL/6J mice," Department of Animal, Dairy and Veterinary Sciences. (August 10, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Presentations Benninghoff, A., University of Tsukuba, Japan, "Diet and bioactives interactions: understanding the impact of different nutritional patterns on the efficacy of functional foods for health." (November 2014)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Rodriguez, D. (Presenter & Author), Neal, C. (Presenter & Author), Phatak, S. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Esquer, H. (Author Only), Contreras, K. (Author Only), Pontsler, K. (Author Only), Armbrust, T. (Author Only), Abercrombie, B. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Lefevre, M. (Author Only), Benninghoff, A. (Author Only), 2016 Student Research Symposium, "Impact of the total Western diet fed over multiple generations on fasting glucose and glucose tolerance in F3 generation offspring.," Department of Animal, Dairy and Veterinary Sciences. (August 10, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Contreras, K. (Presenter & Author), Cuthbert, J. (Author Only), Phatak, S. (Author Only), Larson, D. (Author Only), Benninghoff, A., 2016 ADAS-ASAS Joint Annual Meeting, "Effect of the total western diet via direct or ancestral exposure on estrous cycling in third generation offspring in mice," American Society of Animal Science. (July 19, 2016 - July 23, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Phatak, S. (Presenter & Author), Neal, C. (Author Only), Contreras, K. (Author Only), Armbrust, T. (Author Only), Rodriguez, D. (Author Only), Abercrombie, B. (Author Only), Ponstler, K. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Esquer, H. (Author Only), Cuthbert, J. (Author Only), Hintze, K. J., Ward, R. E., Benninghoff, A., USU Research Week: Student Research Symposium, "Multi-generational effect of western diet on colorectal cancer and impact of green tea on cancer prevention," School of Graduate Studies, Utah State University. (April 14, 2016)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Phatak, S. (Presenter & Author), Larson, D. (Author Only), Hunter, A. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Lefevre, M. (Author Only), Benninghoff, A. (Author Only), Hansen Life Science Retreat 2015, "Multigenerational effect of western diet on colorectal cancer and impact of green tea on cancer prevention," College of Science, Utah State University. (September 19, 2015)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Presentations Benninghoff, A. (Presenter & Author), Monsanto, S. (Author Only), Larson, D. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Lefevre, M. (Author Only), Experimental Biology 2014, "The micronutrient profile of the typical American diet enhances colorectal carcinogenesis in mice." (April 2014)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Benninghoff, A. (Presenter & Author), Hintze, K. J. (Author Only), Phatak, S. (Author Only), Larson, D. (Author Only), Hunter, A. (Author Only), Ward, R. E. (Author Only), Monsanto, S. (Author Only), Hisatome, T. (Author Only), Lefevre, M. (Author Only), Diet and Optimum Health 2015, "Impacts of the new total Western diet on gut health and colon carcinogenesis," Linus Pauling Institute and the Oxygen Club of California, Corvallis, Oregon. (September 9, 2015 - September 11, 2015)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Larson, D. (Author Only), Hintze, K. J. (Author Only), Ward, R. E. (Author Only), Lefevre, M. (Author Only), Hunter, A. (Author Only), Quarnberg, S. (Author Only), Benninghoff, A. (Author Only), Experimental Biology 2015, "Promotion of inflammation-associated colon tumorigenesis by the total Western diet in the APCmin/+ mouse," San Diego, CA. (March 28, 2015 - April 1, 2015)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Presentations Ward, R. E., Hintze, K. J., Lefevre, M., Benninghoff, A., Li, M., Rompato, G., Experimental Biology 2014, "Effects of green tea on the cecal metagenome of mice fed either the AIN-93 or Total Western Diet," American Society for Nutrition, San Diego, CA. (April 26, 2014)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Presentations Benninghoff, A., University of Illinois at Urbana-Champaign, "Impact of the new total Western diet on colorectal carcinogenesis in mice: importance of the micronutrient profile and interaction with green tea bioactives." (February 2014)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Conference Proceedings Phatak, S., Larson, D., Hunter, A., Neal, C., Contreras, K., Pontsler, K., Armbrust, T., Rodriguez, D., Abercrombie, B., Hintze, K. J., Ward, R. E., & Benninghoff, A. (2017). Ancestral and multi-generational consumption of the total Western diet in mice promotes colitis-associated colorectal cancer in third-generation offspring. (vol. 31, pp. 435.4). FASEB Journal.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Conference Proceedings Benninghoff, A., Hunter, A., Monsanto, S., Phatak, S., Hintze, K. J., & Ward, R. E. (2017). Consumption of the total Western diet (TWD) enhanced and sustained colonic inflammation and promoted colon tumorigenesis in mice, which led to marked changes in the composition of the gut microbiome in mice. (vol. 31, pp. 435.3). FASEB Journal
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Refereed Journal Articles Ward, R. E., Benninghoff, A., Healy, B. J., Li, M., Vagu, B., & Hintze, K. J. (2017, January 13). Consumption of the total Western diet differentially affects the response to green tea in rodent models of chronic disease compared to the AIN93G diet. Molecular Nutrition and Food Research.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Conference Proceedings Contreras, K., Cuthbert, J., Phatak, S., Larson, D., & Benninghoff, A. (2016). Effect of the total Western diet via direct or ancestral exposure on estrous cycling in third-generation offspring in mice. (S5 ed., vol. 94, pp. 97). Journal of Animal Science.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Conference Proceedings Hintze, K. J., Hisatome, T., Kellen, S., Ward, R. E., Benninghoff, A., & Lefevre, M. (2017). Effects of the Food Matrix and Western Diet on the Mouse Microbiome. (vol. 31, pp. 444.6). FASEB Journal.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Hintze, K. J. (Presenter & Author), Benninghoff, A. (Author Only), Ward, R. E. (Author Only), Utah One Health Symposium, "The intestinal microbiome: I. Me, myself, and 30 trillion of my closest friends." (November 17, 2017)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Refereed Journal Articles Benninghoff, A., Lefevre, M., Hintze, K. J., Ward, R. E., & Broadbent, J. R. (2015, August). Fighting cancer with functional foods: new approaches to investigate the interactions of dietary bioactive chemicals and the gut microbiome. Journal of Developments in Sustainable Agriculture, 10(1), 34-54.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Benninghoff, A. (Presenter & Author), Hintze, K. J. (Presenter & Author), Utah One Health Symposium, "The intestinal microbiome: II. Gut bacteria as critical players in gut health." (November 17, 2017)
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Refereed Journal Articles Hintze, K. J., Benninghoff, A., Cho, C., & Ward, R. E. (2018, April). Modeling the Western diet for pre-clinical investigations. Advances in Nutrition, 9, 1-9.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Refereed Journal Articles Hintze, K. J., Benninghoff, A., & Ward, R. E. (2017, May). Improving animal diets to increase relevance to human populations. Functional Foods in Health and Disease, 7(5), 329-337.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Refereed Journal Articles Monsanto, S. P., Hintze, K. J., Ward, R. E., Larson, D. P., Lefevre, M., & Benninghoff, A. (2016, June 01). The new total Western diet for rodents does not induce an overweight phenotype or alter parameters of metabolic syndrome in mice. Nutrition Research, 36(9), 1031-1044
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Phatak, S. (Presenter & Author), Hintze, K. J., Ward, R. E., Benninghoff, A., American College of Toxicology 38th Annual Meeting, "Multi-generational consumption of a Western diet for rodents promotes colitis-associated colorectal cancer in third-generation offspring," Palm Springs, CA. (November 5, 2017 - November 8, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Phatak, S. (Presenter & Author), Rodriguez, D., Neal, C., Park, E., Brubaker, M., Krepinski, K., Hintze, K. J., Benninghoff, A., 35th Annual Meeting of the Mountain-West Regional Chapter of the Society of Toxicology, "Impact of calcium and vitamin D or methyl donor supplementation on colitis and colon tumorigenesis in mice fed the total Western diet.," Society of Toxicology, Albuquerque, NM. (September 20, 2017 - September 22, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Phatak, S. (Presenter & Author), Neal, C., Armbrust, T., Hunter, A., Contreras, K., Esquer, H., Pontsler, K., Abercrombie, B., Rodriguez, D., Larson, D., Lefevre, M., Ward, R. E., Hintze, K. J., Benninghoff, A., 35th Annual Meeting of the Mountain-West Regional Chapter of the Society of Toxicology, "Multi-generational consumption of a Western diet for rodents promotes colitis-associated colorectal cancer in third-generation offspring," Society of Toxicology, Albuquerque, NM. (September 20, 2017 - September 22, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Phatak, S. (Presenter & Author), Hintze, K. J., Ward, R. E., Benninghoff, A., Environmental Mutagenesis and Genomics Society 48th Annual Meeting, "Ancestral and multi-generational consumption of the total Western diet in mice promotes colitis-associated colorectal cancer in third-generation offspring." (September 9, 2017 - September 13, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Rodriguez, D. (Presenter & Author), Aardema, N. (Author Only), Neal, C. (Author Only), Phatak, S. (Author Only), Armbrust, T. (Author Only), Hunter, A. (Author Only), Contreras, K. (Author Only), Tinsley, H. (Author Only), Yasui, T. (Author Only), Brubaker, M. (Author Only), Park, E. (Author Only), Krepinski, K. (Author Only), Herrera, M. (Author Only), Draper, A. (Author Only), Speas, E. (Author Only), Boomen, R. (Author Only), Campbell, K. (Author Only), Ward, R. E. (Author Only), Hintze, K. J. (Author Only), Benninghoff, A. (Author Only), 2017 ADVS Student Research Symposium, "Impact of basal diet on obesity phenotype of host mice following fecal transfer from obese or lean human donors," Department of Animal, Dairy and Veterinary Sciences. (August 9, 2017)


Progress 01/15/16 to 01/14/17

Outputs
Target Audience:Target Audience Research scientists Dieticians Clinical practitioners Changes/Problems:Changes/Problems As noted in last year's report, one possible concern was personnel management as the laboratory technician resigned. However, that issue did not manifest and the productivity of this research group has been excellent. I foresee no major problems in the coming year. What opportunities for training and professional development has the project provided?Opportunities This project constitutes the dissertation research of Sumira Phatak. As such, Sumira has extensive one-on-one interaction with the project PI, as well as the Co-I's, and is gaining experience in animal husbandry, experiment management, nutrition, animal necropsy and various bench-based assays. Sumira also received professional development training through various workshops on campus, including technical writing and scientific presentation. Additionally, two other graduate students and two undergraduate students have provided technical assistance, and have gained extensive experience in animal husbandry. A new opportunity realized in 2015 was the added focus on reproductive endpoints, in alignment with the research interests of another graduate student, Jocelyn Cuthbert. With no changes to the experiment design, Jocelyn collected data on reproductive outcomes (successful matings, litter size, litter growth, subsequent reproductive performance, estrus cycling in F3 and collected ovarian samples for immunohistochemistry) to evaluate the impact of these diets on reproductive competence, specifically oocyte quality. In 2016, Jocelyn collaborated with an undergraduate student to analyze these data and present the results at local and national scientific meetings. Although not specifically designed for this purpose, we hope to identify some preliminary data that may support further investigation supported through other funding opportunities. How have the results been disseminated to communities of interest?Dissemination An abstract presenting initial results of this project will be presented at the 2017 Experimental Biology meeting in Chicago, IL by the graduate student. Other presentations have been delivered by undergraduates at local meetings and the regional Mountain West Society of Toxicology meeting in September, 2016. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work A no cost extension was requested for an additional year to accommodate the lengthy data analysis process and continued analysis of the molecular endpoints as outlined in the grant proposal, with a focus on immunohistochemistry of the colon and the gene expression/DNA methylation.

Impacts
What was accomplished under these goals? Accomplishments 1) Major activities completed: By the second year of this program, all mouse breeding had been completed to generate all needed mice for the colorectal cancer study to be performed on the F3 generation. This cancer study was also completed, with more than 500 necropsies performed, by November 2015. As of this report, all in vivo aspects of this project have been completed. Also, in 2016, we developed and optimized procedures for extraction of nucleic acids from colon tissue samples and developed a likely target list for analysis DNA methylation and gene expression data. We also developed and tested a set of 96 primer pairs for genes of interest for use in nano-scale qPCR using the Fluidigm Biomark platform. Work to optimize procedures for immunohistochemistry of inflammation and cancer biomarkers was also initiated. 2) Specific objectives met: All animal animal breeding, the F3 generation cancer study, data collection from the in vivo study has been completed. During 2016, data analysis was completed for for parameters including (in part) food intake, energy intake, water intake, mating success, rate of weight gain during pregnancy, liter size at birth, weight of offspring at weaning, rate of weight gain during grow out of offspring for each generation, final body weight at each generation, body composition of offspring, fasting glucose level, glucose tolerance, estrus cycling (F3 generation). Also, we completed analysis for the control arm (no green tea supplementation) tumorigenesis endpoints, including tumor incidence, size and number in F3 offspring. (Analysis of F3 generation data for green tea-supplemented mice still pending). All tissues collected during necropsy have been processed and are ready for further molecular analyses (immunohistochemistry of colons, DNA methylation/gene expression analyses of colons, other markers in blood samples). Personnel training objectives and quality control assessment for upcoming laboratory assays have been met, such that assays can be initiated in the next couple of months. 3) Significant results achieved, including major findings, developments, or conclusions (both positive and negative): Some of our initial results are described below and should be considered preliminary at this time: Cancer endpoints in F3 generation offspring (no green tea exposure) - Notably, incidence of CAC was significantly higher in F3 offspring exposed ancestrally to the TWD (92%) as compared to their F3 counterparts fed AIN93G (56%). Moreover, tumor burden was markedly higher (> 3-fold increase) in F3 offspring exposed to TWD over multiple generations when compared to F3 offspring provided TWD directly. Alternatively, neither ancestral or multi-generation exposure to the DIO diet altered CAC incidence, whereas a small, significant increase in tumor burden in F3 offspring ancestrally-exposed to the DIO was observed as compared to direct-fed mice. Fasting glucose and glucose tolerance - Glucose tolerance (assessed as AUC) in F3 offspring was not significantly affected by diet regimen for the TWD or the DIO groups nor by supplementation with green tea. A significant main effect of green tea on fasting glucose was apparent for the TWD set of diets, though not for the DIO groups. Also, a significant main effect of diet on difference from baseline (an indicator of persistent glucose elevation) was evident for the TWD groups. Strong cage effects were identified, which reduced the statistical power of the analysis. Interestingly, this phenomenon has been previously observed by our research group in a prior study testing the DIO diet. Overall, these observations point to a complex relationship between basal diet and the green tea supplement, even though neither treatment resulted in severe glucose abnormalities. Reproductive endpoints - In F3 offspring, neither gender ratio or litter size were impacted by either the TWD or DIO diets as compared to the control AIN93G diet. With respect to estrus cycling (an added focus described in 2015's report), no significant differences were observed among any of the diet treatment groups for any of the estrous cycle stages (p>0.18). These data disagree with prior observations that a high fat diet prolonged time in diestrus, although the current study used diets with fewer calories from fat (35% for TWD and 45% for DIO) compared to the study by Brothers, et al (2010), which employed a 60% fat diet. 4) Key impacts or other accomplishments realized. As part of this breeding program, we have measured a number of additional parameters beyond the scope of the primary hypothesis to enhance the impact of this work, including continuous assessment of food and energy intakes, frequency of successful matings, litter size, weight gain of dams and pups, etc. to assess impacts of diet on fertility and health of offspring. Over multiple generations, these additional parameters will provide insight into the effect of a western style diet on early life parameters. A likely key impact of this study is the novel observation that ancestral exposure to a Western type diet markedly increased colon cancer incidence and disease severity in F3 offspring that were not fed this diet directly. Also, exposure to TWD over multiple generations markedly exacerbated the disease in F3 offspring as compared to those fed TWD directly. These observations point to the important influence of (grand)parental lifestyle factors on health outcomes in offspring. Put simply, the poor diet of the great-grandparents' appeared to have influenced cancer development in their great-grandchildren, who otherwise ate a healthy diet. Abbreviations: total Western diet, TWD; diet-induced obesity diet, DIO

Publications


    Progress 01/15/15 to 01/14/16

    Outputs
    Target Audience:Worldwide Research scientists Dieticians Clinical practitioners Changes/Problems:Changes/Problems The only problem encountered this year was one of personnel management. The laboratory technician supported by this project resigned her position in October, although this What opportunities for training and professional development has the project provided? Opportunities This project constitutes the dissertation research of Sumira Phatak. As such, Sumira has extensive one-on-one interaction with the project PI, as well as the Co-I's, and is gaining experience in animal husbandry, experiment management, nutrition, animal necropsy and various bench-based assays. Sumira also received professional development training through various workshops on campus, including technical writing and scientific presentation. Additionally, two other graduate students and two undergraduate students have provided technical assistance, and have gained extensive experience in animal husbandry. A new opportunity realized in 2015 was the added focus on reproductive endpoints, in alignment with the research interests of another graduate student, Jocelyn Cuthbert. With no changes to the experiment design, Jocelyn collected data on reproductive outcomes (successful matings, litter size, litter growth, subsequent reproductive performance, estrus cycling in F3 and collected ovarian samples for immunohistochemistry) to evaluate the impact of these diets on reproductive competence, specifically oocyte quality. Although not specifically designed for this purpose, we hope to identify some preliminary data that may support further investigation supported through other funding opportunities. How have the results been disseminated to communities of interest?Dissemination Not yet applicable. Although we anticipated preparing an abstract for the Experimental Biology 2016 meeting, the breeding schedule did not allow us to meet the 6-month advance deadline for abstracts. I prefer for the student to present a complete story based on statistical evaluation, not conjecture based on an incomplete data set. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work In 2016, we will proceed with the analysis of molecular endpoints as outlined in the grant proposal, with a focus on immunohistochemistry of the colon and the gene expression/DNA methylation analyses. Also, the student will continue to complete post-necropsy assessment of tumor outcome. Also, we will complete all statistical analyses of other endpoints assessed concurrent with the multi-generation mouse study. Because execution of the mouse study took a bit longer than anticipated and because it fully occupied the students' time, I do expect that completion of the molecular analyses will require at least the full year. Thus, it is possible that a no-cost extension may be requested to facilitate the end analyses and write up of this project.

    Impacts
    What was accomplished under these goals? Accomplishments 1) Major activities completed: By the second year of this program, all mouse breeding had been completed to generate all needed mice for the colorectal cancer study to be performed on the F3 generation. This cancer study was also completed, with more than 500 necropsies performed, by November 2015. As of this report, all in vivo aspects of this project have been completed. 2) Specific objectives met: We completed all animal breeding, the F3 generation cancer study, and collection of a very large set of data gathered during the study and at necropsy to elucidate the effect of dietary treatment on many key parameters. These include (in part) food intake, energy intake, water intake, mating success, rate of weight gain during pregnancy, liter size at birth, weight of offspring at weaning, rate of weight gain during grow out of offspring for each generation, final body weight at each generation, body composition of offspring, fasting glucose level, glucose tolerance, estrus cycling (F3 generation), and the most important measures, tumor incidence, size and number in F3 offspring. All tissues collected during necropsy have been processed and are ready for further molecular analyses (immunohistochemistry of colons, DNA methylation/gene expression analyses of colons, other markers in blood samples). Curation of the data collected during the course of the animal study was recently completed, and statistical analyses will begin January 2016; we expect this analysis to be rather complex given the abundant number of endpoints to be assessed. 3) Significant results achieved, including major findings, developments, or conclusions (both positive and negative): A significant observation was that the TWD did not overtly negatively impact breeding success, as assessed by frequency of successful pregnancies, weight gain during pregnancy, litter size, weight gain during nursing or offspring weight at weaning. This observation persisted throughout all generations, although statistical analyses must be performed to support this general observation. Also, preliminary analyses suggest that, as expected, mice consuming the TWD did not experience excess weight gain compared to mice consuming the DIO diet across multiple generations. Curation of the data collected during the course of the animal study was recently completed, and statistical analyses will begin January 2016; we expect this analysis to be rather complex given the abundant number of endpoints to be assessed. 4) Key impacts or other accomplishments realized. As part of this breeding program, we have measured a number of additional parameters beyond the scope of the primary hypothesis to enhance the impact of this work, including continuous assessment of food and energy intakes, frequency of successful matings, litter size, weight gain of dams and pups, etc. to assess impacts of diet on fertility and health of offspring. Over multiple generations, these additional parameters will provide insight into the effect of a western style diet on early life parameters. Curation of the data collected during the course of the animal study was recently completed, and statistical analyses will begin January 2016; we expect this analysis to be rather complex given the abundant number of endpoints to be assessed.

    Publications


      Progress 01/15/14 to 01/14/15

      Outputs
      Target Audience: Target Audience worldwide Changes/Problems: Changes/Problems As of now, we have not encountered major delays that would adversely impact the timeline. As expected, the breeding program took quite a while to ramp up to the scale needed. Thus, while some mice are old enough to start the cancer study shortly, it will be several months before we have completed populating all of the experimental groups. No changes to ACUP are needed at this time. What opportunities for training and professional development has the project provided? Opportunities This project constitutes the dissertation research of Sumira Phatak. As such, Sumira has extensive one-on-one interaction with the project PI, as well as the Co-I's, and is gaining experience in animal husbandry, experiment management, nutrition, animal necropsy and various bench-based assays. Sumira also received professional development training through various workshops on campus, including technical writing and scientific presentation. Additionally, two other graduate students and two undergraduate students have provided technical assistance, and have gained extensive experience in animal husbandry. How have the results been disseminated to communities of interest? Dissemination Not yet applicable. We anticipate submitting an abstract to the Experimental Biology meeting for 2016 later this year. What do you plan to do during the next reporting period to accomplish the goals? Plan of Work As outlined in the grant, we plan to proceed with the cancer trial without deviation from the original protocol. The 4-month study will likely be concluded for all experimental animals by the end of the summer. Thus, we expect preliminary cancer outcomes by fall 2015. Also, pathology should be completed this year. This fall, we will also initiate the other molecular analyses, including assessment of DNA methylation by ultra-deep pyrosequencing.

      Impacts
      What was accomplished under these goals? Accomplishments 1) Major activities completed: In the first year of this project, we have nearly completed the breeding program to generate mice for a colorectal cancer study to be performed on F3 generation mice. The breeding program includes three separate mouse colonies, each with different basal diets (the optimal AIN93G diet, the new Total Western Diet and the 60% fat DIO diet. From these colonies, ultimately 7 breeding groups were generated, and then used to produce nearly 600 offspring for the cancer study. As of December 31, 2014, we are only a few weeks away from completing the breeding program and initiating the first mice as part of the cancer trial. 2) Specific objectives met; We have established all standard operating procedures for execution of this complicated, multi-generation cancer study; we have successfully ramped up the breeding program to generate the number of mice needed for the cancer trial in the 3rd generation. Additionally, satisfactory progress has been made in preparing assays for endpoints to be measured at the conclusion of the cancer study this year. 3) Significant results achieved, including major findings, developments, or conclusions (both positive and negative); A significant observation was that the TWD did not overtly negatively impact breeding success, as assessed by frequency of successful pregnancies, weight gain during pregnancy, litter size, weight gain during nursing or offspring weight at weaning. As mentioned in the proposal, this was a potential pitfall that may have caused problems in ramping up the breeding program to generate sufficient numbers of animals. This pitfall was, in fact, not realized and breeding proceeded normally. 4) Key impacts or other accomplishments realized. As part of this breeding program, we have measured a number of additional parameters beyond the scope of the primary hypothesis to enhance the impact of this work, including continuous assessment of food and energy intakes, frequency of successful matings, litter size, weight gain of dams and pups, etc. to assess impacts of diet on fertility and health of offspring. Over multiple generations, these additional parameters will provide insight into the effect of a western style diet on early life parameters. As expected, though, the key impact of assessing cancer outcome will not be realized until later in year 2 of the project.

      Publications