Thesis (M.S.)--Oklahoma State University, 2008. / Vita. Includes bibliographical references.

Thesis (M.S.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (M.S.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (M.S.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2004. / Vita. Includes bibliographical references (p. 79-86).

<p>Consumption of soy foods has been shown to provide beneficial health outcomes such as reduction of menopause symptoms, reduced risk of breast cancer and prostate cancer, improved cardiovascular health, and improved bone health. The mechanism hypothesized to be driving these outcomes is the conversion of the soy isoflavone daidzein into the metabolite equol by bacteria in the gut microbiome. Equol is an exclusively microbially produced metabolite with a high binding affinity to mammalian estrogen receptors. Not all humans harbor equol-producing microbes in their gut, and less than half of the population can be classified as equol producers. To date, soy feeding research published suffers from confounding factors that make assessing the causal impact of equol production in health difficult due to: (i) large interpersonal variation of the human microbiome and human genomes and that (ii) all lab-raised rodent models harboring natural microbiomes are highly efficient equol producers. In this study, we sought to establish a gnotobiotic mouse model harboring synthetic bacterial communities with divergent equol-producing capacities by designing two communities: the Equol(-) community and Equol(+) community. The Equol(-) community was designed to include ten bacterial strains commonly found within a human microbiome without equol-producing capacity<em>.</em> To create the Equol(+) community, the equol-producing bacteria <em>Adlercreutzia equolifaciens</em> was added to the Equol(-) community<em>.</em> Female and male germ-free C57BL/6 mice were colonized with either the Equol(-) or Equol(+) community for 4 weeks. Daidzein was administered by dietary supplementation (1.5% wt/wt daidzein) in a semi-purified diet containing fermentable fiber starting two weeks prior to bacterial colonization. As expected, equol was detected in the serum of mice colonized with the Equol(+) community, but not detectable in those colonized with the Equol(-) community. There were no sex differences detected in equol production. 16S rRNA gene sequencing of mouse cecal content revealed that ~50-80% of the strains from each community colonized within the mice at detectable levels. Strain-specific qPCR improved the detection of strains not observed consistently through 16S rRNA gene sequencing. Our results demonstrated that this model is reliable in producing the expected equol producing and non-equol producing phenotypes when colonized with the Equol(+) and Equol(-) communities, respectively. This model system can be utilized in a broad range of future studies to conclusively determine the causal impact of endogenous equol production in many areas, such as cardiometabolic health and bone health.</p>

<p>  </p> <p><strong>Background information:</strong> </p> <p>Lactose is a disaccharide found in milk and milk products. Lactose is digested by the enzyme lactase. Lactase non-persistence is a genetic trait in which individuals have low lactase activity. Approximately 70 percent of the world population is lactase non-persistent. It is a major cause of lactose maldigestion. An increase of 20 ppm hydrogen in breath within six hours of a lactose challenge dose (0.5 g of lactose per kg bodyweight) indicates lactose maldigestion. On the other hand, lactose intolerant individuals experience abdominal pain, bloating, diarrhea, and flatulence on consuming dairy. Therefore, lactose intolerant individuals avoid milk, which is a rich source of calcium. Consequently, lactose intolerance has been associated with reduced calcium intake and low bone mineral density. There are two mechanisms for lactose intolerance. The first mechanism is dependent on lactose dose and the second one is independent of lactose. Recently, A1 and A2 β-caseins have been associated with lactose intolerance. Studies conducted in China, New Zealand and Australia demonstrated fewer symptoms on consumption of milk containing only A2 β-casein as compared to milk containing both A1 and A2 β-casein. However, no study was conducted in the population residing in United States, where crossbred cows producing milk containing both A1 and A2 β-casein is the norm. Moreover, no study compared tolerance and digestion on consuming milk with different proportions of A1 and A2 β-casein. Lactose intolerant individuals can include A2 milk in their diet to meet the calcium requirement, if milk containing only A2 β-casein causes fewer symptoms and less maldigestion. </p> <p><strong>Objectives:</strong></p> <p>1. To determine if a single meal of A2 milk containing only A2 β-casein would be better tolerated, producing fewer GI symptoms and less maldigestion, than conventional milk containing 75 percent A1 β-casein and 25 percent A2 β-casein</p> <p>2. To determine if a single meal of Jersey milk containing 25 percent A1 β-casein and 75 percent A2 β-casein would produce less maldigestion and intolerance, than conventional milk containing 75 percent A1 β-casein and 25 percent A2 β-casein</p> <p>3. To determine if the gastric emptying time of milk containing only A2 β-casein and milk containing 75 percent A1 β-casein and 25 percent A2 β-casein was different </p> <p>4. To determine if inflammation, maldigestion and intolerance is lower with a two-week daily consumption of milk containing only A2 β-casein as compared to milk containing 75 percent A1 β-casein and 25 percent A2 β-casein</p> <p><strong>Methods:</strong> </p> <p>Three randomized, double-blinded, crossover trials were conducted. The first study was conducted to determine tolerance and digestion of milk containing different proportions of A1 β-casein and A2 β-casein in subjects residing in the United States. There were four milk interventions in the study: A2 milk (milk containing 100% A2 β-casein), Jersey milk (milk containing 25%/75% A1/A2 β-casein), conventional milk (milk containing 75%/25% A1/A2 β-casein) and lactose-free milk (milk containing 60%/40% A1/A2 β-casein). Lactose intolerance in subjects was determined using a Qualifying Lactose Challenge Symptom Score after a challenge milk dose of 0.5 g of lactose/kg bodyweight. Subjects were screened for lactose maldigestion with a six-hour Hydrogen Breath Test. Symptoms and madigestion response to a single meal of milk (dose calculated as 0.5 g of lactose/kg bodyweight) containing different proportions of A1 and A2 β-casein were determined in lactose intolerant subjects and lactose maldigesters. </p> <p>The second study was conducted to examine the difference in gastric transit between A2 milk and conventional milk in lactose maldigesters. Magnetic Resonance Imaging (MRI) technique was used to observe gastric emptying. Subjects rated abdominal pain after consumption of the two milk interventions using a six-point Likert scale.</p> <p>The final study was conducted to determine symptoms, maldigestion and inflammation due to consumption of A2 milk and conventional milk for fourteen days on a daily basis in lactose maldigesters. Subjects rated lactose intolerance symptoms every day during the fourteen days using a six-point Likert scale. Breath hydrogen, serum inflammatory markers and serum antioxidant concentrations were measured on day 15 after the two-week milk consumption.  All the studies were registered at clinicaltrials.gov. </p> <p><strong>Results: </strong></p> <p>Results from the first study indicated that abdominal pain due to a single meal of A2 milk in lactose intolerant subjects was lower by twenty-three percent as compared to conventional milk (p=0.004, n=25). Similarly, there was a twenty-eight percent decrease in abdominal pain score on consumption of A2 milk as compared to conventional milk in lactose maldigesters (p=0.001, n=33). All the other symptoms were not different between A2 and conventional milk in lactose intolerant subjects and lactose maldigesters. A single meal of A2 milk reduced the total hydrogen production (p=0.04, n=33) by sixteen percent and total symptoms production (p=0.04, n=33) by eighteen percent in lactose maldigesters as compared to conventional milk. However, the total hydrogen and total symptoms produced were not different in lactose intolerant subjects between A2 and conventional milk. Similarly, individual GI symptoms, total symptoms and total hydrogen were not different between jersey and conventional milk in lactose intolerant subjects and lactose maldigesters.</p> <p>Results from gastric transit study indicated that volume of A2 milk in the stomach was higher by twenty-four percent at 30 (p=0.01, n=10), forty-six percent at 60 (p=0.002, n=10) and one hundred and sixty-one percent at 120 (p<0.001, n=10) minutes as compared to conventional milk in lactose maldigesters. </p> <p>Results from the last study suggested a fecal urgency score lower by thirty-three percent (p=0.033, n=10) in lactose maldigesters due to daily consumption of A2 milk for two weeks as compared to conventional milk. The other symptoms, inflammation markers, antioxidant and breath hydrogen production were not different between A2 and conventional milk due to two-week milk consumption.</p> <p><strong>Conclusion:</strong> </p> <p>Total intolerance symptoms and total hydrogen production were lower due to consumption of a single meal of A2 milk than conventional milk in lactose maldigesters. Gastric transit of A2 milk was slower as compared to conventional milk in lactose maldigesters. Daily consumption of A2 milk for two-weeks reduced fecal urgency as compared to conventional milk in lactose maldigesters. </p> <p>  </p>

<p>Colorectal cancer (CRC) is the third most prevalent cancer in the United States and estimated to affect 151,030 people and kill 52,580 people in 2022. Although some populations are more susceptible for CRC due to inherited cancer-causing mutations or having family history of CRC, most CRC cases occur sporadically with accumulation of a series of somatic mutation in tumor suppressor genes, oncogenes, and DNA repair system in the colon. Mutations in the adenomatous polyposis coli <em>(APC)</em> and the Kirsten rat sarcoma viral oncogene homologue <em>(KRAS)</em> are known as cancer driving mutations which are most frequently identified genetic lesions at early stages of sporadic CRC development. To evaluate effective drugs for prevention and treatment, preclinical models of CRC that resemble key features of human CRCs are crucial. Genetically modified mouse models (GEMM) of CRC bearing loss of <em>Apc</em> with or without other mutations, such as oncogenic <em>Kras</em> have been valuable tools to study pathobiology, treatment, and prevention of CRC. However, a major limitation of most <em>Apc</em>-mutant GEMMs is the predominant distribution of tumors in the small intestine rather than in the colon. Previously, a murine model bearing colon-specific mutations in <em>Apc</em> and <em>Kras</em> has been reported to develop colon-specific tumorigenesis in the colon, utilizing the <em>carbonic anhydrase 1 promoter/enhancer-Cre recombinase (CAC)</em> in Cre-LoxP system to restrict <em>Apc</em> knockout and a latent expression of oncogenic <em>Kras</em> in the colon tissue. However, only limited features of this model, so called AKC mouse, have been characterized so far. The lack of in depth understanding of this model could potentially hamper its utility for cancer research. Therefore, in Chapter 2 of this dissertation, I first characterized key aspects of disease-related phenotypes including clinical and histopathological features, tumor-elicited inflammation, the transcriptomic profiles, the gut microbial profiles in the AKC mice. Further, comparative analysis has been made on the transcriptomic profiles between AKC mice and human colon cancers with mutations in <em>APC</em> and <em>KRAS</em> at cancer stage II or below to evaluate the utility of the mouse model for studying human CRCs.</p> <p>Chemoprevention is the use of drugs or natural substances to inhibit initiation and delay of the progression of tumorigenesis, which could be a promising approach to reduce the incidence, mortality, and morbidity of CRC. Delta-tocotrienol (𝛿TE) is a natural analogue of vitamin E which has been shown to have antioxidant, anti-inflammatory and anticancer activities. Although its anticancer effects have been studied in different models of CRCs, including carcinogen-induced models, carcinogen-induced colitis-associated models, and colon cancer xenograft models, it has not been tested in a genetic model of sporadic CRC harboring <em>Apc</em> and <em>Kras</em> mutations. Therefore, in Chapter 3, the antitumor effects of 𝛿TE-rich tocotrienols (𝛿TE/gTE) and the potential mechanisms were investigated in AKC mice. 𝛿TE/𝛾TE-supplementation significantly improved the survival of AKC mice and suppressed tumorigenesis in association with inhibition of cell proliferation in the tumors. Further, the anti-tumor effects were correlated with reduction of pro-inflammatory and pro-tumorigenic cytokines, such as interleukin-1b and granulocyte-macrophage colony stimulating factor (GM-CSF), transcriptional enrichment of pathways involved in fatty acid metabolism, and reduction of diacylglycerol (DG) level in the colon tissue.</p> <p>Finally, AKC mice were used for screening the efficacies of other potential chemoprevention candidates, including aspirin, sulindac, and resveratrol in Chapter 4. Aspirin and sulindac are nonsteroidal anti-inflammatory drugs (NSAIDs) and they are among the most studied chemoprevention agents for CRC. However, the long-term regular use of NSAIDs may cause bleedings in the gastrointestinal organ system or hemorrhagic stroke. For aspirin, although extensive studies have shown its beneficial role in the prevention of primary CRC, there are mixed results for its benefit and harms, which may require further identification of populations who will benefit from the regular use of aspirin for prevention of CRC. Resveratrol is a naturally derived polyphenol, which is known for its antioxidant, anti-inflammatory, and antimicrobial activities with generally good safety profile. In our study, when the dietary supplementation of three compounds alone or in combination with 𝛿TE/gTE were examined for its antineoplastic effects in the AKC mice, only aspirin significantly suppressed tumorigenesis with decreased pro-inflammatory and pro-tumorigenic cytokines in the colon without overt toxicity. We found that sulindac induced serious gastric lesions and potential liver toxicity with some animals died earlier than the rest over the study period. Additionally, in this model, resveratrol was not effective in reducing tumorigenesis, in contrast to a previous study where the workgroup used similar genetic model of CRC but different modality to induce the mutations. Our findings add lines of evidence that depending on the use of different models the test compounds, aspirin, sulindac, and resveratrol may exhibit varying cancer prevention effects. Further research is warranted to identify underlying mechanisms that could explain the heterogenous responses to the test compounds and to optimize the interventions. </p>

<p>   </p> <p>Ulcerative colitis is a chronic disease that affects more than 770,000 U.S individuals and the number will increase to 1 million by 2025, resulting in $7 billion cost to manage the disease. Ulcerative colitis is characterized by inflammation along the colon and is a risk factor for the deadly colitis-associated colon cancer (CAC). Emerging research shows that gut microbes, the microorganisms living in our intestine, regulate colon inflammation. Specifically, an imbalanced microbial community may promote the growth of pathogens that invade the host to cause or exacerbate colitis. Therefore, researchers have been searching for safe and cost-effective approaches to keep gut microbes balanced in a long run and thus to control colitis. To this end, my research investigates the microbial modulatory capacities of dietary phytochemicals including polyphenols and vitamin E forms, delineates the role of microbial interaction in their protective effects against colon inflammation and further utilizes such interactions to develop anti-colitis therapies. To address the research questions, I have performed three independent projects and discussed them separately in chapters 2-4. </p> <p>The first project (chapter 2) focused on the anti-CAC and anti-colitis effects of grape polyphenols supplemented through a whole grape powder. Polyphenols are natural chemicals found in plants and have been shown to alleviate colon inflammation in both clinical and animal studies, but the underlying mechanisms are not completely understood. In particular, the role of microbial modulation in polyphenol-mediated benefits is not fully established. Here we hypothesized that, polyphenols may attenuate colon inflammation via interacting with gut microbes. Through two animal studies, we found that 10% grape powder (10GP) diet, which contains 0.033% polyphenols, attenuated colitis-associated tumorigenesis, and prevented disease-induced microbial dysbiosis. Moreover, 10GP diet only mitigated colitis in conventional animals, but not antibiotic-treated, gut microbe-depleted animals. Collectively, these two studies demonstrated that the interaction with gut microbes played a causative role in the protective effects of 10GP against colon inflammation. </p> <p>Like polyphenols, vitamin E forms are also phytochemicals with phenolic structures and undergo liver metabolism followed by biliary excretion to the gut. In the second project, we investigated the anti-colitis effects of vitamin E-based synbiotics therapies. Previously, we found that d-tocotrienol 13-carboxychromanol (dTE-13’), a metabolite of the natural vitamin E form dTE, inhibited colitis-associated tumorigenesis in mice, modulated their gut microbiota and increased the relative abundance of a lactic acid bacterium, which is commonly used in food industry. Interestingly, a subspecies of this bacterium, named <em>Lactococcus. lactis</em> subsp. <em>cremoris</em> (<em>L. cremoris</em>), has been reported to attenuate ulcerative colitis in mice. Therefore, we reasoned that combining dTE-13’with <em>L. cremoris</em> may offer synergistic protection against ulcerative colitis by modulating gut microbes. Through two animal studies coupled with anaerobic cell culture, we found that combining <em>L. cremoris</em> with dTE-13’, not the parental dTE, showed superior anti-colitis effects, rendered gut microbes resistant to disease-associated dysbiosis and facilitated the microbial reduction of a double bond on dTE-13’ into dTE-13’ (2DB). Overall, these data suggested that dTE-13’ interacted with <em>L. cremoris</em> to benefit the host. </p> <p>To further corroborate the microbial metabolism of vitamin E forms under <em>in vivo</em> settings, we launched the third project (chapter 4) where we compared the metabolites formation of dTE and dTE-13’ between antibiotic-treated mice that had reduced gut bacterial load and conventional ones. We found that in dTE-gavaged animals, antibiotics treatment decreased the fecal amounts of dTE and its metabolites by 61% and 98%, respectively, while increased dTE level in the adipose tissue. Similarly, in animals gavaged with dTE-13’, antibiotics treatment led to a 98% reduction in its downstream metabolites. More importantly, antibiotics treatment reduced the ratio of the parental dTE and dTE-13’ to their metabolites in feces, especially the reduction from dTE-13’ (3DB) to dTE-13’ (2DB), suggesting the active role of gut microbes in the metabolism of dTE and dTE-13’. This observation is consistent with the results from the anaerobic study performed in the second project.</p> <p>In summary, we showed that grape polyphenols and vitamin E form-based synbiotics offered strong protection against colon inflammation and their interaction with gut microbes likely contributed to the observed benefits. In the study of grape polyphenols, we proved the causal role of gut microbes in polyphenol-mediated alleviation of colitis. In the subsequent study of vitamin E forms, we presented evidence that the superiority of the synbiotics might be rooted in the enhanced microbial metabolism of vitamin E forms. Together, these results supported the central role of gut microbes in the management of colitis and proposed two different classes of dietary phytochemicals that can manipulate gut microbes to benefit the host. Natural bioactive compounds like polyphenols and vitamin E forms are ideal candidates for long-term preventive measures as they have less side effects and are more cost-effective compared to drugs. Moreover, by understanding the targeting microbes of different phytochemical compounds, hopefully we will be able to customize phytochemical supplementation based on individual microbial profile and dietary habits. For instance, we may optimize the dosage and type used based on the microbes present in the gut, or add in probiotics to design more effective synbiotics just like the combination of dTE-13’ and <em>L.cremoris</em>.</p> <p>  </p>