Non Technical Summary
1. Current Issue or Problem and Its ImportanceThe research addresses the health implications of consuming foods prepared in thermally abused oils (TAOs), which are prevalent in the fast-food industry. These oils, when repeatedly heated, degrade and generate harmful lipid oxidation products (LOPs). Consuming foods fried in these oils is linked to increased risks of chronic diseases, including gut health disorders and colorectal cancer. This research is essential because a significant portion of the population consumes fast food regularly, and understanding the health impacts of TAOs can lead to improved dietary guidelines and healthier commercial food preparation methods. Beyond individual health, this research has broader implications: economically, by potentially reducing healthcare costs and improving productivity; community-wise, by enhancing public health; environmentally, by promoting more sustainable cooking practices; and in agriculture, by influencing the production of healthier cooking oils.2. Methods and ApproachesIn our study, we will explore the effects of thermally oxidized oils (TAOs) across three key objectives using C57BL/6J mice to mimic human dietary patterns. The first objective investigates how different durations of TAO exposure--acute, subchronic, and chronic--affect gut inflammation, microbiome composition, and immune responses. Mice will be fed diets with varying levels of oxidized oils and will undergo thorough health monitoring, including analysis of colon tissue and blood for various biomarkers, along with regular fecal sampling to observe microbiome changes over time. The second objective assesses the impact of chronic TAO exposure on colitis and colon cancer development using a mouse model of colitis-associated colorectal cancer to simulate human disease progression, with comprehensive evaluations of disease markers, gut microbiome alterations, and immune response. The third objective involves laboratory studies to determine the direct effects of TAOs and lipid oxidation products on intestinal epithelial cells and gut microbiota, using both mouse-derived and human donor samples in fermentation systems to study microbiome composition and function. These integrated approaches aim to elucidate the multifaceted impact of TAOs on gut health and disease mechanisms.3. Ultimate Goals and Expected ImpactThe ultimate goal of this project is to delineate the health risks associated with the consumption of TAOs and to use this knowledge to inform public health guidelines and commercial food preparation practices. Achieving these goals can lead to significant health improvements, reducing the incidence of diet-related diseases such as gut disorders and colorectal cancer. This would not only enhance the quality of life for individuals but also lead to economic benefits through decreased healthcare costs and increased productivity. Societally, the project aims to raise public awareness about the risks associated with fried foods and influence policy changes that promote healthier food preparation practices. These efforts are expected to foster a broader understanding of nutrition and health, encouraging more informed dietary choices and potentially leading to national and global changes in food safety regulations.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	3840	1010	100%

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
Thermal processing of vegetable oils high in unsaturated fatty acids generates lipid oxidation products (LOPs) that can adversely affect human health. The consumption of fried foods represents a significant route of exposure to these toxic compounds. However, the impact of dietary intake of LOPs from thermally abused oils (TAOs) on the gut microbiome and the associated effects on gut health remain largely unexplored. Our overarching objective is to investigate the interrelationships for dietary intake of LOPs from TAOs, the gut microbiome, and gut health. Our specific objectives are: 1) Determine the impacts of acute, subchronic, or chronic exposure to dietary TAOs on biomarkers of gut inflammation, the composition of the gut microbiome, and underlying molecular pathways mediating mucosal inflammatory and immune response in healthy C57BL6/J mice. 2) Determine the role of chronic exposure to dietary TAOs in modulating symptoms of colitis and progression to colon tumorigenesis, shifting the composition of the gut microbiome, and altering mucosal immune and inflammation pathways using the AOM/DSS model of colitis-associated colorectal carcinogenesis. 3) Determine the direct relationship of dietary TAOs on intestinal epithelial cells and the gut microbiome using in vitro cell culture and batch fermentation methods. The findings will provide valuable insights into the relationship between the dietary intake of TAOs and the composition and metabolic function of the gut microbiome in healthy mice and a mouse model of colitis-associated colorectal cancer.Milestones and TimelineYear 1: Diet preparation, Perform Experiment 1Year 2: Experiment 1 sample processing; Experiment 2 analysis; Experiment 2 diet preparation; perform Experiment 2; manuscript preparation.Year 3: Experiment 2 sample processing; Experiment 2 analysis; Experiment 3 (complete); manuscript preparation.Year 4: Experiments 4 and 5 (complete); manuscript preparation.

Project Methods
Objective 1.Objective 1 of the grant aims to investigate the effects of acute (7 days), subchronic (4 weeks), and chronic (18 weeks) exposure to dietary Thermally Oxidized Oils (TAOs) on biomarkers of gut inflammation, gut microbiome composition, and molecular pathways underlying mucosal inflammatory and immune responses in healthy C57BL6/J mice. Male C57BL/6J mice will be acquired from a single colony to maintain consistent initial microbiome conditions. Mice will be randomly assigned to experimental groups and fed one of three diets: a control diet with no oxidized oils, a diet with 50% oxidized oil mixture, and a diet with 100% oxidized oil mixture, with TAO concentrations reflecting real-world human intakes. Throughout the study, mice will have ad libitum access to their respective diets, and food intake will be monitored. Mice will be euthanized at various time points for analysis of colon tissue, including histopathology, fatty acid profiles, gene expression, and microbiome sequencing. Blood plasma will also be analyzed for various biomarkers. Fecal samples will be collected to track changes in microbiome composition over time. The experimental design encompasses acute, subchronic, and chronic exposures to elucidate the dynamic responses to TAOs.Objective 2.Objective 2 aims to investigate the impact of chronic exposure to dietary Thermally Oxidized Oils (TAOs) on symptoms of colitis and progression to colon tumorigenesis in C57BL/6J mice using the AOM/DSS model of colitis-associated colorectal carcinogenesis. This model combines AOM-induced colon tumorigenesis with DSS-induced colitis, mimicking human colorectal cancer progression. Male C57BL/6J mice will be used, and experimental groups will receive different diets as described previously. Mice will be monitored for food intake and body weight. After 28 days, mice will receive AOM and then DSS to induce colitis and tumorigenesis. Multiple time points will be included to assess disease development and gut microbiome changes. Colon tissues will be analyzed for histopathology, immunohistochemistry, fatty acid profiles, and gene expression. Blood plasma will be examined for various biomarkers. Fecal and cecal samples will be collected for microbiome analysis. The study design allows for the comprehensive evaluation of the effects of chronic TAO exposure on colitis and tumorigenesis progression, microbiome composition, and mucosal immune response pathways.Objective 3.Objective 3 focuses on determining the direct impact of dietary Thermally Oxidized Oils (TAOs) on intestinal epithelial cells and the gut microbiome using in vitro cell culture and batch fermentation methods. Experiment 3 aims to assess the effects of Lipid Oxidation Products (LOPs) derived from TAOs on the expression of inflammation and oxidative stress biomarker genes in normal human colon cells, human colon adenocarcinoma and carcinoma cells, and mouse macrophages. Cell lines will be cultured and treated with TAO polar lipids and specific LOPs, with or without lipopolysaccharide (LPS), to evaluate inflammation response. Cytotoxicity and intracellular reactive oxygen species (ROS) levels will be assessed, and gene expression will be analyzed using qRT-PCR. Experiment 4 will employ an in vitro fermentation system using cecal or fecal material from healthy male C57BL/6J mice to evaluate the impact of TAO supplementation on microbiome composition and short-chain fatty acid (SCFA) concentrations. Experiment 5 will extend this investigation to human donors, utilizing a similar fermentation approach to assess the effects of TAOs on human gut microbiota and SCFA production. These experiments will provide insights into the direct effects of TAOs on intestinal cells and the gut microbiome, shedding light on their potential role in gut health and disease.