Non Technical Summary
Avocado (Persea americana) has become a popular fruit in human nutrition in recent years. According to the USDA, avocado consumption per capita has increased 833% in the last threedecades from 0.8 lb in 1979 to 7.5 lb in 2017, with 2.45 billion pounds of avocados consumed in 2018. Consumers have incorporated avocados into their diets in both raw and processed forms (i.e., raw fruit or cooking oil). Avocado oil also can be found in cosmetics and other personal care products. The rapid increase in the popularity of avocado as a food item has been mainly driven by its association with health and wellness. Avocado pulp is an excellent source of macro and micronutrients, and rich in bioactive lipids. According to the U.S. Nutrition Labeling and Education Act (NLEA) a serving size of avocado (50 g) provides: 80 kcal, 7 g of total lipids (comprised of approximately 67% of mono- and 12% of polyunsaturated fatty acids), 3.4 g of total dietary fiber, and only 0.3 g of sugar. A number of studies have demonstrated that consumption of avocado may improve lipid profile, decrease risk of metabolic syndrome, and aid in weight management, mitigate age-related oxidative DNA damage, and have anti-carcinogenic effects. Although most of the scientific literature has focused on nutraceutical effects of avocado based on its lipid and fatty acid compositionand high antioxidant capacity, recent interest has been devoted to a fatty acid derivative, called persin. The latter is synthesized in idioblast oil cells present in the avocado fruit and leaves with suggested insecticidal and fungicidal activity. Persin is classified as an acetogenin; derived from the biosynthesis of long-chain fatty acids and with similar structure of linoleic acid. In vitro, persin has shown cytotoxic and proapoptotic effects in human breast cancer cell lines. In contrast with the human literature, consumption of avocado by small ruminant (goats and sheep) and monogastric animal species (avians, reptiles, dogs, and cats) is discouraged, due to acute signs of toxicity (i.e., labored breathing, decreased appetite, lethargy, congestion in the lungs, cardiomyopathy, and nephrosis) and eventual death. The toxic effects of avocado have been attributed to the presence of persin, even though this compound has not been quantified in any of those studies. A recent research project in Dr. Godoy's laboratory (PI) in collaboration with Dr. David Sarlah, Assistant Professor of Chemistry have quantified persin in different parts of the raw and cooked avocado fruit (e.g., peel, pulp, and pit). We learned that raw avocado has greater concentrations of persin, and that the peel and pulp are the most concentrated sources of this acetogenin. We are, therefore, interested in evaluating if consumption of avocado oil (extracted from the pulp) could result in unattended health implications due to inherent ingestion of persin. We are proposing to determine whether acute and chronic adult exposure to persin would result in cytotoxic effects in male and female mice, leading to increased oxidative stress and inflammation, and negatively impacting their health and reproductive status. We also plan to evaluate alterations in blood and gastrointestinal metabolic profiling and gut microbiome due to acute and chronic adult exposure to persin using the mouse model.
Animal Health Component
Research Effort Categories
Goals / Objectives
The overall goal of this research is to evaluate the role of persin, a phytochemical in avocado, as a functional or toxic compound using a dose-response approach. The specific aims of this proposal are: 1. To synthesized labeled-persin and determine its metabolism in vivo, 2. To examine the cytostatic and cytotoxic effects of persin in reproductive organs, mammary glands, liver and gastrointestinal tissues, 3. To determine systemic metabolic and gut microbial alterations due to acute and chronic exposure to persin, 4. To evaluate whether metabolic and physiological responses differ when mice are orally dosed with synthetic persin or with equivalent dose of persin using avocado oil. Our central hypothesis is that adult exposure to persin will stimulate oxidative and inflammatory processes in reproductive and gastrointestinal tissues, and will decrease microbial diversity and concentration of short-chain fatty acids, used as proxy of gut health. We also hypothesize that the presence of natural antioxidants, mono- and poly-unsaturated fatty acids present in avocado oil will potential attenuate the cytotoxic effects of persin, or potentially alter its physiological effects in vivo.
Labeled persin synthesis and analysis: High Performance LC/MS will be used to quantify (+)-persin content. Chromatographic separation will be done on a Kinetex® EVO C-18 column (1.7 µm) with a binary gradient from 20% to 90% MeCN in water over twominutes, then 90% to 95% over twominutes at 0.4 mL/minute. Chemical detection will be done with selected ion monitoring (SIM) with ESI ionization on Shimadzu LCMS-2020 instrument. Signal intensities correspond to integrated ion count traces. A five-point linear calibration curve will be built at concentrations of 10, 25, 50, 75 and 100 uM. Experimental design: All experimental protocols will be reviewed and approved by the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign prior to their initiation. CD-1 female and male mice will be used in a complete randomized design and orally dosed with either vehicle (tocopherol- stripped corn oil, 0% persin), avocado oil or labeled-persin at 10 (low) or 100 (high) mg of persin/kg/day for a total of either 10 days or 30 days to reflect acute vs. chronic exposure to this toxin. All mice will be fed ad libitum a chow diet containing approximately 20% protein, 70% carbohydrate, 10% fat, and 3,85 kcal/g. After 10 or 30 days of receiving the assigned treatments, mice will be euthanized and ovaries, uteri, mammary gland, testes, liver, segments, and content of gastrointestinal tract and blood samples will be collected. All tissues collected will be processed for histological evaluation, whereas ovaries, uteri, mammary gland, and testes will also be evaluated for proliferation and inflammatory markers using immunohistochemistry. Digesta from distal small intestine, cecum, and distal colon will be collected from each mice. DNA extraction, amplification of bacterial, archaeal, and fungal DNA will be performed using targeted primers and a Fluidigm Access Array and sequenced using MiSeq Illumina with v3 reagents. Sequence data will be analyzed using QIIME 2.0. Targeted short-chain and branched-chain fatty acid concentrations will be done using gas chromatography. Serum samples from mice in the same treatment will be pared and analyzed for untargeted serum metabolic profiling at the Metabolomics Center at University of Illinois. Multivariate supervised and unsupervised methods will be used to uncover simultaneous changes of metabolites across experimental factors. Among the supervised strategies that use the experimental conditions as input, k-nearest neighbor, discriminant analysis, and partial least squares methods will be investigated.