NOVEL METHODS FOR MITIGATION OF DIETARY RISK FACTORS FOR DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1005916
• Project No.: TEX0-1-6215
• Project Start Date: Mar 16, 2015
• Project End Date: Jan 28, 2020

Project Director
Phillips, T.

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Veterinary Integrative Biosciences

Non Technical Summary
Polycyclic Aromatic Hydrocarbons (PAHs) are unavoidable environmental pollutants produced from the incomplete combustion of organic materials. People are frequently exposed to PAHs through the food they eat, especially foods that have been grilled, barbecued or smoked. A variety of PAHs, which normally exist in complex mixtures, have been recognized as carcinogenic and capable of eliciting negative health effects in humans. The association between high PAH exposure and the risk of cancer has been established in various epidemiological studies. PAHs are also harmful to the human immune system and can significantly interfere with development of a fetus and/or child resulting in brain defects. Furthermore, early life exposures to PAHs are thought to increase incidence of liver cancer later in life. While a portion of PAH exposures result from inhalation of airborne contaminants, dietary exposure has also been shown to play a significant role in the formation of DNA damage, which could lead to cancer. Our recent survey in a US city with high incidence of liver cancer in San Antonio, TX indicated that approximately half of study participants had detectable levels of a PAH biomarker in their urine, suggesting considerable exposure to one or more PAHs. In addition, our laboratory reported increased levels of a urinary aflatoxin (AF) biomarker in the same community, which correlated with consumption of corn tortillas, rice, and nuts. AFs are naturally occurring toxins produced by Aspergillus fungi that frequently contaminate commodities such as maize, peanuts and tree nuts and exposure to the most prevalent and potent type of aflatoxin, AFB1, can cause liver toxicity, suppression of the immune system, and decreased growth rate in animals and humans. Chronic exposure to AFs is a major risk factor for the development of liver cancer. The aflatoxin problem is especially pronounced in traditionally underserved communities and high risk areas due to the frequent consumption of moldy foods or disproportionate consumption of foods containing aflatoxin-contaminated ingredients. In fact, a study recently completed by our laboratory indicated that approximately 85% of the Texas study population exhibited detectable aflatoxin B1-albumin adducts, an exposure biomarker found in the blood. This is increased from 21% in 2009 and likely a result of recent droughts (unpublished data). Thus, strategies that reduce multiple exposures to PAHs and AFs are highly desirable for populations at risk for both agents. The PAH, benzo[a]pyrene (BaP), is similar to AFB1 in that both AFB1 and BaP are toxic to the immune system and are strong carcinogens, leading to the development of tumors. Among various strategies that have been successfully used to reduce human exposures to mycotoxins, the binding of AFs in the gastrointestinal tract by a specific type of clay has been shown to be a safe and viable approach. This conclusion is based on previous studies by our laboratry using multiple animal models and clinical intervention trials in humans in the US and Ghana where it was shown that NovaSil (NS) clay, administered by capsule or in food, significantly reduced exposure to AFs. Although no successful clinical interventions have been reported for PAHs using similar clay enterosorbent strategies, certain food ingredients, including powdered young barley leaves, rice bran fiber, lignin and other plant-based components such as quercitin and chlorogenic acid may bind to a variety of PAHs, thereby reducing their absorption in the gastrointestinal tract. The findings from this research will be of direct relevance to vulnerable populations where consumption of a diet contaminated with PAHs and AF occurs frequently.

Animal Health Component

25%

Research Effort Categories

Basic

0%

Applied

100%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	5230	1150	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5230 - Feed and feed additives;

Field Of Science
1150 - Toxicology;

Keywords

aflatoxin

pah

maize

smoked foods

enterosorbents

clay

dietary risk factor mitigation

Goals / Objectives
In the proposed project, our aims are based on the central hypothesis that intervention with a mixture of sorbents will decrease dietary exposures to both PAHs and AF. Our aims focus on characterizing and selecting the optimal IDF and clay mixtures that will reduce exposure to PAHs and AF based on in vitro, Cnidarian, and mammalian models. We postulate that reduced PAH and AF exposure from the diet of animals will mitigate environmental risk factors for disease. In addition, we hypothesize that dried blood spots can be used as a non-invasive method to quantitate PAH and AF biomarkers, eliminating the need for cold shipment and storage of urine or blood. These hypotheses will be addressed by determining optimal sorbents for PAHs and aflatoxins in vitro and in vivo, confirming the ability of sorbent mixtures to modulate toxicity of Benzo[a]pyrene and Aflatoxin B1 in a long-term rodent study, and detecting and quantifying PAHs and AF in dried bloodspots.

Project Methods
Specific Aim 1: Determination of optimal sorbents for PAHs and aflatoxins in vitro and in vivo. Isothermal Analyses. Equilibrium adsorption data for test sorbents will be fit to multiple isotherm equations (i.e., Langmuir, Freundlich, Langmuir-Freundlich combinations, Toth, and various transforms of these equations) . These data will be used to verify the sorption and selectivity of ligands (PAHs and AF) onto the surfaces of IDF (i.e. powdered young barley leaves, psyllium husk, cellulose, chitosan) and clay-based materials. Endpoints will include: i) type and number of potential adsorption sites, ii) affinity constants (Kd), iii) capacity constants (Qmax), iv) adsorption energy distribution (rH (kJ/mol)), v) enthalpy of adsorption (rG (kJ/mol)), and vi) heterogeneity coefficients (n). Also, an in vitro model for sorbents, previously developed in our laboratory, will be used to assess the binding of BaP and AFB1 (singly and in combination) under simulated conditions of the GI tract.Hydra Bioassay. Results from isothermal analysis indicating sorbent efficacy will be verified in a sensitive freshwater Cnidarian model. Combinations of IDF, clay, and IDF + clay at concentrations of ≤2% will be incubated with 10 μg/mL BaP and/or 10 μg/mL AFB1. These test solutions will be shaken for 2 hr, then centrifuged (2000 rpm) for 20 min prior to incubation (18° C) for 20 min. Efficacy will be evaluated at seven time points from 0-92 hr. The average toxicity rating will be determined by calculating the average morphological score for a specific toxin concentration and sorbent at each time point.Bioavailability of PAHs & AF in Rats. Male Sprague-Dawley rats (240-250 g) will be purchased and maintained on AIN-76A powdered feed [50] and water ad libitum at the Laboratory Animal Research Facility, Texas A&M University. Experimental diets containing combinations of the IDF and mineral-based material selected from the initial in vitro studies at concentrations of 2.0%, 0.5% and 0.25% (w/w) will be prepared. PAHs and AFB1 will be added to diet. Treatment groups (5 rats/group) will be randomly selected to be dosed with PAHs, AFB1 (150 µg/kg) or both toxins with or without sorbents in the diet. Urine will be collected at selected intervals during the study. Body weights and feed intake will also be monitored throughout the study. 1-OHP and Aflatoxin M1 (AFM1) levels in urine will be determined to confirm differences in total PAH and AFB1 bioavailability between treatment groups.Specific Aim 2: Confirm the ability of sorbent mixtures to modulate toxicity of Benzo[a]pyrene and Aflatoxin B1 in a long-term rodent study. Diet Preparation/Treatment Model. Experimental diets will be prepared as described in Aim 1, except the concentrations of BaP and AFB1 will be 2 mg/kg and 150 µg/kg, respectively. The control diet will consist of AIN-76 alone. In addition, diets containing the IDF + clay which exhibited the greatest binding affinity from Aim 1 will be combined at concentrations of 0.5% and 0.25% (w/w) and will be prepared from each of the respective spiked and control feeds. The treatment regimen will consist of the following: 1) Absolute control diet; 2) BaP; 3) AFB1; 4) BaP and AFB1; 5) BaP and AFB1 with 0.5% IDF + clay in the diet; 6) BaP and AFB1 with 0.25% IDF + clay in the diet. The experimental period will be 180 days. Bloodspots will also be collected from each treatment group on days 0 and 180 for analysis in Aim 3.Biomarkers of PAH and AF Exposure (with and without IDF + clay). The ability of an IDF + clay mixture (2 doses) to reduce the bioavailability of AF to the blood will be assessed using the serum AFB1-lysine (AFB1-lys) adduct exposure biomarker. AFB1-lys will be measured by HPLC-fluorescence detection. The results of AFB1-lys concentration will be adjusted by serum albumin level and will be expressed as the amount of AFB1-lys/mg albumin.Urinary 1-OHP levels will be measured using an HPLC-fluorescence method. Briefly, urine samples (4.0 mL) will be adjusted to pH 5.0 with an equal volume of 1.0 M acetate buffer (pH 5.0). β-Glucuronidase (50 μl) possessing sulfatase activity will be added into the sample and incubated for 4 h at 37 °C. The hydrolyzed samples will be purified through primed Sep-Pak C18 columns. 1-OHP will be eluted from the column with 100% MeOH; the eluates will be evaporated to dryness under N2 and reconstituted in MeOH. The analyses will be conducted using HPLC with fluorescence detection. Excitation and emission parameters will be set at 240 and 388 nm, respectively. A 125×4.6 mm Spherisorb ODS2 HPLC column with a particle size of 3 μm will be used with a mobile phase comprised of 75% MeOH in water. Chromatographic separation will be achieved by isocratic elution at a flow rate of 1.1 mL/min for 15 min. The 1-OHP peak will be detected at a retention time of approximately 6.2 min, and the limit of detection is approximately 0.25 nmol/L of urine. 1-OHP levels will be adjusted by urinary creatinine concentration.Hematology, Serum Biochemistry, Vitamin/Mineral Analysis and Analysis of Immune Parameters. Serum biochemical parameters will be monitored throughout the study. Vitamins and other micronutrients will be assessed at the beginning and conclusion of the study, as described previously. In addition to hematological parameters, rat spleen lymphocyte phenotypes of CD4+, CD8+ T cells and CD3-CD8+ NK cells and intracellular cytokine secretion functions of IL-4, IFN-γ, and TNF-α will be measured by flow cytometric analyses as described previously.Organ Toxicology and Microscopic Identification of FAH by Histopathology and Immunohistochemistry. At the conclusion of the study, final body weights of rats will be determined followed by CO2 asphyxiation. Major organs will be fixed in neutral buffered formalin (pH 7.4) for routine processing and staining with hematoxylin and eosin (H&E). Severity of microscopic changes will be graded. Sections will be further evaluated for preneoplastic and tumorigenic alterations and necrosis for major organs, including liver, lung, stomach, esophagus, kidney, intestines, and testis/ovary.Foci of altered hepatocytes (FAH) will be assessed via H&E stained liver sections and confirmed by immunohistochemistry. FAH detected will be classified according to their different cell types . Dewaxed sections of liver will be stained using a standard three stage indirect strepavidin-biotin technique; peroxidase activity will be developed using the chromagen substrate diamino-benzidine and GST-P+ staining will be used to identify initiated hepatocytes. The number and size of the 'enzyme altered' foci will be quantified microscopically (×10 magnification) and categorized according to the number of cells per focus or 'minifocus' and expressed as GST-P+ lesions per cm2 of liver section.Specific Aim 3: Detect and quantify PAHs and AF in dried bloodspots.Sample Preparation and Extract Analysis. To investigate the feasibility of monitoring PAH and AF exposures using bloodspot analysis, samples taken from animals during Aim 2 will be extracted from bloodspot cards and analyzed via HPLC-MS/MS. Blood spot discs will be extracted overnight. Extraction of samples will include the addition of methanol and water, vortexing, and centrifugation to collect supernatant, which will be evaporated to dryness under a gentle stream of N2 gas. The samples will then be reconstituted and subjected to HPLC-MS/MS analysis. A Waters Acquity Ultra High Performance LC coupled to a dual quadrupole MS with electrospray ionization capability will be utilized to detect and quantify PAHs, as well as several major AF congeners, in sample extracts.

Progress 03/16/15 to 01/28/20

Outputs
Target Audience:1) Preprofessional undergraduate students in the Biomedical Sciences Program at Texas A&M University and graduate students in Toxicology, Engineering, Food Science and Nutrition, and Animal Science at Texas A&M University. 2) Environmental toxicologists, Materials and soil scientists, computational chemists, public health professionals, food and feed industry, USDA, USAID, NIH, NIEHS, FDA, and CDC. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has facilitated the training of graduate studentsfrom the Toxicology Program at Texas A&M University. How have the results been disseminated to communities of interest?Yes. Twenty six (26)papershave been published in appropriate scientific journals from 2016through2020. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In earlier work,calcium montmorillonite clay (NovaSil)in the diet of animals wasshown to tightly bind aflatoxin B1 (AfB1) and reduce the symptoms of aflatoxicosis. To investigate and improve the capacity and efficacy of clay-based materials as aflatoxin sorbents, we have developed and tested calcium and sodium montmorillonite clays and a variety of novel, clay-based materials including claysamended with nutrients, plant biopolymers, chlorophylland other natural products.Also, we have determined the sorption of AfB1 by isothermal analysis and tested the ability of thesesorbent materialsto protect a living organism (adult hydra) againstAfB1 toxicity. The results have shownthat exchanging montmorillonite clays with carnitine and choline inhibited swelling of the clays, enhanced their organophilic properties,and increased the sorption capacity and efficacy of clay surfaces for AfB1 and other hazardous toxicants.Results from dehydroxylated and heat-collapsed clays suggested that AfB1 was primarily adsorbed in the clay interlayer, as predicted from thermodynamic calculations and computational modeling. The hydra bioassay further indicated thatmodified clays could significantly protect adult hydra from AfB1 and other chemicals with inclusion rates as low as0.005%. Thus,enterosorbent therapy for aflatoxins may also be applicable tohazardous chemicals such as pesticides and PAHs based on their similar sorption mechanisms and the use of broad-acting sorbents.Importantly,enterosorbent therapy using edible claysfor aflatoxins andenvironmental chemical pollutantscanbe delivered in nutritional supplements, foods that are vulnerable to contamination, andflavored liquids and animal feeds during disasters, emergencies and outbreaks where the threat of exposure is high.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang M, Bera G, Mitra K, Wade TL, Knap AH, Phillips TD. 2020. Tight sorption of arsenic, cadmium, mercury, and lead by edible activated carbon and acid-processed montmorillonite clay. Environ. Sci. Pollut. Res. 33009611.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang M and Phillips TD. 2020. Edible clay inclusion in the diet of oysters can reduce tissue residues of polychlorinated biphenyls. J. Toxicol. Environ. Health Sci. https://doi.org/10.1007/s13530-020-00058-2.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Orr AA, He S, Wang M, Goodall A, Hearon SE, Phillips TD, Tamamis P. 2020. Insights into the interactions of bisphenol and phthalate compounds with unamended and carnitine-amended montmorillonite clays. Comput. Chem. Eng.; 143, 107063
• Type: Journal Articles Status: Published Year Published: 2020 Citation: 222. Wang M and Phillips TD. 2020. Edible clay inclusion in the diet of oysters can reduce tissue residues of polychlorinated biphenyls. J. Toxicol. Environ. Health Sci. (Impact Factor: .8).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: 223. Orr AA, He S, Wang M, Goodall A, Hearon SE, Phillips TD, Tamamis P. 2020. Insights into the interactions of bisphenol and phthalate compounds with unamended and carnitine-amended montmorillonite clays. Comput. Chem. Eng.; 143, 107063 (Impact Factor: 4.0).

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientists and academicians interested in sorbent materials. Scientists and academicians interested in the chemical and physical characterization of these materials when they are used to bind environmental toxins in drinking water and contaminated food. Scientists and academicians interested in methods to mitigate toxin exposures following disasters such as hurricanes, floods, chemical spills, fires and acts of terror. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two doctoral graduate students. How have the results been disseminated to communities of interest?Yes, through scientific publications and presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue our work on the development of broad-acting sorbents for environmental chemicals.

Impacts
What was accomplished under these goals? In our ongoingstudies we have developed novel, broad-actingsorbent materialsthat tightly bind diverse PAHs, pesticides, metals, plasticizers, commercial solvents and mycotoxins. These sorbents can be added to food and water to reduce unintentional exposuresfrom environmental chemicals following disasters. These new materialsmay facilitate "enterosorbent" therapy for first responders and vulnerable humans and animals living near the site of disasters.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, M., Safe, S.H., Hearon, S.E., Phillips, T.D. (2019). Strong adsorption of polychlorinated biphenyls by processed montmorillonite clays: potential applications as toxin enterosorbents during disasters and floods. Environmental Pollution. 255(1), 113210, PMID 31542671.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, M., Orr, A.A., He, S., Dalaijamts, C., Chiu, W.A., Tamamis, P., Phillips, T.D. (2019). Montmorillonite clays can tightly sorb glyphosate and paraquat onto interlamellar surfaces and protect against exposure and toxicity. ACS Omega. 4(18), 17702-17713, PMID 31681876
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, M., Hearon, S.E., Johnson, N.M., Phillips, T.D. (2019). Development of broad-acting clays for the tight adsorption of benzo[a]pyrene and aldicarb. Applied Clay Science, 168, 196-202
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, M., Hearon, S.E., Phillips, T.D. (2019). Development of enterosorbents that can be added to food and water to reduce toxin exposures during disasters. Journal of Environmental Science and Health, Part B, 54(6), 514-524, PMID 31014207
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hearon, S.E., Wang, M., Phillips, T.D. (2019). Strong adsorption of dieldrin by parent and processed montmorillonite clays. Environmental Toxicology and Chemistry. Nov. 22 doi:10.1002/etc.4642, PMID 31756776
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, M., Hearon, S.E., Phillips, T.D. (2019). A high capacity bentonite clay for the sorption of aflatoxins. Food Addit Contam Part A. Dec. 2:1-10. PMID: 31790632.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Scientists and academicians interested in sorbent materials that will decrease exposures to aflatoxins and environmentalchemicals in vulnerable humans and animals during emergencies and disasters such as droughts and floods. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have presented these findings at various scientific meetings, including the National Meeting of the Society of Toxicology,NIEHS Superfund meetings in North Carolina and Sacramento, California, and local student training forums and symposiumsat Texas A&M University and the University of Texas in Austin. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To investigate and improve the capacity and efficacy of clay-based materials as aflatoxin and environmental chemicalsorbents, we developed and tested calcium and sodium montmorillonite clays amended with nutrients including L-carnitineand choline. Also, we determined the sorption of AfB1 by isothermal analysis and tested the ability of these amendedsorbents to protect adult hydra from AfB1 toxicity. The results showed that exchanging montmorillonite clays with L-carnitineand choline inhibited swelling of the clays and increased the sorption capacity and efficacy of clay surfaces for AfB1. Resultsfrom dehydroxylated and heat-collapsed clays suggested that AfB1 was primarily adsorbed in the clay interlayer, as predictedfrom thermodynamic calculations and computational modeling. The hydra bioassay further indicated that the modi?ed clayscan signi?cantly protect adult hydra from AfB1 with as low as 0.005% clay inclusion. This enterosorbent therapy may also beapplied to screen hazardous chemicals such as pesticides and PAHs based on similar sorption mechanisms. Taken together,enterosorbent therapy could be delivered in nutritional supplements, foods that are vulnerable to aflatoxin contamination,flavored liquids and animal feeds during emergencies and outbreaks of acute aflatoxicosis, and as a screening model forhazardous environmental chemicals.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Awuor AO, Yard E, Daniel JH, Martin C, Bii C, Romoser A, Oyugi E, Elmore S, Amwayi S, Vulve J, Zitomer NC, RybakME, Phillips TD, Montgomery JM, Lewis L.Evaluation of the efficacy, acceptability and palatability of calcium montmorillonite clay used to reduce aflatoxin B1 dietaryexposure in a crossover study in Kenya. Food Add. Contam. 34:93-102.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Onel M, Beykal B, Wang M, Grim, FA, Zhou L, Wright FA, Phillips TD, Rusyn I, Pistikopoulos EN. 2018. Optimal chemicalgrouping and sorbent material design by data analysis, modeling, and dimensionality reduction techniques. ESCAPE, 43:421-426.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Xue KS, Qian G, Lin S, Su J, Tang L, Gelderblom WCA, Riley RT, Phillips TD, Wang JS. 2018. Modulation of pre-neoplastic biomarkers induced by sequential aflatoxin B1 and fumonisin B1 exposure in F344 rats treated with UPSN clay.Food Chem. Toxicol. 114: 316-324.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang M, Hearon SE, Johnson NM, Phillips TD. 2018. Development of broad-acting clays for the tight adsorption ofbenzo[a]pyrene and aldicarb. Appl. Clay Sci. 168: 196-202.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Scientists and academicians interested in sorbent materials that will decrease exposures to aflatoxins and environmentalchemicals in vulnerable humans and animals during emergencies and disasters such as droughts and floods. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have presented these findings at various scientific meetings, including the National Meeting of the Society of Toxicology,NIEHS Superfund meetings in North Carolina and Sacramento, California, and local student training forums and symposiumsat Texas A&M University and the University of Texas in Austin. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To investigate and improve the capacity and efficacy of clay-based materials as aflatoxin and environmental chemicalsorbents, we developed and tested calcium and sodium montmorillonite clays amended with nutrients including L-carnitineand choline. Also, we determined the sorption of AfB1 by isothermal analysis and tested the ability of these amendedsorbents to protect adult hydra from AfB1 toxicity. The results showed that exchanging montmorillonite clays with L-carnitineand choline inhibited swelling of the clays and increased the sorption capacity and efficacy of clay surfaces for AfB1. Resultsfrom dehydroxylated and heat-collapsed clays suggested that AfB1 was primarily adsorbed in the clay interlayer, as predictedfrom thermodynamic calculations and computational modeling. The hydra bioassay further indicated that the modi?ed clayscan signi?cantly protect adult hydra from AfB1 with as low as 0.005% clay inclusion. This enterosorbent therapy may also beapplied to screen hazardous chemicals such as pesticides and PAHs based on similar sorption mechanisms. Taken together,enterosorbent therapy could be delivered in nutritional supplements, foods that are vulnerable to aflatoxin contamination,flavored liquids and animal feeds during emergencies and outbreaks of acute aflatoxicosis, and as a screening model forhazardous environmental chemicals.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Maki C, Allen S, Wang M, Ward SH, Rude BJ, Bailey RB, Phillips TD. 2017. Calcium montmorillonite clay for the reductionof aflatoxin residues in milk and dairy products. Dairy and Vet Sci J. 2:1-8.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cui H, Gu X, Chen J, Xie Y, Ke S, Wu J, Golovko A, Morpurgo B, Yan C, Phillips TD, Xie W, Luo J, Zhou Z, Tian Y. 2017.Pregnane X receptor regulates the AhR/CypA1 pathway and protects liver cells from benzo[a]pyrene-induced DNAdamage. Toxicol. Lett. 275:67-76.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang M, Maki CR, Deng Y, Tian Y, Phillips TD. 2017. Development of high capacity enterosorbents for aflatoxin B1 andother hazardous chemicals. Chem. Res. Toxicol. 30(9): 1694-1701.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists and academicians interested in sorbent materials that will decrease exposures to aflatoxins and environmental chemicals in vulnerable humans and animals during emergencies and disasters such as droughts and floods. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have presented these findings at various scientific meetings, including the National Meeting of the Society of Toxicology, NIEHS Superfund meetings in North Carolina and Sacramento, California, and local student training forums and symposiums at Texas A&M University and the University of Texas in Austin. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To investigate and improve the capacity and efficacy of clay-based materials as aflatoxin and environmental chemical sorbents, we developed and tested calcium and sodium montmorillonite clays amended with nutrients including L-carnitine and choline. Also, we determined the sorption of AfB1 by isothermal analysis and tested the ability of these amended sorbents to protect adult hydra from AfB1 toxicity. The results showed that exchanging montmorillonite clays with L-carnitine and choline inhibited swelling of the clays and increased the sorption capacity and efficacy of clay surfaces for AfB1. Results from dehydroxylated and heat-collapsed clays suggested that AfB1 was primarily adsorbed in the clay interlayer, as predicted from thermodynamic calculations and computational modeling. The hydra bioassay further indicated that the modi?ed clays can signi?cantly protect adult hydra from AfB1 with as low as 0.005% clay inclusion. This enterosorbent therapy may also be applied to screen hazardous chemicals such as pesticides and PAHs based on similar sorption mechanisms. Taken together, enterosorbent therapy could be delivered in nutritional supplements, foods that are vulnerable to aflatoxin contamination, flavored liquids and animal feeds during emergencies and outbreaks of acute aflatoxicosis, and as a screening model for hazardous environmental chemicals.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Awuor AO, Yard E, Daniel JH, Martin C, Bii C, Romoser A, Oyugi E, Elmore S, Amwayi S, Vulve J, Zitomer NC, Rybak ME, Phillips TD, Montgomery JM, Lewis L. Evaluation of the efficacy, acceptability and palatability of calcium montmorillonite clay used to reduce aflatoxin B1 dietary exposure in a crossover study in Kenya. Food Add. Contam. 34:93-102. Maki C, Allen S, Wang M, Ward SH, Rude BJ, Bailey RB, Phillips TD. 2017. Calcium montmorillonite clay for the reduction of aflatoxin residues in milk and dairy products. Dairy and Vet Sci J. 2:1-8. Cui H, Gu X, Chen J, Xie Y, Ke S, Wu J, Golovko A, Morpurgo B, Yan C, Phillips TD, Xie W, Luo J, Zhou Z, Tian Y. 2017. Pregnane X receptor regulates the AhR/CypA1 pathway and protects liver cells from benzo[a]pyrene-induced DNA damage. Toxicol. Lett. 275:67-76. Onel M, Beykal B, Wang M, Grim, FA, Zhou L, Wright FA, Phillips TD, Rusyn I, Pistikopoulos EN. 2018. Optimal chemical grouping and sorbent material design by data analysis, modeling, and dimensionality reduction techniques. ESCAPE, 43: 421-426. Xue KS, Qian G, Lin S, Su J, Tang L, Gelderblom WCA, Riley RT, Phillips TD, Wang JS. 2018. Modulation of pre-neoplastic biomarkers induced by sequential aflatoxin B1 and fumonisin B1 exposure in F344 rats treated with UPSN clay. Food Chem. Toxicol. 114: 316-324. Wang M, Maki CR, Deng Y, Tian Y, Phillips TD. 2017. Development of high capacity enterosorbents for aflatoxin B1 and other hazardous chemicals. Chem. Res. Toxicol. 30(9): 1694-1701. Wang M, Hearon SE, Johnson NM, Phillips TD. 2018. Development of broad-acting clays for the tight adsorption of benzo[a]pyrene and aldicarb. Appl. Clay Sci. 168: 196-202.

Progress 03/16/15 to 09/30/15

Outputs
Target Audience:Target audience: Preprofessionalundergraduate students in the Biomedical Sciences Program at Texas A&M University and graduate students in Toxicology, Engineering, and Food Science and Nutrition at Texas A&M University. Efforts: Capstone course (VIBS Food Toxicology and Safety) is designed to educate students in the basic tenets of food safety and research to promote an awareness of the interdisciplinary contributions of toxicology, chemistry, microbiology and public health. An understanding of emerging issues in food safety and environmental and dietary risk factors for diseases are encouraged. Critical thinking and problem solving are emphasized in classroom instruction and student presentations. Other target audiences: Environmental toxicologists,Dairy scientists, Materialsand soil scientists, computational chemists,public health professionals, food and feed industry, USDA, USAID, FDA, CDC, NIH. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This multi-state projectprovided forthe training of graduate students from the Toxicology Program at Texas A&M University and the Dairy Science Programs at Tarleton State University, the University of Georgia, and Mississippi State University. How have the results been disseminated to communities of interest?Yes. Two papers have been published in appropriate scientific journals in 2016, with a third in preparation for 2017. What do you plan to do during the next reporting period to accomplish the goals?Publish our clay intervention study in Kenya, and continue our research with mycotoxins and environmental chemicals.

Impacts
What was accomplished under these goals? Aflatoxin (AF) is a hazardous chemical produced by Aspergillus flavus and A. parasiticus fungi. It has been shown to be toxic and carcinogenic in humans and animals. Importantly, the young of all species are the most susceptible to AF. When contaminated crops are fed to lactating animals, AF is metabolized to hydroxylated derivatives such as aflatoxin M1 (AFM1). These AF metabolites are excreted into the milk. A practical and inexpensive strategy to reduce AF exposures from milk is the inclusion of high affinity AF enterosorbents such as Novasil Plus, or NSP, in the diets of dairy animals. In dairy cows, NSP has been reported to sequester and tightly bind AF in the gastrointestinal tract, reducing the carryover of AFM1 into milk. To determine if NSP can mitigate AF without interfering with milk composition and quality, independent (multistate) studies were designed using 15 lactating dairy cows randomly assigned to one of 5 treatment groups in 5 X 5 Latin square experimental designs. The cows were treated with diets containing AF at 0 or approximately 100 μg/kg and three different levels of NSP (0.0, 0.5 and 1%) of the diet. Milk samples were collected and analyzed for AFM1, milk composition, minerals, vitamin A, and riboflavin. In both studies, AFM1 concentrations in milk were significantly reduced by the 0.5% and 1% NSP treatment levels. The low dose of NSP (0.5%) resulted in a decrease in the level of AFM1 in the milk ranging from: 47-55%; whereas, the high dose (1%) reduced the level of AFM1 in the milk by 68-70.9%. Milk composition, minerals, vitamin A, and riboflavin concentrations were unaffected by any of the dietary treatments. Additionally, dry matter intake (DMI) and milk yield were not different among treatments. Results from this study demonstrated that NSP clay effectively reduced the excretion of AFM1 in milk from lactating cows fed diets containing 117-121 μg/kg AF without interfering with DMI, milk production, milk composition and milk quality.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Pollock B, Elmore S, Romoser A, Tang L, Kang M, Xue K, Rodriquez M, Dierschke N, et al. Intervention trial with calcium montmorillonite clay in a south Texas population exposed to aflatoxin. Food Additives & Contaminants. 2016;33:1346-54.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Afum C, Cudjoe L, Hills J, Hunt R, Padilla L, Elmore S, Afriyie A, Opare-Sem O, et al. Association between aflatoxin M1 and liver disease in HBV/HCB infected persons in Ghana. International Journal of Environmental Research in Public Health. 2016;29:377.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Maki C, Monteiro A, Elmore S, Tao S, Bernard J, Harvey R, Romoser A, Phillips T. Calcium montmorillonite clay in dairy feed reduces aflatoxin concentrations in milk without interfering with milk quality, compsition or yield. Animal Feed Science and Technology. 2016;214:130-135.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Qian G, Tang L, Lin S, Xue KS, Mitchell NJ, Su J, et al. Sequential dietary exposure to aflatoxin B1 and fumonisin B1 in F344 rats increases liver preneoplastic changes indicative of a synergistic interaction. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association. 2016;95:188-95.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Maki CR, Thomas AD, Elmore SE, Romoser AA, Harvey RB, Ramirez-Ramirez HA, et al. Effects of calcium montmorillonite clay and aflatoxin exposure on dry matter intake, milk production, and milk composition. Journal of dairy science. 2016;99(2):1039-46.