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<p>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.</p>
<p>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.</p>