Location: Food Animal Metabolism Research
2024 Annual Report
Objectives
Objective 1: Determine the absorption, distribution, metabolism, and excretion of emerging and legacy chemicals in food animals.
Sub-objective 1.A: Determine the metabolism and disposition of [14C]-nitrofurazone in broiler chickens.
Sub-objective 1.B: Determine the ADME of [14C]-PBDEs 47, 99, and 153 in laying turkeys.
Sub-objective 1.C: Determine the ADME of 1,3,7,8-tetrabromo [14C]-dibenzo-p-dioxin in laying hens.
Sub-objective 1.D: Determine the ADME of a defined mix of PFAS, including perfluorohexane sulfonic acid (PFHxS) in lactating cattle.
Sub-objective 1.E: Determine the fate of PFAS originating in a contaminated water source during the life cycle of laying hens.
Sub-objective 1.F: Determine the ADME of [14C]-(-)-trans-'9-tetrahydrocannabinol (THC) and/or [14C]-cannabidiol (CBD) in lactating dairy goats.
Sub-objective 1.G: Determine the accumulation and depuration kinetics of THC and CBD in feedlot cattle supplemented with dietary hemp.
Sub-objective 1.H: Evaluation of cellular uptake, translocation, and toxicity of microplastics using cell models.
Sub-objective 1.I: Determination of the fate of microplastics in laying hens.
Sub-objective 1.J: Determination of the uptake and depuration of microplastics in lactating dairy goats.
Objective 2: Develop and validate sensitive and accurate rapid analytical tools to detect emerging and legacy residues in food animals and food animal systems.
Sub-objective 2.A: Develop ambient ionization mass spectrometric detection and quantitation techniques of chemicals in matrices easily collected from live animals (blood, hair, urine, saliva).
Sub-objective 2.B: Develop ambient ionization mass spectrometric detection and quantitation techniques of chemicals in postmortem matrices (blood, tissues).
Objective 3: Determine levels and sources of emerging and legacy chemical or biological residues in the domestic food supply.
Sub-objective 3.A: In cooperation with regulatory agencies, determine the levels of dioxins, furans, and PBDEs in the U.S. meat supply.
Sub-objective 3.B: Determine the source(s) contributing to high background levels of PBDEs in commercial turkey.
Approach
Consumers loathe the idea of chemical residues in milk, meat, and eggs even though quantifiable risk of harm from chemicals in U.S. livestock products is exceedingly low. Regardless, consumers equate trace-levels of chemical residues in food with poor product quality and safety. Consequently, producers, regulatory officials, industry representatives, and consumers agree that chemical residues in food should be minimized to the greatest extent possible.
We propose to conduct absorption, distribution, metabolism, and excretion (ADME) studies on legacy and emerging chemicals for which significant data gaps exist. These chemicals include hemp-derived cannabinoids, a legacy antibiotic (nitrofurazone), halogenated persistent pollutants, and environmentally relevant microplastic contaminants (Objective 1). Basic ADME studies will allow the science-based selection of target matrices (saliva, urine, milk, liver, kidney, fat, etc.) and ‘marker compounds’ (parent compound or metabolites) of critical importance to the development of practical rapid screening technologies (Objective 2). ADME studies also provide data from which pre-harvest residue accumulation rates and post-exposure depuration rates can be calculated. Such data will facilitate the marketing of essentially residue-free animals in instances of known animal exposures. In some cases, especially for highly potent halogenated hydrocarbons and emerging contaminants, the U.S. government has a vested interest in ensuring that residues in remain well below regulatory thresholds. Under Objective 3, we propose a continuation of a 25-year cooperative effort with the USDA Food Safety and Inspection Service (FSIS) to survey the U.S. meat supply for dioxins and dioxin-like chemical residues. This survey has been critical to the discovery of environmental sources of dioxins and has been critical to reducing food animal exposures. We also propose to continue discovery efforts to elucidate contamination sources of livestock-based foods.
Collectively, the goal of this proposal is to develop science-based solutions that minimize consumer exposures to chemical residues in food animal products.
Progress Report
Research efforts relating to Objective 1, “Determine the absorption, distribution, metabolism, and excretion of emerging and legacy chemicals in food animals”. The analytical phase of a cooperative study on the fate of 1- and 2-monobutyrins in broiler chickens was completed. Heavy isotope labeled standards of monocaprylin and monocaprin were synthesized for use as analytical internal standards. Synthetic (unlabeled) monocaprylic- and monocarpic glyceride esters were used in a live phase study with broiler chickens to determine whether they are absorbed intact. The analytical phase of that experiment is in progress.
Additional quantities of heavy-isotope-labeled nitrofurazone internal standard and heavy-isotope cyano metabolite standards were synthesized and shared with federal cooperators. The distribution phase of a [14C]-nitrofurazone metabolism study in broiler chickens was completed. Extracellular and intracellular distributions [14C]-residue were measured as were extractable and bound residues. Efforts to determine the identity(ies) of protein- and/or nucleic acid bound metabolites have not yet been successful and the identification of a unique nitrofurazone marker residue remains elusive.
A study designed to investigate the effects of a perfluoro alkyl substances (PFAS)-specific sorbent included in the diets of broilers on residues of PFAS was completed. The analytical phase was initiated in the 1st quarter of FY2024 and continues. Initial plasma results indicate partial success in reducing perfluoro sulfonic acid residues in broilers, but perfluoro carboxylic acids were little impacted by inclusion of the sorbent. An additional study in broilers to determine the effects of naturally produced feed components on PFAS distribution is in progress. The analytical phase is expected to begin in the 4th quarter of FY2024 and will continue into FY2025.
The analytical phase of a study investigating rates of perfluoroalkyl substances (PFAS; 13 perfluorinated carboxylic acids and 12 perfluorinated sulfonates) accumulation in broiler chickens continued. Whereas PFAS in plasma and liver have been quantified; skeletal muscle, gizzard, and skin have been processed and are being extracted for PFAS analyses. Likewise, the analytical phase of a 9-month laying hen study, started in FY2022, continues. Laying hens (n = 72), exposed to PFAS containing water, were harvested during growth, maturation, and during the egg-laying period. A depuration phase was included in the laying hen study so that PFAS depletion in tissues and eggs could be measured. Analysis of tissues from both studies, and eggs from the laying hen study, is in progress.
A study was performed to evaluate the biological effects of poly(methyl methacrylate) (PMMA) micro/nanoplastics in liver cells. PMMA was less toxic than the polystyrene micro/nanoplastics that were previously studied. PMMA micro/nanoplastics were not cytotoxic for concentration as high as 100 µg/mL with exposure time up to 24 hours. However, PMMA micro/nanoplastics were able to translocate into liver cells, produce an indicator inflammatory mediator, and release an enzyme that associated with programmed cell death.
A study was initiated to evaluate cytotoxicity produced by microplastics and PFAS separately and combined. The effects of PFAS chain length and functional group are being investigated as are microplastic particle size and surface function. Interactions of PFAS and particles in cell model systems are being investigated.
Synthetic methods for [14C]-incorporation into polyethylene terephthalate (PET) were developed and methods for making micro/nanosized PET particles have been developed and validated. Animal protocols to investigate [14C]-PET in laying hens and lactating ruminants have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
Objective 2 is focused on the development of analytical methods capable of rapidly and sensitively measuring chemical analytes in food-animal matrices. A cooperative study with the U.S. Food and Drug Administration and USDA’s Food Safety and Inspection Service was initiated to validate a multi-residue method applicable to the measurement of 32 perfluoroalkyl substances including precursors, carboxylates, and sulfonates in matrices including feed, egg, milk, liver, muscle and blood plasma. The method utilizes a one-step extraction and internal standardization with limits of quantitation goals being approximately 50 ng/kg (parts per trillion) in each matrix. Cross-laboratory validation of the method is currently in progress.
In cooperative studies, an analytical method was developed to accurately measure inverse sugars occurring in sugar beet pulp. The formation of inverse sugars is a measure of beet quality and is correlated to beet infection by pathogens. The method was used to measure invert sugars in studies conducted by cooperators.
An analytical method was developed to determine the mycotoxins patulin and ochratoxin A in numerous fungal isolates from sugar beets collected by collaborators. Patulin has not previously been reported to be produced by fungal pathogens isolated from sugar beets.
The goal of Objective 3 is to determine the levels of dioxins, furans, and polybrominated diphenyl ethers (PBDEs) in the U.S. meat supply. Collection of pork fat, beef (fat and liver), and siluriformes samples (~400 total), under the auspices of an Interagency Agreement with the USDA Food Safety and Inspection Service, began in Q1 2024 and will continue through Q1 2025. Analytical work quantifying dioxins, furans, and PBDEs in the collected samples began in Q1 2024 and will continue through Q2 2025. In addition, the validation of the dioxin/furan analytical method on a new gas chromatograph – tandem mass spectrometer (GC-MS/MS) was completed after replacing an aging magnetic sector GC-MS instrument. The new instrument provides decreased detection limits (improved sensitivity), ease of use, and lower maintenance costs.
Accomplishments
1. Microplastic elimination in milk of lactating sheep. Microplastics (MP) are environmental contaminants that have been frequently measured in feed and/or water used for human consumption. Because microplastics have been hypothesized to cause adverse effects in humans, there is interest in the extent of their transfer from feed to animal products which may be used as human food. Unfortunately, only limited information is available on the fate of MP in domestic food animals and their transfer to edible food products. ASR scientists in Fargo, North Dakota, employed radiolabeled polystyrene microparticles to determine the extent of microplastic absorption and routes of elimination in lactating sheep. Low, but measurable quantities, of radiotracer were present in blood, milk, and urine throughout the 72-hour study period indicating that some of the microparticles were absorbed. However, most of them were excreted in feces. The study provides compelling evidence that while polystyrene microparticles are poorly absorbed by ruminants, trace quantities are eliminated in milk.
2. Perfluoroalkyl substance (PFAS) depletion in dairy cattle. Poly-and perfluorinated substances (PFAS) are becoming a concern to consumers in the United States and other countries due to negative health effects that have been associated with their presence in food and water. In some states, dairy animals exposed to feed and water contaminated with PFAS have themselves become contaminated and milk from the cows could not be marketed, at great expense to farmers who produced the cattle. ARS scientists in Fargo, North Dakota, characterized the rates of PFAS depletion from milk and edible tissues of cattle sourced from a contaminated farm. Although microscopic evaluation did not reveal tissue abnormalities in exposed animals, sulfate containing PFAS residues remained in milk and most tissues for the duration of the 22-week study. In contrast, carboxylate containing PFAS residues did not accumulate in the cattle. Data generated from the study will aid regulatory agencies in determining the relative risks of consuming milk or meat from cattle inadvertently exposed to PFAS in feed and/or water.
Review Publications
Shelver, W.L., McGarvey, A.M., Holthusen, J.E., Young, J.M., Byrd, C.J., Smith, D.J. 2024. Comparison of immunoassay and LC-tandem mass spectrometry analyses of ractopamine in hog oral fluid. Food additives & contaminants. Part A: Chemistry, analysis, control, exposure & risk assessment. 41:162-174. https://doi.org/10.1080/19440049.2023.2300738.
Singh, A., Smith, D.J. 2023. Facile synthesis of 14C-nitrofurazone from 14C-urea. Journal of Labelled Compounds and Radiopharmaceuticals. https://doi.org/10.1002/jlcr.4068.
Singh, A., Smith, D.J., Strahan, G.D., Lehotay, S.J. 2023. Synthesis and spectroscopic characterization of 13C4-labelled 4-cyano-2-oxobutyraldehyde semicarbazone: A metabolite of nitrofurazone. Journal of Labelled Compounds and Radiopharmaceuticals. 67:18-24. https://doi.org/10.1002/jlcr.4077.
Lupton, S.J., Ochoa, C., Domesle, A., Duverna, R. 2024. Dietary exposure levels to polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and non-ortho-polychlorinated biphenyls in U.S. meat, poultry, and siluriform fish from 2018-2019. Food Additives & Contaminants. Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment. 41:303-312. https://doi.org/10.1080/19440049.2024.2306924.
Shelver, W.L., McGarvey, A.M., Billey, L.O., Banerjee, A. 2023. Fate and disposition of [14C]-polystyrene microplastic after oral administration to laying hens. Science of the Total Environment. 909. Article 168512. https://doi.org/10.1016/j.scitotenv.2023.168512.
Johnston, J.J., Ebel, E., Williams, M., Esteban, E., Lupton, S.J., Scholljegerdes, E., Ivey, S., Powell, M., Smith, D.J. 2023. A blood-based ante-mortem method for estimating PFOS in beef from contaminated dairy cattle. ACS Agricultural Science and Technology. https://doi.org/10.1021/acsagscitech.3c00102?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as.