Progress 03/21/01 to 02/07/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Persistent organic pollutants such as polychlorinated dioxins, furans, and biphenyls are ubiquitous in the environment and enter the food chain as animals are exposed through their surroundings and feed. These toxic contaminants are concentrated in animal products containing fats (i.e. meat, dairy, and eggs) and are ultimately consumed by humans. The health risk to humans of these low-level contaminants is not adequately known, but dioxins and related compounds have been shown to have numerous adverse health effects in animals. One of the most potent dioxins (2,3,7, 8-tetrachlorodibenzo-p-dioxin or TCDD) has been classified as a known human carcinogen by the World Health Organization (WHO) and U.S. National Toxicology Program (NTP). WHO and the European Union (EU) have set limits on
the acceptable levels of these contaminants in food products. Recent recalls of chickens in the U.S. and Belgium with higher than acceptable levels demonstrates the potential for exposures. Our research efforts are directed at reducing exposure to dioxins and dioxin-like compounds from the food supply by 1) developing rapid, lower cost methods to analyze food products for these chemicals, 2) identifying and reducing potential sources of animal exposure to these toxic chemicals, 3) exploring methods which may prevent or lower the accumulation of these chemicals in animal fats, and 4) determining background levels currently found in domestic animal products. Less expensive methods of analysis will allow widespread monitoring for dioxins in foods minimizing the risks associated with episodic contaminations. Investigation into potential sources of dioxins, such as pentachlorophenol-treated wood or agricultural burning practices, will help determine the contribution of these sources to
animal contamination and whether changes in certain farming practices may be needed. Remediation methods for animals already exposed or methods which decrease uptake could help to decrease background levels in animals or toxic levels after a serious contamination has occurred. More data on the current levels of these contaminants in livestock will provide regulators with a better idea of the safety of the U.S. food supply and its acceptability under the new EU regulations. The research relates to Food Safety, National Program 108 (100%). 2. List by year the currently approved milestones (indicators of research progress) Objective 1 (Rapid assays). Year 1 (2001): Design and synthesize new antigens for dioxin immunochemical methods and obtain a commercial partner for a promising method (DD3 IAC). Evaluate commercial ELISA assays for dioxin analysis of fat samples. Year 2 (2002): Isolate and characterize new antibodies for dioxin analytical methods. Develop an immunoaffinity
chromatography (IAC) application for milk samples. Improve fat cleanup for ELISA. Year 3 (2003): Prepare and test new antibodies for dioxin IACs & ELISAs. Develop IAC application for fat. Year 4 (2004): Optimize IAC and ELISA conditions for rapid analysis of dioxins in food and patent any promising antibodies. Year 5 (2005): Automate and commercialize best IACs and ELISAs. Objective 2 (Identify sources). Year 1 (2001): Conduct PCP-wood combustion studies. Conduct absorption, disposition, metabolism, and excretion (ADME) studies of a hydroxy-dioxin metabolite and selected polybrominated diphenyl ethers (PBDEs). Year 2 (2002): Conduct ADME studies of a predioxin and an HxCDD. Year 3 (2003): Develop methods to analyze for predioxins in environment. Conduct ADME studies on selected organo- phosphate flame retardants. Year 4 (2004): Conduct ADME and half-life studies of HxCDD in beef animals. Year 5 (2005): Conduct ADME study on a brominated naphthalene in rats. Objective 3
(Remediation strategies). Year 1 (2001): Complete clenbuterol remediation study in rats. Year 2 (2002): Characterize a dioxin-binding biliary protein and enzyme systems involved in dioxin metabolism. Year 3 (2003): Conduct ractopamine remediation study in chickens. Conduct ADME studies in knock-out mice. Year 4 (2004): Investigate effect of enzyme induction and dioxin body burdens in whole animals. Year 5 (2005): Conduct clenbuterol prophylaxis study in rats. Conduct thyroid disruption toxicology study in rats. Objective 4 (Current levels in food). Year 1 (2001): Establish sample collection protocol for food surveys. Year 2 (2002): Develop analyses for co-planar PCBs and PBDEs in fat and food samples. Year 3 (2003): Collect and begin analysis of food samples. Year 4 (2004): Finish analyses. Year 5 (2005): Compile survey results into database and publish results. 4a List the single most significant research accomplishment during FY 2006. Debromination and persistence of the
flame retardant Deca-BDE. Program 108, Food Safety, Component 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships. Deca-BDE is an additive flame retardant produced in high volume and incorporated into numerous consumer goods ranging from upholsteries to electronics. Recently other polybrominated diphenyl ether formulations (BDE-types of flame retardants) have been withdrawn from production due to persistence and toxicity issues. The persistence and toxicity of Deca-BDE is currently being re-evaluated. In a six-week feeding study in rats, we investigated the fate and persistence of the major component of Deca-BDE, namely BDE-209. Results of the study showed that BDE-209 is minimally bioavailable but is more persistent than previously reported and is debrominated in rats to more highly persistent lower brominated compounds. These data will be of interest to regulatory agencies and the scientific community evaluating the safety of Deca-BDE 5. Describe the major
accomplishments to date and their predicted or actual impact. The research reported for this CRIS is conducted under National Program 108, Food Safety, and directly contributes to the Chemical Residue Component. Research activities are further related to ARS Strategic Plan Goal 3, Enhance protection and safety of the Nations agriculture and food supply; Objective 3.1, Provide science-based knowledge on the safe production, storage, processing, and handling of plant and animal products and on the detection and control of toxin-producing and/or pathogenic bacteria and fungi parasites, mycotoxins, chemical residues, and plant toxins so as to assist regulatory agencies and the food industry in reducing the incidence of foodborne illnesses. Development of an immunoassay for polybrominated diphenyl ethers. Polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants that can accumulate through the food chain, but rapid inexpensive methods of detection are not
available. Antibodies were generated and incorporated into a rapid screening assay (magnetic particle immunoassay) with the collaboration of Abraxis, LLC. The assay is successful in detecting PBDEs in human milk, chicken meat, fish, water, and soil samples with high specificity and sensitivity. The cost and turn-around time for this new assay is significantly less than that of the traditional PBDE analysis; and, therefore, its use may result in affordable, widespread, monitoring of these persistent pollutants. (ARS performance measures 3.1.1.1 and 3.1.2.1) Fate and transport of dioxins in soils. Dioxins are persistent pollutants principally formed by combustion and incineration processes and deposited on the ground; however, little is known about their potential to migrate through the soil into ground water. The fate and transport of two particular dioxins through soil was studied in various soil columns. The dioxins adhered to the soils based on the organic content and surface
area of the soil, but 15-42% of the dioxins were transported through the soil columns with water infiltration. These results suggest that dioxins deposited on the surface may reach ground water reservoirs by preferential flow or colloidal transport. (ARS performance measure 3.1.1.3). Surveys of dioxins and other environmental contaminants in U.S. meat. Five hundred ten meat samples (beef, market hogs, young chickens, and young turkeys) were collected from slaughter houses across the U.S. and analyzed for dioxins. Polybrominated diphenyl ethers (PBDEs) and dioxins were also analyzed in a smaller market basket study that consisted of bacon, chicken, ground beef, sirloin steak, and pork chops collected from grocery stores across the U.S. Dioxins in domestic meat products were low and at similar levels as found in Europe and Japan. Dioxins in chicken, turkey, and pork appeared to have declined in the past ten years while beef did not show as much change. In this first report on
PBDEs in U.S. meats, PBDEs in chicken averaged ten-times higher in the U.S. than other countries. Knowledge of current contaminant levels in foods are important to track trends of these contaminants in the food supply, to provide more accurate estimates of human dietary intakes for risk assessment purposes, and to demonstrate the quality of U.S. foods, especially for export purposes. Individual food data can also be valuable to industry and regulatory agencies in tracking down sources of contamination. EPA scientists plan to use this data in their reassessment of dioxin. (ARS performance measures 3.1.2.1 and 3.1.2.7) Transfer of dioxins from feed into cows milk. In 2002 a dioxin- contaminated mineral supplement was discovered which had been used by poultry, pork, and dairy producers in the U.S. A study was conducted to determine how much of the dioxin contamination may have been incorporated into food products. Dairy cows fed a dioxin-contaminated mineral supplement excreted
30% of the consumed dioxins into their milk; however, milk from Minnesota dairies that had purchased the contaminated supplement did not show elevated levels of dioxins. It was concluded that although dioxins from the mineral supplement were readily transferred into cows milk, the level of dioxins in the finished feeds used on MN farms was low enough to not cause a significant increase in the dioxin levels in milk, thus leading to no elevated risk to the consumer. Studies on the transfer of dioxins from feed or feed ingredients into animal products, such as milk, provide important information (e.g. bioconcentration factors and withdrawal rates) to regulatory agencies when assessing the potential risk of a contamination incident. (ARS performance measures 3.1.1.3 and 3.1.1.4). Pharmacokinetic studies of polybrominated diphenyl ethers. The bioavailability and bioaccumulation of PBDEs were determined by feeding studies in rats using two commercial mixtures: one contained tetra- to
hexa-BDEs and one contained hexa- to octa-BDEs. Purification methods and GC/MS detection methods were developed to analyze the PBDEs in excreta and tissues. Results of the studies showed that all the congeners were bioavailable to varying extents (15-60%) and were supported by other studies conducted in rats using single compounds (BDEs-99, -100, -153, and 209). All studies showed that the potential for these contaminants to accumulate through the food supply decreased with increasing bromine content, supporting the decision by industry to cease production of the penta- and octa-formulations while retaining only the decaBDE product. BDEs-47, 99, 100, and 153 were identified as potential markers for PBDE contamination in the food supply. (ARS performance measures 3.1.1.1 and 3. 1.1.3) The role of Cytochrome P450-1A2 in dioxin excretion. Toxic dioxin congeners are extremely persistent in animal systems and methods to increase clearance from the body are needed. The roles of
Cytochrome P450-1A2 (Cyp 1A2) and metabolism in the sequestration and clearance of dioxins were investigated using knockout mice (designed to possess no Cyp 1A2). A comparison of the metabolism and excretion of two toxic dioxins (TCDD and PeCDD) in regular and knockout mice showed that even in the absence of Cyp 1A2, a dioxin-sequestering protein, no increase in metabolism or excretion occurred. The inherent resistance of dioxins to metabolic change and, thereby, excretion means that other approaches to decrease body burdens may need to be explored. (ARS performance measures 3.1.1.3 and 3.1.1.4) Remediation of dioxins with ractopamine. In a remediation study, rats lost 30% of their body fat when fed clenbuterol (a leanness-enhancing agent) in their diet compared to controls; the amount of dioxin in the fat stores after a dioxin exposure also decreased by 30% when clenbuterol was fed. These results are encouraging and suggest similar dioxin body burden decreases could be achieved
in animals raised for food provided approved leanness-enhancing agents are available. Lowering dioxin levels in the food supply is strongly recommended by the NAS Committee on the Implications of Dioxins in the Food Supply. (ARS performance measures 3.1. 1.3 and 3.1.1.4) 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A Trust Fund Cooperative Agreement has been established between the ARS and a commercial vendor for Development of a Magnetic Particle Based Immunoassay for triclosan (No. 58-3K95-5-1106; (Oct 2005). The vendor has commercialized the triclosan ELISA. A Material Transfer Agreement (MTA) was established with a commercial vendor to evaluate ractopamine monoclonal antibodies. (November 2005)
Impacts
(N/A)
Publications
- Larsen, G.L., Zhaosheng, F., Casey, F., Hakk, H. 2004. Sorption, mobility and fate of 1,4,7,8-tetrachlorodibenzo-p-dioxin in soils. Organohalogen Compounds. 66:2292-2296.
- Hu, X., Shelver, W.L., Wettstein, G.W., Gholami, K., Shelver, W.H. 2004. Studies of intramolecular hydrogen bonding in protonated and non- protonated hexahydrophyridinobenzodioxins. Journal of Molecular Structure (Theochem)686:7-14.
- Shelver, W.L., Keum, Y., Kim, H., Rutherford, D., Hakk, H., Bergman, A., Li, Q.X. 2005. Hapten syntheses and antibody generation for the development of a polybrominated flame retardant ELISA. [abstract] 229th American Chemical Society (ACS) National Meeting, San Diego, CA, 3/13- 17/2005, Picogram No. 68, Abstract No. 210.
- Shelver, W.L., Keum, Y., Kim, H., Rutherford, D., Hakk, H., Bergman, A., Li, Q.X. 2005. Hapten syntheses and antibody generation for the development of a polybrominated flame retardant elisa. Journal of Agricultural and Food Chemistry 53(10):3840-3847.
- Hakk, H., Larsen, G.L., Bowers, J. 2004. Metabolism, tissue disposition, and excretion, of 1,2-bis(2,4,6-tribromophenoxy)ethane (btbpe) in male sprague-dawley rats. Chemosphere 54:1367-1374.
- Huwe, J.K., Hoffman, M.K., Deyrup, C., Hulebak, K., Larsen, G.L., Zaylskie, R., Lorentzsen, M.K., Clinch, N. 2004. A survey of pcdd/fs and co-planar pcbs in the us meat and poultry supply in 2002-2003. Organohalogen Compounds 66:1942-1946.
- Larsen, G.L., Huwe, J.K., Low, M., Rutherford, D., Hakk, H. 2004. Binding of 2,2',4,4',6-pentabromodiphenyl ether (bde-100) and/or its metabolites to mammalian biliary carrier proteins. Organohalogen Compounds 66:3830- 3835.
- Huwe, J.K., Smith, D.J. 2005. Laboratory and on-farm studies on the bioavailability of dioxins from a contaminated mineral supplement fed to dairy cows. Journal of Agriculture and Food Chemistry 53:2362-2370.
- Huwe, J.K., Larsen, G.L. 2005. Polychlorinated dioxins, furans, and biphenyls, and polybrominated diphenyl ethers in a U.S. meat market basket and estimates of dietary intake. Environmental Science and Technology 39(15):5606-5611.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Persistent organic pollutants such as polychlorinated dioxins, furans, and biphenyls are ubiquitous in the environment and enter the food chain as animals are exposed through their surroundings and feed. These toxic contaminants are concentrated in animal products containing fats (i.e. meat, dairy, and eggs) and are ultimately consumed by humans. The health risk to humans of these low-level contaminants is not adequately known, but dioxins and related compounds have been shown to have numerous adverse health effects in animals. One of the most potent dioxins (2,3,7, 8-tetrachlorodibenzo-p-dioxin or TCDD) has been classified as a known human carcinogen by the World Health Organization (WHO) and U.S. National Toxicology Program (NTP). WHO and the European Union (EU) have set limits on the acceptable
levels of these contaminants in food products. Recent recalls of chickens in the U.S. and Belgium with higher than acceptable levels demonstrates the potential for exposures. Our research efforts are directed at reducing exposure to dioxins and dioxin-like compounds from the food supply by 1) developing rapid, lower cost methods to analyze food products for these chemicals, 2) identifying and reducing potential sources of animal exposure to these toxic chemicals, 3) exploring methods which may prevent or lower the accumulation of these chemicals in animal fats, and 4) determining background levels currently found in domestic animal products. Less expensive methods of analysis will allow widespread monitoring for dioxins in foods minimizing the risks associated with episodic contaminations. Investigation into potential sources of dioxins, such as pentachlorophenol-treated wood or agricultural burning practices, will help determine the contribution of these sources to animal
contamination and whether changes in certain farming practices may be needed. Remediation methods for animals already exposed or methods which decrease uptake could help to decrease background levels in animals or toxic levels after a serious contamination has occurred. More data on the current levels of these contaminants in livestock will provide regulators with a better idea of the safety of the U.S. food supply and its acceptability under the new EU regulations. The research relates to Food Safety, National Program 108 (100%). 2. List the milestones (indicators of progress) from your Project Plan. Objective 1 (Rapid assays). Year 1 (2001): Design and synthesize new antigens for dioxin immunochemical methods and obtain a commercial partner for a promising method (DD3 IAC). Evaluate commercial ELISA assays for dioxin analysis of fat samples. Year 2 (2002): Isolate and characterize new antibodies for dioxin analytical methods. Develop an immunoaffinity chromatography (IAC)
application for milk samples. Improve fat cleanup for ELISA. Year 3 (2003): Prepare and test new antibodies for dioxin IACs & ELISAs. Develop IAC application for fat. Year 4 (2004): Optimize IAC and ELISA conditions for rapid analysis of dioxins in food and patent any promising antibodies. Year 5 (2005): Automate and commercialize best IACs and ELISAs. Objective 2 (Identify sources). Year 1 (2001): Conduct PCP-wood combustion studies. Conduct absorption, disposition, metabolism, and excretion (ADME) studies of a hydroxy-dioxin metabolite and selected polybrominated diphenyl ethers (PBDEs). Year 2 (2002): Conduct ADME studies of a predioxin and an HxCDD. Year 3 (2003): Develop methods to analyze for predioxins in environment. Conduct ADME studies on selected organo- phosphate flame retardants. Year 4 (2004): Conduct ADME and half-life studies of HxCDD in beef animals. Year 5 (2005): Conduct ADME study on a brominated naphthalene in rats. Objective 3 (Remediation strategies).
Year 1 (2001): Complete clenbuterol remediation study in rats. Year 2 (2002): Characterize a dioxin-binding biliary protein and enzyme systems involved in dioxin metabolism. Year 3 (2003): Conduct ractopamine remediation study in chickens. Conduct ADME studies in knock-out mice. Year 4 (2004): Investigate effect of enzyme induction and dioxin body burdens in whole animals. Year 5 (2005): Conduct clenbuterol prophylaxis study in rats. Conduct thyroid disruption toxicology study in rats. Objective 4 (Current levels in food). Year 1 (2001): Establish sample collection protocol for food surveys. Year 2 (2002): Develop analyses for co-planar PCBs and PBDEs in fat and food samples. Year 3 (2003): Collect and begin analysis of food samples. Year 4 (2004): Finish analyses. Year 5 (2005): Compile survey results into database and publish results. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially
met, or not met. If not met, why. 1. Automate and commercialize best IACs and ELISAs. Milestone Substantially Met 2. Conduct ADME study on a brominated naphthalene in rats. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 3. Conduct clenbuterol prophylaxis study in rats. Conduct thyroid disruption toxicology study in rats. Milestone Not Met Other 4. Compile survey results into database and publish results. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This CRIS project will expire 3/20/2006. A new project has been written and is under review by an OSQR panel of experts. The new project is scheduled to be implemented 3/21/2006. FY 2006. Evaluate Cytochrome P450s as specific binding proteins for dioxin-like compounds for potential use in rapid assays (objective 1). Develop cleanup
methods for dioxins in soils for future evaluation of the impact of agricultural burning on dioxin levels in the environment (objective 2). Conduct absorption, disposition, metabolism, and excretion (ADME) studies of BDE-47, an environmental contaminant, in chickens to evaluate its fate and persistence in a food-producing animal (objective 2). Analyze data from a metabonomic study in rat urine to elucidate biological changes occurring after dioxin or PBDE exposure (objective 3). Begin a feeding study in swine to determine the impact of a growth promoter, ractopamine, on the uptake and disposition of dioxins from the feed into the animals (objective 3). 4a What was the single most significant accomplishment this past year? Development of an immunoassay for polybrominated diphenyl ethers. Polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants that can accumulate through the food chain, but rapid inexpensive methods of detection are not available. Antibodies
were generated and incorporated into a rapid screening assay (magnetic particle immunoassay) with the collaboration of Abraxis, LLC. The assay is successful in detecting PBDEs in fish, water, and soil samples with high specificity and sensitivity. The cost and turn-around time for this new assay is significantly less than that of the traditional PBDE analysis; and, therefore, its use may result in affordable, widespread, monitoring of these persistent pollutants. 4b List other significant accomplishments, if any. Assessment of the persistence of two flame retardants in mammals. A particular group of polybrominated diphenyl ethers (a class of flame retardants) are persistent in the environment and have been shown to be accumulating in humans. Two of these compounds (BDE-154 and BDE-209) were studied in laboratory rats to examine the absorption, disposition, metabolism, and excretion characteristics of this group of pollutants in animals. The results of the studies show that BDE-154
is well absorbed and persistent in the animals, but BDE-209 is poorly absorbed and quickly excreted. Because BDE-209 is the predominant member of this flame retardant class (soon to be the only member), this data lends scientific support to the decision to allow continued use of BDE-209 while discontinuing use of products containing BDE-154. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The research reported for this CRIS is conducted under National Program 108, Food Safety, and directly contributes to the Chemical Residue Component. Research activities are further related to ARS Strategic Plan Goal 3, Enhance protection and safety of the Nations agriculture and food supply; Objective 3.1, Provide science-based knowledge on the safe production, storage, processing, and handling of plant and animal products and on the detection and control of toxin-producing and/or pathogenic bacteria and fungi parasites, mycotoxins,
chemical residues, and plant toxins so as to assist regulatory agencies and the food industry in reducing the incidence of foodborne illnesses. In a remediation study, rats lost 30% of their body fat when fed clenbuterol (a leanness-enhancing agent) in their diet compared to controls; the amount of dioxin in the fat stores after a dioxin exposure also decreased by 30% when clenbuterol was fed. These results are encouraging and suggest similar dioxin body burden decreases could be achieved in animals raised for food provided approved leanness-enhancing agents are available. Lowering dioxin levels in the food supply is strongly recommended by the NAS Committee on the Implications of Dioxins in the Food Supply. (First year milestone for objective 3, ARS performance measures 3.1.1.3 and 3.1.1.4) The bioavailability and bioaccumulation of PBDEs were determined by feeding studies in rats using two commercial mixtures: one contained tetra- to hexa-BDEs and one contained hexa- to octa-BDEs.
Purification methods and GC/MS detection methods were developed to analyze the PBDEs in excreta and tissues. Results of the studies showed that all the congeners were bioavailable to varying extents (15-60%) and were supported by other studies conducted in rats using single compounds (pentaBDEs-99 and -100, and decaBDE). All studies showed that the potential for these contaminants to accumulate through the food supply decreases with increasing bromine content, supporting the decision by industry to cease production of the penta- and octa-formulations while retaining only the decaBDE product. BDEs-47, 99, 100, and 153 were identified as potential markers for PBDE contamination in the food supply. (Second year milestone for objective 4, ARS performance measures 3.1.1.1 and 3.1.1.3) Toxic dioxin congeners are extremely persistent in animal systems and methods to increase clearance from the body are needed. The roles of Cytochrome P450-1A2 (Cyp 1A2) and metabolism in the
sequestration and clearance of dioxins were investigated using knockout mice (designed to possess no Cyp 1A2). A comparison of the metabolism and excretion of two toxic dioxins (TCDD and PeCDD) in regular and knockout mice showed that even in the absence of Cyp 1A2, a dioxin-sequestering protein, no increase in metabolism or excretion occurred. The inherent resistance of dioxins to metabolic change and, thereby, excretion means that other approaches to decrease body burdens may need to be explored. (Third year milestone for objective 3, ARS performance measures 3.1.1.3 and 3.1.1.4) Domestic meat samples, including bacon, chicken, ground beef, sirloin steak, and pork chops from grocery stores, were analyzed for dioxins. All the samples had dioxin levels below the regulatory limits set by the European Union for food products. Polybrominated diphenyl ethers (PBDEs) were also analyzed in these meat products and found to be similar to levels reported in Europe and Japan, except for
chicken which averaged ten-times higher in the U.S. This is the first report on PBDEs in U.S. foods. Knowledge of current contaminant levels in foods are important to track trends of these contaminants in the food supply, to provide more accurate estimates of human dietary intakes for risk assessment purposes, and to demonstrate the quality of U.S. foods, especially for export purposes. Individual food data can also be valuable to industry and regulatory agencies in tracking down sources of contamination. (Third year milestone for objective 4, ARS performance measures 3.1.2.1 and 3.1. 2.7) In 2002 a dioxin-contaminated mineral supplement was discovered which had been used by poultry, pork, and dairy producers in the U.S. A study was conducted to determine how much of the dioxin contamination may have been incorporated into food products. Dairy cows fed a dioxin- contaminated mineral supplement excreted 30% of the consumed dioxins into their milk; however, milk from Minnesota
dairies that had purchased the contaminated supplement did not show elevated levels of dioxins. It was concluded that although dioxins from the mineral supplement were readily transferred into cows milk, the level of dioxins in the finished feeds used on MN farms was low enough to not cause a significant increase in the dioxin levels in milk, thus leading to no elevated risk to the consumer. Studies on the transfer of dioxins from feed or feed ingredients into animal products, such as milk, provide important information (e.g. bioconcentration factors and withdrawal rates) to regulatory agencies when assessing the potential risk of a contamination incident. (Additional milestone to investigate sources of dioxins in the food supply, Objective 2, ARS performance measures 3.1.1.3 and 3.1.1.4). Dioxins are persistent pollutants principally formed by combustion and incineration processes and deposited on the ground; however, little is known about their potential to migrate through the
soil into ground water. The fate and transport of two particular dioxins through soil was studied in various soil columns. The dioxins adhered to the soils based on the organic content and surface area of the soil, but 15-42% of the dioxins were transported through the soil columns with water infiltration. These results suggest that dioxins deposited on the surface may reach ground water reservoirs by preferential flow or colloidal transport. (Additional milestone to investigate sources of dioxins in the food supply, Objective 2, ARS performance measure 3.1.1.3). 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Established trust CRADAs with Abraxis LLC: 58-3K95-5-1106 Development of a magnetic particle based immunoassay for triclosan and
58-5442-5-409 Development of a magnetic particle immunoassay and immunoaffinity columns for PBDEs. The magnetic particle immunoassay for PBDEs is commercially available through Abraxis, LLC, and Environmental Assurance Monitoring. The current applications are for fish, water, and soils samples. The magnetic particle immunoassay for triclosan is still in the development stages and would not be available for at least another six months. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Fargo Forum, 9/13/2004, wrote an article about our research into polybrominated diphenyl ethers Local Researchers Studying Metabolism of Fire Retardant. USDA, ARS, News service, 1/26/2005, carried an article about our research into polybrominated diphenyl ethers Flame Retardants Examined in Animal Tissues.
Impacts
(N/A)
Publications
- Huwe, J.K., Larsen, G.L. 2005. Polychlorinated dioxins, furans, and biphenyls, and polybrominated diphenyl ethers in a U.S. meat market basket and estimates of dietary intake. Environmental Science and Technology 39(15):5606-5611.
- Hakk, H., Larsen, G.L., Bowers, J. 2004. Metabolism, tissue disposition, and excretion, of 1,2-bis(2,4,6-tribromophenoxy)ethane (btbpe) in male sprague-dawley rats. Chemosphere 54:1367-1374.
- Huwe, J.K., Hoffman, M.K., Deyrup, C., Hulebak, K., Larsen, G.L., Zaylskie, R., Lorentzsen, M.K., Clinch, N. 2004. A survey of pcdd/fs and co-planar pcbs in the us meat and poultry supply in 2002-2003. Organohalogen Compounds 66:1942-1946.
- Larsen, G.L., Huwe, J.K., Low, M., Rutherford, D., Hakk, H. 2004. Binding of 2,2',4,4',6-pentabromodiphenyl ether (bde-100) and/or its metabolites to mammalian biliary carrier proteins. Organohalogen Compounds 66:3830- 3835.
- Larsen, G.L., Zhaosheng, F., Casey, F., Hakk, H. 2004. Sorption, mobility and fate of 1,4,7,8-tetrachlorodibenzo-p-dioxin in soils. Organohalogen Compounds. 66:2292-2296.
- Hu, X., Shelver, W.L., Wettstein, G.W., Gholami, K., Shelver, W.H. 2004. Studies of intramolecular hydrogen bonding in protonated and non- protonated hexahydrophyridinobenzodioxins. Journal of Molecular Structure (Theochem)686:7-14.
- Shelver, W.L., Keum, Y., Kim, H., Rutherford, D., Hakk, H., Bergman, A., Li, Q.X. 2005. Hapten syntheses and antibody generation for the development of a polybrominated flame retardant ELISA. [abstract] 229th American Chemical Society (ACS) National Meeting, San Diego, CA, 3/13- 17/2005, Picogram No. 68, Abstract No. 210.
- Shelver, W.L., Keum, Y., Kim, H., Rutherford, D., Hakk, H., Bergman, A., Li, Q.X. 2005. Hapten syntheses and antibody generation for the development of a polybrominated flame retardant elisa. Journal of Agricultural and Food Chemistry 53(10):3840-3847.
- Huwe, J.K., Smith, D.J. 2005. Laboratory and on-farm studies on the bioavailability of dioxins from a contaminated mineral supplement fed to dairy cows. Journal of Agriculture and Food Chemistry 53:2362-2370.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Persistent organic pollutants such as polychlorinated dioxins, furans, and biphenyls are ubiquitous in the environment and enter the food chain as animals are exposed through their surroundings and feed. These toxic contaminants are concentrated in animal products containing fats (i.e. meat, dairy, and eggs) and are ultimately consumed by humans. The health risk to humans of these low-level contaminants is not adequately known, but dioxins and related compounds have been shown to have numerous adverse health effects in animals. One of the most potent dioxins (2,3,7, 8-tetrachlorodibenzo-p-dioxin or TCDD) has been classified as a known human carcinogen by the World Health Organization (WHO) and U.S. National Toxicology Program (NTP). WHO and the European Union (EU) have set limits on the acceptable
levels of these contaminants in food products. Recent recalls of chickens in the U.S. and Belgium with higher than acceptable levels demonstrates the potential for exposures. Our research efforts are directed at reducing exposure to dioxins and dioxin-like compounds from the food supply by 1) developing rapid, lower cost methods to analyze food products for these chemicals, 2) identifying and reducing potential sources of animal exposure to these toxic chemicals, 3) exploring methods which may prevent or lower the accumulation of these chemicals in animal fats, and 4) determining background levels currently found in domestic animal products. Less expensive methods of analysis will allow widespread monitoring for dioxins in foods minimizing the risks associated with episodic contaminations. Investigation into potential sources of dioxins, such as pentachlorophenol-treated wood or agricultural burning practices, will help determine the contribution of these sources to animal
contamination and whether changes in certain farming practices may be needed. Remediation methods for animals already exposed or methods which decrease uptake could help to decrease background levels in animals or toxic levels after a serious contamination has occurred. More data on the current levels of these contaminants in livestock will provide regulators with a better idea of the safety of the U.S. food supply and its acceptability under the new EU regulations. The research relates to Food Safety, National Program 108 (100%). 2. List the milestones (indicators of progress) from your Project Plan. Objective 1 (Rapid assays). Year 1 (2001): Design and synthesize new antigens for dioxin immunochemical methods and obtain a commercial partner for a promising method (DD3 IAC). Evaluate commercial ELISA assays for dioxin analysis of fat samples. Year 2 (2002): Isolate and characterize new antibodies for dioxin analytical methods. Develop an immunoaffinity chromatography (IAC)
application for milk samples. Improve fat cleanup for ELISA. Year 3 (2003): Prepare and test new antibodies for dioxin IACs & ELISAs. Develop IAC application for fat. Year 4 (2004): Optimize IAC and ELISA conditions for rapid analysis of dioxins in food and patent any promising antibodies. Year 5 (2005): Automate and commercialize best IACs and ELISAs. Objective 2 (Identify sources). Year 1 (2001): Conduct PCP-wood combustion studies. Conduct absorption, disposition, metabolism, and excretion (ADME) studies of a hydroxy-dioxin metabolite and selected polybrominated diphenyl ethers (PBDEs). Year 2 (2002): Conduct ADME studies of a predioxin and an HxCDD. Year 3 (2003): Develop methods to analyze for predioxins in environment. Conduct ADME studies on selected organo- phosphate flame retardants. Year 4 (2004): Conduct ADME and half-life studies of HxCDD in beef animals. Year 5 (2005): Conduct ADME study on a brominated naphthalene in rats. Objective 3 (Remediation strategies).
Year 1 (2001): Complete clenbuterol remediation study in rats. Year 2 (2002): Characterize a dioxin-binding biliary protein and enzyme systems involved in dioxin metabolism. Year 3 (2003): Conduct ractopamine remediation study in chickens. Conduct ADME studies in knock-out mice. Year 4 (2004): Investigate effect of enzyme induction and dioxin body burdens in whole animals. Year 5 (2005): Conduct clenbuterol prophylaxis study in rats. Conduct thyroid disruption toxicology study in rats. Objective 4 (Current levels in food). Year 1 (2001): Establish sample collection protocol for food surveys. Year 2 (2002): Develop analyses for co-planar PCBs and PBDEs in fat and food samples. Year 3 (2003): Collect and begin analysis of food samples. Year 4 (2004): Finish analyses. Year 5 (2005): Compile survey results into database and publish results. 3. Milestones: A. FY 2004 milestones. Objective 1: Optimize IAC and ELISA conditions for rapid analysis of dioxins in food and patent
any promising antibodies. This milestone was not met due to the inability to generate improved antibodies for dioxin assays. The objective has been hampered by difficult chemistries and lack of personnel to commit to the research. Objective 2: Conduct ADME and half-life studies of HxCDD in beef animals. Due to difficulties and delays in the synthesis of radiolabelled-HxCDD for the ADME studies, this milestone has been postponed. Instead, the absorption, disposition, excretion, and half- life of dioxin mixtures (including HxCDDs) were studied in dairy cattle and hogs. Objective 3: Investigate effect of enzyme induction and dioxin body burdens in whole animals. A preliminary experiment was completed in rats that showed pre-dosing with certain non-toxic dioxins can increase the excretion of the most toxic dioxin (2,3,7,8-TCDD). Further studies are planned to characterize enzyme systems involved with excretion. Objective 4: Finish analyses for surveys. This milestone has been
substantially met with the completion of a survey of U.S. poultry and meat for dioxins and PCBs conducted with FSIS and the near completion of a study on the levels of dioxins, PCBs, and PBDEs in meats and poultry collected at major grocery stores throughout the U.S. B. List the milestones that you expect to address over the next 3 years. What do you expect to accomplish, year by year, under each milestone? This CRIS project will expire after 2005 and a new project will be written within the next year. FY 2005. Validate or continue to improve rapid screening assays, such as ELISAs, for environmentally-relevant brominated flame retardants (added milestone, objective 1). Measure the levels of dioxins formed in 'backyard burning' situations or agricultural burns (added milestone, objective 2). Conduct a pilot remediation study with beta-agonists in farm animals (delayed year 3 milestone, objective 3). Complete disposition and withdrawal experiments in hogs using dioxin-contaminated
feed (added milestone, objective 2). Begin ADME studies in laboratory animals on brominated flame retardants, such as hexabromocyclododecane (HBCD), BDE-47 or BDE-209 (these flame retardants are currently the most interesting, year 5 milestones, objective 2). Publish data from food surveys (year 5 milestone, objective 4). Conduct sorption and transport studies of two polychlorinated biphenyls in soil (added milestone, objective 2). FY 2006. Determine the metabolism and disposition of 1,2,3,6,7,8-HxCDD, an important contaminant in pentachlorophenol formulations, in laboratory animals (delay experiments in cattle, year 2 milestone, objective 3). Continue ADME studies of environmentally-relevant brominated flame retardants (HBCD, BDE-47, and BDE-209) (year 5 milestone, objective 2). Quantitate the release of dioxin and mixed chlorinated/brominated diphenyl ethers from the incineration of electronic wastes (added milestone, objective 2). Design experiments and enlist partners to
investigate sources of dioxins in beef cattle feed components (objective 2). Analyze data from a metabonomic study in rats to elucidate biological changes occurring after dioxin exposure (objective 3). Investigate the use of Cytochrome P450s in immunoaffinity columns for dioxin analysis of fatty matrices (objective 1). FY 2007. Conduct ADME studies of dioxins in turkeys or beef cattle (objective 2). Analyze samples of beef cattle feed components collected from various locations to determine major sources of dioxin (objective 2). Continue/finish remediation studies with beta-agonists in farm animals (objective 3). Expand metabonomic studies to other persistent compounds (objective 3). Investigate metabolism pathways of brominated diphenyl ethers using in vitro systems i.e. cells or individual isozymes (objective 3). 4. What were the most significant accomplishments this past year? A. A particular group of polybrominated diphenyl ethers (a class of flame retardants) are
persistent in the environment and have been shown to be accumulating in humans. One of these compounds (BDE-100) was studied in laboratory rats to examine the absorption, disposition, metabolism, and excretion characteristics of this potential group of pollutants in mammals. The results of the study show that BDE-100 is well absorbed, is not metabolized extensively, and is deposited mainly in the skin. Although production of BDE-100 will soon cease voluntarily, environmental reservoirs of this class of compounds may continue to contribute to animal and human body burdens. B. Almost no information exists about the levels of polybrominated diphenyl ethers (a class of persistent flame retardants) in the U.S. food supply; although levels in humans have been shown to be higher in North America than in Europe or Japan and on the rise. In order to assess whether food could be a source of human exposure, meat samples from large grocery stores across the U.S. were analyzed for
polybrominated diphenyl ethers (PBDEs). The meat samples contained PBDEs in highly variable amounts, but most averaged no higher than meats in Europe or Japan. By itself, diet may not explain the higher levels of PBDEs in the U.S. population (added milestone for objective 4). C-D. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Under this CRIS project, the following major goals have been accomplished: The usefulness of a leanness enhancing agent, clenbuterol, as a remediation protocol to lower an animal's dioxin body burden after an exposure was investigated. The preliminary study was conducted in rats and showed body fat and dioxin levels decreased by 30% compared to controls when clenbuterol was fed after a dioxin exposure. These results are encouraging and suggest similar body burden decreases could be achieved in livestock species. (First year milestone for objective 3) Polybrominated diphenyl ethers
(PBDEs), a class of flame retardants and emerging persistent pollutant, were investigated for their occurrence in food products. Purification methods and GC/MS detection methods were developed and applied to the analysis of 13 chickens fat samples. All the samples analyzed contained PBDEs with levels reaching 45 ng/g lipid. These results show the potential for human exposure to these contaminants through the food supply. (Second year milestone for objective 4) Toxic dioxin congeners are extremely persistent in animal systems and methods to increase clearance from the body are needed. The roles of Cytochrome P450-1A2 (Cyp 1A2) and metabolism in the sequestration and clearance of dioxins were investigated at the Biosciences Research Laboratory using knock out mice developed by the U.S. EPA laboratory in Research Triangle Park, NC. A comparison of the metabolism and excretion of two toxic dioxins (TCDD and PeCDD) in regular and knockout mice (no Cyp 1A2) showed that even in the
absence of Cyp 1A2, a dioxin- sequestering protein, no increase in metabolism or excretion occurred. The inherent resistance of dioxins to metabolic change and, thereby, excretion means that other approaches to decrease body burdens may need to be explored. (Third year milestone for objective 3) Updated information is needed on the levels of dioxins in the U.S. food supply in order to determine more accurate exposure estimates and the associated risks. Meat products were collected from large grocery stores by ARS employees across the U.S. and shipped to the Biosciences Research Laboratory where they were analyzed for dioxins. Seventy six samples including bacon, chicken, ground beef, sirloin steak, pork chops, and bison burger were analyzed for dioxins. Except for one bison sample, all the samples tested had dioxin levels below the regulatory limits set by the EU for food products. (Third year milestone for objective 4) In 2002 a dioxin-contaminated mineral supplement was
discovered which had been used by poultry, pork, and dairy producers in the US. In order to determine how much of the dioxin contamination may have been incorporated into food products, a study was conducted at the ARS Biosciences Research Laboratory in Fargo, ND, in which dairy cows were fed a diet containing the contaminated mineral supplement and levels of dioxin were measured in the milk. In addition, milk from farms in Minnesota which had used the contaminated supplement was sampled by the MN Department of Agriculture and analyzed for dioxin levels at the ARS Biosciences Research Laboratory. Cows fed the contaminated diet excreted 30% of the consumed dioxins into their milk; however, milk from MN dairies did not show elevated levels of dioxins. The conclusion of this study is that although dioxins from the mineral supplement were readily transferred into cows= milk, the level of dioxins in the finished feeds used on MN farms must have been low enough to not cause a
significant increase in the dioxin levels in milk thus leading to no elevated risk to the consumer. (Additional milestone to investigate sources of dioxins in the food supply, Objective 2). Dioxins are persistent pollutants principally formed by combustion and incineration processes and deposited on the ground; however, little is known about their potential to migrate through the soil into ground water. The fate and transport of two particular dioxins through various soil columns was studied at the ARS Biosciences Research Laboratory in Fargo, ND. The dioxins adhered to the soils based on the organic content and surface area of the soil, but 15-42% of the dioxins were transported through the soil columns with water infiltration. These results suggest that dioxins deposited on the surface may reach ground water reservoirs by preferential flow or colloidal transport. (Additional milestone to investigate sources of dioxins in the food supply, Objective 2). 6. What science and/or
technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Survey of dioxin levels in beef, pork, and poultry from throughout the U. S. to Food Safety Inspection Service. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Gave presentation, "A survey of PCDD/Fs and co-planar PCBs in the U.S. meat and poultry supply in 2002-2003," to the FSIS/ARS Annual Research Workshop in Shepherdstwon, WV, February 18-20, 2004.
Impacts
(N/A)
Publications
- Huwe, J.K., Clark, G., Chu, A., Garry, V. 2003. Calux and high resolution gc/ms analysis of dioxin-like compounds in chlorophenoxy pesticide formulations. Organohalogen Compounds. 60:227-230.
- Hakk, H., Letcher, R.J. 2003. Metabolism in the toxicokinetics and fate of brominated flame retardants (bfrs)-a review. Environment International. 29:801-828.
- Huwe, J.K., Smith, D.J. 2003. Uptake and excretion of pcdd/fs from a magnesium mineral supplement fed to dairy cows. Organohalogen Compounds. 64:419-422.
- Larsen, G.L., Casey, F., Fan, Z., Hakk, H. 2003. Sorption, mobility and fate of 1,2,7,8-tetrachlorodibenzo-p-dioxin in soils and sand. Organohalogen Compounds. 60:397-400.
- Huwe, J.K., Davison, K., Feil, V.J., Larsen, G.L., Lorentzsen, M.K., Zaylskie, R., Tiernan, T.O. 2004. Levels of polychlorinated dibenzo-p- dioxins and dibenzofurans in cattle raised at agricultural research facilities across the USA and the influence of pentachlorophenol-treated wood. Journal of Food Additives & Contaminants 21(2):182-194.
- Morck, A., Hakk, H., Orn, U., Klasson-Wehler, E. 2003. Decabromodiphenyl ether in the rat: absorption, distribution, metabolism, and excretion. Drug Metabolism and Disposition. 31:900-907.
- Huwe, J.K. 2004. Polybrominated diphenyl ethers in meat samples collected from supermarkets across the US. [abstract] Abstracts of the 3rd International Workshop on Brominated Flame Retardants, June 6-9, 2004, Toronto, ON, Canada. pp. 41-44.
- Hakk, H., Huwe, J.K., Low, M., Rutherford, D., Larsen, G.L. 2004. Tissue disposition, excretion, and metabolism of 2,2'4,4',6-pentabromodiphenyl ether (BDE-100) in male Sprague-Dawley rats [abstract]. Abstracts of the 3rd International Workshop on Brominated Flame Retardants, June 6-9, 2004, Toronto, ON, Canada. pp. 429-432.
- Hakk, H., Diliberto, J. 2003. Comparison of overall metabolism of 1,2,3,7, 8-pecdd in cyp1a2 (-/-) knockout and c57bl/6n parental strains of mice. Organohalogen Compounds. 64:293-296.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Persistent organic pollutants such as polychlorinated dioxins, furans, and biphenyls are ubiquitous in the environment and enter the food chain as animals are exposed through their surroundings and feed. These toxic contaminants are concentrated in animal products containing fats (i.e. meat, dairy, and eggs) and are ultimately consumed by humans. The health risk to humans of these low-level contaminants is not adequately known. Our research efforts are directed at reducing this exposure from the food supply by 1)determining background levels currently found in domestic animal products, 2) identifying and reducing potential sources of animal exposure to these toxic chemicals, 3)exploring methods which may prevent or lower the accumulation of these chemicals in animal fats, and 4) developing rapid, lower cost methods to analyze food products for these chemicals. 2. How serious is the
problem? Why does it matter? Dioxins and related compounds have been shown to have numerous adverse health effects in animals. One of the most potent dioxins (2,3,7,8- tetrachlorodibenzo-p-dioxin or TCDD) has been classified as a known human carcinogen by the World Health Organization (WHO) and U.S. National Toxicology Program (NTP). WHO and the European Union (EU) have set limits on the acceptable levels of these contaminants in food products. Recent recalls of chickens in the U.S. and Belgium with higher than acceptable levels demonstrates the potential for exposures. More data on the current levels of these contaminants in livestock will provide regulators with a better idea of the safety of the U.S. food supply and its acceptability under the new EU regulations. Investigation into potential sources of dioxins, such as pentachlorophenol-treated wood or agricultural burning practices, will help determine the contribution of these sources to animal contamination and whether
changes in certain farming practices may be needed. Remediation methods for animals already exposed or methods which decrease uptake could help to decrease background levels in animals or toxic levels after a serious contamination has occurred. Less expensive methods of analysis will allow widespread monitoring for dioxins in foods minimizing the risks associated with episodic contaminations. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Regarding Food Safety, National Program 108 (100%), our research seeks to reduce residues of dioxins and dioxin-like compounds in the food supply. 4. What were the most significant accomplishments this past year? A. In 2002 a dioxin-contaminated mineral supplement was discovered which had been used by poultry, pork, and dairy producers in the US. In order to determine how much of the dioxin contamination may have been incorporated into food products, studies were conducted at the
ARS Biosciences Research Laboratory in Fargo, ND, in which levels of dioxin were measured in the milk of dairy cows fed a diet containing the contaminated mineral supplement and in milk from farms in Minnesota which had used the contaminated supplement (sampled by the MN Department of Agriculture). Cows fed the contaminated diet excreted 30% of the consumed dioxins into their milk; however, milk from MN dairies did not show elevated levels of dioxins. The conclusion of this study is that although dioxins from the mineral supplement were readily transferred into cows' milk, the level of dioxins in the finished feeds used on MN farms must have been low enough to not cause a significant increase in the dioxin levels in milk thus leading to no elevated risk to the consumer. B. Dioxins are persistent pollutants principally formed by combustion and incineration processes and deposited on the ground; however, little is known about their potential to migrate through the soil into ground
water. The fate and transport of two particular dioxins through various soil columns was studied at the ARS Biosciences Research Laboratory in Fargo, ND in collaboration with North Dakota State University, Soil Science Department. The dioxins adhered to the soils based on the organic content and surface area of the soil, but 15-42% of the dioxins were transported through the soil columns with water infiltration. These results suggest that dioxins deposited on the surface may reach ground water reservoirs by preferential flow or colloidal transport. C-D. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Under this CRIS project, the following major goals have been accomplished: The usefulness of a leanness enhancing agent, clenbuterol, as a remediation protocol to lower an animal's dioxin body burden after an exposure was investigated. The preliminary study was conducted in rats and showed body fat and dioxin levels
decreased by 30% compared to controls when clenbuterol was fed after a dioxin exposure. These results are encouraging and suggest similar body burden decreases could be achieved in livestock species. (First year milestone for objective 3) Polybrominated diphenyl ethers (PBDEs), a class of flame retardants and emerging persistent pollutant, were investigated for their occurrence in food products. Purification methods and GC/MS detection methods were developed and applied to the analysis of 13 chickens fat samples. All the samples analyzed contained PBDEs with levels reaching 45 ng/g lipid. These results show the potential for human exposure to these contaminants through the food supply. (Second year milestone for objective 4) Toxic dioxin congeners are extremely persistent in animal systems and methods to increase clearance from the body are needed. The roles of Cytochrome P450-1A2 (Cyp 1A2) and metabolism in the sequestration and clearance of dioxins were investigated at the
Biosciences Research Laboratory using knock out mice developed by the U.S. EPA laboratory in Research Triangle Park, NC. A comparison of the metabolism and excretion of two toxic dioxins (TCDD and PeCDD) in regular and knockout mice (no Cyp 1A2) showed that even in the absence of Cyp 1A2, a dioxin- sequestering protein, no increase in metabolism or excretion occurred. The inherent resistance of dioxins to metabolic change and, thereby, excretion means that other approaches to decrease body burdens may need to be explored. (Third year milestone for objective 3) Updated information is needed on the levels of dioxins in the U.S. food supply in order to determine more accurate exposure estimates and the associated risks. Meat products were collected from large grocery stores by ARS employees across the U.S. and shipped to the Biosciences Research Laboratory where they were analyzed for dioxins. Seventy six samples including bacon, chicken, ground beef, sirloin steak, pork chops, and
bison burger were analyzed for dioxins. Except for one bison sample, all the samples tested had dioxin levels below the regulatory limits set by the EU for food products. (Third year milestone for objective 4) 6. What do you expect to accomplish, year by year, over the next 3 years? During FY 2004, we plan to continue investigating the use of our immunoaffinity column for milk samples; to develop new antibodies to environmentally-relevant brominated flame retardants which will be incorporated into rapid screening assays; to report survey data on the background levels of dioxins and related compounds in certain meats (chicken, turkey, pork, and beef); to determine the metabolism and disposition of 1,2,3,6,7,8-HxCDD, an important contaminant in pentachlorophenol formulations, in laboratory animals; to extend the investigation of clenbuterol or ractopamine remediation methods to farm animals i.e. hogs; to continue looking at disposition and withdrawal rates of dioxins in farm animals
(poultry and hogs) using a contaminated feed supplement; to study the absorption, disposition, metabolism, excretion, and potential toxicologic response in laboratory animals of certain polybrominated diphenyl ethers which are considered persistent organic pollutants (BDE-100). During FY 2005, we plan to validate or continue to improve rapid screening assays for environmentally-relevant brominated flame retardants; to measure the levels of dioxins formed in 'backyard burning' situations or agricultural burns (delayed due to other pressing research issues); to complete remediation studies with beta-agonists in farm animals; to complete disposition and withdrawal experiments in farm animals using dioxin-contaminated feed; to begin ADME studies in laboratory animals of predioxins and other brominated flame retardants, such as brominated naphthalene or BDE-47 (delayed due to synthesis time and resources required for these compounds). During FY 2006, we plan to determine the metabolism and
disposition of 1, 2,3,6,7,8-HxCDD, an important contaminant in pentachlorophenol formulations, in cattle; to continue with the metabolism studies of environmentally-relevant brominated flame retardants (brominated naphthalene and BDE-47); to quantitate the release of dioxin and mixed chlorinated/brominated diphenyl ethers from the incineration of electronic wastes; to evaluate the future direction of the CRIS project and write a new five-year research plan. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Published an article 'Transfer and Bioconcentration of Dioxins from Feeds into Beef Cattle and Dairy Cows' by Janice K. Huwe on the Feedinfo News Service, www.feedinfo.com 6/2003.
Impacts
(N/A)
Publications
- Huwe, J.K. 2002. Comparison of soxhlet and accelerated solvent extractions in the analysis of dioxins and furans from liver samples. Organohalogen Compounds. 58:229-232.
- Huwe, J.K., Hakk, H., Lorentzsen, M.K. 2002. A mass balance feeding study of a commercial octabromodiphenyl ether mixture in rats. Organohalogen Compounds. 58:229-232.
- Hakk, H., Diliberto, J.J. 2003. Comparison of overall metabolism of 1,2,3, 7,8-PeCDD in CYP1A2 (-/-) knockout and C57BL/6N parental strains of mice. Organohalogen Compounds. 64:293-296.
- Hakk, Heldur, Casey, Francis, and Larsen, Gerald. Sorption, mobility and fate of 1,3,7,8-tetrachlorodibenzo-p-dioxin in soils and sand. Josep Rivera, et al., editors. Barcelona, Spain. Organohalogen Compounds, Dioxin 2002, 22nd International Symposium on Halogenated Environmental Organic Pollutants and POPs. 2002. v. 57. p. 301-304.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Persistent organic pollutants such as polychlorinated dioxins, furans, and biphenyls are ubiquitous in the environment and enter the food chain as animals are exposed through their surroundings and feed. These toxic contaminants are concentrated in animal products containing fats (i.e. meat, dairy, and eggs) and are ultimately consumed by humans. The health risk to humans of these low-level contaminants is not adequately known. Our research efforts are directed at reducing this exposure from the food supply by 1)determining background levels currently found in domestic animal products, 2) identifying and reducing potential sources of animal exposure to these toxic chemicals, 3)exploring methods which may prevent or lower the accumulation of these chemicals in animal fats, and 4) developing rapid, lower cost methods to analyze food products for these chemicals. 2. How serious is the
problem? Why does it matter? Dioxins and related compounds have been shown to have numerous adverse health effects in animals. One of the most potent dioxins (2,3,7,8- tetrachlorodibenzo-p-dioxin or TCDD) has been classified as a known human carcinogen by the World Health Organization (WHO) and U.S. National Toxicology Program (NTP). WHO and the European Union (EU) have set limits on the acceptable levels of these contaminants in food products. Recent recalls of chickens in the U.S. and Belgium with higher than acceptable levels demonstrates the potential for exposures. More data on the current levels of these contaminants in livestock will provide regulators with a better idea of the safety of the U.S. food supply and its acceptability under the new EU regulations. Investigation into potential sources of dioxins, such as pentachlorophenol-treated wood or agricultural burning practices, will help determine the contribution of these sources to animal contamination and whether
changes in certain farming practices may be needed. Remediation methods for animals already exposed or methods which decrease uptake could help to decrease background levels in animals or toxic levels after a serious contamination has occurred. Less expensive methods of analysis will allow widespread monitoring for dioxins in foods minimizing the risks associated with episodic contaminations. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? Regarding Food Safety, National Program 108 (100%), our research seeks to reduce residues of dioxins and dioxin-like compounds in the food supply. 4. What was your most significant accomplishment this past year? A. Toxic dioxin congeners are extremely persistent in animal systems and methods to increase clearance from the body are needed. The roles of Cytochrome P450-1A2 (Cyp 1A2) and metabolism in the sequestration and clearance of dioxins were investigated at the Biosciences
Research Laboratory in Fargo, ND using knockout mice developed by the U.S. EPA Laboratory in Research Triangle Park, NC. A comparison of the metabolism and excretion of TCDD in regular and knockout mice (no Cyp 1A2) showed that even in the absence of Cyp 1A2, a TCDD sequestering protein, no increase in TCDD metabolism or excretion occurred. The inherent resistance of dioxins to metabolic change and, thereby, excretion means that other approaches to decrease body burdens may need to be explored. B. Updated information is needed on the levels of dioxins in the U.S. food supply in order to determine more accurate exposure estimates and the associated risks. Meat products were collected from large grocery stores by ARS employees across the U.S. and shipped to the Biosciences Research Laboratory in Fargo, ND where they were analyzed for dioxins. Seventy six samples including bacon, chicken, ground beef, sirloin steak, pork chops, and bison burger were analyzed for dioxins. Except for
one bison sample, all the samples tested had dioxin levels below the regulatory limits set by the EU for food products. C-D. None 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? During the one and a half years of this CRIS, the following has been accomplished: The usefulness of a leanness enhancing agent, clenbuterol, as a remediation protocol to lower an animal's dioxin body burden after an exposure was investigated. The preliminary study was conducted in rats and showed body fat and dioxin levels decreased by 30% compared to controls when clenbuterol was fed after a dioxin exposure. These results are encouraging and suggest similar body burden decreases could be achieved in livestock species. (First year milestone for objective 3). Polybrominated diphenyl ethers (PBDEs), a class of flame retardants and emerging persistent pollutant, were investigated for their occurrence in food products. Purification methods and
GC/MS detection methods were developed and applied to the analysis of 13 chickens fat samples. All the samples analyzed contained PBDEs with levels reaching 45 ng/g lipid. These results show the potential for human exposure to these contaminants through the food supply. (Second year milestone for objective 4). 6. What do you expect to accomplish, year by year, over the next 3 years? During FY 2003, we plan to continue investigating the use of our immunoaffinity column for milk samples; to establish a CRADA with Paracelsian, Inc. for the purpose of exploring coupling an immunoaffinity column to a receptor-based assay for the rapid purification and detection of dioxins from various matrices; to purify, digest, sequence, and study a rat bile protein which is vital for the excretion of dioxins and related compounds; to continue analyzing food samples to establish background levels in U.S. foods; to study the sorption, mobility, and fate of dioxins in soil using non-toxic dioxin
congeners as models (a new direction of research); to investigate dioxin levels in milk from MN farms involved in a recent feed contamination episode and to gather information for risk assessment purposes by conducting disposition and withdrawal studies in cows using this dioxin-contaminated feed (new research arising from immediate needs). During FY 2004, we plan to report survey data on the background levels of dioxins and related compounds in certain meats (chicken, turkey, pork, and beef); to report dioxin levels found in MN milk from a contamination episode; to determine the metabolism and disposition of 1,2,3,6,7,8-HxCDD, an important contaminant in pentachlorophenol formulations, in laboratory animals and cattle; to extend the investigation of clenbuterol or ractopamine remediation methods to farm animals i.e. hogs; to continue looking at disposition and withdrawal rates of dioxins in farm animals (chickens and/or beef) using a contaminated feed supplement; to study the
absorption, disposition, metabolism, and excretion (ADME)in laboratory animals of certain polybrominated diphenyl ethers which are considered persistent organic pollutants. During FY 2005, we plan to measure the levels of dioxins formed in "backyard burning" situations or agricultural burns (delayed due to other pressing research issues); to complete remediation studies with beta- agonists in farm animals; to complete disposition and withdrawal experiments in farm animals using dioxin-contaminated feed; to begin ADME studies in laboratory animals of predioxins and other brominated flame retardants, such as brominated naphthalene (delayed due to synthesis time and resources required for these compounds). 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Presented a talk entitled "Dioxins in Foods and Feeds" before the National Academy of Science Committee on the
Implications of Dioxin in the Food Supply. 2/19/2002 Article, "Protecting the U.S. food," written by Mikkel Pates, reporter from the Grand Forks Herald, Grand Forks, ND, about Dr. Janice Huwe's work on dioxin's in the U.S. food supply. Grand Forks Herald. October 20, 2001. p. 1A-2A. Article, "North Dakota Lab could see security role," written by Mikkel Pates, reporter from the Grand Forks Herald, Grand Forks, ND, about the dioxin work conducted in the Animal Metabolism-Agricultural Chemicals Research Unit under the direction of Dr. Janice Huwe and the role the Unit might play in protecting the U.S. food supply. Agweek. October, 22, 2001. p 24.
Impacts
(N/A)
Publications
- Hakk, H., Larsen, G.L., Feil, V.J. The metabolism of [14C]1,2,7,8- tetrachlorodibenzo-p-dioxin in a ruminating Holstein calf. Xenobiotica. 2001. v. 31(7). p. 443-455.
- Fries, G.F., Feil, V.J., Zaylskie, R.G., Bialek, K.M., Rice, C.P. Treated wood in livestock facilities: relationships among residues of pentachlorophenol, dioxins, and furans in wood and beef. Environmental Pollution. 2002. v. 116. P. 301-307.
- Huwe, J.K., Lorentzsen, M., Thuresson, K., Bergman, A. Analysis of mono- to deca-brominated diphenyl ethers in chickens at the part per billion level. Chemosphere. 2002. v. 46. p. 635-640.
- Huwe, J., Zaylskie, R., Feil, V. Distribution of dioxins into beef lipid stores: another look at levels in typical sampling matrices and retail meat cuts. Jae-Ho Yang, editor. Gyeongju, Korea. Organohalogen Compounds, Dioxin 2001, 21st International Symposium on Halogenated Environmental Organic Pollutants and POPs. 2001. v. 51. p. 310-313.
- Hakk, H., Huwe, J., Lorentzsen, M. A mass balance study of a commercial pentabromodiphenyl ether mixture in male Sprague-Dawley rats. Jae-Ho Yang, editor. Gyeongju, Korea. Organohalogen Compounds, Dioxin 2001, 21st International Symposium on Halogenated Environmental Organic Pollutants and POPs. 2001. v. 52. p. 5-8.
- Shappell, N.W., Billey, L.O., Feil, V. Effect of clenbuterol on liver stores of dioxins and furans in rats. Jae-Ho Yang, editor. Gyeongju, Korea. Organohalogen Compounds, Dioxin 2001, 21st International Symposium on Halogenated Environmental Organic Pollutants and POPs. 2001. v. 53. p. 460- 463.
- Hakk, H., Larsen, G. Klasson-Wehler, E., Orn, U., Bergman, A. Tissue disposition, excretion, and metabolism of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) in male Sprague-Dawley rats. Xenobiotica. 2002. v. 32(5). p. 369-382.
- Shelver, W.L., Shan, G., Gee, S.J., Stanker, L.H., Hammock, B.D. Comparison of retention patterns of PCDDs/PCDFs on immunoaffinity columns generated with different monoclonal antibody clones and polyclonal antibodies produced using different hapten conjugates. American Chemical Society 223rd National Meeting. 2002. Abstract No. 39.
- Huwe, J.K. Dioxins in food: a modern agricultural perspective. Journal of Agricultural and Food Chemistry. 2002. v. 50(7). p. 1739-1750.
- Shelver, W.L., Shan, G., Gee, S.J., Stanker, L.H., Hammock, B.D. Comparison of immunoaffinity column retention patterns of PCDDs/PCDFs on columns generated with different monoclonal antibody clones and polyclonal antibodies produced using different hapten conjugates. Analytica Chimica ACTA 2002. v. 457. p. 199-209.
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Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
Persistent organic pollutants such as polychlorinated dioxins, furans, and biphenyls are ubiquitous in the environment and enter the food chain as animals are exposed through their surroundings and feed. These toxic contaminants are concentrated in animal products containing fats (i.e. meat, dairy, and eggs) and are ultimately consumed by humans. The health risk to humans of these low-level contaminants is not adequately known. Our research efforts are directed at reducing this exposure from the food supply by 1)determining background levels currently found in domestic animal products, 2) identifying and reducing potential sources of animal exposure to these toxic chemicals, 3)exploring methods which may prevent or lower the accumulation of these chemicals in animal fats, and 4)developing rapid, lower cost methods to analyze food products for these chemicals.
2. How serious is the problem? Why does it matter?
Dioxins and related compounds have been shown to have numerous adverse health effects in animals. One of the most potent dioxins (2,3,7,8- tetrachlorodibenzo-p-dioxin) has been classified as a known human carcinogen by the World Health Organization (WHO) and U.S. National Toxicology Program (NTP). WHO and several countries have set limits on the acceptable levels of these contaminants in food products. Recent recalls of chickens in the U.S. and Belgium with higher than acceptable levels demonstrates the potential for exposures. More data on the current levels of these contaminants in livestock will provide regulators with a better idea of the safety of the U.S. food supply. Investigation into potential sources of dioxins, such as pentachlorophenol-treated wood or agricultural burning practices, will help determine the contribution of these sources to animal contamination and whether changes in certain farming practices may be needed. Remediation methods for animals already
exposed or methods which decrease uptake could help to decrease background levels in animals or toxic levels after a serious contamination has occurred. Less expensive methods of analysis will allow widespread monitoring for dioxins in foods minimizing the risks associated with episodic contaminations.
3. How does it relate to the National Program(s) and National Component(s)?
Regarding Food Safety, National Program 108 (100%), our research seeks to reduce residues of dioxins and dioxin-like compounds in the food supply.
4. What were the most significant accomplishments this past year?
A. Polybrominated diphenyl ethers (PBDEs), a class of flame retardants, have been found to be increasing in the environment; however, little is known about the effects of these low environmental levels on animals. In our laboratory, we have examined the bioavailability and bioaccumulation of low levels of PBDEs by conducting a controlled feeding study in rats using a commercial pentabromodiphenyl ether mixture containing tetra- to hexa- BDEs. Purification methods and GC/MS detection methods were developed to analyze PBDEs in excreta and whole body compartments in order to provide a total mass balance calculation. Results of the study showed that 30-60% of the dose was accumulated in the animals, but no single congener showed preferentially higher accumulation. B-D. None
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
The following accomplishments were achieved under the previous related CRIS. A geographical survey of domestic beef showed low levels of dioxins and furans in general, but indicated that when beef samples were contaminated with significant levels of dioxins and furans it was due to pentachlorophenol treated wood in the feeding facilities. The absorption and distribution of dioxins and furans in animals was determined through a controlled feeding study and related metabolism studies in calves and model animals. The data indicated that an animal exposed to the toxic dioxins retains 30-50% of the dose until it is harvested for food and bioconcentrates the dioxins 10-fold in adipose tissues. Distribution of dioxins into different tissue matrices is equivalent on a fat weight basis; except for liver which accumulated them to a higher degree. Strategies to reduce dioxin intake by humans include trimming fat, cooking food and discarding the fats and juices (which can remove 45% of the
dioxins from hamburger as determined by our studies), and avoiding eating liver. An immunoaffinity column has been developed for the isolation of dioxins and furans from serum. This column recognizes seven of the most toxic congeners, decreases cleanup time and solvent consumption, and, consequently, decreases the cost of dioxin analyses. A preliminary study on the use of clenbuterol, a leanness enhancing agent, as a means to remediate dioxin-contaminated animals was conducted in rats. The data showed that body fat and dioxin levels decreased by 30% compared to controls when clenbuterol was fed after a dioxin exposure. These results are encouraging and suggest similar body burden decreases could be achieved in livestock.
6. What do you expect to accomplish, year by year, over the next 3 years?
During FY 2002, we plan to investigate the use of our immunoaffinity column for milk samples; to begin a widespread sampling project to establish background dioxin levels in the U.S. food supply; to investigate the role of predioxins in dioxin exposure; to investigate the pharmacokinetics of two toxic dioxin congeners in a CYP1A2 knockout mouse system; to measure dioxin levels resulting from the agricultural practice of burning fields and the role of chlorinated pesticide usage on this process; to look at clenbuterol as a means to prevent dioxin uptake and deposition in laboratory animals. During FY 2003, we plan to purify, digest, sequence, and study a rat bile protein which is vital for the excretion of dioxins and related compounds; to continue to explore promising methods for rapid detection or purification of dioxin in foods including the use of antibodies, lipophilic proteins, receptors, or bioassays; to continue analyzing food samples to establish background levels in U.S.
foods; to measure the levels of dioxins formed in "backyard burning" situations. During FY 2004, we plan to report survey data on the background levels of dioxins and related compounds in certain meats (chicken, pork, and beef); to determine the metabolism and disposition of 1,2,3,6,7,8-HxCDD, an important contaminant in pentachlorophenol formulations, in laboratory animals and cattle; to extend the investigation of clenbuterol or ractopamine remediation methods to turkeys.
7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product?
None
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
"Monitoring Dioxins" in Agricultural Research, Jan. 2001 issue, pp.14-15. "Dioxins in Food: An Agricultural Perspective" a white paper written for the USDA, 1/2001.
Impacts
(N/A)
Publications
- Feil, V.J., Larsen, G.L. Dioxins in food from animal sources. IN: Persistent, Bioaccumulative, and Toxic Chemicals I: Fate and Exposure. Lipnick, Robert, et al., editors. American Chemical Society Symposium Series 772. 2001. p. 245-251.
- Hakk, Heldur, Larsen, Gerald, Feil, Vernon. Tissue distribution, excretion, and metabolism of 1,2,7,8-tetrachlorodibenzo-p-dioxin in the rat. Chemosphere. 2001. v.42. p. 975-983.
- Hakk, H., Larsen, G., Bergman, A., Orn, U. Metabolism, excretion, disposition, and enterohepatic circulation of the flame retardant tetrabromobisphenol-A in conventional and bile-duct cannulated rats. Xenobiotica. 2000. v. 30(9). p. 881-890.
- Feil, Vernon J., Huwe, Janice K., Zaylskie, Richard G., Davison, Kenneth L., Anderson, Vernon L., Marchello, Martin, Tiernan, Thomas. Chlorinated dibenzo-p-dioxin and dibenzofuran concentrations in beef animals from a feeding study. Journal of Agricultural and Food Chemistry. 2000. v. 48(12). p. 6163-6173.
- Huwe, Janice K., Shelver, Weilin L., Stanker, Larry, Patterson, Jr., Donald G., Turner, Wayman E. A simplified method for the isolation of polychlorinated dibenzo-p-dioxins and furans from serum samples using immunoaffinity chromatgraphy prior to high resolution GC-MS detection. Journal of Chromatography B. 2001. v.757. p. 285-293.
- Hakk, Heldur, Larsen, Gerald Bowers, Joseph. Tissue disposition, excretion, and metabolism of 1,2-Bis(2,4,6-tribromophenoxy)ethane (BTBPE) in male Sprague-Dawley rats. Abstracts of the 2nd International Workshop on Brominated Flame Retardants. 2001. p. 99-102.
- Hakk, Heldur, Huwe, Janice, Larsen, Gerald. Preliminary results of a mass balance study of a commercial pentabromodiphenyl ether mixture in male Sprague-Dawley rats. Abstracts of the 2nd International Workshop on Brominated Flame Retardants. 2001. p. 283-285.
- Larsen, G., Casey, F., Bergman, A., Hakk H. Mobility, sorption, and fate of tetrabromobisphenol A (TBBPA) in loam soil and sand. Abstracts of the 2nd International Workshop on Brominated Flame Retardants. 2001. p. 213-216.
- Garber, E.A.E., Larsen, G.L., Hakk, H., Bergman, A. Frog Embryo Teratogenic Assay: Xenopus (FETAX) analysis of the biological activity of tetrabromobisphenol A (TBBPA). Abstracts of the 2nd International Workshop on Brominated Flame Retardants. 2001. p. 259-262.
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