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United States Department of Agriculture

Agricultural Research Service

Research Project: THE TOXICITY OF PYRROLIZIDINE ALKALOID-CONTAINING PLANTS AND OTHER HEPATOTOXIC AND NEUROTOXIC PLANTS

Location: Poisonous Plant Research

2008 Annual Report


1a.Objectives (from AD-416)
Objective I: Develop diagnostic techniques and biomarkers to better identify animals poisoned by pyrrolizidine alkaloids (PA's) and their subsequent metabolites. Objective II: Determine pyrrole toxzicity and carcinogenicity and compare pyrrole toxicity with that of PA and PA-N-oxides. Characterize the risk to fetuses and neonates that are exposed by maternal PA ingestion. 2.1 Determine pyrrole toxicity and carcinogenicity. 2.2 Characterize transplacental and transmammary toxicity of various PA's. Objective III: Describe the gross,histological and ultrastructural lesions of Rayless goldenrod (Isocoma plurifora or Haplopappus heterophyllus) and white snakeroot (Eupatorium rugosum) intoxication and determine the effect on fetal and neonatal development. Objective IV: Describe the clinical, morphological, and molecular alterations of certain hepatotoxic and neurotoxic plant-induced toxicosis in animals. Develop better techniques to monitor chlorophyll and phylloerythrin metabolism and correlate them with photosensitivity in livestock.


1b.Approach (from AD-416)
Pyrrolizidine alkaloid (PA) metabolites (pyrrole) adducts such as pyrrole-thiamidine, pyrrole-guanine, pyrrole-methionine or pyrrole-glutathione will be linked to an immunogenic proteins and used as the immunogens to generate pyrrole specific antibodies. These same pyrrole-specific antibodies will be used to develop immunohistochemistry, ultrastructural immunochemistry and ELISA diagnostic techniques. Cellular kinetic will be documented and described. Additional biomarkers of poisoning will be developed using proteomic and genomic techniques. Tissue bound pyrroles or adducts that are likely to contaminate animal products, will be tested in mouse models for toxicity and carcinogenicity. The molecular events of hepatic carcinogenesis including altered expression or activation of various oncogenes, tumor suppressor genes and cell proliferation mediators will be evaluated. Similar sensitive mouse models will be used to test the fetal and neonatal effects of individual PA-toxicity. PA’s likely to cross the placenta or to be excreted in milk will be identified and the risk of such poisoning described. The toxicity of specific PA’s will be compared with PA chemical structure to identify those functional groups that are likely to lead to transplacental and transmammary transfer and poisoning. As rodent placentation is unique, these results (transmammary and transplacental PA transfer) will be verified in livestock. Rayless goldenrod and white snakeroot poisoning will be characterized by exposing horses to varying plant doses. The clinical, physiologic and pathologic response to poisoning will be monitored daily using clinical evaluations, exercise tolerance via treadmill evaluation of physical strength and endurance, electrocardiograms, echocardiography, hematology and serum biochemistry. The progression and lesions of poisoning will be described using biopsy, post mortem examination, histologic and ultrastructural evaluations. A dose response study using pregnant mares will be used to characterize fetal and neonatal poisoning and to identify which lesions are reversible. Photosensitization will be studied using clinical surveys. Clinical findings, histologic changes and hepatic function data will be collected, characterized and correlated with the serum and dermal phylloerythrin concentrations. Risk models of feed-related photosensitivity will be developed to predict susceptible populations and risk.


3.Progress Report
This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component II, Subcomponent Rangeland Poisonous Plants, Problem Statement P.

Hepatotoxic and neurotoxic plants, especially those containing pyrrolizidine alkaloids (PA’s) are invasive weeds that poison livestock, wildlife, and humans throughout the world. Techniques are needed to monitor PA contamination in foods and feeds, identify poisoned animals, and predict the fate of poisoned animals. Another toxic plant, rayless goldenrod (Isocoma spp.) poisons livestock and wildlife. Better descriptions of Isocoma toxicity, especially the effects of sub-clinical poisoning on fetuses and neonates, are needed. Other plants containing photodynamic chromophores or toxins that alter liver function result in dermal accumulation of photodynamic plant metabolites. These molecules react with sunlight producing photosensitization. Information is needed to better understand the pathogenesis of photosensitivity and predict when animals are at risk. The objectives are to: 1)Develop specific biomarkers and diagnostic techniques to identify PA-poisoned animals and monitor foods, feed, and animal products for contamination, 2.1)Study PA-induced carcinogenesis using genetically modified mice, 2.2)Increase PA bioactivation to develop a highly sensitive mouse model of PA toxicity. This model will be used to evaluate the toxicity of dietary PA's, PA-metabolites, as well as further define fetal and neonatal toxicity via transplacental and transmammary intoxication. 3)Better describe and characterize the effect of consuming rayless goldenrod in horses. 4)Develop additional techniques to monitor chlorophyll metabolite kinetics and correlate these metabolites with plant induced photosensitivitiy. Accomplishments of this CRIS include: PA containing plants have been collected and analyzed. Alkaloids from these collections will be used to synthesize PA metabolites and various conjugates for toxicity and immunogenicity testing. P53 knockout mice were obtained and pilot studies to study the carcinogenicity of PA's in this sensitive model were initiated. Rayless goldenrod and whitesnake root was collected. Mass spectrometry protocols were developed to analyze plant material for tremetone like compounds. A treadmill for large animal exercise, electromyography, and electrocardiographic monitoring was built and tested. Initial pilot studies were conducted and methodology to monitor cardiac function and exercise tolerance was developed. Pilot studies indicated that rayless goldenrod at doses of 2-3% of body weight for 6 days are toxic for goats. Doses of approximately 1.5 to 2% of body weight for 7 days are toxic for horses. The information obtained from these pilot doses will be used to design subsequent dose response studies. Phylloerythrin was obtained and tested. These methodologies need additional optimization as the assay lacks sensitivity. The proposed work will enhance diagnosis and monitoring of poisoned livestock and wildlife and will better ensure quality and safety of foods, feed and herbal products.


4.Accomplishments
1. PA Metabolites and Conjugates synthesized. Plants containing pyrrolizidine alkaloids (PA's) are global in distribution and they are the most wide spread poisonous plant problems in the world. As PA's and their metabolites can contaminate feeds, foods, and herbal products, some countries have regulated exposure limits and imposed trade restrictions. To better manage this problem, sensitive and accurate techniques are needed to ensure food safety and to identify poisoned animals. PA containing plants were collected and analyzed. Alkaloids from these collections will be used to synthesize PA metabolites and various conjugates for toxicity and immunogenicity testing. As PA-containg plants often contaminate feed and food, these techniques will be used to ensure feed/food safety, identify poisoned animals and prognose the potential outcome of poisoning. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component 11, Subcomponent Rangeland Poisonous Plants, Problem Statement P.

2. Modification of PA Metabolism. Hepatotoxic and neurotoxic plants, especially those containing pyrrolizidine alkaloids (PA's) are invasive weeds that poison livestock, wildlife, and humans throughtout the world. Techniques are needed to monitor PA contamination in foods and feed, identify poisoned animals, and predict the fate of poisoned animals. P53 knockout mice were obtained and pilot studies to study the carcinogenicity of PA’s in this sensitive model were initiated. Animal protocols and IACUC approvals to study the modification of PA metabolism were developed and are under review. These studies will help understand the effects and risks of low dose PA poisoning. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component 11, Sub-component Rangeland Poisonous Plants, Problem Statement P.

3. Rayless goldenrod analysis for tremetone and other tremetone-like compounds. Rayless goldenrod (Isocoma spp.) poisons livestock and wildlife when other forages are depleted. Better descriptions of Isocoma toxicity, especially the effects of sub-clinical poisoning on fetal and neonate health are needed. Rayless goldenrod and white snakeroot was collected. Animal protocols and IACUC approvals were developed and approved. Mass spectrometry protocols were developed to analyze plant material for tremetone, hydroxytremetone, dehydrotremetone, and other tremetone like compounds. A treadmill for large animal exercise, electromyography, and electrocardiographic monitoring was built and tested. Initial pilot studies were conducted and methodology to monitor cardiac function and exercise tolerance was developed. Pilot studies indicated rayless goldenrod at doses of 2-3% of body weight for 6 days are toxic for goats. Doses of approximately 1.5 to 2% of body weight for 7 days are toxic for horses. The information obtained from these pilot doses will be used to design subsequent dose response studies. These studies will provide information on the toxicity, mechanism of poisoning, and the risk of intoxication. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component 11, Subcomponent Rangeland Poisonous Plants, Problem Statement P.

4. Phylloerythrin Assay Methodologies. Plants containing photodynamic chromophores or toxins that alter liver function result in dermal accumulation of photodynamic plant metabolites. These molecules react with sunlight producing photosensitization. Information is needed to better understand the pathogenesis of photosensitivity and predict when animals are at risk. Methodologies need to be developed. Phylloerythrin assays using flourometric spectrometry was initiated. Control phylloerythrin was obtained and tested. These methodologies need additional optimization as they lack sensitivity. These assays will be used to determine the extent of chlorophyl metabolism as it relates to photosensitivity. This research relates to NP 215 Rangeland, Pasture and Forage, Action Plan Component 11, Subcomponent Rangeland Poisonous Plants, Problem Statement P.


5.Significant Activities that Support Special Target Populations
A series of 4 publications were published that target horse owners.


6.Technology Transfer

Number of Web Sites Managed1
Number of Newspaper Articles and Other Presentations for Non-Science Audiences14
Number of Other Technology Transfer2

Review Publications
Soto-Blanco, B., Stegelmeier, B.L., Pfister, J.A., Gardner, D.R., Panter, K.E. 2007. Comparative effects of prolonged administration of cyanide, thiocyanate and chokecherry (Prunus virginiana) to goats. Journal of Applied Toxicology, 28:356-363

Molyneux, R.J., Lee, S.T., Gardner, D.R., Panter, K.E., James, L.F. 2007. Phytochemicals: The Good, the Bad, and the Ugly?. Phytochemistry.68(22-24):2973-2985.

Pimentel, L., Correa, F.R., Gardner, D.R., Panter, K.E., Dantas, A.F., Medeiros, R.A., Mota, R.A., Araujo, J.A. 2007. Mimosa tenuiflora as a Cause of Malformations in Ruminants in the Northeastern Brazilian Semiarid Rangelands. Veterinary Pathology.

Zhang, J., Garrossian, M., Gardner, D.R., Garrossian, A., Chang, Y., Kim, Y.K., Chang, C.T. 2008. Synthesis and anticancer activity studies of cyclopamine derivatives. Bioorganic and Medicinal Chemistry Letters. Online 18(2008) 1359-1363.

Last Modified: 9/22/2014
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