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United States Department of Agriculture

Agricultural Research Service


Location: Poisonous Plant Research

2009 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
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 feed, 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. PA containing plants have been collected and analyzed for their pyrrolizidine alkaloid content. Alkaloids from these collections will be used to synthesize PA metabolites and various conjugates for toxicity and immunogencitiy testing. P53 knockout mice were also obtained and pilot studies to determine the carcinogenicity of PA’s in this sensitive model were initiated and several mice developed neoplasms. Animal protocols and IACUC approvals to study the modification of PA metabolism were developed and are under review. Rayless goldenrod and white snakeroot were collected. Quantitative HPLC protocols were developed to analyze plant material for tremetone, 3-hydroxytremetone, 6-hydroxytremetone, dehydrotremetone and other tremetone-like compounds. Plant material from eleven different locations have been analyzed for their benzofuran ketone compound chemotype and concentrations. 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. Development of phylloerythrin assays using flourometric spectrometry was initiated. Control phylloerythrin was obtained and tested. These methodologies need additional optimization as the assay lacks sensitivity. This research 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. Development of a quantitative method for the measurement of benzofuran ketones in rayless goldenrod and white snakeroot. White snakeroot (Ageratina altissima) and rayless goldenrod (Isocoma pluriflora) can cause “trembles” and “milk sickness” in livestock and humans, respectively. Tremetol, a complex mixture of sterols and derivatives of methyl ketone benzofuran has been extracted from white snakeroot and rayless goldenrod and is reported to be the toxic substance in plant material. In this research, the three major benzofuran ketones, tremetone, dehydrotremetone, and 3-oxyangeloyl-tremetone were isolated from rayless goldenrod. Using these compounds as standards, a quantitative HPLC method was developed to measure these compounds in white snakeroot and rayless goldenrod. Concentrations of tremetone, dehydrotremetone, and 3-oxyangeloyl-tremetone were found to vary considerably among the different white snakeroot and rayless goldenrod plant collections. Differences in concentrations of tremetone, dehydrotremetone and 3-oxyangeloyl-tremetone in white snakeroot and rayless goldenrod plants may explain the historical, sporadic and unpredictable toxicity of these plants to livestock and humans. The quantitative HPLC method will be used to make risk assessments of plant populations of white snakeroot and rayless goldenrod that may be toxic to livestock.

2. Analytical methods for the detection of the pyrrolizidine alkaloids were investigated. The detailed methods have been explored for the plant extraction, alkaloid isolation using solid phase extraction procedures and detection and quantitation of alkaloids using liquid chromatography/mass spectrometry. The specific alkaloid content of the two main comfrey species has been characterized. These analytical methods will improve the detection of dangerous PA's in herbal products and seed grains to reduce the risk of poisoning in animals and humans.

Review Publications
Davis, T.Z., Lee, S.T., Ralphs, M.H., Panter, K.E. 2009. Selected Common Poisonous Plants of the United States' Rangelands. Rangelands. 31(1):38-44 DOI:10.2111/1551-501X-31.1.38

Lee, S.T., Green, B.T., Welch, K.D., Pfister, J.A., Panter, K.E. 2008. Steroselective Potencies and Relative Toxicities of Coniine Enantiomers. Chemical Research in Toxicology. 21(10): 2061-2064. DOI 10.1021/tx800229w

Lee, S.T., Mitchell, R., Wang, Z., Heiss, C., Gardner, D.R., Azadi, P. 2009. Isolation, Characterization, and Quantification of Steroidal Saponins in Switchgrass (Panicum virgatum L.). Journal of Agricultural and Food Chemistry. 57(6):2599-2604. DOI: 10.1021/jf803907y

Provenza, F.D., Villalba, J.J., Wiedmeier, R.W., Layman, T., Owens, J., Lisonbee, L., Clemensen, A., Welch, K.D., Gardner, D.R., Lee, S.T. 2009. Value of Plant Diversity for Diet Mixing and Sequencing in Herbivores. Rangelands. 31(1):45-49. DOI:10.2111/1551-501X-31.1.45

Stegelmeier, B.L., Gardner, D.R., Davis, T.Z. 2009. Livestock Poisoning with Pyrrolizidine Alkaloid Containing Plants (Senecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium and Echium spp.). Rangelands. 31(1):25-37.

Stegelmeier, B.L., Green, B.T., Panter, K.E., Welch, K.D., Hall, J.O. 2009. Identifying Plant Poisoning in Livestock. Rangelands 31(1):5-9

Stegelmeier, B.L., Sani, Y., Pfister, J.A. 2009. Baccharis Pteronioides Toxicity in Livestock and Hamsters. Journal of Veterinary Diagnostic Investigation. 21:208-213

De Almeida, M.B., Schild, A.L., Brasil, N.D., Quevedo, P., Fiss, L., Pfister, J.A., Riet-Correa, F. 2009. Conditioned Aversion in Sheep Induced by Baccharis coridifolia. Applied Animal Behaviour Science. 117:197-200 DOI: 10.1016/japplanim.2008.12.006

Lee, S.T., Davis, T.Z., Gardner, D.R., Stegelmeier, B.L., Evans, T.J. 2009. A Quantitative Method for the Measurement of Three Benzofuran Ketones in Rayless Goldenrod (Isocoma pluriflora) and White Snakeroot (Ageratine altissima) by HPLC. Journal of Agricultural and Food Chemistry. 57(12):5639-5642

Last Modified: 09/18/2017
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