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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

2010 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
Pyrrolizidine alkaloids (PA’s) are toxins found in a wide variety of plants worldwide and are responsible for poisoning in livestock, wildlife and humans. Analytical techniques and diagnostic procedures have been developed, however improved methods to monitor PA contamination in foods and feed, identify poisoned animals, and predict the fate of poisoned animals are needed. PA containing plants have been collected and analyzed, and PA’s have been isolated for metabolite synthesis and protein conjugation to complete toxicity and immunogenicity studies.Small animal models are being developed to understand PA toxicity and evaluate PA metabolism. P53 knockout mice (sensitive to neoplasm formation) have been used to study PA carcinogenicity and initial results indicate that riddelliine (a PA toxin) will induce vascularneoplasms in these mice. Pilot studies of the transplacental and transmammary toxicity of various PA’s have been done and PA-induced cP450 up-regulation has been verified in mice. Rayless goldenrod and white snakeroot cause poisoning in livestock in the southwest and midwest, respectively. A series of benzofuran ketones have been isolated and characterized from these plants and toxicity testing in a rodent and small ruminant model will proceed. Feeding trials with plant material have been done in horses and goats and initial physiographic and electrocardiographic studies indicate that significant changes occur in exercise tolerance that is reflected in biochemical and histologic lesions and pathology. Chemical techniques to analyze rayless goldenrod and dose response studies in horses and goats are completed and published. The effects of these plants and their toxins on fetal and neonatal development and health will be done. Phylloerythrin is a plant metabolite that causes photosensitivity (sunburn) in animals. Assays using flourometric and uv spectrometry have been developed and will be used to better understand induction of photosensitivity and its relationship with different diets. Better techniques to monitor chlorophyll and phylloerythrin metabolism and correlate them with photosensitivity in livestock is progressing.


4.Accomplishments
1. Benzofuran Ketones Chemistry. Further enhancement of quantitative methods for benzofuran ketones were made to screen white snakeroot and rayless goldenrod populations, which cause “trembles” and “milk sickness” in livestock and humans. As similar benzofuran ketones are found in both plants, it has been speculated that their toxicities are similar. Research results by ARS scientists in Logan, UT show that toxin concentrations varied considerably among different plant populations. Additional collections and taxonomic studies suggest that some populations may be different subspecies and there are toxic and non-toxic populations. This information will be useful as specific populations can be identified, tested and the risk of poisoning can be more closely evaluated for livestock producers in the mid west and south west. This will also lead to additional studies to understand when and why plants produce these toxins.

2. Rayless goldenrod toxicity. Rayless goldenrod toxicity in horses and goats was described and characterized by ARS scientists in Logan, UT. Dose response studies using large animal exercise physiology, electromyography, and electrocardiographic monitoring have been developed and used in initial dose response studies. These 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. Poisoning results in characteristic myonecrosis of many skeletal and cardiac muscles. Additional recovery and ultrastructural studies of these changes are underway. This information is essential for livestock producers and veterinarians in identifying and avoiding these plant-induced diseases. As these toxins are lipid soluble and easily transferred in milk this is also important in human health. Specific toxins will also be purified and tested for toxicity.

3. Development of Pyrrolizidine alkaloid (PA) Chemistry and Analysis Methods. Analytical methods were developed by ARS scientists in Logan, UT and included various extraction methods to analyze PA-containing plants such as comfrey (Symphytum spp.). Comfrey is an herbal product used by people and may be a contaminant of other herbal products. These methods will improve the detection of PA in herbal products, feed and food to reduce the risk of poisoning in animals and humans.

4. Identification of Pyrrolizidine alkaloid (PA) Carcinogenicity. Many PA’s are carcinogenic and sensitive models to characterize the carcinogenicity of different PA’s are needed. Pilot studies by ARS scientist in Logan, UT using P53 knockout mice to study the carcinogenicity of PA’s have been completed. These studies will be used in subsequent studies to characterize the carcinogenicity of different PA’s and better predict the risk of these different alkaloids. This will also be useful to identify the chemical characteristics that are carcinogenic and better identify PA metabolism.

5. Toxicity of water hemlock seed. Analysis of toxicity data in mice has been completed. ARS scientists at Logan, UT determined that immature seed of Ciuta maculata is of equal potency as the highly toxic tubers. This research confirmed that ingestion of immature water hemlock seed was the cause of cattle deaths in central Utah. Chemical analysis demonstrated that immature seed contained equal amounts of cicutoxin and 9.5 and 22 times more cicutol #1 and cicutol #2 respectively, when compared to highly toxic archived tubers tested in earlier experiments. This information will alert veterinarians, extension agents and ranchers of the potential toxicity of water hemlock seed.


5.Significant Activities that Support Special Target Populations
Producer meetings were held in colorado, Utah and Idaho targeting small farmers and ranchers.

Four user publications in proceedings of veterinary medical association proceedings, zoological veterinary association proceedings and several extension publications were published. These outreach efforts target small livestock owners and veterinarians.


Review Publications
Landau, S.Y., Provenza, F.D., Gardner, D.R., Pfister, J.A., Knoppel, E.L., Peterson, C., Kababya, D., Needham, G.R., Villalba, J.J. 2009. Neem-Tree (Azadirachta indica Juss.) Extract as a Feed Additive Against the American Dog Tick (Dermacentor variabilis) in Sheep (Ovis aries). Veterinary Parasitology. 165:311-317

Jani, A.J., Faeth, S.H., Gardner, D.R. 2010. Asexual Endophytes and Associated Alkaloids Alter Arthropod Community Structure and Increase Herbivore Abundances on a Native Grass. Ecology Letters. 13(1): 106-17.

Stegelmeier, B.L., Davis, T.Z., Green, B.T., Lee, S.T., Hall, J.O. 2010. Experimemntal Rayless Goldenrod (Isocoma pluriflora) Toxicosis in Goats. Journal of Veterinary Diagnostic Investigation 22:570-577.

Cerqueira, V.D., Riet-Correa, G., Barbosa, J.D., Duarte, M.D., Oliveira, C.M., De Oliverira, C.A., Tokarnia, C., Lee, S.T., Riet-Correa, F. 2009. Colic Caused by Panicum maximum Toxicosis in Equidae in Northern Brazil. Journal of Veterinary Diagnostic Investigation, 21:882-888

Last Modified: 7/28/2014
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