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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
2012 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 (PAs) are toxins found in a wide variety of plants throughout the world and they are responsible for poisoning of livestock, wildlife, and humans. There is a need to better understand the PAs as toxins as well as to develop methods to identify PA contamination of food and feed. We successfully applied for a federal military grant to support the dissertation work of Lt. Colonel Ammon Brown in our laboratory. Colonel Brown is a board certified veterinary pathologist. He will be in the laboratory for the next 3 years completing his dissertation work entitled “the comparative pathology and carcinogenicity of pyrrolizidine alkaloids”. Two PAs that are potentially toxic to livestock were found in two cryptantha species for the first time, as well as a new chemotype of the PA-containing Amsinckia (fiddleneck). Both a cell culture and a small animal model were developed to test small concentrations of PAs, PA metabolites and PA adducts for their ability to produce PA poisoning. As these PA compounds are those that are likely to contaminate feed and food, these models are essential to determine the toxicity and subsequent risk of these compounds. Additional work was done in studies to characterize the carcinogenic potential of PAs as well as the histological lesions caused by ingestion of PA-containing plants. Research was performed to characterize the effects of less common PA-containing plants including various Ageratum spp. These plants have been associated with several different incidents of livestock and human poisonings in Nepal, Africa and Afghanistan.

White snakeroot and rayless goldenrod contain benzofuran ketones which are believed to be the toxins responsible for causing “trembles” in livestock and “milk sickness” in humans that are exposed to tainted milk. Studies were conducted to compare the toxicity of white snakeroot populations with different chemical profiles to identify which of the benzofuran ketones is or are responsible for causing the associated diseases. Various extracts of white snakeroot were dosed using a goat model to identify the potential toxins. Studies indicate that tremetone is present in all plant populations that have resulted in poisoning, however preliminary data indicates that there may be an additional active compound(s) that is at least partially responsible for the toxicity of the plant.

Monofluoroacetate (MFA) poisoning causes significant losses in the livestock industry throughout the world. A quantitative assay was developed to determine the concentration of MFA in plant material. The assay was used to assay Mascagnia, Amorimia, and Palicourea species that were potentially involved in cases of poisoning in cattle in Brazil.


4.Accomplishments
1. Analysis of pyrrolizidine alkaloid (PA) contaminated grain in Ethiopian communities. "Unidentified Liver Disease" has been reported in communities of the Northwest region of Tigray, Ethiopia since 2001 and it is suspected that the disease is related to natural toxins in food. In cooperation with the Centers for Disease Control and Prevention, samples were collected from the Tigray region of Ethiopia that included stores of local grain (millet, teff, sorghum, maize and sesame), tella (a locally brewed alcoholic beverage), and honey. Samples were analyzed by ARS researchers in Logan, UT for the presence of toxic PAs which can cause liver disease. The concentrations of toxic PAs were higher in grains from “case” household versus "non-case" households. These results are significant in identifying the cause of "Unidentified Liver Disease" affecting Ethiopian households. Currently the CDC is working with Ethiopian communities to identify the source of grain contamination and educate local farmers on PA-containing plants found in these regions.

2. Toxins involved in white snakeroot poisoning. Differences in chemical profiles and concentrations of toxins present in white snakeroot may explain the historically sporadic and unpredictable toxicity of these plants. White snakeroot populations having distinct chemical profiles of the putative toxins were dosed to goats by ARS scientists in Logan, Utah. All of the plant populations that contained significant concentrations of the suspected toxin, tremetone, resulted in toxicity when dosed for up to 10 days. However additional studies indicated that other toxins may also be involved or be required to cause toxicosis. Data generated from such studies are essential for livestock producers and veterinarians to identify and avoid exposure to toxic populations of white snakeroot.

3. Effects of drying on the putative toxins in white snakeroot and rayless goldenrod. ARS researchers in Logan, UT determined the concentrations of the suspected toxins in white snakeroot and rayless goldenrod before and after various drying conditions. The suspected toxins are most stable upon freeze drying, followed by air drying and least stable upon oven drying (60 °C). The putative toxin is stable under air-dried conditions, thus dried white snakeroot and rayless goldenrod are capable of inducing toxicosis in livestock. This knowledge is being conveyed to veterinarians and producers to avoid the risk of poisoning livestock with dried plant material that was previously thought to be of reduced risk.

4. Identification of rangeland plants potentially toxic to livestock. Two Cryptantha species were collected in the vicinity of a suspected poisoning outbreak in cattle. Analyses of the samples by ARS researchers at Logan, Utah determined that the samples contained potentially toxic levels of a specific class of alkaloids termed dehydropyrrolizidine alkaloids (DHPAs). The chemical structures for several new DHPAs were proposed while two new alkaloids were unambiguously elucidated using state-of-the-art spectroscopic techniques. Awareness of the toxic potential of these plants will help producers and extension agents manage livestock on rangelands with Cryptantha species.

5. Development of a quantitative monofluoroacetate (MFA) assay. A method to detect and quantify MFA was developed by ARS researchers in Logan, UT and was used to investigate plant material from field collections and/or herbarium specimens of various plant species suspected of causing sudden death in cattle in Brazil. MFA was detected and quantitated in seven different species. MFA concentrations differed significantly between species which may explain the incidence of poisoning and the amount of plant material required to cause sudden death between these taxa. The information obtained can be used to make risk assessments of plant populations that can cause MFA toxicity.

6. Isolation of standards for analytical quantitation of toxic dehydropyrrolizidine alkaloids (DHPAs) in comfrey. Comfrey and comfrey-derived products, used as herbal medications, contain potentially toxic DHPAs which are the same as, or similar to, those that cause livestock poisoning. ARS scientists at Logan, UT developed a new technique in the “clean-up” of comfrey extracts. Utilization of specially designed purification columns enabled larger scale separation of closely related DHPAs for eventual use as certified analytical standards. Certified analytical standards of DHPAs are essential to the development of validated methods of analysis for DHPAs in animal feed, human food, and animal or human herbal products.

7. Identification of a new chemotype of Amsinckia intermedia (fiddleneck). Fiddleneck is commonly found growing on rangeland in the western United States. ARS researchers in Logan, UT investigated a recent cattle poisoning incident in Arizona that potentially involved fiddleneck. The investigation included an analysis that revealed the population to be a unique chemotype (a plant that produces specific chemicals) that produced the toxin, lycopsamine, which is normally associated with other plant species. This fiddlneck chemotype is being used as a source of lycopsamine to purify for use in toxicity studies and as an analytical standard. Additionally, livestock producers are being advised of the potential risk involved with grazing fiddleneck with this chemotype.

8. Identification of toxic doses of fireweed in cattle. Senecio madagascariensis (fireweed) is an invasive toxic plant that contaminates much of the Hawaiian Islands rangelands. The plant contains a complex mix of toxic PAs. A study was conducted by ARS scientists in Logan, UT to determine the effect of chronic dosing of the PA mix on toxicity to cattle. Cattle that were dosed with medium to high doses of fireweed for 100 days developed liver damage that would affect production efficiencies. This research identified specific serum enzymes that are significantly elevated in fireweed poisoned animals. The information can be used by livestock producers to identify chronically poisoned animals, as well as to make risk assessments of fireweed contaminated rangelands.


Review Publications
Almeida, M.B., Assis-Brasil, N.D., Schild, A.L., Riet-Correa, F., Pfister, J.A., Soares, M.P.S. 2011. Conditioned aversion induced by Baccharis coridifolia in sheep and cattle. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA: CAB International. p. 613-6.

Colegate, S.M., Stegelmeier, B.L., Edgar, J.A. 2012. Dietary exposure of livestock and humans to hepatotoxic natural products. In: Fink-Gremmels, J., editor. Animal Feed Contamination: Effects on Livestock and Food Safety. Philadelphia, PA: Woodhead Publishing Series in Food Sciences, Technology and Nutrition. p. 352-82.

Gardner, D.R., Riet-Correa, F. 2011. Analysis of the toxic amino acid indospicine by liquid chromatography-tandem mass spectrometry. International Journal of Poisonous Plant Research. 1(1):20-7.

Karam, F.S.C., Haraguchi, M., Gardner, D.R. 2011. Seasonal variation in pyrrolizidine alkaloid concentration and plant development in Senecio madagascariensis poir. (Asteraceae) in Brazil. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA: CAB International. p. 179-85.

Lee, S.T., Cook, D., Riet-Correa, F., Pfister, J.A., Anderson, W.R., Lima, F.G., Gardner, D.R. 2012. Detection of monofluoroacetate in Palicourea and Amorimia species. Toxicon. 60(5): 791-6.

Lee, S.T., Davis, T.Z., Cook, D., Stegelmeier, B.L. 2012. Evaluation of drying methods and toxicity of rayless goldenrod (Isocoma pluriflora) and white snakeroot (Ageratina altissima) in goats. Journal of Agricultural and Food Chemistry. 60(19): 4849-53.

Lima, E.F., Riet-Correa, F., Gardner, D.R., Barros, S.S., Medeiros, R.M., Soares, M.P., Riet-Correa, G. 2012. Poisoning by Indigofera lespedezioides in horses. Toxicon. 60: 324-8.

Lima, E.F., Riet-Correa, B., Riet-Correa, F., Medeiros, R.M.T., Gardner, D.R., Riet-Correa, G. 2011. Poisonous plants affecting the central nervous system of horses in Brazil. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA: CAB International. p. 290-4.

Stegelmeier, B.L. 2011. Pyrrolizidine alkaloid-containing toxic plants (Scenecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium, and Echium spp.). Veterinary Clinics of North America. 27(2):419-28.

Stegelmeier, B.L., Panter, K.E. 2012. Poisonous plants that are likely to contaminate hay and prepared feed in the western United States. Rangelands. 34(2): 2-11.

Last Modified: 12/20/2014
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