2013 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.
This is the final report for 5428-32000-015-00D. Dehydro-pyrrolizidine alkaloid (PA) containing plants have global distribution and they often poison livestock and humans by contaminating pastures, feeds, food, and herbal or medicinal products. Toxicity is generally characterized by liver failure though several PAs are carcinogenic. There is a need to better understand the PA’s as toxins as well as to develop methods to identify PA contamination of food and feed. ARS researchers at the PPRL in Logan, UT have identified two additional PAs in two Cryptantha species for the first time, as well as a new chemotype of the PA-containing Amsinckia (fiddleneck). Toxic doses of the PA-containing plant fireweed in cattle have also been determined. Additionally, researchers have developed several in vitro and in vivo models of PA poisoning including a transgenic model of carcinogenesis. Initial carcinogenesis studies have shown that several additional PAs are carcinogenic and that subclinical low PA doses increased the incidence of cancer development. Cell culture models have been developed and used to better compare toxicity of PA compounds. Researchers at the PPRL determined that several PAs are significantly more cytotoxic than riddelliine, the PA that has been listed as a potential human carcinogen. Toxicity appears to be alkaloid specific with little relationship to the base type or class of side chain. As the mechanisms of carcinogenesis and toxicity are probably the same, it is likely that the highly cytotoxic PAs present a larger neoplastic risk than riddelliine, the only one identified as a potential human carcinogen. This information will be used to better understand the mechanisms of intoxication and to better predict the risk of poisoning and carcinogenic potential in both animals and man.
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. Assays were developed to identify and quantitate the benzofuran ketones in white snakeroot and rayless goldenrod. Studies were conducted to determine the relative toxicity of the various benzofuran ketones and to definitively determine the toxin in white snakeroot and rayless goldenrod. The data generated from dosing plant material and extracts indicates that in addition to tremetone, there is likely an additional active compound(s) that is at least partially responsible for the toxicity of white snakeroot and rayless goldenrod. Once these additional toxins are identified assays will be developed to diagnose poisoning and to make risk assessments of plant populations.
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 and to identify potentially toxic plants. The research will be continued in project 5428-32630-012-00D.
Potential toxins involved in white snakeroot poisoning. The differences in chemical profiles and concentrations of potentially toxic compounds present in white snakeroot plants may explain the historically sporadic and unpredictable toxicity. White snakeroot populations having distinct chemical profiles of the putative toxins were dosed to goats by ARS scientists in Logan, Utah. Tremetone, which is believed to be the toxic compound in white snakeroot, did not correlate with poisoning using a Spanish goat model. Instead, dehydrotremetone concentrations correlated with toxicosis. Dosing of plant extracts indicates that other unidentified toxins have a significant role in causing white snakeroot poisoning. Data generated from such studies are essential for livestock producers and veterinarians to identify and avoid exposure to toxic populations of white snakeroot.
Identification of cytotoxic and potentially carcinogenic dehydro-pyrrolizidine alkaloids. The PA, riddelliine has been listed as a potential human carcinogen. ARS researchers in Logan, Utah used cell culture models to determine that several PAs (lasiocarpine, seneciophyline, senecionine, and heliotrine) were significantly more cytotoxic than riddelliine. Toxicity appears to be alkaloid specific with little relationship to the base type or class of side chain. As the mechanisms of carcinogenesis and toxicity are probably the same, it is likely that the highly cytotoxic PAs present a larger neoplastic risk than riddelliine, the only PA currently identified as a potential human carcinogen. Identification of PAs which are cytotoxic and potentially carcinogenic are essential to the identification and development of methods of analysis for PAs in animal feed, human food, and animal or human herbal products that will be used to prevent poisoning.
Cao, Y., Colegate, S.M., Edgar, J.A. 2013. Persistence of echimidine, a hepatotoxic pyrrolizidine alkaloid, from honey into mead. Journal of Food Composition and Analysis. 29(2): 106-9.
Colegate, S.M., Gardner, D.R., Davis, T.Z., Betz, J.M., Panter, K.E. 2013. Dehydropyrrolizidine alkaloids in two Cryptantha species: Including two new open chain diesters one of which is amphoteric. Phytochemical Analysis. 24: 201-12.
Colegate, S.M., Gardner, D.R., Davis, T.Z., Welsh, S.L., Betz, J.M., Panter, K.E. 2013. Identification of a lycopsamine-N-oxide chemotype of Amsinckia intermedia. Biochemical Systematics and Ecology. 48: 132-5.
Colegate, S.M., Gardner, D.R., Joy, R.J., Betz, J.M., Panter, K.E. 2012. Dehydropyrrolizidine alkaloids, including monoesters with an unusual esterifying acid, from cultivated Crotalaria juncea (Sunn Hemp cv. 'Tropic Sun'). Journal of Agricultural and Food Chemistry. 60: 3541-50.
Lee, S.T., Cook, D., Molyneux, R.J., Marcolongo-Pereira, C., Stonecipher, C.A., Gardner, D.R. 2013. The alkaloid profiles of Sophora nuttalliana and Sophora stenophylla. Biochemical Systematics and Ecology. 48: 58-64.