2011 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.
Pyrrolizidine alkaloids (PA’s) are toxins found in a wide variety of plants worldwide and are responsible for poisoning livestock, wildlife, and humans. Some analytical techniques and diagnostic procedures have been developed, however improved methods to monitor PA contamination of food and feed, identify poisoned animals, and predict the fate of poisoned animals are needed. The chemistry of producing the didehydropyrrolizidine alkaloids (“pyrroles”) has been investigated and a post-Doctoral candidate was recently hired to continue with the project by investigating the conjugation chemistry. To further investigate the conjugation chemistry a human hepatocyte cell line has been cultured and cells have been successfully transferred to OpticellsTM that will allow “real time” microscopic monitoring of the effects of the dehydropyrrolizidine alkaloids on the cell monolayers. Additionally several high purity samples of several dehydropyrrolizidine alkaloids of varying structure and some N-oxides have been prepared and structurally characterized in readiness for the in vitro investigation of effects on cultured animal and human hepatocytes, and for in vivo studies of their relative carcinogenic potential with a recently developed model utilizing p53 knockout mice. Histological studies of the microscopic lesions comparing toxicity of PA compounds in the p53 knockout mice are being conducted. Grazing studies were successfully completed in Hawaii to determine how cattle grazed the PA containing plant fireweed. PA analyses of forages that are used to improve PA-plant infested rangelands were also successfully completed.
Rayless goldenrod and white snakeroot cause many cases of poisoning in the southwestern and midwestern United States, respectively. Chemical techniques and procedures were developed and the best methods for handling, and storing plant material and its extracts for studies to further investigate the toxins were determined. Animal studies, in both pregnant and nonpregnant animals, comparing the toxicity of white snakeroot collections with differing chemical profiles were conducted. Physiographic and electrocardiographic studies and biochemical analysis of blood indicate that the toxicities of the plant populations are significantly different. The histological analyses of tissues from the animals in these studies have commenced.
Phylloerythrin and other plant metabolites cause photosensitivity in animals. The tropical grass species Brachiaria and Panicum sp.can cause hepatogenous photosensitization grazed by livestock. It is believed that the steroidal saponin may be the primary toxin. Assays to measure the saponin concentrations in the plants were developed and the saponin concentrations were monitored in Brachiaria sp. over a 12 month period to determine when grazing the grasses was the highest risk.
Stability of benzofuran ketones in "tremetol"-containing plants. Rayless goldenrod and white snakeroot are plants that contain tremetol toxins that cause skeletal and cardiac muscles lesions in cattle, horses and goats. The suspected toxins were previously thought to be unstable when plants were dried as in harvested hay or when the toxins were extracted from the plants. ARS scientists in Logan, Utah, determined that the compounds are stable in dried plant and in crude extracts. This information confirms the risk of feeding hay contaminated with white snakeroot or rayless goldenrod to livestock.
Concentrations of pyrrolyzidine alkaloids (PAs) in Crotalaria juncea L. cultivars. Cultivation of Crotalaria juncea L. (sunn hemp cv.Tropic Sun) is recommended as a green manure crop in a rotation cycle to improve soil condition, help control erosion, suppress weeds and reduce soil nematodes. Because C. juncea belongs to a genus that is known for the production of toxic dehydropyrrolizidine alkaloids (PAs), ARS researchers in Logan, Utah, in collaboration with the NCRS in Hawaii, analyzed extracts of the roots, stems, leaves and seeds of the Tropic Sun cultivar for the presence of PA’s and determined this cultivar to be low risk for animal poisoning. As fertilizer costs increase and environmental concerns continue green manures are a significant source of natural soil conditioning and Crotalaria appears to be a safe and good source of natural nitrogen.
Quantitative assay for indospicine in Indogofera species. Some Indigofera species contain a toxic non-protein amino acid known as indospicine which is poisonous to livestock. Cases of secondary poisonings in dogs that consume the meat of animals that previously grazed indospicine-containing forage have been reported. ARS scientists in Logan, UT, developed a quantitative analytical method for indoscipine in plants. This method was also applied to the analysis of several samples of Indigofera that were collected in Brazil associated with poisoning of horses. Furthermore, this analytical method was applied to a different species of Indigofera responsible for poisoning and "floppy trunk syndrome" in elephants in Africa. This assay is available to Diagnostic Labs and scientists throughout the world where Indogofera plants are a risk to livestock, wildlife, pets and people.
Conditions that cause cattle to graze pyrrolizidine (PA)-containing fireweed in Hawaii. Fireweed is a toxic, invasive weed across much of the Hawaiian Islands. Fireweed contains toxic PAs that poison livestock when ingested. Fireweed is responsible for large economic losses to the cattle industry in Hawaii as virtually all livers from cattle grazing Hawaiian rangelands are condemned at slaughter. A grazing study was conducted to quantify the consumption of fireweed (Senecio madagascariensis) by cattle during spring in Hawaii (Maui). ARS researchers in Logan, Utah, determined that cattle will only graze fireweed under conditions of forage scarcity, and that good pasture management will prevent consumption by cattle. The information from the study will be used by cattle producers to better manage their cattle in pasture rotations in order to prevent consumption of fireweed and improve economic viability of the rural regions of Hawaii.
Saponin concentrations in Brachiaria species. Many tropical grasses cause hepatogenous photosensitization, among them are several species of Brachiaria. There is evidence that steroidal saponins present in these plants may be the primary cause. ARS scientists in Logan, Utah, determined saponin concentrations in two species of Brachiaria (B. decumbens cv. Basilisk and B. brizantha cv. Marandu) from Brazil over the grazing season. In both species, young plants had a higher concentration of saponin than mature plants while B. decumbens contained higher concentrations of saponins than B. brizantha. Researchers concluded that young plants pose a higher risk of causing photosensitization in livestock grazing Brachiaria than do mature plants. This information will be used by cattle producers to manage grazing programs to reduce losses.
Water hemlock poisoning in cattle. Water hemlock is one of the most poisonous plants in North America. Generally, poisoning occurs in early spring when the plants are first starting to emerge from the tubers. The tubers and plants are very palatable and poisoning often occurs when animals go to water and ingest early growth plants or disturb the highly toxic tubers and subsequently ingest them. Water hemlock seed has not been reported to be toxic until now. In one field case of poisoning, 11 cows ingested the immature seed heads of water hemlock and died within 2 days. ARS researchers in Logan, UT, evaluated the toxins and potential poisoning of green seeds of water hemlock and determined the seeds to be extremely toxic and equal to, or of greater toxicity than the tubers. Therefore, recommendations to livestock producers now includes removing mature water hemlock plants and avoiding grazing pastures where water hemlock is in the early seed stage of growth.
Toxin identified in an endangered plant. Pyrrolizidine alkaloids (PAs) are toxins found in a wide variety of plants worldwide and are responsible for poisoning livestock, wildlife, and humans. ARS researchers in Logan, UT, investigated the endangered plant called Terlingua Creek cryptantha (Cryptantha crassipes) from rangelands in Texas and reported the presence of these toxins at levels that would be poisonous to livestock or wildlife if consumed. Researchers also identified a PA in this endangered species common among other Cryptantha species that grow in less arid climates of the west and postulated that these toxins may be critical for these genera to survive in harsh environmental conditions.
Latorre, A.O., Caniceiro, B.D., Wysocki, H.L., Haraguchi, M., Gardner, D.R., Gorniak, S.L. 2011. Selenium reverses Pteridium aquilinum-induced immunotoxic effects. Food and Chemical Toxicology. 49(2):464-70.
Meyerholtz, K.A., Burcham, G.N., Miller, M.A., Wilson, C.R., Hooser, S.B., Lee, S.T. 2011. Development of a gas chromatography-mass spectrometry technique to diagnose white snakeroot (Ageratina altissima) poisoning in a cow. Journal of Veterinary Diagnostic Investigation. 23:775-9.
Molyneux, R.J., Gardner, D.R., Colegate, S.M., Edgar, J.A. 2011. Pyrrolizidine alkaloid toxicity in livestock: A paradigm for human poisoning. Journal of Food Additives & Contaminants. Part A. 28(3): 293-307.
Lee, S.T., Mitchell, R., Gardner, D.R., Tokarnia, C.H., Riet-Correa, F. 2011. Measurement of steroidal saponins in Panicum and Brachiaria grasses in the USA and Brazil. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge,MA. CAB International. 20:142-4.
Pal, B.B., Singh, D.K., Stegelmeier, B.L. 2011. Buffalo calves intoxicated with Ageratum houstonianum mill. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 27:186-9.
Stegelmeier, B.L. 2010. Bracken fern poisoning. In: Kahn, C.M., Line, S., editors. Merck Veterinary Manual. 10th Edition. Rahway, N.J. Merck Publishing Group. p. 2670-2.
Stegelmeier, B.L. 2010. Sweet clover poisoning. In: Kahn, C.M., Line, S., editors. Merck Veterinary Manual. Tenth Edition. Rahway, N.J. Merck Publishing Group. p.2731-2.
Stegelmeier, B.L., Sani, Y., Pfister, J.A. 2011. Baccharis pteronioides toxicity. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 72:433-6.
Williams, M.T., Warnock, B.J., Betz, J.M., Beck, J.J., Gardner, D.R., Lee, S.T., Molyneux, R.J., Colegate, S.M. 2011. Detection of high levels of pyrrolizidine-N-oxides in the endangered plant cryptantha crassipes (Terlingua creek cat's-eye)using HPLC-ESI-MS. Phytochemical Analysis. 22(3) DOI: 10.1002/pca.1314.
Edgar, J.A., Colegate, S.M., Boppre, M., Molyneux, R.J. 2011. Pyrrolizidine alkaloids in food: A spectrum of potential health consequences. Journal of Food Additives & Contaminants. Part A. 28(3):308-24.
Barbosa-Ferreira, M., Pfister, J.A., Gotardo, A.T., Raspantini, P.C.F., Gorniak, S.L. 2011. Effects of Senna occidentalis seeds ingested during gestation on kid behavior. In: Riet-Correa, F., Pfister, J., Schild, A.L., Wierenga, T., editors. Poisoning by Plants, Mycotoxins, and Related Toxins. Cambridge, MA. CAB International. 40:264-9.