Location: Poisonous Plant Research2009 Annual Report
1a. Objectives (from AD-416)
Objective I: Pine Needles 1.1 Determine if isocupressic acid (ICA; the abortifacient compound in pine needles) concentration in pine needles is modulated by the environment. 1.2 Identify the matabolites of isocupressic acid in pine needles that cause abortions in cattle. Determine the biological mechanism, develop diagnostic techniques, and therapeutic procedures. 1.3 Determine factors that influence cattle consumption of pine needles and develop management practices to prevent abortion. Objective II: Broom Snakeweed 2.1 Identify the toxic and abortifacient compounds in broom snakeweed. 2.2 Describe the ecology of broom snakeweed, develop management and control guidelines to reduce incidence of poisoning and abortion in livestock. Objective III: Lupine 3.1 Isolate, identify, and evaluate toxicity and teratogenicity of lupine alkaloids which cause birth defects in calves born to cows that graze these plants. 3.2 Evaluate the role of genotype and environment on lupine alkaloids, and thus the relative toxicity of various species and populations of lupine. 3.3 Determine the physiological mechanism of lupine-induced birth defects and evaluate the maternal and fetal toxicokinetics of alkaloids. 3.4 Evaluate the influence of climate on population cycles of lupine. 3.5 Determine the importance of lupines as nutritional components for cattle during critical times of the year. 3.6 Identify conditions under which cattle graze various lupine species. Objective IV: Veratrum 4.1 Develop models to study the toxicokinetics, including clearance times, and toxicity of steroidal alkaloids in Veratrum californicum.
1b. Approach (from AD-416)
1.1 Data on environmental conditions will be collected at each site using local weather stations. ICA levels and environmental conditions will be correlated to determine if any patterns emerge. Soil samples will be collected at each site for future evaluation. 1.2 Samples of maternal and fetal tissues will be collected for histologic analysis and determination of ICA concentrations using existing ELISA’s and GC/MS methods. Proteomic analyses via LC/MS/MS techniques will be done. 1.3 Pen and field studies using cattle in high, medium and low body condition will be done to determine effects on needle consumption and grazing times. Nutrient supplements will be offered to determine if pine needle consumption will be altered. 2.1 The diterpene acid “fingerprint” of broom snakeweed from various populations in Arizona, New Mexico and Utah will be determined by chemical analysis. Subsequent in vitro and in vivo studies will be done to determine abortifacient activity. 2.2 A grazing study will be conducted to determine if various management practices can be implemented to force cattle to graze snakeweed as a biological control. A clipping study will be conducted to further describe the effects of defoliation on snakeweed and the surrounding plant community. 3.1 Alkaloids will be isolated by chemical methods and identified by chromatography, NMR, mass spectrometry, and elemental analysis. Toxicology will be evaluated using a mouse bioassay and cell lines that express nicotinic acetylcholine receptors. 3.2 A chemical fingerprint of Lupinus sulphureus collected from different locations will be generated using chemical methods. Fingerprints will be analyzed via cluster analysis and phylogenetic analysis will be performed using AFLPs (Amplified Fragment Length Polymorphisms) to determine the genetic relationship of the populations. 3.3 Pregnant goats in late gestation will be used to determine the rate of absorption, distribution and elimination of the teratogenic alkaloids. The pharmacokinetic profiles of the alkaloids will be compared between maternal and fetal systems. 3.4 Established transects will be monitored over the next 5 years to determine the influence of weather patterns on lupine density. Correlations of lupine age, class, density, and trends will be made with seasonal precipitation and temperature. 3.5 Consumption of lupines by cattle on rangelands dominated by low quality forages may be related to nutrient content. Twelve yearling heifers in a field study will be supplemented with different levels of protein to compare lupine ingestion. 3.6 Short-duration and high intensity grazing studies in early, mid, and late summer will be used to determine what role grazing pressure has on lupine intake during different seasons of the year. 4.1 A monogastric model (swine) will be used to determine the kinetics (clearance and metabolism) of a well known teratogenic alkaloid from Veratrum. This pilot project will be a model for testing the clearance of other plant toxins from animal tissues to evaluate food safety of animal products. Clearance rates between the monogastric model and small ruminant model will be compared.
3. Progress Report
Grazing of ponderosa pine needles, broom snakeweed, specific juniper species, lodgepole pine and Monterey cypress by pregnant cows results in abortion/premature parturition, retained placentas, endometritis, death in cows, and dead calves. Isocupressic acid (ICA) has been identified as the toxin in pine needles, lodgepole pine and Monterey cypress responsible for the abortion and other associated complications. ICA has been shown to be metabolized in the rumen and in the liver. Initial collections to evaluate if ICA concentrations change as a function of the environment have been made. An ELISA assay has been developed for ICA and ICA metabolites and has been used to detect these compounds in cow tissues. Pen studies showed that cattle on low protein diets consume lower amounts of fresh pine needles. Analytical methods to profile labdane diterpene in broom snakeweed plants have been investigated. Both gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry methods have been established to profile the diterpene acids. These methods will be used to examine differences in broom snakeweed populations and examine metabolism of diterpene acids to possible abortifacient compounds in the rumen or hepatic system. Several herbicides were applied to broom snakeweed at Monticello and Nephi UT in May and August 2008. Plots were evaluated in June 2009. Picloram was most effective, Picloram + 2,4-D1/4, Aminopyralid and Metsulfuron were acceptable. In the western United States lupines cause sporadic and often large losses to cattle from “crooked calf syndrome”. When pregnant cattle graze lupine containing select piperdine and quinolizidine alkaloids during days 40-100 of gestation calves with congenital deformities such as crooked legs, neck and back and cleft palate can be born. A pharmacodynamic comparison of the alkaloids anabasine, anabaseine, anagyrine, ammodendrine, and coniine and their enantiomers in two cell lines and in a murine model was performed and the alkaloids and enantiomers were rank ordered in terms of potency (toxicity). In three grazing trials, cattle were reluctant to graze lupine in May and June when cheatgrass and forbs were green and succulent. In July, lupine was preferred, because cheatgrass was dry and annual forbs had matured. Increasing grazing pressure can force cattle to graze lupine in any season as desirable forages are depleted.
1. Goat cleft palate model to elucidate mechanism of lupine induced "crooked calf" syndrome. Using a goat cleft palate model developed at the Poisonous Plant Research Laboratory, new techniques and surgical models have been developed to improve treatment of the cleft palate condition in children. Fetal cleft palate repair at day 85 of gestation clearly demonstrates the potential for successful repair in utero without scar tissue formation. The molecular mechanism for cleft palate induction by piperidine alkaloids found in poisonous plants remains elusive, however, we have demonstrated an association between alkaloid-induced reduction in fetal movement and palatal clefting using the congenital goat model. It is probable that the alkaloids that cause cleft palate formation act at the nicotinic receptors resulting in loss of activity. Fetal cleft palate repair at day 85 of gestation in goats demonstrates the potential for successful repair in utero in children without scar tissue formation. This model serves to train surgeons in cleft palate repair and has ended the controversy over whether abnormal jaw structure in children is due to the clefting anomaly itself and not a result of the surgical repair.
2. Efficacy-dependent actions of piperidine alkaloid teratogens and enantiomers. Lupines and other plants such as Conium maculatum L., Nicotiana glauca and Nicotiana tabaccum contain toxic and teratogenic piperdine alkaloids in mixtures of enantiomers. Although, the enantiomers have the same physical properties the enantiomers have different physiological properties. A pharmacodynamic comparison of the alkaloids anabasine, anabaseine, anagyrine, ammodendrine, and coniine in two cell lines was made. These alkaloids and their enantiomers were more effective in depolarizing TE-671 cells which express the human fetal-muscle type nicotinic cholinergic receptor (nAChR) relative to SHSY-5Y cells which predominately express human autonomic nAChRs. The rank order of potency in TE-671 cells was: anabaseine > (+)-anabasine > (-)-anabasine > (±)-anabasine > anagyrine > (-)-coniine > (±)-coniine > (+)-coniine > (±)-ammodendrine > (+)-ammodendrine. The rank order potency in SHSY-5Y cells was: anabaseine > (+)-anabasine > (-)-coniine > (+)-coniine > (+)-ammodendrine > anagyrine > (-)-anabasine > (±)-coniine > (±)-anabasine > (-)-ammodendrine. These findings support the hypothesis that the mechanism behind the teratogenic potential of these compounds is the stimulation of muscle-type nAChR followed by desensitization and finally inhibition of fetal movement. This information provides a pharmacodynamic comparison of the alkaloids anabasine, anabaseine, anagyrine, ammodendrine, and coniine in cell lines demonstrated the difference in toxicity between enantiomers.
3. High intensity grazing systems force cattle to graze lupine. Increasing grazing pressure in intensive grazing systems may force cattle to graze lupine when it is most toxic and during the critical period of gestation. Three, 10 day grazing trials were conducted in May, June and July 2007 and 2008. Forage was restricted to allow only enough to last for the 10 day grazing trials. Increasing grazing pressure as the trials progressed forced cattle to graze lupine towards the end of the trial in May, and in the middle of the trial in June. In July, lupine was the most palatable forage available, and cattle selected it at the beginning of the trial. Results indicate that lupine is relatively palatable in July and August, but cattle can be forced to consume it in intensive grazing systems earlier in the season. Results will guide ranchers using intensive grazing systems when lupine is present in the plant community.
4. Influence of dietary protein on pine needle consumption in cattle. Pregnant cattle often ingest ponderosa pine needles during winter leading to abortions and retained placentas. Previous work at PPRL has shown that dietary protein concentration may influence intake of fresh pine needles by cattle. Pen feeding studies were conducted to determine the influence of high, moderate, or low concentrations of dietary nitrogen (5, 10, and 15 % crude protein; isocaloric diets) on consumption of fresh ponderosa pine needles. Pine needles contain high concentrations of terpenes, and previous work has indicated that cattle on a low protein diet may consume lower quantities of needles because they have insufficient N for effective detoxification or elimination. Rank order of consumption by cattle in the treatment groups were High > Moderate > Low dietary protein concentrations. A second study was conducted to determine if graded amounts of condensed tannin (quebracho tannin; CT) added to a moderate protein diet would alter pine needle consumption. Condensed tannins often bind to dietary protein, and alter N metabolism in ruminants. There was no influence of dietary CT on the amount of pine needles eaten by cattle. This study confirmed and extended previous work showing that cattle on low protein diets consume lower amounts of fresh pine needles in pen studies. Results will be of interest to scientists working with herbivores grazing or browsing on high terpene forages or browse. In addition, livestock producers may be able to reduce incidence of pine needle abortion by manipulating the diets of susceptible cattle during winter periods. Understanding dietary factors important in consumption of pine needles will assist in developing and refining management recommendations to reduce losses.
5. The Alkaloid profiles of Lupinus sulphureus. Lupines are common plants on the rangelands in the western United States. The Lupinus genus is notoriously complex and difficult to classify taxonomically. The general problem is that few species have received detailed study regarding alkaloid composition, taxonomic delineations, and/or phylogenetic relationships. Lupines contain alkaloids that can be toxic and teratogenic causing congenital birth defects (crooked calf syndrome). One such lupine, Lupinus sulphureus, occurs in parts of Oregon, Washington, and British Columbia. Specimens of L. sulphureus from field collections and herbaria were evaluated taxonomically and by chemical means. A total of seven distinct alkaloid profiles and the individual alkaloids associated with each profile were identified. Each alkaloid profile was unique in its geographical distribution and its potential risk to livestock. Taxonomic characterization of the alkaloids must also be performed. These results are being used to define geographical regions where lupines can be or not be teratogenic. Livestock producers will be able to graze cattle in areas that do not contain the teratogenic alkaloids with no risk of losses from lupine, thus providing valuable summer forage.
Cook, D., Lee, S.T., Gardner, D.R., Pfister, J.A., Welch, K.D., Green, B.T., Davis, T.Z., Panter, K.E. 2009. The Alkaloid Profiles of Lupinus sulphureus. Journal of Agriculture and Food Chemistry, 57(4):1646-1653.