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Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" (CCS) that occurs when pregnant cattle graze teratogenic lupines. A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to determine if an intermittent schedule of ten continuous days of lupine treatment followed by 5 d off treatment would be sufficient to decrease, or prevent, the incidence of lupine-induced malformations. Continuous dosing of the teratogenic lupine ( Lupinus leucophyllus ) to pregnant cows for 30 d during the most susceptible stage of pregnancy (gestation days 40 to 70) resulted in severe skeletal birth defects in their calves. However, intermittent dosing of the teratogenic lupine demonstrated that interrupted intake of lupine reduced the severity, or eliminated, permanent skeletal malformations in calves born to cows dosed lupine. Toxicokinetic and ultrasound data demonstrated a clear inverse correlation between serum anagyrine (the primary teratogenic alkaloid in some lupines) concentrations in the dam and fetal movement. In the intermittent group, fetal movement quickly returned to normal after lupine feeding stopped and remained normal until lupine treatment resumed. Therefore, interrupting lupine intake for at least 5 d through an intermittent grazing program could reduce the severity of the CCS. Furthermore, this method would allow ranchers to move cattle back into lupine pastures after a brief interruption, which would allow for more efficient utilization of forage resources., Competing Interests: There is no conflict of interest for this work., (Published by Oxford University Press for the American Society of Animal Science 2024.)

Salvia reflexa (lance-leaf sage)-contaminated alfalfa hay was fed to ~500 mixed-breed beef cattle. Within hours of exposure, nearly half of the cattle developed lethargy, anorexia, depression, and recumbency, followed by bellowing, colic, and death. Even though the uneaten contaminated hay was removed the first day, nearly 100 animals died within the first 48 h. Three of these cattle were examined postmortem, and tissues and hay samples were collected for microscopic and chemical analysis. Several days later, a smaller number of the clinically poisoned cattle developed neurologic disease with aberrant behavior, aggression, icterus, blindness, exhaustion, and death. A total of 165 cattle were fatally poisoned. Poisoned cattle had swollen, dark, mottled livers that had a prominent nutmeg-like lobular pattern on cut section. Histologically, there was severe centrilobular-to-panlobular hepatic necrosis with marked hepatocellular swelling, degeneration, and necrosis. The surviving cattle developed liver disease characterized by altered serum biochemical analyses and microscopic hepatocellular degeneration and necrosis. In subsequent biopsies and analysis, these lesions resolved within 6-7 mo. After confirming toxicity of the hay in cattle, goats, and mice, followed by a mouse bioassay-guided chemical fractionation process, Salvia reflexa was identified as the contaminant in the hay responsible for the hepatotoxicity. S. reflexa has not been reported previously to cause fatal hepatotoxicity in livestock in North America, to our knowledge.

A case of baled alfalfa hay contaminated with multiple weeds induced hepatotoxicity and death in cattle. The hepatotoxic compounds were isolated by bioassay-guided fractionation using a mouse model and identified as salviarin, salvianduline D, rhyacophiline, and 7-hydroxyrhyacophiline. The structure of 7-hydroxyrhyacophiline has not been previously reported. All compounds were found to induce severe acute hepatic necrosis within 24-48 h after a single oral dosage (260-280 mg/kg). The identified diterpenes are known to be found among different Salvia species which led to finding dried plant parts of Salvia reflexa within bales of weedy hay and subsequently a population of S. reflexa was found along the field edges and irrigation ditch banks of the alfalfa hay field. It was thus determined that S. reflexa was responsible for the hepatotoxicity observed in cattle fed the contaminated hay.

The livestock industry in the western United States loses an estimated $500 million annually from livestock production losses due to poisonous plants. Poisoning of livestock by plants often goes undiagnosed because there is a lack of appropriate or available specimens for analysis. The Lupinus species represent an important toxic plant in western North America that can be toxic and/or teratogenic to livestock species due to the quinolizidine alkaloids. The objective of this study was to evaluate the potential of using earwax, hair, oral fluid, and nasal mucus as noninvasive specimens to determine livestock exposure to the teratogenic Lupinus species. Quinolizidine alkaloids were detected in these four matrices in cattle that were administered a single dose of Lupinus leucophyllus. In addition, quinolizidine alkaloids from lupine were detected in the earwax of cattle that grazed on lupine-infested rangelands. This study demonstrates the potential of earwax, hair, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by plants.

Introduction: Pro-toxic dehydropyrrolizidine alkaloids are associated with liver disease in humans. The potential for long-term, low-level or intermittent exposures to cause or contribute to chronically-developing diseases is of international concern. Eupatorium perfoliatum is a medicinal herb referred to as boneset. While the presence of dehydropyrrolizidine alkaloids in some Eupatorium species is well-established, reports on Eupatorium perfoliatum are scant and contradictory., Objective: To investigate the presence of dehydropyrrolizidine alkaloids in a survey of boneset samples and related alcoholic tinctures, and hot water infusions and decoctions., Methods: Methanol, hot water or aqueous ethanol extracts of Eupatorium perfoliatum and three closely-related species were subjected to HPLC-ESI(+)MS and MS/MS analysis using three complementary column methods. Dehydropyrrolizidine alkaloids were identified from their MS data and comparison with standards., Results: Forty-nine samples of Eupatorium perfoliatum were shown to contain dehydropyrrolizidine alkaloids (0.0002-0.07% w/w), the majority dominated by lycopsamine and intermedine, their N-oxides and acetylated derivatives. Alcoholic tinctures and hot water infusions and decoctions had high concentrations of the alkaloids. Different chemotypes, hybridisation or contamination of some Eupatorium perfoliatum samples with related species were suggested by the co-presence of retronecine- and heliotridine-based alkaloids., Conclusions: Sampling issues, low and high alkaloid chemotypes of Eupatorium perfoliatum or interspecies hybridization could cause the wide variation in dehydropyrrolizidine alkaloid concentrations or the different profiles observed. Concerns associated with dehydropyrrolizidine alkaloids provide a compelling reason for preclusive caution until further research can better define the toxicity and carcinogenicity of the dehydropyrrolizidine alkaloid content of Eupatorium perfoliatum. [Correction added on 12 July 2018, after first online publication: The 'Conclusions' section in the abstract has been added.]., (© 2018 John Wiley & Sons, Ltd.)

Phantasmidine, a rigid congener of the well-known nicotinic acetylcholine receptor agonist epibatidine, is found in the same species of poison frog ( Epipedobates anthonyi). Natural phantasmidine was found to be a 4:1 scalemic mixture, enriched in the (2a R,4a S,9a S) enantiomer by chiral-phase LC-MS comparison to the synthetic enantiomers whose absolute configurations were previously established by Mosher's amide analysis. The major enantiomer has the opposite S configuration at the benzylic carbon to natural epibatidine, whose benzylic carbon is R. Pharmacological characterization of the synthetic racemate and separated enantiomers established that phantasmidine is ∼10-fold less potent than epibatidine, but ∼100-fold more potent than nicotine in most receptors tested. Unlike epibatidine, phantasmidine is sharply enantioselective in its activity and the major natural enantiomer whose benzylic carbon has the 4a S configuration is more active. The stereoselective pharmacology of phantasmidine is ascribed to its rigid and asymmetric shape as compared to the nearly symmetric conformations previously suggested for epibatidine enantiomers. While phantasmidine itself is too toxic for direct therapeutic use, we believe it is a useful platform for the development of potent and selective nicotinic agonists, which may have value as pharmacological tools.

Selenium (Se) poisoning by different forms of Se occurs in the United States. However, the toxicokinetics of different selenocompounds after oral ingestion is not well documented. In this study the toxicokinetics of Se absorption, distribution and elimination were determined in serum and whole blood of lambs that were orally dosed with increasing doses of Se as sodium selenite (inorganic Se) or selenomethionine (SeMet, organic Se). Thirty-two lambs were randomly assigned to eight treatment groups, with four animals per group. Se was administered at 1, 2 or 3 mg kg -1 body weight, as either sodium selenite or SeMet with proper control groups. Blood and serum were collected at predetermined time points for 7 days post-dosing. Resulting Se concentrations in both serum and whole blood from SeMet treatment groups were significantly greater than those given equimolar doses of Se as sodium selenite. Se concentrations in serum and whole blood of lambs dosed with SeMet peaked at significantly greater concentrations when compared with lambs dosed with equimolar doses of sodium selenite. Based on the serum and whole blood kinetics, the rate of Se absorption was greater for SeMet than for sodium selenite although rates of absorption for both Se forms decreased with increasing dose. The rates of Se elimination increased with dose. These results demonstrate that SeMet has a greater absorption rate and a similar retention time resulting in a greater area under the curve and thus bioavailability than sodium selenite, which must be considered in both overdose and nutritional exposures. Published 2016. This article is a U.S. Government work and is in the public domain in the USA., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (<3 mm) were examined for maturation and in vivo developmental competence after SCNT. Significantly greater maturation rate was observed in oocytes derived from large follicles compared with that of small follicles (51.6% and 33.7%, P < 0.05). Greater percent of large follicle oocytes exhibited a low glucose-6-phosphate dehydrogenase activity at germinal vesicle stage compared with small follicle oocytes (54.9% and 38.7%, P < 0.05). Relative mRNA expression analysis of 48 genes associated with embryonic and fetal development revealed that three genes (MATER, IGF2R, and GRB10) had higher level of expression in metaphase II oocytes from large follicles compared with oocytes from small follicles. Nevertheless, no difference was observed in pregnancy rates (33.3% vs. 47.1%) and birth rates (22.2% vs. 16.7%) after SCNT between the large and small follicle groups). These results indicate that metaphase II cytoplasts from small and large follicles have similar developmental competence when used in goat SCNT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Swainsonine, an indolizidine alkaloid, is an α-mannosidase and mannosidase II inhibitor that causes lysosomal storage disease and alters glycoprotein processing. Swainsonine is found in a number of plant species worldwide, and is produced by associated endophytic fungi. Prolonged consumption of swainsonine-containing plants by livestock causes a condition characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death. In contrast, Astragalus and Oxytropis that do not contain swainsonine may present a valuable food source for grazing livestock in regions where palatable forage is scarce. This study tested the hypothesis that swainsonine concentrations may be reduced by fungicide treatment or by clipping, thus reducing plant toxicity. Additionally we hypothesized that clipping plants may provide a mechanism for horizontal transmission of the endophyte. To this end, four different fungicides were applied to render the endophyte non-viable, and plant vegetative tissues were periodically clipped. Treatment of Oxytropis sericea with any of four different fungicides did not alter swainsonine concentrations in plants at any of three harvest times. Additionally, we found that individual or multiple clippings had no effect on swainsonine concentrations; plants that contained swainsonine maintained concentrations, and plants low or absent in swainsonine also remained as such at each harvest. These results suggest that there is no evidence of horizontal transmission of the endophyte among individual plants due to clipping., (Published by Elsevier Ltd.)

Introduction: Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-β1 and investigated the changes in the cardiac structure and function leading to AF., Methods and Results: Transgenic goats with cardiac specific overexpression of constitutively active TGF-β1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12 months of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (>2 minutes), whereas none of 6 controls displayed sustained AF (P < 0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687 ± 212.02 seconds vs. 2.50 ± 0.88 seconds, P < 0.0001), but no persistent or permanent AF was observed., Conclusion: A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-β1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility., (© 2016 Wiley Periodicals, Inc.)

Medusahead [ (L.) Nevski] has become a major invasive plant on the annual grass-dominated rangelands within the Channeled Scablands of eastern Washington. Livestock typically avoid grazing medusahead, and forage alternatives are becoming limited in the region. Our hypothesis was that supplying a high-CP supplement would provide a nutritional context that complements the nutritional composition of medusahead and other annual grasses and thus aid cattle in utilizing this vegetation component, making grazing a more effective method of weed control. Cattle grazed annual grass-infested rangelands dominated with medusahead for 10-d periods during June, July, and August over 2 consecutive years. Eight separate pastures were grazed by cattle pairs during each of the 3 grazing periods. Cattle in 4 control pastures received no supplement and cattle in another 4 pastures received a supplement of canola meal that supplied 75% of the daily recommended CP requirement. Bite counts were used to determine diet composition. Forage categories consisted of annual grasses, perennial grasses, and forbs. Bites taken of annual grasses were similar between treatment groups during the first 5 d of the grazing period ( > 0.05), and then cattle supplemented with canola meal increased consumption of annual grasses, during d 6 to 10 of the grazing periods, over nonsupplemented animals ( < 0.05). Consumption of annual grasses was greater during the second year of the grazing study ( < 0.05), likely due to a decline in abundance of forage alternatives in the plant community. The percentage of medusahead in the annual grass forage class tended to decrease in grazed pastures over the 3 yr of the study ( = 0.056): 87 ± 4.2, 64 ± 3.6, and 50 ± 3.6%, respectively. The percentage of medusahead in the annual grasses was similar across years in nongrazed pastures ( > 0.05). Forb production was greatest the first year of grazing and declined the second year of grazing and continued to decline the following year with no grazing ( < 0.05). Perennial grass production was low throughout the study. The effects of grazing on medusahead abundance suggest cattle may be used to graze this weed after it has matured in an integrated management program with other forms of control to reduce infestation prior to seeding with desirable forage species.

Comfrey (Symphytum officinale), a commonly used herb, contains dehydropyrrolizidine alkaloids that, as a group of bioactive metabolites, are potentially hepatotoxic, pneumotoxic, genotoxic and carcinogenic. Consequently, regulatory agencies and international health organizations have recommended comfrey be used for external use only. However, in many locations comfrey continues to be ingested as a tisane or as a leafy vegetable. The objective of this work was to compare the toxicity of a crude, reduced comfrey alkaloid extract to purified lycopsamine and intermedine that are major constituents of S. officinale. Male, California White chicks were orally exposed to daily doses of 0.04, 0.13, 0.26, 0.52 and 1.04 mmol lycopsamine, intermedine or reduced comfrey extract per kg bodyweight (BW) for 10 days. After another 7 days chicks were euthanized. Based on clinical signs of poisoning, serum biochemistry, and histopathological analysis the reduced comfrey extract was more toxic than lycopsamine and intermedine. This work suggests a greater than additive effect of the individual alkaloids and/or a more potent toxicity of the acetylated derivatives in the reduced comfrey extract. It also suggests that safety recommendations based on purified compounds may underestimate the potential toxicity of comfrey., (Published 2015. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Dehydropyrrolizidine alkaloids (DHPA) are a large, structurally diverse group of plant-derived protoxins that are potentially carcinogenic. With worldwide significance, these alkaloids can contaminate or be naturally present in the human food supply. To develop a small animal model that may be used to compare the carcinogenic potential of the various DHPAs, male heterozygous p53 knockout mice were administered a short-term treatment of riddelliine 5, 15 or 45 mg kg(-1) bodyweight day(-1) by oral gavage for 14 days, or dosed a long-term treatment of riddelliine 1 mg kg(-1) bodyweight day(-1) in pelleted feed for 12 months. Exposure to riddelliine increased the odds of tumor development in a dose-responsive manner (odds ratio 2.05 and Wald 95% confidence limits between 1.2 and 3.4). The most common neoplastic process was hepatic hemangiosarcoma, which is consistent with published lifetime rodent riddelliine carcinogenesis studies. Angiectasis (peliosis hepatis) and other previously unreported lesions were also identified. The results of this research demonstrate the utility of the heterozygous p53 knockout mouse model for further investigation of comparative carcinogenesis of structurally and toxicologically different DHPAs and their N-oxides., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Calves with congenital defects born to cows that have grazed teratogenic Lupinus spp. during pregnancy can suffer from what is termed crooked calf syndrome. Crooked calf syndrome defects include cleft palate, spinal column defects and limb malformations formed by alkaloid-induced inhibition of fetal movement. In this study, we tested the hypothesis that there are differences in fetal activity of fetuses carried by Holstein verses Angus heifers orally dosed with 1.1 g/kg dried ground Lupinus leucophyllus. Fetal activity was monitored via transrectal ultrasonography and maternal serum was analyzed for specific lupine alkaloids. There were more (P < 0.05) movements in fetuses of Holstein heifers than those in Angus heifers at eight and 12 h after oral dosing. In addition to serum alkaloid toxicokinetic differences, the Holstein heifers had significantly lower serum concentrations of anagyrine at 2, 4, and 8 h after oral dosing than Angus heifers. Holstein heifers also had significantly greater serum concentrations of lupanine at 12, 18 and 24 h after dosing than the Angus heifers. These results suggest that there are breed differences in susceptibility to lupine-induced crooked calf syndrome. These differences may also be used to discover genetic markers that identify resistant animals, thus facilitating selective breeding of resistant herds., (Published by Elsevier Ltd.)

Selenium-accumulator plants are reputed to be unpalatable to large ungulates. Elk (Cervus canadensis) populations in southeastern Idaho overlap with populations of Se-rich plants, but there is no information on the influence of plant Se concentration on elk dietary preferences. The objective of this study was to determine, under controlled conditions, the preference of elk for feeds varying in Se concentrations. Seven yearling female elk (128 ± 5 kg) were purchased from a commercial elk farm in southeastern Idaho and adapted to low-Se alfalfa pellets. Three feeding trials using pellets with predetermined Se concentrations were conducted. Alfalfa pellets were commercially made with the addition of Symphyotrichum ascendens (western aster) so that the pellets contained 0.4, 5, 20, 50, or 100 mg/kg (DM basis) Se. In trial 1, 5 Se-containing alfalfa pellets (0.4, 5, 20, 50, and 100 mg/kg Se) were offered for 10 d; trial 2 used 4 Se-containing alfalfa pellet choices (0.4, 20, 50, and 100 mg/kg), and trial 3 used 3 pellet choices (0.4, 50, and 100 mg/kg) for 6 d. In trial 1, consumption of the control pellets by elk was greater than each of the other pellet choices (P < 0.001). Similarly, consumption of the 5-mg/kg Se pellet differed from control pellet and all other Se-containing pellets (P < 0.0001). There were no differences (P > 0.50) in consumption of the 20-, 50-, or 100-mg/kg Se pellets. In trial 2, elk consumed more (P < 0.0001) of the control pellet than the 20-, 50-, and 100-mg/kg Se pellets. Similarly, elk consumed more (P < 0.0001) of the 20-mg/kg Se pellet than the 50- and 100-mg/kg Se pellets. There were no differences (P > 0.99) in elk consumption of the 50- and 100-mg/kg Se pellets. In trial 3, elk consumption of the control and 50- and 100-mg/kg Se pellets differed (P ≤ 0.03) from one another each day except that on d 1 and 2, where elk consumption of the 50- and 100-mg/kg Se pellets did not differ (P ≥ 0.32). Elk clearly discriminated against pellets with the highest Se concentrations when they were given pellets with differing Se concentrations. These results suggest that elk are not likely to select forages with high Se concentrations, particularly when high-Se plants are present in a rangeland situation with numerous other forage choices.

Numerous evergreen trees and shrubs contain labdane acids, including isocupressic acid, which can cause late-term abortions in cattle. Recent research has shown that the bark from western juniper trees can also cause late-term abortions in cattle. Additionally, ranchers have observed that cattle in western juniper-infested rangelands tend to have decreased conception rates. The objective of this study was to determine if western juniper alters the estrous cycle of cattle. Fourteen heifers (10 treated and 4 control) were monitored for 74 days for signs of normal estrous behavior, with a 21 day feeding trial with western juniper bark from days 28-48, after which the cattle were bred naturally with a bull. The cattle were checked for pregnancy 30 days after all cattle had been bred. The data from this study indicate that exposure to western juniper bark does not affect normal estrus, estrous cycle or conception rates of cattle., (Published by Elsevier Ltd.)

Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement., (Published by Elsevier Ltd.)

Numerous plants are known to be teratogenic in livestock. In addition to causing malformations, several plants can also cause embryonic death. These losses decrease the reproductive efficiency of animals exposed to these plants. The aim of this study was to determine if teratogenic plants such as lupines or tobaccos cause embryonic losses. A goat model using the plant Nicotiana glauca was used in this study, as this model has been used to characterize the mechanism of Lupinus, Conium, and Nicotiana-induced terata. Four groups of goats were dosed from gestational day 1-10, 11-20, 21-30, and 31-40. Goats were evaluated via ultrasound imaging for pregnancy after completion of the dosing regimen and kids were evaluated for malformations at the time of parturition. Overall, there was no evidence from this study that N. glauca (anabasine) at this dose (2 g/kg/day) would cause embryonic losses in goats. However, the dose of N. glauca used in this study was at the lower threshold that would be expected to produce terata. Therefore it is possible that higher doses of anabasine could cause embryonic loss. Further work is also needed to characterize the kinetic profile of anabasine, and other teratogenic alkaloids, in the fetal compartments., (Published by Elsevier Ltd.)

Introduction: The diversity of structure and, particularly, stereochemical variation of the dehydropyrrolizidine alkaloids can present challenges for analysis and the isolation of pure compounds for the preparation of analytical standards and for toxicology studies., Objective: To investigate methods for the separation of gram-scale quantities of the epimeric dehydropyrrolizidine alkaloids lycopsamine and intermedine and to compare their NMR spectroscopic data with those of their heliotridine-based analogues echinatine and rinderine., Methods: Lycopsamine and intermedine were extracted, predominantly as their N-oxides and along with their acetylated derivatives, from commercial samples of comfrey (Symphytum officinale) root. Alkaloid enrichment involved liquid-liquid partitioning of the crude methanol extract between dilute aqueous acid and n-butanol, reduction of N-oxides and subsequent continuous liquid-liquid extraction of free base alkaloids into CHCl3 . The alkaloid-rich fraction was further subjected to semi-automated flash chromatography using boronated soda glass beads or boronated quartz sand., Results: Boronated soda glass beads (or quartz sand) chromatography adapted to a Biotage Isolera Flash Chromatography System enabled large-scale separation (at least up to 1-2 g quantities) of lycopsamine and intermedine. The structures were confirmed using one- and two-dimensional (1) H- and (13) C-NMR spectroscopy. Examination of the NMR data for lycopsamine, intermedine and their heliotridine-based analogues echinatine and rinderine allowed for some amendments of literature data and provided useful comparisons for determining relative configurations in monoester dehydropyrrolizidine alkaloids. A similar NMR comparison of lycopsamine and intermedine with their N-oxides showed the effects of N-oxidation on some key chemical shifts. A levorotatory shift in specific rotation from +3.29° to -1.5° was observed for lycopsamine when dissolved in ethanol or methanol respectively., Conclusion: A semi-automated flash chromatographic process using boronated soda glass beads was standardised and confirmed as a useful, larger scale preparative approach for separating the epimers lycopsamine and intermedine. The useful NMR correlations to stereochemical arrangements within this specific class of dehydropyrrolizidine alkaloid cannot be confidently extrapolated to other similar dehydropyrrolizidine alkaloids. Published 2014. This article is a U.S. Government work and is in the public domain in the USA., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

A number of plant toxins have been shown to be teratogenic to livestock. The teratogenic action of some of these alkaloids is mediated by nicotinic acetylcholine receptors (nAChR). However, for many of these alkaloids it is difficult to obtain sufficient quantities of individual alkaloids to perform teratology studies in livestock species. Therefore the objective of this study was to determine if a rat model can be utilized to characterize the teratogenic nature of individual plant toxins that are nAChR agonists. In this study, we evaluated the teratogenicity of anabasine by feeding pregnant rats anabasine-containing rodent chow from gestational day (GD) 6-21. On GD21, the dams were euthanized and the gravid uteri were removed. The gravid uteri and individual pups were weighed. The pups were evaluated for bone malformations including cleft palate and scoliosis. Overall, the results of this study suggest that the rat is not a good model to study the teratogenicity of plant toxins that are nAChR agonists. It is possible that in the rat model, anabasine administered orally via the chow may not result in sufficient reduction in fetal movement to cause the significant malformations observed in livestock species., (Published by Elsevier Ltd.)

In the late 1960s, the steroidal alkaloid cyclopamine was isolated from the plant Veratrum californicum and identified as the teratogen responsible for craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in the western United States. Cyclopamine was found to inhibit the hedgehog (Hh) signaling pathway, which plays a critical role in embryonic development. More recently, aberrant Hh signaling has been implicated in several types of cancer. Thus, inhibitors of the Hh signaling pathway, including cyclopamine derivatives, have been targeted as potential treatments for certain cancers and other diseases associated with the Hh signaling pathway. A brief history of cyclopamine and cyclopamine derivatives investigated for the treatment of cancer is presented.

Species of the Amsinckia genus (Boraginaceae) are known to produce potentially hepato-, pneumo-, and/or genotoxic dehydropyrrolizidine alkaloids. However, the taxonomic differentiation of Amsinckia species can be very subtle and there seems to be marked differences in toxicity toward grazing livestock. Methanol extracts of mass-limited leaf samples from herbarium specimens (collected from 1899 to 2013) of 10 Amsinckia species and one variety were analyzed using HPLC-esi(+)MS and MS/MS for the presence of potentially toxic dehydropyrrolizidine alkaloids and/or their N-oxides. Dehydropyrrolizidine alkaloids were detected in all specimens examined ranging from about 1 to 4000 μg/g of plant. Usually occurring mainly as their N-oxides, the predominant alkaloids were the epimeric lycopsamine and intermedine. Also sometimes observed in higher concentrations were the 3'- and 7-acetyl derivatives of lycopsamine/intermedine and their N-oxides. Within a designated species, an inconsistent profile was often observed that may be due to natural variation, taxonomic misassignment, or nonuniform degradation due to plant collection and storage differences.

When livestock are poisoned by plants in a range setting, there is normally more than one poisonous plant in that area. Additionally, many plants contain more than one compound that is toxic to livestock. Frequently, much is known regarding the toxicity of the individual plants and their individual toxins; however, little is known regarding the effect of coexposure to multiple toxic plants or even the effect of multiple toxins from an individual plant. This review discusses some basic principles of mixture toxicology with a focus on recent research that examined the effect of co-administering multiple plant toxins from the same plant and the effect of co-administration of two different poisonous plants, each with different types of toxins. As combined intoxications are likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of multiple poisonous plants to livestock.

The 9th International Symposium on Poisonous Plants (ISOPP9) was held July 15-21, 2013, at the Inner Mongolia Agricultural University in Hohhot, Inner Mongolia Autonomous Region of China. The symposium consisted of three days of oral and poster presentations, followed by a tour of the Xilinhot Region of the Mongolian Grasslands, encompassing grazing conditions consisting of desert, grassland, and steppes. This was the first time that an ISOPP meeting has been held in Asia and provided an opportunity for visitors from outside China to become aware of livestock poisonings caused by plant species with which they were previously not familiar while at the same time demonstrating that many of the problems experienced around the world have a common etiology. Presentations focused on botany, veterinary science, toxicology, mechanism of action, and chemistry. As is appropriate for the Journal of Agricultural and Food Chemistry, this cluster of papers consists of selected oral and poster presentations in which the chemistry of the toxins played a significant role. The symposium revealed that there is considerable scope for isolation, structural elucidation, and analysis of the toxins from the numerous poisonous plant species that have been identified in China. It became apparent that there are abundant opportunities for chemists both within China and abroad to collaborate with Chinese scientists working on biological aspects of livestock poisonings.

In most cases where livestock are poisoned by plants in a range setting, there is more than one potential poisonous plant in the same area. Two poisonous plants that are often found growing simultaneously in the same location are death camas (Zigadenus spp.) and low larkspur (Delphinium spp.). Sheep are known to be susceptible to death camas poisoning while they are thought to be resistant to larkspur. The objective of this study was to determine if co-administration of low larkspur would exacerbate the toxicity of death camas in sheep. A dose finding study was performed to find a dose of death camas that caused minimal clinical signs of poisoning. Sheep were observed for clinical signs of poisoning as well as changes in heart rate and muscle fatigue. Sheep dosed with 1.14 g of death camas per kg BW showed slight frothing and lethargy, whereas sheep dosed with death camas and low larkspur showed slightly more noticeable clinical signs of poisoning. Sheep dosed with only low larkspur, at 7.8 g/kg BW, showed no signs of poisoning. Although we observed a qualitative difference in clinical signs of intoxication in sheep co-treated with death camas and low larkspur we did not detect any quantitative differences in heart rate, exercise-induced muscle fatigue, or differences in serum zygacine kinetics. Consequently, the results from this study suggest that low larkspur does not affect the toxicity of death camas in sheep. The results from this study increase knowledge and understanding regarding the acute toxicity of death camas and low larkspur in sheep. As combined intoxications are most likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of death camas to other livestock species., (Published by Elsevier Ltd.)

The exposure of a developing embryo or fetus to alkaloids from plants, plant products, or plant extracts has the potential to cause developmental defects in humans and animals. These defects may have multiple causes, but those induced by piperidine and quinolizidine alkaloids arise from the inhibition of fetal movement and are generally referred to as multiple congenital contracture-type deformities. These skeletal deformities include arthrogyrposis, kyposis, lordosis, scoliosis, and torticollis, associated secondary defects, and cleft palate. Structure-function studies have shown that plant alkaloids with a piperidine ring and a minimum of a three-carbon side-chain α to the piperidine nitrogen are teratogenic. Further studies determined that an unsaturation in the piperidine ring, as occurs in gamma coniceine, or anabaseine, enhances the toxic and teratogenic activity, whereas the N-methyl derivatives are less potent. Enantiomers of the piperidine teratogens, coniine, ammodendrine, and anabasine, also exhibit differences in biological activity, as shown in cell culture studies, suggesting variability in the activity due to the optical rotation at the chiral center of these stereoisomers. In this article, we review the molecular mechanism at the nicotinic pharmacophore and biological activities, as it is currently understood, of a group of piperidine and quinolizidine alkaloid teratogens that impart a series of flexure-type skeletal defects and cleft palate in animals., (Copyright © 2013 Wiley Periodicals, Inc.)

Poisonous plant research in the United States began over 100 years ago as a result of livestock losses from toxic plants as settlers migrated westward with their flocks, herds, and families. Major losses were soon associated with poisonous plants, such as locoweeds, selenium accumulating plants, poison-hemlock, larkspurs, Veratrum, lupines, death camas, water hemlock, and others. Identification of plants associated with poisoning, chemistry of the plants, physiological effects, pathology, diagnosis, and prognosis, why animals eat the plants, and grazing management to mitigate losses became the overarching mission of the current Poisonous Plant Research Laboratory. Additionally, spin-off benefits resulting from the animal research have provided novel compounds, new techniques, and animal models to study human health conditions (biomedical research). The Poisonous Plant Research Laboratory has become an international leader of poisonous plant research as evidenced by the recent completion of the ninth International Symposium on Poisonous Plant Research held July 2013 in Hohhot, Inner Mongolia, China. In this article, we review plants that negatively impact embryo/fetal and neonatal growth and development, with emphasis on those plants that cause birth defects. Although this article focuses on the general aspects of selected groups of plants and their effects on the developing offspring, a companion paper in this volume reviews current understanding of the physiological, biochemical, and molecular mechanisms of toxicoses and teratogenesis., (Copyright © 2013 Wiley Periodicals, Inc.)

The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research suggested a correlation between the lethal dose (LD50 ) of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. The objective of this study was to characterize the role of the α7 subunit in the acute toxicosis of MLA by evaluating the acute toxicity of MLA in mice lacking the α7 subunit. The LD50 values for MLA were 4.2 ± 0.9, 3.7 ± 1.1 and 3.3 ± 0.9 mg kg(-1) body weight (BW) for wild-type, heterozygous knockout and homozygous knockout mice, respectively. We also evaluated the response of anabasine in these mice. The LD50 values for anabasine were 1.6 ± 0.3, 2.0 ± 0.4 and 1.8 ± 0.3 mg kg(-1) BW for wild-type, heterozygous knockout and homozygous knockout mice, respectively. The protein expresson of various nAChR subunits was compared to determine if mice lacking the α7 subunit compensate by over expressing other nAChR subunits. There were no significant differences in the protein expression of the α3 , α4 , α5 , β2 and β4 subunits amongst the three genotypes of mice in brain or skeletal muscle. The results of this study suggest that α7 nAChR does not play an integral role in the acute toxicosis of MLA or anabasine. Consequently other nAChR subunits of nAChRs found in the neuromuscular junction are probably the primary target for MLA and anabasine resulting in acute toxicosis., (Published 2013. This article is a US Government work and is in the public domain in the USA.)

The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. However, recent research demonstrated that there was no difference in the acute lethality (LD50 ) to MLA in mice lacking the α7 nAChR subunit compared with wild-type mice. The objective of this study was to determine if the α7 nAChR subunit plays a role in motor coordination deficiencies that result from exposure to nAChR antagonists and agonists. We compared the motor function and coordination in wild-type mice to mice lacking the α7 subunit of the nAChR, after treating them with a non-lethal dose of MLA or anabasine, using the following tests: balance beam, grip strength, rotarod, open field and tremor monitor. Analysis of the data indicated that overall there was no difference between the wild-type and knockout mice (P = 0.39 for grip strength; P = 0.21 for rotarod; P = 0.41 for balance beam; P = 0.22 for open field; and P = 0.62 for tremors). Thus results from this study suggest that α7 nAChR does not play an integral role in the acute effects of MLA or anabasine on motor function/coordination. Consequently other subunits of nAChRs found in the neuromuscular junction are likely the primary target for MLA and anabasine resulting in motor coordination deficiencies and acute toxicosis., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Consumption of Se accumulator plants by livestock can result in Se intoxication. Recent research indicates that the Se forms most common in Se accumulator plants are selenate and Se-methylselenocysteine (MeSeCys). In this study the absorption, distribution, and elimination kinetics of Se in serum and whole blood of lambs dosed with a single oral dose of (1, 2, 3, or 4 mg Se/kg BW) of sodium selenate or MeSeCys were determined. The Se concentrations in serum and whole blood for both chemical forms of Se followed simple dose-dependent relationships. Se-methylselenocysteine was absorbed more quickly and to a greater extent in whole blood than sodium selenate, as observed by a greater peak Se concentration (Cmax; P < 0.0001), and faster time to peak concentration (Tmax; P < 0.0001) and rate of absorption (P < 0.0001). The rate of absorption and Tmax were also faster (P < 0.0001) in serum of lambs dosed with MeSeCys compared with those dosed sodium selenate at equimolar doses; however, Cmax in serum was greater (P < 0.0001) in lambs dosed with sodium selenate compared with those dosed MeSeCys at equimolar doses. The MeSeCys was absorbed 4 to 5 times faster into serum and 9 to 14 times faster into whole blood at equimolar Se doses. There were dose-dependent increases in the area under the curve (AUC) for Se in serum and whole blood of lambs dosed with both sodium selenate and MeSeCys. In whole blood the MeSeCys was approximately twice as bioavailable as sodium selenate at equimolar doses as observed by the AUC, whereas in serum there were no differences (P > 0.05) in AUC at the same doses. At 168 h postdosing the Se concentration in whole blood remained much greater (P < 0.0001) in lambs dosed with MeSeCys as compared with lambs dosed with sodium selenate; however, the serum Se concentrations were not different between treatments at the same time point. The results presented in this study demonstrate that there are differences between the kinetics of different selenocompounds when orally dosed to sheep. Therefore, in cases of acute selenosis, it is important to understand the chemical form to which an intoxicated animal was exposed when determining the importance and meaning of Se concentration in serum or whole blood obtained at various times postexposure.

Plants produce a wide variety of chemical compounds termed secondary metabolites that are not involved in basic metabolism, photosynthesis, or reproduction. These compounds are used as flavors, fragrances, insecticides, dyes, hallucinogens, nutritional supplements, poisons, and pharmaceutical agents. However, in some cases these secondary metabolites found in poisonous plants perturb biological systems. Ingestion of toxins from poisonous plants by grazing livestock often results in large economic losses to the livestock industry. The chemical structures of these compounds are diverse and range from simple, low molecular weight toxins such as oxalate in halogeton to the highly complex norditerpene alkaloids in larkspurs. While the negative effects of plant toxins on people and the impact of plant toxins on livestock producers have been widely publicized, the diversity of these toxins and their potential as new pharmaceutical agents for the treatment of diseases in people and animals has also received widespread interest. Scientists are actively screening plants from all regions of the world for bioactivity and potential pharmaceuticals for the treatment or prevention of many diseases. In this review, we focus the discussion to those plant toxins extensively studied at the USDA Poisonous Plant Research Laboratory that affect the nicotinic acetylcholine receptors including species of Delphinium (Larkspurs), Lupinus (Lupines), Conium (poison hemlock), and Nicotiana (tobaccos).

Inhibition of fetal movement is one mechanism behind the development of multiple congenital contracture-type defects in developing fetuses of humans and animals. We tested the alkaloids anabasine, lobeline, and myosmine for agonist actions, and sensitivity to alpha conotoxins EI and GI blockade at fetal muscle-type nicotinic acetylcholine receptors (nAChR) expressed by TE-671 cells. We also determined if the alkaloids decreased fetal movement in an IV dosed, day 40 pregnant goat model. In TE-671 cells, all three alkaloids elicited concentration-dependent changes in membrane potential sensing dye fluorescence. 1.0 μM alpha conotoxin GI shifted the concentration-effect curves of anabasine and myosmine to the right, and decreased maximal responses. Neither of the conotoxins blocked the actions of lobeline in TE-671 cells. In the day 40 pregnant goats, 0.8 mg/kg anabasine abolished fetal movement at 30 and 60 min after dosing and fetal movement was reduced by lobeline and myosmine. The blockade of anabasine and myosmine actions in TE-671 cells by alpha conotoxin GI indicates that they are agonists at fetal muscle-type nAChR. All three alkaloids did significantly decrease fetal movement in the day 40 pregnant goat model suggesting a potential for these alkaloids to cause multiple congenital contracture-type defects in developing fetuses., (Published by Elsevier Ltd.)

Introduction: A livestock poisoning outbreak near Kingman, Arizona, USA, potentially linked to dehydropyrrolizidine alkaloids, prompted an evaluation of some local plants for the presence of these hepatotoxic alkaloids., Objective: To qualitatively and quantitatively examine two species of Cryptantha, a Boraginaceous genus previously shown to produce potentially toxic pyrrolizidine alkaloids, collected from the vicinity of Kingman, Arizona., Method: Plant extracts were analysed using HPLC-electrospray ionisation (+)-MS and MS/MS to determine the presence of dehydropyrrolizidine alkaloid esters. Identities were confirmed by comparison of chromatographic and MS data with authenticated standards and, in the case of the previously undescribed alkaloids, using one- and two-dimensional NMR spectroscopy and high-resolution mass measurement., Results: Cryptantha inequata and C. utahensis were shown to produce retronecine-based dehydropyrrolizidine alkaloids at approximately 0.05% and 0.09% w/w respectively. Cryptantha inequata produced mainly echimidine, acetylechimidine and echiuplatine; dehydropyrrolizidine alkaloids that were previously associated with Echium plantagineum. The previously undescribed structure of echiuplatine was elucidated as an amphoteric, open chain diester with angelic acid and 3-hydroxy-3-methylglutaric acid. Along with lycopsamine, intermedine and dihydroxyechiumine, C. utahensis produced cryptanthine, a previously undescribed open chain diester alkaloid esterified with angelic acid and 2,3-dihydroxy-2-methylbutanoic acid. All pyrrolizidine alkaloids detected were present in the plants mainly as their N-oxides., Conclusion: The retronecine-based alkaloids detected in both Cryptantha species herein investigated aligns them within the Krynitzkia subgenus. The dehydropyrrolizidine alkaloids detected are expected to be toxic but the low levels in the plants potentially mitigate the risk. The identification of the amphoteric echiuplatine provides a cautionary note with respect to the analysis of total dehydropyrrolizidine alkaloid content., (Published 2012. This article is a US Government work and is in the public domain in the USA.)

γ-Coniceine, coniine, and N-methylconiine are toxic alkaloids present in poison hemlock (Conium maculatum). We previously reported the comparison of the relative potencies of (+)- and (-)-coniine enantiomers. In this study, we synthesized γ-coniceine and the enantiomers of N-methylconiine and determined the biological activity of γ-coniceine and each of the N-methylconiine enantiomers in vitro and in vivo. The relative potencies of these piperidine alkaloids on cells expressing human fetal muscle-type nicotinic acetylcholine receptors had the rank order of γ-coniceine > (-)-N-methylconiine > (±)-N-methylconiine > (+)-N-methylconiine. The relative lethalities of γ-coniceine and (-)-, (±)-, and (+)-N-methylconiine in vivo using a mouse bioassay were 4.4, 16.1, 17.8, and 19.2 mg/kg, respectively. The results from this study suggest γ-coniceine is a more potent agonist than the enantiomers of N-methylconiine and that there is a stereoselective difference in the in vitro potencies of the enantiomers of N-methylconiine that correlates with the relative toxicities of the enantiomers in vivo.

Coniine is an optically active toxic piperidine alkaloid and nicotinic acetylcholine receptor (nAChR) agonist found in poison hemlock (Conium maculatum L.). Coniine teratogenicity is hypothesized to be attributable to the binding, activation, and prolonged desensitization of fetal muscle-type nAChR, which results in the complete inhibition of fetal movement. However, pharmacological evidence of coniine actions at fetal muscle-type nAChR is lacking. The present study compared (-)-coniine, (+)-coniine, and nicotine for the ability to inhibit fetal movement in a day 40 pregnant goat model and in TE-671 cells that express fetal muscle-type nAChR. Furthermore, α-conotoxins (CTx) EI and GI were used to antagonize the actions of (+)- and (-)-coniine in TE-671 cells. (-)-Coniine was more effective at eliciting electrical changes in TE-671 cells and inhibiting fetal movement than was (+)-coniine, suggesting stereoselectivity by the receptor. The pyridine alkaloid nicotine did not inhibit fetal movement in a day 40 pregnant goat model, suggesting agonist specificity for the inhibition of fetal movement. Low concentrations of both CTxs potentiated the TE-671 cell response and higher concentrations of CTx EI, and GI antagonized the actions of both coniine enantiomers demonstrating concentration-dependent coagonism and selective antagonism. These results provide pharmacological evidence that the piperidine alkaloid coniine is acting at fetal muscle-type nAChR in a concentration-dependent manner.

Isocupressic acid (ICA) is the abortifacient compound in ponderosa pine (Pinus ponderosa L.) needles, which can cause late-term abortions in cattle (Bos taurus). However, cattle rapidly metabolize ICA to agathic acid (AGA) and subsequent metabolites. When pine needles are dosed orally to cattle, no ICA is detected in their serum, whereas AGA is readily detected. Recent research has demonstrated that AGA is also an abortifacient compound in cattle. The observation has been made that when cattle are dosed with labdane acids for an extended time, the concentration of AGA in serum increases for 1 to 2 d but then decreases to baseline after 5 to 6 d even though they are still being dosed twice daily. Therefore, in this study we investigated whether cattle conditioned to pine needles metabolize ICA, and its metabolites, faster than naïve cattle. Agathic acid was readily detected in the serum of naïve cattle fed ponderosa pine needles, whereas very little AGA was detected in the serum of cattle conditioned to pine needles. We also compared the metabolism of ICA in vitro using rumen cultures from pine-needle-conditioned and naïve cattle. In the rumen cultures from conditioned cattle, AGA concentrations were dramatically less than rumen cultures from naïve cattle. Thus, an adaptation occurs to cattle conditioned to pine needles such that the metabolism AGA by the rumen microflora is altered.

Objective: To determine the toxicokinetics of N-(methylsuccinimido)anthranoyllycoctonine-type low larkspur alkaloids in beef cattle., Animals: 5 Black Angus steers and 35 Swiss Webster mice., Procedures: -Low larkspur (Delphinium andersonii) was collected, dried, ground, and administered to 5 steers via oral gavage to provide a dose of 12 mg of N-(methylsuccinimido)-anthranoyllycoctonine alkaloids/kg. Steers were housed in metabolism crates for 96 hours following larkspur administration; heart rate was monitored continuously, and blood samples were collected periodically for analysis of serum concentrations of 16-deacetylgeyerline, methyllycaconitine, geyerline, and nudicauline and assessment of kinetic parameters. The LD(50) of a total alkaloid extract from D andersonii was determined in Swiss Webster mice., Results: -The alkaloids were quickly absorbed, with a maximum serum concentration achieved within 18 hours after administration. Geyerline and nudicauline coeluted as 1 peak and were considered together for toxicokinetic analysis. Mean ± SD elimination half-life was 18.4 ± 4.4 hours, 15.6 ± 1.5 hours, and 16.5 ± 5.1 hours for 16-deacetylgeyerline, methyllycaconitine, and geyerline and nudicauline, respectively. There were significant differences in maximum serum concentration, amount absorbed, and distribution half-life among the 4 alkaloids. The mouse LD(50) was 9.8 mg/kg., Conclusions and Clinical Relevance: -Results suggested that clinical poisoning was likely to be most severe approximately 18 hours after exposure. Cattle should be closely monitored for at least 36 hours after initial exposure. Additionally, a withdrawal time of approximately 7 days would be required to clear > 99% of the toxic alkaloids from the serum of cattle that have ingested low larkspur.

Cyclopamine (1) was isolated from the plant Veratrum californicum Durand (Liliacea) and identified as the teratogen responsible for severe craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in central Idaho. More recently, cyclopamine (1) was found to inhibit the hedgehog (Hh) signaling pathway which plays a critical role in embryonic development and is implicated in several types of cancer. Thus, cyclopamine (1) and cyclopamine derivatives have been targeted as potential pharmaceutical treatments for certain cancers and other diseases associated with the Hh signaling pathway. A monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay was developed to detect and measure cyclopamine (1) and cyclopamine derivatives in biological samples. The limits of detection of the assay for cyclopamine (1), 3-keto-N-aminoethyl aminocaproyl digyrocinnamoyl-cyclopamine (8), and N-(4-l-rhamnopyranosyl-1H-1,2,3-triazol-1-yl)-methylcyclopamine (11) were 2.9 pg, 0.41 pg and 2.6 pg, respectively. This assay was also found to be useful for the detection and measurement of cyclopamine (1) in sera from mice that had been dosed with cyclopamine (1). The simple ELISA method described herein demonstrates the potential of using these techniques for the rapid screening of biological samples for the presence and levels of cyclopamine (1) and other cyclopamine derivatives that are Hh inhibitors with anticancer potential., (Published by Elsevier B.V.)

Delphinium spp. contain numerous norditerpenoid alkaloids which are structurally delineated as 7, 8-methylenedioxylycoctonine (MDL) and N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids. The toxicity of many tall larkspur species has been primarily attributed to their increased concentration of MSAL-type alkaloids, such as methyllycaconitine (MLA), which are typically 20 times more toxic than MDL-type alkaloids. However, the less toxic MDL-type alkaloids are often more abundant than MSAL-type alkaloids in most Delphinium barbeyi and Delphinium occidentale populations. Previous research demonstrated that MDL-type alkaloids increase the acute toxicity of MSAL-type alkaloids. In this study, we examined the role of MDL-type alkaloids on the overall toxicity of tall larkspur plants to cattle while controlling for the exact dose of MSAL-type alkaloids. Cattle were dosed with plant material from 2 different populations of tall larkspur containing either almost exclusively MDL- or MSAL-type alkaloids. These 2 plant populations were combined to create mixtures with ratios of 0.3:1, 1:1, 5:1, and 10:1 MDL- to MSAL-type alkaloids. The dose that elicited similar clinical signs of poisoning in mice and cattle was determined for each mixture on the basis of the MSAL-type alkaloid content. As the ratio of MDL- to MSAL-type alkaloids increased, the amount of MSAL-type alkaloids required to elicit clinical signs decreased. These results indicate that the less toxic MDL-type alkaloids in tall larkspur exacerbate the toxicity of the MSAL-type alkaloids. Consequently, both the amount of MSAL-type alkaloids and the amount of total alkaloids should be fully characterized to determine more accurately the relative toxicity of tall larkspur plant material.

Piperidine alkaloids are acutely toxic to adult livestock species and produce musculoskeletal deformities in neonatal animals. These teratogenic effects include multiple congenital contracture (MCC) deformities and cleft palate in cattle, pigs, sheep, and goats. Poisonous plants containing teratogenic piperidine alkaloids include poison hemlock (Conium maculatum), lupine (Lupinus spp.), and tobacco (Nicotiana tabacum) [including wild tree tobacco (Nicotiana glauca)]. There is abundant epidemiological evidence in humans that link maternal tobacco use with a high incidence of oral clefting in newborns; this association may be partly attributable to the presence of piperidine alkaloids in tobacco products. In this review, we summarize the evidence for piperidine alkaloids that act as teratogens in livestock, piperidine alkaloid structure-activity relationships and their potential implications for human health., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This article provides an overview of the Poisonous Plant Research Laboratory (PPRL), about the unique services and activities of the PPRL and the potential assistance that they can provide to plant poisoning incidences. The PPRL is a federal research laboratory. It is part of the Agricultural Research Service, the in-house research arm of the U.S. Department of Agriculture. The mission of the PPRL is to identify toxic plants and their toxic compounds, determine how the plants poison animals, and develop diagnostic and prognostic procedures for poisoned animals. Furthermore, the PPRL's mission is to identify the conditions under which poisoning occurs and develop management strategies and treatments to reduce losses. Information obtained through research efforts at the PPRL is mostly used by the livestock industry, natural resource managers, veterinarians, chemists, plant and animal scientists, extension personnel, and other state and federal agencies. PPRL currently has 9 scientists and 17 support staff, representing various disciplines consisting of toxicology, reproductive toxicology, veterinary medicine, chemistry, animal science, range science, and plant physiology. This team of scientists provides an interdisciplinary approach to applied and basic research to develop solutions to plant intoxications. While the mission of the PPRL primarily impacts the livestock industry, spinoff benefits such as development of animal models, isolation and characterization of novel compounds, elucidation of biological and molecular mechanisms of action, national and international collaborations, and outreach efforts are significant to biomedical researchers. The staff at the PPRL has extensive knowledge regarding a number of poisonous plants. Although the focus of their knowledge is on plants that affect livestock, oftentimes, these plants are also poisonous to humans, and thus, similar principles could apply for cases of human poisonings. Consequently, the information provided herein could be of benefit to healthcare providers for human cases as well.

Introduction: Many species in the Lupinus genus are poorly defined morphologically, potentially resulting in improper taxonomic identification. Lupine species may contain quinolizidine and/or piperidine alkaloids that can be acutely toxic and/or teratogenic, the latter resulting in crooked calf disease., Objective: To identify characteristic alkaloid profiles of Lupinus sabinianus, L. garfieldensis and L. sericeus which would aid in discriminating these species from each other and from L. sulphureus., Methods and Materials: Quinolizidine and piperidine alkaloids were extracted from herbarium specimens and recent field collections of L. sabinianus, L. garfieldensis and L. sericeus. The alkaloid composition of each species was defined using GC-FID and GC-MS and compared using multivariate statistics., Results: Each of the three species investigated contained a diagnostic chemical fingerprint composed of quinolizidine and/or piperidine alkaloids., Conclusion: The alkaloid profiles of Lupinus sabinianus, L. garfieldensis and L. sericeus can be used as a tool to discriminate these species from each other and L. sulphureus as long as one considers locality of the collection in the case of L. sabinianus., (Published 2011. This article is a US Government work and is in the public domain in the USA.)

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, extracts of the roots, stems, leaves, and seeds of 'Tropic Sun' were analyzed for their presence using HPLC-ESI/MS. Qualitative analysis identified previously unknown alkaloids as major components along with the expected macrocyclic dehydropyrrolizidine alkaloid diesters, junceine and trichodesmine. The dehydropyrrolizidine alkaloids occurred mainly as the N-oxides in the roots, stems, and, to a lesser extent, leaves, but mainly as the free bases in the seeds. Comprehensive spectrometric and spectroscopic analysis enabled elucidation of the unknown alkaloids as diastereoisomers of isohemijunceine, a monoester of retronecine with an unusual necic acid. The dehydropyrrolizidine alkaloid contents of the roots, stems, and leaves of immature plants were estimated to be 0.05, 0.12, and 0.01% w/w, respectively, whereas seeds were estimated to contain 0.15% w/w.

Sixteen of approximately 500 yearling steers died of acute selenium (Se) toxicosis after grazing on a Se-contaminated range for only a few days. Field studies and chemical analyses identified the predominant toxic plant as western aster (Symphyotrichum ascendens, previously Aster ascendens), which contained over 4,000 ppm Se (dry weight). Several dead animals that were necropsied had acute severe myocardial necrosis characterized by edema and myocyte swelling, with hypereosinophilia, clumping, and coagulation of myocardial proteins. Whole blood from 36 surviving steers was collected and analyzed, and 10 steers with elevated Se concentrations were selected for close monitoring and clinical evaluations. Each steer was weighed, and serum, blood, liver, skeletal muscle, and hair were regularly collected after removal from the Se-contaminated range. One animal that died 18 days after exposure was necropsied and exhibited severe multifocal myocardial fibrosis with extensive hepatic congestion, degeneration, and hemosiderosis. At 180 days postexposure, 2 of the 10 steers were euthanized, and tissue samples were collected. Both steers had rare, small fibrotic foci in their hearts. The Se elimination half-lives from serum, whole blood, liver, and muscle of the recovering steers were 40.5 ± 8.2, 115.6 ± 25.1, 38.2 ± 5.0, and 98.5 ± 19.1 days, respectively. The Se concentration in hair reached a peak of 11.5 ± 5.3 ppm at 22 days postexposure. The findings indicate that cattle are sensitive to acute Se toxicosis caused by ingestion of Se-accumulator plants, with myocardial necrosis as the primary lesion. Some poisoned animals may develop congestive heart failure weeks after the toxic exposure, and in the current study, Se was slowly excreted requiring a relatively long withdrawal time.

As man-made chemicals (anthropogenic) are increasing in number and amount of use, it is not uncommon for farmers, ranchers, consultants, or veterinarians to suspect a xenobiotic (strange or foreign substance) as the cause of reproductive failures. In this article, the authors discuss toxicants that have been shown to affect reproduction, with emphasis on food-producing animals and fowl. The discussion is brief, and written to provide a resource for clinicians, students, and scientists by focusing on toxicant-induced reproductive dysfunction relevant to the toxicant, source, clinical effects, and livestock species known or suspected to be affected., (Copyright © 2011. Published by Elsevier Inc.)

Death camas (Zigadenus spp.) is a common poisonous plant on foothill rangelands in western North America. The steroidal alkaloid zygacine is believed to be the primary toxic component in death camas. Poisonings on rangelands generally occur in the spring when death camas is abundant, whereas other more desirable forage species are limited in availability. In most cases where livestock are poisoned by plants in a range setting, there is more than one potential poisonous plant in that area. One common poisonous plant that is often found growing simultaneously in the same area as death camas is low larkspur (Delphinium nuttallianum). Consequently, the objectives of this study were to conduct acute toxicity studies in mice and to determine if coadministration of low larkspur will exacerbate the toxicity of death camas. We first characterized the acute toxicity of zygacine in mice. The LD(50) of zygacine administered intravenously (i.v.) and orally was 2.0 ± 0.2 and 132 ± 21 mg/kg, respectively. The rate of elimination of zygacine from whole blood was determined to be 0.06 ± 0.01/min, which corresponds to an elimination half-life of 13.0 ± 2.7 min. The i.v. LD(50) of total alkaloid extracts from a Utah and a Nevada collection were 2.8 ± 0.8 and 2.2 ± 0.3 mg/kg, respectively. The i.v. LD(50) of methyllycaconitine (MLA), a major toxic alkaloid in low larkspur, was 4.6 ± 0.5 mg/kg, whereas the i.v. LD(50) of a 1:1 mixture of MLA and zygacine was 2.9 ± 0.7 mg/kg. The clinical signs in mice treated with this mixture were very similar to those of mice treated with zygacine alone, including the time of onset and death. These results suggest that there is an additive effect of coadministering these 2 alkaloids i.v. in mice. The results from this study increase knowledge and understanding regarding the acute toxicity of death camas. As combined intoxications are most likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of death camas to livestock.

Objective: To compare the toxic effects of a Delphinium occidentale chemotype containing N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids and a D occidentale chemotype lacking MSAL-type alkaloids in mice and cattle., Animals: 225 male Swiss Webster mice and 11 Black Angus steers., Procedures: 4 collections of larkspur containing MSAL-type alkaloids and 4 collections of larkspur lacking MSAL-type alkaloids were used. From each collection, total alkaloid extracts (0.05 to 0.20 mL) were administered via tail-vein injection in 27 to 29 mice. Dried, finely ground plant material from 1 collection with and 1 collection without MSAL-type alkaloids (doses equivalent to 37.6 mg of total alkaloids/kg) were each administered to 8 cattle via oral gavage in a crossover experiment; 3 cattle received a single dose equivalent to 150.4 mg of total alkaloids/kg (no MSAL-type alkaloids). In mice, clinical effects were monitored; in cattle, heart rate was monitored before (baseline) and 24 hours after treatment. At the 24-hour time point, cattle were exercised as a measure of muscle weakness., Results: In mice, mean LD(50) associated with alkaloid extracts prepared from plants that did or did not contain MSAL-type alkaloids was 2.3 and 54.2 mg/kg, respectively. In cattle at 24 hours after treatment, plant material containing MSAL-type alkaloids significantly increased heart rate from baseline and was associated with exercise-induced collapse; plant material lacking MSAL-type alkaloids had no similar effects., Conclusions and Clinical Relevance: Taxonomic classification of D occidentale alone was not a good indicator of the toxic risk to grazing cattle.