Review

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Plant Toxins That Affect Nicotinic Acetylcholine Receptors: A Review Benedict T. Green,* Kevin D. Welch, Kip E. Panter, and Stephen T. Lee

USDA/ARS Poisonous Plant Research Laboratory, 1150 East 1400 North, Logan, Utah 84341, United States

ABSTRACT: 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).

■ CONTENTS pollination or seed dispersal or in some cases, such as alkaloids in seeds, may act as a nitrogen reservoir for germination and Introduction 1129 early growth. Given the significance of plant compounds to Nicotinic Acetylcholine Receptors 1129 people and animals, the primary function of secondary Plant Toxins 1131 metabolites in plants remains a topic of extensive discussion Delphinium 1131 and research.1 The chemical structures of these compounds are Lupinus 1132 diverse and range from simple, low molecular weight toxins Conium 1133 such as oxalate in halogeton to the highly complex norditerpene Nicotiana 1134 alkaloids in larkspurs. While the negative effects of plant toxins Tertogenicity 1134 on people and the impact of plant toxins on livestock producers Author Information 1136 have been widely publicized, the diversity of these toxins and Corresponding Author 1136 their potential as new pharmaceutical agents for the treatment Funding 1136 of diseases in people and animals has also received widespread Notes 1136 interest. Scientists are actively screening plants from all regions Acknowledgments 1136 of the world for bioactivity and potential pharmaceuticals for Abbreviations 1136 the treatment or prevention of many diseases. In this review, we References 1136 focus the discussion on those plant toxins extensively studied at the USDA Poisonous Plant Research Laboratory that affect the nicotinic acetylcholine receptors (nAChRs) including species of INTRODUCTION ■ Delphinium (Larkspurs), Lupinus (Lupines), Conium (poison Plants produce a wide variety of chemical compounds termed hemlock), and Nicotiana (tobaccos). secondary metabolites. These compounds are considered “secondary” because they are not involved in basic metabolism, ■ NICOTINIC ACETYLCHOLINE RECEPTORS photosynthesis, or reproduction and are thought to be an evolutionary adaptation to selective pressures in the environ- Nicotinic acetylcholine receptors are ligand-gated cation ment such as herbivory. For centuries, people have used these channels that are members of the Cys-loop family of receptors fl (for review, see ref 2). For nAChRs in particular, there are 17 compounds for avors, fragrances, insecticides, dyes, halluci- fi α β γ δ ε identi ed genetically distinct subunits: 1−10, 1−4, , , and nogens, nutritional supplements, poisons, and pharmaceutical fi agents. Secondary compounds (toxins) may discourage (for review, see ref 3). Functional nAChRs comprise ve predation by herbivores, including insects, microorganisms, subunits arranged symmetrically around a central cation- wildlife, livestock and humans; however, in some cases these channel pore, and the subunit composition of a nAChR can toxins (from poisonous plants) result in large economic losses to the livestock industry. Secondary compounds may also be Received: May 3, 2013 signals to attract insects, birds, or other animals to enhance Published: June 28, 2013

This article not subject to U.S. Copyright. Published 2013 by the American Chemical 1129 dx.doi.org/10.1021/tx400166f | Chem. Res. Toxicol. 2013, 26, 1129−1138 Society Chemical Research in Toxicology Review

Figure 1. Image of Delphinium species and the structures of MLA and deltaline, two prototypical norditerpenoid alkaloids found in high concentrations in toxic larkspur populations. The bottom image is reprinted with permission from Photo Al Schneider. Copyright 2006 Al Schneider. The top image is reprinted with permission from USDA/ARS Poisonous Plant Research Laboratory.

α 6−8 α β vary from a homopentamer like the 7 nAChR to various from Erythrina spp. 4 2 nAChRs are responsible for heteropentamer combinations (each with distinct functional approximately 90% of the nicotine binding in the brain,3 are up- differences) depending on where the receptor is expressed in regulated in rat fetal brain cells that have been chronically the body (for review, see ref 4). For example, the fetal muscle- exposed to nicotine,9 and are thought to play a role in the type nAChR expressed in the muscle of the developing fetus reinforcement of smoking behavior.10 In contrast, the α β γδ α comprises 1(2) 1 nAChR subunits and is sensitive to homopentamer 7 nAChR, which may have a role in the teratogenic plant alkaloids. These receptors exist in multiple etiology of schizophrenia,11 is selectively activated by choline12 conformation states including a resting closed-channel state, an or the quinolizidine alkaloid cytisine from Laburnum spp.13,14 open-channel state, and a desensitized state, all of which have and blocked by methyllycaconitine (MLA) a diterpenoid differing allosterically linked affinities for agonists and alkaloid from Delphinium spp. In addition to agonists and antagonists (for review, see ref 5). Depending on their subunit antagonists like acetylcholine and MLA (both classified as composition, the homo- and heteropentamer combinations also orthosteric ligands of nAChR), there are other compounds have differing affinities for agonists and antagonists at the ligand which can bind to transmembrane domain locations of the α β ffi ff binding sites. For example, 4 2 nAChRs have a high a nity for nAChR to a ect receptor function. These compounds are nicotine, a pyridine alkaloid from N. tabacum,andare known as allosteric modulators.15 Allosteric modulators can be selectively antagonized by dihydro-β-erythroidine, an alkaloid either positive (increase the agonist response) or negative

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Figure 2. Relationship between serum MLA concentrations and heart rate. There is a significant correlation between MLA serum concentrations and heart rate in cattle (P = 0.0001; Spearman r = 0.75). Data points represent MLA serum concentrations (ng/mL), left axis, and heart rate (beats per minute), right axis, over 96 h in five steers that received an equivalent oral dose of 10.4 mg/kg MLA in the form of dried ground larkspur. Each data point represents the mean ± standard error. (Data are from Green et al.101)

(decrease the agonist response) (for review, see ref 16). For type and the N-(methylsuccinimido) anthranoyllycoctonine α 28 example, ivermectin is a positive allosteric modulator of 7 (MSAL)-type. Although the MSAL-type alkaloids are much − nAChR,17 and progesterone can act as a negative allosteric more toxic (typically >20x),29 31 the MDL-type alkaloids are α β 16,18 32,33 modulator of 4 2 nAChR. Functionally, allosteric generally more abundant in many larkspur populations. modulators are thought to change either the affinity of the The three MSAL-type alkaloids that are most important due to ffi receptor for the orthosteric ligand or the intrinsic e cacy of the their toxicity in livestock are MLA, 14-deacetylnudicauline − 19 agonist receptor interaction. In addition to allosteric (DAN), and nudicauline (NUD).30 There are two structural modulation, nAChR agonists and antagonists at high features necessary for toxicity: (1) an N-ethyl bicyclo tertiary concentrations (100 μM−1 mM) can directly block the ion 34 20 alkaloid nitrogen atom and (2) a C-18 anthranilic acid ester. channel of the receptor in a noncompetitive manner. Structure−function studies have shown that the aromatic ester nAChRs are found throughout the body and serve to mediate functional group on MLA is a significant haptophore35 and that diverse physiological functions. Specific locations of nAChRs the succinimide group imparts significant toxicity to alka- include the central nervous system, autonomic ganglia, sensory loids.36,37 In addition, substitution of differing functional groups ganglia, and neuromuscular junctions. These receptors can also ff 30 be located pre- or postsynaptically. Presynaptic nAChRs serve at the C-14 carbon can a ect the toxicity of these alkaloids. to modulate the release of neurotransmitters including biogenic The primary result of larkspur toxicosis in livestock is − amines and amino acids.21 24 Postsynaptic nAChRs mediate neuromuscular paralysis from nAChR blockade at the 38,39 ff excitatory neurotransmission. For example, muscle-type postsynaptic neuromuscular junction. These e ects are nAChRs located in the motor end plate region of the muscle species dependent because in mice and rats, MLA elicits central ff fiber are activated by acetylcholine released from the motor nervous system e ects, and in large animals such as cattle, MLA 40,41 nerve terminal. The opening of muscle-type nAChRs in the elicits primarily peripheral effects. Moreover, in Anolis postjunctional membrane increases the permeability of the cell carolinesis lizards, larkspur alkaloids act as postsynaptic membrane to cations, leading to membrane depolarization and competitive inhibitors of acetylcholine at muscle-type ultimately muscle contraction.25 nAChR.42 MLA is a potent and selective blocker of α7 nAChRs that block acetylcholine evoked currents in rat fetal ■ PLANT TOXINS hippocampal neurons at picomolar concentrations.3,40,43 MLA strongly competes with the high affinity nAChR ligand α- The deleterious effects of many poisonous plants are due to bungarotoxin for binding to nAChRs.34 MLA has been shown toxins produced by the plants that bind to nAChRs. These to displace 125I α-bungarotoxin binding in rat brain membranes plant toxins can be either competitive agonists or antagonists of 125 α with a Ki of 1.4 nM and displaces I -bungarotoxin binding nAChRs. For the remainder of this review, we will focus on μ in human muscle extracts with a Ki of 7.8 M suggesting it is plant toxins from the genera Delphinium, Lupinus, Conium, and 44 Nicotiana. selective for neuronal nAChR. Moreover, the binding of Delphinium. larkspur alkaloids to nAChRs appears to be correlated with There are over 80 wild species of larkspurs 34 (Delphinium) in North America26 (Figure 1). Larkspur species toxicity and may explain the tolerance of sheep to larkspur if 41,44 are divided into three general categories based primarily on the toxins bind with lower affinity at sheep receptors. There mature plant height and geographical distribution: low, tall, and is also a significant correlation between serum MLA plains larkspurs.27 The toxicity of larkspur plants is due to concentration and the physiological effects of MLA such as norditerpenoid alkaloids, which occur as one of two chemical elevated heart rate (Figure 2). Drugs that increase the structural types, the 7,8-methylenedioxylycoctonine (MDL)- persistence of acetylcholine at the neuromuscular junction

1131 dx.doi.org/10.1021/tx400166f | Chem. Res. Toxicol. 2013, 26, 1129−1138 Chemical Research in Toxicology Review such as the acetylcholinesterase inhibitor neostigmine can reverse larkspur toxicosis or reduce susceptibility.45 Lupinus. The genus Lupinus contains more than 500 species of annual, perennial, or soft woody-shrub-like lupines world- wide and are members of the Leguminosae family (Figure 3).46

Figure 4. Concentration−effect relationships with best-fit lines for the actions of anagyrine on membrane potential sensing dye fluorescence Figure 3. Images of Lupinus leucophyllus (top panel) and Lupinus in TE-671 cells and SH-SY5Y cells. In each experiment, the membrane sulphureus (bottom panel). The chemical structure of the piperidine depolarization resulting from the addition of epibatidine or anagyrine in log10 molar concentrations was measured and displayed as a alkaloid ammodendrine and the quinolizidine alkaloid anagyrine are μ included for comparison. Reprinted with permission from USDA/ARS percentage of the maximal epibatidine response (10 M epibatidine Poisonous Plant Research Laboratory. for both cell lines). Each datum represents six experiments of duplicate wells. (Data are from Green et al.102) The toxicity of Lupinus plants is due to the presence of quinolizidine and piperidine alkaloids. More than 150 hydroxylated lupanines or alkaloids of the multiflorine series fi μ 49 quinolizidine alkaloids have been structurally identi ed from (IC50 values of >500 M ). Recent research on the in vivo the legume family.47 Eighteen Lupinus species have been shown disposition of lupanine and 13-hydroxylupanine in humans has to contain the quinolizidine alkaloid anagyrine, and 14 of these demonstrated that the urine t1/2 after oral administration was contain teratogenic amounts of anagyrine.48 Only a small 6.5 and 5.9 h, respectively; and 95% to 100% of the total number of the lupine alkaloids have been investigated in detail. alkaloid administered was recovered unchanged within 72 h.50 For example, 14 alkaloids isolated from Lupinus albus, L. Lupanine, 13- hydroxylupanine, and sparteine have been shown mutabilis, and Anagyris fetida were analyzed for the displace- to block ganglionic neurotransmission (predominantly α3β4, ment of 3H-nicotine (a nAChR selective ligand) or 3H- nAChR), decrease cardiac contractility, and contract uterine quinuclidinyl benzilate (a muscarinic acetylcholine receptor smooth muscle which suggests actions at β-adrenergic selective ligand) in a porcine brain membrane preparation.47 Of receptors.51 Lupanine and sparteine have been shown to the 14 lupine alkaloids tested, the α-pyridones (N-methyl block the effects of pneumogastric nerve stimulation in dogs cytisine and cytisine) had the highest affinities for nAChR and cats suggesting ganglionic effects due to actions at μ α β 52 (IC50s of 0.05 and 0.14 M, respectively), while several predominantly 3 4 nAChR, and both compounds act as quinolizidine alkaloid types including the teratogen anagyrine weak antagonists at muscarinic cholinergic receptors. This is μ 3 (IC50 = 132 M) were also potent displacers of H-nicotine. inconsistent with information from porcine brain membrane Functionally, the lupine alkaloid anagyrine is a partial agonist at experiments,47 as they reported that sparteine had the second α β γδ α β ffi 12 1 nAChR expressed by TE-671 cells and 3 4 nAChR highest a nity for muscarinic acetylcholine receptors of the 14 expressed by SHSY-5Y cells with percent maximum activations quinolizidine alkaloids tested, while lupanine had the 12th ± ± ffi μ of 39 18 and 27 4% activation for TE-671 and SHSY-5Y highest a nity (IC50 = 21 and 190 M for sparteine and cells, respectively (Figure 4). Lupanine, which is widely lupanine, respectively, based on the displacement of 3H- ffi distributed in legumes and lupine species, has an IC50 of 5 quinuclidinyl benzilate (QNB) and micromolar a nity for μ μ M at porcine brain nAChRs and is more active than nAChR (IC50 = 331 and 5 M for sparteine and lupanine,

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Figure 5. Images of poison hemlock and chemical structures of predominant alkaloids. Reprinted with permission from USDA/ARS Poisonous Plant Research Laboratory. respectively based on the displacement of 3H-nicotine)). The from Europe and has become widespread throughout this toxicity of lupanine administered by intraperitoneal injection to country.26 Historically, poison-hemlock has been associated rodents was less than that of sparteine; with LD50 values of 175 with human poisoning more than livestock poisonings and is vs 36 mg/kg, respectively.52,53 believed to be the source of the tea used to execute In lupine, alkaloids are produced in the leaves of the plant by Socrates.58,59 Because of its popular use as an execution potion chloroplasts and translocated via the phloem to be stored in and its pharmaceutical properties, coniine was the first alkaloid epidermal cells and seeds of the plant.46 The synthesis of these characterized and the first alkaloid to be prepared synthetically. alkaloids is regulated by light and fluctuates diurnally.54,55 In In addition to the early chemistry, alkaloids of poison hemlock addition to diurnal alkaloid fluctuations, the piperidine and were the first alkaloids to have their biosynthetic pathways in quinolizidine alkaloid content can vary in plants depending on the plant determined.60,61 Eight piperidine alkaloids are known environmental conditions, season of the year, stage of growth, in poison-hemlock (coniine, N-methylconiine, γ-coniceine, and species of lupine.56 Alkaloid content is typically highest conhydrine, conhydrinone, pseudoconhydrine, N-methylpseu- during early growth stages, decreasing through the flower stage doconhydrine, and 2-methyl piperidine), five of which are while increasing in the seeds and pods. In addition to the above commonly discussed in the literature.62 Two alkaloids, coniine variables influencing alkaloid concentration, elevation has also and γ-coniceine, are most prevalent in the plant and are likely been shown to play a role in alkaloid concentrations. For responsible for toxicity and teratogenicity in animals. γ- example, in Lupinus argenteus plants collected above 3500 m Coniceine is the predominant alkaloid in the early vegetative there was a 6-fold increase in alkaloid concentrations compared stage of plant growth and is a biochemical precursor to the to that in those collected at 2700 m.57 Interestingly, in the same other Conium alkaloids.61,63 Coniine is the predominate study, the researchers saw no difference in herbivory between alkaloid in the seeds of the late growth stage of the plant. γ- ff the lupine populations, and the alkaloid di erences persisted Coniceine (LD50 4.4 mg/kg) is more toxic than coniine (LD50 even when seedlings from the highest and lowest elevations 7.7 mg/kg), which is in turn more toxic than N-methylconiine 64,65 were grown under identical greenhouse conditions, suggesting (LD50 17.8 mg/kg) in mice. The mechanism of action of genetic differences as plants adapted to elevation. the Conium alkaloids is 2-fold. The most clinically significant Conium. Poison-hemlock (Conium maculatum) (Figure 5) effect occurs at the neuromuscular junction where they act as was introduced into the United States as an ornamental plant nondepolarizing blockers like curare.66 Systemically, the toxins

1133 dx.doi.org/10.1021/tx400166f | Chem. Res. Toxicol. 2013, 26, 1129−1138 Chemical Research in Toxicology Review cause biphasic nicotinic effects, including salivation, mydriasis, nAChR.70 As a drug, nicotine has pharmacological actions in and tachycardia followed by bradycardia due to their actions at muscle, the peripheral nervous system, central nervous system, autonomic ganglia. The teratogenic effects are undoubtedly and the cardiovascular system. Nicotine has been shown to related to the neuromuscular effects on the fetus and have been have effects on behavior and cognitive function (for review, see shown to be related to reduction in fetal movement.67 refs 71 and 72). Nicotine has been extensively studied as an Differences in chemical structure impart significant differences addictive substance, and its effects are mediated by actions at in toxicity/teratogenicity (γ-coniceine > coniine > N-methyl nAChR (for more information, see refs 25 and 73). Romano coniine), but the teratogenic potency of the alkaloids is and Goldstein74 demonstrated the stereoselectivity of nicotine unknown, although we believe it is related to toxicity.68 The binding sites in rat brain membranes (stereoselectivity is a clinical signs of poisoning from ingestion of Conium and N. property of receptors). Nicotine has binding affinities in the low glauca are similar in all animal species tested and appear to be nanomolar range at α4β2 nAChR, binding affinities of the same as those caused by lupine. They include early signs of micromolar and above at α7 nAChR, and in the nanomolar nervousness, occlusion of the eyes by the nictitating membrane, range at α3β4 nAChR.3,71,75 progressing quickly through a pattern of nervous system ff stimulation with peripheral and local e ects including frequent ■ TERTOGENICITY urination and defecation, dilated pupils, trembling, incoordina- tion, and excessive salivation. The stimulation soon passes to The teratogenic effects of Lupine, Conium, and Nicotiana spp. depression resulting in relaxation, recumbency, and eventually are discussed together because the malformations associated death from respiratory paralysis at high doses. with these plants are similar, if not the same, and the Nicotiana. The Nicotiana genus (Figure 6) consists of about mechanism of action, the complete inhibition of fetal 60 species in North and South America, Australia, and the movement (Figure 7), is believed to be the same for each of the species.76 The syndrome known as “crooked calf disease” associated with lupine ingestion by the pregnant mother was first reported in the late 1950s. Crooked calf disease is associated with various skeletal contracture-type birth defects and occasionally cleft palate.49,70,77 This appears to be similar to an inherited genetic condition where the same type of birth defects are reported in Charolais cattle.78 Through epidemio- logic evidence and chemical comparison of teratogenic and nonteratogenic lupines, the quinolizidine alkaloid anagyrine was determined to be the teratogen.79 A second teratogen, the piperidine alkaloid ammodendrine, was found in Lupinus formosus and induces the same type of skeletal birth defects.80,81 Further research determined that the anagyrine-containing lupines only caused birth defects in cattle and did not affect sheep or goats, while the piperidine alkaloid containing lupine, L. formosus, induced similar birth defects experimentally in 76,80 Figure 6. Images of cultivated tobacco (top left), tree tobacco (bottom cattle and goats. This led to speculation about the possible two panels), and chemical structures of two common tobacco metabolism or absorption differences between cattle and small alkaloids. Reprinted with permission from USDA/ARS Poisonous ruminants. Keeler and Panter80 hypothesized that the cow Plant Research Laboratory. might metabolize the quinolizidine alkaloid anagyrine to a complex piperidine, meeting the structural characteristics South Pacific. Many of these species are poisonous to livestock determined for the simple teratogenic piperidine alkaloids in 82 and humans. The annual plant, Nicotiana tabacum, is cultivated poison-hemlock. This was supported by feeding trials with as a cash crop, which forms an extensive tobacco industry, while other piperidine alkaloid-containing plants, extracts, and pure N. glauca is a perennial tree or shrub with no commercial value compounds. Even though comparative studies support the but is poisonous to livestock. Burley tobacco (N. tabacum)is hypothesis that the cow may convert the quinolizidine alkaloid native to South America. It was introduced and subsequently anagyrine to a complex piperidine by ruminal metabolism, cultivated in Virginia by European settlers. In 1597, Gerad recent evidence reporting the absorption and elimination believed that burley tobacco relieved discomfort from head- patterns of many of the quinolizidine alkaloids, including aches, toothaches, skin problems, burns, wounds, dropsy, piles, anagyrine, in cattle, sheep, and goats does not support this colic, and deafness. It was also used as a purgative, emetic, and theory.83 Keeler and Balls82 fed commercially available antihelminitic.69 The major constituent, nicotine, was named structural analogues of coniine to pregnant cows to compare after Jean Nicot who helped popularize the use of tobacco in structural relationships to teratogenic effects. Results from the 16th century as a treatment for headache. Nicotine was first Keeler and Balls82 suggested that piperidine alkaloids must isolated and characterized in 1828, but its pharmacological meet certain structural criteria to be teratogenic. On the basis of action was not demonstrated until 1898. Langley, a Cambridge these data, Keeler and Balls82 speculated that the piperidine University physiologist, exposed autonomic ganglia from alkaloids with either a saturated ring or a single double bond different animal species to nicotine and observed an initial with a side chain of at least three carbon atoms in length stimulation followed by inhibition of nervous transmission. adjacent to the nitrogen atom might be considered potential Langley was the first to propose the idea of the autonomic teratogens. Additionally, piperidine alkaloids with a double nervous system with its sympathetic and parasympathetic bond adjacent to the N atom of the piperidine ring are more components. This early research led to the recognition of toxic than either the saturated or N-methyl derivatives.68

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Figure 7. Effect of nicotine, (−)-coniine mandelic acid, and anabasine on ultrasound monitored fetal movement in the day 40 pregnant goat model. The bars represent the mean ± SEM number of fetal movements detected during a 5 min fetal ultrasound monitoring period of seven fetuses in seven anabasine dosed does (0.8 mg/kg anabasine), six fetuses in six does dosed with (−)-coniine mandelic acid (4.4 mg/kg (−)-coniine mandelic acid), and eight nicotine (0.4 mg/kg) dosed does. Fetal movements were measured at time zero just prior to i.v. injection and 30 and 60 min after i.v. dosing. There were significant differences among the treatments (*P < 0.05). (Data are from Green et al.103,104)

One piperidine alkaloid, which meets the structural require- Frequently, minor contractions such as “buck knees” may be ments for teratogenesis, is anabasine, the major alkaloid in attributed to lupine, but the malformations will resolve on their Nicotiana glauca (tree tobacco). By comparison, in N. tabacum own, and the calf will appear normal. The critical gestational (cultivated tobacco) nicotine is the major alkaloid component period for exposure in cattle is 40−70 days with susceptible of tobacco leaves and anabasine a major component of stalks.84 periods extending to 100 days.96,97 The condition has been In a goat model, nicotine does not abolish fetal movement experimentally induced with dried ground lupine at 1 g/kg while anabasine does (Figure 7). Research from the late 1960s body wt and with semipurified preparations of anagyrine at 30 and early 1970s with N. tabacum stalks that have high mg anagyrine/kg body wt fed daily from 30 to 70 days concentrations of anabasine identified that the alkaloid to be gestation.98,99 Crooked calf disease has also been induced by responsible for the outbreaks of malformed pigs not nicotine feeding the piperidine alkaloid-containing lupine L. formosus.80 − from the leaves.85 87 Research has focused on N. glauca, using The teratogenic compounds from this plant are believed to be dried ground plant and solvent extracts from the plant in a goat ammodendrine,80 N-acetyl hystrine, and N-methyl ammoden- model, to study the mechanism of teratogenesis with drine.68 These teratogenic alkaloids are absorbed quickly after application to other livestock species and humans.88 Recent ingestion and can be detected in blood plasma by 0.5 h with research demonstrated the relative order of toxicity of three peak levels of N-methyl ammodendrine and N-acetyl hystrine structurally related piperidine alkaloids as anabaseine > attained by 2 h postgavage and peak levels of ammodendrine 68 83,100 anabasine > N-methyl anabasine. Moreover, coniine, a simple attained in about 24 h. The plasma elimination t1/2 in the piperidine from poison hemlock, and anabasine, a simple cow was about 12 h for ammodendrine and less than 2 h for N- piperidine from tree tobacco (Nicotiana glauca), have been methyl ammodendrine. N-Acetyl hystrine demonstrated a fi shown to induce the same fetal defects in cattle, sheep, pigs, biphasic elimination pattern with a rapid rst phase, t1/2 less 67,89 and goats. The related alkaloid anabaseine (a piperidine than 2 h, and a much slower second phase, t1/2 greater than 12 alkaloid structurally similar to anabasine) has a double (imine) h.100 Toxicological comparison in a mouse bioassay demon- bond between positions 1 and 2 of the piperidine ring and is strated that N-acetyl hystrine is most toxic > N methyl found in certain marine worms and in ants.90,91 Anabasine and ammodendrine > ammodendrine.68 anabaseine are both potent agonists at nAChRs,92 and Plants produce a variety of secondary compounds, and many benzylidene-anabaseine analogues which act as selective partial of them are bioactive in animals. If these compounds possess agonists of α7 nAChR have received much attention for their the correct chemical structure, then they can act as neurotoxins − potential as cognitive enhancers.93 95 at nAChRs. For example, piperidine alkaloids from Nicotiana Arthrogryposis is the most common malformation caused by spp. can activate and ultimately desensitize nAChR at the potent piperidine alkaloid nAChR agonists like anabasine. The neuromuscular junction of developing fetuses.88 The desensi- observed terata include one or more of the following: scoliosis, tization and resulting inhibition of fetal movement results in torticollis, kyphosis, or cleft palate. The elbow joints are often arthrogryposis and other fetal defects. The norditerpenoid immobile because of misalignment of the ulna with the articular alkaloids found in larkspurs have a different chemical structure surfaces of the distal extremity of the humerus and front limbs and pharmacological mode of action, but they too act as rotated laterally. In crooked calf disease, the osseous changes neurotoxins. Norditerpenoid alkaloids act as antagonists to are permanent and become progressively worse as the calf block the ligand binding sites of nAChR and cause acute grows and its limbs are subjected to greater load-bearing stress. toxicosis in adult animals, which can result in death. Even

1135 dx.doi.org/10.1021/tx400166f | Chem. Res. Toxicol. 2013, 26, 1129−1138 Chemical Research in Toxicology Review though the compounds have deleterious effects in mammals, chronic exposure of rats to mainstream cigarette smoke or alpha 4 beta they must provide some advantage to the plants that produce 2 receptors to nicotine. Biochem. Pharmacol. 50, 2001−2008. them. The likely purpose of these compounds is to discourage (10) Dani, J. A., Jenson, D., Broussard, J. I., and De Biasi, M. (2011) predation, enhance pollination and seed dispersal, or serve as a Neurophysiology of nicotine addiction. J. Addict. Res. Ther., nitrogen reservoir for germination and early growth. Regardless DOI: 10.4172/2155-6105.S1-001. of the true purpose of plant secondary compounds, the (11) Lloyd, G. K., and Williams, M. (2000) Neuronal nicotinic acetylcholine receptors as novel drug targets. J. Pharmacol. Exp Ther. bioactivity of these compounds makes them useful tools in 292, 461−467. human and veterinary medicine or potentially harmful toxins. (12) Papke, R. L., Bencherif, M., and Lippiello, P. (1996) An evaluation of neuronal nicotinic acetylcholine receptor activation by ■ AUTHOR INFORMATION quaternary nitrogen compounds indicates that choline is selective for − Corresponding Author the alpha 7 subtype. Neurosci. Lett. 213, 201 204. *Phone: 435-752-2941. Fax: 435-753-5681. E-mail: Ben. (13)Alkondon,M.,Pereira,E.F.,Eisenberg,H.M.,and Albuquerque, E. X. (1999) Choline and selective antagonists identify [email protected]. two subtypes of nicotinic acetylcholine receptors that modulate GABA Funding release from CA1 interneurons in rat hippocampal slices. J. Neurosci. This work was funded by the United States Department of 19, 2693−2705. Agriculture, Agricultural Research Service. (14) Papke, R. L., and Porter Papke, J. K. (2002) Comparative Notes pharmacology of rat and human alpha7 nAChR conducted with net charge analysis. Br. J. Pharmacol. 137,49−61. The mention of trade names or commercial products in this “ ” fi (15) Changeux, J. P. (2011) 50th anniversary of the word allosteric . publication is solely for providing speci c information and does Protein Sci. 20, 1119−1124. not imply recommendation or endorsement by the U.S. (16) Bertrand, D., and Gopalakrishnan, M. (2007) Allosteric Department of Agriculture. modulation of nicotinic acetylcholine receptors. Biochem. Pharmacol. The authors declare no competing financial interest. 74, 1155−1163. (17) Collins, T., and Millar, N. S. (2010) Nicotinic acetylcholine ■ ACKNOWLEDGMENTS receptor transmembrane mutations convert ivermectin from a positive to a negative allosteric modulator. Mol. Pharmacol. 78, 198−204. We thank Isabelle McCollum and Terrie Wierenga for technical (18) Valera, S., Ballivet, M., and Bertrand, D. (1992) Progesterone assistance. modulates a neuronal nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. U.S.A. 89, 9949−9953. ■ ABBREVIATIONS (19) Ehlert, F. J. (2005) Analysis of allosterism in functional assays. J. − nAChR, nicotinic acetylcholine receptor; MLA, methyllycaco- Pharmacol. Exp. Ther. 315, 740 754. (20) Lape, R., Krashia, P., Colquhoun, D., and Sivilotti, L. G. (2009) nitine; MDL-type alkaloid, 8-methylenedioxylycoctonine alka- Agonist and blocking actions of choline and tetramethylammonium on loid; MSAL-type alkaloid, N-(methylsuccinimido) anthranoyl- human muscle acetylcholine receptors. J. Physiol. 587, 5045−5072. lycoctonine alkaloid; DAN, 14-deacetylnudicauline; NUD, (21) Marshall, D. L., Redfern, P. H., and Wonnacott, S. 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