Plant alkaloids that cause developmental defects through the disruption of cholinergic neurotransmission

Birth Defects Research (Part C) 99:235–246 (2013) REVIEW Plant Alkaloids that Cause Developmental Defects through the Disruption of Cholinergic Neurotransmission Benedict T. Green,* Stephen T. Lee, Kevin D. Welch , and Kip E. Panter 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 a 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 flexuretype skeletal defects and cleft palate in animals. Birth Defects Research (Part C) 99:235–246, 2013. Published 2013 Wiley Priodicals, Inc. Key words: piperidine alkaloids; pyridine alkaloids; nicotinic acetylcholine receptor; desensitization; TrpB INTRODUCTION The maternal consumption of plants, plant products, or extracts from plants that contain piperidine, pyridine, or quinolizidine alkaloids has the potential to cause developmental defects in humans and animals. Many of the actions of these alkaloids, found in lupines, poison hemlock, Nicotiana spp., and others (if they possess the correct structural features required to activate the receptor), are mediated by nicotinic acetylcholine receptors (nAChR), which are ligand-gated cation channels. These teratogenic ligands bind to nAChR ligand- binding sites, activate the receptor to open the cation channel, and ultimately desensitize the receptor to close the cation channel. In the developing fetus, teratogenic piperidine alkaloid-mediated desensitization of fetal muscle-type nAChR is postulated to inhibit fetal movement, resulting in skeletal flexure defects and cleft palate. The inhibition of fetal movement disrupts the normal developmental process to cause multiple congenital contracture-type (MCC-type) deformities (arthrogyrposis, kyposis, lordosis, scoliosis, and torticollis) and cleft palate (Panter and Keeler, 1992; Weinzweig et al., 2008). Nicotinic acetylcholine receptor-mediated fetal movement is essential for normal development, and disruption of this movement causes fetal defects. For example, when chick embryo movement is prevented with the depolarizing neuromuscular blocking agent decamethonium, the correct development of joints is disrupted (Drachman and Sokoloff, 1966). Muscle contraction is essential in early development because it activates the WNT/b-catenin signaling pathway to initiate joint formation (Kahn et al., 2009). Later in development, the inhibition of fetal movement causes deformation, simply through abnormal alignment and position of the fetus in utero (Panter et al., 1999). The association between the inhibition of fetal movement by plant alkaloids and the formation of MCCtype defects is well-documented in livestock. When plants, such as lupine (Lupinus spp.), tobacco (Nicotiana spp.), or poison hemlock (Conium spp.), are consumed by pregnant females at the sensitive stage of development, the teratogenic alkaloids from the plants cross the placenta into the fetal compartment to act at fetal muscle-type nAChR and inhibit fetal movement. This has been documented with ultrasound imaging studies of fetuses in pregnant livestock dosed i.v. with the piperidine Benedict T. Green, Stephen T. Lee, Kevin D. Welch and Kip E. Panter United States Department of Agriculture, Poisonous Plant Research Laboratory, Agricultural Research Service, 1150 E 1400 N, Logan, Utah, 84321 *Correspondence to: Benedict T. Green, Poisonous Plant Research Laboratory, 1150 E 1400 N, Logan, UT 84321. E-mail Ben.Green@ars.usda.gov View this article online at (wileyonlinelibrary.com). DOI: 10.1002/bdrc.21049 Published 2013. This article is a U.S. Government work and is in the public domain in the USA. 236 GREEN ET AL. Figure 1. The effects of coniine mandelic acid enantiomers on fetal movement in day 40 pregnant goats. (A) The effects of mandelic acid enantiomers (4.4 mg/kg) and saline (0.8 ml/kg) on fetal movement at 0.5, 1, 2, and 8 hr after i.v. dosing. The concentrations of mandelic acid enantiomers were dosed on an equimolar basis to those used in the coniine mandelic acid combinations in panel (B), and did not significantly affect fetal movement at any of the time points (p > 0.05, mixed model repeated measures analysis, (1) and (2)-mandelic acid verses saline control). Each datum point represents mean6 SEM of the number of fetal movements in three to six fetuses. (B) The effects of coniine mandelic acid enantiomers (8 mg/kg) and saline (0.8 ml/kg) on fetal movement at 0.5, 1, 2, and 8 hr after i.v. dosing of the pregnant dam. Fetal movements were measured at time zero just prior to i.v. injection, and at 0.5, 1, 2, and 8 hr after i.v. dosing. Each datum point represents mean 6 SEM of the number of fetal movements in three to six fetuses. (*p < 0.05, mixed model repeated measures analysis, coniine mandelic acid enantiomer combination vs. saline control). Data from Green et al. (2013a). alkaloid coniine (Fig. 1), or oral dosing studies with poison hemlock (C. maculatum) and tree tobacco (N. glauca). These studies have documented the stereoselective inhibition of fetal movement and Birth Defects Research (Part C) 99:235–246 (2013) the formation of MCC-type defects and cleft palates (Panter et al., 1990; Panter and Keeler, 1992, 1993; Green et al., 2013a). In humans, these deformities are known as congenital arthrogyrposis, and are attributed to disorders of the neuromuscular system caused by a variety of genetic and environmental factors, including tobacco use by pregnant women (Polizzi et al., 2000; O’Flaherty, 2001; Steinlein, 2007; Shi et al., 2008). In this review, we focus on mechanism of action of alkaloids that act as agonists at fetal muscletype nAChR to inhibit fetal movement, that have been shown in vivo to form MCC-type defects in the developing fetus of livestock. The developmental effects of cholinergic drugs and plant alkaloids that act at nAChR have long been the subject of research. For example, Bueker and Platner (1956) compared the actions of the endogenous nAChR ligand acetylcholine, to those of physostigmine (from the calabar bean, Physostigma venenosum) on the development of chick embryos, and found that physostigmine caused spinal defects. By the 1970s, it was recognized that chemicals that interfere with cholinergic neurotransmission cause deformities in chick embryos (Landaulet, 1975). These chemicals included organophosphate insecticides that cause arthrogyrposis (at the time termed “Type II defects”; Moscioni et al., 1977; Seifert and Casida, 1978). Concurrently, Dr. Richard Keeler at the Poisonous Plant Research Laboratory was exploring the structure– activity relationships of piperidine and pyridine nAChR agonists from poisonous plants. STRUCTURE–ACTIVITY RELATIONSHIPS Richard Keeler, a Chemist at the Poisonous Plant Research Laboratory in Logan, Utah, pioneered the structure–teratogenicity relationships of piperidine alkaloid livestock teratogens (Keeler and Balls, 1978; Keeler, 1988). Dr. Keeler dosed pregnant cattle with eight DEVELOPMENTAL DEFECTS DUE TO DISRUPTION OF CHOLINERGIC NEUROTRANSMISSION 237 Figure 2. Chemical structures of piperidine, pyridine, and quinolizidine alkaloids. structurally related piperidines (piperidine, 2-methylpiperidine, 2ethylpiperidine, 3-methylpiperidine, N-methylpiperidine, 2-piperidineethanol, 2n-propyl-D1-piperidine (cconiceine), and 2-propylpiperidine (coniine). Both coniine (Fig. 2) and c-coniceine produced MCC-type defects in the offspring of the cows. These results led Dr. Keeler to conclude that the formation of terata in livestock is due to the position and length of the side-chain on the piperidine ring, that is, piperidine alkaloids with a carbon side-chain of at least three carbons or larger attached to the carbon a to the piperidine nitrogen have teratogenic activity. The presence of a double bond adjacent to the nitrogen like that in c-coniceine or anabaseine (Fig. 2) also confers increased toxicity and teratogenicity. The effects of a double bond are best illustrated by the results of a mouse bioassay in which cconiceine had a LD50 value of 4.4 mg/kg, (6)-coniine 7.7 mg/kg, and (6)-N-methyl coniine 17.8 mg/kg (Lee et al., 2013b). The influence of structure– activity relationships on teratogenicity identified by Keeler and Balls (1978) also provided an explanation for the field reports of MCCtype terata in offspring of sows that had consumed waste tobacco stalks (Nicotiana tabacum) when pregnant (Crowe, 1969; Menges et al., 1970; Crowe and Pike, 1973; Crowe and Swerczek, 1974). Results from this research found that the pyridine alkaloid nicotine, which is present at high concentrations in the leaves of the tobacco plant, does not cause MCC-type defects. However, the piperidine alkaloid anabasine which is at high concentrations in the pulp of tobacco stalks did cause MCC-type terata (Crowe, 1978; Keeler and Crow 1983, 1984; Keeler et al., 1984). This result was repeated using tree tobacco (Nicotiana glauca), which contains predominately anabasine (Keeler et al., 1981a; Keeler and Crowe, 1984; Panter et al., 1999). These results suggest that the structure of nAChR agonists determines their teratogenicity, and that piperidine but not pyridine alkaloids cause MCC-type terata and cleft palate in livestock. In addition to the effect of structure–activity relationships on teratogenicity, there may also be differences in the agonist-potency of nAChR ligands that cause terata in livestock. A cell-based pharmacodynamic comparison of piperidine, pyridine, and quinolizidine alkaloids actions was made to determine the rank order of potency of these teratogens (Green et al., 2010). In this model, piperidine alkaloids were more in TE-671 cells (as shown by percent maximal response, Table 1) which express human fetal muscle-type nAChR (a1b1cd subunits) than SHSY-5Y cells that express autonomic-type nAChR (predominantly a3b4 subunits). These results, and studies involving experiments with day 40 pregnant goats (Figs. 1 and 3), support the hypothesis that the mechanism behind MCC-type defects is the inhibition of fetal movement. The inhibition of fetal movement is postulated to be due to the agonistdependent desensitization of fetal muscle-type nAChRs by piperidine but not pyridine alkaloids (Panter et al., 1990, 2000; Green et al., 2010). Moreover, the extent of the desensitization is agonist-dependent, and only complete inhibition of fetal movement will cause MCCtype defects. A more detailed description of the research on poisonous plants that contain these toxins is reported in a separate paper by Panter et al. (2013) in this issue. Individual alkaloids that act as agonists at nAChR are discussed below. PIPERIDINE ALKALOIDS Anabasine Anabasine is an optically active piperidine alkaloid which has been the basis for much of the research on alkaloid-associated MCC-type defect formation in livestock (Keeler and Crowe, 1983; Lee et al., 2006). Anabasine is a full nAChR agonist and nicotine-like natural product (Daly, 2005; Lesarri et al., 2010). This alkaloid is a minor tobacco alkaloid found at low concentrations in tobacco, and acts synergistically with other tobacco alkaloids to facilitating smoking behavior (Clemens et al., 2009), and is the principal piperidine alkaloid in tree tobacco. Tree tobacco has been mistaken for wild spinach and it’s consumption in “wild salads” is responsible for human fatalities (Furer et al., 2011). Anabasine is in tobacco and Birth Defects Research (Part C) 99:235–246 (2013) 238 GREEN ET AL. TABLE 1. EC50 Values of Selected Teratogenic Compounds in TE-671 Cells and SH-SY5Y Cells Compound (6)-Ammodendrinea (1)-Ammodendrinea (2)-Ammodendrinea Anabaseinea (6)-Anabasinea (1)-Anabasinea (2)-Anabasinea Anagyrinea (6)-Coniinea (1)-Coniinea (2)Coniinea Lobelined Myosmined Nicotinea SH-SY5Y SH-SY5Y TE-671 cell TE-671 cell EC50 (mM), cell percent EC50 (mM), cell percent (95% C.I.) maximum (95% C.I.) maximum NAb 11.0c 121.9c 0.3 (0.1–0.6) 95.5a (44.7–203.6) 2.4(1.2–4.8) 19.7 (4.0–97.5) 18.1 (0.5–663) 51.4 (0.03–77.44) 10.2 (3.4–30.6) 9.6 (7.4–12.6) NT NT 56.0 (28.5–11.0) 7 6 3% 14 6 4 % 13 6 4% 79 6 8% 539.2c 1101c NAb 0.2 (0.001–22.7) 10.1 (28.7–34.9) 0.7 (0.4–1.3) 1.7 (0.01–247.8) 19.1 (0.9–3.91) 208 (10.7–40.6) 900 (41.4 – 19.60) 115 (5.3–24.7) 219 (5–9.07) 307 (16.7–56.2) 43.7 (6.1–31.4) 27 6 8 % 17 6 6% 362 % 91 6 10 % 64 6 8 % 45 6 3 % 32 6 2% 27 6 4% 35 6 6% 31 6 13% 33 6 2% NT NT 68 6 7% 100 6 5% 91 6 6 % 63 6 7 % 39 6 18% 68 6 5% 47 6 5% 79 6 4% 41 6 7 95 6 4 60 6 8% a Data from Green et al., 2010. Due to lack of potency and efficacy the EC50 value was not calculated. c 95% confidence interval not calculated. d Data from Green et al., 2013b. b tobacco products, such as oral snuff and cigarettes, as well as in biological samples from smokers (Jacob et al., 1999; Brunnemann et al., 2002; Djordjevic and Doran, 2009; Miller et al., 2010a,b). In cell culture models, anabasine is a potent agonist, with an EC50 value in the micromolar range in both TE-671 cells and SHSY-5Y cells (Table 1), and a potent inducer of MCC-type deformities, as shown by its complete inhibition of fetal movement dosed either i.v. (Fig. 3) or orally (in the form of dried ground tree tobacco; Fig. 4). Interestingly, the presence of a double bond adjacent to the nitrogen in the piperidine ring of the closely related alkaloid anabaseine (Fig, 2) shifts the EC50 values into the nanomolar range in both TE-671 cells and SHSY-5Y cells. Coniine Coniine is a piperidine alkaloid and nAChR agonist found in poison hemlock (C. maculatum L.). Poison hemlock was introduced into the United States from Europe. It is often mistaken for wild carrot and is responsible for acute human poisonings (for more information, see Marion, 1950; Frank et al., 1995; Lopez et al., 1999; Vetter, 2004; Lee et al., 2008). Poison hemlock also causes acute poisoning in livestock and MCC-type defects in the offspring of grazing livestock that have consumed the plant when pregnant (Panter et al., 1999). Pregnant livestock typically consume mature poison hemlock plant material in contaminated feed. If the female survives initial exposure to poison hemlock, coniine will cross the placenta and inhibit the movement of the fetus to cause MCC-type deformities (Edmonds et al., 1972; Panter et al., 1988; West et al., 2009; Schep et al., 2009; Swerczek 2012). The teratogenic actions of coniine are species specific, with only minor teratogenicity in rabbits and no teratogenicity in rats (Forsyth and Frank, 1993; Forsyth Birth Defects Research (Part C) 99:235–246 (2013) et al., 1994). In a cell culture model, coniine is a more potent agonist at fetal muscle-type nAChR expressed by TE-671 cells, than at autonomic-type nAChR expressed by SHSY-5Y cells (Table 1), and coniine stereoselectively inhibits fetal movement in the day 40 pregnant goat model (Fig. 1; Green et al., 2013a). Lobeline Lobeline is the principal piperidine alkaloid in Indian tobacco (Lobelia inflata; Felpin and Lebreton, 2004). Lobelia plant preparations have been used for the treatment of asthma, as an emetic, the treatment of tobacco smoking, and as a proposed treatment for psychostimulant abuse (Dwoskin and Crooks, 2002; Felpin and Lebreton 2004). Lobeline displaces 3 [H]-nicotine binding from rat brain membranes with nanomolar affinity, but is insensitive to the noncompetitive ganglionic-blocking agent mecamylamine and the DEVELOPMENTAL DEFECTS DUE TO DISRUPTION OF CHOLINERGIC NEUROTRANSMISSION 239 with (1)-ammodendrine being more effective at depolarizing TE671 cells and more likely to cause MCC-type defects in livestock (Table 1; Lee et al., 2005). Stereoselectivity is a requirement for pharmacological specificity of a receptor and is an important feature of ligand-binding to nAChR (Romano and Goldstein, 1980). PYRIDINE ALKALOIDS In contrast to piperidine alkaloids which inhibit fetal movement to cause MCC-type terata, pyridine alkaloids do not inhibit fetal movement and do not form the correct molecular interactions at the ligand-binding site of fetal muscletype nAChR. Figure 3. The effect of anabasine, lobeline, and myosmine on fetal movement in the day 40 pregnant goat. The bars represent the mean 6 SEM number of fetal movements detected during a five minute fetal ultrasound monitoring period of nine fetuses dosed with saline (0.4 ml/kg), seven anabasine (0.8 mg/kg), eight lobeline (4.0 mg/kg) eight myosmine (5.0 mg/kg), and eight nicotine (0.4 mg/kg) dosed does. Fetal movements were measured at time zero, just prior to i.v. injection and at 0.5 and 1 hr after i.v. dosing. There were significant differences among the treatments (*p < 0.05, one-way ANOVA, Tukey’s multiple comparison test). Data from Green et al. (2013b). neuronal nAChR competitive antagonist dihydro-b-erythroidine in mouse pharmacological assays of motor function and body temperature (Damaj et al., 1997). Lobeline has central nervous system effects in rodents (Brioni et al., 1993; Briggs and McKenna, 1998; Roni and Rahman, 2011). In day 40 pregnant goats, lobeline significantly decreases but did not completely inhibit fetal movement, suggesting that it has a low probability to cause MCC-type defects (Fig. 3). In TE-671 cells, lobeline is a partial agonist (Table 1) and is insensitive to a conotoxin (CTx) EI and GI blockade of the receptor (Green et al., 2013b). a CTxs EI and GI are venom toxins isolated from cone snails (Conus spp.) that selectively target the nAChR ligand-binding sites to block the actions of agonists at the receptor (Gray et al., 1981; Groebe et al., 1995). Blockade of agonism by an antagonist is required for the pharmacological confirmation of a receptor-mediated biological event, and lack of antagonism suggests an alternate mechanism of action. Thus, the exact mechanism of action of lobeline cell membrane depolarization in TE-671 cells is unknown. Ammodendrine Ammodendrine is a piperidine alkaloid teratogen, found in Lupinus spp., that causes MCC-type defects in cattle (Keeler and Panter, 1989). Ammodendrine is present in Lupinus formosus as enantiomeric mixtures, which can vary in the optical rotations of plane polarized light, depending on the ratios of each enantiomer present in the plant material (Lee et al., 2005). This is significant because the ratio of (1) to (2) enantiomers will affect potency and efficacy at nAChR. As the ratios of (1) to (2) enantiomers change from plant population to population or season, the toxicity and teratogenicity of the plants will change as well as the severity of the MCC-type defects. In TE-671 cells, the actions of ammodendrine are stereoselective Nicotine The pyridine alkaloid nicotine is a prototypical ligand for nAChR, and is involved in tobacco use and dependence. The effects of nicotine on behavior and cognitive function are well-known (for review, see Decker et al., 1995; Yakel, 2012). Nicotine has been extensively studied as an addictive substance, and its effects are mediated by actions at nAChR in the central and peripheral nervous systems (for more information, see Taylor, 2001; Leslie et al. 2013). Nicotine has binding affinities for a3b4, and a4b2 nAChR in the nanomolar range, and in the micromolar range at a7 nAChR (Decker et al., 1995; Jensen et al., 2003; Wonnacott and Barik, 2007). In TE-671 cells which express fetal muscle-type nAChR, nicotine acts as a partial agonist (Table 1) and at 1.0 mM concentrations does not desensitize the cells (Green et al., 2010). Nicotine does not inhibit fetal movement in day 40 pregnant goats dosed IV, because it lacks the correct ring structure associated with MCC-type terata formation (Figs. 2 and 3). Myosmine Myosmine is a pyridine alkaloid found in tobacco (N. tabacum) and other plants, such as the pituri bush (Duboisia hopwoodii), peanuts (Arachus hypogaea), and Birth Defects Research (Part C) 99:235–246 (2013) 240 GREEN ET AL. plete blockade of fetal movement did not result. Thus, myosmine does not likely cause MCC-type terata, as complete inhibition of fetal movement is required. QUINOLIZIDINE ALKALOIDS Anagyrine Figure 4. The alkaloids present in Sophora stenophyla collected in Hildale, Utah (Data from Lee et al., 2013a) (A) and the effects of orally dosed Sophora stenophyla on fetal movement in a day 40 pregnant goat model (B). Alkaloids depicted in panel (A) include (percent alkaloid content 6 SD): (1) sparteine (0.14 6 0.06), (2) N-methylcytisine (0.42 6 0.06), (3) cytisine (0.26 6 0.06, (4) sophorocarpine (coeluted with matrine, not calculated), (5) matrine (coeluted with sophorocarpine, not calculated), and (6) sophoramine (0.33 6 0.03; Data from Lee et al., 2013a). The estrus cycles in 20 Spanish-type female goats were synchronized and then bred by a Spanish-type buck as previously described (Panter et al., 1990). On day 40 of pregnancy, the goats were ultrasounded as previously described for three minutes and the number of fetal movements counted (Green et al., 2013a). The doses were then averaged with 4 g/kg body weight Sophora (n 5 7), 2 g/kg body weight Nicotiana (5.5 mg/kg anabasine) as a positive control (n 5 7), or 5 g/kg body weight dried ground grass hay as a negative control (n 5 6). The bars represent the mean 6 SEM of fetal movements detected during a 3min fetal ultrasound monitoring period. Fetal movements were measured at time zero just prior to dosing and 2 hr after oral dosing. There were significant differences among the treatments (*p < 0.05, one-way ANOVA, Tukey’s multiple comparison test). All animal work was approved by the Utah State University IACUC. hazelnuts (Corylus avellana; Luanratana and Griffin, 1982; Zwickenpflug et al., 1998; Zwickenpflug and Tyroller, 2006). Myosmine is genotoxic in adenocarinoma cells, human mucosal epithelial cells, and lymphocytes (Kleinsasser et al., 2003; Vogt et al., 2006). In TE-671 cells, myosmine is a full agonist (Table 1), and its actions are concentration-dependent and sensitive to blockade by the nAChR antagonist a CTx GI (Green et al., 2013b). Although myosmine significantly decreased fetal movement in a day 40 pregnant goat, com- Birth Defects Research (Part C) 99:235–246 (2013) The quinolizidine alkaloid anagyrine is a partial agonist at nAChR, expressed by both TE-671 cells and SHSY-5Y cells (Table 1). Anagyrine causes MCC-type deformities and cleft palate in cattle, but not in sheep, goats, or hamsters (Keeler and Panter, 1989; Panter and Keeler, 1993). It is hypothesized that anagyrine is metabolized in the cow to a piperidine alkaloid, which is then absorbed as a complex piperidine with teratogenic activity. However, data from kinetic experiments in cattle, sheep, and goats do not demonstrate clear differences in biotransformation (Keeler et al., 1976; Keeler and Panter, 1989; Gardner and Panter, 1994). Anagyrine is a partial agonist with low percent maximal responses in both TE-671 cells and SHSY-5Y cells (Table 1). More research is needed to elucidate if metabolic transformation of anagyrine is responsible for its pharmacological activity in cattle. N-Methylcytisine One quinolizidine alkaloid that has significant human exposure is N-methylcytisine, found in herbal preparations of blue cohosh (Caulophyllum thalictroides). Blue cohosh has been reported to cause perinatal stroke, acute myocardial infarction, and congestive heart failure in humans, and in vitro evidence suggests that blue cohosh is teratogenic in rat embryo culture experiments (Kennelly et al., 1999; Finkel and Zarlengo, 2004; Dugoua et al., 2008). When blue cohosh is used as an abortifacient, there are symptoms consistent with nicotine toxicity that are attributed to the quinolizidine alkaloid, N-methylcytisine (Rao and Hoffman, 2002). Blue cohosh also DEVELOPMENTAL DEFECTS DUE TO DISRUPTION OF CHOLINERGIC NEUROTRANSMISSION 241 contains the piperidine alkaloid, caulophyllum B, and the quinolizidine alkaloids, O-acetylbaptifoline, lupanine, and anagyrine (Madgula et al., 2009). In a rat embryo culture assay N-methylcytisine, at concentrations of 20 mg/ml, caused terata (neural tube defects, defects in eye development, and a twisted tail; Kennelly et al., 1999). However, when N-methylcyctisine was orally dosed in the form of dried ground Sophora stenophylla to day 40 pregnant goats there were no significant decreases in fetal movement (Fig. 4), suggesting that MCC-type defects are unlikely to occur from ingestion of S. stenophylla. LIGAND-BINDING TO nAChR The actions of the alkaloids listed above are mediated by nAChR, which are members of the cys-loop receptor family. There are 17 genetically distinct nAChR subunits; a1–10, b1–4, c, d, and e identified (for review see, Wonnacott and Barik, 2007). These receptors are important drug targets, and drugs that act at nAChR are currently being developed for the treatment of Alzheimer’s disease, schizophrenia, Parkinson’s disease, and epilepsy (Levin and Rezvani, 2002; Yakel, 2012). Nicotinic acetylcholine receptors are present throughout the body, including the central nervous system, autonomic ganglia, sensory ganglia, and neuromuscular junctions. These receptors can be presynaptic or postsynaptic, with presynaptic nAChR modulating the release of neurotransmitters such as biogenic amines (Marshall et al., 1997; Sershen et al., 1997; Rao et al., 2003; Zappettini et al., 2010). When nAChR are located postsynaptically, like the motor endplate region of the neuromuscular junction, they mediate excitatory neurotransmission. Each functional receptor consists of five subunits arranged around a central cationchannel pore. The subunit composition of nAChRs can vary from the a7 nAChR homopentamer, to heteropentamers with the subunit composition dependent on where the receptor is expressed in the body (for review, see Albuquerque et al., 2009). The teratogenic effect of plant alkaloids in livestock is thought to be due to agonist actions at fetal muscle-type nAChR expressed in the muscle of the developing fetus. Fetal muscle-type nAChR are comprised of a1(2)b1cd nAChR subunits and the a subunits contain the orthosteric ligandbinding sites for acetylcholine (one per a subunit; Arias, 2000). The ligand-binding sites of nAChR are extensively studied, and models of these sites termed “pharmacophores” have been developed to describe specific interactions of agonists with the receptor. A pharmacophore can be defined as a group of chemical features (including intermolecular forces such as H-bonding, cation-p bonds, and hydrophobic and electrostatic interactions) required for a ligand to interact with the ligandbinding site of a receptor to elicit a biological response (Wermut et al., 1998; Glennon and Dukat, 2004). The precursor to the current nAChR pharmacophore model was proposed by Beers and Reich (1970). They described the minimum features required for ligand binding and activation of nAChR, including a cationic center, and a hydrogen bond acceptor. Modifications of the nAChR pharmacophore model have been made to account for experimental results (for more information, see Sheridan et al., 1986; Glennon and Dukat, 2004; Blum et al., 2010; Tavares et al., 2012). Recent research suggests that the cationic center in nAChR agonists plays an important role in bond formation between the receptor and ligands at the ligandbinding site. Differences in receptor binding to the cationic center of the ligand can account for agonist potency and efficacy. For example, a tryptophan amino acid at position 149 of the a subunit (Trp a149 also known as TrpB) in the muscletype receptor forms a cation-p bond with acetylcholine, but not nicotine. Nicotine, which is less potent at muscle-type receptors, forms a hydrogen-bond with the backbone carbonyl of TrpB (Dougherty, 2013). The specific interactions between the nAChR agonists and TrpB have been disrupted with unnatural amino acid mutagenesis (Nowak, et al., 1995). In fetal muscle-type nAChR expressed in Xenopus oocytes, the progressive fluoridation of the TrpB ring side-chains shifts acetylcholine EC50 values rightward, with the amount of shift correlating with the number of fluoride side chains on the aromatic ring of the tryptophan molecule. Conversely, the EC50 values of nicotine were not shifted in a parallel manner (Zhong et al., 1998; Beene et al., 2002; Dougherty, 2008). The lack of nicotine EC50 shift from progressive fluoridation suggests that nicotine does not form a cation-p bond interaction at the aromatic ring of TrpB (Cashin, et al., 2005). Interestingly, nicotine is more potent at neuronal a4b2 nAChR, and the fluoridation of TrpB results in parallel EC50 value shifts for nicotine and acetylcholine, suggesting that both agonists form a cation-p bond at TrpB in a4b2 nAChR (Xiu et al., 2009). The differences in nicotine binding between muscle-type nAChR and a4b2 nAChR are attributed to a glycine at position 153 on the a subunit of the receptor, which affects the interactions between nicotine and TrpB by preventing the cation-p interaction (Xiu et al., 2009; Dougherty, 2013). In humans, an aG153S mutation that converts glycine to serine in muscle-type nAChR results in a congenital myasthenic syndrome. This syndrome is associated with profound muscle weakness due to receptor desensitization, altered ligand affinity, and altered gating of the receptor cation channel (Sine et al., 1995). Moreover, experimental in vitro conversion of the glycine to lysine in the muscle-type receptor results in the restoration of a strong cation-p bond interaction between nicotine and TrpB (Xiu, et al., 2009). Differences in cation-p bond formation between agonists at TrpB in the muscle receptor have been used to classify ligands as either nicotinic (lack cation-p interaction) or cholinergic (formation of a cation-p bond between the agonist and TrpB; Cashin et al., 2005). Birth Defects Research (Part C) 99:235–246 (2013) 242 GREEN ET AL. Figure 5. Schematic models of nAChR receptor conformation states. (A) Desensitizaiton model proposed by Katz and Thesleff (1957); acetylcholine (ACh), receptor (R), ACh bound to the nAChR ligand-binding site with an open cation channel (AChR), ACh bound to the nAChR ligand-binding site with a closed cation channel in a desensitized state (AChR’), and an nonreactive desensitized receptor state with ACh unbound (R’). (B) Two-state cyclic nAChR model from Sine and Taylor, (1982); ligand/agonist (L), activatable receptor (R), high-affinity desensitized state (R’) allosteric constant (M). For more information on the modeling of nAChR–ligand interactions, see Edelstein et al., (1996). It is tempting to speculate that the lack of a strong cation-p interaction at TrpB is responsible for the differences in teratogenicity observed between the piperidine alkaloids like coniine and pyridine alkaloids like nicotine. AGONIST MEDIATED nAChR DESENSITZATION The molecular mechanism behind the inhibition of fetal movement described above is postulated to be the formation of a cation-p bond between TrpB and the teratogenic alkaloid. The result of this ligand/ligand-binding site interaction is activation of the receptor and ultimately prolonged receptor desensitization. Nicotinic acetylcholine receptor desensitization was first described from experiments with frog sartorius muscle (Katz and Thesleff, 1957). They described desensitization as the state of the frog muscle fibers which becomes unresponsive to continued agonist exposure, and from which the muscle fibers recover only after complete with- drawal of the agonist. Results from these early experiments led to the formation of a hypothesis suggesting the existence of multiple receptor states, one of which is desensitized and unresponsive to further stimulus by agonist (Fig. 5a). This initial model by Katz and Thesleff (1957) has been expanded to account for multiple ligand-binding sites of nAChR and positive allosterism with the desensitized state having the highest affinity for agonists (Fig. 5b). The progressive allosteric transition from a low affinity resting state to higher affinity open state and finally much higher affinity desensitized state provides the driving force for the transition between receptor states (Sine and Taylor, 1982), and can occur at different rates from fast to slow (for review see Ochoa et al., 1989). Modeling of the nAChR ligandbinding sites suggests multiple states of the receptor with varying affinities toward ligands with the ion channel open or closed. Desensitization can also be modulated through a cAMP-dependent protein Birth Defects Research (Part C) 99:235–246 (2013) kinase A-mediated phosphorylation of the receptor (Lu et al., 1993; Fu, 1993; Paradiso and Brehm, 1998). For example, the diterpenoid alkaloid forskolin from Coleus forskohlii activates adenylyl cyclase to increase intracellular cAMP concentrations that results in the phosphorylation of the intracellular domains of the nAChR subunits by protein kinase A. Experimentally, treatment of 1-day-old embryonic Xenopus muscle cells with forskolin increases the rate of acetylcholineinduced nAChR desensitization (Fu, 1993). Desensitization is also dependent on the structure of the agonist. For example, fetal muscle-type nAChR desensitized with nicotine recover faster than receptors desensitized with acetylcholine (Reitstetter et al., 1999). The difference in recovery between nicotine and acetylcholine at the fetal muscletype receptors is most likely due to bond formation between nicotine and acetylcholine at TrpB of the fetal muscle-type receptor, as described above. Agonist dependency has also been shown in experiments with day 40 pregnant goats. Intravenous dosing of day 40 pregnant goats with the piperidine alkaloid coniine results in prolonged inhibition of fetal movement, whereas the pyridine alkaloid nicotine does not inhibit fetal movement (Figs. 1 and 3; Green et al., 2013b). Biologically, nAChR desensitization may be a means to protect against excitotoxicity by reducing the number of receptors and molecules of agonist available for receptor activation by trapping them at the ligand-binding sites of desensitized high-affinity receptors (Giniatullin et al., 1997, 2001, 2005). Ultimately, the likely mechanism behind MCC-type defects is the inhibition of fetal movement due to stimulation followed by desensitization of fetal muscle-type nAChR by piperidine alkaloids. HUMAN HEALTH Tobacco use by pregnant women is associated with many birth cheiloschisis and palatoschisis (Shaw et al., 2009; Hackshaw et al., DEVELOPMENTAL DEFECTS DUE TO DISRUPTION OF CHOLINERGIC NEUROTRANSMISSION 243 2011). One tobacco alkaloid in particular, nicotine, has received much research attention. For example, prenatal nicotine causes a variety of adverse pregnancy outcomes, including low birth weight, spontaneous abortion, and cognitive impairment, including attention deficit/hyperactivity disorder (Milberger et al., 1996; Ernst et al., 2001; Counotte et al., 2012). Results from in vitro experiments with human granulosa cells and choriocarcinoma cells suggest that anabasine, nicotine, and cotinine can inhibit the production of estrogen and progesterone, both of which are involved in the maintenance of pregnancy (Barbieri et al., 1986; Gocze et al., 1999; Albrecht et al., 2000). Maternal smoking in humans has also been associated with orofacial clefts. The offspring of heavy smokers who used tobacco products during the periconceptional period are about two times more likely to have a cleft lip or palate, and 4% of all orofacial clefts can be attributed to maternal smoking (Honein et al., 2007). A meta-analysis study by Little et al. (2004) calculated a 30% increased risk for cleft lip with or without cleft palate, and attributed this risk to a combination of environmental and genetic factors. The tobacco alkaloid anabasine causes MCCtype skeletal defects and cleft palate in livestock (Panter et al., 1999). Anabasine is in tobacco and tobacco products, such as oral snuff and cigarettes, as well as in biological samples from smokers, suggesting that there is potential for fetal exposure to this teratogenic alkaloid in humans (Jacob et al., 1999; Brunnemann et al., 2002; Djordjevic and Doran, 2009; Miller et al., 2010a,b). CONCLUSIONS Plant alkaloids that meet the certain structural requirements (i.e., a piperidine ring with a three carbon side-chain a to the piperidine nitrogen) are teratogenic in livestock and human teratogenesis is associated with tobacco use. These alkaloids act through the disruption of cholinergic neurotransmission at the neuromuscular junction in livestock species, such as cattle and goats. This disruption is mediated by a cation-p bond formation between agonists and TrpB in the ligand binding sites of the muscletype nAChR in the developing fetus. Differences in cation-p interactions at TrpB may be responsible for the differences in teratogenicity observed between piperidine and pyridine alkaloids. Ultimately, the formation of a cation-p bond between TrpB and teratogenic alkaloids results in activation and then desensitization of the fetal muscle-type nAChR at the fetal neuromuscular junction which inhibits movement. If the interaction between piperidine alkaloid agonists and the receptor is prolonged, MCC-type defects and cleft palates are the result. ACKNOWLEDGEMENTS The authors thank Clint Stonecipher, Edward L. Knoppel, Terrie Wierenga, and Rex Probst for technical assistance. The authors thank Isabelle McCollum for technical assistance and helpful comments about the manuscript. REFERENCES Albrecht ED, Aberdeen GW, Pepe GJ. 2000. 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