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.
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