effects of ipomoea carnea aqueous fraction gorniak
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Effects of Ipomoea carnea aqueous fraction intake by dams during
pregnancy on the physical and neurobehavioral development
of rat offspring
A. Schwarz, S.L. Gorniak, M.M. Bernardi, M.L.Z. Dagli, H.S. Spinosa*
Department of Pathology, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87,
CEP 05508-900 Sao Paulo, Sao Paulo, Brazil
Received 1 October 2002; received in revised form 24 December 2002; accepted 15 May 2003
Abstract
The effects of daily prenatal exposure to 0.0, 0.7, 3.0 and 15.0 mg/kg of the aqueous extract (AQE) of Ipomoea carnea dried leaves on
gestational days 521 were studied in rat pups and adult offspring. The physical and reflex developmental parameters, open-field, plus-maze,
social interaction, forced swimming, catalepsy and stereotyped behaviors, as well as striatal, cortical and hypothalamic monoamine levels (at
140 days of age) were measured. Maternal and offspring body weights were unaffected by exposure to the different doses of the AQE. High
postnatal mortality, smaller size at Day 1 of life, reversible hyperflexion of the carpal joints and delay in the opening of both ears and in
negative geotaxis were observed in the offspring exposed to the higher dose of AQE. At 60 and 90 days of age, open-field locomotion
frequency was quite different between 0.0 and animals treated with 0.7 and 3.0 mg/kg AQE. No changes were observed in the plus-maze,
social interaction, forced swimming, catalepsy, stereotyped behavior and central nervous system monoamines concentrations. Dams treated
with the higher AQE dose showed severe cytoplasmic vacuolation in liver, kidney, pancreas and thyroid tissues, in contrast to the mild
vacuolation observed in the other experimental groups. No alterations were observed in the histopathological study of the offspring of all
experimental groups at 140 days of age. During adulthood, behavior was not modified in offspring exposed to the higher dose of AQE as well
as no changes occurred in central nervous system neurotransmitters. The present data show that the offspring development alterations were
not severe enough to produce behavioral and central monoamine level changes.
D 2003 Elsevier Inc. All rights reserved.
Keywords: Animal behavior; Ipomoea carnea; Perinate; Neurotransmitter
1. Introduction
Ipomoea carnea, a tropical plant of the Convolvulaceae
family, is a shrubby, quickly growing toxic plant, widely
distributed throughout Brazil [48]. During periods ofdrought, animals graze on this plant which grows even in
the presence of adverse climatic conditions [30].
After prolonged periods of plant intake, the animals
exhibit a variety of clinical signs like depression, general
weakness, loss of body weight, staggering gait, muscle
tremors, ataxia, posterior paresis, and paralysis [10,11,
26,49]. These toxic effects are attributed to the polyhy-
droxylated alkaloids swainsonine, calystegines B1, B2, C1
and 2a-2b-dihydroxynortropane detected in I. carnea [3],chemical toxins present mainly in the leaves of the plant.
Swainsonine, an indolizidine alkaloid, is a potent inhib-
itor of lysosomal a-mannosidase and Golgi a-mannosidase-
II, resulting in lysosomal accumulation of incompletelyprocessed oligosaccharides and alteration of the synthesis,
processing and transport of glycoproteins [9,46]. Calyste-
gines, nortropanic alkaloids, inhibit the activity of glucosi-
dases, galactosidases and xylosidases, lysosomal enzymes,
which act on oligosaccharide metabolism [2].
The lysosomal storage disorder induced in animals graz-
ing on I. carnea [11,47,49] is very similar to the rare human
genetic mannosidosis [13], characterized by cytoplasmic
vacuolization of nervous and peripheral cells as a conse-
quence of the inhibition or absence of this enzyme [46].
It has long been known that I. carnea intake for a
prolonged period of time induces neurobehavioral effects
0892-0362/$ see front matterD 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0892-0362(03)00078-3
* Corresponding author. Tel.: +55-11-3091-7656; fax: +55-11-3091-
7829.
E-mail address: [email protected] (H.S. Spinosa).
www.elsevier.com/locate/neutera
Neurotoxicology and Teratology 25 (2003) 615 626
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in goats, cattle and sheep [4749]. However, there are no
reports about its effects on offspring if grazed by animals
during the gestation period. It is also known that swainso-
nine and calystegines are weakly basic compounds. Thus,
how can they cross the brain barrier and cause neurologic
effects? And how can they cross the placental barrier too?
Perhaps these alkaloids can interact with barrier functionand sites, thus being able to enter the brain and contact the
fetus. Hydrophilic compounds can cross these barriers at
high concentrations and accumulate in subcellular compart-
ments with a low pH [2].
Swainsonine may be actively accumulated in liver and
kidney because of the extensive sugar scavenging systems
of metabolism and conservation present in these organs [6].
The same does not occur in the brain or placenta, which
require high swainsonine doses to develop lesions, and
whose clearance rates are somewhat slower than in others
organs [45]. The concentrations of swainsonine in nervous
system and placenta may be lower because the brain andplacental barrier is relatively rich in lipids and swainsonine
is less lipid soluble. In addition, cell damage is a conse-
quence of both time of exposure to swainsonine and
quantity of the compound. Thus, this damage is determined
by the duration of exposure [35].
The literature extensively reports that swainsonine
causes, in addition to increased embryo mortality during
early exposure, delayed placentation and deformities of the
carpal joints of newborn lambs [2830], a fact that, in
view of the inhibition of lysosomal a-D-mannosidase bythe toxin, may be equivalent to the mannosidosis found in
Angus cattle and humans [30]. Later exposure causes
reductions in uterine and placental vascularity, hydrops
amnii, hydrops allantois and disruption of fetal fluid
balance [8].
It is known that exposure of dams to xenobiotics
during the gestation or lactation periods can impair the
physical and neural development of the offspring. This
developmental neurotoxicity can occur in different man-
ners because it involves alterations in dam and offspring
behavior, neurohistology, neurochemistry and gross dys-
morphology of the offspring central nervous system [31].
The main objective of the present investigation was to
study the possible toxic effects of I. carnea aqueous
extract (AQE) on rats exposed to it during the organo-genesis and fetal development periods of gestation. Phys-
ical and reflex development, neurobehavioral aspects and
central nervous system monoamine levels of the offspring
were determined.
2. Material and methods
2.1. Plant
I. carnea was planted in a 1,500 m2 field of the Centro de
Pesquisa em Toxicologia Veterina
ria, Pirassununga, Sao
Paulo state, Brazil. When the plants were mature, the leaves
were taken (AprilJune 2001) for extraction.
2.2. Preparation of the aqueous fraction extract (AQE)
First the fresh leaves were triturated with ethyl alcohol
(97 Gay Lussac) in a blender and then macerated for 72 h inethyl alcohol (97 Gay Lussac) and filtered using a Buchnerfunnel. Next, the filtrate was evaporated under reduced
pressure and the product obtained was reserved. The recov-
ered ethyl alcohol was again returned to the leaf residue for
a 24-h maceration period, a new filtration and evaporation
were performed under reduced pressure, and the product
was again reserved. This procedure was repeated two more
times and the four products obtained were pooled, forming
the final extract. The final extract was diluted in distilled
water and filtered through filter paper. The filtered portion,
ethanolic fraction, was treated with butanolic alcohol and
separated with a decantation funnel. This procedure origi-nated the AQE that was stored at 20 C.
A previous study showed that this AQE contains swain-
sonine and calystegines A3, B1, B2, B3 and C1 [24]. Dr. Dale
Gardner, Utah State University (USDA-ARS Poisonous
Plants Research Laboratory, USA), using high-performance
liquid chromatography and mass spectrometry, later showed
quantitatively that the AQE used in this study (samples were
sent off) contained 0.09% swainsonine, 0.11% calystegine
B2, 0.14% calystegine B1, and 0.06% calystegine C1 (per-
sonal communication).
2.3. Animals
Wistar rats from the Department of Pathology (Faculdade
de Medicina Veterinaria e Zootecnia, Universidade de Sao
Paulo), weighing 180 200 g and aged approximately 90
days, were used. The animals were housed in polypropylene
cages (50 40 20 cm). The animals employed for the
evaluation of stereotyped behavior were housed in wire
cages (50 30 17 cm) 8 days before the test. The animals
were kept under controlled temperature (2224 C) on a12:12 light/dark schedule (lights on at 6:00 a.m.), with free
access to food and water. The animals used in this study
were maintained in accordance with The Guide for the Care
and Use of Laboratory Animal, National Research Council,USA (1996).
2.4. Procedures
2.4.1. Treatment, reproductive parameters and maternal
data
Sexually naive female rats (n = 58) were mated with
males previously tested as fertile (2 females and 1 male
per cage). Pregnancy was determined by the presence of
spermatozoa in vaginal smears on the following morning,
designated as gestation day 1 (GD1). Pregnant rats were
removed and kept in separate cages. On GD5, the dams
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were divided into five groups. Four experimental groups
(n = 12) were treated orally by gavage once a day from GD5
to GD21 with 0.0, 0.7, 3.0 or 15.0 mg/kg AQE. One no
gavage control group (n = 10) was used (this group was
employed to study the possible effects caused by the stress
of the gavage procedure).
The pregnant rats were weighed at GD2, GD4, GD6,GD8, GD10, GD12, GD14, GD16, GD18, GD20 and
GD21. Food and water consumption during pregnancy,
length of gestation, litter size, anogenital distance of the
pups, sex ratio and postnatal death at PN1 were also
assessed.
2.4.2. Offspring studies
All the pregnant rats were allowed to give birth and
nurture their offspring normally. No cross-fostering proce-
dure was used. Parturition day was defined as PN0. On PN1,
all the litters were examined externally and sexed. Litters
were organized into groups of eight pups each, four malesand four females, and the remaining pups were discarded.
One male and one female pup of each litter were marked
daily with colored felt tip pens. The same male and female
pups of each litter were used for all the physical and
developmental evaluations. They were weighed individually
every day until weaning (PN21) and then on PN30, PN60,
PN75 and PN90. The following parameters were observed:
coat appearance (beginning on PN2), pinna detachment
(beginning on PN2), eruption of incisor teeth (beginning
on PN3), adult gait (when the pups walk without propping
their ventral portion on the floor, beginning on PN10), ears
open (determined by visualization of the open auditory
meatus, beginning on PN10), eye opening (determined by
the visualization of a longitudinal eyelid fissure, beginning
on PN10), testis descent (considered as the scrotum purse
touching the testis, beginning on PN16), vaginal opening
(when the vaginal hole is visualized, beginning on PN30),
day of appearance of the surface righting reflex (first day
when the normal ventral position is assumed successfully
within a period of time not exceeding 15 s after the pup is
placed on its back, beginning on PN5), day of appearance of
negative geotaxis (first day when a 180 turn is assumedwithin a period of time not exceeding 30 s, after the pup is
placed face down on a 45 inclined platform, beginning on
PN6) and palmar grasp reflex (pup grasps a paper clip withforepaws if stroked; pups are born with this reflex that
usually disappears between PN8 and PN10). Pups were
observed daily between 8:00 and 11:00 a.m., separated from
the mothers at the time of observation (no more than 3 min),
and immediately returned to their home cages. Mean day of
appearance for each of the above parameters was calculated.
All data were analyzed considering the litter as the smallest
unit.
On PN21, the offspring were weaned and the littermates
separated and housed together by sex. The same male and
female pups chosen from each litter for the observation of
all developmental parameters cited above were used for the
open field observation at PN21, PN30, PN60 and PN90 as
well as for the plus-maze analysis at PN90. After these
procedures, they were reserved for the histopathology study.
Thus, in summary, one male and one female animal from
each litter (20 animals per group) were used for these tests.
Due to the high mortality rate of the 15.0 mg/kg group (six
litters survived), only 12 animals (1 male and 1 female pupfrom each litter) were employed. For the same reason, the
animals of the 15.0 mg/kg group were not examined for
stereotyped behavior or catalepsy and were not submitted to
the forced swimming and social interaction tests.
For the stereotyped behavior test, another 12 animals per
group (6 males and 6 females), not handled before at any
experimental situation, were employed. Not necessarily one
male and one female per litter was employed. At least one
animal (a male or a female) from each litter was used.
For the catalepsy and forced swimming tests, another 10
animals per group (5 males and 5 females), not handled
before at any experimental situation, were used in each test.Only one animal (a male or a female) from each litter was
used.
For the social interaction test, another 12 animals per
group (6 males and 6 females), not handled before at any
experimental situation, were employed. Pairs of animals
(strangers to each other) of the same sex and from the same
group but from different litters were organized for this
evaluation.
For the determination of monoamine levels, another 10
animals per group (5 males and 5 females), not handled
before at any experimental situation, were employed. Only
one animal (a male or a female) from each litter was used. In
the 15.0 mg/kg group, at least two animals (one male and
one female) per litter were used for this evaluation because
of the low number of surviving litters (only six).
2.4.3. Open-field studies
The open-field behaviors of male and female offspring
were measured at weaning (PN21) and at PN30, PN60 and
PN90; the same animals (1 male and 1 female pup from
each litter employed for evaluation of physical develop-
ment) were used on these different days. The device was
similar to that described by Broadhurst [7], i.e., a round
arena 40 cm in diameter for pups and 96 cm in diameter for
adults surrounded by a 25 cm high enclosure painted whiteand subdivided into 25 parts painted black. During the
experiments, a 40-W white bulb located 72 cm above the
floor provided continuous illumination of the arena.
For the observations, each animal was individually
placed in the center of the arena and the following param-
eters were measured over a period of 5 min: locomotion
frequency (number of floor units entered with both feet),
rearing frequency (number of times the animal stood on its
hind legs), immobility time (total number of seconds with
no movement) and defecation (number of fecal pellets).
Hand-operated counters and stopwatches were employed to
score these behaviors. To minimize possible influences of
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circadian changes on open-field behaviors, control and
experimental animals were alternated. The device was
washed with a 5% alcohol/water solution before placing
the animals on it in order to obviate possible biasing effects
due to odor clues left by previous rats.
2.4.4. Plus-maze studiesThe plus-maze behaviors of male and female offspring
were measured at PN90, after the open-field test, using the
same animals. Thus, the animals were removed from the
open field and immediately placed in the center of the plus-
maze and observed for 5 min.
The device consisted of two opposite open arms (50 cm
long 10 cm wide) and two opposite closed arms (50 cm
long 10 cm wide 40 cm high) arranged at 90 angles.The floor of the maze was made of wood, painted gray and
located 50 cm above the floor. The center of the maze was
open and the walls of the closed arms started 2 cm from the
center of the maze.For the observations, each animal was individually
placed in the center of the maze with the head facing one
of the open arms, and the following parameters were
measured over a period of 5 min: number of entries into
the open arms, number of entries into the closed arms, time
spent in the open arms, time spent in the closed arms and
time spent in the center of the plus-maze. Hand-operated
counters and stopwatches were employed to score these
behaviors. To minimize the influence of possible circadian
changes on plus-maze behaviors, control and experimental
animals were alternated. The device was washed with a 5%
alcohol/water solution before placing the animals on it to
obviate possible biasing effects due to odor clues left by
previous rats.
2.4.5. Stereotyped behavior
Due to the high mortality rate of the 15.0 mg/kg group, a
sufficient number of pups was not available for the stereo-
typed behavior test. Thus, other pups from control and
experimental groups not previously used in any test were
employed.
The stereotyped behavior of male and female offspring
was measured at PN95. After being initially housed socially
four to a cage at PN21, the animals were housed individ-
ually in wire cages through 8 days for habituation to theirnew home, avoiding interference of exploratory behavior in
the new cage. After the isolation period of eight days, the
animals received 0.6 mg/kg apomorphine hydrochloride
(Sigma) subcutaneously. Stereotypy was quantified every
10 min (with an observation time not exceeding 10 s per
animal) for 2 h after apomorphine treatment by the scoring
system proposed by Setler et al. [42]. Briefly, scores ranging
from 0 (asleep or stationary) to 6 (continuous licking and
gnawing of cage grids) were recorded for animal behavior
using stopwatches to measure the duration of the behaviors.
Time effect curves were constructed using the scores
obtained during a total of 2 h after apomorphine treatment.
2.4.6. Catalepsy
Due to the high mortality rate of the 15.0 mg/kg group, a
sufficient number of pups was not available for the catalepsy
behavior test. Thus, other pups from control and experimen-
tal groups not previously used in any test were employed.
Catalepsy was measured at PN90 in male and female
offspring 20, 40, 60, 80, 100, 120, 140, 160 and 180 minafter intraperitoneal administration of haloperidol (1 mg/
kgJanssen Farmaceutica) on the basis of the duration of
animal immobility (total number of seconds of lack of
movement). The maximal test period allowed per animal
was 20 min. Rats were tested individually for permanence in
the upright position, with their forepaws flattened on a
horizontal bar placed 10 cm above the bench. Each rat
was tested three times for catalepsy at each time interval and
the sum of three immobility episodes at each time was used
to construct the timeeffect curves. Stopwatches were used
to score this behavior.
2.4.7. Social interaction
Due to the high mortality rate of the 15.0 mg/kg group, a
sufficient number of pups was not available for the social
interaction behavior test. Thus, other pups from control and
experimental groups not previously used in any test were
employed.
The social interaction behaviors of male and female
offspring were measured by direct observation of pairs of
rats strangers to one each other, of the same experimental
group, but from different litters, in the open-field at PN95.
Rats initially housed socially four to a cage since PN21, as
explained above, were, before this test, housed individually
in propylene cages for 5 days. This procedure is needed to
increase the motivation for social investigation. Then, each
rat was allowed to spend 10 min alone in the arena for
habituation. One day later, the rats of a same pair, strangers
to each other (they never stayed together, in any circum-
stance), were placed together in the device for 10 min for
familiarization and the test was performed 24 h later.
Stopwatches were used to measure the following behaviors:
total time spent smelling and following each other, and in
genital investigation and licking over a period of 10 min.
Aggressive and passive behaviors were extremely rare in
this test and were not quantified.
2.4.8. Forced swimming test
Due to the high mortality rate of the 15.0 mg/kg group, a
sufficient number of pups was not available for the forced
swimming behavior test. Thus, other pups from control and
experimental groups not previously used in any test were
employed.
The test was performed as previously described [3739].
At PN95, rats were plunged individually into a vertical glass
cylinder (height 40 cm; diameter 22 cm) containing 25 cm of
water at 20 C. After 15 min in the cylinder, animals wereremoved and allowed to dry for 30 min in a heated enclosure
(28 C) before being placed in their individual cages again.
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One day later, rats were plunged in the cylinder again, and
latency to start floating, as well as total immobility time, in
seconds, was quantified during the following 5 min.
2.4.9. Determination of monoamine levels
At PN140, male and female rats from control and exper-
imental groups, not used for any behavior test, were decap-itated. Brains were dissected on dry ice and prepared as
described elsewhere [15]. Briefly, the striatum, cortex and
hypothalamus were weighed and stored at 80 C untilneurochemical analysis were carried out. Following sample
collections, perchloric acid was added to the tissues, which
were then homogenized by sonication for immediate deter-
mination of monoamine levels. Dopamine (DA) and its
metabolites [3,4-dihydroxyphenylacetic acid (DOPAC) and
homovanillic acid (HVA)], serotonin (5HT) and its metabo-
lite [5-hydroxyindolacetic acid (5HIAA)] and norepineph-
rine (NOR) and its metabolite [vanilmandelic acid (VMA)]
were measured by HPLC (Shimadzu, model 6A) using a C-1column (Shimpak-ODS), an eletrochemical detector (Shi-
madzu, model 6A), a sample injector (15 and 20 ml valve)and an integrator (Shimadzu, model 6A Chromatopac). Each
sample was run for 18 min. The detection limit was 2 pg for
DA, DOPAC, NOR, 5HT and 5HIAA, and 20 pg for HVA.
2.4.10. Histopathology
On the day of weaning, the dams (10 per group) were
anesthetized with ethyl ether and parts of the liver, pancreas,
thyroid, kidney and brain were collected and maintained in
10% formalin for later histopathological study. The same
procedure was used for the experimental and control off-
spring groups (10 per group) at PN 140. The animals used
here were the same as those used before in the open field
and plus-maze behavior tests.
2.5. Statistical analysis
Results are expressed as litter means S.E.M. as the
maternal unit to avoid litter effects. Bartletts test was used
to determine data homogeneity. ANOVA followed by the
Dunnett test was used to analyze the parametric data of
the dams, the perinatal death, testis descent, vaginal open
and neurotransmitters levels of male and female offspring.
The nonparametric data were analyzed by the Kruskal
Wallis test followed by the Dunn test for multiple com-
parisons. For all the others parameters analysed, a two-way ANOVA was employed. The t test was used as a post
hoc test when no interactions were observed between
factors. In the case of a significant interaction, one-way
ANOVA was applied. In all cases, results were considered
significant for P< .05.
3. Results
3.1. Reproductive parameters and maternal data
No group differences in water ingestion, food consump-tion or weight gain during gestation (Table 1) and lacta-
tion (data not shown) were observed between control and
experimental dams, suggesting the absence of deleterious
effects promoted by the gavage procedure. A lower weight
gain was observed only in the dams exposed to the higher
dose of the AQE during the first week (Days 0 7) of
gestation [F(4,41) = 2.882, P=.0342; Dunnetts Q = 2.769]
when compared with the no gavage control dams. On the
other hand, the histopathologic study showed a dose-
dependent frequency of cytoplasmic vacuoles in liver,
kidney, pancreas and thyroid tissues of experimental
dams. In fact, rare cytoplasmic vacuoles were observed
in the 0.7 mg/kg treated rats, while moderate to extensive
cell vacuole appearance was observed in animals treated
with the 3.0 and 15.0 mg/kg doses, respectively (Fig. 1).
Analysis of brain tissue did not reveal any alteration in
either the experimental or control groups. Also, no
histopathological lesions were detected in the offspring
of the control and experimental groups observed during
adulthood.
Table 1
Effects of I. carnea AQE on water ingestion, food intake and weight gain of rats exposed to different doses during the gestation period (Day 5 to 21)
Day No gavage AQE (mg/kg)
(n =10)0.0 (n = 12) 0.7 (n = 12) 3.0 (n = 12) 15.0 (n =12)
Water ingestion (ml/day) 5 40.1 5.2 45.6 3.6 40.1 1.5 38.5 1.3 42.3 3.8
12 47.2 3.0 49.0 3.1 47.3 2.0 54.2 4.0 48.5 3.9
20 53.6 4.8 48.4 2.5 57.6 4.5 52.9 3.1 53.7 4.1
Food intake (g/day) 5 19.4 1.1 22.2 0.8 22.6 0.9 20.2 1.1 20.8 0.9
12 26.6 0.9 25.3 0.6 24.3 0.7 22.8 1.1 25.3 1.1
20 26.0 1.8 25.3 0.9 29.5 1.7 26.2 1.1 23.6 1.9
Weight gain (g/interval) 0 7 21.5 2.3 16.8 1.5 21.7 3.9 15.4 1.5 10.7 1.9 *
7 14 25.5 1.7 24.5 2.4 20.7 2.0 18.6 1.8 18.8 2.9
14 21 57.1 5.2 63.4 4.9 69.1 6.0 67.8 4.6 59.0 9.7
0 21 98.7 4.1 100.9 5.8 103.3 10.8 97.1 4.6 81.6 8.4
Data are presented as means S.E.M.
* P< .05 compared to 0.0 mg/kg group.
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3.2. Offspring studies
Table 2 shows the effects of AQE on the offspring of rats
exposed to different doses during the gestation period.
Pregnancy duration, total number of pups per litter (born
dead or alive) and weight gain (measured from PN1 to PN21
and on PN30, PN60, PN75 and PN90) (data not shown) were
not different between the control and experimental groups
(data not shown). The postnatal deaths were observed at
PN1. Statistical analysis showed significant differences in
death rate only between 0.0 mg/kg group and group treated
with the 15.0 mg/kg AQE dose [F(4,45) = 21.154, P< .0001;
Dunnetts Q = 7.455]. Whenever possible, neurobehavioral
and reflexologic features were assessed in litters with four
female and four male pups. No differences were observed
between the 0.0 mg/kg AQE and no gavage control group in
any of the tests performed, again showing the absence of
deleterious effects promoted by the gavage procedure.
The two-way ANOVA applied showed offspring bodysize, ear opening day and appearance day of negative
geotaxis altered results. Treatment and sex interfered in
the results of body size [treatment: F(4,90) = 6.61, P=.001;
sex: F(1,90) = 5.69, P=.0192; interaction: F(4,90) = 0.15,
P=.9624] but no interaction was observed between both
factors. Only the treatment interfered in the ear opening
results [treatment: F(4,90) = 5.60, P=.0011; sex: F(1,90) =
0.08, P=.7838; interaction: F(4,90)= 0.16, P=.9568], as in
the appearance day of negative geotaxis results [treatment:
F(4,90) = 5.23, P=.0008; sex: F(1,90) = 0.01, P=.9039; in-
teraction: F(4,90)= 0.34, P=.8471]. Since no interaction was
observed between factors on these parameters, the t test was
applied between data as post hoc test. Thus, a smaller body
size on offspring of dams treated with 15.0 mg/kg/day of
AQE, as well as a decrease in body size in female offspring
of both experimental and control groups in relation to male
offspring was observed. Delay of ear opening as well as a
delay in the appearance day of negative geotaxis on off-
spring of dams treated with 15.0 mg/kg/day of AQE was
observed when compared to the 0.0 mg/kg and the no
gavage control groups (the gavage procedure did not pro-
voke alterations in offspring development).
During pup development a few days after birth, alteration
of the thoracic limbs was observed in some pups exposed to
the higher AQE dose, which, however disappeared between
10 and 12 days of age as the pups grew. This reversible
deformity of carpal joints appeared in all pups of six litters
(60%) from dams exposed to the higher AQE dose (P
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tion frequency of male 15.0 mg/kg group and female 3.0
mg/kg group when compared to the 0.0 mg/kg male and
female groups, and reduced locomotion frequency of males
when compared to female rats.
In the same way, the two-way ANOVA applied between
immobility time data in each day of male and female
offspring exposed or not to the AQE showed that on
PN21 the immobility time was affected only by sex and
that interaction by both factors occurred [treatment:
F(4,90) = 1.38, P=.2476; sex: F(1,90) = 6.51, P=.0124; in-
teraction: F(4,90) = 3.02, P=.0217]. Since interaction was
observed between factors on immobility time at PN21, an
ordinary ANOVA was applied between data. Thus, reduc-
tion in immobility time was observed in male [F(4,42) = 4.222, P=.0058] and in less extend in female [F(4,
42) = 2.139, P=.0298] offspring exposed to 15.0 mg/kg/day
when compared to the 0.0 mg/kg group.
3.2.2. Additional neurobehavioral studies
Fig. 3 show the results of social interaction, forced
swimming test, stereotyped behavior, catatonia and elevated
plus-maze. No alterations were observed in any of these
tests. As pointed out previously, certain behavioral tests
were not performed with the litters exposed to the 15 mg/kg
dose because of the small number of pups consequent to the
high postnatal death rate.
3.2.3. Monoamine levels
Neurotransmitter and metabolite levels were determined
at 140 days of age. The data showed differences in
neurotransmitter levels and metabolite/neurotransmitter ra-
tios between the 0.0 mg/kg and no gavage control groups
as well as between the 0.0 mg/kg group and the experi-
mental groups. The statistical differences are presented in
Table 3.
3.2.3.1. Effects of gavage. The 0.0 mg/kg group showed
decreased striatal [F(4,45) = 9.557, P< .0001; Dunnetts:
Q = 4.469] DOPAC levels, increased striatal [F(4,45) =
6.841, P=.0002; Dunnetts: Q = 4.112] VMA levels and
increased cortical [F(4,45) = 7.585, P< .0001; Dunnetts:Q = 4.063] 5HT levels when compared to the no gavage
control group.
3.2.3.2. Effects of treatment. With respect to the effects of
treatment, changes were observed in experimental groups
compared to the 0.0 mg/kg group.
The 0.7 and 3.0 mg/kg groups showed increased striatal
[ F(4,45) = 9.557, P< .0001; Dunnetts: Q (0.7 mg/
kg)= 4.414, Q (3.0 mg/kg) = 5.267] DOPAC levels, de-
creased striatal VMA levels [F(4,45)= 6.841, P=.0002;
Dunnetts: Q (0.7 mg/kg) = 3.885, Q (3.0 mg/kg) = 3.869]
and decreased cortical [F(4,45) = 7.585, P< .0001; Dun-
Table 2
Effects of I. carnea AQE on the offspring of rats exposed to different doses during the gestation period (day 5 to 21)
Parameters Sex No gavage AQE (mg/kg)
(n = 10)0.0 (n = 10) 0.7 (n = 10) 3.0 (n = 10) 15.0 (n = 6)
Postnatal death 0.6 0.2 0.4 0.3 0.3 0.2 0.5 0.4 5.2 0.9***
Size (cm) on M 6.0 0.1 5.9 0.1 6.0 0.08 5.9 0.04 5.5 0.2 *
Postnatal day 1 F 5.9 0.1 5.7 0.1 5.7 0.1 5.8 0.1 5.3 0.1 **Weight (g) on M 5.75 0.2 5.9 0.2 5.9 0.1 5.9 0.2 5.5 0.1
Postnatal day 1 F 5.5 0.1 5.6 0.1 5.6 0.1 5.6 0.1 5.1 0.2
Ear opening M 14.0 0.3 13.6 0.2 14.0 0.2 13.6 0.2 15.0 0.6 *
Appearance day F 13.6 0.2 13.7 0.2 14.0 0.2 13.6 0.2 15.0 0.6 **
Coat appearance M 5.6 0.5 5.6 0.4 5.8 0.5 5.5 0.5 5.3 0.7
Day F 5.5 0.5 5.5 0.6 5.8 0.5 5.5 0.5 5.3 0.7
Pinna detachment M 3.6 0.2 3.7 0.3 3.7 0.2 3.5 0.2 3.3 0.4
Day appearance F 3.7 0.3 3.8 0.1 3.6 0.2 3.5 0.2 3.5 0.6
Tooth eruption M 10.5 0.5 10.5 0.5 10.8 0.2 10.4 0.4 9.7 0.9
Appearance day F 10.8 0.5 10.4 0.5 10.4 0.3 10.1 0.3 9.8 0.9
Adult gait M 13.9 0.3 14.1 0.3 13.8 0.3 13.7 0.4 14.7 0.3
Appearance day F 13.9 0.2 13.9 0.4 14.0 0.3 13.8 0.4 14.5 0.2
Eye opening M 14.9 0.2 14.8 0.3 15.3 0.2 14.7 0.2 15.2 0.5
Appearance day F 14.8 0.3 14.7 0.3 14.5 0.3 14.8 0.2 15.0 0.5
Palmar grasp M 9.0 0.4 9.4 0.3 9.0 0.4 8.8 0.4 10.0 0.6Reflex disappearance day F 8.9 0.4 9.1 0.3 9.0 0.4 8.9 0.3 9.8 0.5
Surface right reflex M 5.1 0.1 5.2 0.1 5.1 0.1 5.1 0.1 5.3 0.2
Appearance day F 5.1 0.1 5.1 0.1 5.1 0.1 5.1 0.1 5.3 0.2
Testis descent day 21.8 0.3 21.6 0.4 21.9 0.4 21.5 0.5 21.8 0.8
Vaginal opening day 37.8 0.6 37.7 0.5 37.4 0.7 36.2 0.7 38.3 0.4
Negative geotaxis M 6.9 0.2 6.9 0.2 6.9 0.2 6.8 0.4 7.7 0.3
Appearance day F 7.2 0.3 6.7 0.2 6.8 0.2 6.6 0.2 7.8 0.3 *
Data are presented as means S.E.M.
* P< .05.
** P< .01.
*** P< .001 compared to 0.0 mg/kg group.
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Fig. 2. Effects of the I. carnea AQE on locomotion and rearing frequencies, immobility time and defecation of rats exposed to different doses during the
gestation period (day 5 to 21). Data are presented as means S.E.M.; n = 10 rats per group; P>.05, two-way ANOVA and ordinary ANOVA test.
Fig. 3. Effects of I. carnea AQE on behaviors of adult rats exposed to different doses during the gestation period (Days 6 to 21). Data are presented as
means S.E.M. or as median; n = 10 rats per group; P>.05, two-way ANOVA and ordinary ANOVA or Kruskal Wallis test.
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netts: Q (0.7 mg/kg) = 2.753, Q (3.0 mg/kg) = 5.155] 5HT
levels when compared to the 0.0 mg/kg group.
No alterations were observed at the central monoamine
activity systems, determined by the relation between me-
tabolite and neurotransmitter.
4. Discussion
Our results indicate that exposure to the I. carnea AQE
during gestation induced maternal toxicity, as shown by
cytoplasmic vacuolation in dam cells. Offspring exposed tothe higher dose showed some physical and developmental
alterations during early life but a few behavioral alterations
during adulthood. In addition, alterations in the levels of
neurotransmitters and their metabolites did not occur.
It is well known that stress during the critical period of
organogenesis promotes changes in maternal behavior and
in postnatal offspring development [4]. So, the no gavage
control group was employed in this study to evaluate the
possible noxious effects of stress induced by the gavage
procedure.
Administration of different doses of I. carnea AQE did
not alter food or water intake of dams. But histopathological
data showed the occurrence of cell vacuolation, reflecting
maternal toxicity of the extract. In addition, a high postnatal
death rate was observed after treatment of the dams with the
high AQE concentration.
Pup body size and weight at birth are important variables
that may alter the physical and neurobehavioral develop-
ment of the animals [12,32]. In the present study, offspring
exposed to the higher dose of I. carnea aqueous fraction
(15.0 mg/kg/day) showed high perinatal mortality and
smaller body size at birth. Deformity of carpal joints was
also observed in offspring exposed to the higher dose
during gestation, although this condition gradually disap-peared, supporting a possible reversibility of lesions. This
reversible deformity was also observed in sheep offspring
exposed to locoweeds (plants with swainsonine) during
gestation [2830].
Swainsonine inhibits a-mannosidase II and lysosomal a-mannosidase, while calystegines inhibit a- and b-glucosi-dases and a-galactosidase; both prevent oligosaccharidematuration and complexation and glycoprotein formation
[11,25]. Cytoplasmic vacuolation is a classical effect of
intoxication with polyhydroxylated alkaloids like swainso-
nine and calystegines, the main toxic agents of I. carnea
[11]. Thus, accumulation of oligosaccharides in cytoplasmic
Table 3
Effects ofI. carnea AQE on central neurotransmitters (ng/g) and metabolite/neurotransmitter ratio of adult rats exposed to different doses during the gestation
period (Days 5 to 21)
Region No gavage AQE (mg/kg)
(n =10)0.0 (n = 10) 0.7 (n = 10) 3.0 (n = 10) 15.0 (n =10)
DOPAC Cortex 36.3 8.5 21.0 4.3 30.1 4.6 41.6 9.0 32.1 11.0
Striatum 1935.9 181.1 871.2 177.5aa 1922.8 152.4bb 2125.9 115.3bb 1339.8 202.6Hypothalamus 111.3 24.8 77.5 9.3 133.8 23.4 127.9 35.2 108.8 29.8
DA Cortex 58.5 18.9 29.8 3.5 38.9 2.8 25.1 5.7 33.6 5.0
Striatum 5464.2 897.0 5341.0 1060.6 4962.2 1008.4 4539.7 892.0 5985.3 1072.1
Hypothalamus 220.4 20.3 206.6 8.9 183.8 15.8 167.1 16.1 243.8 24.2
DOPAC/DA Cortex 0.31 0.06 0.26 0.03 0.23 0.01 0.28 0.04 0.25 0.03
Striatum 0.22 0.04 0.19 0.02 0.20 0.01 0.17 0.01 0.18 0.02
Hypothalamus 0.33 0.05 0.31 0.02 0.28 0.01 0.30 0.02 0.29 0.01
VMA Cortex 38.5 11.8 80.0 29.2 60.4 21.1 79.8 36.6 25.6 16.4
Striatum 97.5 11.8 330.8 54.9aa 110.4 19.8bb 111.3 17.7bb 270.2 73.2
Hypothalamus 71.9 17.1 125.4 23.3 134.7 30.6 121.9 45.0 63.0 16.0
NE Cortex 59.2 5.4 61.1 3.4 56.3 6.1 73.1 7.2 55.5 6.0
Striatum 89.9 33.5 133.6 42.5 62.3 22.0 46.3 16.2 122.7 42.6
Hypothalamus 207.6 9.8 209.2 14.6 214.6 14.3 215.9 16.1 226.2 16.0
VMA/NE Cortex 0.06 0.02 0.08 0.30 0.05 0.02 0.09 0.05 0.09 0.03
Striatum 2.28 0.02 2.31 0.04 2.33 0.06 2.30 0.03 2.32 0.04Hypothalamus 0.13 0.01 0.15 0.03 0.17 0.04 0.14 0.02 0.13 0.02
5HIIA Cortex 232.6 13.9 179.1 9.4 228.0 22.6 258.7 39.4 199.8 35.0
Striatum 410.2 30.5 332.7 32.8 376.8 24.6 417.2 70.5 375.0 35.8
Hypothalamus 414.6 47.6 324.7 28.0 431.3 40.4 322.4 46.3 337.5 50.8
5HT Cortex 349.7 25.4 581.7 43.5aa 424.5 50.1b 287.3 36.6bb 447.9 42.1
Striatum 243.8 63.2 305.2 21.6 154.3 38.8 164.6 48.6 285.8 59.1
Hypothalamus 599.2 47.0 566.0 60.7 414.3 54.2 462.6 62.4 541.4 71.8
5HIAA/5HT Cortex 0.20 0.01 0.50 0.03 0.40 0.03 0.30 0.02 0.50 0.04
Striatum 1.31 0.2 1.27 0.10 1.25 0.08 1.28 0.15 1.30 0.18
Hypothalamus 1.58 0.01 1.62 0.02 1.60 0.03 1.62 0.01 1.59 0.02
Data are presented as means S.E.M. 0.01.a,bP< .05; aa,bbP< .01; where a = statistical significance by manipulation (0.0 mg/kg group in relation to no gavage control group) and b = statistical significance
by treatment (in relation to the 0.0 mg/kg group).
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vacuoles is observed and adequate cell growth is disrupted,
explaining, at least in part, the smaller body size at birth, the
higher postnatal death rate and the reversible deformity of
carpal joints of the offspring exposed to the higher dose of
the extract during gestation. The lesion reversibility can be
explained by the fact that swainsonine and calystegines are
weakly basic compounds that cannot cross the placental barrier (lipid rich), and, once in the body, are rapidly
cleared. Thus, no toxin remains in the poisoned animal
and cellular repair begins soon after the end of exposure
[44].
Delay in ear opening was detected in the offspring
exposed to the higher dose of the plant aqueous fraction
during gestation. In this case, again we may speculate about
the importance of glycosidases for cell growth and devel-
opment. Male and female litter weight and weight gain were
similar for the experimental and control groups throughout
the experimental period.
Prenatal exposure to the higher dose of the I. carneaAQE caused a delayed day appearance of negative geotaxis
in the 15.0 mg/kg female pups group when compared to
the other experimental and control female groups. Com-
pared to 15.0 mg/kg male pups, this difference did not
occur, suggesting that male pups exposure to testosterone
that occurs prenatally, triggering the masculinization of
male fetuses [22] and the existence of different sex
steroid-binding sites in many of the male and female brain
areas [5], can explain this difference only in the females
groups.
Some significant alterations were observed in locomotion
frequency in the open-field, suggesting that exposure to I.
carnea alkaloids during the gestation period interfered with
the serotonergic and mesolimbic dopaminergic systems,
which are strongly related to the neuromotor system and
to locomotor activity, respectively [1,20,21,23,27].
The plus-maze [17] and the social interaction behavior
[19] were initially proposed as models for testing anxiolytic
drugs. It is also known that anxiogenic agents can be
indirectly mediated by the activation of the hypothalam-
ic pituitary adrenal axis, which modulates the action of
corticotrophic, corticosterone and adreno-corticotrophic hor-
mones [18]. The AQE of I. carnea did not alter the
parameters observed in the plus-maze and in the social
interaction of experimental offspring compared to control,suggesting that the alkaloids of the plant may not increase
anxiogenic behavior. To determine if I. carnea alkaloids
definitely modulate the HPA axis it will be necessary to
measure the levels of the hormones cited above and of other
related ones, and to determine the levels of a broad range of
neurotransmitters [18].
The forced swimming test shows an immobility behavior
(depression) as a result of the severe stress to which the
animals are exposed [3739]. In the present study, no
interference with the parameters of the forced swimming
test was observed in the experimental animals compared to
the 0.0 mg/kg group.
Stereotyped behavior is related to progressive activation
of nigrostriatal dopaminergic receptors [14]. Catalepsy can
be induced by administration of dopaminergic receptor
blockers [36], indicating the participation of central dopa-
minergic systems in motor manifestations [16,40]. Data
show no changes in catatonia and stereotyped behavior.
The adverse effects observed during the postnatal perioddid not promote significant neurobehavioral changes in the
animals when they reached adult age. Also, no changes were
observed in the central monoamines activity systems.
It is well known that maternal stress affects physiological
and behavioral functions in the offspring, and that in the
immature brain many neurotransmitters act as developmen-
tal signals or regulators, resulting in permanent changes in
the densities of several neurotransmitter receptors once the
brain has matured [34]. In addition, some studies have
revealed that prenatal stress reduces dopamine neurotrans-
mission in the ventral striatum [50], and that changes in
catecholamine and especially brain noradrenaline levelshave been associated with behavioral deficits caused by
stress exposure [33,43]. On this basis, the no gavage control
group was employed to evaluate the stress effects from the
gavage procedure. Since the alterations observed did not
affect the monoamine activity systems, we can speculate
about alternative pathways developed by the CNS of these
animals, promoting a normal life. Several studies have
demonstrated that during ontogeny, when the central ner-
vous system is incompletely developed, paradoxical and
enduring effects of exposure to agonists and antagonists
occur. Rat pups with serotonin depletion following methyl-
enedioxymethamphetamine (MDMA) exposure, for exam-
ple, show no changes in weight gain or locomotor activity
[51]. In this case, the target system is still developing and
the property of central nervous system plasticity may persist
through this period. In addition, metabolic central nervous
system enzymes and the bloodbrain barrier are maturing
postnatally, ensuring very different pharmacokinetic profiles
during development [41].
The administration of different doses of I. carnea AQE
had some effects on the levels of neurotransmitters and their
metabolites, but did not alter these activity systems. No
alterations were detected during adult age in rats exposed
during pregnancy to the different AQE doses. Probably, a
compensatory mechanism may be developed by the centralnervous system to permit animal survival in their environ-
ment. It is well known, as cited above, that the central
nervous system presents plasticity and the ability to find
alternative pathways for survival when some physiological
functions are compromised. In addition, comparative anal-
ysis of male and female neurotransmitter and metabolite
levels revealed the persistence of sexual dimorphism (data
not shown).
In summary, the present data show that the deleterious
effects observed in the offspring exposed to the higher dose
of AQE during gestation did not alter the neurobehavior and
the monoamine activity systems analyzed during adulthood.
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Perhaps there was little transfer of the toxic agents of the
plant to the fetus and the fetotoxic effects were maternally
mediated. An alternative hypothesis is that, in rats, the main
effects of swainsonine and calystegines occur outside the
central nervous system. In any case, a single study cannot
completely rule out the effects of I. carnea on the central
nervous system as a whole.
Acknowledgements
This research was supported by grants from Fundacao
de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)
and is part of the Masters thesis presented by Aline
Schwarz to the Faculdade de Ciencias Farmaceuticas da
Universidade de Sao Paulo. Special thanks are to Prof. Dr.
Jorge Camilo Florio for HPLC technical assistance and Dr.
Mitsue Haraguichi for the leaves extraction technical
assistance.
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