Effects of Ipomoea Carnea Aqueous Fraction Gorniak

8/4/2019 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


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


sufficient number of pups was not available for the social

interaction behavior test. Thus, other pups from control and


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


sufficient number of pups was not available for the forced

swimming behavior test. Thus, other pups from control and


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.



11/12

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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Effects of Ipomoea Carnea Aqueous Fraction Gorniak

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