Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
Research Article
Neurobiology
International Journal of Pharma and Bio Sciences
ISSN
0975-6299
BISPHENOL A INHIBITS DUODENAL MOVEMENT IN RAT BY INCREASING
ACETYLCHOLINESTERASE ACTIVITY AND DECREASING AVAILABILITY OF
FREE Ca2+IN SMOOTH MUSCLE CELLS.
KAUSHIK SARKAR, PANCHALI TARAFDER, PARTHA P. NATH AND GOUTAM PAUL*
Toxicology Unit, Environmental Physiology Division, Department of Physiology,
University of Kalyani, Kalyani, West Bengal-741235, India.
ABSTRACT
We report here the toxic effects of Bisphenol A (BPA) on the function of duodenum in
vitro of rats. We found significant depression of amplitude (in 20 days exposure groups)
and frequency of contraction of duodenum (in 30 days exposure groups). We also
observed a significant increase in acetylcholinesterase (AChE) activity in duodenal
tissues in both the exposure periods. From this result it is suggested that BPA
depresses the amplitude and frequency of duodenal movement presumably by
increasing the AChE activity in the endplate membrane of smooth muscle and efferents
of local Myenteric Auerbach’s plexus. Further, we observed an increase in deposition of
calcium salts in Von Kossa’s stained duodenal tissue section in both BPA exposure
groups. From this study it is concluded that BPA inhibits duodenal movement by
increasing AChE activity at the local neuromuscular junction and decreasing the
availability of free Ca2+ in smooth muscle cells and local Myenteric terminals.
KEY WORDS: BPA, Duodenal movement, AChE, Calcium salts, Myenteric Auerbach’s plexus.
GOUTAM PAUL
Professor, Department of Physiology,
University of Kalyani, Kalyani, West Bengal-741235, India.
This article can be downloaded from www.ijpbs.net
B - 679
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
INTRODUCTION
Bisphenol A (BPA) is commercially used for
coating the inner lining of food and beverage
cans, manufacturing polycarbonate plastic
bottles (for example water bottles, baby
bottles etc.) and composite dental sealants
formulas 1, 2, 3, 4. The primary source of human
exposure to BPA is the leachate from food
and beverage containers, water bottles and
dental sealants under normal conditions of
use. Contamination of water and food with
BPA is increased when the polymer in
container wall is heated 5, 6, 7, 8, 9. Some
reports about the BPA induced health hazards
have been reported discriminately in animal
models. BPA exerts reproductive and
developmental toxicity in animal models due
to its endocrine disrupting function 10, 11, 12.
Moreover, BPA induces the onset of puberty,
ovarian malformations, formation of cysts in
the ovaries, premature disruption of estrus
cycle rhythmicity, defective sperm production
with a decrease in sperm count in rat models
13, 14, 15, 16, 17, 18
. It has also been reported that
BPA induces oxidative stress induced injury in
hepatic cells and is metabolized to BPAglucuronide in the liver and excreted in bile 19,
20, 21, 22
. But the report about the possible toxic
effects of BPA on the function of intestine has
not been reported till date. It is known that
single unit smooth muscle in the intestine is
the principal motor for intestinal motility. The
pacemaking activity of smooth muscle is
controlled by local reflex of Myenteric
Auerbach’s and Meissner’s plexuses; and the
local reflex in turn are regulated by the higher
centres through autonomic nerves. So, the
present study was designed to examine the
effects of BPA on the movement of duodenum
in vitro of rats.
MATERIALS AND METHODS
Reagents and Chemicals
All common chemicals were of analytical
grade. Dimethyl sulfoxide (DMSO), sodium
chloride (NaCl), potassium chloride (KCl),
magnesium chloride (MgCl2), calcium chloride
(CaCl2), sodium bicarbonate (NaHCO3),
sodium dihydrogen phosphate (NaH2PO4),
glucose, neutral red, silver nitrate, and sodium
thiosulphate were procured from E.Merck,
India. Bisphenol A (BPA) was purchased from
Sigma Chemicals Co. (USA).
Animals
Studies were carried out on 14-16 weeks old
White Albino rats of Sprague Dawley Strain
weighing about 110-150 gm. The rats were
fed standard laboratory chow and water, and
were maintained in Animal House as per
recommendations of the Kalyani University
Animal Ethics Committee.
Experimental design
After one week of acclimatization, the rats
were randomly distributed to three groups of
seven animals each. The rats of the first group
were received 0.5 ml 20% DMSO by oral
gavages and was designated as a vehicle
control. The rats of the second and third
groups were received 50mgBPA/kg body
weight/day for 20 days and 30 days durations
and were marked as treated groups. After
completion of treatment the rats were
sacrificed on three post treatment durations
i.e. on 24 hours, 5th and 10th day of each
group. For functional studies of duodenal
movement the rats were sacrificed by cervical
dislocation, the abdomen was immediately
opened and the duodenal segments of 3cm
each were removed by transverse incision.
Functional studies on duodenal movement
For the functional studies duodenal segments
of the rat’s intestine were used. Duodenal
segments were placed in Tyrode solution
consisting of 8.0 g/l NaCl, 0.2 g/l KCl, 0.2 g/l
CaCl2, 0.1 g/l MgCl2, 1.0 g/l NaHCO3, 0.05 g/l
NaH2PO4 and 1.0 g/l Glucose (pH 7.4). The
luminal content of each segment was gently
flushed out. Whole full thickness duodenal
part was placed longitudinally in 40ml organ
bath containing Tyrode solution and
continuously bubbled with 95 % O2 and 5%
CO2, and temperature was maintained within
a range of 37ºC ± 0.5. Continuous recording
of duodenal movement was achieved with
isotonic transducer (IT-2245) coupled with
RMS-Polyrite D software (RMS, India). Before
This article can be downloaded from www.ijpbs.net
B - 680
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
recording, the preparation underwent repeated
and prolonged washed with Tyrode solution to
avoid accumulation of metabolites in the organ
bath.
dehydrated with graded ethanol, cleared with
xylene and the whole stained section was
mounted with DPX for observation under the
microscope.
Assay of Acetylcholinesterase (AChE)
activity
AChEactivity was assayed by the method of
Ellman et. al., 1961 modified by Srikumar et.
al., 2004 23, 24. In this method 0.4ml aliquot of
the duodenal tissue homogenate was added
to a cuvette containing 2.6ml phosphate buffer
(0.1M, pH 8) and 100µl of DTNB. The
contents of the cuvette were mixed thoroughly
and absorbance was measured at 412nm in
visible spectrophotometer. When absorbance
reaches a stable value, it was recorded as the
basal reading. Then 20µl of substrate
(acetylthiocholine iodide) was added and
change in absorbance was recorded for a
period of 10 minutes at intervals of 2 minutes
and change in the absorbance per minute was
determined. The enzyme activity was
expressed as a rate in micromoles of
substrate hydrolyzed/minute/mg protein.
Statistical analysis
The data were expressed as mean± SEM of
the value of each experimental group. Force
of contractions was measured in terms of
amplitude and frequency. For functional
assays, the values of the treated preparations
were expressed as percent change of the
basal
(or
control)
values.
Statistical
comparisons between the values obtained in
control and in treated rats were carried out by
using a Student’s t test for paired values.
P≤0.05 was considered significant. The
number of the duodenal preparation used in
each experiment is indicated by the alphabet
‘n’ in the results.
Von Kossa’s staining techniques for
detection of calcium deposition in
duodenal smooth muscle cells and muscle
layer
Cytoplasmic calcium deposits in duodenal
smooth muscle cells and extracellular spaces
of muscle layer were detected by Von Kossa’s
staining technique 25. 5-7µm paraffin sections
of duodenal tissue fixed in neutral buffered
formalin were prepared (from control and
experimental groups). A tissue section was
treated with xylene to remove the paraffin and
thus graded ethanol for rehydration. After that
the section was flooded with 5% silver nitrate
solution and placed in a petri dish with a
suitable support for holding the slide. The
whole set up was placed under a 100 watt
lamp for a period of 45-60 minutes. Then the
section was washed with distilled water for
removing the silver nitrate solution and then
placed into 5% sodium thiosulphate solution
for 5 minutes. The section was then washed
properly with running tap water for 5 minutes
and after that the section was counter stained
with 1% neutral red solution. At the last stage
the counterstained tissue section was
RESULTS AND DISCUSSION
Effects of BPA on the movement of
duodenum in vitro of BPA treated rats
In order to elucidate the neurophysiological
basis of the effect of BPA on the movement of
the isolated intestine of rats, movements of
duodenum of DMSO (vehicle control) and
BPA treated rats were recorded. The mean
amplitudes of contractions of duodenum of 20
days BPA treated rats were significantly
decreased (p< 0.05) in 24hrs. and 5th day post
treatment durations with respect to vehicle
control amplitudes (Panel-1 of Fig.1 & Fig.2).
No significant changes were observed in
mean frequency of duodenal contractions at
all post treatment durations (i.e. in 24hrs., 5th
and 10th day) (Panel-1 of Fig.1 & Fig.3). In 30
days treatment, the mean amplitudes of the
duodenal contractions at all post treatment
durations were not altered significantly (Panel2 of Fig.1 & Fig.4). We observed significant
changes in mean frequency of isolated
duodenal contractions in 24 hrs. post
treatment duration of BPA treated rats when
compared to vehicle control (Panel-2 of Fig.1&
Fig. 5). From the results it is suggested that
BPA depresses the movement of duodenum
presumably by inducing the release of
inhibitory neurotransmitters (norepinephrine,
NO etc.) or by inhibiting the release of
This article can be downloaded from www.ijpbs.net
B - 681
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
facilitatory neurotransmitters (glutamate, Ach
etc.) from the efferents at synapse-en-pasant
of local Myenteric Auerbach’s plexus
innervating the duodenum. From the results it
is also revealed that the frequency of the
movement is not altered significantly for 20
days treatment periods and amplitude of the
movement for 30 days treatment periods. This
might be due to sustainability of contractile
tone of duodenal smooth muscles as a result
of inherent intracellular mechanisms like latch
bridge mechanism.
Recording of movements of isolated duodenum
Figure 1
Representative recordings of the movement of isolated duodenum of DMSO (vehicle of BPA) and BPA treated rats in vitro.
Panel 1: Recording of the movement of duodenum of DMSO (vehicle control, 20% DMSO-0.5ml for 20 days duration)
treated rats (A), recording of movement of duodenum of BPA (50mg/kgBW/day for 20 days duration) treated rat of 24 hrs.
post treatment duration (B), 5th day post treatment duration (C) and 10th day post treatment duration (D). Panel 2:
Recording of the movement of duodenum of DMSO (vehicle control, 20% DMSO-0.5ml for 30 days duration) treated rats
(A), recording of movement of duodenum of BPA (50mg/kgBW/day for 30 days duration) treated rat of 24hrs. post
th
th
treatment duration (B), 5 day post treatment duration (C) and 10 day post treatment duration (D).
Amplitude of duodenal contractions for 20 days exposure group of rats
Figure 2
Showing changes in mean amplitudes of isolated duodenal movements of rats, treated with BPA and DMSO (vehicle) for
20 days duration. The values are represented as mean± S.E.M. (n=5), *p<0.05 vs. vehicle control.
This article can be downloaded from www.ijpbs.net
B - 682
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
Frequency of duodenal contractions for 20 days exposure group of rats
Figure 3
Showing changes in mean frequency of isolated duodenal movements of rats, treated with BPA and DMSO (vehicle) for 20
days duration. The values are represented as mean± S.E.M. (n=5).
Amplitude of duodenal contractions for 30 days exposure group of rats
Figure 4
Showing changes in mean amplitudes of isolated duodenal movements of rats, treated with BPA and DMSO (vehicle) for
30 days duration. The values are represented as mean± S.E.M. (n=5).
Frequency of duodenal contractions for 30 days exposure group of rats
Figure 5
Showing the changes in mean frequency of isolated duodenal movements of rats, treated with BPA and DMSO (vehicle)
for 30 days duration. The values are represented as mean± S.E.M. (n=5), *p<0.05 vs. vehicle control.
This article can be downloaded from www.ijpbs.net
B - 683
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
Effects of BPA on the activity of AChE
enzyme of duodenum in BPA treated rats
for both 20 and 30 days exposures
In order to study the mechanism of action of
BPA in duodenal movement, AChE activity
was measured in three post treatment
durations for both 20 and 30 days
exposures.The rats were sacrificed in three
post treatment durations i.e. in 24hrs., 5th and
10th day after the application of last dose of
BPA. The activities of AChE were increased in
24hrs. and 5th day post treatment durations of
20 days group of BPA treated rats with
respect to DMSO (vehicle) treated rats of 20
days treatment groups. But no significant
changes in AChE activity was observed in 10th
day post treatment duration of 20 days
treatment group of rats (Fig. 6). In 30 days
BPA exposed rat groups the AChE activity
was increased significantly in 24hrs. post
treatment duration (p<0.05, n=5), but the
activities of AChE were decreased gradually in
5th and 10th days post treatment durations
when compared to vehicle control for 30 days
treatment group of rats. From the results it is
suggested that BPA induced inhibition of the
duodenal movement might be due to increase
in activity of AChE at the local synapse on to
the smooth muscle.
AChE activity of 20 days exposure group of rats
Figure 6
Showing the alterations in AChE activity in 20 days DMSO (vehicle) and BPA exposures groups of rats in three post
treatment durations. The values are represented as mean± S.E.M. (n=5), *p<0.05 vs. vehicle control.
AChE activity of 30 days exposure group of rats
Figure 7
Showing the alterations in AChE activity 30 days DMSO (vehicle) and BPA exposure groups of rat in three post treatment
durations. The values are represented as mean± S.E.M. (n=5), *p<0.05 vs. vehicle control.
This article can be downloaded from www.ijpbs.net
B - 684
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
Detection of calcium deposits in
duodenal smooth muscle layers of BPA
treated rats
In order to study the effect of BPA on
calcium homeostasis in duodenal smooth
muscle cells, the calcium salts deposits were
detected in BPA and DMSO (vehicle) treated
duodenal sections in both 20 and 30 days
exposure groups. We found an increase in
deposition of calcium salts in the
submucosal and muscularis layers of Von
Kossa’s stained duodenal transverse
sections in a duration dependent manner for
both 20 days (Fig. 8) and 30 days (Fig. 9)
exposure groups of rats; and maximum
deposition was observed in 24 hrs. post
treatment durations of both 20 and 30 days
exposure groups (section B of Fig. 8 & Fig.
9). This result suggests that BPA may
decrease the availability of the Ca2+ in
efferent nerve terminals of local Myenteric
Auerbach’s plexus in smooth muscle cells of
mucularis mucosa and muscularis externa
presumably by forming lesions in the
duodenal smooth muscle layer through
deposits of calcium salts.
Von Kossa’s stained duodenal tissue sections of 20 days exposure groups
Figure 8
Von Kossa’s stained duodenal tissue section for 20 days exposure groups. Images were obtained by digital SLR Olympus
Camera (E-620) fitted with Olympus light microscope (CH20i) (100X magnification). Control sections (vehicle control): Ath
th
24hrs. post treatment duration, C- 5 day post treatment duration and E-10 day post treatment duration. BPA treated
th
sections: B-24hrs. post treatment duration, D-5 day post treatment duration, F-10th day post treatment duration. Arrow
sign indicates the sites of calcium salts deposition. Submucosal smooth muscle layer is indicated by alphabet ‘S’.
This article can be downloaded from www.ijpbs.net
B - 685
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
Von Kossa’s stained duodenal tissue sections of 30 days exposure groups
Figure 9
Von Kossa’s stained duodenal tissue section for 30 days exposure groups. Images were obtained by digital SLR Olympus
Camera (E-620) fitted with Olympus light microscope (CH20i) (100X magnification). Control sections (vehicle control): Ath
th
24hrs. post treatment duration, C- 5 day post treatment duration and E-10 day post treatment duration. BPA treated
th
th
sections: B-24hrs. post treatment duration, D-5 day post treatment duration, F-10 day post treatment duration. Arrow
sign indicates the sites of calcium salts deposition. Submucosal smooth muscle layer and muscularis smooth muscle
layer are indicated by alphabet ‘S’ and ‘M’ respectively.
CONCLUSION
BPA inhibits the movement of the duodenum
in vitro of rat presumably by depressing the
myoneural transmission at synapse-enpassant junction of local Myenteric Auerbach’s
plexus of duodenum or by inhibiting the crossbridge formations in smooth muscle cell.
Depression of myoneural transmissions at
synapse-en-passant or inhibition of crossbridge formations in smooth muscles might be
due to the non availability of the Ca2+ as a
result of the formation of deposits of calcium
salts in submucosal and muscularis smooth
muscle layers of the duodenum.
ACKNOWLEDGEMENTS
Financial grant from UGC [F.No.- 36-260/2008(SR) dated 27.03.09] is gratefully acknowledged to
carry out this study.
This article can be downloaded from www.ijpbs.net
B - 686
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
REFERENCES
1.
Ranjit N, Siefert K and Padmanabhan V,
Bisphenol-A and disparities in birth
outcomes: a review and directions for
future research, Journal of Perinatology,
30: 2–9, (2010).
2. Biles JE, McNeal TP, Begley TH and
Hollifield HC, Determination of bisphenolA in reusable polycarbonate food contact
plastics and migration to food simulating
liquids, J Agric Food Chem, 45:35413544, (1997).
3. Brotons JA, Olea-Serrano MF, Villalobos
M, Pedraza V and Olea N, Xenoestrogens
released from lacquer coating in food
cans, Environ Health Perspect, 103: 608612, (1995).
4. Erickson and Britt E, Bisphenol A under
scrutiny. Chemical and Engineering
News, American Chemical Society,
86(22): 36-39, (2008).
5. Le HH, Carlson EM, Chua JP and Belcher
SM, Bisphenol A is released from
polycarbonate drinking bottles and mimics
the neurotoxic actions of estrogen in
developing cerebellar neurons, Toxicology
Letters,176: 149–156, (2008).
6. Olea N, Pulgar R, Perez P, Olea-Serrano
F, Rivas A, Novillo-Fertrell A, Pedraza V,
Soto
AM
and
Sannenschein
C,
Estrogenicity of resin-based composites
and sealants used in dentistry, Environ
Health Perspect, 104: 298-305, (1996).
7. Ranjini R and Padmavathi T, Phenol
tolerance and degradation profile of novel
edible mushroom hypsizygus ulmarius in
ligninolytic and nonligninolytic media, Int J
Pharm Bio Sci, 3(4): (B) 987–994, (2012).
8. Vom Saal FS and Meyers JP, Bisphenol A
and risk of metabolic disorders, JAMA,
300, (2008).
9. Vom Saal FS, Akingbemi BTa and Becler
SM, Chapel Hill bisphenol A expert panel
consensus statement: integration of
mechanisms, effects in animals and
potential to impact human health at
current levels of exposure, Reprod
Toxicol, 24(2): 131-138, (2007).
10. NTP-CERHR Expert Panel Report on the
Reproductive and Developmental Toxicity
of Bisphenol A, National Toxicology
11.
12.
13.
14.
15.
16.
17.
18.
Program U.S. Department of Health and
Human Services. NTP-CERHR-BPA-07,
(2007).
Savabieasfahani M, Kannan K, Astapova
O, Evans NP and Padmanabhan V,
Developmental programming: differential
effects of prenatal exposure to bisphenolA or methoxychlor on reproductive
function, Endocrinology, 147: 5956–5966,
(2006).
Krishnan AV, Stathis P, Permuth SF,
Tokes L and Feldman D, Bisphenol-A: An
estrogenic substance is released from
polycarbonate flasks during autoclaving,
Endocrinology, 132: 2279–2286, (1993).
Patisaul HB, Fortino AE and Polston EK,
Neonatal
genistein
or
bisphenol-A
exposure alters sexual differentiation of
the AVPV, Neurotoxicol Teratol, 28:111–
118, (2006).
Patisaul HB, Todd KL, Mickens JA and
Adewale HB, Impact of neonatal exposure
to the ERα agonist PPT, bisphenol-A or
phytoestrogens
on
hypothalamic
kisspeptin fiber density in male and
female rats, Neurotoxicology, 30:350–
357, (2009).
Chitra KC, Rao KR and Mathur PP, Effect
of experimental varicocele on structure
and function of epididymis in adolescent
rats, Asian J Androl, 5(3): 203-208,
(2003).
Tan BL, Kassim NM and Mohd MA,
Assessment of pubertal development in
juvenile male rats after sub-acute
exposure to bisphenol A and nonyphenol,
Toxicol Lett, 143: 261–270, (2003).
Tinwell H, Haseman J, Lefevre PA, Wallis
N and Ashby J, Normal sexual
development of two strains of rat exposed
in utero to low doses of bisphenol A,
Toxicol Sci, 68: 339–348, (2002).
Tyl RW, Myers CB, Marr MC, Thomas BF,
Keimowitz AR, Brine DR, Veselica MM,
Fail PA, Chang TY, Seely JC, Joiner RL,
Butala JH, Dimond SS, Cagen SZ,
Shiotsuka RN, Stropp GD and Waechter
JM, Three‐generation reproductive toxicity
study of dietary bisphenol A in CD
This article can be downloaded from www.ijpbs.net
B - 687
Int J Pharm Bio Sci 2013 Apr; 4(2): (B) 679 - 688
19.
20.
21.
22.
Sprague‐Dawley rats, Toxicol Sci, 68:
121–146, (2002).
Bindhumol V, Chitra KC and Mathur PP,
Bisphenol A induces reactive oxygen
species generation in the liver of male
rats, Toxicol, 188: 117–124, (2003).
Inoue H, Yuki G, Yokota H and Kato S,
Bisphenol
A
glocuronidation
and
absorption in rat intestine, Drug Metab
Dispos, 31(1): 140-144(2003).
Nakagawa Y and Tayama S, Metabolism
and cytotoxicity of bisphenol A and other
bisphenols in isolated rat hepatocytes,
Arch Toxicol, 74(2): 99-105, (2000).
Sakamoto H, Yokoto H, Kibe R, Sayama
Y and Yussa A, Excretion of bisphenol Aglocuronide into the small intestine and
deconjugation in the cecum of the rat,
Biochim Biophys Acta, 1573(2): 171-176,
(2002).
23. Ellman GL, Courtney KD, Andres V and
Jr. Feather-StoneRM, A new and rapid
colorimetric
determination
of
acetylcholinesterase activity, Biochem
Pharmacol, 7:88-95, (1961).
24. Srikumar BN, Ramkumar K, Raju TR and
Shankaranarayana Rao BS, Assay of
acetylcholinesterase activity in the brain.
Brain and Behavior, National Institute of
Mental Health and Neuro Sciences,
Bangalore, India. 142-144, (2004).
25. Von Kossa J, Ueber die im Organismus
kuenstlich erzeugbren Verkakung, Beitr
Path Anat, 29:62, (1901).
This article can be downloaded from www.ijpbs.net
B - 688