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Endosulfan induced inhibition of 3h-5-hydroxytryptamine uptake in platelets
Toxicology Letters, 32 (1986) 203-208
203
Elsevier
TOXLett.
1615
ENDOSULFAN INDUCED UPTAKE IN PLATELETS
(Serotonin;
MOHINI
aggregation;
ANAND,
MAHIMA
GOPAL,
Industrial Toxicology Research
February
(Revision
received
(Accepted
CHAPLA
AGRAWAL,
S.V.
CHANDRA,
P.K.
RAY,
and K. SHANKER*
macology and Therapeutics, (Received
OF 3H-5-HYDROXYTRYPTAMINE
platelets; platelet-rich plasma; endosulfan)
KRISHNA
VERMA*
INHIBITION
9th, May
June 20th,
Centre,
P.B. No. SO, Lucknow-
001, and *Department
of Phar-
King George’s Medical College, Lucknow (India)
1986) lOth, 1986)
1986)
SUMMARY Endosulfan,
a chlorinated
pesticide,
of [‘HIS-hydroxytryptamine treated
with endosulfan
aggregatory
responses
(1 and 3 mg/kg) of the platelets.
min at 37°C also resulted discusses
is widely used to control
various
insect pests.
Rats exposed
to
for periods of 10, 30, and 60 days showed significant (P< 0.05) inhibition (5HT) uptake by platelet-rich plasma (PRP) in an ex vivo study. Rats
I mg and 3 mg endosulfan/kg
in significant
the use of rat blood
platelets
up to 60 days elucidated Incubation
of PRP
(P< 0.05) inhibition as a model
a marked
with 10 pM and of platelet
for the study
inhibition
aggregation
of neuro-
of ADP-induced
100 PM endosulfan in vitro.
and cardiovascular
for 15
The paper toxicity
of endosulfan.
INTRODUCTION
Endosulfan (6,7,8,9,9a hexachloro-1,5,5a,6,9,9a, hexahydro-6,9-methano-2,4,3benzodioxythepin-3 oxide, a chlorinated hydrocarbon pesticide is frequently used for crop protection as well as public health purposes. Several incidents of endosulfan poisoning in nontarget species along with its persistence in the environment have been reported [l-3]. Endosulfan is known to have neurotoxic effects, such as Abbreviations: PRP,
CNS, central
platelet-rich
plasma.
nervous
system;
5-HT,
5-hydroxytryptamine;
PPP,
platelet-poor
plasma;
204
hyperactivity, irritability, tremors, convulsions, loss of consciousness and paralysis [4-61. Involvement of CNS and 5-HT have been shown in endosulfan toxicity along with certain behavioural alterations [7]. Blood platelets are akin to CNS neurons, which can take up as well as store serotonin. An alteration in 5-HT uptake is reported farction
in various diseases, viz., ischaemic heart disease [lo], acute myocardial in[ll], hypertension, etc. We have investigated the endosulfan-5-HT interac-
tion by studying the 5-HT uptake explore the cardiotoxic potentials MATERIALS
AND
mechanism in rat blood of the pesticides.
platelets
with a view to
METHODS
Male albino rats (250-300 g) of the Industrial Toxicology breeding colony, fed a pellet diet ad lib were used in the study.
Research
Centre
Drug and chemicals 5-Hydroxytryptamine [3H]binoxalate obtained from the New England Nuclear Corporation (spec. act. 26.4 mCi/mmol), was dissolved in normal saline, stored cold in the dark. Trisodium citrate was used for citrate buffer. Scintillant’s composition: naphthaline 21 g; (1,4-bis 2,5-phenyl-oxazolyl)benzene (POPOP), 0.024 g; 2,5-diphenyl oxazole (PPO), 1.3 g; toluene 100 ml; methanol, 60 ml, and dioxane, 100 ml. Endosulfan (Hoechst Pharmaceutical Ltd., F.R.G., 95% pure) was used for the study. Rats were divided into 3 groups. The first group of experimental rats were treated with endosulfan 1 mg/kg daily (i.p.) dissolved in propylene glycol, while the second group received a daily dose of 3 mg/kg. The third group served as control. It received propyleneglycol (99% pure) in identical quantity. Blood of 6 rats from each group was obtained after 10, 30 and 60 days of exposure by heart puncture following light ether anaesthesia. It was immediately transferred to cellulose acetate centrifuge tubes containing 0.129 M sodium citrate buffer (1 ml buffer; 9 ml blood). Tubes were cooled and centrifuged at 1000 rev./min for 10 min at 0°C. The supernatant PRP was taken out carefully [12]. Platelet counts were done by fixing 0.1 ml PRP with 1.9 ml of fixative solution (1% w/v of formaldehyde in 3% sodium citrate) in a Naebour haemocytometer. Serotonin uptake was measured by determination of content of radioactivity in platelets incubated with 5-HT[3H]binoxalate (spec. act. 26.4 mCi/mmol). Solution of labelled amine was made in 0.9% saline. Duplicate samples of PRP (1 ml) were preincubated for 5 min at 37°C. After addition of 5-HT (1 pM per 25 ~1 volume). The reaction mixture was further incubated for 5 min with constant stirring. The uptake process was terminated by adding ice-cooled formaldehyde solution (4% w/v, 2 ml). Tubes were chilled and centrifuged at 10000 rev./min for 10 min at 0°C. Supernatant was decanted and inner side of the tube was wiped off with filter paper. The pellet was digested in 0.2 ml formic acid (21 mol/l) and 0.3 ml distilled water for 2 h and then transferred to counting vials containing 10 ml scintillant [13].
205
Radioactivity 33% efficiency
was measured
in an automatic
Packard
Scintillation
with endosulfan (1 PM, 10 PM and 100 FM) for 15 min at responses to ADP were quantitated on a Payton Associated
Dual Channel Aggregometer using PRP as the 0% transmission transmission standards. In ex vivo experiments PRP of treated aggregation responses to ADP were again studied. AND
having
for 3H.
PRP was incubated 37°C and aggregation
RESULTS
counter
and PPP as 100% rats was taken and
DISCUSSION
Exposure of rats to endosulfan 1 mg and 3 mg/kg for 10 days showed 55 to 47% inhibition of [3H]5-HT uptake by platelets. Similarly rats exposed to same compound for 30 days revealed a statistically significant inhibition as compared to controls (P
of endosulfan
on circulating
platelets
ACKNOWLEDGEMENTS
Authors are thankful to Mr G.S.D. Gupta for his valuable help during the course of experiments. Thanks are also due to Miss Shameem Naaz for technical help. Mr S.H. Mehdi for photography and Miss Sangeeta Chandra for statistical data analysis.
207
%
ot rat platelets
Agqreqdon blood
Fig. 4. ADP induced vitro.
*P <0.05:
aggregation
of platelets
after endosulfan
(1 pM, 10 pM and 100 PM) exposure
in
**P
REFERENCES 1 S.S.
Nicholson
Assoc.,
and G.W.
Copper,
Apparent
endosulfan
toxicosis
in valves,
.I. Ann.
Vet. Med.
170 (1977) 319.
P.J. Fox, J. Doutbeaik and B.A. Wood, 2 P. Mathiessen, and their predator after aerial spraying for the control
Accumulation
of endosulfan
of Tsetse fly in Botswana,
residue Pestic.
in fish Sci.,
13
(1982) 39-48. 3 K. Gopal, freshwater 4 G.E.
R.N.
Khanna,
organisms,
Thompson,
M. Anand
Toxicol.
Poisoning
Lett.,
and
G.S.D.
Gupta,
The acute
toxicity
of endosulfan
to
7 (1981) 453-456.
of cattle following
accidental
spraying
with thiodan,
Afr.
Vet. Assoc.,
37 (1966) 81-85. 5 G. Terziev and N.R. Dimitrova, ing with Thiodan, 6 P.K. Gupta,
Endosulfan
induced
15 (1976) 708-713. 7 M. Anand, S. Mehrotra, dosulfan
induced
8 K.l. Bentlay tion,
Forensic
medical
P. Med. Plv. Div.,
and P.D.
neurotoxicity
K. Gopal,
aggressive
R.N.
behaviour
Henry,
and forensic
chemical
study of acute lethal poison-
15 (1974) 708-713. in rats and mice, Bull. Environ.
Sur and S.V. Chandra,
in normal
Serotonin:
and lesioned
a potent
Toxicol.,
Role of neurotransmitters
rats, Toxicol.
constriction
Contam.
of human
Lett.,
coronary
in en-
24 (1985) 79-84. artery,
Circula-
12 (1981) 641-643.
G. Majno vascular
and G.E.
Palade,
permeability
study,
Studies
H.H.H. Oei and W.E. Hughes, J., 106 (1983) 1077-1081. V. Kumar, Lucknow G.V.R. Physiol.,
A study University,
Born
and
Platelet
of platelet India,
R.E.
Biochem. serotonin
biochemistry
I. The effect of histamine Cytol.,
uptake
in acute
and serotonin
on
11 (1961) 571-576. during
myocardial
myocardial
ischaemia,
infarction,
Thesis
Am. Heart for
M.D.,
1983.
Gilson,
146 (1959) 472-491.
on inflammation,
.I. Biophys.
Studies
on uptake
of 5-hydroxytryptamine
by blood
platelets,
J.
208
13 V.R. Cujrati, aemia
K. Shanker,
of pregnancy,
14 M. Anand,
A.C. Akveild
flow in cats,
including
15 K.P. Bhargava, Johnson
dimeform, 17 A.K.
Agarwal,
dosulfan 18 P.M.
Knowles,
Lett.,
M. Anand,
and K.P. Bhargava,
Effect of neurotoxic
circulation,
Vet. Hum.
pesticide Toxicol.,
and S. Vrat, Uptake
in hypertension,
Inhibition
Serotonin
in tox-
207 (1985) 11-18. endosulfan
on tissue blood
23 (4) (1981) 252-258.
of serotonin
by human
platelets
Life Sci., 25 (1979) 195-198.
of rat platelet
5-hydroxytryptamine
uptake
by chlor-
9 (1981) 1-4. N.F. Zaidi and P.K. Seth,
Biochem.
and R.A.
Pharmacol.,
Physiol.,
N. Misra, K. Shanker
and C.O.
neurotoxicity,
Vanhoutte
Biochem.
cerebral
to CNS involvement
Toxicol.
S. Vrat, Chandrawati
and P.R. Saxena, regional
N. Raina,
and its relevance 16 T.L.
S.S. Parmar,
Clin. Exp. Pharmacol.
Cohen,
Pharmacol., The elusory
32 (1983) 3671-3674.
Involvement
of serotonergic
receptors
inen-
32 (1983) 3591-3593. role of serotonin
in vascular
function
and disease,
203
Elsevier
TOXLett.
1615
ENDOSULFAN INDUCED UPTAKE IN PLATELETS
(Serotonin;
MOHINI
aggregation;
ANAND,
MAHIMA
GOPAL,
Industrial Toxicology Research
February
(Revision
received
(Accepted
CHAPLA
AGRAWAL,
S.V.
CHANDRA,
P.K.
RAY,
and K. SHANKER*
macology and Therapeutics, (Received
OF 3H-5-HYDROXYTRYPTAMINE
platelets; platelet-rich plasma; endosulfan)
KRISHNA
VERMA*
INHIBITION
9th, May
June 20th,
Centre,
P.B. No. SO, Lucknow-
001, and *Department
of Phar-
King George’s Medical College, Lucknow (India)
1986) lOth, 1986)
1986)
SUMMARY Endosulfan,
a chlorinated
pesticide,
of [‘HIS-hydroxytryptamine treated
with endosulfan
aggregatory
responses
(1 and 3 mg/kg) of the platelets.
min at 37°C also resulted discusses
is widely used to control
various
insect pests.
Rats exposed
to
for periods of 10, 30, and 60 days showed significant (P< 0.05) inhibition (5HT) uptake by platelet-rich plasma (PRP) in an ex vivo study. Rats
I mg and 3 mg endosulfan/kg
in significant
the use of rat blood
platelets
up to 60 days elucidated Incubation
of PRP
(P< 0.05) inhibition as a model
a marked
with 10 pM and of platelet
for the study
inhibition
aggregation
of neuro-
of ADP-induced
100 PM endosulfan in vitro.
and cardiovascular
for 15
The paper toxicity
of endosulfan.
INTRODUCTION
Endosulfan (6,7,8,9,9a hexachloro-1,5,5a,6,9,9a, hexahydro-6,9-methano-2,4,3benzodioxythepin-3 oxide, a chlorinated hydrocarbon pesticide is frequently used for crop protection as well as public health purposes. Several incidents of endosulfan poisoning in nontarget species along with its persistence in the environment have been reported [l-3]. Endosulfan is known to have neurotoxic effects, such as Abbreviations: PRP,
CNS, central
platelet-rich
plasma.
nervous
system;
5-HT,
5-hydroxytryptamine;
PPP,
platelet-poor
plasma;
204
hyperactivity, irritability, tremors, convulsions, loss of consciousness and paralysis [4-61. Involvement of CNS and 5-HT have been shown in endosulfan toxicity along with certain behavioural alterations [7]. Blood platelets are akin to CNS neurons, which can take up as well as store serotonin. An alteration in 5-HT uptake is reported farction
in various diseases, viz., ischaemic heart disease [lo], acute myocardial in[ll], hypertension, etc. We have investigated the endosulfan-5-HT interac-
tion by studying the 5-HT uptake explore the cardiotoxic potentials MATERIALS
AND
mechanism in rat blood of the pesticides.
platelets
with a view to
METHODS
Male albino rats (250-300 g) of the Industrial Toxicology breeding colony, fed a pellet diet ad lib were used in the study.
Research
Centre
Drug and chemicals 5-Hydroxytryptamine [3H]binoxalate obtained from the New England Nuclear Corporation (spec. act. 26.4 mCi/mmol), was dissolved in normal saline, stored cold in the dark. Trisodium citrate was used for citrate buffer. Scintillant’s composition: naphthaline 21 g; (1,4-bis 2,5-phenyl-oxazolyl)benzene (POPOP), 0.024 g; 2,5-diphenyl oxazole (PPO), 1.3 g; toluene 100 ml; methanol, 60 ml, and dioxane, 100 ml. Endosulfan (Hoechst Pharmaceutical Ltd., F.R.G., 95% pure) was used for the study. Rats were divided into 3 groups. The first group of experimental rats were treated with endosulfan 1 mg/kg daily (i.p.) dissolved in propylene glycol, while the second group received a daily dose of 3 mg/kg. The third group served as control. It received propyleneglycol (99% pure) in identical quantity. Blood of 6 rats from each group was obtained after 10, 30 and 60 days of exposure by heart puncture following light ether anaesthesia. It was immediately transferred to cellulose acetate centrifuge tubes containing 0.129 M sodium citrate buffer (1 ml buffer; 9 ml blood). Tubes were cooled and centrifuged at 1000 rev./min for 10 min at 0°C. The supernatant PRP was taken out carefully [12]. Platelet counts were done by fixing 0.1 ml PRP with 1.9 ml of fixative solution (1% w/v of formaldehyde in 3% sodium citrate) in a Naebour haemocytometer. Serotonin uptake was measured by determination of content of radioactivity in platelets incubated with 5-HT[3H]binoxalate (spec. act. 26.4 mCi/mmol). Solution of labelled amine was made in 0.9% saline. Duplicate samples of PRP (1 ml) were preincubated for 5 min at 37°C. After addition of 5-HT (1 pM per 25 ~1 volume). The reaction mixture was further incubated for 5 min with constant stirring. The uptake process was terminated by adding ice-cooled formaldehyde solution (4% w/v, 2 ml). Tubes were chilled and centrifuged at 10000 rev./min for 10 min at 0°C. Supernatant was decanted and inner side of the tube was wiped off with filter paper. The pellet was digested in 0.2 ml formic acid (21 mol/l) and 0.3 ml distilled water for 2 h and then transferred to counting vials containing 10 ml scintillant [13].
205
Radioactivity 33% efficiency
was measured
in an automatic
Packard
Scintillation
with endosulfan (1 PM, 10 PM and 100 FM) for 15 min at responses to ADP were quantitated on a Payton Associated
Dual Channel Aggregometer using PRP as the 0% transmission transmission standards. In ex vivo experiments PRP of treated aggregation responses to ADP were again studied. AND
having
for 3H.
PRP was incubated 37°C and aggregation
RESULTS
counter
and PPP as 100% rats was taken and
DISCUSSION
Exposure of rats to endosulfan 1 mg and 3 mg/kg for 10 days showed 55 to 47% inhibition of [3H]5-HT uptake by platelets. Similarly rats exposed to same compound for 30 days revealed a statistically significant inhibition as compared to controls (P
of endosulfan
on circulating
platelets
ACKNOWLEDGEMENTS
Authors are thankful to Mr G.S.D. Gupta for his valuable help during the course of experiments. Thanks are also due to Miss Shameem Naaz for technical help. Mr S.H. Mehdi for photography and Miss Sangeeta Chandra for statistical data analysis.
207
%
ot rat platelets
Agqreqdon blood
Fig. 4. ADP induced vitro.
*P <0.05:
aggregation
of platelets
after endosulfan
(1 pM, 10 pM and 100 PM) exposure
in
**P
REFERENCES 1 S.S.
Nicholson
Assoc.,
and G.W.
Copper,
Apparent
endosulfan
toxicosis
in valves,
.I. Ann.
Vet. Med.
170 (1977) 319.
P.J. Fox, J. Doutbeaik and B.A. Wood, 2 P. Mathiessen, and their predator after aerial spraying for the control
Accumulation
of endosulfan
of Tsetse fly in Botswana,
residue Pestic.
in fish Sci.,
13
(1982) 39-48. 3 K. Gopal, freshwater 4 G.E.
R.N.
Khanna,
organisms,
Thompson,
M. Anand
Toxicol.
Poisoning
Lett.,
and
G.S.D.
Gupta,
The acute
toxicity
of endosulfan
to
7 (1981) 453-456.
of cattle following
accidental
spraying
with thiodan,
Afr.
Vet. Assoc.,
37 (1966) 81-85. 5 G. Terziev and N.R. Dimitrova, ing with Thiodan, 6 P.K. Gupta,
Endosulfan
induced
15 (1976) 708-713. 7 M. Anand, S. Mehrotra, dosulfan
induced
8 K.l. Bentlay tion,
Forensic
medical
P. Med. Plv. Div.,
and P.D.
neurotoxicity
K. Gopal,
aggressive
R.N.
behaviour
Henry,
and forensic
chemical
study of acute lethal poison-
15 (1974) 708-713. in rats and mice, Bull. Environ.
Sur and S.V. Chandra,
in normal
Serotonin:
and lesioned
a potent
Toxicol.,
Role of neurotransmitters
rats, Toxicol.
constriction
Contam.
of human
Lett.,
coronary
in en-
24 (1985) 79-84. artery,
Circula-
12 (1981) 641-643.
G. Majno vascular
and G.E.
Palade,
permeability
study,
Studies
H.H.H. Oei and W.E. Hughes, J., 106 (1983) 1077-1081. V. Kumar, Lucknow G.V.R. Physiol.,
A study University,
Born
and
Platelet
of platelet India,
R.E.
Biochem. serotonin
biochemistry
I. The effect of histamine Cytol.,
uptake
in acute
and serotonin
on
11 (1961) 571-576. during
myocardial
myocardial
ischaemia,
infarction,
Thesis
Am. Heart for
M.D.,
1983.
Gilson,
146 (1959) 472-491.
on inflammation,
.I. Biophys.
Studies
on uptake
of 5-hydroxytryptamine
by blood
platelets,
J.
208
13 V.R. Cujrati, aemia
K. Shanker,
of pregnancy,
14 M. Anand,
A.C. Akveild
flow in cats,
including
15 K.P. Bhargava, Johnson
dimeform, 17 A.K.
Agarwal,
dosulfan 18 P.M.
Knowles,
Lett.,
M. Anand,
and K.P. Bhargava,
Effect of neurotoxic
circulation,
Vet. Hum.
pesticide Toxicol.,
and S. Vrat, Uptake
in hypertension,
Inhibition
Serotonin
in tox-
207 (1985) 11-18. endosulfan
on tissue blood
23 (4) (1981) 252-258.
of serotonin
by human
platelets
Life Sci., 25 (1979) 195-198.
of rat platelet
5-hydroxytryptamine
uptake
by chlor-
9 (1981) 1-4. N.F. Zaidi and P.K. Seth,
Biochem.
and R.A.
Pharmacol.,
Physiol.,
N. Misra, K. Shanker
and C.O.
neurotoxicity,
Vanhoutte
Biochem.
cerebral
to CNS involvement
Toxicol.
S. Vrat, Chandrawati
and P.R. Saxena, regional
N. Raina,
and its relevance 16 T.L.
S.S. Parmar,
Clin. Exp. Pharmacol.
Cohen,
Pharmacol., The elusory
32 (1983) 3671-3674.
Involvement
of serotonergic
receptors
inen-
32 (1983) 3591-3593. role of serotonin
in vascular
function
and disease,