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Catalytic potential of field mouse Mus booduga brain acetylcholinesterase during repeated hexachlorophene treatment
Toxicology Letters, 23 (1984) 177-182 Elsevier
177
TOXLett . 1303
CATALYTIC POTENTIAL ACETYLCHOLINESTERASE TREATMENT (Chlorinated
OF FIELD MOUSE MUS BOODUGA BRAIN DURING REPEATED HEXACHLOROPHENE
biphenol; AChE; inhibition; brain; field mouse)
G. VENKATESWARA
PRASAD, W. RAJENDRA and K. INDIRA*
Department of Zoology, S. V. University, Tirupati-517502 (A.P.) (India) (Received February lst, 1984) (Revision received April 24th, 1984) (Accepted June 13th, 1984)
SUMMARY The catalytic efficiency of the field mouse (Mus booduga) brain acetyl cholinesterase (AChE, EC 3.1.1.7) was significantly (PcO.001) decreased probably through the reduction in the active site density of the enzyme content and elevation in the activation energy (AE) requirements during repeated hexachlorophene (HCP) treatment. Fall in the activity potential of AChE may account for the interference of HCP or its reactive metabolites with the acetylcholine (ACh)-AChE system and deserve consideration in contributing to the neurotoxicity.
INTRODUCTION
Since the proposal of the brain membranes as the target of HCP action [l] work has been carried out on the effects of HCP on several membrane-associated enzymes and processes [2-41. AChE, which occurs at high specific activity in the brain [5], is intimately associated with the membranes [6] and reflects one of several facets of neuronal activity. Our unpublished results show that HCP strongly inhibits AChE in vitro. Although HCP is known to accumulate in the brains of animals during chronic exposure [7-81 there are no reports on its effects on the modulation of AChE. The present investigation was undertaken to study the kinetic response of mouse (Mus boo&go) brain AChE to repeated HCP treatment. * To whom correspondence Abbreviations:
should be sent.
ACh, acetyl cholinesterase; AChE, acetylcholine; HCP, hexachlorophene.
0378-4274/84/$ 03.00 0 Elsevier Science Publishers B.V.
178
MATERIALS
AND METHODS
Animals Field mice (Mus booduga) in the weight range of 13 + 2 g (mean + S.D) collected from local fields were maintained under laboratory conditions (temperature 30 + 2°C; relative humidity 75%; and a light period of 12 h) for 10 days before being subjected to HCP treatment. Chemical Technical grade (99.8% w/w) HCP (2,2’-methylenebis-(3,4,6-trichlorophenol) obtained from Sigma Chemicals (U.S.A.) was dissolved in the minimum volume of corn oil and was administered orally at a dose of 65 mg/kg/day for 7 days. The controls were given an isovolumetric amount (0.02 ml) of corn oil. Preparation of homogenates Brain tissue from both control and experimental animals was rapidly isolated after decapitation, chilled in ice-cold 0.9% NaCI, blotted, weighed and subjected to 10% (w/v) homogenisation in 0.25 M ice-cold sucrose solution using a motordriven Yorko-speed control homogenizer. The crude homogenate was employed as the enzyme source. Enzyme assay AChE was assayed by the method of Metcalf [9]. The assay mixture containing 100 PM sodium phosphate buffer (pH 7.4), varied concentrations of ACh (0.5 - 5 mM), 0.2 ml of the enzyme source, in a total volume of 2.0 ml was incubated at 37’C for 30 min. The reaction was arrested with 2 ml of alkaline hydroxylamine hydrochloride followed by 1:l HCl-water. The contents were centrifuged at 2000 x g for 10 min. The aliquots were checked for metabolized ACh concentration spectrophotometrically using ferric chloride. Protein content Protein content of the enzyme preparation was determined by the method of Lowry et al. [lo] using crystalline bovine serum albumin as the standard. Kinetic parameters AChE activity was assayed over a range of ACh concentrations. Kinetic parameters, V,,, and Km, were determined using least squares as the best fit. Temperature-dependent activity The effect of temperature on the enzyme activity was studied from 27°C to 52“C at intervals of 5°C at pH 7.4 (50 mM of sodium phosphate buffer) and at a substrate concentration of 5 mM. Other conditions of enzymic assay remain the same as
179
described already. The activation energy (AE) values were determined using Arrhenius equation [AE = 4.576 TrTz (log K “-log K’ /Tz-Tr) cal/mol] as given by Dixon and Webb [l 11. RESULTS
The results obtained from the substrate kinetic study are summarized in Table I. Mouse (MU booduga) brain AChE activity was significantly (P< 0.001) decreased during repeated HCP treatment. The metabolism of ACh was dependent on its concentration up to 4 mM in the controls, whereas in experimental mice the substrate dependence was only up to 3.2 mM. The maximal velocity (I/m& was decreased by 33.7% (P< O.OOl), whereas the Michaelis-Menten constant (Km) was unaltered during HCP intoxication. The activation energy of the enzymic reaction (Table II) was elevated significantly (P
TABLE I KINETIC ANALYSIS OF AChE IN CONTROL AND HCP-TREATED Sample
Vmaxa
Control HCP-treated
MUS BOODUGA
BRAIN
Kinetic parameters
2.49 f
Klllb 0.35’
1.65 + 0.14 P
1.0 + 0.061’ I.0 f 0.032 (-)d
‘Represented in pmol ACh hydrolysed/mg protein/h. bRepresented in mM of ACh. ‘Values are mean + S.D. of six experiments. d% deviations from control.
TABLE II ACTIVATION ENERGY VALUES OF CONTROL AND HCP-TREATED MUS BOODUGA AChE Sample
Control HCP-treated
Activation energy (AE) values in cal/mol 27-32°C
32-31°C
2500 f 3ooa 3300 + 400 P
1400 f 100 2200 + 200 P< 0.001 (+ 58.84)
‘Values are mean f SD. of six experiments. b% deviations from control.
BRAIN
180
-25
-20
-15
-10
-05
05
10
15
20
25
ACh (mM)-’
Fig. 1. Double HCP-treated
reciprocal (0)
Mus
plots of the substrate
concentration
versus AChE
activity
of control
(0) and
booduga brains.
DISCUSSION
The enzyme kinetic studies (Table I; Fig. 1) demonstrated that the mouse (MUS booduga) brain AChE activity was significantly (P
181
zyme is increased with the rise in temperature. At each given temperature range, the AChE from HCP-treated MUS booduga brain demanded higher than the normal amount of activation energy suggesting that the enzyme is catalytically not as efficient as that of the controls. Very little is known about the biochemical basis of HCP neurotoxicity. The observed changes in the kinetic parameters and AE requirements show that the AChE is strongly inhibited during HCP intoxication. As the majority of the neurones in mouse brain adopt cholinergic transmission, the decrease in the catalytic potential of AChE may play an important role in the animals’ susceptibility to the neurotoxic effects of HCP. However, strong inhibition of other enzymes like adenylate cyclase and ATPase [3] suggests that interaction of HCP and/or its metabolites [15] with AChE and other membrane-associated enzymes may all contribute to the expression of neurotoxicity. ACKNOWLEDGEMENTS
We are indebted to Dr. V. Mohanachari for helpful discussions. This work has been supported by CSIR, New Delhi through a Junior Research Fellowship to one of the authors (GVP).
REFERENCES
1 R.D. Kimbrough
and T.B. Gains,
light and electron 2 H.C.
Powell
Gruevic
and P.W.
(Eds.),
3 P. Mavier,
microscopy,
Lampert,
Neurotoxicology,
D. Stengel
rat liver plasma
Environ.
effects on the rat brain:
Health,
Hexachlorophene Raven
and .I. Hanoune,
membrane
Press,
neurotoxicity,
Inhibition
a mechanism
Effects
study of high doses by
23 (1971) 114-l 18.
New York,
by hexachlorophene,
4 T.L. Miller and D.R. Buhler, erythrocytes,
Hexachlorophene
Arch.
of adenylate Biochem.
in 1. Raizin, cyclase
and ATPase
Pharmacol.,
of hexachlorophenol
and N.
hemolysis,
activities
from
2.5 (1976) 305-309.
on monovalent
for hexachlorophene-induced
H. Shiraki
1977, p. 381.
cation
Biochim.
transport
in human
Biophys.
Acta,
352
(1974) 86-96. 5 A.G.E.
Pearse,
Histochemistry,
6 D. Nachmansohn, 7 A.G. Ulsamir,
Handbook
Stratton 9 R.L.
and biochemistry,
and N.K. Gonatas, (Eds.),
Metcalf,
acetyl choline, 1951, p. 43. 10 O.H. Lowry,
Livingstone, Physiology,
P.D. Renate and F.N. Marzulli,
ty, tissue concentrations 8 J. Tofighi
Churchil of Sensory
Progress Assay
methods
N.J.
for cholinesterases
Rosebrough,
A.L.
Cosmet.
and the nervous based
Farr and R.J.
Randall,
Venkateswara
acetylcholinesterase
Prasad,
V.
Mohanachari,
by hexachlorophene,
W.
Bull. Environ.
rats: toxici-
13 (1975) 69-80. in H. Zimmerman
New York,
chemical
Analysis,
1971, pp. 18-102.
on developing
system,
Press,
upon
of Biochemical
phenol reagent, J. Biol. Chem. 193 (1951) 265-275. 11 M. Dixon and C.E. Webb, Enzymes, Longman, London, 12 G.
New York,
Toxicol.,
Vol. 3, Academic
in D. Click (Ed.), Methods
1972, pp. 761-807.
Effects of hexachlorophene
Food,
Hexachlorophene
in Neuropathy,
London,
Spinger-Verlag,
determination
of unreacted
Vol. 5, Interscence,
Protein
and Cl.
1976, p. 297.
measurement
New York,
with the Folin
1979, p. 171.
Rajendra Contam.
and
K.
Toxicol.,
Indira,
Inhibition
(1984) (in press).
of
182 13 G. Flores and R.D. Buhler, In vitro hemolysis by chlorinated biphenyl. The role of hexachlorophene-protein interaction in the toxicity of chlorinated biphenols, Fed. Proc., 31 (1972) 520-526. 14 J.L. Wang and D.R. Buhler, Inhibition of dehydrogenase enzymes by hexachlorophene, Biochem. Pharmacol., 27 (1978) 2947-2953. 15 A. Miller, M.C. Henderson and D.R. Buhler, Cytochrome P-450 mediated covalent binding of hexachlorophenol to rat tissue proteins, Mol. Pharmacol., 14 (1977) 323-336.
177
TOXLett . 1303
CATALYTIC POTENTIAL ACETYLCHOLINESTERASE TREATMENT (Chlorinated
OF FIELD MOUSE MUS BOODUGA BRAIN DURING REPEATED HEXACHLOROPHENE
biphenol; AChE; inhibition; brain; field mouse)
G. VENKATESWARA
PRASAD, W. RAJENDRA and K. INDIRA*
Department of Zoology, S. V. University, Tirupati-517502 (A.P.) (India) (Received February lst, 1984) (Revision received April 24th, 1984) (Accepted June 13th, 1984)
SUMMARY The catalytic efficiency of the field mouse (Mus booduga) brain acetyl cholinesterase (AChE, EC 3.1.1.7) was significantly (PcO.001) decreased probably through the reduction in the active site density of the enzyme content and elevation in the activation energy (AE) requirements during repeated hexachlorophene (HCP) treatment. Fall in the activity potential of AChE may account for the interference of HCP or its reactive metabolites with the acetylcholine (ACh)-AChE system and deserve consideration in contributing to the neurotoxicity.
INTRODUCTION
Since the proposal of the brain membranes as the target of HCP action [l] work has been carried out on the effects of HCP on several membrane-associated enzymes and processes [2-41. AChE, which occurs at high specific activity in the brain [5], is intimately associated with the membranes [6] and reflects one of several facets of neuronal activity. Our unpublished results show that HCP strongly inhibits AChE in vitro. Although HCP is known to accumulate in the brains of animals during chronic exposure [7-81 there are no reports on its effects on the modulation of AChE. The present investigation was undertaken to study the kinetic response of mouse (Mus boo&go) brain AChE to repeated HCP treatment. * To whom correspondence Abbreviations:
should be sent.
ACh, acetyl cholinesterase; AChE, acetylcholine; HCP, hexachlorophene.
0378-4274/84/$ 03.00 0 Elsevier Science Publishers B.V.
178
MATERIALS
AND METHODS
Animals Field mice (Mus booduga) in the weight range of 13 + 2 g (mean + S.D) collected from local fields were maintained under laboratory conditions (temperature 30 + 2°C; relative humidity 75%; and a light period of 12 h) for 10 days before being subjected to HCP treatment. Chemical Technical grade (99.8% w/w) HCP (2,2’-methylenebis-(3,4,6-trichlorophenol) obtained from Sigma Chemicals (U.S.A.) was dissolved in the minimum volume of corn oil and was administered orally at a dose of 65 mg/kg/day for 7 days. The controls were given an isovolumetric amount (0.02 ml) of corn oil. Preparation of homogenates Brain tissue from both control and experimental animals was rapidly isolated after decapitation, chilled in ice-cold 0.9% NaCI, blotted, weighed and subjected to 10% (w/v) homogenisation in 0.25 M ice-cold sucrose solution using a motordriven Yorko-speed control homogenizer. The crude homogenate was employed as the enzyme source. Enzyme assay AChE was assayed by the method of Metcalf [9]. The assay mixture containing 100 PM sodium phosphate buffer (pH 7.4), varied concentrations of ACh (0.5 - 5 mM), 0.2 ml of the enzyme source, in a total volume of 2.0 ml was incubated at 37’C for 30 min. The reaction was arrested with 2 ml of alkaline hydroxylamine hydrochloride followed by 1:l HCl-water. The contents were centrifuged at 2000 x g for 10 min. The aliquots were checked for metabolized ACh concentration spectrophotometrically using ferric chloride. Protein content Protein content of the enzyme preparation was determined by the method of Lowry et al. [lo] using crystalline bovine serum albumin as the standard. Kinetic parameters AChE activity was assayed over a range of ACh concentrations. Kinetic parameters, V,,, and Km, were determined using least squares as the best fit. Temperature-dependent activity The effect of temperature on the enzyme activity was studied from 27°C to 52“C at intervals of 5°C at pH 7.4 (50 mM of sodium phosphate buffer) and at a substrate concentration of 5 mM. Other conditions of enzymic assay remain the same as
179
described already. The activation energy (AE) values were determined using Arrhenius equation [AE = 4.576 TrTz (log K “-log K’ /Tz-Tr) cal/mol] as given by Dixon and Webb [l 11. RESULTS
The results obtained from the substrate kinetic study are summarized in Table I. Mouse (MU booduga) brain AChE activity was significantly (P< 0.001) decreased during repeated HCP treatment. The metabolism of ACh was dependent on its concentration up to 4 mM in the controls, whereas in experimental mice the substrate dependence was only up to 3.2 mM. The maximal velocity (I/m& was decreased by 33.7% (P< O.OOl), whereas the Michaelis-Menten constant (Km) was unaltered during HCP intoxication. The activation energy of the enzymic reaction (Table II) was elevated significantly (P
TABLE I KINETIC ANALYSIS OF AChE IN CONTROL AND HCP-TREATED Sample
Vmaxa
Control HCP-treated
MUS BOODUGA
BRAIN
Kinetic parameters
2.49 f
Klllb 0.35’
1.65 + 0.14 P
1.0 + 0.061’ I.0 f 0.032 (-)d
‘Represented in pmol ACh hydrolysed/mg protein/h. bRepresented in mM of ACh. ‘Values are mean + S.D. of six experiments. d% deviations from control.
TABLE II ACTIVATION ENERGY VALUES OF CONTROL AND HCP-TREATED MUS BOODUGA AChE Sample
Control HCP-treated
Activation energy (AE) values in cal/mol 27-32°C
32-31°C
2500 f 3ooa 3300 + 400 P
1400 f 100 2200 + 200 P< 0.001 (+ 58.84)
‘Values are mean f SD. of six experiments. b% deviations from control.
BRAIN
180
-25
-20
-15
-10
-05
05
10
15
20
25
ACh (mM)-’
Fig. 1. Double HCP-treated
reciprocal (0)
Mus
plots of the substrate
concentration
versus AChE
activity
of control
(0) and
booduga brains.
DISCUSSION
The enzyme kinetic studies (Table I; Fig. 1) demonstrated that the mouse (MUS booduga) brain AChE activity was significantly (P
181
zyme is increased with the rise in temperature. At each given temperature range, the AChE from HCP-treated MUS booduga brain demanded higher than the normal amount of activation energy suggesting that the enzyme is catalytically not as efficient as that of the controls. Very little is known about the biochemical basis of HCP neurotoxicity. The observed changes in the kinetic parameters and AE requirements show that the AChE is strongly inhibited during HCP intoxication. As the majority of the neurones in mouse brain adopt cholinergic transmission, the decrease in the catalytic potential of AChE may play an important role in the animals’ susceptibility to the neurotoxic effects of HCP. However, strong inhibition of other enzymes like adenylate cyclase and ATPase [3] suggests that interaction of HCP and/or its metabolites [15] with AChE and other membrane-associated enzymes may all contribute to the expression of neurotoxicity. ACKNOWLEDGEMENTS
We are indebted to Dr. V. Mohanachari for helpful discussions. This work has been supported by CSIR, New Delhi through a Junior Research Fellowship to one of the authors (GVP).
REFERENCES
1 R.D. Kimbrough
and T.B. Gains,
light and electron 2 H.C.
Powell
Gruevic
and P.W.
(Eds.),
3 P. Mavier,
microscopy,
Lampert,
Neurotoxicology,
D. Stengel
rat liver plasma
Environ.
effects on the rat brain:
Health,
Hexachlorophene Raven
and .I. Hanoune,
membrane
Press,
neurotoxicity,
Inhibition
a mechanism
Effects
study of high doses by
23 (1971) 114-l 18.
New York,
by hexachlorophene,
4 T.L. Miller and D.R. Buhler, erythrocytes,
Hexachlorophene
Arch.
of adenylate Biochem.
in 1. Raizin, cyclase
and ATPase
Pharmacol.,
of hexachlorophenol
and N.
hemolysis,
activities
from
2.5 (1976) 305-309.
on monovalent
for hexachlorophene-induced
H. Shiraki
1977, p. 381.
cation
Biochim.
transport
in human
Biophys.
Acta,
352
(1974) 86-96. 5 A.G.E.
Pearse,
Histochemistry,
6 D. Nachmansohn, 7 A.G. Ulsamir,
Handbook
Stratton 9 R.L.
and biochemistry,
and N.K. Gonatas, (Eds.),
Metcalf,
acetyl choline, 1951, p. 43. 10 O.H. Lowry,
Livingstone, Physiology,
P.D. Renate and F.N. Marzulli,
ty, tissue concentrations 8 J. Tofighi
Churchil of Sensory
Progress Assay
methods
N.J.
for cholinesterases
Rosebrough,
A.L.
Cosmet.
and the nervous based
Farr and R.J.
Randall,
Venkateswara
acetylcholinesterase
Prasad,
V.
Mohanachari,
by hexachlorophene,
W.
Bull. Environ.
rats: toxici-
13 (1975) 69-80. in H. Zimmerman
New York,
chemical
Analysis,
1971, pp. 18-102.
on developing
system,
Press,
upon
of Biochemical
phenol reagent, J. Biol. Chem. 193 (1951) 265-275. 11 M. Dixon and C.E. Webb, Enzymes, Longman, London, 12 G.
New York,
Toxicol.,
Vol. 3, Academic
in D. Click (Ed.), Methods
1972, pp. 761-807.
Effects of hexachlorophene
Food,
Hexachlorophene
in Neuropathy,
London,
Spinger-Verlag,
determination
of unreacted
Vol. 5, Interscence,
Protein
and Cl.
1976, p. 297.
measurement
New York,
with the Folin
1979, p. 171.
Rajendra Contam.
and
K.
Toxicol.,
Indira,
Inhibition
(1984) (in press).
of
182 13 G. Flores and R.D. Buhler, In vitro hemolysis by chlorinated biphenyl. The role of hexachlorophene-protein interaction in the toxicity of chlorinated biphenols, Fed. Proc., 31 (1972) 520-526. 14 J.L. Wang and D.R. Buhler, Inhibition of dehydrogenase enzymes by hexachlorophene, Biochem. Pharmacol., 27 (1978) 2947-2953. 15 A. Miller, M.C. Henderson and D.R. Buhler, Cytochrome P-450 mediated covalent binding of hexachlorophenol to rat tissue proteins, Mol. Pharmacol., 14 (1977) 323-336.