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Phorate-Induced Enzymological Alterations in Mouse Olfactory Bulb
Brain Research Bulletin, Vol. 44, No. 3, pp. 247–252, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 1 .00
PII S0361-9230(97)00116-0
Phorate-Induced Enzymological Alterations in Mouse Olfactory Bulb S. VANDANA*1 AND S. ZZAMAN *Department of Zoology, Toxicology and Cell Biology Unit, College of Science, M. L. S. University, Udaipur-313001, India [Received 20 December 1996; Accepted 15 May 1997] ABSTRACT: The organophosphate pesticide, phorate, is an extremely hazardous insecticide. Not much experimental study is available on effects of phorate on different brain areas. We report in this study the alterations induced by phorate on enzyme profile of mouse olfactory bulb. Olfactory bulb, the first processing centre after the sensory cells in the olfactory pathway, has connections with the other higher centres of the brain like hippocampus and hypothalamus. Phorate was administered orally in the diet at the doses of 1.0 mg and 1.5 mg/kg body weight to adult albino mice. After 32 weeks of exposure animals were sacrificed and cryosections were processed for acetylcholinesterase and butyrylcholinesterase (AChE and BChE, respectively) enzyme localization. Significant reduction occurs in AChE and BChE activity at higher dose level, whereas reduced BChE activity was found at both dose levels. Our results shows an obvious effect on cholinesterase enzyme profile of olfactory bulb of mice after systemic administration of low doses of phorate for long terms. © 1997 Elsevier Science Inc.
olfactory system has been proposed as a early causative factor in Alzheimer’s disease [18]. Although many compounds are purported to cause olfactory dysfunction, little experimental work has been done with chronic systemically administered pesticides. A considerable amount of research has addressed the OP-induced CNS toxicity in various brain regions, but very little attention has been given to its effect on the olfactory bulb. Neurotoxic consequences of long-term low-level exposure have been poorly addressed experimentally [7]. According to Hungerford et al. [11], chronic toxicity study plays an important role in identifying OP insecticides that are risk factors in human diseases. Because OP causes irreversible inhibition of cholinesterase during chronic exposure, it would have an obvious effect on central cholinergic functions. Therefore, phorate {O,O-diethyl S-(ethylthiomethyl)phosphorodithioate} an extremely hazardous OP insecticide [14,31], causes poisoning in formulating plants even after taking protective measures [17,42], is taken up for the present chronic toxicity study in mice.
KEY WORDS: Acetylcholinesterase, Butyrylcholinesterase, Olfactory bulb, Organophosphate.
MATERIALS AND METHODS INTRODUCTION
Experimental Animal
Organophosphate (OP) pesticides constitute an increasing problem in industrial and agricultural toxicology. These pesticides are highrisk compounds and are mostly involved in occupational health hazards [8,22]. The wide-spread effects of environmental contaminants [36] and caution about occupational health and safety [29] has provided the impetus for research concerning the centres in the brain that direct the behaviour of the subject. Specialised motor and sensory system enable the organism to make an appropriate behavioral response to adapt the continuously changing external environment [21]. Olfactory system works as a defense mechanism against environmental toxicants. Detection of toxicants by this system direct the behaviour of subjects to minimise further exposure [1]. The olfactory bulb, the first processing centre after the sensory cells in the olfactory pathways, is an important component of olfactory system. It plays a role not only in olfactory adaptation [9], sensitivity enhancement by motivation [21], but also in memory and cognition [28]. This brain area is also a target of toxic insult during pre- and postnatal development [2,30,40]. The degeneration of 1
Inbred Swiss albino male mice (Mus musculus) 10 weeks of age, were selected as a biomodel for the present study. The mice were caged and housed in well ventilated room and were maintained at 246 2°C temperature with a 12-h dark and light phase. A standard diet and water were made available ad lib to the mice. Materials Phorate (thimet), technical grade (97.5%) {o,o-diethyl S-(ethlythiomethyl) phosphorodithioate} was procured from Pesticide India Limited, Udaipur, India. All other chemicals used in the present study were obtained from Sigma (St. Louis, MO) and BDH (India). Experimental Procedure Dose selection. The doses were selected after conducting a 12-week pilot study for Maximum Tolerated Dose based on clinical symptoms and other variables like body weight, mortality, acceptability of diet, etc. The doses selected for chronic toxicity
Requests for reprints should be addressed to Dr. Vandana, Department of Anatomy, All India Institute of Medical Sciences, New Delhi-110029, India.
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248 study (32 weeks), i.e., 1.0 mg/kg body weight/day and 1.5 mg/kg body weight/day were lower than the maximum tolerated dose (2.3 mg/kg body weight in diet) and were approximately 15 and 20% of the LD50, respectively. Administration of the test chemical. The solubility of the test chemical, phorate was determined to find out appropriate vehicle for administration. Ethyl alcohol was selected as the vehicle for phorate. Phorate was fed in the diet, i.e., 6 g diet/day/mouse. An appropriate dose of phorate was initially dissolved in ethyl alcohol, then phorate-ethyl alcohol concentration was added to a finely grounded diet of known composition, after which alcohol was allowed to evaporate. The use of ethyl alcohol prevents heterogenous distribution of the test chemical in an otherwise dry diet. The diet containing the test chemical was prepared daily to minimize the loss of chemical through instability or volatility. Fresh diet was placed in the feed containers (hoppers) fixed in individual cages. In a similar way a diet was prepared without the test chemical for the control group. The body weights of experimental mice were monitored every forthnight throughout the study. The mice were divided into three groups of 10 each. The mice of test groups were orally exposed (in diet) to the two level of phorate viz.:1.0 mg/kg body weight (P1) and 1.5 mg/kg body weight (P2). The control group (P0) was fed the same diet but without the test chemical. The experimental groups were exposed to phorate for 32 weeks (1/3 of the life span of a mouse). Four days after completion of exposure (to prevent immediate effects of phorate) eight mice in each test group including control were sacrificed by cervical dislocation. Brains were immediately dissected out in chilled calcium-formol and fixed for 12 to 14 h at 4°C. The tissues were washed in distilled water and serial cryostat sections of 16 mm were cut coronally. Alternate freefloating sections of the olfactory bulb from each mouse were processed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme by the method of Karnovsky and Roots [15]. Selective AChE and BChE inhibitors were used (0.05 mM 1,5-bis(4-Allyldimethylammoniumphenyl)pentan-3-one dibromide and 1024 M tetraisopropyl pyrophosphoramide, respectively), including negative control. Substrate was prepared in stock for all the groups, and conditions like reaction time, temperature, quantity of media, number of sections, and area of vessels (to prevent sections from overlap) were kept constant. The sections were mounted on glass slides and coded. All coded slides were evaluated under a light microscope. Data analysis. The neurotoxic effects of phorate were evaluated by comparing the distribution of AChE and BChE enzyme staining in the different layers of the olfactory bulb of the control and all test animals. The inhibition of enzyme activity was interpreted as effect of phorate exposure. For assessment of enzyme activity or inhibition, a visual estimation of reaction gradation were scored in randomly selected 10 sections/enzyme/mouse. The gradation of enzyme activity was 2no activity, 6 negligible activity, 1 weak activity, 11 mild activity, 111 moderate activity, 1111 strong activity, 11111 intense activity. Scored data from test groups were collected. Decoded scores were averaged according to enzyme, test group, layer, and displayed in a bar diagram. No visual variability in scores for enzyme reactions were noticed in mice within the group. For photomicrographs representative sections were photographed.
VANDANA AND ZZAMAN
FIG. 1. Schematic representation of different layers of olfactory bulb of mouse brain. The boxed area representing the photomicrographs of histoenzymology in Figs. 2 and 3. Light micrograph of coronal sections from mouse olfactory bulb stained for AChE enzyme activity (Fig. 2A–F).
RESULTS Observations No hypercholinergic symptoms were apparent in the test groups; however, there was increased frequency of defecation and micturation in last few weeks (nearly 4 weeks) in test group mice given 1.5 mg/kg body weight of phorate. Other variables like grooming, acceptability of diet, breeding performance were found to be within control levels throughout the exposure period. There was no significant change in body weight, which could be attributed to the phorate exposure given as it was within the control range. Examination of histoenzymologically stained sections of mice olfactory bulb indicate that phorate exposure has an obvious effect on the AChE and BChE enzyme profile. Figure 1 shows schematic representation of the layers of the olfactory bulb observed for histoenzymological studies. The distinct lamination of olfactory bulb is evident by AChE staining in the control animals (Fig. 2A and B). The outermost nerve fibre layer (NF) was devoid of AChE enzyme activity, but the glomerular layer (GL) was very distinct with intense enzyme staining. The other layers intensely stained were mitral cell layer (ML) external and internal plexiform layer (EP and IP, respectively). The internal granule cell layer (IG) showed irregular mesh work of intense AChE activity. The innermost layer of olfactory bulb, the olfactory tract (OT), showed a mild to strong AChE staining. The staining intensity of AChE at the dose level 1.0 mg/kg body weight of phorate did not appear to be different from control (Fig. 2C and D). An overall reduced AChE enzyme staining in the olfactory layers was observed in group P2, which was very distinct in the external plexiform layer, granule cell layer, and olfactory tract (Fig. 2E and F). The mitral cell layer and inner plexiform layer showed moderate reaction. The normal BChE activity is exhibited in Fig. 3A and B. The outermost nerve fibre layer was negligibly stained for BChE, while a strong enzyme staining was seen in the mitral cell layer, inner plexiform layer, and olfactory tract. The other layers of the glomerular and granule cell layer also showed moderate to strong reaction. The external plexiform layer was also negligible for BChE enzyme activity. The population of blood vessels stained for BChE varies in olfactory layers. It was relatively high in the
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FIG. 2. (A, B) Normal pattern of AChE enzyme activity (bar 5 125 mm). (C, D) Phorate treatment (1.0 mg/kg body weight) showing no change in enzyme activity compared to the control group (bar 5 125 mm). (E, F) The phorate-treated (1.5 mg/kg body weight) group showing an overall decreased enzyme activity (bar 5 119 mm). Photomicrographs of olfactory bulb stained for BChE enzyme (Fig. 3A–F).
glomerular layer, intermediate in the external plexiform layer, and sparse in the granule cell layer. Unlike AChE, BChE enzyme inhibition in olfactory layers was evident in mice exposed to 1.0 mg/kg body weight phorate (Fig. 3C and D). The least-affected layer seem to be the olfactory tract, where an intense BChE activity was evident. The blood vessels in different layers showed BChE activity. The mitral cell, internal plexiform and glomerular layer showed moderate enzyme activity, while mild BChE activity was noticed in the granule cell layer. Complete absence of BChE staining in the olfactory layers even in blood vessels make it very difficult to
differentiate the layers of olfactory bulb in group P2 (Fig. 3E and F). Figure 4 shows comprehensive histochemical data quantified on the basis of qualitative estimation. The results suggest a direct correlation between enzyme inhibition and dose of phorate administered. DISCUSSION This study presents the histoenzymological analysis of neurotoxic effects of chronic low-level phorate exposure on olfactory bulb of mouse brain.
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FIG. 3. (A, B) Normal pattern of BChE activity (bar 5 119 mm). (C, D) An overall decreased activity after 1.0 mg/kg body weight phorate treatment (bar 5 125 mm). (E, F) The olfactory layers are devoid of BChE enzyme activity after 1.5 mg/kg body weight phorate treatment (bar 5 125 mm).
The distinct lamination of the olfactory bulb contain several types of neurons, and the input have terminals at different levels in the bulb. The layers of the olfactory bulb can be differentiated by the AChE enzyme staining (Fig. 2A and B). The outer-most layer, i.e., nerve fibre layer, is devoid of cholinergic innervations and, hence, negative for AChE activity. The glomerular layer intensely positive for AChE is normally rich in cholinergic terminals [10] and the synapses in the glomerular layer are both adrenergic and cholinergic in nature [35]. Ravel et al. [27] suggested the existence of strong cholinergic control on the olfactory input at the level of the first synapse, i.e., glomeruli in the system. These glomeruli also abstract or enhance the sensory informations before transmitting for further processing in the deeper layers of the olfactory bulb [6].
The phorate administration at the dose level of 1.5 mg/kg body weight shows some decrease in AChE enzyme activity in glomerular layer, whereas a lower dose of phorate does not have any apparent effect in this layer and in external plexiform layer. The tuffted neurons present in external plexiform layer sends cholinergic fibres to the olfactory tract. These neurones and fibres in the external plexiform layer were strongly AChE positive even after 1.0 mg/kg body weight of phorate exposure. As the dose of the phorate was increased, i.e., 1.5 mg/kg body weight, the enzyme activity was totally absent in the neurones and fibres of external plexiform layer. Not much variation in AChE activity in the other layers—mitral, internal plexiform, granule cell layer, and olfactory tract—was noticed after phorate treatment at the lower dose, but a
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FIG. 4. AChE and BChE activity in various layers of mice olfactory bulb. Each value represent the average of qualitative gradation scores. Abbreviation used: NF—nerve fibre layer; GL— glomerular layer; EP— external plexiform layer; ML—mitral cell layer; IP—internal plexiform layer; IG—internal granular cell layer; OT— olfactory tract/white matter.
marked decrease in enzyme activity was noticed in these layers at a higher dose level, i.e., 1.5 mg/kg body weight of phorate reflecting dose-dependent response. Contrary to AChE inhibition, BChE activity can be seen even at a lower dose of phorate. This suggests that BChE is a sensitive indicator of phorate poisoning, which is in accordance with some other organophosphate poisoning [20,29]. Compared to other layers the presence of a strong BChE positivity in the blood vessels of glomerular layer is supposed to be for hydrolysis of acetylcholine, which has escaped from AChE at the glomerular synapse [4,19]. The complete absence of BChE activity in the layers including blood vessels at the higher dose group may be due to the impairment of the synthesis of this enzyme in the liver because phorate was also found to be hepatotoxic at the same dose level, whereas a lower dose of phorate (1.0 mg/kg body weight) does not have significant alterations in the liver (unpublished work). Similarly, Saxena and Sarin [32] also reported hepatotoxicity of phorate. Butyrylcholinesterase is also reported to be decreases in liver diseases [14] and by action of certain other OP pesticides [37]. The other probable reason of decrease in BChE enzyme activity at lower dose group (P1) and total absence at higher dose group (P2) may be due to the proposed scavenger function of this enzyme towards OP compounds [5]. Sensory system plays major role in adaptative changes in animal behaviour to lower down the further exposure to harmful pollutants [21,24]. Recently, Yamamoto et al. [41] suggested that the olfactory bulb plays an important role in the learning and memory processes necessary for both a working memory task and a reference memory task. Similarly, Richardson and Zucco [28] also suggested its role in memory and cognition. According to
Hunter and Murray [12], cholinergic mechanism appears to be involved in olfactory learning, and impairment in cholinergic functions mediates impairment in the olfactory bulb [3]. The impairment of cholinergic control at the information processing centre may occur due to the OP pesticide toxicity, as the present study suggest. There are reports that point strongly to olfactory influences on the hypothalamus [26,34], and these connections are probably important in some of the behavioural and endocrinological functions of the hypothalamus. Because there are functional relationship between olfactory bulb and hippocampus [38], any changes in the former would have an effect on the latter, which itself is a target of neurotoxicants. The chronic low-level exposure to OP pesticides may also hamper the other functions mediated by the olfactory bulb [23,25,39]. Cholinesterases are target of OP pesticides. The OP pesticiderelated poisoning symptoms observed in occupationally exposed population indicate purturbations in cholinergic functions [29] and investigations has proved that workers exposed occupationally (long term) to OP showed the chronic neurologic sequelae and lower cholinesterase level [13,16,36]. Individuals exposed occpationally to OP pesticides long term showed not only neuropsychological symptoms but other side effects also [22]. The experimental studies have shown that decreased AChE activity in the brain reflects in the behavioural profile of the OP-exposed rats [33]. Kashyap et al. [17] reported clinical manifestations and decreased cholinesterase activity in formulators following 2 weeks of exposure to phorate. In the exposed formulators, 60% of them showed gastrointestinal symptoms and significant reduction in plasma BChE activity. In present study, test group mice exposed to
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1.5 mg/kg body weight for 32 weeks showed increased frequency of defecation and micturation in the last few weeks. Significant reduction (100%) in BChE activity in the olfactory bulb was observed. Taking this data into account, the effect of phorate we observed histochemically, reflects impairment of cholinergic transmission in the cortical structures involved in olfactory stimuli processing. It provides neurochemical evidence of damage that occurs in the olfactory bulb due to phorate insecticide. This study suggests that chronic (part of life span) low-level exposure to OP may alter neurochemical indices in the olfactory bulb, which may leads to further changes at other higher brain centres connected with the olfactory bulb. ACKNOWLEDGEMENTS
This work was supported by grant from Indian Council of Medical Research No. J/3-5 (6) 84 BMS No. 8400950.
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PII S0361-9230(97)00116-0
Phorate-Induced Enzymological Alterations in Mouse Olfactory Bulb S. VANDANA*1 AND S. ZZAMAN *Department of Zoology, Toxicology and Cell Biology Unit, College of Science, M. L. S. University, Udaipur-313001, India [Received 20 December 1996; Accepted 15 May 1997] ABSTRACT: The organophosphate pesticide, phorate, is an extremely hazardous insecticide. Not much experimental study is available on effects of phorate on different brain areas. We report in this study the alterations induced by phorate on enzyme profile of mouse olfactory bulb. Olfactory bulb, the first processing centre after the sensory cells in the olfactory pathway, has connections with the other higher centres of the brain like hippocampus and hypothalamus. Phorate was administered orally in the diet at the doses of 1.0 mg and 1.5 mg/kg body weight to adult albino mice. After 32 weeks of exposure animals were sacrificed and cryosections were processed for acetylcholinesterase and butyrylcholinesterase (AChE and BChE, respectively) enzyme localization. Significant reduction occurs in AChE and BChE activity at higher dose level, whereas reduced BChE activity was found at both dose levels. Our results shows an obvious effect on cholinesterase enzyme profile of olfactory bulb of mice after systemic administration of low doses of phorate for long terms. © 1997 Elsevier Science Inc.
olfactory system has been proposed as a early causative factor in Alzheimer’s disease [18]. Although many compounds are purported to cause olfactory dysfunction, little experimental work has been done with chronic systemically administered pesticides. A considerable amount of research has addressed the OP-induced CNS toxicity in various brain regions, but very little attention has been given to its effect on the olfactory bulb. Neurotoxic consequences of long-term low-level exposure have been poorly addressed experimentally [7]. According to Hungerford et al. [11], chronic toxicity study plays an important role in identifying OP insecticides that are risk factors in human diseases. Because OP causes irreversible inhibition of cholinesterase during chronic exposure, it would have an obvious effect on central cholinergic functions. Therefore, phorate {O,O-diethyl S-(ethylthiomethyl)phosphorodithioate} an extremely hazardous OP insecticide [14,31], causes poisoning in formulating plants even after taking protective measures [17,42], is taken up for the present chronic toxicity study in mice.
KEY WORDS: Acetylcholinesterase, Butyrylcholinesterase, Olfactory bulb, Organophosphate.
MATERIALS AND METHODS INTRODUCTION
Experimental Animal
Organophosphate (OP) pesticides constitute an increasing problem in industrial and agricultural toxicology. These pesticides are highrisk compounds and are mostly involved in occupational health hazards [8,22]. The wide-spread effects of environmental contaminants [36] and caution about occupational health and safety [29] has provided the impetus for research concerning the centres in the brain that direct the behaviour of the subject. Specialised motor and sensory system enable the organism to make an appropriate behavioral response to adapt the continuously changing external environment [21]. Olfactory system works as a defense mechanism against environmental toxicants. Detection of toxicants by this system direct the behaviour of subjects to minimise further exposure [1]. The olfactory bulb, the first processing centre after the sensory cells in the olfactory pathways, is an important component of olfactory system. It plays a role not only in olfactory adaptation [9], sensitivity enhancement by motivation [21], but also in memory and cognition [28]. This brain area is also a target of toxic insult during pre- and postnatal development [2,30,40]. The degeneration of 1
Inbred Swiss albino male mice (Mus musculus) 10 weeks of age, were selected as a biomodel for the present study. The mice were caged and housed in well ventilated room and were maintained at 246 2°C temperature with a 12-h dark and light phase. A standard diet and water were made available ad lib to the mice. Materials Phorate (thimet), technical grade (97.5%) {o,o-diethyl S-(ethlythiomethyl) phosphorodithioate} was procured from Pesticide India Limited, Udaipur, India. All other chemicals used in the present study were obtained from Sigma (St. Louis, MO) and BDH (India). Experimental Procedure Dose selection. The doses were selected after conducting a 12-week pilot study for Maximum Tolerated Dose based on clinical symptoms and other variables like body weight, mortality, acceptability of diet, etc. The doses selected for chronic toxicity
Requests for reprints should be addressed to Dr. Vandana, Department of Anatomy, All India Institute of Medical Sciences, New Delhi-110029, India.
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248 study (32 weeks), i.e., 1.0 mg/kg body weight/day and 1.5 mg/kg body weight/day were lower than the maximum tolerated dose (2.3 mg/kg body weight in diet) and were approximately 15 and 20% of the LD50, respectively. Administration of the test chemical. The solubility of the test chemical, phorate was determined to find out appropriate vehicle for administration. Ethyl alcohol was selected as the vehicle for phorate. Phorate was fed in the diet, i.e., 6 g diet/day/mouse. An appropriate dose of phorate was initially dissolved in ethyl alcohol, then phorate-ethyl alcohol concentration was added to a finely grounded diet of known composition, after which alcohol was allowed to evaporate. The use of ethyl alcohol prevents heterogenous distribution of the test chemical in an otherwise dry diet. The diet containing the test chemical was prepared daily to minimize the loss of chemical through instability or volatility. Fresh diet was placed in the feed containers (hoppers) fixed in individual cages. In a similar way a diet was prepared without the test chemical for the control group. The body weights of experimental mice were monitored every forthnight throughout the study. The mice were divided into three groups of 10 each. The mice of test groups were orally exposed (in diet) to the two level of phorate viz.:1.0 mg/kg body weight (P1) and 1.5 mg/kg body weight (P2). The control group (P0) was fed the same diet but without the test chemical. The experimental groups were exposed to phorate for 32 weeks (1/3 of the life span of a mouse). Four days after completion of exposure (to prevent immediate effects of phorate) eight mice in each test group including control were sacrificed by cervical dislocation. Brains were immediately dissected out in chilled calcium-formol and fixed for 12 to 14 h at 4°C. The tissues were washed in distilled water and serial cryostat sections of 16 mm were cut coronally. Alternate freefloating sections of the olfactory bulb from each mouse were processed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme by the method of Karnovsky and Roots [15]. Selective AChE and BChE inhibitors were used (0.05 mM 1,5-bis(4-Allyldimethylammoniumphenyl)pentan-3-one dibromide and 1024 M tetraisopropyl pyrophosphoramide, respectively), including negative control. Substrate was prepared in stock for all the groups, and conditions like reaction time, temperature, quantity of media, number of sections, and area of vessels (to prevent sections from overlap) were kept constant. The sections were mounted on glass slides and coded. All coded slides were evaluated under a light microscope. Data analysis. The neurotoxic effects of phorate were evaluated by comparing the distribution of AChE and BChE enzyme staining in the different layers of the olfactory bulb of the control and all test animals. The inhibition of enzyme activity was interpreted as effect of phorate exposure. For assessment of enzyme activity or inhibition, a visual estimation of reaction gradation were scored in randomly selected 10 sections/enzyme/mouse. The gradation of enzyme activity was 2no activity, 6 negligible activity, 1 weak activity, 11 mild activity, 111 moderate activity, 1111 strong activity, 11111 intense activity. Scored data from test groups were collected. Decoded scores were averaged according to enzyme, test group, layer, and displayed in a bar diagram. No visual variability in scores for enzyme reactions were noticed in mice within the group. For photomicrographs representative sections were photographed.
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FIG. 1. Schematic representation of different layers of olfactory bulb of mouse brain. The boxed area representing the photomicrographs of histoenzymology in Figs. 2 and 3. Light micrograph of coronal sections from mouse olfactory bulb stained for AChE enzyme activity (Fig. 2A–F).
RESULTS Observations No hypercholinergic symptoms were apparent in the test groups; however, there was increased frequency of defecation and micturation in last few weeks (nearly 4 weeks) in test group mice given 1.5 mg/kg body weight of phorate. Other variables like grooming, acceptability of diet, breeding performance were found to be within control levels throughout the exposure period. There was no significant change in body weight, which could be attributed to the phorate exposure given as it was within the control range. Examination of histoenzymologically stained sections of mice olfactory bulb indicate that phorate exposure has an obvious effect on the AChE and BChE enzyme profile. Figure 1 shows schematic representation of the layers of the olfactory bulb observed for histoenzymological studies. The distinct lamination of olfactory bulb is evident by AChE staining in the control animals (Fig. 2A and B). The outermost nerve fibre layer (NF) was devoid of AChE enzyme activity, but the glomerular layer (GL) was very distinct with intense enzyme staining. The other layers intensely stained were mitral cell layer (ML) external and internal plexiform layer (EP and IP, respectively). The internal granule cell layer (IG) showed irregular mesh work of intense AChE activity. The innermost layer of olfactory bulb, the olfactory tract (OT), showed a mild to strong AChE staining. The staining intensity of AChE at the dose level 1.0 mg/kg body weight of phorate did not appear to be different from control (Fig. 2C and D). An overall reduced AChE enzyme staining in the olfactory layers was observed in group P2, which was very distinct in the external plexiform layer, granule cell layer, and olfactory tract (Fig. 2E and F). The mitral cell layer and inner plexiform layer showed moderate reaction. The normal BChE activity is exhibited in Fig. 3A and B. The outermost nerve fibre layer was negligibly stained for BChE, while a strong enzyme staining was seen in the mitral cell layer, inner plexiform layer, and olfactory tract. The other layers of the glomerular and granule cell layer also showed moderate to strong reaction. The external plexiform layer was also negligible for BChE enzyme activity. The population of blood vessels stained for BChE varies in olfactory layers. It was relatively high in the
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FIG. 2. (A, B) Normal pattern of AChE enzyme activity (bar 5 125 mm). (C, D) Phorate treatment (1.0 mg/kg body weight) showing no change in enzyme activity compared to the control group (bar 5 125 mm). (E, F) The phorate-treated (1.5 mg/kg body weight) group showing an overall decreased enzyme activity (bar 5 119 mm). Photomicrographs of olfactory bulb stained for BChE enzyme (Fig. 3A–F).
glomerular layer, intermediate in the external plexiform layer, and sparse in the granule cell layer. Unlike AChE, BChE enzyme inhibition in olfactory layers was evident in mice exposed to 1.0 mg/kg body weight phorate (Fig. 3C and D). The least-affected layer seem to be the olfactory tract, where an intense BChE activity was evident. The blood vessels in different layers showed BChE activity. The mitral cell, internal plexiform and glomerular layer showed moderate enzyme activity, while mild BChE activity was noticed in the granule cell layer. Complete absence of BChE staining in the olfactory layers even in blood vessels make it very difficult to
differentiate the layers of olfactory bulb in group P2 (Fig. 3E and F). Figure 4 shows comprehensive histochemical data quantified on the basis of qualitative estimation. The results suggest a direct correlation between enzyme inhibition and dose of phorate administered. DISCUSSION This study presents the histoenzymological analysis of neurotoxic effects of chronic low-level phorate exposure on olfactory bulb of mouse brain.
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FIG. 3. (A, B) Normal pattern of BChE activity (bar 5 119 mm). (C, D) An overall decreased activity after 1.0 mg/kg body weight phorate treatment (bar 5 125 mm). (E, F) The olfactory layers are devoid of BChE enzyme activity after 1.5 mg/kg body weight phorate treatment (bar 5 125 mm).
The distinct lamination of the olfactory bulb contain several types of neurons, and the input have terminals at different levels in the bulb. The layers of the olfactory bulb can be differentiated by the AChE enzyme staining (Fig. 2A and B). The outer-most layer, i.e., nerve fibre layer, is devoid of cholinergic innervations and, hence, negative for AChE activity. The glomerular layer intensely positive for AChE is normally rich in cholinergic terminals [10] and the synapses in the glomerular layer are both adrenergic and cholinergic in nature [35]. Ravel et al. [27] suggested the existence of strong cholinergic control on the olfactory input at the level of the first synapse, i.e., glomeruli in the system. These glomeruli also abstract or enhance the sensory informations before transmitting for further processing in the deeper layers of the olfactory bulb [6].
The phorate administration at the dose level of 1.5 mg/kg body weight shows some decrease in AChE enzyme activity in glomerular layer, whereas a lower dose of phorate does not have any apparent effect in this layer and in external plexiform layer. The tuffted neurons present in external plexiform layer sends cholinergic fibres to the olfactory tract. These neurones and fibres in the external plexiform layer were strongly AChE positive even after 1.0 mg/kg body weight of phorate exposure. As the dose of the phorate was increased, i.e., 1.5 mg/kg body weight, the enzyme activity was totally absent in the neurones and fibres of external plexiform layer. Not much variation in AChE activity in the other layers—mitral, internal plexiform, granule cell layer, and olfactory tract—was noticed after phorate treatment at the lower dose, but a
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FIG. 4. AChE and BChE activity in various layers of mice olfactory bulb. Each value represent the average of qualitative gradation scores. Abbreviation used: NF—nerve fibre layer; GL— glomerular layer; EP— external plexiform layer; ML—mitral cell layer; IP—internal plexiform layer; IG—internal granular cell layer; OT— olfactory tract/white matter.
marked decrease in enzyme activity was noticed in these layers at a higher dose level, i.e., 1.5 mg/kg body weight of phorate reflecting dose-dependent response. Contrary to AChE inhibition, BChE activity can be seen even at a lower dose of phorate. This suggests that BChE is a sensitive indicator of phorate poisoning, which is in accordance with some other organophosphate poisoning [20,29]. Compared to other layers the presence of a strong BChE positivity in the blood vessels of glomerular layer is supposed to be for hydrolysis of acetylcholine, which has escaped from AChE at the glomerular synapse [4,19]. The complete absence of BChE activity in the layers including blood vessels at the higher dose group may be due to the impairment of the synthesis of this enzyme in the liver because phorate was also found to be hepatotoxic at the same dose level, whereas a lower dose of phorate (1.0 mg/kg body weight) does not have significant alterations in the liver (unpublished work). Similarly, Saxena and Sarin [32] also reported hepatotoxicity of phorate. Butyrylcholinesterase is also reported to be decreases in liver diseases [14] and by action of certain other OP pesticides [37]. The other probable reason of decrease in BChE enzyme activity at lower dose group (P1) and total absence at higher dose group (P2) may be due to the proposed scavenger function of this enzyme towards OP compounds [5]. Sensory system plays major role in adaptative changes in animal behaviour to lower down the further exposure to harmful pollutants [21,24]. Recently, Yamamoto et al. [41] suggested that the olfactory bulb plays an important role in the learning and memory processes necessary for both a working memory task and a reference memory task. Similarly, Richardson and Zucco [28] also suggested its role in memory and cognition. According to
Hunter and Murray [12], cholinergic mechanism appears to be involved in olfactory learning, and impairment in cholinergic functions mediates impairment in the olfactory bulb [3]. The impairment of cholinergic control at the information processing centre may occur due to the OP pesticide toxicity, as the present study suggest. There are reports that point strongly to olfactory influences on the hypothalamus [26,34], and these connections are probably important in some of the behavioural and endocrinological functions of the hypothalamus. Because there are functional relationship between olfactory bulb and hippocampus [38], any changes in the former would have an effect on the latter, which itself is a target of neurotoxicants. The chronic low-level exposure to OP pesticides may also hamper the other functions mediated by the olfactory bulb [23,25,39]. Cholinesterases are target of OP pesticides. The OP pesticiderelated poisoning symptoms observed in occupationally exposed population indicate purturbations in cholinergic functions [29] and investigations has proved that workers exposed occupationally (long term) to OP showed the chronic neurologic sequelae and lower cholinesterase level [13,16,36]. Individuals exposed occpationally to OP pesticides long term showed not only neuropsychological symptoms but other side effects also [22]. The experimental studies have shown that decreased AChE activity in the brain reflects in the behavioural profile of the OP-exposed rats [33]. Kashyap et al. [17] reported clinical manifestations and decreased cholinesterase activity in formulators following 2 weeks of exposure to phorate. In the exposed formulators, 60% of them showed gastrointestinal symptoms and significant reduction in plasma BChE activity. In present study, test group mice exposed to
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1.5 mg/kg body weight for 32 weeks showed increased frequency of defecation and micturation in the last few weeks. Significant reduction (100%) in BChE activity in the olfactory bulb was observed. Taking this data into account, the effect of phorate we observed histochemically, reflects impairment of cholinergic transmission in the cortical structures involved in olfactory stimuli processing. It provides neurochemical evidence of damage that occurs in the olfactory bulb due to phorate insecticide. This study suggests that chronic (part of life span) low-level exposure to OP may alter neurochemical indices in the olfactory bulb, which may leads to further changes at other higher brain centres connected with the olfactory bulb. ACKNOWLEDGEMENTS
This work was supported by grant from Indian Council of Medical Research No. J/3-5 (6) 84 BMS No. 8400950.
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