Cellular and Molecular Mechanisms of Dichlorvos Neurotoxicity: Cholinergic, Nonchlolinergic, Cell Signaling, Gene Expression and Therapeutic Aspects

Indian Journal of Experimental Biology Vol 48, July 2010, pp. 697-709

Review Article

Cellular and molecular mechanisms of dichlorvos neurotoxicity: Cholinergic, nonchlolinergic, cell signaling, gene expression and therapeutic aspects

B K Binukumar & K D Gill * Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India 160 012

Inappropriate use of toxic chemicals is common in developing countries, where it leads to excessive exposure and high risks of unintentional poisoning. Risks are particularly high with the pesticides used in agriculture, poor rural populations live and work in close proximity to these compounds and often store these compounds in and around their homes. It is estimated that most of the death from pesticide poisoning occur in developing countries. Organophosphate insecticides have been extensively used in agriculture in developing countries. Dichlorvos is a synthetic insecticide and belongs to a family of chemically related organophosphate pesticides (OP). Toxicity of dichlorvos has been documented in accidental human poisoning, epidemiological studies, and animal models. In this review, molecular mechanisms of dichlorvos neurotoxicity have been described. Usage, biotransformation, environmental levels, general population and occupational exposure, effects on cell signaling receptors, mitochondrial metabolism, oxidative stress and gene expression of dichlorvos have been reviewed. Assessment of acute and chronic exposures as well as neurotoxicity risk for lifetime exposures to dichlorvos have also been considered. In addition special emphasis has been given to describe, the role of dichlorvos in the chronic neurotoxicity and its molecular targets that ultimately lead to neurodegeneration.

Keywords : Behavioral, Dichlorvos, Neurotoxicity, Nicotinic receptors, Organophosphate

Organic derivatives of phosphorus-containing acids Indonesia, South Africa, and Brazil 3. Acute OP are chiefly familiar as organophosphate (OP) compounds poisoning is an important cause of pesticides. These compounds are anticholinesterase morbidity and mortality in India 4. Among the insecticides, widely used in agriculture, horticulture, numerous pesticides that can result in death, veterinary medicine, public hygiene, and also used as organophosphate insecticides are the most common nerve agents in chemical warfare 1,2 . Lack of culprits because of their high toxicity. biopersistance of OPs in comparison with the Dichlorvos, an organophosphate, is a direct-acting organochlorines has made most countries to replace cholinesterase (ChE) inhibitor. Although first the organochlorines with OPs. Consequently their synthesized in the late 1940s 5, its commercial scale of use has increased in recent years. Pesticide manufacture started much later, in 1961. The annual poisoning remains a serious public health problem production of dichlorvos was as high as 4.2 million worldwide. According to the World Health pounds (lbs) in the late 1970s, and fell to 992,000 lbs Organization’s estimate, 3 million cases of pesticide by 1989. More recent estimates are not available, but poisoning occur every year, resulting in more than are likely to be lower due to many recent 250,000 deaths. This number also accounts for a cancellations of its use 6. Dichlorvos can be released substantial fraction of the almost 900,000 people into the environment as a major degradation product worldwide who die by suicide every year. However, of other OP insecticides, such as trichlorfon, naled, in developing countries, where the use of OP and metrifonate 6-8. Dichlorvos can kill insects when compounds is particularly widespread because of the ingested, or absorbed through the integument or via hot climatic conditions, the number of deaths may be spiracles 9. It is commercially available under the trade high. For example, pesticide poisonings are relatively names viz., Dedevap, Nogos, Nuvan, Phosvit and common in countries such as Sri Lanka, Venezuela, Vapona. Some common synonyms are Bayer 19149, DDVF, DDVP, ENT-20738, OMS 14, SD 1750 and ______C-177. The major mechanism for the action of *Correspondent author-Telephone: 0172-2747585 Fax: +91 172 2744401; 2745078. dichlorvos is inhibition of acetylcholinesterase E-mail: [email protected]. (AChE), leading to an increase in the level of 698 INDIAN J EXP BIOL, JULY 2010

acetylcholine in the synaptic cleft and hence released to any or all environmental media producing both nicotinic and muscarinic signs and (air, surface water, groundwater, and soil). The symptoms of intoxication in the peripheral and central general population is exposed to dichlorvos primarily nervous system like nausea, vomiting, lacrimation, through inhalation of contaminated indoor air, either salivation, bradycardia, miosis and finally death may during and/or immediately after application or occur due to respiratory failure 5. through the use of polyvinyl chloride resin strips. The In agriculture, dichlorvos has been used to protect second major route of exposure to dichlorvos for the stored crops from insect damage. It was used in general population is through direct dermal contact livestock industry to control external parasites on with the chemical spray during domestic applications, animals such as fleas and ticks. Dichlorvos was also contact with dichlorvos treated plant materials such as added in animal feed as an anthelmintic (worming grass or ornamental plants or contact with other agent) for swine, horses and dogs 6. It was used in treated surfaces (e.g. furniture) in domestic or office mushroom houses to control flies and insects 10 and buildings. In developing countries dichlorvos may was added directly to the water in fish farms, to still be applied by professional exterminators for control fish parasites 11 . The annual agricultural use of insect control in buildings and in turf grass treatments. dichlorvos was estimated as 248,000 lbs during 1982 6. These applications create potential for some exposure Estimates done in late 1980s indicate that 60% of for the general population through inhalation, dermal dichlorvos used worldwide was for plant protection, contact and oral intake, especially in children. 30% was for public hygiene and vector control, and In addition to the individuals occupationally 10% to protect stored crops 11 . Dichlorvos was among exposed to dichlorvos, several groups within the the several organophosphate pesticides (OP) shipped general population may receive potentially higher by the Department of Defense for use during the Gulf inhalation exposures to dichlorvos. These groups War 12 . include individuals living near factories where In water, dichlorvos hydrolyzes into dichlorvos is produced or processed and those dimethylphosphoric acid and dichloroacetaldehyde individuals living near hazardous waste sites where (DCA). Dichlorvos has also been reported to this compound is present. Although dichlorvos is not penetrate into the waxy layers of plant tissues, where tightly bound to soil particles, ingestion of dichlorvos it persists longer and undergoes hydrolysis to DCA 13 . contaminated soil or soil where polyvinyl chloride The occurrence of dichlorvos residues in the resin strips have been disposed off, might be a route environment does not necessarily originate from the of exposure particularly for children. Ingestion of use of dichlorvos. They may also occur as conversion contaminated groundwater by individuals living in the products of another widely used OP pesticide, vicinity of hazardous waste sites may be another trichlorfon. The air concentration of dichlorvos varies possible source of exposure for both adults and according to the method of application (strips, spray children if these individuals use untreated well water cans or fogging), temperature and humidity. as their primary source of drinking water. Concentration has been reported to be in the range of 0.1-0.3 mg/m 3 in the first week after using resin strips Acute cholinergic toxicity (1 strip/30 m 3)14 . Food samples, meals and unwrapped Dichlorvos exerts its toxic effects in humans and ready to eat foodstuffs exposed under practical animals by inhibiting neural acetyl cholinesterase. If conditions to dichlorvos generated by resin strips this enzyme is inhibited, acetylcholine accumulates in showed residues with in a range of the synapse, resulting in increased firing of the 0.01-0.1 mg/kg 14,15 . Food and beverages exposed to postsynaptic neuron or increased neuroeffector experimental air concentrations of 0.04-0.58 mg/m 3 activity. The consequences of increased cholinergic for 30 min, contained dichlorvos residues in the range activity in the parasympathetic autonomic nervous of 0.005-0.5 mg/kg, with the exception of margarine, system (muscarinic receptors) can include increased which contained up to 106 mg/kg 16 . salivation, lacrimation, perspiration, miosis, nausea, vomiting, diarrhoea, excessive bronchial secretions General population exposure to dichlorvos etc. The effects of increased neuroeffector activity on No quantitative information is available on the skeletal muscles (nicotinic receptors) can include percentage of dichlorvos released to different muscle fasciculations, cramps, muscle weakness and environmental compartments. Dichlorvos can be depolarization type paralysis. Effects on cholinergic BINUKUMAR & GILL: DICHLORVOS NEUROTOXICITY 699

synapses in the central nervous system period of 6 months, even when the strips were (predominantly muscarinic) can result in drowsiness, replaced at shorter intervals than that normally fatigue, mental confusion, headache, convulsions and recommended. The maximum average concentration coma. These classical symptoms of organophosphate in the air was approximately 0.1 mg/m 3. In factory neurotoxicity increase in severity and rapidity of onset workers exposed to an average of 0.7 mg/m 3 for in a dose dependent manner. In addition to this, 8 months, significant inhibition of plasma and red decreased vigilance, hallucinations, defects in blood cell ChE activity was found 11 . expressive language and cognitive function, impaired memory, depression, anxiety or irritability and Delayed neurotoxicity of dichlorvos psychosis have been well reported after dichlorvos Neurotoxic potential of organophosphorus exposure 5. compounds have already been described in details 11 . Several studies have shown that dichlorvos does not Clinical studies of acute and chronic toxicity produce delayed neurotoxicity in pre-medicated hens, The clinical signs and symptoms of dichlorvos are whether it is administered orally or generally attributable to acetylcholine accumulation subcutaneously 17-19 . Contradictory to these studies and are commonly divided into three groups; Choudhary et al.20 reported, single subcutaneous dose muscarinic, nicotinic and central. There have been of dichlorvos (200 mg/kg body weight) resulted in two clinical reports describing four patients suffering marked changes in the dopaminergic neurotransmitter from severe poisoning from dichlorvos, taken orally, system in terms of increased levels of both dopamine who survived after treatment and who showed and norepinephrine along with significant increase in delayed neurotoxic effects. Thus, although the the activity of both the catecholamine synthesizing possibility of neuropathy in human beings cannot be enzymes, tyrosine hydroxylase (TH) and dopamine- excluded, it is likely to occur only after almost lethal beta-hydroxylase. This increase was accompanied oral doses. When dichlorvos was administered orally with a concomitant decrease in the activity of major to human volunteers (single or repeated doses of a degradative enzyme, monoamine oxidase. Scatchard slow-release PVC formulation), significant inhibition plot analysis revealed a significant decrease in both of red blood cell ChE activity was found at 4 mg/kg K(d) and B(max) for dopamine D2 receptors. body weight or more. At 1 mg/kg body weight or Administration of nimodipine, a centrally acting more, plasma ChE activity was significantly inhibited. calcium channel blocker, along with dichlorvos Daily oral doses of 2 mg dichlorvos/person for restricted all these alterations to within control values 28 days reduced plasma ChE activity by 30%, but red and could also ameliorate certain behavioral deficits cell ChE activity was unaffected 11 . by maintaining the dopaminergic neurotransmitter Human volunteers who were exposed to dichlorvos system. The study underlines the importance of by inhalation for a certain period per day for a number alterations in the dopamine system as a possible of consecutive days or weeks showed ChE inhibition causative mechanism behind the behavioral and at a concentration of 1 mg/m 3 or more, but not at functional changes associated with delayed 0.5 mg/m 3. These results were confirmed in studies neurotoxicity. In addition to this, dichlorvos treatment with pesticide operators who came into contact with also led to a consistent increase in the activity of both dichlorvos. Hospitalized patients showed similar microtubule associated protein kinases viz. results after oral administration or exposure by Ca 2+ /Calmodulin-dependent and cAMP dependent inhalation. Sick adults and children and healthy protein kinases, at all post exposure intervals pregnant women and babies in hospital wards treated (day 7, 15 and 21) as compared to that of controls. with dichlorvos strips (1 strip/30 or 40 m 3) displayed Autoradiography followed by micro-densitometric normal ChE activity. Only subjects exposed 24 h/day studies demonstrated enhanced phosphorylation of to concentrations above 0.1 mg/m 3 or patients with 55 and 280 kDa proteins in dichlorvos-exposed liver insufficiency showed a moderate decrease in animals. These two proteins were confirmed to be plasma ChE activity. No significant effects on plasma tubulin and microtubule associated protein-2 (MAP-2) or red blood cell ChE activity were observed in by western blotting. The hyperphosphorylation of people exposed to the recommended rate of one these two proteins was shown to interfere with the dichlorvos strip per 30 m 3 in their homes over a assembly of neuronal microtubules as shown by 700 INDIAN J EXP BIOL, JULY 2010

electron microscopic studies that may eventually lead chronic OP neurotoxicity is a general malaise lacking to possible disruption of neuronal cyto-architecture in specificity and related to mild cognitive resulting in axonal degeneration 21 . dysfunction, similar to that described for Gulf War The inhibition of brain neurotoxic esterase (NTE) syndrome 26 . The mechanisms underlying these effects without signs of ataxia has been observed. Johnson et are not known, and the role of AChE inhibition is al.22 reported mild signs of ataxia in pre- medicated controversial 26,27 and may vary depending on the hens 2 weeks after a single massive subcutaneous exposure parameters. Chronic neurotoxicity dichlorvos dose (100 mg/kg body wt) and severe subsequent to a single acute exposure to OPs may be inhibition of NTE in peripheral nerve, spinal cord, triggered by AChE inhibition. In fact, acute sub lethal and brain. However, no ataxia was in pre-medicated doses of OPs were shown to have long-term effects in hens given the same dose through the same route. humans 28,29 . Oral administration of dichlorvos to rat These hens showed severe inhibition of brain NTE (70 mg/kg) inhibited not only AChE but also but far less inhibition of spinal cord NTE. It appears hexokinase, phosphofructokinase, lactate dehy- that ataxia arises from the inhibition of spinal cord drogenase and glutamate dehydrogenase. Dichlorvos NTE. When the dose was repeated 1-3 days after the administration also caused significant depletion in the first dose, spinal cord NTE inhibition increased and brain glycogen content along with increased glycogen the hens became ataxic. phosphorylase activit 30 . Repeated administration of We also reported delayed neurotoxicity potential of 50% of LD 50 (i.e., 40 mg/kg body wt per day for 10 - dichlorvos 23 . In vitro , dichlorvos caused a 21 days), caused myelin pallor and micro-vacuolation concentration and time-dependent decrease in the of the white matter. It seems likely that primary activity of NTE. The Ki of dichlorvos for NTE was degeneration of axons and secondary myelin sheath calculated to be 1.28 ×10 3 M/min. In vitro reactivation abnormalities caused the spongy tissue loosening and ageing studies revealed that dichlorvos-inhibited observed under the electron microscope 31,32 . NTE became refractory to activation by potassium In studies by Ali et al.33 male rats were given 3 mg fluoride after 5 min in the ageing medium, thus dichlorvos/kg per day intraperitoneally for 10 days. indicating the formation of an aged complex between Following perfusion-fixation, sections of cerebellum dichlorvos and NTE. In vivo also, dichlorvos and spinal cord were studied with the electron (200 mg/kg body wt.) given as a single subcutaneous microscope. An abnormal increase in the number of dose inhibited NTE in brain at various intervals after mitochondria in the spinal cord was found. Myelin exposure (24 h, 10 days, and 21 days). The delayed degeneration was detected in the spinal cord and neurotoxicity potential of dichlorvos was finally myelin figures were occasionally noted within confirmed by the rota rod test, which revealed severe oedematous dendrite profiles. Another study of motor deficit in all the exposed animals. dichlorvos neurotoxicity involved the investigation of lipid peroxidation. This entails the direct reaction Chronic neurotoxicity between oxygen and lipids to form free-radical Previous years have seen a continuous interest in intermediates and semi- stable peroxides. Major studies concerning the effects of sub lethal acute cellular components, such as membranes and exposure to various OPs24 or chronic contact with subcellular organelles, are damaged by these free organophosphate pesticides 25 . There is increasing radicals. Dose-dependent increase in the rate of lipid evidence that OPs may also cause a long-term, peroxidation in various regions of the brain of persistent chronic neurotoxicity following either a the rat after ip administration of dichlorvos single acute high-dose exposure or repeated exposures (at concentrations ranging from 0.6 to 3 mg/kg body to low-level, subclinical doses of OPs. The clinical wt, daily) for 10 days have been reported 34 . Also, and epidemiological data in support of chronic OP there was an increased incidence of lipofuscin-like neurotoxicity presents with pathological lesions in pigment in the Purkinje cells of the cerebellar cortex. both the PNS and CNS, but it is the latter that is Julka et al.35 observed dichlorvos exposure (5 mg/kg- primarily responsible for presenting neurologic body wt, ip) cause significant increase in the activities symptoms and changes in neurobehavioral of the antioxidant enzymes superoxide dismutase performance, reflecting cognitive and psychomotor (SOD) and catalase. Dichlorvos exposure also dysfunction. The most sensitive manifestation of resulted in a significant decrease in glutathione BINUKUMAR & GILL: DICHLORVOS NEUROTOXICITY 701

peroxidase activity. The decreased levels of both metabolism of catecholamines and 5-HT may be reduced and oxidized glutathione as observed on disturbed by dichlorvos. dichlorvos exposure affected the GSH/GSSG ratio. These results indicate that the enzymes SOD and Dichlorvos and cell signaling receptors catalase may enhance the disposal of potentially toxic Due to the ubiquitous distribution of both nicotinic radicals. Furthermore, the decrease in GSH levels and muscarinic cholinergic receptors throughout the may be a mechanism for the detoxification of body, exposure to OP compounds has widespread dichlorvos in the brain. Maslinska et al.36 found that toxic consequences in several target organs. Virtually dichlorvos (dose levels of 4-8 mg/kg body weight for all cholinergic synapses can be affected by exposure 10 days) affected the phospholipid-protein balance in to anticholinesterase compounds such as OPs. These the brain of rabbits. The animals were exposed during include autonomic postganglionic parasympathetic the postnatal "critical" life period, which constitutes a nerve endings, sympathetic and parasympathetic turning point in the development of the brain. At this ganglia, motor end plates of skeletal muscle, and, of time, the neurons have already undergone course, various regions of the CNS. Hyperactivity at considerable arborization, and myelination and these synapses due to accumulation of ACh causes a vascularization are expanding rapidly. In addition, the variety of symptoms mediated by overstimulation of overall oxygen consumption is reaching its steepest muscarinic and nicotinic receptors. rate of increase. In the myelin sheaths under Density of receptors with a stereo specific binding formation, several phospholipids are deposited. The site for nicotine in the mammalian brain is only 1% of authors found changes in the phospholipid-protein that of muscarinic receptors 42 . In the brain, the highest ratio which correlated well with the observed delay in concentrations of nicotinic receptors are found in the myelin sheath formation. Ultrastructural changes in thalamus, cortex, superior colliculus, and striatum, certain subcellular organelles may be connected with whereas the lowest concentrations occur in the the change in this ratio, since it is crucial to the piriform cortex and hippocampus. Thus, the structural and functional properties of the membranes distribution of nicotinic receptors in the CNS clearly and enzymes bound to them. differs from that of muscarinic receptors. It is also Kobayashi et al.37,38 investigated the concentration quite evident that most of the cholinergic effects of of total, free, labile-bound and stable-bound ACh in OPs in the CNS are mediated via muscarinic rather the brain of rats given single or multiple subcutaneous than nicotinic receptors. This is important because the injections of dichlorvos (0.2-4 mg/kg body weight). most dramatic toxic actions of OPs are mediated via The results suggest that alterations in the mobilization their effects on cholinergic receptors in the CNS and and storage of ACh in the central cholinergic nerves subsequent stimulation of other neurotransmitter may be induced. The time course for ACh systems in the brain, as well as via cholinergic accumulation was measured in rat brain regions after receptor stimulation in other target organs, subsequent intravenous treatment with 15 mg dichlorvos/kg body to the initial effects of OPs on AChE and the weight 39 . The striatum had the highest rate of cholinergic systems. accumulation and the cerebellum the lowest. The In addition to their indirect effects on muscarinic calculated turnover time for the different regions of receptors through AChE-mediated changes in ACh the brain was between 0.9 and 5.6 min. levels, many OPs can affect their expression and In studies by Ali and Hasan 40 and Ali et al.34 rats function directly. Paraoxon, dichlorvos, and tetraethyl were given ip 3 mg dichlorvos/kg body weight per pyrophosphate (TEPP) were found to be non- day for 10 or 15 days. The concentrations of competitive antagonists of muscarinic receptors in dopamine, norepinephrine, and 5-hydroxytryptamine bovine caudate nuclei labeled with [ 3H] quinuclidinyl (5-HT) were significantly decreased in different parts benzilate ([ 3H]-QNB) at concentrations that had no of the brain, and 5-HT was significantly increased in effect on AChE activity. Direct effects of OP the spinal cord. A single dose or short-term compounds on muscarinic receptors were studied by (12 weeks) treatment of rats with high concentrations using rat brain membranes or cultures of human of dichlorvos, which produced brain ChE inhibition, neuroblastoma N1E-115 cells 43 . Recently, it has been resulted in decreased norepinephrine levels in the reported that dichlorvos exposure causes significant 41 brain . From these studies, it was suggested that the reduction in the expression of M 1, M 2 and M 3 702 INDIAN J EXP BIOL, JULY 2010

muscarinic receptor subtypes in high dose group The balance between the production of free radicals animals whereas, in low dose group animals where and antioxidant defenses in the body has important AChE is not inhibited, the expression of only M 2 health implications: if there are too many free radicals receptor was found to be reduced significantly 44 . QNB or too few antioxidants for protection, a condition of binding to the muscarinic receptors were carried out oxidative stress develops, which may cause chronic in the synaptic plasma membranes of the rat brain in and permanent damage. Pesticides may induce the presence of varying concentrations of [ 3H] QNB oxidative stress, leading to generation of free radicals (0–2 n M). Non-specific binding was carried out in the and alteration in antioxidants, oxygen free radicals, presence of atropine (1 µM). The Bmax for the the scavenging enzyme system and lipid peroxidation. 6.0 mg/kg body wt. dichlorvos exposed group was There is growing evidence supporting the 26% and for 1.0 mg/kg body wt. dichlorvos exposed involvement of ROS and reactive nitrogen species in group was 22% lower than the control group excitotoxicity injury. Excessive activation of (982.8 fmol/mg) animals. The affinity constants (Kd) cholinergic and glutamatergic receptor is thought to for control, low and high dose group animals were be responsible for excitotoxicity 45 . During normal 0.23, 0.23 and 0.022 nM, respectively. So, marked respiration, small amounts of ROS are produced as by reductions in [ 3H] QNB binding were seen products of the ETC process. However, perturbations immediately after the cessation of the exposure, in mitochondrial respiration can lead to excessive indicating a marked reduction in muscarinic receptor ROS generation and inundate cellular antioxidant numbers. capacity, leading to DNA damage, lipid peroxidation, protein modification, and eventually cell death 46 . Effects of mitochondrial metabolism and oxidative Common initiating mechanism of excitotoxicity is stress thought to be frequent stimulation of nicotinic Reactive oxygen species and their destructive acetylcholine receptor at the mammalian nature is known for along, but their diverse neuromuscular junction and muscuranic, nicotic and pathophysiological effects on vital organs are still of glutamatergic receptor in brain. It has been great interest. Oxidative stress can be defined as the hypothesized that increased ACh levels following imbalance between the production of free radicals AChE inhibition activate glutamatergic neurons capable of causing peroxidation of the lipid layer of causing the release of glutamate, which ultimately cells and the body’s antioxidant defense. Free radicals results in excitotoxicity via increased intracellular are defined as atoms or molecules that contain one or calcium and activation of nitric oxide synthase more unpaired electrons. The toxicity of many following NMDA receptor activation 47 . There is a xenobiotics is associated with the production of free support for the proposed mechanism of chronic OP radicals, which are not only toxic themselves, but are neurotoxicity in that it has been demonstrated that also implicated in the pathophysiology of many nitric oxide synthesis inhibitors block OP-induced diseases. For example, there is extensive evidence for seizures 48 . However, the role of AChE inhibition in oxidative stress as an important mechanism of this sequence of events has yet to be established. In neurodegeneration in Alzheimer’s disease. Other contrast, chronic OP neurotoxicity induced by diseases include Parkinson’s disease, cataracts, repeated exposures to subclinical OP doses has been atherosclerosis, neoplastic diseases, diabetes, chronic reported to occur in the absence of AChE inhibition, inflammatory diseases of the gastrointestinal tract, suggesting that mechanisms other than aging of skin, asthma, and many others. anticholinesterase activity mediate the neurotoxic Pesticide induced oxidative stress has also been a effects elicited by this exposure scenario 26,27 . focus of toxicological research for the last decade as a However, what these mechanisms are has yet to be possible mechanism of toxicity. Several studies have established. Our results also indicated decreased been conducted to determine whether oxidative stress mitochondrial electron transfer activities of in humans or animals results from various agents in cytochrome oxidase (complex IV) along with altered this group and is associated with their toxic effects. mitochondrial complex I, and complex II activity, To understand the exact nature of oxidative stress, it which might have resulted from elevated is necessary to describe the principles of free radical mitochondrial calcium uptake. The alterations in the production and the body’s normal defense system. mitochondrial calcium uptake and mitochondrial BINUKUMAR & GILL: DICHLORVOS NEUROTOXICITY 703

electron transfer enzyme activities in turn might have other’s results 27 and suggest an intriguing possibility caused an increase in malondialdehyde, protein that repeated exposures to sublethal or subclinical carbonyl and 8-hydoxydeoxyguanosine formation as a doses of OPs increases apoptotic neuronal death via result of enhanced lipid peroxidation, and as well as oxidative stress. protein and mtDNA oxidation. All this could have The experimental evidence supports the hypothesis been because of enhanced oxidative stress, decreased that OPs modulate intracellular signaling pathways GSH levels and also decreased Mn-SOD activity in downstream of receptors and suggests that the diverse the mitochondria isolated from dichlorvos treated rat neurotoxic effects of many OPs may reflect their brain. In addition to disruption of cellular antioxidant influence on multiple intracellular signaling defense system, chronic dichlorvos exposure also pathways. Functional studies examining the effects of triggers the release of cytochrome c from dichlorvos on signaling events downstream of mitochondria to cytosol as well as activates caspase-3 muscarinic receptor activation further support the also 49 . Low-level long-term dichlorvos exposure hypothesis that dichlorvos can interact directly with 44 finally resulted in oligonucleosomal DNA M2 receptors . Activation of M 2 and M 4 receptors fragmentation, a hallmark of apoptosis. These studies generally reduces the activity of adenylyl cyclase, provide an evidence of impaired mitochondrial which decreases cAMP production, whereas bioenergetics and apoptotic neuronal degeneration activation of M 1, M 3, or M 5 receptors increases after chronic low-level exposure to dichlorvos, phosphoinositide-specific phospholipase C activity, (Fig. 1). These results are also in accordance with which increases release of inositol triphosphate,

Fig. 1  Hypothetical model for chronic dichlorvos neurotoxicity. Chronic dichlorvos exposure leads to decreased activities of cytochrome oxidase, mitochondrial complex I, complex II, elevated mitochondrial calcium uptake, increased malondialdehyde, protein carbonyl, 8-hydoxydeoxyguanosine formation, decreased GSH levels, decreased Mn-SOD activity, release of cytochrome c from mitochondria to cytosol, activates caspase-3 and finally oligonucleosomal DNA fragmentation, a hallmark of apoptosis 704 INDIAN J EXP BIOL, JULY 2010

(Fig. 2). Chronic dichlorvos exposure (6 mg/kg body rise in the intrasynaptosomal calcium levels. The wt./day) for a period of 8 weeks caused significant activity of major calcium expelling enzyme i.e. reduction in both high affinity (HA) and low affinity Ca 2+ ATPase was found to be reduced. Also, the (LA) choline uptake (CU), with maximal effect being depolarization induced calcium uptake via voltage observed in the brain stem followed by cerebellum operated calcium channels increased significantly. and cerebrum. Muscarinic receptor binding was Concomitant to the increase in intrasynaptosomal significantly decreased in brain stem and cerebellum calcium, calpain activity was found to be increased. as reflected in the decreased receptor number (Bmax), Dichlorvos could mediate through modifications in without any change in the binding affinity [K(d)] of the intracellular calcium homeostasis which may lead the receptors. Dichlorvos treatment caused marked to impaired neuronal function. inhibition in cAMP synthesis as indicated by decreased adenylate cyclase activity as well as cAMP Effects on signaling and gene expression levels in cerebrum, cerebellum and brain stem. These Signaling pathways identified as potential targets studies show that dichlorvos may interact with in OP neurotoxicity can modulate gene expression muscarinic receptor-linked second messenger system via alterations in the expression levels or activational and this could be a potential mechanism for the status of transcription factors. One transcription factor of considerable interest in OP neurotoxicity is neurotoxic effects observed after repeated exposure to 2+ low levels of dichlorvos, which are unexplainable on Ca /cAMP response element binding protein the basis of cholinergic hyperactivity 50 . Interestingly, (CREB), which is activated via phosphorylation by a the mechanism of action for the OP steroidogenesis variety of signaling pathways, including cAMP/PKA, MAP kinase/ERK, p38, and CaM inhibitor, diethylumbelliferyl phosphate, is also 52 believed to be mediated through an interaction with kinase II . Numerous studies have indicated that the cAMP/PKA pathway 3. OPs can activate CaM CREB is critical to several forms of use-dependent kinase II 27 . Raheja and Gill 51 have also reported that synaptic plasticity and transcription-dependent forms chronic dichlorvos administration caused significant of memory, and evidence supports a major role for CREB in cell survival and differentiation during brain development 52,53 . Since impairments of brain development and memory function are two primary neurological effects observed in laboratory studies with OPs, Schuh et al. 54 have hypothesized that the mechanisms underlying these effects may include alterations in the expression or activational status of CREB. OPs caused similar effects in primary cultures of hippocampal neurons. Mechanism(s) by which OPs activate CREB is not known, but is probably not mediated by OP effects on adenylyl cyclase activity, which are predominantly inhibitory. There is documentation of OP effects on other transcription factors important in neurodevelopment and synaptic plasticity. Thus, OPs elevate levels and activation of c-fos 55,56 cause developmental stage- specific changes in AP-1 and Sp-1 expression and DNA binding activity 57 and stimulate phosphorylation of c-Jun 58 . Recently Verma et al. 59 have reported that dichlorvos at low dose exposure leads to reduction in the signal transduction cascade linked to muscuranic receptor subtypes and adenylyl cyclase-linked signaling pathway was impaired. This  Fig. 2 Dichlorvos interacts directly with M2 receptors. finally leads to significant reduction in the Activation of M 2 receptor reduces the activity of adenylyl cycla se, which decreases cAMP production, which leads to decreased phosphorylation of CREB in both low dose and high production of cAMP dependent protein kinase dose group animals. BINUKUMAR & GILL: DICHLORVOS NEUROTOXICITY 705

Neurobehavioral implication of dichlorvos reported chronic dichlorvos administration caused a exposure marked decrease in both the ambulatory and Importance of neurobehavioral toxicity in risk stereotypic components of spontaneous locomotor assessment lies in the fact that behaviour can be activity of rats. The muscle strength and coordination regarded as the net output of the sensory, motor and of the dichlorvos-treated animals was also cognitive functions occurring in the nervous system significantly impaired. Besides, a marked and can serve as potentially sensitive endpoints of deterioration in the memory function assessed in chemical induced neurotoxicity. Neurobehavioral terms of the conditioned avoidance response was sequelae of acute and chronic organophosphate discernible at the end of the treatment schedule in the exposure have been described in the literature for experimental animals. Verma et al.59 also explained decades 60, 61. the molecular mechanism of neurobehavioral Due to the almost ubiquitous nature of cholinergic impairments, seen after chronic dichlorvos exposure, pathways and synaptic networks, organophosphate may be due to significant reduction of M 2 muscarinic pesticide such as dichlorvos by virtue of their being receptor linked adenylyl cyclase signaling pathway antichlolinergic agent may exert the widest range of and reduction in the phosphorylation of CREB behavioural effects. Schulz et al.62 have reported dose (Fig. 3). related alterations in open field behaviour of animals along with impairment in cognitive functions Recent advances in treatment of organophosphate following dichlorvos exposure during gestation. Dose poisoning and time related exposure to dichlorvos has been Therapeutic treatment against OP poisoning has reported to alter the EEG pattern and the nerve been studied extensively since World War II. The conduction velocity in treated animals, with characteristic signs and symptoms of acute OP concomitant decrease in the AchE levels. intoxication are generally thought to arise from the Significant motor deficit in terms of altered motor inhibition of acetylcholinesteras and the ensuing functions and coordination following a delayed cholinergic crisis. Nevertheless, the currently adopted neurotoxic insult by dichlorvos have been reported 63 . treatments (such as those supplied by atropine-oxime While the effects of acute dichlorvos poisoing are autoinjectors) are not fully satisfactory under actual well characterized, significant lacunae exist as regards life threatening conditions 64 . Atropine is suitable to the chronic effects, particularly those associated with counteract the muscarinic effects of excess behavioural aspects of dichlorvos exposure. We acetylcholine, as bronchospasm, bronchorrhoea and

Fig. 3Chronic dichlorvos exposure, leads to significant reduction of M 2 muscarinic receptor linked adenylyl cyclase signaling pathway and reduction in the phosphorylation of CREB 706 INDIAN J EXP BIOL, JULY 2010

pulmonary oedema. But it is ineffective at nicotinic shown to have protective effects against receptor sites, e.g. at respiratory muscles. To cope organophosphate poisoning. Harrison et al.69 have with the respiratory problems, antidotes reactivating investigated the ability of several adenosine A(1) inhibited AChE have been developed. Their clinical receptor agonists to inhibit epileptiform activity effectiveness, however, is still a matter of induced by the organophosphate sarin, in CA1 controversy. The reason for this uncertainty is caused stratum pyramidale of guinea pig hippocampal slice. by the difficulty in clearly assessing oxime effects due Efforts have now been expanded to identify a to both the high complexity of the various catalytic protein capable of not only binding, but also microscopic reactions involved and problems in rapidly hydrolyzing the standard threat nerve agents. recording the distinct clinical changes in sedated and Recent work has focused on paraoxonase-1(PON1). artificially ventilated patients during cholinergic The specific hydrolytic activity of PON1 crisis. In order to counteract the toxic effects of OP at paraoxonase/arylesterase enzymes in liver and blood striated muscles additional therapeutic measures are provide a natural barrier against the entry of indispensable. Here reactivating oximes can be organophosphate toxins into the central and peripheral expected to act as specific antidotes 65,66 . The search nervous systems. Cowan et al .70 have reported that for oxime-based reactivators dates back to the early boosting serum levels of PON1 enzymes by a gene 1950s, starting with hydroxylamine and hydroxamic delivery vector raises the threshold for acids 66 . Later on, ketoximes and aldoximes were organophosphate toxicity by hydrolytic destruction investigated. Meanwhile, more than 1500 compounds before the chemical can enter the brain. Moreover, have been tested, but only few have been studied for recently Stevens et al.71 have reported that engineered human use. One of the major drawbacks in the recombinant human PON1 purified from Escherichia development of oximes as antidotes against poisoning coli protects against organophosphate poisoning. is the inability to perform clinical studies in humans. Therefore, different in vitro and in vivo models have Conclusion been used to evaluate the efficacy of oximes. One of As dichlorvos displays relatively limited selectivity the major problems in the assessment of oxime between insects and non target species, including efficacy in humans is the difficulty to extrapolate humans, concerns on their potential adverse effects in animal data to humans. human populations will continue. Further Presently, the main focus in oxime research is on investigation of the low level long-term exposure the development of broad spectrum reactivators being leading to dichlorvos neurotoxicity should be able to counteract the effects of structurally different undertaken. More information is required to properly OPs. While 2-PAM is a generally weak reactivator of evaluate the chronic exposure to humans leading to OP-inhibited AChE, obidoxime effectively reactivates neuropsychological manifestations. Neurotoxicity human AChE inhibited by different OP pesticides. HI studies at low concentrations with large groups of 6 has been shown to be effective with most nerve rodents must be undertaken to settle the present agents, including cyclosarin, but is a weak reactivator dilemma of acetylcholine dependent neurotoxicity vs. of pesticide-inhibited AChE and fails to reactivate acetylcholine independent neurotoxicity. Moreover, tabun-inhibited enzyme. MMB-4 is superior to an epidemiological study of long-term exposed 2-PAM, but mostly less potent than obidoxime and HI workers might reveal information concerning the 6. Presently, Hagedorn oxime HLö 7 may be toxicology of dichlorvos as well as its possible considered as a broad spectrum reactivator active neurotoxicity to humans. The issues discussed in this against organophosphonates, phosphates and review represent real-life problems, with clinical, phosphoramidates, but this oxime is difficult to societal and legal ramifications. Continuing research synthesize and rather unstable in solution 67 . Data with in all these areas, and others not mentioned, is newer oximes are scarce and are mostly generated warranted. The extreme toxicity of dichlorvos using animal AChE 68 . highlights the need for a more complete Organophosphate poisoning can result in seizures understanding of it’s mechanisms of toxic actions. and subsequent neuropathology. One possible This information also provides new insights into therapeutic approach would be to employ adenosine neurotoxicity and opens new vistas for research to A(1) receptor agonists, which have already been explore mechanisms of OP toxicity. Even though, the BINUKUMAR & GILL: DICHLORVOS NEUROTOXICITY 707

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