Chlorpyrifos Toxicity in Mouse Cultured Cerebellar Granule Neurons at Different Stages of Development: Additive Effect on Glutamate-Induced Excitotoxicity (original) (raw)
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Toxicology and Applied Pharmacology, 2002
Previous studies suggest that low doses of the organophosphate insecticide chlorpyrifos (CPF) disrupt brain development and cognitive function by mechanisms that do not involve the inhibition of acetylcholinesterase (AChE). In the present study we tested the hypothesis that CPF and its metabolites alter the Ca 2؉ /cAMP response element binding protein (CREB), a critical molecule in brain development and cognitive function. We further tested the hypothesis that changes in CREB occur independent of AChE inhibition. Western blot analysis of lysates from primary cultures of cortical neurons exposed to CPF, CPF-oxon, or trichloropyridinol (TCP) for 1 h and cultures exposed to trichloropyridinol (TCP) for 7 days indicated that all exposures increased the level of the phosphorylated (activated) form of CREB (pCREB), without significant changes in total CREB or ␣-tubulin. Remarkably, pCREB in cortical neurons was elevated by 300-400% of control levels with estimated EC50s of 60 pM, <30 fM, and <30 pM for CPF, CPF-oxon, and TCP, respectively. AChE activity and cell viability were not affected by organophosphate concentrations that caused significant increases in pCREB (up to 100 nM, 100 pM, and 10 M of CPF, CPF-oxon, and TCP, respectively). The level of pCREB in hippocampal neurons was also elevated after exposure to CPF, but pCREB in cultured astrocytes was not affected. Inclusion of the cytochrome P-450 inhibitor SKF-525A did not inhibit the effects of CPF on pCREB levels, indicating that metabolism of CPF to CPF-oxon was not necessary to cause the increase in pCREB. The increases in neuronal pCREB observed in this study provide biochemical evidence that CPF and its metabolites are active at critical sites within the nervous system at levels far below those required to inhibit AChE, which could explain many of the reported neurodevelopmental and behavioral changes attributed to CPF toxicity.
O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate; Dursban) is a widely used broad-spectrum organophosphorus (OP) insecticide. Because some OP compounds can cause a sensory-motor distal axonopathy called OP compound-induced delayed neurotoxicity (OPIDN), CPS has been evaluated for this paralytic effect. Early studies of the neurotoxicity of CPS in young and adult hens reported reversible leg weakness but failed to detect OPIDN. More recently, a human case of mild OPIDN was reported to result from ingestion of a massive dose (about 300 mg/kg) in a suicide attempt. Subsequent experiments in adult hens (the currently accepted animal model of choice for studies of OPIDN) showed that doses of CPS in excess of the LDso in atropine-treated animals inhibited brain neurotoxic esterase (NTE) and produced mild to moderate ataxia. Considering the extensive use of CPS and its demonstrated potential for causing OPIDN at supralethal doses, additional data are needed to enable quantitative estimates to be made of the neuropathic risk of this compound. Previous work has shown that the ability of OP insecticides to cause acute cholinergic toxicity versus OPIDN can be predicted from their relative tendency to inhibit the intended target, acetylcholinesterase i Portions of this research were presented at the 28th annual meeting of the Society of Toxicology, (ACHE), versus the putative neuropathic target, NTE, in brain tissue. The present study was designed to clarify the magnitude of neuropathic risk associated with CPS exposures by measuring hen brain AChE and NTE inhibition following dosing in vivo and determining the bimolecular rate constant of inhibition (ki) for each enzyme by the active metabolite, CPS oxon (CPO), in vitro. CPS administered to atropine-treated adult hens at 0, 75, 150, and 300 mg/kg po in corn oil produced mean values for brain AChE inhibition 4 days after dosing of 0, 58, 75, and 86%, respectively, and mean values for brain NTE inhibition of 0, 21, 40, and 77%, respectively. Only the high dose (six times the unprotected LDso in hens) produced NTE inhibition above the presumed threshold of 70%, and these animals were in extremis from cholinergic toxicity at the time of euthanization despite continual treatment with atropine. When 150 mg/kg CPS po in corn oil was given to atropine-treated hens on Day 0, inhibition on Days 1, 2, 4, 8, and 16 for brain AChE was 86, 82, 72, 44, and 29%, respectively, and for brain NTE was 30, 28, 38, 29, and 6%, respectively. No signs of OPIDN were observed in any of the animals during the 16-day study period. Kinetic studies of the inhibition of hen brain AChE and NTE by CPO in vitro demonstrated that CPO exhibits high potency and extraordinary selectivity for its intended target, ACHE. The ki values were 15.5/~M -1 min -1 for AChE and 0.145/.tM -1 min -I for NTE. The calculated fixed-time (20-min) 15o values were 2.24 nM for AChE and 239 nM for NTE, yielding an /so ratio for NTE/ AChE of 107. These results may be compared with data compiled for other OP compounds with respect to NTE/AChE 15o ratios and the corresponding doses required to produce OPIDN relative to the LDs0. In general, NTE/AChE/5o ratios greater than 1 indicate that the dose required to produce OPIDN is greater than the LDs0. Taken together, the results of this study indicate that acute exposures to CPS would not be expected to cause OPIDN except under extreme conditions such as attempted suicides involving medically assisted survival of doses considerably in excess of the LDso. 9 1993 Society of Toxicology.
Chlorpyrifos Leads to Oxidative Stress-Induced Death of Hippocampal Cells in Vitro
Neurophysiology, 2013
Chlorpyrifos (ClPF) is a broad-spectrum organophosphate insecticide widely used in agriculture, industry, and at home. Like all organophosphates, ClPH affects the nervous system by inhibiting the enzyme acetylcholinesterase (AChE). In addition, it is transformed in higher animals into ClPFoxon that is about 3000 times more potent against the nervous system than ClPF itself. As was found recently, the action on ACh is not the only mechanism of ClPF toxicity. One other mechanism of this organophosphate is induction of oxidative stress leading to generation of free radicals. We investigated the effects of ClPF on hippocampal cells of the rat in vitro and focused our attention on mediation of its cytotoxic effect related to the production of reactive oxygen species. Transfection of cultured hippocampal cells by green fluorescent protein (GFP) was used. We studied the dose dependence of the intensity of ClPF-induced damage and cell death of hippocampal neurons in vitro and the dependence on the duration of ClPF action. We also observed survival of the cells incubated in the media with only ClPF and under the same conditions but with the addition of Trolox as an antioxidant. It was found that Trolox demonstrated clear neuroprotective effects at all concentrations of ClPF tested during the research period. It is concluded that the negative effect of ClPF on hippocampal neurons results, to a considerable extent, in the development of oxidative stress.
Biological effects assessment of chlorpyrifos and some aspects of its neurotoxicity
In the present study a general survey is made of chlorpyrifos - one of the most commonly used organophosphorus pesticides in the world. The review does not give an exhaustive treatment of the biochemistry of chlorpyrifos. This question has been discussed at length in many reviews and will be briefly dealt with here. An attempt was made to review the vast amount of information concerning mechanisms of chlorpyrifos neurotoxicity. It has long been known, that chlorpyrifos is acetylcholinesterase inhibitor and that high sensitivity of the cholinesterases inhibitors makes them highly toxic to the central nervous system. Chlorpyrifos continues to receive considerable research interest. In the past five years there have been some innovations in the question concerning mechanisms of chlorpyrifos neurotoxicity, specifically it was repeatedly demonstrated that chlorpyrifos toxicity is not limited to cholinesterase inhibition alone but can act by other mechanisms. Here we summarize the existen...
Mitochondrial complex I inhibition as a possible mechanism of chlorpyrifos induced neurotoxicity
Background: Organophosphates (OPs) represent the most widely used class of pesticides. Although perceived as low toxicity compounds compared to the previous organochlorines, they still possess neurotoxic effects both on acute and delayed levels. Delayed neurotoxic effects of OPs include OPIDN and OPICN. The mechanisms of these delayed effects have not been totally unraveled yet. One possible contributor for neurotoxicity is mitochondrial complex I (CI) inhibition. Purpose: in the present study we evaluated the contributing role of (CI) inhibition in chlorpyrifos (CPF) induced delayed neuropathy in hens. Methods: Experimented birds received 150 mg/kg of CPF, and evaluated behaviorally and biochemically. Results: CPF treated hens received 150 mg/kg and developed signs of delayed neurotoxicity, which were verified by NTE inhibition. These effects were paralleled by CI inhibition and decrease in ATP level. Conclusions: The data confirms the possible role of CI inhibition in CPF induced delayed neuropathy.
Developmental Brain Research, 2005
Neurochemical and behavioral studies indicate that the widely used organophosphorus insecticide, chlorpyrifos (CPF), evokes neurobehavioral teratogenicity with a wide window of vulnerability, ranging from embryonic life through postnatal development. Few studies have detailed morphological damage that corresponds to the operational deficits. We administered 5 mg/kg of CPF sc daily on postnatal days (PN) 11-14, a regimen that is devoid of systemic toxicity, but that elicits long-term cognitive impairment and disruption of cholinergic, catecholaminergic, and serotonergic synaptic function. On PN15 and 20, we conducted quantitative morphologic examinations of neurons and glia in CA1, CA3, and dentate gyrus regions of the hippocampus. Although hippocampal morphology after CPF exposure was normal on gross observation, morphometric analysis revealed a significant overall reduction in the total number of neurons and glia. Superimposed on this basic effect, CPF elicited a delayed-onset increase in the neuron/glia ratio that emerged by PN20, connoting selective gliotoxicity. The alterations in cell numbers were accompanied by significant perikaryal swelling and by enhanced development of astrocytic processes. Layer thickness also showed delayed-onset effects of CPF, with thinning of the CA1 and CA3 layers and enlargement of the dentate gyrus. Our results indicate that there are subtle morphological changes in the juvenile rat brain after neonatal CPF exposure that are detectable only with quantitative analysis and that correlate with regional and cell-specific targets identified earlier in neurochemical studies. The simultaneous targeting of neurons and glia by CPF is likely to play an important role in its developmental neurotoxicant effects.
Toxicology, 2018
Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75 and α-nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aβ and tau proteins production and activation of GSK3β enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of ...
The Journal of Toxicological Sciences, 2013
-Organophosphates (OPs) are important toxic compounds commonly used for a variety of purposes in agriculture, industry and household settings. It has been well established that the main mechanism of acute toxic action of OP is the inhibition of acetylcholinesterase (AChE). However, we observed long term deficit after acute subcutaneous exposure to Chlorpyrifos (CPF) even when AChE activity is restored. In fact, besides AChE inhibition, non-AChE targets have also been proposed as an alternative mechanism involved in the acute lethal action and side effects of short or long-term exposure. In this context, our main aim in this research was to establish a dose-response curve of Acylpeptide hydrolase (APH) and AChE regional brain activity after acute CPF administration that could explain these long term effects observed in the literature. Moreover, since available data suggest that long term effects of OPs exposure could involve neuronal cell death, our second aim was to evaluate, assessing by Fluoro-Jade B (FJB) staining, whether CPF produces induced cell death. Our results show that an acute exposure to 250 mg/kg CPF does not induce neuronal death as measured by FJB but produces highest AChE regional brain inhibition after administration. In addition, APH seems to be more sensitive than AChE to CPF exposure because after 31 days of exposure, complete recovery was seen only for APH activity at Frontal Cortex, Cerebellum and Brain Stem.
Pharmacology Biochemistry and Behavior, 1993
Neonatal (7 days old) rats are markedly more sensitive than adults (3 months old) to the acute toxic effects of the insecticide, chlorpyrifos (CPF). In the present study, we have compared the effects of subacute CPF exposures in these same age groups. Repeated doses of CPF (40 mg/kg, SC, every 4 days, total of 4 doses) caused extensive inhibition of cortical, hippocampal, and striatal cholinesterase (ChE) activity in adult rats at 4 (90-92%) and 14 (71-78%) days after the last treatment. Rats treated similarly during postnatal maturation (beginning on day 7) showed a much lower degree of ChE inhibition (21-60%) at these time points. Muscarinic ([3H]quinuclidinyl benzilate, QNB) receptor binding in cortex, hippocampus, and striatum was reduced in adult brain at 4 (30-43%) and 14 (22-32%) days after the final treatment, whereas receptor densities were only marginally affected (5-11% reduction) in young rats. Basal motor activity levels were not affected in either young or adult rats as a function of CPF exposure. CPF-treated adult rats exhibited higher activity levels after challenge with scopolamine (1 mg/kg, IP) at 2, 4, 6, and 8 weeks after treatment, whereas CPF exposure did not affect the motoric response to scopolamine in rats treated during postnatal maturation. These data suggest that although neonatal rats are more sensitive to acute lethal effects from high doses of CPF, adult rats exhibit more persistent neurochemical and neurobehavioral alterations following repeated, lower-level exposures.
Chlorpyrifos-induced alterations in rat brain acetylcholinesterase, lipid peroxidation and ATPases
Indian journal of biochemistry & biophysics, 2005
The effect of chlorpyrifos (O, O'-diethyl-3, 5, 6-trichloro-2-pyridyl phosphorothionate, CPF) exposure on acetylcholinesterase (AChE) activity, lipid peroxidation and different ATPases activities was studied in rats. CPF caused significant inhibition of synaptosomal AChE activity in different regions of brain (fore, mid and hind) and inhibition ranged from 36 to 82% in rats receiving 20-100 mg CPF/kg body wt for 3 days. It also produced oxidative stress, resulting in marked increase in peroxidative damage of membrane lipids in a dose-dependent manner. The levels of malondialdehyde (MDA) and 4-hydroxy-2-nonanal (4-HNE), two major end products of lipid peroxidation were significantly increased in all the regions of brain. Increase in MDA levels was 66%, 117% and 172% in fore brain, 70%, 108% and 170% in mid brain and 40%, 110% and 169% in hind brain of rats given 20, 50 and 100 mg CPF/kg body wt for 3 days. The maximum increase in 4-HNE levels in all the three regions of brain was...