Brain Cholinesterase Inhibition Research Articles

First meta-analysis study of cholinesterase inhibition in experimental animals by organophosphate or carbamate insecticides under the influence of diphenhydramine.

Diphenhydramine is an H1-antihistamine that counteracts the toxic effects of organophosphate and carbamate insecticides that inhibit cholinesterase (ChE) activity. This meta-analysis aimed to investigate the effects of diphenhydramine on ChE inhibition induced by these insecticides in the plasma, erythrocytes, or whole brain of experimental animals. A data search was performed on erythrocyte, plasma, and brain ChE inhibition caused by organophosphate and carbamate insecticides in experimental animals (mice, rats, and chicks) treated with the antihistamine diphenhydramine in accordance with preferred reporting items for systematic reviews and meta-analysis, which was done by the two-group random-effects model meta-analysis. The meta-analysis included 18 records extracted from six studies that, appeared from 1996 to 2022. Using the random-effects model, a two-group meta-analysis revealed that the combined effect size (ChE inhibition) was significantly more favorable in the control group than in the diphenhydramine intervention, as shown by a forest plot. The combined effect size (standardized mean difference) was 0.67, with a standard error of 0.3, a lower limit of 0.04, and an upper limit of 1.29 (p = 0.025). The heterogeneity was moderate, as I2 of the combined effect size was 74%, with a significant Cochrane Q-test result (Q = 65, p < 0.0001). Subgroup analysis indicated that, with brain ChE inhibition, the heterogeneity (I2) became 5%, which was lower than ChE inhibition in plasma (84%) and erythrocytes (78%). No publication bias was identified using the funnel plot and Egger's test. This meta-analysis suggests that, in addition to its documented antidotal action against ChE-inhibiting insecticides, diphenhydramine can also reduce the extent of ChE inhibition, especially in the brain, which is the main site of toxicity of these insecticides. There is a need for additional studies to assess such enzyme inhibition in different parts of the brain.

Assessment of Cholinesterase inhibition activity of birds inhabiting pesticide exposed croplands and protected area in hot semi-arid region of Pakistan.

Acetylcholinesterase (AChE) activity levels can be used as an indicator for AChE inhibition due to pesticide poisoning in bird species. We assessed the comparative brain cholinesterase (AChE) activity level of five bird species inhabiting pesticide exposed croplands and Protected Area i.e. Deva Vatala National Park (DVNP), Bhimber by using a spectrophotometric method. AChE activity levels ranged from 56.3 to 85.9 µmol/min/g of brain tissue of birds representing DVNP. However, AChE activity levels ranged from 27.6 to 79.9 µmol/min/g of brain tissue of birds representing croplands. AChE activity levels observed in Jungle babbler, Common babbler, and Red-vented bulbul showed significant differences (P < 0.05) at two sites. However, White wagtail and Black drongo demonstrated non-significant differences (P > 0.05). Maximum inhibition was recorded in Jungle babbler (53%) followed by Common babbler (35%), Red-vented bulbul (18%), White wagtail (15%), and Black drongo (7%). The brain cholinesterase inhibition levels under-protected ecosystems (DVNP, Bhimber) and agricultural landscape suggest insecticidal contamination and its impact on avifauna diversity. The study also emphasizes on the importance of pesticide-free zones to protect the biodiversity of birds.

Persistent proteomic changes in glutamatergic and GABAergic signaling in the amygdala of adolescent rats exposed to chlorpyrifos as juveniles

Chlorpyrifos (CPF) remains one of the most widely used organophosphorus insecticides (OPs) despite the concerns about its developmental neurotoxicity. Developmental exposure to CPF has long-lasting negative impacts, including abnormal emotional behaviors. These negative impacts are observed at exposure levels do not cause inhibition of acetylcholinesterase, the canonical target of OPs. Exposure to CPF at these levels inhibits the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) but it is not clear what the persistent effects of this inhibition are. To investigate this, male rat pups were exposed orally to either corn oil, 0.75 mg/kg CPF, or 0.02 mg/kg PF-04457845 (PF; a specific inhibitor of FAAH) daily from postnatal day 10 (PND10) - PND16. This dosage of CPF does not inhibit brain cholinesterase activity but inhibits FAAH activity. On PND38 (adolescence), the protein expression in the amygdala was determined using a label-free shotgun proteomic approach. The analysis of control vs CPF and control vs PF led to the identification of 44 and 142 differentially regulated proteins, respectively. Gene ontology enrichment analysis revealed that most of the proteins with altered expression in both CPF and PF treatment groups were localized in the synapse-related regions, such as presynaptic membrane, postsynaptic density, and synaptic vesicle. The different biological processes affected by both treatment groups included persistent synaptic potentiation, glutamate receptor signaling, protein phosphorylation, and chemical synaptic transmission. These results also indicated disturbances in the balance between glutamatergic (↓ Glutamate AMPA receptor 2, ↓ Excitatory amino acid transporter 2, and ↑ vesicular glutamate transporter 2) and GABAergic signaling (↑ GABA transporter 3 and ↑ glutamate decarboxylase 2). This imbalance could play a role in the abnormal emotional behavior that we have previously reported. These results suggest that there is a similar pattern of expression between CPF and PF, and both these chemicals can persistently alter emotional behavior as a consequence of inhibition of FAAH.

Perinatal diazinon exposure compromises the development of acetylcholine and serotonin systems

Organophosphate pesticides are developmental neurotoxicants. We gave diazinon via osmotic minipumps implanted into dams prior to conception, with exposure continued into the second postnatal week, at doses (0.5 or 1 mg/kg/day) that did not produce detectable brain cholinesterase inhibition. We evaluated the impact on acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT) systems in brain regions from adolescence through full adulthood. Diazinon produced deficits in presynaptic ACh activity with regional and sex selectivity: cerebrocortical regions and the hippocampus were affected to a greater extent than were the striatum, midbrain or brainstem, and females were more sensitive than males. Diazinon also reduced nicotinic ACh receptors and 5HT1A receptors, with the same regional and sex preferences. These patterns were similar to those of diazinon given in a much more restricted period (postnatal day 1–4) but were of greater magnitude and consistency; this suggests that the brain is vulnerable to diazinon over a wide developmental window. Diazinon’s effects differed from those of the related organophosphate, chlorpyrifos, with regard to regional and sex selectivity, and more importantly, to the effects on receptors: chlorpyrifos upregulates nicotinic ACh receptors and 5HT receptors, effects that compensate for the presynaptic ACh deficits. Diazinon can thus be expected to have worse neurodevelopmental outcomes than chlorpyrifos. Further, the disparities between diazinon and chlorpyrifos indicate the problems of predicting the developmental neurotoxicity of organophosphates based on a single compound, and emphasize the inadequacy of cholinesterase inhibition as an index of safety.

Efficacy of novel phenoxyalkyl pyridinium oximes as brain-penetrating reactivators of cholinesterase inhibited by surrogates of sarin and VX

Pyridinium oximes are strong nucleophiles and many are effective reactivators of organophosphate-inhibited cholinesterase (ChE). However, the current oxime reactivators are ineffective at crossing the blood-brain barrier and reactivating brain ChE in the intact organism. Our laboratories have developed a series of substituted phenoxyalkyl pyridinium oximes (US patent 9,227,937 B2) with the goal of identifying reactivators effective in crossing the blood-brain barrier. The first 35 of the series were found to have similar in vitro efficacy as reactivators of ChE inhibited by a sarin surrogate (phthalimidyl isopropyl methylphosphonate, PIMP) or a VX surrogate (nitrophenyl ethyl methylphosphonate, NEMP) in bovine brain preparations as previously observed in rat brain preparations. A number of these novel oximes have shown the ability to decrease the level of ChE inhibition in the brains of rats treated with a high sublethal dosage of either a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the VX surrogate NEMP. Levels of reactivation at 2 h after oxime administration were up to 35% while the currently approved therapeutic, 2-PAM, yielded no reduction in brain ChE inhibition. In addition, there was evidence of attenuation of seizure-like behavior with several of the more effective novel oximes, but not 2-PAM. Therefore these novel oximes have demonstrated an ability to reactivate inhibited ChE in brain preparations from two species and in vivo data support their ability to enter the brain and provide a therapeutic action. These novel oximes have the potential to be developed into improved antidotes for nerve agent therapy.

Novel substituted phenoxyalkyl pyridinium oximes enhance survival and attenuate seizure-like behavior of rats receiving lethal levels of nerve agent surrogates.

Novel substituted phenoxyalkyl pyridinium oximes, previously shown to reactivate brain cholinesterase in rats treated with high sublethal dosages of surrogates of sarin and VX, were tested for their ability to prevent mortality from lethal doses of these two surrogates. Rats were treated subcutaneously with 0.6mg/kg nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate) or 0.65mg/kg nitrophenyl ethyl methylphosphonate (NEMP; VX surrogate), dosages that were lethal within 24h to all tested rats when they received only 0.65mg/kg atropine at the time of initiation of seizure-like behavior (about 30min). If 146mmol/kg 2-PAM (human equivalent dosage) was also administered, 40% and 33% survival was obtained with NIMP and NEMP, respectively, while the novel Oximes 1 and 20 provided 65% and 55% survival for NIMP and 75 and 65% for NEMP, respectively. In addition, both novel oximes resulted in a highly significant decrease in time to cessation of seizure-like behavior compared to 2-PAM during the first 8h of observation. Brain cholinesterase inhibition was slightly less in novel oxime treated rats compared to 2-PAM in the 24h survivors. The lethality data indicate that 24h survival is improved by two of the novel oximes compared to 2-PAM. The cessation of seizure-like behavior data strongly suggest that these novel oximes are able to penetrate the blood-brain barrier and can combat the hypercholinergic activity that results in seizures. Therefore this oxime platform has exceptional promise as therapy that could both prevent nerve agent-induced lethality and attenuate nerve agent-induced seizures.

Decreased anxiety in juvenile rats following exposure to low levels of chlorpyrifos during development

Exposure to chlorpyrifos (CPF) during the late preweanling period in rats inhibits the endocannabinoid metabolizing enzymes fatty acid hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resulting in accumulation of their respective substrates anandamide (AEA) and 2-arachidonylglycerol (2-AG). This occurs at 1.0mg/kg, but at a lower dosage (0.5mg/kg) only FAAH and AEA are affected with no measurable inhibition of either cholinesterase (ChE) or MAGL. The endocannabinoid system plays a vital role in nervous system development and may be an important developmental target for CPF. The endocannabinoid system plays an important role in the regulation of anxiety and, at higher dosages, developmental exposure to CPF alters anxiety-like behavior. However, it is not clear whether exposure to low dosages of CPF that do not inhibit ChE will cause any persistent effects on anxiety-like behavior. To determine if this occurs, 10-day old rat pups were exposed daily for 7 days to either corn oil or 0.5, 0.75, or 1.0mg/kg CPF by oral gavage. At 12h following the last CPF administration, 1.0mg/kg resulted in significant inhibition of FAAH, MAGL, and ChE, whereas 0.5 and 0.75mg/kg resulted in significant inhibition of only FAAH. AEA levels were significantly elevated in all three treatment groups as were palmitoylethanolamide and oleoylethanolamide, which are also substrates for FAAH. 2-AG levels were significantly elevated by 0.75 and 1.0mg/kg but not 0.5mg/kg. On day 25, the latency to emerge from a dark container into a highly illuminated novel open field was measured as an indicator of anxiety. All three CPF treatment groups spent significantly less time in the dark container prior to emerging as compared to the control group, suggesting a decreased level of anxiety. This demonstrates that repeated preweanling exposure to dosages of CPF that do not inhibit brain ChE can induce a decline in the level of anxiety that is detectable during the early postweanling period.

The effects of novel 7-MEOTA-donepezil like hybrids and N-alkylated tacrine analogues in the treatment of quinuclidinyl benzilate-induced behavioural deficits in rats performing the multiple T-maze test.

The number of approved drugs for the clinical treatment of Alzheimer disease remains limited. For this reason, there is extensive search for novel therapies. Of these, cholinesterase inhibitors have some proven benefit in slowing the disease progression and still remain the first-line therapeutic approach. In this study, the pro-cognitive effect of four novel tacrine-related inhibitors was evaluated and compared with the standards, tacrine and donepezil. Wistar rats trained to perform the multiple T-maze were treated intra-peritoneally with the anticholinergic agent 3-quinuclidinyl benzilate (QNB, 2.0 mg/kg), followed 30 min later by another injection containing a therapeutic dose of standard or novel cholinesterase inhibitor. The rats were repeatedly subjected to the multiple T-maze task at several time points following QNB administration (1, 24, 48 and 72 h). The passage time and number of errors were recorded. The inhibitory potential of selected therapeutic doses was assessed in a separate in vivo experiment using a spectrophotometric method. QNB significantly impaired the performance of the rats within 48 h. The four novel cholinesterase inhibitors attenuated the effect of QNB at 1 h, 24 h and 48 h test intervals. The novel compounds resulted in brain cholinesterase inhibition ranging from 5.4 to 11.3 %, and their effect on the QNB-induced deficit recorded in the T-maze performance was comparable to that of the standards or higher at some time points. The best result was achieved with derivative 4, followed by derivatives 2 and 3, suggesting that these compounds could be candidates for the treatment of Alzheimer disease.

Prenatal nicotine changes the response to postnatal chlorpyrifos: Interactions targeting serotonergic synaptic function and cognition.

Nicotine and chlorpyrifos are developmental neurotoxicants that target serotonin systems. We examined whether prenatal nicotine exposure alters the subsequent response to chlorpyrifos given postnatally. Pregnant rats received nicotine throughout gestation at 3mg/kg/day, a regimen designed to achieve plasma levels seen in smokers; chlorpyrifos was given to pups on postnatal days (PN) 1-4 at 1mg/kg, just above the detection threshold for brain cholinesterase inhibition. We assessed long-term effects from adolescence (PN30) through full adulthood (PN150), measuring the expression of serotonin receptors and serotonin turnover (index of presynaptic impulse activity) in cerebrocortical brain regions encompassing the projections that are known targets for nicotine and chlorpyrifos. Nicotine or chlorpyrifos individually increased the expression of serotonin receptors, with greater effects on males than on females and with distinct temporal and regional patterns indicative of adaptive synaptic changes rather than simply an extension of initial injury. This interpretation was confirmed by our finding an increase in serotonin turnover, connoting presynaptic serotonergic hyperactivity. Animals receiving the combined treatment showed a reduction in these adaptive effects on receptor binding and turnover relative to the individual agents, or even an effect in the opposite direction; further, normal sex differences in serotonin receptor concentrations were dissipated or reversed, an effect that was confirmed by behavioral evaluations in the Novel Objection Recognition Test. In addition to the known liabilities associated with maternal smoking during pregnancy, our results point to additional costs in the form of heightened vulnerability to neurotoxic chemicals encountered later in life.

In vitro inhibition of plasma and brain cholinesterases of growing chicks by chlorpyrifos and dichlorvos

This study assessed in vitro inhibition of plasma and brain cholinesterases (ChE) by the organophosphate insecticides chlorpyrifos and dichlorvos in chicks at ages of 2, 10, 20 and 30 days. Plasma samples and whole-brain homogenates of each age group were pooled and ChE activity was determined by an electrometric method with or without the organophosphate (0.5 and 1.0 µM). ChE inhibitions were the highest in 2- and 10-day-old chicks (20–83%), followed by 5–73% inhibitions at ages of 20 and 30 days. Cholrpyrifos decreased plasma ChE activity only at the ages of 2 and 10 days by 21–75%. Dichlorvos decreased plasma ChE activity at all the ages tested (2–30 days) by 39–83%. The highest percentage of plasma ChE inhibition by chlorpyrifos and dichlorvos was in 2-day-old chicks. In the brain homogenate, chlorpyrifos decreased the ChE activity between ages 2 and 20 days by 20–62%. The highest percentage of brain ChE inhibition occurred at the age of 10 days and the least was at 20 and 30 days. Dichlorvos decreased brain ChE activity between ages 2 and 20 days by 24–74%. At the age of 30 days, only the higher concentration of dichlorvos (1 µM) decreased brain ChE activity by 27%. The data suggest an age-related differential in vitro inhibition of plasma and brain ChE s in chicks. In vitro ChE inhibition, though it does not exclude interfering factors, could be useful for initial screening of the inhibitory actions of organophosphate insecticides in chickens.

Low level chlorpyrifos exposure increases anandamide accumulation in juvenile rat brain in the absence of brain cholinesterase inhibition

The prevailing dogma is that chlorpyrifos (CPF) mediates its toxicity through inhibition of cholinesterase (ChE). However, in recent years, the toxicological effects of developmental CPF exposure have been attributed to an unknown non-cholinergic mechanism of action. We hypothesize that the endocannabinoid system may be an important target because of its vital role in nervous system development. We have previously reported that repeated exposure to CPF results in greater inhibition of fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide (AEA), than inhibition of either forebrain ChE or monoacylglycerol lipase (MAGL), the enzyme that metabolizes the endocannabinoid 2-arachidonylglycerol (2-AG). This exposure resulted in the accumulation of 2-AG and AEA in the forebrain of juvenile rats; however, even at the lowest dosage level used (1.0mg/kg), forebrain ChE inhibition was still present. Thus, it is not clear if FAAH activity would be inhibited at dosage levels that do not inhibit ChE. To determine this, 10 day old rat pups were exposed daily for 7 days to either corn oil or 0.5mg/kg CPF by oral gavage. At 4 and 12h post-exposure on the last day of administration, the activities of serum ChE and carboxylesterase (CES) and forebrain ChE, MAGL, and FAAH were determined as well as the forebrain AEA and 2-AG levels. Significant inhibition of serum ChE and CES was present at both 4 and 12h. There was no significant inhibition of the activities of forebrain ChE or MAGL and no significant change in the amount of 2-AG at either time point. On the other hand, while no statistically significant effects were observed at 4h, FAAH activity was significantly inhibited at 12h resulting in a significant accumulation of AEA. Although it is not clear if this level of accumulation impacts brain maturation, this study demonstrates that developmental CPF exposure at a level that does not inhibit brain ChE can alter components of endocannabinoid signaling.

Carbaryl and 1-Naphthol Tissue Levels and Related Cholinesterase Inhibition in Male Brown Norway Rats from Preweaning to Senescence

Studies incorporating both toxicokinetic and dynamic factors provide insight into chemical sensitivity differences across the life span. Tissue (brain, plasma, liver) levels of the N-methyl carbamate carbaryl, and its metabolite 1-naphthol, were determined and related to brain and RBC cholinesterase (ChE) inhibition in the same animals. Dose-response (3, 7.5, 15, or 22.5 mg/kg, 40–45 min postdosing) and time course (3 or 15 mg/kg at 30, 60, 120, or 240 min postdosing) of acute effects of carbaryl (oral gavage) in preweanling (postnatal day [PND] 18) and adult male Brown Norway rats from adolescence to senescence (1, 4, 12, 24 mo) were compared. At all ages there were dose-related increases in carbaryl and 1-naphthol in the dose-response study, and the time-course study showed highest carbaryl levels at 30 min postdosing. There were, however, age-related differences in that the 1- and 4-mo rats showed the lowest levels of carbaryl and 1-naphthol, and PND18 and 24-mo rats had similar, higher levels. The fastest clearance (shortest half-lives) was observed in 1- and 4-mo rats. Carbaryl levels were generally higher than 1-naphthol in brain and plasma, but in liver, 1-naphthol levels were similar to or greater than carbaryl. Brain ChE inhibition closely tracked brain carbaryl concentrations regardless of the time after dosing, but there was more variability in the relationship between RBC ChE and plasma carbaryl levels. Within-subject analyses suggested somewhat more brain ChE inhibition at lower carbaryl levels only in the PND18 rats. These findings may reflect maturation followed by decline in kinetic factors over the life span.

Dose-dependent Effects of Ladostigil on Microglial Activation and Cognition in Aged Rats

The current study determined the effects of chronic treatment of aging rats with ladostigil, a cholinesterase (ChE) and monoamine oxidase (MAO) inhibitor, at doses of 1 and 8.5mg/kg/day, on novel object recognition (NOR) and reference memory in the Morris water maze (MWM). A dose of (1mg/kg/day) did not inhibit ChE or MAO but prevented the loss of NOR and reference memory in the MWM that occurs at 20.5months of age. This anti-aging effect was associated with a reduction in the expression of CD11b, a marker of microglial activation, in the fornix and parietal cortex and restoration of microglial morphology to that in young adult rats. Ladostigil (8.5mg/kg/day) inhibited brain ChE by ≈30% and MAO A and B by 55-59%, and had a similar, or greater effect than the low dose on microglia, but was less effective in preventing the decline in NOR. Ladostigil (8.5mg/kg/day) may have caused too much cortical ChE inhibition and acetylcholine elevation at 16months when NOR was intact. In support of this suggestion we showed that acute administration of ladostigil (8.5mg/kg) worsened NOR at this age. However, at 20months, when NOR was impaired and brain acetylcholine levels are 40% below normal, ladostigil (8.5mg/kg) reversed the memory deficit. Ladostigil (1mg/kg/day) prevents the development of age-related memory deficits by a combination of immunomodulatory and antioxidant effects. A dose causing 30% ChE inhibition is necessary in order to reverse existing memory deficits at 20months of age.

Oxidative stress and cholinesterase inhibition in plasma, erythrocyte and brain of rats’ pups following lactational exposure to malathion

The organophosphorus (OP) pesticide malathion is a highly neurotoxic compound. Some studies have reported neurotoxicity signs after in utero exposure to OP pesticides. However there is no evidence of the exclusive contribution of the lactational exposure to malathion as a possible cause of neurotoxicity in rats’ pups. In this respect, we investigated the exclusive contribution of malathion (200mg/kg, b.w.) exposure through maternal milk in rat pups during lactation. We evaluated the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), as well as on biochemical parameters related to the oxidative stress such lipoperoxidation and antioxidant enzyme activities as superoxide dismutase (SOD) and catalase (CAT) in the brain, plasma and erythrocytes of rats’ pups at 21st postnatal day (Pnd). These parameters were also evaluated in the same tissues but at 51 Pnd. Our results showed that the malathion exposure during lactation induced a high inhibitory effect of the brain, plasma and erythrocyte AChE and BChE activities in rat pups. Many changes were observed in the biochemical parameters related to the oxidative stress for pups brain, plasma and erythrocyte. The present study shows, for the first time, that the exposure of postnatal pups to malathion via lactation inhibits the activity of brain, plasma and erythrocytes cholinesterase in the pups. These findings suggest that malathion exposure during lactation induced a cerebral alterations and oxidative stress in rat pups.

Impact of Chemical Proportions on the Acute Neurotoxicity of a Mixture of Seven Carbamates in Preweanling and Adult Rats

Statistical design and environmental relevance are important aspects of studies of chemical mixtures, such as pesticides. We used a dose-additivity model to test experimentally the default assumptions of dose additivity for two mixtures of seven N-methylcarbamates (carbaryl, carbofuran, formetanate, methomyl, methiocarb, oxamyl, and propoxur). The best-fitting models were selected for the single-chemical dose-response data and used to develop a combined prediction model, which was then compared with the experimental mixture data. We evaluated behavioral (motor activity) and cholinesterase (ChE)-inhibitory (brain, red blood cells) outcomes at the time of peak acute effects following oral gavage in adult and preweanling (17 days old) Long-Evans male rats. The mixtures varied only in their mixing ratios. In the relative potency mixture, proportions of each carbamate were set at equitoxic component doses. A California environmental mixture was based on the 2005 sales of each carbamate in California. In adult rats, the relative potency mixture showed dose additivity for red blood cell ChE and motor activity, and brain ChE inhibition showed a modest greater-than additive (synergistic) response, but only at a middle dose. In rat pups, the relative potency mixture was either dose-additive (brain ChE inhibition, motor activity) or slightly less-than additive (red blood cell ChE inhibition). On the other hand, at both ages, the environmental mixture showed greater-than additive responses on all three endpoints, with significant deviations from predicted at most to all doses tested. Thus, we observed different interactive properties for different mixing ratios of these chemicals. These approaches for studying pesticide mixtures can improve evaluations of potential toxicity under varying experimental conditions that may mimic human exposures.

Cholinesterase inhibition and toxicokinetics in immature and adult rats after acute or repeated exposures to chlorpyrifos or chlorpyrifos–oxon

The effect of age or dose regimen on cholinesterase inhibition (ChEI) from chlorpyrifos (CPF) or CPF–oxon (CPFO) was studied in Crl:CD(SD) rats. Rats were exposed to CPF by gavage in corn oil, rat milk (pups), or in the diet (adults) or to CPFO by gavage in corn oil. Blood CPF/CPFO levels were measured. With acute exposure, ChEI NOELs were 2mg/kg CPF for brain and 0.5mg/kg CPF for red blood cells (RBCs) in both age groups. In pups, ChEI and blood CPF levels were similar using either milk or corn oil vehicles. Compared to gavage, adults given dietary CPF (12h exposure) had greater RBC ChEI, but lower brain ChEI at corresponding CPF doses, indicating an effect of dose rate. With repeated CPF exposures, ChEI NOELs were the same across ages (0.5 and 0.1mg/kg/day for brain and RBCs, respectively). With CPFO dosing, the ChEI NOELs were 0.1mg/kg (acute) and 0.01mg/kg/day (repeated doses) for RBCs with no ChEI in brain at CPFO doses up to 0.5 (pup) or 10mg/kg (adult) for acute dosing or 0.5mg/kg/day for both ages with repeat dosing. Thus, there were no age-dependent differences in CPF ChEI via acute or repeated exposures. Pups had less ChEI than adults at comparable blood CPF levels. Oral CPFO resulted in substantial RBC ChEI, but no brain ChEI, indicating no CPFO systemic bioavailability to peripheral tissues.

Acetylcholinesterase inhibition dose–response modeling for chlorpyrifos and chlorpyrifos-oxon

This paper evaluates new data for cholinesterase inhibition with chlorpyrifos (CPF). Marty et al. (2012) recently conducted a CPF cholinesterase inhibition study in rats that included testing of males and females, dosing by gavage or diet, administration in corn oil or milk, and with pups and adults. Additionally, the study included cholinesterase inhibition testing for CPF-oxon, the active moiety that inhibits cholinesterase. The study included 5–6 dose groups with eight animals/sex/group for most of the tests. This paper provides a benchmark dose (BMD) analysis of the data from Marty et al. (2012), including a BMD meta-analysis that includes CPF cholinesterase inhibition data from different assays within the Marty et al. (2012) study and, in one case, from another study. From the meta-analysis, the recommended BMD10s, based on brain acetylcholinesterase inhibition, are 1.7mg/kg/day (BMDL10=1.3mg/kg/day) for acute doses to children and adults, and 0.67mg/kg/day (BMDL10=0.53mg/kg/day) for repeat doses to children and adults. At the dose levels considered in this analysis, there was no evidence of a difference in responses between males and females, corn oil versus milk administration, or pups versus adults. The data on pups versus adults show that an extra safety factor to protect the young is not needed for CPF. CPF data from the literature suggest that brain cholinesterase inhibition is the most appropriate metric for cholinesterase inhibition risk assessment.

Tacrine and its analogues impair mitochondrial function and bioenergetics: a lipidomic analysis in rat brain

Tacrine is an acetylcholinesterase (AChE) inhibitor used as a cognitive enhancer in the treatment of Alzheimer's disease (AD). However, its low therapeutic efficiency and a high incidence of side effects have limited its clinical use. In this study, the molecular mechanisms underlying the impact on brain activity of tacrine and two novel tacrine analogues (T1, T2) were approached by focusing on three aspects: (i) their effects on brain cholinesterase activity; (ii) perturbations on electron transport chain enzymes activities of non-synaptic brain mitochondria; and (iii) the role of mitochondrial lipidome changes induced by these compounds on mitochondrial bioenergetics. Brain effects were evaluated 18 h after the administration of a single dose (75.6 μmol/kg) of tacrine or tacrine analogues. The three compounds promoted a significant reduction in brain AChE and butyrylcholinesterase (BuChE) activities. Additionally, tacrine was shown to be more efficient in brain AChE inhibition than T2 tacrine analogue and less active than T1 tacrine analogue, whereas BuChE inhibition followed the order: T1 > T2 > tacrine. The studies using non-synaptic brain mitochondria show that all the compounds studied disturbed brain mitochondrial bioenergetics mainly via the inhibition of complex I activity. Furthermore, the activity of complex IV is also affected by tacrine and T1 treatments while FoF(1) -ATPase is only affected by tacrine. Therefore, the compounds' toxicity as regards brain mitochondria, which follows the order: tacrine >> T1 > T2, does not correlate with their ability to inhibit brain cholinesterase enzymes. Lipidomics approaches show that phosphatidylethanolamine (PE) is the most abundant phospholipids (PL) class in non-synaptic brain mitochondria and cardiolipin (CL) present the greatest diversity of molecular species. Tacrine induced significant perturbations in the mitochondrial PL profile, which were detected by means of changes in the relative abundance of phosphatidylcholine (PC), PE, phosphatidylinositol (PI) and CL and by the presence of oxidized phosphatidylserines. Additionally, in both the T1 and T2 groups, the lipid content and molecular composition of brain mitochondria PL are perturbed to a lesser extent than in the tacrine group. Abnormalities in CL content and the amount of oxidized phosphatidylserines were associated with significant reductions in mitochondrial enzymes activities, mainly complex I. These results indicate that tacrine and its analogues impair mitochondrial function and bioenergetics, thus compromising the activity of brain cells.

Evaluation of the effect of Ferula asafoetida Linn. gum extract on learning and memory in Wistar rats.

Objective:Memory loss is universal and is the first symptom to manifest in majority of the patients suffering from Alzheimer's disease. This study is designed to investigate the effect of Ferula asafoetida linn. (F. foetida) extract on learning and memory in rats.Materials and Methods:Learning and memory were evaluated using elevated plus maze and passive avoidance paradigm after the oral administration of two doses (200 mg/kg and 400 mg/kg) of F. foetida aqueous extract with rivastigmine as positive control. Brain cholinesterase activity, serum thiols and cholesterol were also estimated.Results:Extract produced significant improvement in memory score i.e. step through latency at 400 mg/kg dose in passive avoidance model (P< 0.05) and dose-dependent improvement of transfer latency in elevated plus maze model (P< 0.001). Dose-dependent inhibition of brain cholinesterase (P< 0.001) and significant improvement in antioxidant levels (P< 0.05) were also noted.Conclusions:Memory enhancing potential of F. foetida can be attributed to acetylcholinesterase inhibiting and antioxidant properties. Hence, dietary usage of F. foetida is beneficial and can also be employed as an adjuvant to existing anti-dementia therapies.

213 HDL-ASSOCIATED PARAOXONASE 1 (PON1) HYDROLYZING ACTIVITY AGAINST MACROPHAGE OXIDIZED LIPIDS PROTECTS AGAINST MACROPHAGE FOAM CELL FORMATION AND ATHEROGENESIS

Individual differences in detoxication capacities for specific organophosphorous (OP) compounds are due largely to differences in catalytic efficiency or abundance of the HDL-associated enzyme, paraoxonase (PON1). First, we provide evidence that children less than 2 years of age represent a particularly susceptible population for OP exposure due to low abundance of PON1 and variable onset of plasma PON1 activity. Second, we describe studies examining the neurotoxic effects of chronic, low-level OP pesticide exposure in mice. PON1 knockout (PON1−/−) and wild-type mice were exposed chronically (PN4 to PN21) to low levels of chlorpyrifos oxon (CPO). Endpoints included cholinesterase activity, histopathology, gene expression, and behavior. Even at PN4, when PON1 levels were low in wild-type mice, PON1−/− mice were more sensitive to inhibition of brain cholinesterase by CPO. At PN22, and persisting as long as 4 months, chronic developmental exposure to 0.18 mg/kg/d or 0.25 mg/kg/d CPO resulted in perinuclear vacuolization of cells in a discrete area of the neocortex and irregular distribution of neurons in the cortical plate, with an increase in the number of affected cells at 0.25 mg/kg/d. Third, we describe a transgenic mouse model in which human transgenes encoding either hPON1Q192 or hPON1R192 were expressed at equal levels in place of mouse PON1. The developmental onset of expression followed the mouse time course and was identical for the two transgenes, allowing these mice to be used to assess the importance of the Q192R polymorphism during development. Adult mice expressing hPON1R192 were significantly more resistant than hPON1Q192 mice to CPO toxicity. Our studies indicate that children less than 2 years old, especially those homozygous for PON1Q192, would be predicted to be particularly susceptible to CPO toxicity.