Short-term behavioral and histological findings following a single concussive and repeated subconcussive brain injury in a rodent model.

PRIMARY OBJECTIVE: It is unclear of the correlation between a mild traumatic brain injury (mTBI) and repeated subconcussive (RSC) impacts with respect to injury biomechanics. Thus, the present study was designed to determine the behavioral and histological differences between a single mTBI impact and RSC impacts with subdivided cumulative kinetic energies of the single mTBI impact. RESEARCH DESIGN: Adult male Sprague-Dawley rats were randomly assigned to a single mTBI impact, RSC impact, sham, or repeated sham groups. METHODS AND PROCEDURES: Following a weight drop injury, anxiety-like behavior and general locomotive activity and were assessed using the open field test, while motor coordination was evaluated using a rotarod unit. Neuronal loss, astrogliosis, and microgliosis were assessed using NeuN, GFAP and Iba-1 immunohistochemistry. All assessments were undertaken at 3- and 7-days post impact. MAIN OUTCOMES AND RESULTS: No behavioral disturbances were observed in injury groups, however, both injury groups did lead to microgliosis following 3-days post-impact. CONCLUSIONS: No pathophysiological differences were observed between a single mTBI impact and RSC impacts of the same energy input. Even though a cumulative injury threshold for RSC impacts was not determined, a threshold still may exist where no pathodynamic shift occurs.

Effects of Chlorpyrifos on Serine Hydrolase Activities, Lipid Mediators, and Immune Responses in Lungs of Neonatal and Adult Mice.

Chlorpyrifos (CPF) is an organophosphate (OP) pesticide that causes acute toxicity by inhibiting acetylcholinesterase (AChE) in the nervous system. However, endocannabinoid (eCB) metabolizing enzymes in brain of neonatal rats are more sensitive than AChE to inhibition by CPF, leading to increased levels of eCBs. Because eCBs are immunomodulatory molecules, we investigated the association between eCB metabolism, lipid mediators, and immune function in adult and neonatal mice exposed to CPF. We focused on lung effects because epidemiologic studies have linked pesticide exposures to respiratory diseases. CPF was hypothesized to disrupt lung eCB metabolism and alter lung immune responses to lipopolysaccharide (LPS), and these effects would be more pronounced in neonatal mice due to an immature immune system. We first assessed the biochemical effects of CPF in adult mice (≥8 weeks old) and neonatal mice after administering CPF (2.5 mg/kg, oral) or vehicle for 7 days. Tissues were harvested 4 h after the last CPF treatment and lung microsomes from both age groups demonstrated CPF-dependent inhibition of carboxylesterases (Ces), a family of xenobiotic and lipid metabolizing enzymes, whereas AChE activity was inhibited in adult lungs only. Activity-based protein profiling (ABPP)-mass spectrometry of lung microsomes identified 31 and 32 individual serine hydrolases in neonatal lung and adult lung, respectively. Of these, Ces1c/Ces1d/Ces1b isoforms were partially inactivated by CPF in neonatal lung, whereas Ces1c/Ces1b and Ces1c/BChE were partially inactivated in adult female and male lungs, respectively, suggesting age- and sex-related differences in their sensitivity to CPF. Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) activities in lung were unaffected by CPF. When LPS (1.25 mg/kg, i.p.) was administered following the 7-day CPF dosing period, little to no differences in lung immune responses (cytokines and immunophenotyping) were noted between the CPF and vehicle groups. However, a CPF-dependent increase in the amounts of dendritic cells and certain lipid mediators in female lung following LPS challenge was observed. Experiments in neonatal and adult Ces1d-/- mice yielded similar results as wild type mice (WT) following CPF treatment, except that CPF augmented LPS-induced Tnfa mRNA in adult Ces1d-/- mouse lungs. This effect was associated with decreased expression of Ces1c mRNA in Ces1d-/- mice versus WT mice in the setting of LPS exposure. We conclude that CPF exposure inactivates several Ces isoforms in mouse lung and, during an inflammatory response, increases certain lipid mediators in a female-dependent manner. However, it did not cause widespread altered lung immune effects in response to an LPS challenge.

Inhibition of Endocannabinoid-Metabolizing Enzymes in Peripheral Tissues Following Developmental Chlorpyrifos Exposure in Rats.

Repeated developmental exposure to the organophosphate (OP) insecticide chlorpyrifos (CPF) inhibits brain fatty acid amide hydrolase (FAAH) activity at low levels, whereas at higher levels, it inhibits brain monoacylglycerol lipase (MAGL) activity. FAAH and MAGL hydrolyze the endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG), respectively. Peripherally, AEA and 2-AG have physiological roles in the regulation of lipid metabolism and immune function, and altering the normal levels of these lipid mediators can negatively affect these processes. Exposure to CPF alters brain endocannabinoid hydrolysis activity, but it is unclear whether low-level exposure alters this activity in peripheral tissues important in metabolic and immune function. Therefore, rat pups were exposed orally from day 10 to 16 to 0.5, 0.75, or 1.0 mg/kg CPF or 0.02 mg/kg PF-04457845 (a specific FAAH inhibitor). At 12 hours postexposure, FAAH, MAGL, and cholinesterase (ChE) activities were determined. All treatments inhibited FAAH activity in brain, spleen, and liver. CPF inhibited ChE activity in spleen and liver (all dosages) and in brain (highest dosage only). CPF inhibited total 2-AG hydrolysis and MAGL-specific activity in brain and spleen (high dosage only). In liver, total 2-AG hydrolysis was inhibited by all treatments and could be attributed to inhibition of non-MAGL-mediated 2-AG hydrolysis, indicating involvement of other enzymes. MAGL-specific activity in liver was inhibited only by the high CPF dosage, whereas PF-04457845 slightly increased this activity. Overall, exposure to low levels of CPF and to PF-04457845 can alter endocannabinoid metabolism in peripheral tissues, thus potentially affecting physiological processes.

Cnr1-/- has minimal impact on chlorpyrifos-mediated effects in the mouse endocannabinoid system, but it does alter lipopolysaccharide-induced cytokine levels in splenocytes.

Chlorpyrifos (CPF) is an organophosphate pesticide that can inhibit endocannabinoid (eCB) metabolizing enzymes in animal models at levels that do not significantly alter acetylcholinesterase (AChE) in the central nervous system (CNS). Previous studies indicated that repeated low-level CPF exposure in developing rats increased the levels of eCBs in the brain. Because eCBs play a role in immune homeostasis through their engagement with cannabinoid receptors, we investigated the role of cannabinoid receptor 1 (CB1, encoded by the Cnr1 gene) on the CPF-mediated effects in the spleen and lung of neonatal and adult female mice. We treated neonatal and adult female Cnr1-/- mice with 2.5 mg/kg oral CPF or vehicle for 7 days. Tissues were harvested 4 h after the last CPF dose to evaluate eCB metabolic enzyme activity, levels of eCBs, and tissue immunophenotype. There were a small number of genotype-dependent alterations noted in the endpoints following CPF treatment that were specific to age and tissue type, and differences in eCB metabolism caused by CPF treatment did not correlate to changes in eCB levels. To explore the role of CB1 in CPF-mediated effects on immune endpoints, in vitro experiments were performed with WT murine splenocytes exposed to chlorpyrifos oxon (CPO; oxon metabolite of CPF) and challenged with lipopolysaccharide (LPS). While CPO did not alter LPS-induced pro-inflammatory cytokine levels, inactivation of CB1 by the antagonist SR141716A augmented LPS-induced IFN-γ levels. Additional experiments with WT and Cnr1-/- murine splenocytes confirmed a role for CB1 in altering the production of LPS-induced pro-inflammatory cytokine levels. We conclude that CPF-mediated effects on the eCB system are not strongly dependent on CB1, although abrogation of CB1 does alter LPS-induced cytokine levels in splenocytes.

Inhibition of Serum Esterases in Juvenile Rats Repeatedly Exposed to Low Levels of Chlorpyrifos.

Chlorpyrifos (CPF) is an organophosphorus insecticide that has gained significant attention cue to the reported toxicity associated with developmental exposure. While the canonical mechanism of toxicity of CPF involves the inhibition of brain acetylcholinesterase (AChE), we have reported that exposure of juvenile rats to levels of CPF that do not yield any inhibition of brain AChE results in neurobehavioral alterations at later ages. However, it is unclear what effect exposure to these low levels of CPF has on blood esterase activities which are frequently used not only as biomarkers of exposure but also to set exposure levels in risk assessment. To determine this, male and female rat pups were exposed orally from postnatal day 10 to 16 to either corn oil (vehicle) or 0.5, 0.75, or 1.0 mg/kg CPF. At 12 h after the final exposure, serum cholinesterase (ChE), butyrylcholinesterase (BChE), and carboxylesterase (CES), and red blood cell (RBC) and brain AChE activities were determined. There were no differences between sexes in either the controls or individual treatments for all enzymes. Only the highest dosage of 1.0 mg/kg CPF yielded significant brain AChE inhibition (22-24%) but all dosages significantly inhibited the blood esterases with inhibition being highest with serum CES (65-85%) followed by serum BChE (57-76%), RBC AChE (35-65%), and then serum ChE (16-32%). Our data verify that blood esterases are inhibited at dosages of CPF that alter neurobehavioral performance in the absence of effects on brain AChE activity.

The mechanistic basis for the toxicity difference between juvenile rats and mice following exposure to the agricultural insecticide chlorpyrifos.

At high exposure levels, organophosphorus insecticides (OPs) exert their toxicity in mammals through the inhibition of brain acetylcholinesterase (AChE) leading to the accumulation of acetylcholine in cholinergic synapses and hyperactivity of the nervous system. Currently, there is a concern that low-level exposure to OPs induces negative impacts in developing children and the chemical most linked to these issues is chlorpyrifos (CPF). Our laboratory has observed that a difference in the susceptibility to repeated exposure to CPF exists between juvenile mice and rats with respect to the inhibition of brain AChE. The basis for this difference is unknown but differences in the levels of the detoxification mechanisms could play a role. To investigate this, 10-day old rat and mice pups were exposed daily for 7 days to either corn oil or a range of dosages of CPF via oral gavage. Four hours following the last administration of CPF on day 16, brain, blood, and liver were collected. The inhibition of brain AChE activity was higher in juvenile rats as compared to juvenile mice. The levels of activity of the detoxification enzymes and the impact of CPF exposure on their activity were determined in the two species at this age. In blood and liver, the enzyme paraoxonase-1 (PON1) hydrolyzes the active metabolite of CPF (CPF-oxon), and the enzymes carboxylesterase (CES) and cholinesterase (ChE) act as alternative binding sites for CPF-oxon removing it from circulation and providing protection. Both species had similar levels of PON1 activity in the liver and serum. Mice had higher ChE activity in liver and serum than rats but, following CPF exposure, the percentage inhibition was similar between species at an equivalent dosage. Even though rats had slightly higher liver CES activity than mice, the level of inhibition following exposure was higher in rats. In serum, juvenile mice had an 8-fold higher CES activity than rats, and exposure to a CPF dosage that almost eliminated CES activity in rats only resulted in 22% inhibition in mice suggesting that the high serum CES activity in mice as compared to rats is a key component in this species difference. In addition, there was a species difference in the sensitivity of CES to inhibition by CPF-oxon with rats having a lower IC50 in both liver and serum as compared to mice. This greater enzyme sensitivity suggests that saturation of CES would occur more rapidly in juvenile rats than in mice, resulting in more CPF reaching the brain to inhibit AChE in rats.

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.

In vitro age-related differences in rats to organophosphates.

The mechanism of toxic action for organophosphates (OPs) is the persistent inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and subsequent hyperstimulation of the nervous system. Organophosphates display a wide range of acute toxicities. Differences in the OP's chemistries results in differences in the compound's metabolism and toxicity. Acute toxicities of OPs appear to be principally dependent on compound specific efficiencies of detoxication, and less dependent upon efficiencies of bioactivation and sensitivity of AChE. Serine esterases, such as carboxylesterase (CaE) and butyrylcholinesterase (BChE), play a prominent role in OP detoxication. Organophosphates can stoichiometrically inhibit these enzymes, removing OPs from circulation thus providing protection for the target enzyme, AChE. This in vitro study investigated age-related sensitivity of AChE, BChE and CaE to twelve structurally different OPs in rat tissues. Sensitivity of esterases to these OPs was assessed by inhibitory concentration 50s (IC50s). The OPs displayed a wide range of inhibitory potency toward AChE with IC50s in the low nM-µM range with no differences among ages; however, the CaE IC50s generally increased with age reflecting greater protection in adults. These results suggest age-related differences in acute toxicities of OPs in mammals are primarily a result of their detoxication capacities.

Inhibition of fatty acid amide hydrolase by chlorpyrifos in juvenile rats results in altered exploratory and social behavior as adolescents.

The organophosphorus insecticide chlorpyrifos (CPF) is suspected to cause developmental neurotoxicity in children leading to long term effects. Developmental exposure of rat pups to CPF at low levels disrupts degradation of the brain endocannabinoids through the inhibition of fatty acid amide hydrolase (FAAH) and decreases the reactivity of juvenile rats in an emergence test. In this study, we further investigated the effects of developmental CPF exposure on behavior but also included exposure to PF-04457845, a specific inhibitor of FAAH, for comparison of behavior altered by FAAH inhibition with behavior altered by CPF. Ten day old rat pups were exposed orally either to 0.5, 0.75, or 1.0 mg/kg CPF or 0.02 mg/kg PF-04457845 daily for 7 days. In an open field (day 23), the high CPF and PF-04457845 groups exhibited increased motor activity but no differences in the time spent in the field's center. In an elevated plus maze (day 29), all treatment groups had increased open arm activity but ethological behaviors associated with anxiety were not altered. Behaviors in the maze associated with increased general activity and exploratory drive were increased. Social interactions (day 36) were measured and all treatment groups exhibited increased levels of play behavior. The similarities in behavior between PF-04457845 and CPF suggest that enhanced endocannabinoid signaling during the exposure period plays a role in the persistent alteration of behavior observed following developmental CPF exposure.

Adolescent rat social play: Amygdalar proteomic and transcriptomic data.

This data article contains the proteomic and transcriptomic data of the amygdala of adolescent rats involved in social play compared to non-behavioural animals. Social play was performed on male Sprague Dawley rats on postnatal day 38 and protein and gene expression in the amygdala was determined following behavioural testing. The protein expression was measured by analysing trypsin digested protein samples using a LTQ Orbitrap Velos mass spectrometer equipped with an Advion nanomate ESI source. The obtained tandem mass spectra were extracted by Thermo Proteome Discoverer 1.3 and the data were displayed with Scaffold v 4.5.1. The transcriptomic data were generated by llumina HiSeq 4000 system. Cuffdiff (v2.2.1) program was used to calculate RNA-seq based gene expression levels. For further interpretation of data presented in this article, please see the research article 'Proteomic and Transcriptional Profiling of Rat Amygdala Following Social Play' (Alugubelly et al. 2019).

Proteomic and transcriptional profiling of rat amygdala following social play.

Social play is the most characteristic form of social interaction which is necessary for adolescents to develop proper cognitive, emotional, and social competency. The information available on neural substrates and the mechanism involved in social play is limited. This study characterized social play by proteomic and transcriptional profiling studies. Social play was performed on male Sprague Dawley rats on postnatal day 38 and protein and gene expression in the amygdala was determined following behavioral testing. The proteomic analysis led to the identification of 170 differentially expressed proteins (p ≤ 0.05) with 67 upregulated and 103 downregulated proteins. The transcriptomic analysis led to the identification of 188 genes (FDR ≤ 0.05) with 55 upregulated and 133 downregulated genes. DAVID analysis of gene/protein expression data revealed that social play altered GABAergic signaling, glutamatergic signaling, and G-protein coupled receptor (GPCR) signaling. These data suggest that the synaptic levels of GABA and glutamate increased during play. Ingenuity Pathway Analysis (IPA) confirmed these alterations. IPA also revealed that differentially expressed genes/proteins in our data had significant over representation of neurotransmitter signaling systems, including the opioid, serotonin, and dopamine systems, suggesting that play alters the systems involved in the regulation of reward. In addition, corticotropin-releasing hormone signaling was altered indicating that an increased level of stress occurs during play. Overall, our data suggest that increased inhibitory GPCR signaling in these neurotransmitter pathways occurs following social play as a physiological response to regulate the induced level of reward and stress and to maintain the excitatory-inhibitory balance in the neurotransmitter systems.

Effect of repeated juvenile exposure to Δ9­tetrahydrocannabinol on anxiety-related behavior and social interactions in adolescent rats.

The gestational and adolescent periods are critically important for brain development and exposure to Δ9­tetrahydrocannabinol (Δ9-THC) during these periods results in long term behavioral and biochemical abnormalities in laboratory animals. However, recent reports indicate a dramatic rise in oral Δ9-THC exposure in young children but the effects of this exposure scenario have not been adequately investigated. Using a model designed to mimic childhood exposure, male and female rat pups were orally exposed to either corn oil or 10 mg/kg Δ9-THC daily from postnatal days 10-16. On day 29, rats were tested in the elevated plus maze under both low and high illumination with no differences in anxiety-related parameters observed between controls and treated rats. Under high but not low illumination, male Δ9-THC rats exhibited increased anxiolytic behavior as compared to female Δ9-THC rats suggesting a sexual dimorphic effect that was only observed under increased aversiveness. In addition, male Δ9-THC rats had increased activity levels as compared to control males. On day 38, social interactions were determined and both male and female Δ9-THC rats exhibited lower levels of social exploration behaviors but increased episodes of social play behaviors and increased time spent engaged in play. These data suggest that oral exposure to Δ9-THC during a period similar to childhood in humans can result in altered social behavior once adolescence is reached.

Alteration of neurotrophins in the hippocampus and cerebral cortex of young rats exposed to chlorpyrifos and methyl parathion.

Exposure to either chlorpyrifos (CPS) or methyl parathion (MPS) results in the inhibition of acetylcholinesterase and leads to altered neuronal activity which normally regulates critical genes such as the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The effects of postnatal exposure to CPS and MPS on the expression of messenger RNA (mRNA) and protein levels for NGF and BDNF were investigated in the frontal cerebral cortex (cortex) and hippocampus of rats. Oral administration of CPS (4.0 or 6.0 mg/kg), MPS (0.6 or 0.9 mg/kg), or the safflower oil vehicle was performed daily from postnatal day 10 (PND10) through PND20. Exposure induced significant effects on growth and cholinesterase activity. Increased NGF protein levels were observed in the hippocampus but not the cortex on PND20 with some reduction occurring on PND28 in both regions. These changes did not correlate with the changes in NGF mRNA. BDNF mRNA was increased in both regions on PND20 and PND28, whereas BDNF protein levels were increased on PND20. On PND12, c-fos mRNA, a marker of neuronal activation, was increased in both regions. Total BDNF protein was increased in the hippocampus but decreased in the cortex. No changes in NGF protein were observed. These results indicate that repeated developmental OP exposure during the postnatal period alters NGF and BDNF in the cortex and the hippocampus and the patterns of these alterations differ between regions.

Altered muscarinic acetylcholine receptor subtype binding in neonatal rat brain following exposure to chlorpyrifos or methyl parathion.

The neurodevelopmental effects of two organophosphorus (OP) insecticides, chlorpyrifos (CPS) and methyl parathion (MPS), on cholinesterase (ChE) activity and muscarinic acetylcholine receptor (mAChR) binding were investigated in neonatal rat brain. Animals were orally gavaged using an incremental dosing regimen from postnatal day 1 (PND1) until PND8 with a low, medium, and high dosage for both CPS and MPS. On PND4 and PND8, ChE activity was measured in whole brain while the total and subtype densities of mAChRs were measured in three brain sections: area anterior to optic chiasma (anterior forebrain), area from the optic chiasma to the medulla/pons (posterior forebrain); and the medulla/pons excluding the cerebellum. The ligands 3H-pirenzepine, 3H-AF-DX 384, 3H-4-DAMP, and 3H-QNB were used to measure the maximal binding of the M1, M2/M4, and M3 subtypes and total mAChR receptors, respectively. In the anterior and the posterior forebrain, the levels of all mAChRs nearly doubled from PND4 to PND8, while in the medulla/pons, M1- and M3-subtype mAChR densities were low and did not increase and M2/M4 subtype and total mAChR slightly increased from PND4 to PND8. Reduction of ChE activity and mAChR binding by CPS or MPS was more evident in rats at PND8 than at PND4. With respect to mAChR binding, the greatest effects were observed in the medulla/pons and the least effects were observed in the posterior region of the forebrain. These results demonstrate that OPs exert adverse effects on rat central nervous system development through the cholinergic system in an age- and region-dependent manner.

Dopaminergic toxicity of the herbicide atrazine in rat striatal slices.

A possible link between Parkinson's disease and pesticide exposure has been suggested, and recently it was shown that the herbicide atrazine (ATR) modulates catecholamine metabolism in PC12 cells and affects basal ganglia function in vivo. Hence, the objectives of this study were to: (i) determine if ATR is capable of modulating dopamine (DA) metabolism in striatal tissue slices in vitro and (ii) explore possible mechanisms of its effects. Striatal tissues from adult male Sprague-Dawley rats were incubated with up to 500 microM ATR in a metabolic shaker bath at 37 degrees C and an atmosphere of 95% O(2) and 5% CO(2) for 4h. At the end of incubation, samples were collected for both tissue and media levels of DA and its metabolites (3,4-dihydroxyphenylacetic acid, DOPAC and homovanillic acid, HVA), which were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). To gain some mechanistic insight in to the way ATR affects DA metabolism, several pharmacological manipulations were performed. Striata exposed to ATR at concentrations of 100 microM and greater had a dose-dependent decrease of tissue levels of DA. At doses of ATR 50 microM and greater, the DOPAC+HVA/DA ratio was dose-dependently increased. Tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) protein levels and activity were not affected by ATR treatment. However, high potassium-induced DA release into the medium was decreased, whereas the increase in media DA observed in the presence of the DA uptake inhibitor nomifensine was increased even further by ATR in a dose-dependent manner. All of these effects of ATR were observed at levels that were not toxic to the tissue, as LDH release into the medium (lactate dehydrogenase, an index of non-specific cytotoxicity) was not affected by ATR. Taken together, results from this study suggest that ATR decreases tissue DA levels not by affecting TH activity, but possibly by interfering with the vesicular storage and/or cellular uptake of DA.

Effects of Chlorpyrifos or Methyl Parathion on Regional Cholinesterase Activity and Muscarinic Receptor Subtype Binding in Juvenile Rat Brain.

The effects of developmental exposure to two organophosphorus (OP) insecticides, chlorpyrifos (CPF) and methyl parathion (MPS), on cholinesterase (ChE) activity and muscarinic acetylcholine receptor (mAChR) binding were investigated in preweanling rat brain. Animals were orally gavaged daily with low, medium, and high dosages of the insecticides using an incremental dosing regimen from postnatal day 1 (PND1) to PND20. On PND12, PND17 and PND20, the cerebral cortex, corpus striatum, hippocampus, and medulla-pons were collected for determination of ChE activity, total mAChR density, and the density of the individual mAChR subtypes. ChE activity was inhibited by the medium and high dosages of CPF and MPS at equal levels in all four brain regions at all three ages examined. Exposure to both compounds decreased the levels of the M1, M2/M4, and M3 subtypes and the total mAChR level in all brain regions, but the effects varied by dosage group and brain region. On PND12, only the high dosages induced receptor changes while on PND17 and PND20, greater effects became evident. In general, the effects on the M1 subtype and total receptor levels appeared to be greater in the cerebral cortex and hippocampus than in the corpus striatum and medulla-pons. This did not appear to be the case for the M2/M4 and M3 subtypes effects. The differences between CPF and MPS were minimal even though in some cases, CPF exerted statistically greater effects than MPS did. In general, repeated exposure to organophosphorus insecticides can alter the levels of the various mAChR subtypes in various brain regions which could induce perturbation in cholinergic neurochemistry during the maturation of the brain regions.

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.

Effect of developmental exposure to chlorpyrifos on the expression of neurotrophin growth factors and cell-specific markers in neonatal rat brain.

Chlorpyrifos (CPS), a known neurotoxicant, is a widely used agricultural organophosphorus insecticide. The effects of postnatal exposure to CPS on the expression of mRNA for two factors critical to brain development, nerve growth factor (NGF) and reelin, were investigated in the forebrain of rats. In addition, the expression of mRNA for the muscarinic acetylcholine receptor (mAChR) M(1) subtype and cell-specific markers for developing neurons (beta-III tubulin), astrocytes (glial fibrillary acidic protein, GFAP), and oligodendrocytes (myelin-associated glycoprotein, MAG) was also investigated. Oral administration of CPS (1.5 or 3.0 mg/kg) or the corn oil vehicle was performed daily from postnatal days (PNDs) 1 through 6. No signs of overt toxicity or of cholinergic hyperstimulation were observed after CPS administration. Body weight was significantly different from controls on PND7 in both males and females exposed to 3.0 mg/kg CPS. Quantitative PCR was performed on the forebrain. The expression of NGF, reelin, and M(1) mAChR mRNA was significantly reduced with both dosages of CPS in both sexes. beta-III Tubulin mRNA expression remained unchanged after exposure, whereas MAG mRNA expression was significantly decreased with both dosages of CPS in both sexes, suggesting effects on the developing oligodendrocytes. In contrast, GFAP mRNA levels were significantly increased with both dosages of CPS in both sexes, suggesting increased astrocyte reactivity. Our findings indicate that dosages of CPS which cause significant cholinesterase inhibition but do not exert overt toxicity can adversely affect the expression levels of critical genes involved in brain development during the early postnatal period in the rat.

Comparison of inhibition kinetics of several organophosphates, including some nerve agent surrogates, using human erythrocyte and rat and mouse brain acetylcholinesterase.

Because testing of nerve agents is limited to only authorized facilities, our laboratory developed several surrogates that resemble nerve agents because they phosphylate the acetylcholinesterase (AChE) with the same moiety as the actual nerve agents. The inhibition kinetic parameters were determined for AChE by surrogates of cyclosarin (NCMP), sarin (NIMP, PIMP and TIMP) and VX (NEMP and TEMP) and other organophosphorus compounds derived from insecticides. All compounds were tested with rat brain and a subset was tested with mouse brain and purified human erythrocyte AChE. Within the compounds tested on all AChE sources, chlorpyrifos-oxon had the highest molecular rate constant followed by NCMP and NEMP. This was followed by NIMP then paraoxon and DFP with rat and mouse brain AChE but DFP was a more potent inhibitor than NIMP and paraoxon with human AChE. With the additional compounds tested only in rat brain, TEMP was slightly less potent than NEMP but more potent than PIMP which was more potent than NIMP. Methyl paraoxon was slightly less potent than paraoxon but more potent than TIMP which was more potent than DFP. Overall, this study validates that the pattern of inhibitory potencies of our surrogates is comparable to the pattern of inhibitory potencies of actual nerve agents (i.e., cyclosarin>VX>sarin), and that these are more potent than insecticidal organophosphates.

Species differences in paraoxonase mediated hydrolysis of several organophosphorus insecticide metabolites.

Paraoxonase (PON1) is a calcium dependent enzyme that is capable of hydrolyzing organophosphate anticholinesterases. PON1 activity is present in most mammals and previous research established that PON1 activity differs depending on the species. These studies mainly used the organophosphate substrate paraoxon, the active metabolite of the insecticide parathion. Using serum PON1 from different mammalian species, we compared the hydrolysis of paraoxon with the hydrolysis of the active metabolites (oxons) of two additional organophosphorus insecticides, methyl parathion and chlorpyrifos. Paraoxon hydrolysis was greater than that of methyl paraoxon, but the level of activity between species displayed a similar pattern. Regardless of the species tested, the hydrolysis of chlorpyrifos-oxon was significantly greater than that of paraoxon or methyl paraoxon. These data indicate that chlorpyrifos-oxon is a better substrate for PON1 regardless of the species. The pattern of species differences in PON1 activity varied with the change in substrate to chlorpyrifos-oxon from paraoxon or methyl paraoxon. For example, the sex difference observed here and reported elsewhere in the literature for rat PON1 hydrolysis of paraoxon was not present when chlorpyrifos-oxon was the substrate.