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.

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.

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.

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).

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.

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.