Organophosphate agent action at the fatty acid amide hydrolase enhancing anandamide-induced apoptosis in NG108-15 cells

Abstract

Organophosphate (OP) agents are continuously utilized in large amount throughout the globe for crop protection and public health, thereby creating a potential concern on human health. OP agent as an anticholinesterase also acts on the endocannabinoid (EC)-hydrolases, i.e., fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), to reveal unexpected adverse effects including ADHD-like behaviors in adolescent male rats. The present investigation examines a hypothesis that OP compound inhibiting the EC-hydrolase(s) dysregulates the EC-signaling system, triggering apoptosis in neuronal cells. Ethyl octylphosphonofluoridate (EOPF), as an OP probe, preferably acts on FAAH over MAGL in intact NG108-15 cells. Anandamide (AEA), an endogenous FAAH substrate, is cytotoxic in a concentration-dependent manner, although 2-arachidonoylglycerol, an endogenous MAGL substrate, gives no effect in the concentrations examined here. EOPF pretreatment markedly enhances AEA-induced cytotoxicity. Interestingly, the cannabinoid receptor blocker AM251 diminishes AEA-induced cell death, whereas AM251 does not prevent the cell death in the presence of EOPF. The consistent results are displayed in apoptosis markers evaluation (caspases and mitochondrial membrane potential). Accordingly, FAAH inhibition by EOPF suppresses AEA-metabolism, and accumulated excess AEA overstimulates both the cannabinoid receptor- and mitochondria-mediated apoptotic pathways.

INTRODUCTION

Organophosphate (OP) insecticides are constantly utilized throughout the world for protecting crops, people, and animals from pest insect attack and disease transmission. OPs are top volume accounting for over 50% of the total insecticides by amount but 13% of sales, implying that people are chronically exposed to OP compounds in life (Casida and Bryant, 2017). OP agents are nerve poisons acting at cholinergic neurons by irreversibly inhibiting the acetylcholinesterase (AChE) as primary target of acute toxicity (Casida and Durkin, 2013). These compounds phosphorylate the serine hydroxyl residue at the AChE catalytic triad. Intriguingly, OP agent may also react with many other serine hydrolases to potentially reveal the secondary or unexpected biological and/or toxicological effects (Casida and Durkin, 2013). In particular, the fatty amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), endocannabinoid (EC)-hydrolyzing enzymes, are secondary OP targets associated with neurotoxicity (Quistad et al., 2001, 2002), hypomotility (Quistad et al., 2006; Nomura et al., 2008), plasma hypertriglyceridemia (Ruby et al., 2008; Suzuki et al., 2014), and male reproductive toxicity (Suzuki et al., 2013; Noro et al., 2013; Ito et al., 2014). Recently, interest has been given to whether OP exposure develops attention-deficit hyperactivity disorder (ADHD). Some epidemiological studies have suggested that OP exposure is conceivably a risk factor for ADHD (Rauh et al., 2006; Bouchard et al., 2010; Marks et al., 2010; Yu et al., 2016; Rohlman et al., 2019). In our previous study, ethyl octylphosphonofluoridate (EOPF) (Fig. 1), as an OP probe, elevates ECs level in the rat brain through inhibition of the FAAH and MAGL, and EOPF induces ADHD-like behaviors in adolescent male rats with SLV-319 [a cannabinoid (CB) receptor inverse agonist] reversed manner, therefore indicating that excitation of the EC-signaling system is a plausible etiological mechanism of OP-induced ADHD-like behaviors (Ito et al., 2020).

Fig. 1

Chemical structures of an OP agent EOPF, a CB receptor blocker AM251, and EC agonists AEA and 2-AG.

Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) (Fig. 1), endogenous substrates of FAAH and MAGL, respectively, and agonists of CB receptor, are major ECs that regulate central and peripheral physiological function. Particularly, EC-signaling system modulates the balance between cell death and proliferation (Fonseca et al., 2013). EC in an appropriate level controls cell growth, whereas a massive amount of EC overstimulates cellular signaling through CB receptor, transient receptor potential vanilloid receptor, and receptor-independent pathways, eventually causing apoptosis in various cells (Maccarrone and Finazzi-Agró, 2003). However, the mechanism of OP agent-associated neuronal cell death, via affecting the EC-signaling system, remains unclear. The present investigation examines a hypothesis that OP agent, inhibiting the EC-hydrolase(s), suppresses EC-metabolism, and subsequently excess EC dysregulates the signaling system(s) leading to apoptosis in NG108-15 neuronal cells, thereby rationalizing that the OP-induced cell death is a possible trigger responsible for OP-developed ADHD-like behaviors (Ito et al., 2020).

MATERIALS AND METHODS

Chemicals

The sources of chemicals utilized in the present investigation are listed as the following: AM251, acetylthiocholine iodide, 5,5’-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent), and Immunostar^® Zeta western blotting detection reagent from Wako Pure Chemical (Osaka, Japan); AEA from Abcam (Cambridge, MA, USA); 2-AG from Sigma-Aldrich (St. Louis, MO, USA); [¹⁴C]AEA and [¹⁴C]mono-oleoylglycerol ([¹⁴C]OG) from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA); Dulbecco’s modifed Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and HAT supplement (liquid mixture of sodium hypoxanthine, aminopterin, and thymidine) from Gibco Life Technologies (Grand Island, NY, USA); ε-poly-L-lysine solution from Cosmo Bio Co., Ltd. (Tokyo, Japan); mouse monoclonal anti-FAAH antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); rabbit polyclonal anti-MAGL antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG from Invitrogen (Rockford, IL, USA); HRP-conjugated goat anti-mouse IgG from Medical & Biological Laboratories Co., Ltd. (Tokyo, Japan). EOPF (> 98% purity) was synthesized according to a previous report (Wu and Casida, 1995).

Cell culture and inhibitor treatment

The NG108-15 cell line (mouse neuroblastoma-rat glioma hybrid), expressing functional CB receptor (Tomiyama and Funada, 2011) and obtained from the European Collection of Authenticated Cell Cultures (Salisbury, UK), was maintained in culture medium (DMEM supplemented with 10% FBS, 2% HAT, 100 unit/mL penicillin and 0.1 mg/mL streptomycin) at 37°C in 5% CO₂/95% air atmosphere. For EC-hydrolase inhibitor treatment, the cells were seeded at 200,000 per tissue culture plate (100 mm diameter dish) precoated with ε-poly-L-lysine and maintained for 48 hr in culture medium. The cells were gently washed in Dulbecco’s phosphate buffered saline (PBS) and incubated in serum-free medium for an additional 90 min. EOPF (final concentration 0.001 to 100 nM) was administered to the culture dish for a 30 min incubation. The medium was removed from the culture plate, and the cells were washed twice with ice-cold PBS and then quickly harvested and homogenized on ice in 50 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA. The protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA).

Enzyme assay

FAAH and MAGL activities in NG108-15 cells were assayed by hydrolysis of the corresponding substrate [¹⁴C]AEA and [¹⁴C]OG, respectively (55 mCi/mmol for both substrates), according to previous reports of Quistad et al. (2001, 2006). Briefly, an aliquot of homogenate (containing 100 or 50 µg protein/assay for FAAH or MAGL, respectively) from EOPF-treated or untreated intact NG108-15 cells was incubated with 1 µM [¹⁴C]AEA or [¹⁴C]OG for 30 min at 37°C, and then the reaction terminated by addition of an organic solvent (chloroform:methanol:hexane, 1.25:1.4:1.0) and 200 mM K₂CO₃. Subsequently, the radioactivity in the aqueous upper layer, as the amount of [¹⁴C]arachidonic acid or [¹⁴C]oleic acid produced from the enzymatic reaction, was determined by liquid scintillation counting. AChE activity (with or without EOPF pretreatment for 15 min) in the present cell homogenate (500 µg protein/assay) was measured spectrophotometrically (after 30 min incubation at 37°C) using acetylthiocholine iodide (final concentration 0.16 mM) as the substrate and Ellman’s reagent (final concentration 0.05 mM) as the chromogen.

Immunoblotting

Western blotting was conducted by the procedure described elsewhere. Concisely, the cell homogenate sample (adjusted to 25 μg protein) was subjected to 10% SDS-PAGE and then separated proteins on the SDS-PAGE gel were transferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the nitrocellulose membrane was incubated with anti-FAAH (1:1000) or anti-MAGL (1:5000) for overnight at 4ºC, followed by incubation with suitable HRP-conjugated secondary antibody (1:3000 for FAAH; 1:25000 for MAGL) for 2 hr at room temperature. The protein signals were enhanced using an electrochemiluminescence detection reagent.

Evaluation of cytotoxicity and apoptosis markers

The cytotoxicity and apoptosis markers were determined by Cytotoxicity LDH Assay Kit-WST, JC-1 MitoMP Detection Kit (Dojindo Molecular Technologies, Inc., Rockville, MD, USA), Caspase-Glo^® 3/7 Assay System, and Caspase-Glo^® 9 Assay System (Promega Corporation, Madison, WI, USA) according to the instruction manuals. The cells were seeded at 1,000 per well (on a plate with 96-wells) precoated with ε-poly-L-lysine and incubated for 24 hr in culture medium. The culture medium was replaced by DMEM containing 1% FBS and 2% HAT supplement, and then the cells were maintained for 30 min. The cells were subsequently treated with an appropriate concentration of AEA, 2-AG, and/or EOPF for 4 hr (for mitochondrial membrane potential detection), or 8 hr (for caspase-3/7 and caspase-9 assays), or 24 hr (for cytotoxicity assay). For inhibitor treatment, the cells were preincubated with EOPF (20 nM) and/or AM251 (100 nM) for 30 min. Data was obtained using the VarioSkan^TMLUX multimode microplate reader for detecting the fluorescent and luminescent (Thermo Fisher Scientific, Waltham, MA, USA).

Data analysis

All statistical analyses and calculations were performed using Sigmaplot software 12.3 (SPSS Inc., Chicago, IL, USA). A molar concentration of test chemical necessary for 50% inhibition (IC₅₀) value was calculated by iterative nonlinear least-squares regression. Significant differences among the multiple comparison were determined using one-way analysis of variance followed by Bonferroni’s test.

RESULTS

Effect of EOPF on FAAH and MAGL activities and protein expression levels in intact NG108-15 cells

Hydrolytic activity of FAAH, MAGL, or AChE in NG108-15 cells was 100 ± 10, 90 ± 9, or 4,000 ± 800 nmol/mg/min (± SD, n = 3), respectively. EOPF decreased FAAH and MAGL activities in the present cells in a concentration-dependent manner (Fig. 2A and B). IC₅₀ values (± SD, n = 3) of EOPF against FAAH and MAGL were 0.07 ± 0.01 and 1.1 ± 0.2 nM, respectively. However, EOPF was less potent to AChE (IC₅₀ 150 ± 17 nM). EOPF did not affect protein expression levels of FAAH and MAGL (Fig. 2C and D) in the present cells.

Fig. 2

Effect of EOPF on EC hydrolase (FAAH or MAGL) activity expressed in intact NG108-15 cells. EOPF (> 10 nM) completely inhibits the FAAH and MAGL (A and B). Cells were treated with EOPF for 30 min, and subsequently FAAH or MAGL activity was determined by [¹⁴C]AEA or [¹⁴C]OG hydrolysis, respectively. Each data has an error bar (mean ± SD, n = 3). Asterisks indicate the significant difference (**p < 0.01) among control and treatments. EOPF has no effect on the FAAH (67 KDa) and MAGL (30KDa) expression levels compared with that of untreated NG108-15 cells (C and D). Each image shown is representative of three individual experiments.

Cytotoxicity of ECs and EOPF

The cell death was significantly induced by administration of 20 μM or 50 μM AEA (2.0-fold or 5.1-fold, respectively) relative to that of control, but 2-AG had no effect in the concentrations examined here (Fig. 3A and B). EOPF was not cytotoxic even at 100 nM, a concentration completely inhibiting FAAH and MAGL activities (Fig. 3C).

Fig. 3

Cytotoxicity of ECs and EOPF in NG108-15 cells. AEA induces cell death in a dose-dependent manner (A), whereas 2-AG (B) or EOPF (C) shows no effect. Cytotoxicity was evaluated as lactate dehydrogenase (LDH) release after 24 hr exposure to AEA, 2-AG, or EOPF. Each data has an error bar (mean ± SD, n = 3). Asterisks indicate the significant difference (**p < 0.01) among control and treatments.

Effect of EOPF on EC-induced cell death

AEA (30 μM)-induced cytotoxicity (5.1-fold relative to that of control) was significantly suppressed (by 30%) in the presence of AM251 treatment [100 nM, a concentration that completely blocks CB receptor (Bruno et al., 2014)] (Fig. 4A). The single or dual treatment of AM251 and/or EOPF was not cytotoxic. EOPF pretreatment (20 nM, a concentration completely inhibiting FAAH and MAGL) markedly facilitated the AEA-induced cell death (1.7-fold relative to that of AEA alone). Interestingly, AM251 failed to prevent the AEA-induced cell death enhanced by EOPF. Furthermore, AEA alone at lower concentration (15 μM) did not lead to cell death however, EOPF pretreatment clearly induced cytotoxicity at the above AEA concentration (3.7- or 3.4-fold relative to that of control or AEA alone, respectively), and the cell death was not circumvented in the presence of AM251 (Fig. 4B). 2-AG in the absence and presence of EOPF and/or AM251 showed no effect in NG108-15 cells (Fig. 4C).

Fig. 4

Effect of EOPF on EC-elicited cell death in NG108-15 cells. AEA in the presence of EOPF clearly enhances the cell death relative to that of the AEA alone. Intriguingly, AM251 does not prevent the AEA-induced cell death in the presence of EOPF, although AM251, in the absence of EOPF, diminishes the cell death (A). AEA alone at lower concentration (15 µM) is unable to induce cytotoxicity, while AEA at 15 µM with EOPF pretreatment clearly elicits cell death, and the cell death is not averted by AM251 (B). 2-AG or/and EOPF in the absence or presence of AM251 remains unchanged (C). Cells were pretreated with AM251 (100 nM) and/or EOPF (20 nM) for 30 min, and AEA (15 or 30 μM) or 2-AG (30 μM) was subsequently administered to determine the cytotoxicity as LDH release after 24 hr incubation. Each data has an error bar (mean ± SD, n = 3). Asterisks indicate the significant difference (*p < 0.05 or **p < 0.01) among control and treatments.

Effect of EOPF on AEA-induced apoptosis

AEA (30 μM) significantly led to elevation of the caspase-3/7 (90-fold) and caspase-9 (19-fold) activities compared with those of the control (Fig. 5 A and B). The elevation of these apoptosis markers was remarkably emphasized in the presence of EOPF (20 nM) [caspase-3/7 and caspase-9 were more active (1.8- and 1.5-fold, respectively) than those of AEA alone]. AEA alone enhanced the mitochondrial membrane potential loss (80% loss relative to that of untreated control) (Fig. 5C). The loss induced by AEA alone was further progressive in the presence of EOPF (70 or 90% loss compared to that of AEA alone or untreated control, respectively).

Fig. 5

Effect of EOPF on the AEA-induced apoptosis. AEA increases caspase-3/7 (A) or caspase-9 (B) activity and the elevation is significantly enhanced by EOPF pretreatment. Furthermore, the AEA-induced mitochondrial membrane potential loss (C) is emphasized in the presence of EOPF. Cells were pretreated with EOPF (20 nM) for 30 min, and then AEA (30 μM) was administered to determine apoptosis markers. Each data has an error bar (mean ± SD, n = 3-4). Asterisks indicate the significant difference (*p < 0.05 or **p < 0.01) among control and treatments.

DISCUSSION

The goal of this study is to identify the mechanism(s) of OP-triggered apoptosis in a neuronal cell line, through irreversible inhibition of EC-hydrolase(s), being relevant, in part, to the OP-developed ADHD-like behaviors (Ito et al., 2020). Synthetic CB agonist induces ADHD-like symptoms, altering locomotor activity, anxiety like-behavior, and motivational effect in adolescent spontaneously hypertensive rats, an animal model for ADHD symptoms, whereas CB receptor antagonist diminishes these effects (Pandolfo et al., 2007, 2009). Fascinatingly, synthetic CB agonist causes neuronal cell apoptosis through a direct stimulation of CB receptor (Tomiyama and Funada, 2011, 2014), although the mechanism(s) of OP-elicited cell apoptosis is ambiguous. Children 8 to 15 years of age, representative of the United States population with high levels of urinary OP metabolites, are associated with ADHD prevalence (Bouchard et al., 2010). Our previous paper proposes that EC-signaling system is a conceivable target for OP-developed ADHD-like behaviors in juvenile male rats (Ito et al., 2020). Thus EOPF, as an extremely potent inhibitor of EC-hydrolase(s), leads to an excitation of EC-signaling system via elevation of AEA and 2-AG levels (approximately 3 ng/g brain and 1 µg/g brain, respectively) and subsequent overactivation of the CB receptor (Ito et al., 2020).

The present investigation reports that EOPF preferably inhibits FAAH activity over that of MAGL in NG108-15 cells. AEA, but not 2-AG (at least in the concentrations used here), is cytotoxic and apoptotic in the present cells. AEA in an appropriate concentration promotes neural cell differentiation and proliferation, whereas an excess AEA causes cell apoptosis via overactivation of CB receptor (Maccarrone and Finazzi-Agró, 2003; Aguado et al., 2005; Soltys et al., 2010). In the present investigation, EOPF suppresses AEA-degradation, and the accumulated AEA overstimulates the CB receptor, then leading to apoptotic cell death in NG108-15 cells. AEA-induced cell death is prevented by a CB receptor blocker AM251. In contrast, EOPF pretreatment clearly enhances the cell death without AM251 reversed manner. These results suggest that OP-associated cell death consists of two pathways: CB receptor-mediated and the receptor-independent mechanisms. Mitochondria are thought to be the primary organelles involved in mediating most apoptotic pathways in mammalian cells (Chalah and Khosravi-Far, 2008). AEA directly and dose-dependently facilitates mitochondrial swelling and membrane potential loss in isolated mitochondria and human lung cancer cells (Athanasiou et al., 2007; Catanzaro et al., 2009), revealing that the intracellular uptake of excess AEA directly affects mitochondria functions, triggering toward cell apoptosis (Nunn et al., 2012). EOPF further encourages AEA-induced mitochondrial membrane potential loss and activation of apoptosis initiator and executioner (caspase-9 and caspase-3/7, respectively), therefore supporting that the CB receptor-independent pathway is also involved in OP-triggered apoptosis in NG108-15 cells. Furthermore, CB receptor is present at the membranes of mouse neuronal mitochondria (Bénard et al., 2012), suggesting that elevated EC by OP treatment presumably acts on the mitochondrial CB receptor. However, the physiological roles of mitochondrial CB receptor, including apoptosis induction mechanism, remain to be elucidated (Ye et al., 2019).

The EC-signaling system regulates maturation of excitatory synapse transmission during development (Yasuda et al., 2008), and CB receptor stimulation on the excitatory and inhibitory presynaptic terminals modulates neurotransmitter release and ion channel activation (Szabo and Schlicker, 2005). ECs play a major role in several forms of short- and long-term synaptic plasticity (Freund et al., 2003; Piomelli, 2003; Chevaleyre et al., 2006; Trezza et al., 2012), and a severely controlled EC level (neither excessive nor insufficient) maintains normal synaptic excitation/inhibition balance.

In summary, FAAH inhibition by EOPF suppresses AEA-metabolism, and accumulated excess AEA overstimulates both the CB receptor- and mitochondria-mediated apoptosis pathways. Accordingly, OP agent acting at the EC-signaling system creates a possible hazard disturbing neuronal-network maturation in early developmental period vulnerable to chemicals.

ACKNOWLEDGMENTS

This work was supported, in part, by JSPS KAKENHI 19H03888. This paper is dedicated to the memory of Dr. John E. Casida (1929-2018) (University of California, Berkeley), who was a superstar in the fields of pesticide chemistry and toxicology.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Aguado,  T.,  Monory,  K.,  Palazuelos,  J.,  Stella,  N.,  Cravatt,  B.,  Lutz,  B.,  Marsicano,  G.,  Kokaia,  Z.,  Guzmán,  M. and  Galve-Roperh,  I. (2005): The endocannabinoid system drives neural progenitor proliferation. FASEB J., 19, 1704-1706.
•  Athanasiou,  A.,  Clarke,  A.B.,  Turner,  A.E.,  Kumaran,  N.M.,  Vakilpour,  S.,  Smith,  P.A.,  Bagiokou,  D.,  Bradshaw,  T.D.,  Westwell,  A.D.,  Fang,  L.,  Lobo,  D.N.,  Constantinescu,  C.S.,  Calabrese,  V.,  Loesch,  A.,  Alexander,  S.P.,  Clothier,  R.H.,  Kendall,  D.A. and  Bates,  T.E. (2007): Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death. Biochem. Biophys. Res. Commun., 364, 131-137.
•  Bénard,  G.,  Massa,  F.,  Puente,  N.,  Lourenço,  J.,  Bellocchio,  L.,  Soria-Gómez,  E.,  Matias,  I.,  Delamarre,  A.,  Metna-Laurent,  M.,  Cannich,  A.,  Hebert-Chatelain,  E.,  Mulle,  C.,  Ortega-Gutiérrez,  S.,  Martín-Fontecha,  M.,  Klugmann,  M.,  Guggenhuber,  S.,  Lutz,  B.,  Gertsch,  J.,  Chaouloff,  F.,  López-Rodríguez,  M.L.,  Grandes,  P.,  Rossignol,  R. and  Marsicano,  G. (2012): Mitochondrial CB₁ receptors regulate neuronal energy metabolism. Nat. Neurosci., 15, 558-564.
•  Bouchard,  M.F.,  Bellinger,  D.C.,  Wright,  R.O. and  Weisskopf,  M.G. (2010): Attention-deficit/hyperactivity disorder and urinary metabolites of organophosphate pesticides. Pediatrics, 125, e1270-e1277.
•  Bruno,  A.,  Lembo,  F.,  Novellino,  E.,  Stornaiuolo,  M. and  Marinelli,  L. (2014): Beyond radio-displacement techniques for identification of CB1 ligands: the first application of a fluorescence-quenching assay. Sci. Rep., 4, 3757.
•  Casida,  J.E. and  Durkin,  K.A. (2013): Neuroactive insecticides: targets, selectivity, resistance, and secondary effects. Annu. Rev. Entomol., 58, 99-117.
•  Casida,  J.E. and  Bryant,  R.J. (2017): The ABCs of pesticide toxicology: amounts, biology, and chemistry. Toxicol. Res. (Camb.), 6, 755-763.
•  Catanzaro,  G.,  Rapino,  C.,  Oddi,  S. and  Maccarrone,  M. (2009): Anandamide increases swelling and reduces calcium sensitivity of mitochondria. Biochem. Biophys. Res. Commun., 388, 439-442.
•  Chalah,  A. and  Khosravi-Far,  R. (2008): The mitochondrial death pathway. Adv. Exp. Med. Biol., 615, 25-45. Springer, Dordrecht.
•  Chevaleyre,  V.,  Takahashi,  K.A. and  Castillo,  P.E. (2006): Endocannabinoid-mediated synaptic plasticity in the CNS. Annu. Rev. Neurosci., 29, 37-76.
•  Fonseca,  B.M.,  Costa,  M.A.,  Almada,  M.,  Correia-da-Silva,  G. and  Teixeira,  N.A. (2013): Endogenous cannabinoids revisited: a biochemistry perspective. Prostaglandins Other Lipid Mediat., 102-103, 13-30.
•  Freund,  T.F.,  Katona,  I. and  Piomelli,  D. (2003): Role of endogenous cannabinoids in synaptic signaling. Physiol. Rev., 83, 1017-1066.
•  Ito,  Y.,  Tomizawa,  M.,  Suzuki,  H.,  Okamura,  A.,  Ohtani,  K.,  Nunome,  M.,  Noro,  Y.,  Wang,  D.,  Nakajima,  T. and  Kamijima,  M. (2014): Fenitrothion action at the endocannabinoid system leading to spermatotoxicity in Wistar rats. Toxicol. Appl. Pharmacol., 279, 331-337.
•  Ito,  Y.,  Tomizawa,  M.,  Suzuki,  K.,  Shirakawa,  Y.,  Ono,  H.,  Adachi,  K.,  Suzuki,  H.,  Shimomura,  K.,  Nabeshima,  T. and  Kamijima,  M. (2020): Organophosphate agent induces ADHD-like behaviors via inhibition of brain endocannabinoid-hydrolyzing enzyme(s) in adolescent male rats. J. Agric. Food Chem., 68, 2547-2553.
•  Maccarrone,  M. and  Finazzi-Agró,  A. (2003): The endocannabinoid system, anandamide and the regulation of mammalian cell apoptosis. Cell Death Differ., 10, 946-955.
•  Marks,  A.R.,  Harley,  K.,  Bradman,  A.,  Kogut,  K.,  Barr,  D.B.,  Johnson,  C.,  Calderon,  N. and  Eskenazi,  B. (2010): Organophosphate pesticide exposure and attention in young Mexican-American children: the CHAMACOS study. Environ. Health Perspect., 118, 1768-1774.
•  Nomura,  D.K.,  Blankman,  J.L.,  Simon,  G.M.,  Fujioka,  K.,  Issa,  R.S.,  Ward,  A.M.,  Cravatt,  B.F. and  Casida,  J.E. (2008): Activation of the endocannabinoid system by organophosphorus nerve agents. Nat. Chem. Biol., 4, 373-378.
•  Noro,  Y.,  Tomizawa,  M.,  Ito,  Y.,  Suzuki,  H.,  Abe,  K. and  Kamijima,  M. (2013): Anticholinesterase insecticide action at the murine male reproductive system. Bioorg. Med. Chem. Lett., 23, 5434-5436.
•  Nunn,  A.,  Guy,  G. and  Bell,  J.D. (2012): Endocannabinoids in neuroendopsychology: multiphasic control of mitochondrial function. Philos. Trans. R. Soc. Lond. B Biol. Sci., 367, 3342-3352.
•  Pandolfo,  P.,  Pamplona,  F.A.,  Prediger,  R.D. and  Takahashi,  R.N. (2007): Increased sensitivity of adolescent spontaneously hypertensive rats, an animal model of attention deficit hyperactivity disorder, to the locomotor stimulation induced by the cannabinoid receptor agonist WIN 55,212-2. Eur. J. Pharmacol., 563, 141-148.
•  Pandolfo,  P.,  Vendruscolo,  L.F.,  Sordi,  R. and  Takahashi,  R.N. (2009): Cannabinoid-induced conditioned place preference in the spontaneously hypertensive rat-an animal model of attention deficit hyperactivity disorder. Psychopharmacology (Berl.), 205, 319-326.
•  Piomelli,  D. (2003): The molecular logic of endocannabinoid signalling. Nat. Rev. Neurosci., 4, 873-884.
•  Quistad,  G.B.,  Sparks,  S.E. and  Casida,  J.E. (2001): Fatty acid amide hydrolase inhibition by neurotoxic organophosphorus pesticides. Toxicol. Appl. Pharmacol., 173, 48-55.
•  Quistad,  G.B.,  Sparks,  S.E.,  Segall,  Y.,  Nomura,  D.K. and  Casida,  J.E. (2002): Selective inhibitors of fatty acid amide hydrolase relative to neuropathy target esterase and acetylcholinesterase: toxicological implications. Toxicol. Appl. Pharmacol., 179, 57-63.
•  Quistad,  G.B.,  Klintenberg,  R.,  Caboni,  P.,  Liang,  S.N. and  Casida,  J.E. (2006): Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice. Toxicol. Appl. Pharmacol., 211, 78-83.
•  Rauh,  V.A.,  Garfinkel,  R.,  Perera,  F.P.,  Andrews,  H.F.,  Hoepner,  L.,  Barr,  D.B.,  Whitehead,  R.,  Tang,  D. and  Whyatt,  R.W. (2006): Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life among inner-city children. Pediatrics, 118, e1845-e1859.
•  Rohlman,  D.S.,  Ismail,  A.,  Bonner,  M.R.,  Abdel Rasoul,  G.,  Hendy,  O.,  Ortega Dickey,  L.,  Wang,  K. and  Olson,  J.R. (2019): Occupational pesticide exposure and symptoms of attention deficit hyperactivity disorder in adolescent pesticide applicators in Egypt. Neurotoxicology, 74, 1-6.
•  Ruby,  M.A.,  Nomura,  D.K.,  Hudak,  C.S.,  Mangravite,  L.M.,  Chiu,  S.,  Casida,  J.E. and  Krauss,  R.M. (2008): Overactive endocannabinoid signaling impairs apolipoprotein E-mediated clearance of triglyceride-rich lipoproteins. Proc. Natl. Acad. Sci. USA, 105, 14561-14566.
•  Soltys,  J.,  Yushak,  M. and  Mao-Draayer,  Y. (2010): Regulation of neural progenitor cell fate by anandamide. Biochem. Biophys. Res. Commun., 400, 21-26.
•  Suzuki,  H.,  Tomizawa,  M.,  Ito,  Y.,  Abe,  K.,  Noro,  Y. and  Kamijima,  M. (2013): A potential target for organophosphate insecticides leading to spermatotoxicity. J. Agric. Food Chem., 61, 9961-9965.
•  Suzuki,  H.,  Ito,  Y.,  Noro,  Y.,  Koketsu,  M.,  Kamijima,  M. and  Tomizawa,  M. (2014): Organophosphate agents induce plasma hypertriglyceridemia in mouse via single or dual inhibition of the endocannabinoid hydrolyzing enzyme(s). Toxicol. Lett., 225, 153-157.
•  Szabo,  B. and  Schlicker,  E. (2005): Effects of cannabinoids on neurotransmission. Handb. Exp. Pharmacol., 168, 327-365.
•  Tomiyama,  K. and  Funada,  M. (2011): Cytotoxicity of synthetic cannabinoids found in “Spice” products: the role of cannabinoid receptors and the caspase cascade in the NG 108-15 cell line. Toxicol. Lett., 207, 12-17.
•  Tomiyama,  K. and  Funada,  M. (2014): Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement of cannabinoid CB₁ receptors and apoptotic cell death. Toxicol. Appl. Pharmacol., 274, 17-23.
•  Trezza,  V.,  Campolongo,  P.,  Manduca,  A.,  Morena,  M.,  Palmery,  M.,  Vanderschuren,  L.J. and  Cuomo,  V. (2012): Altering endocannabinoid neurotransmission at critical developmental ages: impact on rodent emotionality and cognitive performance. Front. Behav. Neurosci., 6, 2.
•  Yasuda,  H.,  Huang,  Y. and  Tsumoto,  T. (2008): Regulation of excitability and plasticity by endocannabinoids and PKA in developing hippocampus. Proc. Natl. Acad. Sci. USA, 105, 3106-3111.
•  Ye,  L.,  Cao,  Z.,  Wang,  W. and  Zhou,  N. (2019): New insights in cannabinoid receptor structure and signaling. Curr. Mol. Pharmacol., 12, 239-248.
•  Yu,  C.J.,  Du,  J.C.,  Chiou,  H.C.,  Chung,  M.Y.,  Yang,  W.,  Chen,  Y.S.,  Fuh,  M.R.,  Chien,  L.C.,  Hwang,  B. and  Chen,  M.L. (2016): Increased risk of attention-deficit/hyperactivity disorder associated with exposure to organophosphate pesticide in Taiwanese children. Andrology, 4, 695-705.
•  Wu,  S.Y. and  Casida,  J.E. (1995): Ethyl octylphosphonofluoridate and analogs: optimized inhibitors of neuropathy target esterase. Chem. Res. Toxicol., 8, 1070-1075.