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Toxicology Letters 225 (2014) 153–157 Contents lists available at ScienceDirect Toxicology Letters jou rn al hom epage: www.elsevier.com/locate/toxlet Organophosphate agents induce plasma hypertriglyceridemia in mouse via single or dual inhibition of the endocannabinoid hydrolyzing enzyme(s) Himiko Suzuki a , Yuki Ito a , Yuki Noro a , Mamoru Koketsu b , Michihiro Kamijima a , Motohiro Tomizawa a,c,a Department of Occupational and Environmental Health, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan b Department of Chemistry and Biomolecular Science, Gifu University, Gifu 501-1193, Japan c Faculty of Applied Bioscience, Tokyo University of Agriculture, Setagayaku, Tokyo 156-8502, Japan h i g h l i g h t s Organophosphate insecticide fen- itrothion induces mouse plasma hypertriglyceridemia. The triglyceride elevation is pre- vented by the cannabinoid receptor antagonist AM251. Fenitrothion exposure leads to selec- tive inhibition of the liver fatty acid amide hydrolase. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 29 October 2013 Received in revised form 5 December 2013 Accepted 6 December 2013 Available online 17 December 2013 Keywords: Fatty acid amide hydrolase Fenitrothion Monoacylglycerol lipase Triglyceride a b s t r a c t Diverse serine hydrolases including endocannabinoid metabolizing enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have been suggested as secondary targets for organophos- phate (OP) agents to exert adverse toxic effects such as lipid homeostasis disruption. The goal of this investigation is to verify that a major OP insecticide fenitrothion (FNT) induces plasma hypertriglyc- eridemia through the inhibition of FAAH and/or MAGL in comparison with that elicited by isopropyl dodecylfluorophosphonate (IDFP), a potent FAAH/MAGL inhibitor. Fasted mice were treated intraperi- toneally with FNT or IDFP and were subsequently sacrificed for evaluations of plasma triglyceride (TG) levels and liver FAAH/MAGL activities. Plasma TG levels were significantly enhanced by the FNT or IDFP treatment (1.7- or 4.8-fold, respectively) compared with that of vehicle control. The IDFP expo- sure reduced the liver FAAH and MAGL activities, whereas the FNT exposure led to the preferential FAAH inhibition. The brain acetylcholinesterase was almost unaffected by the FNT or IDFP treatment, thus lead- ing to no neurotoxic sign. Intriguingly, the TG elevations were averted by concomitant administration with the cannabinoid receptor antagonist AM251. The present findings suggest that OP agents induce plasma hypertriglyceridemia in mouse through single or dual inhibition of FAAH or/and MAGL, apparently leading to overstimulation of cannabinoid signal regulating energy metabolism. © 2013 Elsevier Ireland Ltd. All rights reserved. Corresponding author at: Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagayaku, Tokyo 156-8502, Japan. Tel.: +81 3 5477 2529; fax: +81 3 5477 2626. E-mail address: [email protected] (M. Tomizawa). 1. Introduction Organophosphate (OP) agents have been the major insecticides for many decades and are indispensable tools in protecting crops, people, and animals from pest insect attack and disease trans- mission. OP compounds are nerve poison primarily acting at the 0378-4274/$ see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.12.004

Organophosphate agents induce plasma hypertriglyceridemia in mouse via single or dual inhibition of the endocannabinoid hydrolyzing enzyme(s)

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Page 1: Organophosphate agents induce plasma hypertriglyceridemia in mouse via single or dual inhibition of the endocannabinoid hydrolyzing enzyme(s)

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Toxicology Letters 225 (2014) 153– 157

Contents lists available at ScienceDirect

Toxicology Letters

jou rn al hom epage: www.elsev ier .com/ locate / tox le t

rganophosphate agents induce plasma hypertriglyceridemia inouse via single or dual inhibition of the endocannabinoid

ydrolyzing enzyme(s)

imiko Suzukia, Yuki Itoa, Yuki Noroa, Mamoru Koketsub, Michihiro Kamijimaa,otohiro Tomizawaa,c,∗

Department of Occupational and Environmental Health, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, JapanDepartment of Chemistry and Biomolecular Science, Gifu University, Gifu 501-1193, JapanFaculty of Applied Bioscience, Tokyo University of Agriculture, Setagayaku, Tokyo 156-8502, Japan

i g h l i g h t s

Organophosphate insecticide fen-itrothion induces mouse plasmahypertriglyceridemia.The triglyceride elevation is pre-vented by the cannabinoid receptorantagonist AM251.Fenitrothion exposure leads to selec-tive inhibition of the liver fatty acidamide hydrolase.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 29 October 2013eceived in revised form 5 December 2013ccepted 6 December 2013vailable online 17 December 2013

eywords:atty acid amide hydrolaseenitrothiononoacylglycerol lipase

a b s t r a c t

Diverse serine hydrolases including endocannabinoid metabolizing enzymes fatty acid amide hydrolase(FAAH) and monoacylglycerol lipase (MAGL) have been suggested as secondary targets for organophos-phate (OP) agents to exert adverse toxic effects such as lipid homeostasis disruption. The goal of thisinvestigation is to verify that a major OP insecticide fenitrothion (FNT) induces plasma hypertriglyc-eridemia through the inhibition of FAAH and/or MAGL in comparison with that elicited by isopropyldodecylfluorophosphonate (IDFP), a potent FAAH/MAGL inhibitor. Fasted mice were treated intraperi-toneally with FNT or IDFP and were subsequently sacrificed for evaluations of plasma triglyceride (TG)levels and liver FAAH/MAGL activities. Plasma TG levels were significantly enhanced by the FNT orIDFP treatment (1.7- or 4.8-fold, respectively) compared with that of vehicle control. The IDFP expo-

riglyceride sure reduced the liver FAAH and MAGL activities, whereas the FNT exposure led to the preferential FAAHinhibition. The brain acetylcholinesterase was almost unaffected by the FNT or IDFP treatment, thus lead-ing to no neurotoxic sign. Intriguingly, the TG elevations were averted by concomitant administrationwith the cannabinoid receptor antagonist AM251. The present findings suggest that OP agents induceplasma hypertriglyceridemia in mouse through single or dual inhibition of FAAH or/and MAGL, apparentlyleading to overstimulation of cannabinoid signal regulating energy metabolism.

∗ Corresponding author at: Faculty of Applied Bioscience, Tokyo University ofgriculture, 1-1-1 Sakuragaoka, Setagayaku, Tokyo 156-8502, Japan.el.: +81 3 5477 2529; fax: +81 3 5477 2626.

E-mail address: [email protected] (M. Tomizawa).

378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2013.12.004

© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Organophosphate (OP) agents have been the major insecticidesfor many decades and are indispensable tools in protecting crops,people, and animals from pest insect attack and disease trans-mission. OP compounds are nerve poison primarily acting at the

Page 2: Organophosphate agents induce plasma hypertriglyceridemia in mouse via single or dual inhibition of the endocannabinoid hydrolyzing enzyme(s)

1 y Lett

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54 H. Suzuki et al. / Toxicolog

cetylcholinesterase (AChE) as an inhibitor phosphorylating theerine hydroxyl residue of the catalytic triad (Casida, 2009; Casidand Durkin, 2013). Recent attention in OP toxicology has also beenaid to diverse OP-sensitive serine hydrolase secondary targets

ncluding fatty acid amide hydrolase (FAAH) and monoacylglycerolipase (MAGL) (Casida and Quistad, 2004, 2005). FAAH and MAGLre hydrolyzing enzymes of endogenous cannabinoid agonists,nandamide (AEA) and 2-arachidonoylglycerol (2-AG), respec-ively, which activate the cannabinoid type-1 receptor (CB1R).

odulation or overstimulation of the endocannabinoid signalingystem may lead to unfavorable physiological effects: i.e., neurotox-city (Quistad et al., 2001, 2002); hyperactivity (Carr et al., 2011);permatoxicity (Lewis and Maccarrone, 2009; Lewis et al., 2012;oro et al., 2013; Suzuki et al., 2013); energy balance modulation

Cota et al., 2003; Gamage and Lichtman, 2012); and hypoalgesia,atalepsy, or hypomotility (Long et al., 2009; Nomura et al., 2008a;uistad et al., 2006).

A specific concern is given in OP-induced lipid homeostasis dis-uption. The endocannabinoid system plays a pivotal role in energyalance, lipid homeostasis and food intakes (Bluher et al., 2006;olombo et al., 1998; Cota et al., 2003; Engeli et al., 2005; Jbilot al., 2005; Osei-Hyiaman et al., 2005). Actually, an OP agent, iso-ropyl dodecylfluorophosphonate (IDFP) (Fig. 1), enhances plasmariglyceride (TG) levels through inhibition of MAGL activities inhe mouse liver, muscle, and adipose tissues (Ruby et al., 2008).owever, the triggering mechanism underlying the OP-inducedG elevation has not been entirely defined. Therefore, the firstim of the present investigation is to verify that a major OPnsecticide fenitrothion (FNT) (Fig. 1) also induces plasma lipid dis-urbances in mice. The second goal is to clarify the initial event(s)or the OP-induced TG elevation in comparison between actionsf FNT and IDFP. Consequently, this study suggests that the OP-licited plasma hypertriglyceridemia appears to be relevant toingle or dual inhibition of FAAH or/and MAGL perhaps lead-ng to overstimulation of CB1R-mediated signal regulating energy

etabolism.

. Materials and methods

.1. Chemicals

Sources of the chemicals utilized in the present study are listeds follows: FNT and FNT oxon (Fig. 1) from Wako Pure ChemicalsOsaka, Japan); AM251 from Cayman Chemical (Ann Arbor, MI);adiolabeled substrates [14C]AEA and [14C]mono-oleoylglycerolrom American Radiolabeled Chemicals (St. Louis, MO); AEA, and

EA-d4 from Cayman Chemical and Abcam (Cambridge, MA),espectively; 2-arachidonoyl glycerol (2-AG) and 1-O-dodecyl-rac-lycerol (OG) from Cayman Chemical and Santa Cruz BiotechnologySanta Cruz, CA), respectively. IDFP was synthesized according

ig. 1. Chemical structures of OP agents considered in the present investigation. Aajor OP insecticide FNT is transformed by cytochrome P450 to FNT oxon, therefore

nabling to phosphorylate the serine hydroxyl residue at the catalytic triad of diverseerine hydrolases. IDFP is a potent FAAH or MAGL inhibitor (Nomura et al., 2008a;uistad et al., 2006).

ers 225 (2014) 153– 157

to previous report of Segall et al. (2003a,b). For TG and totalcholesterol (T-Chol) assays, enzyme reagent kits, triglyceride E-test and cholesterol E-test, were purchased from Wako PureChemicals.

2.2. Animal studies

Throughout the animal experiments, the study was carried outin accordance with the Guide to Animal Experimentation of NagoyaCity University (approval no. H23M-17). Male ICR mice aged 9weeks were purchased from Japan SLC (Hamamatsu, Japan). Theanimals were housed in cages in the animal room under controlledenvironmental conditions: temperature 23–25 ◦C; relative humid-ity 57–60%; and a 12-h light–dark cycle (lighting: 9:00–21:00).Food and water were provided ad libitum.

Following 1 week of acclimation, the mice were fasted for 4 hand intraperitoneally administered vehicle (dimethyl sulfoxide),FNT (50 mg/kg), IDFP (20 mg/kg), or AM251 (10 mg/kg). In addition,AM251 (10 mg/kg) was simultaneously treated with FNT or IDFP.At the 3 h post-treatment, the fasted animals were sacrificed bydecapitation. Blood was collected into heparinized tubes and thencentrifuged to separate plasma. The resultant plasma and harvestedliver and brain samples were stored at −80 ◦C until analyzed.

2.3. Plasma lipids

The plasma TG and T-Chol levels were assayed spec-trophotometrically based on a glycerol-3-phosphate oxidase-or cholesterol oxidase-peroxidase enzymatic reaction using N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodiumsalt (Allain et al., 1974; Spayd et al., 1978).

2.4. Enzyme assays

FAAH or MAGL activity in mice liver was assayed by hydrolysis ofthe corresponding substrate [14C]AEA or [14C]mono-oleoylglycerol,respectively (55 mCi/mmol for both substrates), according tothe previous methods of Quistad et al. (2001, 2006). In brief,100 mg mouse liver was homogenized in 50 mM Tris–HCl buffer(pH 8.0). The homogenate was centrifuged at 1000 × g and4 ◦C for 10 min and the supernatant was then at 20,000 × gfor 20 min. The 20,000 × g pellet was finally reconstituted inthe Tris buffer. Then, the aliquot was incubated with 1 �M[14C]AEA or [14C]mono-oleoylglycerol for 30 min at 37 ◦C, the enzy-matic reaction was terminated by addition of organic solvent(chloroform:methanol:hexane, 1.25:1.4:1.0) and 200 mM K2CO3.Subsequently, the radioactivity in the aqueous upper phase, as theamount of the [14C]arachidonic acid or [14C]oleic acid producedfrom the enzymatic reaction, was determined by liquid scintilla-tion counter. In vitro potency of FNT oxon as an inhibitor of theFAAH or MAGL in the mouse liver preparation was also evaluated bythe above procedure. Half maximal inhibitory concentration (IC50)values were calculated by iterative nonlinear least-squares regres-sion using Sigmaplot software ver. 8.0 (SPSS, Chicago, IL). Relativeto brain AChE assay, the mouse brain was homogenized in 0.1 MNa2HPO4–HCl (pH 8.0), and then the AChE activity was measuredspectrophotometrically with acetylthiocholine as the substrate anddithionitrobenzoic acid as the chromogen (Quistad et al., 2006).

2.5. LC–MS

Analysis of liver AEA and 2-AG levels was performed on a

LC–MS/MS-8030 system (SHIMADZU, Kyoto, Japan) composed ofa solvent delivery device (LC30AD), an autosampler (SIL-30AC), asystem controller (CBM-20A), and a column thermostat (CTO-20A)based on the method reported by Zoerner et al. (2012) with a slight
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H. Suzuki et al. / Toxicology Letters 225 (2014) 153– 157 155

Fig. 2. Effects of OP treatment (i.p. route) on plasma TG and T-Chol levels inmice (mg/ml ± SD, n = 5–21). Asterisks indicate significant difference (*P < 0.05 or**P < 0.01) between the vehicle and treatment groups or between the exposed groups(indicated by bar with asterisks). Plasma TG levels were significantly elevated bythe 50 mg/kg FNT (1.7-fold) (top left) and 20 mg/kg IDFP treatments (4.8-fold) (topright) relative to the vehicle control. These TG elevations were clearly preventedby simultaneous administration with the cannabinoid receptor antagonist AM251(10 mg/kg). Plasma T-Chol levels were almost identical among the all groups (bot-tom). In our preliminary observations, the plasma TG increase was also evident fromt(

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Table 1Ex vivo inhibition of the liver FAAH and MAGL and the brain AChE caused by FNT orIDFP exposure in mice.

Treatment Enzyme activity (±SD, n = 6–13)

Liver (pmol/mg/min) Brain (nmol/mg/min)

FAAH MAGL AChE

Vehicle 410 ± 62 1800 ± 230 220 ± 37FNT 50 mg/kg 310 ± 39** 1700 ± 51 250 ± 27IDFP 20 mg/kg 12 ± 5** 190 ± 48** 190 ± 30

The FAAH activity was significantly inhibited in the FNT-treated group (76% relativeto the vehicle control) and completely in the IDFP group (3%). In contrast, the MAGLactivity was unaffected by the FNT treatment, however, it was markedly diminishedin the IDFP-exposed group (11%). No discernible change or a slight attenuation inbrain AChE levels was detected in FNT- or IDFP-treated group, consistently leading

groups (23 ± 5.9 and 27 ± 4.1 for FNT and IDFP, respectively). In liver2-AG levels (pg/mg liver ± SD), which are supposed to be regulatedby MAGL, a marked difference was observed between the vehicle

Fig. 3. Potency of FNT oxon as an inhibitor of mouse liver FAAH or MAGL. FAAH orMAGL activity was assayed by hydrolysis of the corresponding substrate [14C]AEA

14

he result of FNT or chlorpyrifos treatment (both at 20 mg/kg, n = 6 for each OP agent)88 ± 29* or 86 ± 26* mg/ml, respectively).

odification. Concisely, liver tissue (50 mg) was homogenized in50 �l 250 mM sucrose–phosphate buffer (pH 7.4). The internaltandards, 4 ng AEA-d4 and 80 ng OG, were added to the 50 �l of theesultant liver preparations before solvent extraction with toluene.fter toluene evaporation under a stream of nitrogen, the samplesere reconstituted in water–methanol (1:3), and the aliquots of

hese solutions were analyzed by LC–MS using a Kinetex 1.7 �mB-C18 100A column (100 × 2.1 mm) (Phenomenex, Torrance, CA).

.6. Statistics

Statistical analysis was determined by one-way analysis of vari-nce followed by Bonferoni or Sidak correction using the SPSS 10.0JChicago, IL). The values went through a logarithmic conversion tottain a normal distribution if they were not normally distributed.

. Results

.1. Plasma lipids levels

Effects of OP treatment on plasma TG and T-Chol levels arehown in Fig. 2. Plasma TG levels (mg/ml ± SD) were markedlylevated by the FNT (110 ± 25, 1.7-fold) and IDFP treatments310 ± 110, 4.8-fold) relative to that of the vehicle control (64 ± 18).G levels by AM251 alone (70 ± 21) showed no significant dif-erence compared with that of the vehicle. These TG elevationsere clearly prevented by simultaneous administration with the

annabinoid receptor antagonist AM251 (73 ± 22 or 150 ± 60ith FNT or IDFP, respectively). Plasma T-Chol levels remainednchanged among all groups (110–120 mg/ml).

to no neurotoxic sign.** Indicates significant difference (P < 0.01) between the vehicle and treated groups

(i.p. route).

3.2. Enzyme activities

The FAAH activities (pmol/mg/min) in the FNT- and IDFP-treatedgroups were 310 and 12, respectively, compared with 410 forthe vehicle control (Table 1). In contrast, the MAGL activities(pmol/mg/min) for vehicle control and FNT-exposed group werealmost identical: i.e., 1800 and 1700, respectively, whereas the IDFPtreatment greatly reduced the MAGL activity (190). No differencein brain AChE levels (nmol/mg/min) was noted in the vehicle andFNT-treated groups (220 and 250, respectively), although a slightinhibition (190) was observed in the IDFP exposure (the FNT andIDFP treatments did not induce any neurotoxic signs).

3.3. Potency of FNT oxon as inhibitor of liver FAAH or MAGL

FNT oxon was more potent against FAAH than MAGL as indi-cated by their IC50 values: i.e., 8.5 or 140 �M against FAAH or MAGL,respectively (Fig. 3).

3.4. AEA and 2-AG levels

Liver AEA levels (pg/mg liver ± SD) (Fig. 4), which may be mod-ulated by FAAH inhibition, did not show any significant differencebetween the vehicle control (26 ± 7.9) and the both OP-exposed

or [ C]mono-oleoylglycerol, respectively (Quistad et al., 2001, 2006). The IC50 value(±SD, n = 3) of FNT oxon is 8.5 ± 0.5 or 140 ± 22 �M against FAAH or MAGL, respec-tively. IDFP is a strong inhibitor of the mouse FAAH or MAGL (IC50 2 or 0.8 nM,respectively) (Quistad et al., 2006).

Page 4: Organophosphate agents induce plasma hypertriglyceridemia in mouse via single or dual inhibition of the endocannabinoid hydrolyzing enzyme(s)

156 H. Suzuki et al. / Toxicology Lett

Fig. 4. Effects of FNT or IDFP exposure on endocannabinoid level in the mouse liver.Fasted mice were treated with vehicle control, FNT (50 mg/kg), or IDFP (20 mg/kg).AEA and 2-AG levels (pg/mg tissue ± SD, n = 6–10) in the mouse liver homogenatewb

cb

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ere measured by LC–MS. Double asterisks indicate significant difference (P < 0.01)etween the vehicle control and treated groups.

ontrol (57 ± 9.9) and IDFP treatment (233 ± 100) (4.1-fold), but notetween the vehicle and FNT exposure (69 ± 15).

. Discussion

The present investigation focuses on an OP-elicited non-eurotoxic effect specifically on the lipid homeostasis and therimary goal is to clarify a mechanism for OP-induced plasmaypertriglyceridemia in mice through the inhibition of FAAH or/andAGL. A recent study, using CB1R- or apolipoprotein (Apo)E-

nockout mice acutely treated with IDFP, has suggested that IDFPnhibits the MAGL in liver, muscle, or adipose tissues, therebylevating the endocannabinoid 2-AG level which subsequentlyveractivates the endocannabinoid signaling system and conse-uently leads to plasma hypertriglyceridemia. The TG elevation isssociated with reduced plasma TG clearance and an accumulationn plasma of ApoE-depleted TG-rich lipoproteins (Ruby et al., 2008).nterestingly, a FAAH-selective inhibitor, UBR597 (a carbamateompound), does not induce the hypertriglyceridemia, although

synthetic and direct CB1R agonist WIN55212-2 also raises thelasma TG levels (Ruby et al., 2008). Therefore these results indi-ate that the FAAH inhibition and the following AEA elevation aloneay not be responsible for the plasma hypertriglyceridemia (Ruby

t al., 2008). Furthermore, plasma T-Chol levels are also elevated1.3- to 1.4-fold) by IDFP treatment in the CB1R-dependent mannerRuby et al., 2008).

In contrast, the present report demonstrates that a major OPnsecticide FNT (and also chlorpyrifos) induces the plasma hyper-riglyceridemia in mouse through single inhibition of FAAH. FNTxon (bioactive metabolite of FNT) is proved to be more potentgainst FAAH than MAGL in mouse liver. Further, IDFP, in con-rast to FNT, compellingly inhibits both of the FAAH and MAGL in

ouse liver, triggering an extraordinary elevation in plasma TGevel. Either FNT- or IDFP-induced plasma hypertriglyceridemia isnambiguously prevented by concomitant administration with theannabinoid receptor antagonist AM251. Because FAAH or MAGLs responsible for hydrolyzing the endocannabinoid AEA or 2-AG,espectively, FNT or IDFP exposure, to block the FAAH and/or MAGL,hould elevate the AEA and/or 2-AG level. Actually, IDFP treatmentarkedly increases the liver 2-AG level. However, unexpectedly,

ignificant alterations in the liver AEA level by FNT or even by IDFPreatment were indiscernible under the present conditions. Thisnanticipated phenomenon may presumably be attributable to thehysiological lability of AEA. Indeed, the half-life of AEA in vivo haseen suggested to be on the order of minutes in the brain and otherissues (Willoughby et al., 1997). Moreover, a clear AEA elevation

y FAAH blockade is also undetectable or ambiguous in the brainr testis (Nomura et al., 2008a; Nomura and Casida, 2011; Quistadt al., 2001; Suzuki et al., 2013). Collectively, these observationseveal a reasonable possibility that the FAAH and AEA pathway (in

ers 225 (2014) 153– 157

addition to the MAGL and 2-AG route) also has relevance to theOP-induced plasma hypertriglyceridemia. On the other hand, incontradiction to the finding of Ruby et al. (2008), FNT or even IDFPexposure in the present study (using different mouse stain) doesnot induce plasma T-Chol elevation. Segall et al. (2003b) found thatIDFP is a potent inhibitor of [3H]CP-55,940 (a CB1R agonist) bind-ing to the mouse brain membranes. However, Nomura et al. (2008b)subsequently reported that apparent direct OP inhibition of CB1Ragonist binding may be due instead to metabolic stabilization of 2-AG in brain membranes as the actual inhibitor. Moreover, the FNTand IDFP treatments do not inhibit or faintly affect, respectively,the brain AChE, in this consequence leading to no neurotoxicity.

In summary, the present investigation provides an insight intothe molecular events for the OP-elicited plasma lipid disturbanceattributable to single or dual inhibition of FAAH or/and MAGLapparently leading to overstimulation of the endocannabinoid sys-tem regulating energy metabolism. Furthermore, a transient orsustained exposure of OP pesticides may potentially cause lipidhomeostasis disruption.

Conflict of interest statement

None declared.

Acknowledgments

This work was funded in part by JSPS KAKENHI grant number24659303 to M.T. The authors thank Shota Fukuno (Gifu University)for his technical assistance.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2013.12.004.

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