Lisa Chedik

Arnaud Bruyère

Astrid Bacle

Sophie Potin

Marc Le Vée

Dr Olivier Fardel
email: olivier.fardel@univ-rennes1.fr

Arnaud Bruyere

Interactions of pesticides with membrane drug transporters implications for toxicokinetics and toxicity

-Various pesticides belonging to diverse chemical classes, including organochlorines, pyrethroid and organophosphorus pesticides, can inhibit the activity of ATP-binding cassette or solute carrier drug transporters. Activities of the drug efflux pump P-glycoprotein and of the uptake organic cation transporter (OCT) 1 and OCT2 have notably been demonstrated to be impacted by pesticides.

-Concentrations of pesticides that block in vitro transporter activities (in the 1-100 µM range) are commonly much higher than those reached in humans in response to environmental exposure, making the in vivo relevance of transporter inhibition unlikely for most of pesticides.

-A limited number of pesticides has been shown to regulate expression of some drug transporters, notably in hepatic cells.

-Pesticides can additionally be substrates for drug transporters, which may contribute to their toxicokinetics and may also govern their toxicity.

-Overall, pesticides remain poorly characterized with respect to relationships with transporters. A more extensive and systematic characterization of the interactions of pesticides with drug transporters is consequently required. Polymorphisms in transporters,

Introduction

Drug transporters are plasma membrane proteins, implicated in the cellular uptake or efflux of xenobiotics. They belong to the solute carrier (SLC) or ATP binding cassette (ABC) transporter superfamilies [START_REF] Doring | Phase 0 and phase III transport in various organs: combined concept of phases in xenobiotic transport and metabolism[END_REF]. Human SLC transporters are usually implicated in the uptake of drugs, through mediating facilitated diffusion or secondary active transport across the plasma membrane [START_REF] Colas | SLC Transporters: Structure, Function, and Drug Discovery[END_REF], whereas ABC transporters are efflux pumps, responsible for the primary active export of drugs out of cells, through their intrinsic ATPase activity [START_REF] Schinkel | Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview[END_REF]. Both SLC and ABC transporters are expressed at anatomical/histological sites important for xenobiotic disposition, including intestine, blood-tissue barriers like the blood-brain barrier, liver and kidney [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. By this way, drug transporters play a major role in the different steps of pharmacokinetics, including absorption, distribution and hepatic and renal elimination [START_REF] Szakacs | The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME-Tox)[END_REF][START_REF] Shitara | Transporters as a determinant of drug clearance and tissue distribution[END_REF].

They may consequently influence drug efficacy as well as drug toxicity. Moreover, drugmediated inhibition of their activity can cause pharmacokinetic-based drug-drug interactions [START_REF] Konig | Transporters and drug-drug interactions: important determinants of drug disposition and effects[END_REF]. This has led drug regulatory agencies to edict guidances for the study of putative interactions of new molecular entities with clinically-relevant drug transporters [START_REF] Prueksaritanont | Drug-drug interaction studies: regulatory guidance and an industry perspective[END_REF].

In addition to drugs, environmental pollutants can interact with transporters, i.e., they inhibit and/or are handled by them [START_REF] Abu-Qare | A role for P-glycoprotein in environmental toxicology[END_REF][START_REF] Fardel | Environmental chemicals as substrates, inhibitors or inducers of drug transporters: implication for toxicokinetics, toxicity and pharmacokinetics[END_REF]11], which may have consequences in terms of the toxicokinetics and toxicity of pollutants. Environmental chemicals may additionally regulate levels of transporter expression, i.e., they notably enhance transporter expression, which may in turn result in increased transport activity [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF]. Among pollutants which have to be A c c e p t e d M a n u s c r i p t considered for putative interactions with drug transporter activity and/or expression, chemical organic pesticides are likely major ones. Indeed, these compounds, defined as any chemical or mixture of chemical intended for preventing, destroying, repelling, or mitigating pests, and belonging to diverse chemical classes (See Table 1 for a schematic presentation of main classes of organic pesticides), are largely used for occupational (agriculture…) or domestic purposes, for notably their insecticide, herbicide, fungicide or rodenticide properties. They are consequently widely distributed in the environment and humans can be exposed to them in a major way, through the oral, dermal or pulmonary route. Such exposures are thought to promote the development of various pathologies, including cancers, neurologic diseases and endocrine disruption, owing to the diverse toxic effects of pesticides [13,[START_REF] Mostafalou | Pesticides: an update of human exposure and toxicity[END_REF]. In the present review, we have summarized the current knowledges about interactions of human drug transporters with organic pesticides, with special emphasis on possible consequences in terms of pesticide toxicokinetics and toxicity.

Interactions of pesticides with ABC transporter activities

Interactions with P-glycoprotein (P-gp) activity

P-gp, encoded by multidrug resistance gene 1 (MDR1/ABCB1), was historically characterized as an efflux pump for various structurally-unrelated anticancer drugs [15], thus conferring multidrug resistance to cancer cells (See Table 2 for a summary of main drug transporters). Pgp also handles many non-anticancer drugs like the cardiotonic drug digoxin. It is physiologically expressed at various blood-tissue barriers and in absorptive or excretory organs such as the gut, the liver and the kidney; by this way, P-gp plays a major role in pharmacokinetics [START_REF] Zhou | Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition[END_REF].

Various pesticides, belonging to diverse classes, have been shown to inhibit human Pgp activity. This has been mainly demonstrated through analyzing their effects on cellular A c c e p t e d M a n u s c r i p t accumulation or efflux of radiolabeled or fluorescent reference P-gp substrates in P-gp expressing cells [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF]18]. The threshold of at least 50% inhibition of P-gp activity by pesticides used at 100 µM or 250 µM concentration can be retained for considering a pesticide as a P-gp inhibitor. Using this criteria, the organochlorine insecticides chlordecone, heptachlor and heptachlor epoxide, the organophosphate insecticides azinphos-ethyl, chlorpyrifos, coumaphos, phosalone, chlorthiophos, dicapthon, parathion, diazinon and fenamiphos, the avermectins ivermectin, abamectin and emamectin benzoate and the fungicide clotrimazole have been identified as inhibitors of human P-gp [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF][START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF]19,20,21,22,23,[START_REF] Dalzell | Characterization of multidrug transportermediated efflux of avermectins in human and mouse neuroblastoma cell lines[END_REF]. Tetrachlorohydroquinone, a major metabolite of pentachlorophenol, as well as the insecticide hydramethylnon and the fungicide propiconazole and its metabolites, also block Pgp-mediated transport [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF][START_REF] Mazur | P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticidedrug interactions[END_REF]. The dibenzoylhydrazines tebufenozide and methoxyfenozide, which exert their insecticide activity through permanent activation of the ecdysone receptor, constitute additional P-gp inhibitors; they decrease P-gp-mediated transport of the antiarrhythmic agent quinidine [START_REF] Miyata | In vitro and in vivo evaluations of the Pglycoprotein-mediated efflux of dibenzoylhydrazines[END_REF]. With respect to endosulfan, it was found to inhibit human P-gp in three studies [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF]21,[START_REF] Sreeramulu | Interaction of insecticides with mammalian Pglycoprotein and their effect on its transport function[END_REF], but not in two other studies [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF]20], which may reflect differences in the various P-gp activity assays used in these studies. The herbicides acetochlor, alachlor, metolachlor and metazachlor, unlike dimetachlor, propachlor and prynachlor, have also been described as P-gp inhibitors [START_REF] Oosterhuis | Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins[END_REF]. Half maximal inhibitory concentrations (IC 50 ) are available only for a few pesticides inhibiting P-gp. In the study of Bain and al. [19], they range from 7.3 µM (hydramethylnon) to 229.8 µM (parathion). IC 50 values for P-gp inhibition by endosulfan, phosalone and propiconazole are however lower, i.e., around 3 µM [21]; similarly, avermectins block P-gp-mediated efflux at low concentrations, around 0.2-0.6 µM [START_REF] Dalzell | Characterization of multidrug transportermediated efflux of avermectins in human and mouse neuroblastoma cell lines[END_REF]. Other pesticides such as the carbamates aldicarb, aldoxycarb, aminocarb, carbaryl and propoxur, the dithiocarbamate maneb, the organophosphate pesticides mevinphos, dialifos and phosmet, the phenoxy herbicide A c c e p t e d M a n u s c r i p t fluazifop-butyl and various pyrethroids (allethrin, bifenthrin, β-cyfluthrin, λ-cyhalothrin, βcypermethrin, deltamethrin, esfenvalerate, fenpropathrin, fluvalinate, cis-permethrin, transpermethrin, resmethrin, tefluthrin and tetramethrin), failed to inhibit human P-gp activity to a significant extent [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF][START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF]18,19]. It was also the case for atrazine, paraquat, propiconazole, vinclozolin and the organochlorine insecticides 2,4'-dichlorodiphenyltrichloroethane (DDT), chlordane and toxaphene [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF][START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF], as well as for cyperquat [START_REF] Staal | Interactions of 1-methyl-4-phenylpyridinium and other compounds with P-glycoprotein: relevance to toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine[END_REF], a bipyridil compound also known as 1-methyl-4-phenylpyridinium (MPP+), which has been used as an herbicide in the past. Additional organochlorine pesticides such as 4,4'-DDT, dieldrin, lindane, methoxychlor and mirex did not block human P-gp-mediated transport [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF], whereas they inhibited that of mouse P-gp [11], suggesting inter-species differences with respect to interaction of P-gp with pesticides.

2,4'-DDT and 4,4'-DDT as well as their metabolite dichlorodiphenyldichloroethane (DDE) nevertheless decreased human P-gp ATPase activity [START_REF] Bircsak | Inhibition of human MDR1 and BCRP transporter ATPase activity by organochlorine and pyrethroid insecticides[END_REF]. For major classes of pesticides, the relative percentage of chemicals inhibiting drug transporters, including P-gp, as well as the total number of compound tested against transporter activity, according to published data, are indicated in Table 3.

Transport of pesticides by human P-gp has been investigated through direct analysis of their cellular accumulation or efflux in P-gp-positive cells, and also by measuring the stimulation/modulation of P-gp ATPase activity, which constitutes an indirect argument for Pgp-mediated transport. According to experimental data, there is only limited evidence for pesticides as substrates for the efflux pump (See Table 4 for a summary of pesticides handled by drug transporters). P-gp has been demonstrated to handle paraquat and thus protects against cytotoxicity induced by this pesticide [START_REF] Wen | Induction and activation of P-glycoprotein by dihydroxylated xanthones protect against the cytotoxicity of the P-glycoprotein substrate paraquat[END_REF]32,[START_REF] Vilas-Boas | Development of novel rifampicin-derived Pglycoprotein activators/inducers. synthesis, in silico analysis and application in the RBE4 cell model, using paraquat as substrate[END_REF][START_REF] Zerin | Protective effect of methylprednisolone on paraquat-induced A549 cell cytotoxicity via induction of efflux transporter, Pglycoprotein expression[END_REF][START_REF] Dinis-Oliveira | P-glycoprotein induction: an antidotal pathway for paraquat-induced lung toxicity[END_REF], but may fail to contribute to the systemic pharmacokinetics of this herbicide in mice [START_REF] Lacher | Absence of P-glycoprotein transport in the pharmacokinetics and toxicity of the herbicide paraquat[END_REF]. The avermectins ivermectin and selamectin are substrates for P-gp [START_REF] Brayden | Avermectin transepithelial transport in MDR1-and MRPtransfected canine kidney monolayers[END_REF][START_REF] Lespine | Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)[END_REF], notably in mouse where the pump limits the brain penetration of ivermectin [START_REF] Schinkel | Disruption of the mouse mdr1a Pglycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs[END_REF]. Endosulfan may be a weak substrate for P-gp [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF]. The organophosphate pesticide diazinon as well as rotenone have also been suggested to be P-gp substrates, because they stimulated P-gp ATPase activity, unlike dieldrin, endosulfan, ivermectin and maneb, without however inhibiting P-gp-mediated transport of rhodamine 123 [20]. Dibenzoylhydrazine insecticides stimulated P-gp ATPase activity, but are in fact poorly transported by the pump [START_REF] Miyata | In vitro and in vivo evaluations of the Pglycoprotein-mediated efflux of dibenzoylhydrazines[END_REF]. P-gp ATPase activity is additionally stimulated by methylparathion, cypermethrin and fenvalerate [START_REF] Sreeramulu | Interaction of insecticides with mammalian Pglycoprotein and their effect on its transport function[END_REF], whereas P-gp seems to be not involved in resistance of intestinal Caco-2 cells to propoxur, thus likely discarding the hypothesis that the carbamate may be transported by the pump [START_REF] Yazdian | Evaluation of the expression of Pglycoprotein in propoxur-resistant Caco-2 cells[END_REF]. Propiconazole was also not handled by P-gp [START_REF] Mazur | P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticidedrug interactions[END_REF], as well as deltamethrin, cis-permethrin and trans-permethrin [START_REF] Zastre | Lack of P-glycoprotein-mediated efflux and the potential involvement of an influx transport process contributing to the intestinal uptake of deltamethrin, cis-permethrin, and trans-permethrin[END_REF], the rodenticide anti-vitamin K warfarin [42] or the herbicide cyperquat/MPP+ [START_REF] Staal | Interactions of 1-methyl-4-phenylpyridinium and other compounds with P-glycoprotein: relevance to toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine[END_REF][START_REF] Martel | Characterization of the efflux of the organic cation MPP+ in cultured rat hepatocytes[END_REF][START_REF] Martel | Characterization of the transport of the organic cation [3H]MPP+ in human intestinal epithelial (Caco-2) cells[END_REF].

Interactions with multidrug resistance-associated protein (MRP/ABCC) activities

MRPs constitute a group of ABC transporters, comprising nine members in humans. Seven of these nine MRPs, i.e., MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5), MRP7 (ABCC10) and MRP8 (ABCC11), have been unambiguously demonstrated to transport drugs, especially anionic drugs [START_REF] Slot | Mammalian multidrug-resistance proteins (MRPs)[END_REF]. MRP1 was historically the first identified MRP; it was initially characterized as an efflux transporter for anticancer drugs overexpressed in drug-resistant cancer cells. MRP1 was next demonstrated to exhibit a broad tissue distribution and to transport a wide range of xenobiotics, including anionic drugs and drug conjugates [START_REF] Cole | Multidrug resistance protein 1 (MRP1, ABCC1), a "multitasking" ATPbinding cassette (ABC) transporter[END_REF]. MRP2, like MRP1, primarily transports organic anions and is expressed in various tissues, especially in the liver, kidney and gastrointestinal tract [START_REF] Keppler | Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy[END_REF] [START_REF] Malofeeva | Modulation of the ATPase and transport activities of broad-acting multidrug resistance factor ABCC10 (MRP7)[END_REF], as well as MRP8, notably towards nucleoside analogs [START_REF] Oguri | MRP8/ABCC11 directly confers resistance to 5fluorouracil[END_REF],

The avermectins abamectin, emamectin and ivermectin have been reported to inhibit MRP activity; the corresponding IC 50 values are around 1.5-2.0 µM and have been determined

through measuring MRP-mediated efflux of the dye glutathione methylfluorescein in human neuronal SH-SY5Y cells [START_REF] Dalzell | Characterization of multidrug transportermediated efflux of avermectins in human and mouse neuroblastoma cell lines[END_REF]. The fact that ivermectin inhibited MRP1, MRP2 and MRP3

ATPase activities after stimulation by their respective activators [START_REF] Lespine | Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)[END_REF] fully supports this conclusion. Whether ivermectin may be a good substrate for MRP1 remains however to be established, because conflicting data about this point have been reported [START_REF] Brayden | Avermectin transepithelial transport in MDR1-and MRPtransfected canine kidney monolayers[END_REF][START_REF] Lespine | Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)[END_REF]. The pyrethroids allethrin and tetramethrin, unlike bifenthrin, β-cyfluthrin, λ-cyhalothrin, βcypermethrin, deltamethrin, esfenvalerate, fenpropathrin, fluvalinate, cis-permethrin, transpermethrin, resmethrin and tefluthrin, were found to inhibit MRP activity in hepatoma MRP2expressing HuH-7 cells, with IC 50 values around 40-50 µM [18]. The organochlorine pesticides endosulfan, chlordane, heptachlor and chordecone also blocked MRP-mediated efflux in HuH-7 cells, whereas 4,4'-DDT, 2,4'-DDT, dieldrin and lindane were without effect [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF]. None of the chloroacetanilides acetochlor, alachlor, dimetachlor, metazachlor, metolachlor, propachlor and prynachlor interacts with MRP1 or MRP2; MRP1 was however demonstrated to transport an important intermediate of the acetochlor detoxification pathway [START_REF] Oosterhuis | Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins[END_REF]. MRP1 additionally handles the chloroacetanilide herbicide methoxychlor and confers resistance to the organophosphorus insecticide fenitrothion and the carbamate herbicide chlorpropham, suggesting that these two pesticides may also be substrates [START_REF] Tribull | The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury[END_REF].

Interactions with breast cancer resistance protein (BCRP/ABCG2) activity

BCRP is an ABC transporter handling both anticancer drugs and non-anticancer drugs, like statins, as well as environmental chemicals like carcinogenic heterocyclic aromatic amines A c c e p t e d M a n u s c r i p t

[51] and endogenous compounds like urate [START_REF] Matsuo | Common defects of ABCG2, a high-capacity urate exporter, cause gout: a function-based genetic analysis in a Japanese population[END_REF]. It is expressed at blood-tissue barriers and in gut and excretory organs like liver and kidney. BCRP is also present in stem cells. . The Hoechst H33342 accumulation assay also indicated that thirteen widely-used pesticidal active substances including the fungicides azoxystrobin, dimethomorph, dithianon and tolclofos-methyl, the benzidizamole carbendazim, the organophosphate pesticides chlorpyrifos and dimethoate, the herbicides chlormequat, diflufenican and ioxynil, the carbamates methiocarb and propamocarb and the sulfonylurea herbicide rimsulfuron are likely inhibitor of rabbit BCRP; no such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, imazalil (also known as enilconazole), glyphosate and thiacloprid [START_REF] Halwachs | Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model[END_REF]. BCRP was additionally found to transport the rodenticide warfarin [START_REF] Yang | R-and S-Warfarin Were Transported by Breast Cancer Resistance Protein: From In Vitro to Pharmacokinetic-Pharmacodynamic Studies[END_REF],

whereas ivermectin is unlikely to be a substrate for the pump [START_REF] Geyer | Brain penetration of ivermectin and selamectin in mdr1a,b P-glycoprotein-and bcrp-deficient knockout mice[END_REF], even if it inhibits its activity, with a rather low IC 50 value (2.2 µM) [START_REF] Muenster | Characterization of substrates and inhibitors for the in vitro assessment of Bcrp mediated drug-drug interactions[END_REF].

Interactions of pesticides with SLC drug transporter activities

Interactions with activity of SLC drug transporters handling organic cations

SLC drug transporters transporting organic cations mainly correspond to organic cation transporter (OCT) 1 (SLC22A1), OCT2 (SLC22A2) and OCT3 (SLC22A3), acting as A c c e p t e d M a n u s c r i p t electrogenic and membrane potential-sensitive diffusional transporters, and to multidrug and toxin extrusion (MATE) protein 1 (SLC47A1) and MATE2-K (SLC47A2) (a functionally active isoform of MATE2), acting as electroneutral, sodium-independent and pH-dependent proton antiporters [START_REF] Nies | Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy[END_REF]. OCT1 and MATE1 are located at the sinusoidal and canalicular poles of hepatocytes, respectively, whereas OCT2 and MATE2-K are found at the basolateral and apical membranes of renal proximal tubular cells, respectively. OCT3 has a wide tissue distribution. OCT1, OCT2, MATE1 and MATE2-K handle cationic drugs, including metformin and oxaliplatin [START_REF] Yonezawa | Organic cation transporter OCT/SLC22A and H(+)/organic cation antiporter MATE/SLC47A are key molecules for nephrotoxicity of platinum agents[END_REF]. Endogenous compounds such as neurotransmitters are also transported by OCTs.

With a threshold of at least 50% inhibition of transport activity when used at 100 µM for being considered as a transporter inhibitor, the organochlorine pesticides endosulfan, chlordane, heptachlor, dieldrin and lindane, unlike 2-4'-DDT, 4'-DDT and chlordecone, were found to inhibit OCT1 activity in human hepatoma HepaRG cells, with IC 50 values of 0.9 µM (dieldrin) and 1.5 µM (lindane) [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF]. The pyrethroids allethrin, imiprothrin, prallethrin and tetramethrin and the organophosphorus pesticides fenamiphos, fenitrothion, malathion, methyl parathion, parathion, phosmet, profenofos and propetamphos also blocked OCT1 activity, as well as that of OCT2, in HEK293 cells overexpressing these transporters [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]; respectively. These two pyrethroids, which also blocked OCT1-mediated uptake of the endogenous substrate dopamine, however failed to trans-stimulate OCT1 activity, indicating that they are unlikely to be substrates for OCT1 [18] (Table 4). Analysis of accumulation of these two pesticides into control (MOCK) HEK293 cells and OCT1-transduced HEK293 cells by liquid chromatography tandem-mass spectrometry (LC-MS/MS) fully supports this conclusion, through indicating similar levels of pesticides in control HEK293-MOCK cells and OCT1-positive counterparts (Fig. 1A). Allethrin and tetramethrin also similarly accumulated in MOCK-and OCT2-transduced HEK293 cells (Fig. 1A), thus discarding a transport of these two pesticides by OCT2. By contrast, OCT1-transduced cells displayed increased accumulation of the OCT1 reference substrate DAPI when compared to control MOCK-transduced HEK293 cells (Fig. 1B); in the same way, OCT2-transduced cells exhibited increased accumulation of the OCT2 reference substrate rhodamine 123 (Fig. 1B).

MATE1
Moreover, the OCT1 inhibitor verapamil failed to enhance allethrin and tetramethrin accumulation in OCT1-transduced HEK293 cells, whereas it markedly increased that of DAPI (Fig. 1). The OCT2 inhibitor amitriptyline also enhanced cellular level of rhodamine 123 in OCT2-transduced HEK293 cells, but not those of allethrin and tetramethrin (Fig. 1). Like allethrin and tetramethrin, fenamiphos and phosmet are not transported by OCT1 or OCT2 (Table 4) [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]. By contrast, cyperquat/MPP+ has been demonstrated to be transported by human OCT1, OCT2, OCT3, MATE1 and MATE2-K [START_REF] Gorboulev | Cloning and characterization of two human polyspecific organic cation transporters[END_REF][START_REF] Wu | Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain[END_REF][START_REF] Otsuka | A human transporter protein that mediates the final excretion step for toxic organic cations[END_REF][START_REF] Masuda | Identification and functional characterization of a new human kidney-specific H+/organic cation antiporter, kidney-specific multidrug and toxin extrusion 2[END_REF]. MPP+ concomitantly inhibits the transport of the mutagenic vital dye ethidium by OCT1 and OCT2 [START_REF] Lee | Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule[END_REF]. OCT2 and MATE1, unlike OCT1 and OCT3, handle paraquat under its native N,N′-dimethyl-4,4′bipyridinium dichloride form, which harbors two cationic charges [START_REF] Chen | Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family[END_REF]. The monocationic radical form of PQ, coming from redox cycling with cellular diaphorases such as NADPH oxidase and nitric oxide synthase, has nevertheless been shown to be a substrate for mouse Oct3 [START_REF] Rappold | A study reporting the role of the SLC transporter OCT3 in neurotoxicity of paraquat 67. Obaidat A, Roth M, Hagenbuch B. The expression and function of organic anion transporting polypeptides in normal tissues and in cancer[END_REF].

A c c e p t e d M a n u s c r i p t

Interactions with activity of SLC drug transporters handling organic anions

There are two main families of SLC drug transporters handling organic anions. The first one corresponds to organic anion transporting polypeptides (OATPs/SLCOs), notably OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3) and OATP2B1 (SLCO2B1) [67]; the second one is that of organic anion transporters (OATs), comprizing OAT1 (SLC22A6) and OAT3 (SLC22A8) [START_REF] Wang | Renal organic anion transporters (SLC22 family): expression, regulation, roles in toxicity, and impact on injury and disease[END_REF]. OATP1B1 and OATP1B3 are specifically located at the sinusoidal pole of hepatocytes;

OAT1 and OAT3 are present at the basolateral pole of proximal tubular cells, whereas OATP2B1 is more widely distributed. Substrates for OATPs include anionic drugs and endogenous compounds, such as bile acids, bilirubin, prostaglandins and hormones, like thyroxine and steroid conjugates [START_REF] Konig | Uptake transporters of the human OATP family: molecular characteristics, substrates, their role in drug-drug interactions, and functional consequences of polymorphisms[END_REF]. OATs transport anionic compounds, including drugs such as penicillins and cephalosporins, endogenous compounds such as estrone-3-sulfate and environmental contaminants such as perfluorooctanoic acid [START_REF] Burckhardt | Drug transport by Organic Anion Transporters (OATs)[END_REF].

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is a common substrate for human renal OAT1 and OAT3, but its transport by OAT1 is markedly greater than that by OAT3 [START_REF] Nozaki | Characterization of the uptake of organic anion transporter (OAT) 1 and OAT3 substrates by human kidney slices[END_REF]. The organochlorine pesticides endosulfan, chlordane, heptachlor, dieldrin, lindane, 2-4'-DDT, 4-4'-DDT and chlordecone failed to inhibit OATP activity in human hepatoma HepaRG cells [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF]. With respect to pyrethroids, allethrin and tetramethrin inhibited metasystox, whereas dichlorvos, fenitrothion, methamidophos, methyl parathion, monocrotophos, parathion, phosmet, propetamphos and temephos were without effect [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF].

No organophosphorus pesticide blocked OAT1. Activity of OATP1B1 was inhibited by profenofos and temephos, whereas that of OATP2B1 was cis-stimulated by fenamiphos, malathion, parathion, phosmet and profenofos [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF].

Regulation of drug transporter expression by pesticides

Pesticides have previously been shown to regulate expression of drug detoxifying proteins such as hepatic cytochromes P-450 (CYPs) [START_REF] Hodgson | Human metabolic interactions of environmental chemicals[END_REF][START_REF] Abass | Characterization of human cytochrome P450 induction by pesticides[END_REF]. Indeed, pesticides are known activators of nuclear receptors like pregnane X receptor (PXR) and constitutive androstane receptor (CAR) [START_REF] Kretschmer | CAR and PXR: xenosensors of endocrine disrupters?[END_REF], acting as xenobiotic-sensing receptors regulating expression of CYPs. By this way, several pesticides among organophosphate chemicals, pyrethroids, carbamates, organochlorines insecticides and phenylurea compounds up-regulated CYP3A4 and CYP2B6 expression in hepatocytes [START_REF] Abass | Characterization of human cytochrome P450 induction by pesticides[END_REF]. Drug transporters are also targets for nuclear receptors in human and/or rodent cells [START_REF] Staudinger | Regulation of drug transporter gene expression by nuclear receptors[END_REF]. Indeed, PXR activation induces expression of P-gp in intestinal cells [START_REF] Geick | Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin[END_REF]. It also enhances levels of MRP2 in primary human hepatocytes [START_REF] Kast | Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid Xactivated receptor, and constitutive androstane receptor[END_REF],

MRP3 in human hepatoma HuH-7 cells [START_REF] Teng | Induction of ABCC3 (MRP3) by pregnane X receptor activators[END_REF], Bcrp in mouse Sertoli cells [START_REF] Whyte-Allman | Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier[END_REF] and OCT1 in chronic myeloid leukemia cells [START_REF] Austin | Evidence that the pregnane X and retinoid receptors PXR, RAR and RXR may regulate transcription of the transporter hOCT1 in chronic myeloid leukaemia cells[END_REF]. CAR additionally up-regulates P-gp in CAR-transfected hepatoma HepG2 cells [START_REF] Cerveny | Valproic acid induces CYP3A4 and MDR1 gene expression by activation of constitutive androstane receptor and pregnane X receptor pathways[END_REF] and intestinal LS174 cells [START_REF] Burk | A role for constitutive androstane receptor in the regulation of human intestinal MDR1 expression[END_REF]. CAR is also implicated in regulation of MRP2 in primary human hepatocytes [START_REF] Kast | Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid Xactivated receptor, and constitutive androstane receptor[END_REF], Bcrp in mouse and rat brain capillaries [START_REF] Wang | Constitutive androstane receptor-mediated upregulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier[END_REF] and Mrp4 in mouse liver [START_REF] Wagner | CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice[END_REF]. Another nuclear receptor, i.e., the farnesoid X receptor (FXR), increases hepatic expression of MRP2 [START_REF] Kast | Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid Xactivated receptor, and constitutive androstane receptor[END_REF] and of bile salt export pump (BSEP/ABCB11) [START_REF] Ananthanarayanan | Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor[END_REF], a canalicular hepatic transporter involved in biliary acid elimination.

Pesticides activating drug sensing receptors are consequently susceptible to induce expression of drug transporters. In agreement with this hypothesis, the organochlorine A c c e p t e d M a n u s c r i p t insecticides chlordane, heptachlor, dieldrin, lindane, 2,4'-DDT and chlordecone, which are known agonists of PXR [START_REF] Lemaire | A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides[END_REF], increased mRNA expression of P-gp, MRP2 and BCRP in hepatic HepaRG cells when used at 10 µM [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF]. In the same way, chlorpyrifos, which notably activates PXR and CAR [86, 87], enhanced expression of P-gp and BCRP in human villous cytotrophoblast cells when used at concentration 10-100 µM [START_REF] Ridano | Impact of chlorpyrifos on human villous trophoblasts and chorionic villi[END_REF]. The exact role played by PXR and/or CAR in this ABC transporter up-regulation in placental cells remains however unclear because the expression of PXR and CAR is very low in the human placenta [START_REF] Geenes | Characterisation of the nuclear receptors FXR, PXR and CAR in normal and cholestatic placenta[END_REF]. PXR and CAR also contribute to the impact of fipronil on hepatic expression of transporters like MRP2 and MRP3 [START_REF] Roques | The nuclear receptors pregnane X receptor and constitutive androstane receptor contribute to the impact of fipronil on hepatic gene expression linked to thyroid hormone metabolism[END_REF]. Nuclear receptors are additionally likely to participate to regulation of transporters (P-gp, BSEP, BCRP or OATP1B1) in primary human hepatocytes exposed to chemicals from the ToxCast320 chemical library, comprising many pesticide active ingredients [START_REF] Rotroff | A study analyzing the effects of pesticides on drug transporter expression in primary human hepatocytes 92. Menez C, Mselli-Lakhal L, Foucaud-Vignault M, et al. Ivermectin induces Pglycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line[END_REF]. Nuclear-receptor independent ways of drug transporter regulation by pesticides have also been described. Thus, ivermectin induced P-gp expression through mRNA stabilization in murine hepatocyte cell line [92]. The sinusoidal sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1), which is not known to be regulated by PXR or CAR, exhibited decreased mRNA expression in human hepatoma HepaRG cells exposed to organochlorines [START_REF] Bucher | A study describing the interactions of organochlorine pesticides with drug transporter activity and expression 13[END_REF]. Finally, it is noteworthy that transporter induction may result from chronic exposure to pesticides. Thus, P-gp levels are increased in response to repeated exposure of intestinal Caco-2 cells to the organophosphophorus pesticide diazinon [START_REF] Lecoeur | Effect of organophosphate pesticide diazinon on expression and activity of intestinal P-glycoprotein[END_REF].

Pesticides as modulators of drug transporter activity or expression: implications for pesticide toxicity

Toxicity due to transporter activity inhibition by pesticides

Inhibition of ABC or SLC drug transporter activities by a chemical (called the perpetrator) is susceptible to cause alterations of pharmacokinetics of a drug (called the victim) substrate for A c c e p t e d M a n u s c r i p t the incriminated transporter, finally leading to drug toxicity (in the case of increased drug plasma concentration) or drug inefficacy (in the case of decreased drug plasma concentration).

In addition, transport of endogenous substrates may be impaired, which may lead to toxicity, like cholestasis due to inhibition of the bile salt transporter BSEP [START_REF] De Lima | Hepatobiliary transporters in druginduced cholestasis: a perspective on the current identifying tools[END_REF], or endocrine disruption linked to altered elimination of hormones. For such effects, a key point to take into account is whether the perpetrator, whose inhibitory effects towards transporter activity are commonly initially demonstrated in in vitro assays, reaches in vivo concentrations efficient against the considered transporter. When the perpetrator is a drug, its in vitro IC 50 value has to be confronted to its maximum unbound plasma concentration (I 1 ) and to its maximal theoretical gastrointestinal concentration (I 2 ), calculated as the oral dose in a volume of 250 mL; according to 2012 FDA guidance for drug-drug interactions, transporter inhibition may be clinically achievable when I 1 /IC 50 ≥0.1 and/or I 2 /IC 50 ≥10 [START_REF] Zhang | A regulatory viewpoint on transporter-based drug interactions[END_REF]. The application of such thresholds for pesticides may indicate that in vivo inhibition of drug transporter activity in response to environmental exposure to pesticides is very unlikely for most of them. Indeed, in vitro IC 50 values of pesticides inhibiting transporter activity are usually in the 1-100 µM range (see above), whereas their plasma concentrations are often in the 1-100 nM range in humans [START_REF] Glynn | Serum concentrations of organochlorines in men: a search for markers of exposure[END_REF][START_REF] Leng | Pyrethroids used indoors--biological monitoring of exposure to pyrethroids following an indoor pest control operation[END_REF][START_REF] Ruiz-Suarez | Levels of organochlorine pesticides in blood plasma from residents of malaria-endemic communities in Chiapas, Mexico[END_REF]. The pesticide concentration may be even much lower when considering only the unbound free ones. This hypothesis is fully supported by the fact that at least some pesticides, such as dieldrin and atrazine, bind extensively to plasma proteins [START_REF] Gulden | Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro[END_REF]100]. Average oral daily intake of a pesticide in the food is additionally usually low, in the µg or ng range, making unlikely the fact that pesticide IC 50 values against transporter activity may be reached in the gastrointestinal tract. Pesticide-drug interactions as well as inhibition of physiological substrate transport by pesticides may consequently be discarded, for most of pesticides found in the environment. It is however noteworthy that humans are often exposed to mixtures of pesticides or to pesticides and other environmental contaminants, whose inhibitory effects

Toxicity due to transporter expression changes caused by pesticides

Regulation of drug transporter expression by pesticides, i.e., induction or repression, can theoretically result in enhanced or decreased transport of drugs or endogenous substrates, which may in turn alter pharmacokinetics and/or toxicity. One key point to consider is that environmental exposure to pesticides has to result in in vivo pesticide level sufficient for triggering transporter regulation, which may be unlikely owing to the relative low concentrations of pesticides reported in humans exposed to these chemicals, as already discussed above. Moreover, it is noteworthy that whether transporter expression modulation may result in clinically-significant alteration of drug transporter activity remains to be formally established. In fact most, if not all, clinical drug-drug interactions in relation with transporters are linked to transporter activity inhibition by the drug perpetrator, and not to altered transporter expression [START_REF] Konig | Transporters and drug-drug interactions: important determinants of drug disposition and effects[END_REF]102]. The only example is perhaps that of therapeutic proteins like tocilizumab, a humanized monoclonal antibody against the interleukin-6

receptor. Tocilizumab is thought to restore normal expression of transporters, including P-gp, Class 2 drugs are highly permeable, so they will generally be able to enter enterocytes by passive diffusion, unaided by uptake transporters. However, due to low solubility limiting luminal concentration, they are unlikely to saturate efflux transporters. Consequently, class 2

A c c e p t e d M a n u s c r i p t compounds can be pumped out of enterocytes, which can influence bioavailability and absorption rate. Pesticides exhibit limited luminal concentration, like class 2 drugs, even if this probably reflects the low dairy intake of pesticides, and not a poor solubility, as for class 2 drugs. Transport of pesticides by ABC pumps may additionally be implicated in reduced brain penetration of pesticides, through P-gp-and BCRP-mediated efflux into the lumen of brain capillary. The fact that silencing of P-gp led to a 100-fold increase of ivermectin distribution into the central nervous system of transgenic mice fully supports this assertion [START_REF] Schinkel | Disruption of the mouse mdr1a Pglycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs[END_REF].

Implications for toxicity

Handling of pesticides by transporters may decrease or increase their toxicity, depending on the nature of transport. Thus, efflux by ABC transporters out of cells is expected to result in reduced intracellular accumulation of pesticides, and by this way, in decreased toxic effects. This is illustrated by the fact that P-gp expression confers resistance to paraquat, through stimulating its cellular export out of cells [32]. By this mechanism, P-gp notably protects against paraquat-induced toxicity in human and mouse proximal tubule cells [START_REF] Wen | Induction and activation of P-glycoprotein by dihydroxylated xanthones protect against the cytotoxicity of the P-glycoprotein substrate paraquat[END_REF].

Interestingly, the ABC transporter MRP1 may also confer resistance to paraquat; this may be due to inhibition of apoptosis caused by the pesticide [107]. MRP1 also decreased toxicity of fenitrothion, chlorpropham and methoxychlor in MRP1-transfected cells and protects seminiferous tubules from methoxychlor-induced damage [START_REF] Tribull | The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury[END_REF]. Overexpression of ABC transporters has similarly been shown to confer resistance to pesticides in insect cells [108].

Transport of pesticides by ABC transporters at blood-tissue barriers, notably at the bloodbrain barrier, also contributes to protect these tissues, notably the central nervous system, from pesticide toxicity, as already demonstrated for P-gp and neurologic toxicity of ivermectin [START_REF] Schinkel | Disruption of the mouse mdr1a Pglycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs[END_REF]. In the same way, P-gp may play the same protective role towards pesticides at the placental barrier.

A c c e p t e d M a n u s c r i p t

Handling of pesticides by drug transporters such as SLC transporters may also result in decreased toxicity of pesticides. Thus, mouse Oct3 mediates cellular entry of paraquat, but only under its reduced monovalent cation form [START_REF] Rappold | A study reporting the role of the SLC transporter OCT3 in neurotoxicity of paraquat 67. Obaidat A, Roth M, Hagenbuch B. The expression and function of organic anion transporting polypeptides in normal tissues and in cancer[END_REF]. Because Oct3 is mainly expressed by non-dopaminergic cells in the nigrostriatal region, this Oct3-mediated transport of paraquat contributes to reduce its accumulation in dopaminergic neurons; this highlights a buffering capacity by non-dopaminergic cells, which indirectly protects dopaminergic neurons from toxicity of paraquat [START_REF] Rappold | A study reporting the role of the SLC transporter OCT3 in neurotoxicity of paraquat 67. Obaidat A, Roth M, Hagenbuch B. The expression and function of organic anion transporting polypeptides in normal tissues and in cancer[END_REF]. Whether SLC transporters may, by contrast, favor toxicity of pesticides through increasing their intracellular accumulation remains yet unknown. Such a toxicity caused by SLC-transporter mediated uptake of chemicals has nevertheless already been demonstrated for other environmental contaminants such as the microcystin congeners.

Conclusion

Various reports have indicated that pesticides belonging to diverse pharmacological/chemical classes can interact with drug transporters. Most of these studies described inhibition of ABC or SLC drug transporter activities by pesticides; only a few of them concerns the handling of pesticides by transporters or the regulation of transporter expression by pesticides.

Importantly, most, if not all, pesticides required concentrations in the µM range for inhibiting transporter activities; such concentrations are much higher than pesticide concentrations (in the nM range or less) commonly observed in humans exposed to environmental pesticides.

Pesticides are therefore unlikely to cause pesticide-drug interaction based on drug transport alteration in humans. In the same way, putative pesticide-mediated alteration of endogenous A c c e p t e d M a n u s c r i p t substrate transport and possible deleterious consequences in response to environmental exposure may probably be discarded. By contrast, for pesticides recognized as substrates, transporters may be implicated in their toxicokinetics and their toxicity towards human health.

Expert opinion

Drug transporters are now considered as major actors of pharmacokinetics [START_REF] Konig | Transporters and drug-drug interactions: important determinants of drug disposition and effects[END_REF], which fully justify the study of drug-transporter interactions during the development of new molecular entities by pharmaceutical companies. Data reported above unambiguously indicate that some pesticides also interact with transporters, i.e., they can inhibit their activity, regulate their expression or be themselves substrates. This provides proof of concept that transporters should be considered when studying the toxicokinetics and toxicity of pesticides in humans.

For transporter inhibition, the concentrations of pesticides in vitro blocking transporter activities have however to be confronted to the levels of unbound pesticides reached in humans in response to environmental exposure, in order to precise the in vivo relevance of such transporter inhibitions. Indeed, in most cases, the pesticide concentrations required to inhibit transporter may probably be much higher than pesticide blood concentrations reached in exposed humans. This likely discards any in vivo transporter inhibitions in response to environmental exposure to pesticides. In the same way, the in vivo relevance of the cisstimulation of some transporter activities by certain pesticides remains to be clarified.

Chemical pesticides remain much less studied than drugs with respect to interactions with transporters, with no or only limited data available for most pesticides. Further studies are therefore required to characterize pesticide-transporter interactions in a more extensive and systematic manner and to determine their possible implications in deleterious effects of pesticides towards human health. To do this, the use of high-throughput assay panels for human drug transporters may be welcome. Such assays, mainly based on fluorescent probes Environ Res Public Health. 2017;14 [START_REF] Konig | Transporters and drug-drug interactions: important determinants of drug disposition and effects[END_REF].

• A study using in silico approach for predicting the transport of pesticides across

Legend to figure

A

  c c e p t e d M a n u s c r i p t Key-words: Drug transporters; environmental exposure; pesticides; toxicity; toxicokinetics Article highlights -Interactions of organic pesticides with drug transporters are important to consider owing to the major contribution of transporters to toxicokinetics and the well-established toxic effects of pesticides, to which humans are widely exposed.

A c c e p t e d M a n u s c r i p t

  A c c e p t e d M a n u s c r i p t

  OATP1B1 and OAT3 activities, and cis-stimulated that of OATP2B1[18]. Similarly, imiprothrin and prallethrin, unlike phenothrin, cis-stimulated OATP2B1 activity and inhibited that of OAT3. By contrast allethrin and tetramethrin failed to alter in a major way activities of OATP1B3 and OAT1. Similarly, bifenthrin, β-cyfluthrin, λ-cyhalothrin, β-cypermethrin, deltamethrin, esfenvalerate, fenpropathrin, fluvalinate, cis-permethrin, trans-permethrin, resmethrin and tefluthrin did not impair OATP1B3 and OAT1 activities, as well as those of OATP1B1, OATP2B1 and OAT3[18]. Regarding organophosphorus pesticides, OAT3 activity was inhibited by fenamiphos, malathion and profenofos, but cis-stimulated by A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t towards transporters may

  be additive or synergic. This may result in in vivo inhibition of transporters in response to exposure to chemical mixtures, even if concentration of each single pesticide of the mixture is low. The fact that binary mixtures of pesticides, including diazinon, have been shown to exhibit synergistic inhibition of P-gp [21] likely supports this hypothesis.It is also noteworthy that pesticide metabolites may cause transporter inhibition. This may be hypothesized to add or synergize with transporter inhibition triggered by the parental pesticide. Examples of pesticide metabolites acting against transporters correspond to hydroxylated metabolites of propiconazole, active against P-gp[START_REF] Mazur | P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticidedrug interactions[END_REF], and to chlorpyrifos oxon, which inhibits drug labelling of P-gp and stimulates its ATPase activity, in contrast to parental chlorpyrifos[101].

A c c e p t e d M a n u 6 .of pesticides 6 . 1

 661 s c r i p t repressed by IL-6 in inflammatory patients, which may be the source of drug-drug interactions[103]. Pesticides as substrates of drug transporters: implications for toxicokinetics and toxicity Implications for toxicokinetics Handling of pesticides by ABC and/or SLC transporters is likely to contribute to the different steps of the disposition process, i.e., intestinal absorption, drug disposition, notably across the blood-tissue barriers like the blood-brain barrier, and hepatic and renal elimination. Owing to the well-established drug transporter-drug metabolizing enzymes interplay [104], it may also influence pesticide metabolism. In this context, it is noteworthy that some pesticides such as organochlorine insecticides have historically been considered as lipophilic chemicals, which freely diffuses across the plasma membrane according to their concentration gradient, thus ruling out any major role for drug transporters. This assertion may now be questioned by the putative handling of some pesticides by ABC transporters, notably P-gp and BCRP. Indeed, these ABC transporters are present at the apical pole of intestinal cells, where they actively expel their substrates into the digestive lumen, thus preventing their absorption[105].Handling of lipophilic pesticides by ABC transporters like P-gp and BCRP may therefore theoretically limits their intestinal absorption. Moreover, the concentration of pesticides in the gastrointestinal lumen is believed to be low, as already discussed above, which precludes any saturation of the ABC efflux pumps. Such lipophilic pesticides may therefore behave as class 2 compounds in the biopharmaceutics drug disposition classification system (BDDCS)[106].

  These compounds, produced by cyanobacteria, use OATP1B1 and OATP1B3 to enter into cells and, therefore, specifically target OATP1B1-or OATP1B3-expressing cells[109]. Such an OATP-mediated uptake is consequently responsible for the selective hepatic toxicity of microcystins[110].

A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u

  s c r i p t individual variation in physiology and pharmacotherapy[123]. In the context of environmental exposure to pesticides, such transporter polymorphisms may have to retain special attention. Indeed, they may result in inter-individual variation in toxicokinetics and toxicity of pesticides and may thus contribute in a notable way to individual susceptibility to these environmental pollutants. For example, subjects with genetic variants of ABCB1 gene associated with low P-gp activity may have enhanced intestinal absorption and brain penetration of pesticides, by reduction of P-gp-mediated efflux of the chemicals at the intestinal and blood-brain barriers; such subjects may therefore be more susceptible to toxicity of pesticides. This hypothesis is fully supported by the fact that exposure to commonly used pesticides, specifically organochlorine and organophosphate insecticides, and the presence of variant ABCB1 genotypes at two polymorphic sites, jointly increase the risk of Parkinson's disease[124].The fact that pesticides are often used as mixtures of enantiomers has additionally to be taken into account when considering drug transporters and pesticides. Indeed, the stereoselectivity of chiral drug transport is well-established [125] and enantiomers of pesticides may therefore differentially interact with transporters. The handling of pesticides by SLC transporters distinct from main drug transporters, such as the L-type amino acid LAT1/2 (SLC7A5/SLC7A8) transporter or the dopamine transporter (DAT/SLC6A3), which handles glyphosate[126] or paraquat[START_REF] Rappold | A study reporting the role of the SLC transporter OCT3 in neurotoxicity of paraquat 67. Obaidat A, Roth M, Hagenbuch B. The expression and function of organic anion transporting polypeptides in normal tissues and in cancer[END_REF], respectively, also merits attention. In the same way, the molecular nature of the membrane transporters responsible for the established transepithelial transport of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA)[127] and of the pyrethroids deltamethrin, cis-permethrin and trans-permethrin[START_REF] Zastre | Lack of P-glycoprotein-mediated efflux and the potential involvement of an influx transport process contributing to the intestinal uptake of deltamethrin, cis-permethrin, and trans-permethrin[END_REF] across intestinal Caco-2 cells remains to be determined. The functional interplay between drug-metabolizing enzymes and transporters in pesticide absorption and disposition has additionally to be considered, as already done for drugs[128]. This interplay may notably correspond to A c c e p t e d M a n u s c r i p t preferential handling of pesticide metabolites by transporters, which suggests that pesticide metabolites may have to be investigated as potential transporter substrates and/or inhibitors.Additive or synergic effects towards transporters of pesticides in mixture with other pesticides or environmental contaminants constitute another important issue to apprehend. Finally, data about handling of pesticides by drug transporters may help to ameliorate the relevance and the accuracy of physiologically-based pharmacokinetic (PBPK) models, which represent promising approaches for pesticide risk assessment in humans[129, 130].

  s c r i p t 121. Chedik L, Mias-Lucquin D, Bruyere A, et al. In Silico Prediction for Intestinal Absorption and Brain Penetration of Chemical Pesticides in Humans [Journal]. Int J

Figure 1 .

 1 Figure 1. Accumulation of the pyrethroids allethrin and tetramethrin in OCT1-and OCT2transfected HEK293 cells

  substrates for P-gp, MRPs, BCRP, OATPs, OATs and OCTs[111, 112], are fully applicable to large series of compounds tested at various concentrations and permit to reduce analysis costs. These functional assays may additionally provide structure-activity information, which

	may be useful for modeling quantitative structure-activity relationships (QSAR) with respect
	to interactions with transporters. Such QSAR studies may help to in silico predict inhibition
	of transporters by pesticides and/or handling of pesticides by transporters. QSAR approaches
	have already been applied with success to drug-transporter interactions, including those
	related to P-gp [113], MRP2 [114], BCRP [115], OCT1 [116], OATP1B1/1B3 [117] and
	OAT1/OAT3 [118]. Interestingly, molecular descriptors associated with OCT1 inhibition by
	pyrethroids have been determined and combining pairwise some of these descriptors allow to
	graphically and successfully predict interactions of imiprothrin, phenothrin and prallethrin
	with OCT1, OCT2, OATP2B1, OAT1 and OAT3 [18]. Besides, or together with QSAR
	studies, molecular docking analyses may constitute a valuable approach for predicting
	interaction of pesticides with transporters, as already established for drugs [119]. Overall, the
	application to pesticides of in silico tools developed for characterizing membrane
	permeability and transporter interactions with drugs, may represent a promising way,
	extending data and methods initially focused on drugs to the pesticide area. The graphical
	BOILED-Egg/SwissADME online method, based on lipophilicity and polarity and originally
	designed for drugs [120], has thus permitted to predict intestinal absorption and brain
	penetration of a large set of pesticides (n = 338) belonging to various chemical classes [121].
	Genes encoding drug transporters are well-known to exhibit polymorphisms, which
	may have functional consequences, i.e., some genetic variants may display increased or
	decreased transport activity [122]. Such transporter polymorphisms concern most, if not all,
	drug transporters, including P-gp, BCRP, OATPs, OATS and OCTs. They are thought to be
	responsible for population-specific differences in drug transport and considerable inter-

complete study bringing the proof of the concept that structurally diverse pesticides can interact with drug transporters

	18.	Chedik L, Bruyere A, Le Vee M, et al. Inhibition of Human Drug Transporter
		Activities by the Pyrethroid Pesticides Allethrin and Tetramethrin. PLoS One.
		2017;12(1):e0169480. doi: 10.1371/journal.pone.0169480. PubMed PMID: 28099443;
		PubMed Central PMCID: PMC5242521. eng.
		• A study reporting the effects of pyrethroids towards ABC and SLC transporter
		activities
	19.	Bain LJ, McLachlan JB, LeBlanc GA. Structure-activity relationships for xenobiotic
		transport substrates and inhibitory ligands of P-glycoprotein. Environ Health Perspect.
		1997 Aug;105(8):812-8. PubMed PMID: 9347896; PubMed Central PMCID:
		PMC1470197. eng.
	20.	Lacher SE, Skagen K, Veit J, et al. P-Glycoprotein Transport of Neurotoxic
		Pesticides.	J	Pharmacol	Exp	Ther.	2015	Oct;355(1):99-107.	doi:
		10.1124/jpet.115.226373. PubMed PMID: 26272936; PubMed Central PMCID:
		PMC4576670. eng.
	21.	Pivcevic B, Zaja R. Pesticides and their binary combinations as P-glycoprotein
		inhibitors in NIH 3T3/MDR1 cells. Environ Toxicol Pharmacol. 2006 Nov;22(3):268-
		76. doi: 10.1016/j.etap.2006.04.002. PubMed PMID: 21783720; eng.
		• The description of pesticide mixture effects towards P-gp activity
	22.	Didier A, Loor F. The abamectin derivative ivermectin is a potent P-glycoprotein
		inhibitor. Anticancer Drugs. 1996 Sep;7(7):745-51. PubMed PMID: 8949985; eng.
	23.	Pouliot JF, L'Heureux F, Liu Z, et al. Reversal of P-glycoprotein-associated multidrug
		resistance by ivermectin. Biochem Pharmacol. 1997 Jan 10;53(1):17-25. PubMed
		PMID: 8960059; eng.

•• A

cellular barriers 122

  . Sissung TM, Troutman SM, Campbell TJ, et al. Transporter pharmacogenetics: transporter polymorphisms affect normal physiology, diseases, and pharmacotherapy. Discov Med. 2012 Jan;13(68):19-34. PubMed PMID: 22284781; PubMed Central PMCID: PMC4006110. eng. 123. Ahmed S, Zhou Z, Zhou J, et al. Pharmacogenomics of Drug Metabolizing Enzymes and Transporters: Relevance to Precision Medicine. Genomics Proteomics Bioinformatics. 2016 Oct;14(5):298-313. doi: 10.1016/j.gpb.2016.03.008. PubMed PMID: 27729266; PubMed Central PMCID: PMC5093856. eng. 124. Narayan S, Sinsheimer JS, Paul KC, et al. Genetic variability in ABCB1, occupational pesticide exposure, and Parkinson's disease. Environ Res. 2015 Nov;143(Pt A):98-106. doi: 10.1016/j.envres.2015.08.022. PubMed PMID: 26457621; PubMed Central PMCID: PMC4911423. eng. 125. Zhou Q, Yu LS, Zeng S. Stereoselectivity of chiral drug transport: a focus on enantiomer-transporter interaction. Drug Metab Rev. 2014 Aug;46(3):283-90. doi: 10.3109/03602532.2014.887094. PubMed PMID: 24796860; eng. 126. Xu J, Li G, Wang Z, et al. The role of L-type amino acid transporters in the uptake of glyphosate across mammalian epithelial tissues. Chemosphere. 2016 Feb;145:487-94. doi: 10.1016/j.chemosphere.2015.11.062. PubMed PMID: 26701683; eng. 127. Kimura O, Tsukagoshi K, Hayasaka M, et al. Transepithelial transport of 4-chloro-2methylphenoxyacetic acid (MCPA) across human intestinal Caco-2 cell monolayers. Basic Clin Pharmacol Toxicol. 2012 Jun;110(6):530-6. doi: 10.1111/j.1742-7843.2011.00850.x. PubMed PMID: 22181038; eng. 128. Shi S, Li Y. Interplay of Drug-Metabolizing Enzymes and Transporters in Drug Absorption and Disposition. Curr Drug Metab. 2014;15(10):915-41. PubMed PMID: 25828591; eng. 129. Lu C, Holbrook CM, Andres LM. The implications of using a physiologically based pharmacokinetic (PBPK) model for pesticide risk assessment. Environ Health Perspect. 2010 Jan;118(1):125-30. doi: 10.1289/ehp.0901144. PubMed PMID: 20056589; PubMed Central PMCID: PMC2831956. eng. 130. Knaak JB, Dary CC, Zhang X, et al. Parameters for pyrethroid insecticide QSAR and PBPK/PD models for human risk assessment. Rev Environ Contam Toxicol.

	a n u s c r i p t
	M
	A c c e p t e d

2012;219:1-114. doi: 10.1007/978-1-4614-3281-4_1. PubMed PMID: 22610175; eng.

This paper was not funded. , honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

P-gp

Paraquat [START_REF] Wen | Induction and activation of P-glycoprotein by dihydroxylated xanthones protect against the cytotoxicity of the P-glycoprotein substrate paraquat[END_REF], ivermectin [START_REF] Lespine | Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)[END_REF][START_REF] Schinkel | Disruption of the mouse mdr1a Pglycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs[END_REF], endosulfan (weak substrate) [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF] Propiconazole [START_REF] Mazur | P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticidedrug interactions[END_REF], deltamethrin [START_REF] Zastre | Lack of P-glycoprotein-mediated efflux and the potential involvement of an influx transport process contributing to the intestinal uptake of deltamethrin, cis-permethrin, and trans-permethrin[END_REF], cispermethrin [START_REF] Zastre | Lack of P-glycoprotein-mediated efflux and the potential involvement of an influx transport process contributing to the intestinal uptake of deltamethrin, cis-permethrin, and trans-permethrin[END_REF], trans-permethrin [START_REF] Zastre | Lack of P-glycoprotein-mediated efflux and the potential involvement of an influx transport process contributing to the intestinal uptake of deltamethrin, cis-permethrin, and trans-permethrin[END_REF], warfarin [42], cyperquat/MPP+ [29]

MRP1

Methoxychlor [START_REF] Tribull | The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury[END_REF], fenitrothion [START_REF] Tribull | The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury[END_REF], chlorpropham [START_REF] Tribull | The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury[END_REF], ivermectin [START_REF] Lespine | Interaction of ivermectin with multidrug resistance proteins (MRP1, 2 and 3)[END_REF]

BCRP

Warfarin [START_REF] Yang | R-and S-Warfarin Were Transported by Breast Cancer Resistance Protein: From In Vitro to Pharmacokinetic-Pharmacodynamic Studies[END_REF] Ivermectin [START_REF] Geyer | Brain penetration of ivermectin and selamectin in mdr1a,b P-glycoprotein-and bcrp-deficient knockout mice[END_REF] OCT1 Cyperquat/MPP+ [START_REF] Gorboulev | Cloning and characterization of two human polyspecific organic cation transporters[END_REF] Allethrin (present study), tetramethrin (present study), paraquat [START_REF] Chen | Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family[END_REF], fenamiphos [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF], phosmet [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF] OCT2 Cyperquat/MPP+ [START_REF] Gorboulev | Cloning and characterization of two human polyspecific organic cation transporters[END_REF], paraquat [START_REF] Chen | Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family[END_REF] Allethrin (present study), tetramethrin (present study), fenamiphos [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF], phosmet [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]

OCT3

Cyperquat/MPP+ [START_REF] Wu | Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain[END_REF] Paraquat [START_REF] Chen | Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family[END_REF]

MATE1

Cyperquat/MPP+ [START_REF] Otsuka | A human transporter protein that mediates the final excretion step for toxic organic cations[END_REF], paraquat [START_REF] Chen | Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family[END_REF] No data MATE2-K Cyperquat/MPP+ [START_REF] Masuda | Identification and functional characterization of a new human kidney-specific H+/organic cation antiporter, kidney-specific multidrug and toxin extrusion 2[END_REF] No data OAT1 2,4-dichlorophenoxyacetic acid (2,4-D) [START_REF] Nozaki | Characterization of the uptake of organic anion transporter (OAT) 1 and OAT3 substrates by human kidney slices[END_REF] No data OAT3 2,4-dichlorophenoxyacetic acid (2,4-D) [START_REF] Nozaki | Characterization of the uptake of organic anion transporter (OAT) 1 and OAT3 substrates by human kidney slices[END_REF] No data


The widespread use of mobile phones raises the question of the effects of electromagnetic fields (EMF, GSM 900 MHz) on the brain. Effects of these EMFs during development stages and in vulnerability states as inflammation have been only poorly studied. Our aim was to assess EMF effects in adolescent rats subjected to neuroinflammation (Lipopolysaccharide (LPS) or artificial cerebrospinal fluid (aCSF) diffusion in lateral ventricle or in hippocampus (1.25 µg/h for 4 weeks from day 25) or to gestational inflammation (LPS or saline injection in peritoneum (0.1 mg/kg) on gestational day (GD) 15, GD17, and GD19). Sprague Dawley rats (32 days) were repeatedly exposed to various specific absorption rates (0, 1.5 or 6 W/kg; 45 min/d, 5 d/week, for 4 weeks). Endpoints were emotional memory, sensori-motor reactivity, exploration and anxiety (using fear conditioning, startle inhibition, open field and elevated plus maze) as well as cerebral Interleukine-1 beta (IL-1β) and glial fibrillary acidic protein (GFAP). The neuroinflammation model was characterized by increased levels of IL-1beta and GFAP, reduced startle level and by the increase of startle inhibition and the decrease of short term emotional memory. Gestational inflammation was linked to teratological effects, but no behavioral modifications. Effect of GSM exposures were shown by a decrease of GFAP in 1.5 W/kg-LPS compared to 0W/kg-LPS exposed rats. There was no GSM or LPS effect on anxiety. GSM EMFs at 900MHz did not induce neurobiological effects by itself and impacted only one biochemical parameter in vulnerable. These data suggest no specific vulnerability of adolescents with or without pre-existing inflammation in response to mobile phone EMF exposure.

I. INTRODUCTION

The widespread use of mobile phones raises the question of the effects of electromagnetic fields (EMFs) from the Global System for Mobile Communication (GSM) 900 MHz to the brain. Up to date, most experimental researches assessing this question focused on healthy adult rodent models. These studies showed that glial fibrilary acidic protein (GFAP) [START_REF] Mausset-Bonnefont | Acute exposure to GSM 900-MHz electromagnetic fields induces glial reactivity and biochemical modifications in the rat brain[END_REF], [START_REF] Brillaud | Effect of an acute 900MHz GSM exposure on glia in the rat brain: a time-dependent study[END_REF] or cytokines [START_REF] Bouji | Effects of 900 MHz radiofrequency on corticosterone, emotional memory and neuroinflammation in middle-aged rats[END_REF], [START_REF] Megha | Microwave radiation induced oxidative stress, cognitive impairment and inflammation in brain of Fischer rats[END_REF] were increased. Moreover, studies showed a possible impact of EMF ted on emotional memory [START_REF] Bouji | Effects of 900 MHz radiofrequency on corticosterone, emotional memory and neuroinflammation in middle-aged rats[END_REF] and anxiety [START_REF] Daniels | The effect of electromagnetic radiation in the mobile phone range on the behaviour of the rat[END_REF]. However, these biochemical and behavioural results

were not always reproduced [START_REF] Bouji | Effects of 900 MHz radiofrequency on corticosterone, emotional memory and neuroinflammation in middle-aged rats[END_REF], [START_REF] Watilliaux | Effect of exposure to 1,800 MHz electromagnetic fields on heat shock proteins and glial cells in the brain of developing rats[END_REF], [START_REF] Jin | Effects of simultaneous combined exposure to CDMA and WCDMA electromagnetic field on immune functions in rats[END_REF] and [START_REF] Kumlin | Mobile phone radiation and the developing brain: behavioral and morphological effects in juvenile rats[END_REF].

Meanwhile, because teenagers represent an increasing part of the cell phone users, concerns are currently raised in the case of cerebral vulnerability, as development and maturation. Moreover, we may hypothesize that adolescent brain vulnerability to environmental insult would be worsened by a previous developmental toxicological exposure, which is a suspected cause for the etiology of psychopathologies diagnosed during adolescence [START_REF] Patterson | Immune involvement in schizophrenia and autism: Etiology, pathology and animal models[END_REF].

In the same way, adolescent subjected to neuroinflammation, which is the common feature of most neuropathologies may be more vulnerable in response to co-exposed GSM. In this way, we assessed behavioural (fear conditioning, startle reflex inhibition, elevated plus maze and open field) and biochemical (IL-1β and GFAP) effects of repeated EMFs GSM 900 MHz exposures in adolescent rat models of gestational inflammation or neuro-inflammation.

II. MATERIAL AND METHOD

A. Neuroinflammation model

At post-natal day 25 (P25): Sprague Dawley rats were implanted with Alzet osmotic pump (0.25 µl/h for 28 days; 5 µg/µl LPS or artificial cerebrospinal fluid (vehicle)). Infusions were performed in one lateral cerebral ventricle (Antero-posterior: -1.3 mm to Bregma, medo-lateral: ±1.5 mm, and dorso-ventral: -3.5 mm) or in the hippocampus (Anteroposterior: -2.5 mm to Bregma, medo-lateral: -3 mm, and dorso-ventral: 7.0 mm).

B. Gestational inflammation model

Pregnant Sprague Dawley rats were injected in intraperitoneal daily with 0.1 mg/kg of LPS or saline on gestational day (GD) 15, GD17, and GD19 C. GSM 900MHz exposure Exposure set-up was performed as previously described [START_REF] Dubreuil | Does head-only exposure to GSM-900 electromagnetic fields affect the performance of rats in spatial learning tasks?[END_REF]. A radio frequency power source (900-64 type, Radio Frequency Power Amplifier, France) emitting a 900 MHz EMF (1/8duty factor) pulse modulated at 217 Hz was connected to a four-output divider. Each output was connected to a loop antenna allowing local exposure of four animals simultaneously in an anechoic chamber.

Exposure dosimetry was previously performed Leveque et al. [START_REF] Leveque | Dosimetric analysis of a 900-MHz rat head exposure system[END_REF] and confirmed experimentally in our laboratory by Ammari et al. [START_REF] Ammari | Effect of head-only sub-chronic and chronic exposure to 900-MHz GSM electromagnetic fields on spatial memory in rats[END_REF]. Presented Specific Absorption Rate (SAR) values correspond to adult rat's exposure.

From P32 to P62, healthy and vulnerable models of rats were placed in a Plexiglas rocket on which an individual loop antenna was fixed for head exposure. Rats were repeatedly exposed (45 min/day; 5 day/week; during 1 month) to various specific absorption rates (SAR) of EMFs GSM 900MHz (0, 1.5 or 6W/kg).

D. Behavioral tests

Fear conditioning paradigm (P56-P57), startle inhibition paradigm (PPI) (P60 to P62), elevated plus maze (P63) and open field (P63 or P64) were tested at the exposition month end for intracerebroventricular inflammation model and gestational inflammation model.

Behavioral and biochemical effects of GSM 900MHz in neuroinflammation and gestational inflammation models

N. Petitdant, A. Lecomte, F. Robidel, A-S. Villégier INERIS INERIS -Toxicologie Expérimentale, Parc ALATA -BP2, 60550 Verneuil-en-Halatte, FRANCE nicolas.petitdant@ineris.fr, anne-sophie.villegier@ineris.fr

Fear conditioning occurred in four standard fear conditioning apparatus purchased from Bioseb (France). Four freezing boxes (25×25×25cm). Freezing response was defined as complete immobilization of the rat, except for respiration [START_REF] Blanchard | Crouching as an index of fear[END_REF]. It was measured in response to stimuli presentation (context and tone) by amplitude of movement of the animal and was scored as percentage of time spent freezing.

Training phase began with a 2 min habituation period (no auditory or aversive stimulus), followed by five training cycles. For one cycle, the three consecutive steps were: 28 s of sound (Frequency: 2 kHz; 100 dB), 2 s of sound + electrical shock (0.25 mA) and a 30 s exploration period (no auditory or aversive stimulus).

To test contextual memory, animals were introduced for 3 min in the boxes used for the training. To avoid extinction, the 3h contextual memory test ended with two training cycles.

To perform the cue memory test, animals were introduced in boxes with modified environment (different box size, walls color and floor texture) for 2 min and then were exposed to the sound for 3 min and 2 min of exploration period.

Prepulse inhibition occurred in the same four standard fear conditioning apparatus. For this test, rats were placed in a restraining tube. Throughout the session, a background noise level of 60 decibel (dB) was maintained.

Rats were run in squads of four. Each rat was put into the PPI chamber for a 5-min acclimatization period with 60 dB background noise. Following this period, 10 startle pulses (120 dB, 40 msec duration) were presented with an average intertribal interval of 15 sec. Then, no stimulus (background noise, 60 dB), prepulses alone (75, 80 or 85 dB, 20 msec duration), startle pulses alone, and prepulses followed 80 msec later by startle pulses were presented six times randomly distributed over the next 15 min. The percentage of PPI induced by each prepulse intensity was calculated as 100[(SP-SPP)/SP], with SP being the average startle amplitude after the startle pulses and SPP being the average startle response after the combination of a certain prepulse and the startle pulse. Elevated plus maze test was performed according to previously described methods [START_REF] Daniels | Maternal separation in rats leads to anxiety-like behavior and a blunted ACTH response and altered neurotransmitter levels in response to a subsequent stressor[END_REF]. The apparatus was composed of a plus shaped acryl maze with two opposite open arms and two opposite closed arms (50 cm in length, 10 cm width, and 31 cm in height), extending out from a central platform (10 cm × 10 cm) and elevated 50 cm above the floor. Rats were placed in the centre of the maze, the head facing an open arm and were allowed to explore for 5 min. Data were presented as percentage of time spent in the open arm compared to the time spent in the four arms during the first 30 seconds of the test.

Open field test was performed according to previously described methods [START_REF] Prut | The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review[END_REF]. Movements were monitored for 30 min using an automated apparatus consisting of four Plexiglas boxes (60 × 60 × 40 cm) equipped with lower 16 sets and top 8 sets (for vertical activity evaluation) of evenly spaced infrared beams located along two adjacent sides of one chamber (Imetronic, France). The time spent in the centre area of the apparatus, considered as a model of stress-induced inhibition of exploration behavior was monitored simultaneously.

A. Sacrifice

At P64, rats were sacrificed. Brains were quickly removed. The right medio-lateral parts of each brain (containing the injection site) were used for ELISA analyses (IL1β and GFAP).

B. Biochemistry

Brain structures were homogenized by sonication in a Trizma base/NaCl/Triton buffer containing a 4% protease inhibitor (Roche). Samples were centrifuged (13000 rpm/20 min/4 °C) and the supernatant was collected. Bradford test was used to measure total protein content in each sample.

IL-1β were quantified using commercialized sandwich ELISA kits (Rat IL-1β/IL-1 F2 DuoSet ELISA Development system, R&D systems) according to the manufacturer's instructions.

GFAP were quantified a sandwich ELISA assay. Plates were coated in-house with the capture antibody (20334-Dako ), detection antibody (ab 10062-abcam) and as tertiary antibody, we used a poly monoclonal anti-IgG-HRP (ab 6728abcam). Glial fibrillary acidic protein purified from human brain (EMD Biosciences) was used as standard.

C. Statistics

Statistical analyses were performed using the Prism 5 (GraphPad Software, inc). Values were given as mean±standard deviation of mean (SEM) per group. D'Agostino and Pearson test was used for variance homogeneity. Analyses were performed using two-way analysis of variance (ANOVA). The main effects were analyzed using Bonferroni's post-hoc corrected t-test. Effects were considered significant when p<0.05.

III. RESULTS

A. Gestational LPS

LPS gestational injections reduced the viable pups number (t-test: p=0.0291) (Fig: 1).

Viable pups number

B. Behavior

The percentage of time spent freezing during the short-term contextual memory test was decreased (two-way ANOVA: p=0.0252) in the neuroinflammation model compared to the control group, but there was no difference in the gestational inflammation model (Fig 2). There is no difference of time spent freezing during the long term contextual memory (fig. 3) and cue memory (fig. 4) tests. No effect of EMF was detected on freezing behaviour (fig. 2,3 and4). Startle amplitude in rats subjected to neuro-inflammation was decreased compared to control rats (two-way ANOVA: p=0.0055), but there was no effect of gestational inflammation (fig 5). Global analysis of the three days of prepulse inhibition test didn't show any effect of inflammation or EMF for both, gestational inflammation and neuroinflammation model.

Gestational

When the third days was analysed separately, for the neuroinflammation model, the percentage of PPI was increased in LPS treated rats when compared to control rats following the prepulse at 80db (two-way ANOVA: p=0.0029) and at 75db (two-way ANOVA: p=0.0253) (fig 5). No EMF effect was detected for this model (fig 5).

In the gestational model, no LPS effect was detected (Fig 5). No LPS or GSM effect was obtained for both vulnerability models regarding the percentage of time spent in the open arm of the elevated plus maze (fig. 6) and the open field test (fig. 7, 8 and9).

Gestational

Gestational inflammation model

C. Biochemistry

In the model of neuroinflammation using intrahippocampal diffusion, GFAP and IL-1β were significantly increased (two-way ANOVA: p<0.0001) in LPS treated rats when compared to vehicle treated rats (Fig 10).

Significant GSM effect was shown by the decrease of GFAP in the 1.5W/kg-LPS-exposed rats when compared to the 0W/kg-LPS-exposed group ( Fig 10).

Further biochemical data for neuro-inflammation and gestational inflammation are in progress. IV. CONCLUSION Our vulnerability models were characterized and validated by gestational LPS-induced foeto-toxicity, by intrahippocampal LPS-induced elevated cytokins and GFAP and by intracerebroventricular LPS-induced startle response to novelty and reduced short term emotional memory.

We did not show any behavioural effect of GSM in healthy adolescent rats after one month head-only exposure.

Our vulnerability models, gestational inflammation model and intracerebroventricular neuroinflammation model, did not show higher behavioural sensibility to GSM.

These data suggest no specific vulnerability of adolescents with or without pre-existing inflammation in response to mobile phone EMF exposure.

Fig. 1 .

 1 Fig. 1. LPS gestational injections effects on the viable pups number. * p=0,0291 (t-test analyse).

Fig. 2 .

 2 Fig. 2. GSM 900MHz and LPS effects on freezing behaviour during the first minute of short term contextual memory test. ** p<0.01: LPS treatment versus vehicle treatment. (Two-way ANOVA analyses).

Fig. 3 .Fig. 4 .

 34 Fig.3. GSM 900MHz and LPS effects on freezing behaviour during the long term contextual memory test.

Fig. 5 .

 5 Fig. 5. GSM 900MHz and LPS effects on startle and startle inhibition. * p<0.05, ** p<0.01: LPS treatment versus vehicle treatment (Two-way ANOVA analyses).

Fig. 6 .

 6 Fig. 6. GSM 900MHz and LPS effects presence time in open arm of elevated plus maze.

Fig. 7 .Fig. 8 .Fig. 9 .

 789 Fig. 7. GSM 900MHz and LPS effects on horizontal activity in open field test.

Fig. 10 .

 10 Fig. 10. GSM 900MHz and LPS effects on cerebral (A) IL-1β and (B) GFAP. * p<0.05, ** p<0.01, ***p<0.001: LPS treatment versus vehicle treatment; ## p<0.01: LPS 1,5W/kg exposed versus LPS 0W/kg and versus LPS 6 W/kg (post-oc Bonferroni analyses).