Nelly Gueniche

Arnaud Bruyere

Mélanie Ringeval

Elodie Jouan

Antoine Huguet

Ludovic Le Hégarat

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

Differential interactions of carbamate pesticides with drug transporters

Keywords: Carbamate pesticides, drug transporters, chlorpropham, propamocarb, activity, inhibition

1. Pesticides are now recognised to interact with drug transporters, but only few data are available on this issue for carbamate pesticides, a widely-used class of agrochemicals, to which humans are highly exposed. The present study was therefore designed to determine whether four representative carbamate pesticides, i.e., the insecticides aminocarb and carbofuran, the herbicide chlorpropham and the fungicide propamocarb, may impair activities of main drug transporters implicated in pharmacokinetics.

2. The interactions of carbamates with solute carrier and ATP-binding cassette transporters were investigated using cultured transporter-overexpressing cells, reference substrates and spectrofluorimetry-, liquid chomatography/tandem mass spectrometry-or radioactivity-based methods.

3. Aminocarb and carbofuran exerted no or minimal effects on transporter activities, whereas chlorpropham inhibited BCRP and OAT3 activities and propamocarb decreased those of OCT1 and OCT2, but cis-stimulated that of MATE2-K. Such alterations of transporters however required chlorpropham/propamocarb concentrations in the 5-50 µM range, likely not relevant to environmental exposure.

Trans-stimulation assays and propamocarb accumulation experiments additionally suggested that propamocarb is not a substrate for OCT1, OCT2 and MATE2-K.

4. These data indicate that some carbamate pesticides can interact in vitro with some drug transporters, but only when used at concentrations higher than those expected to occur in environmentally-exposed humans.

Introduction

Drug transporters mediate the passage of xenobiotics across membranes, especially the plasma membrane. They belong to the solute carrier (SLC) or the ATP-binding cassette (ABC) transporter subfamilies [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. SLC transporters are commonly implicated in drug uptake into cells through facilitated diffusion or secondary active transport, whereas ABC transporters act as efflux pumps through primary active transport. Transporters are now well-recognised as playing a major role in the different steps of pharmacokinetics, including intestinal absorption, distribution across blood-tissue barriers and biliary and renal elimination [START_REF] Ayrton | Role of transport proteins in drug absorption, distribution and excretion[END_REF][START_REF] Konig | Transporters and drug-drug interactions: important determinants of drug disposition and effects[END_REF]. Inhibition of their activity by some drugs, called « perpetrators », can cause drug-drug interactions due to altered pharmacokinetics profile of co-administrated drugs substrates for the inhibited transporters and termed « victims » [START_REF] Liu | Transporter-mediated drug-drug interactions and their significance[END_REF]. This may also triggers adverse toxic effects, due to inhibition of endogenous substrate transport [START_REF] Nigam | What do drug transporters really do?[END_REF].

There is growing evidence that environmental chemicals, like drugs, can inhibit or be substrates of drug transporters [START_REF] Clerbaux | Membrane transporter data to support kinetically-informed chemical risk assessment using nonanimal methods: Scientific and regulatory perspectives[END_REF][START_REF] Fardel | Environmental chemicals as substrates, inhibitors or inducers of drug transporters: implication for toxicokinetics, toxicity and pharmacokinetics[END_REF]. This notably concerns agrochemicals, belonging to different main chemical classes of pesticides [START_REF] Buss | Interaction of pesticides with p-glycoprotein and other ABC proteins: A survey of the possible importance to insecticide, herbicide and fungicide resistance[END_REF][START_REF] Chedik | Interactions of pesticides with membrane drug transporters: implications for toxicokinetics and toxicity[END_REF]. Thus, some organochlorine, pyrethroid or organophosphorus pesticides, used at concentrations in the 1-100 µM range, in vitro inhibited activities of various ABC and/or SLC transporters [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF][START_REF] Bucher | Regulation of hepatic drug transporter activity and expression by organochlorine pesticides[END_REF][START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]Chedik et al., 2017a;[START_REF] Gueniche | Implication of human drug transporters to toxicokinetics and toxicity of pesticides[END_REF], whereas, by contrast, neonicotinoid pesticides rather poorly blocked drug transporter activities (Le [START_REF] Vee | Neonicotinoid pesticides poorly interact with human drug transporters[END_REF]. Inhibition of transporter activities depends on the nature of the pesticide and of the transporter. Some pesticides, like the pyrethroids allethrin and tetramethrin, the organophosphorus fenamiphos and phosmet or the cyanoimidazole fungicide cyazofamid can inhibit various SLC transporters [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]Chedik et al., 2017a;[START_REF] Song | Interactions between cyazofamid and human drug transporters[END_REF], A c c e p t e d M a n u s c r i p t thus demonstrating some lack of specificity, as already reported for inhibition of transporters by marketed drugs [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. A few pesticides, such as ivermectin and paraquat, have moreover been shown to be substrates for transporters [START_REF] Chen | Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family[END_REF][START_REF] Griffin | Selamectin is a potent substrate and inhibitor of human and canine P-glycoprotein[END_REF].

If the concept that pesticides can interact with drug transporters is now well-established, as illustrated above, only a relatively small fraction of the total number of pesticides presently used in the world (more than 1000) has however been so far studied for potential interactions with drug transporters [START_REF] Gueniche | Implication of human drug transporters to toxicokinetics and toxicity of pesticides[END_REF]. Experimental data with respect to this topic remain thus limited. It is notably the case for carbamate pesticides, extensively used for agricultural and non-agricultural purposes, and presumed to exert various deleterious effects towards human health, including endocrine disruption and carcinogenicity [START_REF] Dhouib | From immunotoxicity to carcinogenicity: the effects of carbamate pesticides on the immune system[END_REF][START_REF] Miranda-Contreras | Occupational exposure to organophosphate and carbamate pesticides affects sperm chromatin integrity and reproductive hormone levels among Venezuelan farm workers[END_REF][START_REF] Patel | Pesticides as the drivers of neuropsychotic diseases, cancers, and teratogenicity among agro-workers as well as general public[END_REF][START_REF] Piel | Agricultural exposures to carbamate herbicides and fungicides and central nervous system tumour incidence in the cohort AGRICAN[END_REF]. Indeed, only few data have been reported with respect to putative interactions of carbamates with transporters; they demonstrated a lack of inhibitory effects of some carbamates towards activity of the human ABC efflux pump P-glycoprotein (P-gp/ABCB1) [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF] and an inhibition of the rabbit ABC transporter breast cancer resistance protein (BCRP/ABCG2) by propamocarb [START_REF] Halwachs | Assessment of ABCG2mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model[END_REF]. The present study was therefore designed to get insights about putative alteration of transporter activities by carbamate pesticides, to which humans may be highly exposed [START_REF] Bouvier | Pesticide exposure of non-occupationally exposed subjects compared to some occupational exposure: a French pilot study[END_REF][START_REF] Bouvier | Insecticide urinary metabolites in nonoccupationally exposed populations[END_REF][START_REF] Mostafalou | Pesticides: an update of human exposure and toxicity[END_REF]. For this purpose, we have analysed the effects of four representative carbamates, i.e., the two N-methyl carbamates aminocarb and carbofuran, acting as insecticides through reversible inhibition of insect cholinesterase activity, the herbicide chlorpropham and the fungicide propamocarb, towards activities of main drug transporters implicated in pharmacokinetics. Such transporters were ABC pumps (P-gp, BCRP and multidrug-resistance associated proteins (MRPs/ABCCs)), SLC transporters of A c c e p t e d M a n u s c r i p t organic anions (organic anion transporting polypeptide (OATP) 1B1/SLCO1B1, OATP1B3/SLCO1B3, OATP2B1/SLCO2B1, organic anion transporter (OAT) 1/SLC22A6 and OAT3/SLC22A8) and SLC transporters of organic cations (organic cation transporter (OCT) 1/SLC22A1, OCT2/SLC22A2, multidrug and toxin extrusion protein (MATE) 1/SLC47A1 and MATE2-K/SLC47A2). The prototypical N-methylcarbamates aminocarb and carbofuran were found to not, or only poorly, inhibit drug transporters, whereas chlorpropham blocked OAT3 and BCRP activities. With respect to propamocarb, it inhibited activities of OCT1 and OCT2 and cis-stimulated that of MATE2-K, without being substrate for these transporters.

Materials and Methods

Chemicals

Carbamate pesticides (aminocarb, carbofuran, chlorpropham and propamocarb, whose chemical structures are indicated in Figure 1), rhodamine 123, 6-carboxyfluorescein (6-CF), 2′,7′dichlorofluorescein (DCF), tetraethylammonium bromide (TEA), verapamil, probenecid, amitriptyline, fumitremorgin C, rifamycin SV, sulfobromophthalein (BSP) and glutarate were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). [1-14 C]-TEA (specific activity 3.5 mCi/mmol) and [6,7-3 H(N)]-estrone-3-sulfate (E3S) (specific activity 51.8 Ci/mmol) were purchased from PerkinElmer (Boston, MA, USA). Hoechst 33342 and carboxy-2′,7′dichlorofluorescein (CDCF) diacetate were obtained from Life Technologies (Villebon-sur-Yvette, France), whereas 8-fluorescein-cAMP (8-FcA) was from BioLog Life Science Institute (Bremen, Germany) and 4-(4-(dimethylamino)styryl)-Nmethylpyridinium iodide (4-DiASP) from Thermo Fisher Scientific (Waltham, MA, USA).

Stocked solutions of chemicals were commonly prepared in dimethyl sulfoxide (DMSO); final concentrations of solvent in transport assay medium did not exceed 0.2 % (vol/vol). According to the PubChem database web-site (U.S. National Library of Medicine, A c c e p t e d M a n u s c r i p t Bethesda, MA, USA) (https://pubchem.ncbi.nlm.nih.gov/), all carbamate pesticides tested in the study were predicted to be water-soluble at 100 μM, which was the carbamate pesticide concentration initially retained for screening their potential inhibitory effects towards transporter activities.

Cell culture

Mammary MCF7R cells, overexpressing human P-gp (Jouan et al., 2016b), and hepatoma HuH-7 cells, expressing human MRP2/ABCC2 and MRP4/ABCC4 (Jouan et al., 2016a), were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/L D-glucose (Life Technologies, Villebon sur Yvette, France), supplemented with 10% (vol/vol) fetal calf serum, 1% (vol/vol) MEM non-essential amino acids solution (Life Technologies), 20 IU/mL penicillin and 20 μg/mL streptomycin, as already reported (Chedik et al., 2017a). Human BCRP-transfected HEK-293 cells (HEK-BCRP cells) [START_REF] Tournier | Interaction of drugs of abuse and maintenance treatments with human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2)[END_REF], kindly donated by Pr X. Decleves (Faculty of Pharmacy, University Paris-Descartes, Paris, France), were cultured in DMEM supplemented with 10% (vol/vol) fetal calf serum, 100 IU/mL amoxicillin, 100 μg/mL erythromycin and 200 μg/mL G418.

Human HEK-293 cells stably overexpressing human SLC transporters, i.e., OCT1 (HEK-OCT1 cells), OCT2 (HEK-OCT2), MATE1 (HEK-MATE1 cells), MATE2-K (HEK-MATE2-K cells), OAT1 (HEK-OAT1 cells), OAT3 (HEK-OAT3 cells), OATP1B1 (HEK-OATP1B1), OATP1B3 (HEK-OATP1B3) and OATP2B1 (HEK-OATP2B1 cells), as well as parental wild-type HEK-293 cells (HEK-wt cells) have been previously described (Chedik et al., 2017a). They were routinely cultured in DMEM medium supplemented with 10% (vol/vol) fetal calf serum, 1% (vol/vol) MEM non-essential amino acids solution, 20 IU/mL penicillin, 20 μg/mL streptomycin, and 1 μg/mL bovine insulin (Sigma-Aldrich).

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

The effects of carbamates on activity of ABC and SLC transporters were determined through measuring cellular accumulation or retention of reference substrates for transporters, in the presence or absence of reference inhibitors, as previously described (Chedik et al., 2017a).

The nature of cells and reference substrates and inhibitors as well as incubation times and analytical methods used for transport assays are summarised in Supplementary Table S1.

For accumulation assays, transporter-expressing cells were first incubated at 37°C with reference substrates in the absence (control) or presence of carbamate pesticides or reference inhibitors, in a well-defined transport assay medium (Chedik et al., 2017a), consisting of 136 mM NaCl, 5.3 mM KCl, 1.1 mM KH 2 PO 4 , 0.8 mM MgSO 4 , 1.8 mM CaCl 2 , 10 mM HEPES, 11 mM D-glucose and adjusted to pH = 7.4 (excepted for MATE transporter assays, for which pH was set to 8.4). After washing twice in phosphate-buffered saline (PBS), cells were lysed in distilled water. Intracellular accumulation of reference substrates was finally determined by scintillation counting (for [ 14 C]-TEA and [ 3 H]-E3S) using a Tri-Carb © 2910TR analyzer (PerkinElmer) or by spectrofluorimetry using a SpectraMax Gemini SX spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) (for rhodamine 123, 6-CF, DCF, 8-FcA and 4-DiASP). Liquid chromatography-tandem mass spectroscopy (LC-MS/MS), based on a highperformance liquid chromatography Aria system (Agilent, Les Ulis, France), equipped with a Poroshell 120 C18 (4.6 × 150 mm) column (Agilent, Les Ulis, France) and coupled to a tandem mass spectrometry TSQ Quantum Ultra (Thermo Fisher Scientific, Villebon sur Yvette, France), fitted with an electrospray ionization source (ESI+), was additionally used for analysing unlabeled TEA; monitored ion transitions were at 130.2 > 86.1 m/z. Intracellular accumulations of substrates were finally normalised to cellular protein content, determined by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

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

For efflux assays, transporter-expressing cells were first incubated at 37°C for 30 min with the reference substrate (CDCF used under its diacetate form or Hoechst 33342) (loading phase), in the transport assay medium described above and as indicated in Supplementary Table S1. After washing in PBS, the cells were re-incubated in substrate-free medium in the absence or presence of carbamate pesticides or reference inhibitors (efflux phase). The cells were then washed twice in ice-cold PBS and cellular levels of substrates were measured by spectrofluorimetry and normalised to cellular protein content.

Data were finally expressed as percentages of substrate accumulation (for accumulation assays) or retention (for efflux assays) found in control cells not exposed to carbamate pesticide or reference inhibitor, arbitrary set at 100%. Alternatively, data were expressed as percentages of transporter activity, according to the following equations:

For SLC transporters:

𝑇𝑟𝑎𝑛𝑠𝑝𝑜𝑟𝑡𝑒𝑟 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = [𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑃𝑒𝑠𝑡𝑖𝑐𝑖𝑑𝑒 ] -[𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟 ] [𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝐶𝑜𝑛𝑡𝑟𝑜𝑙𝑒 ] -[𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟 ] × 100 (𝐴)
For ABC transporters :

𝑇𝑟𝑎𝑛𝑠𝑝𝑜𝑟𝑡𝑒𝑟 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = [𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟 ] -[𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑃𝑒𝑠𝑡𝑖𝑐𝑖𝑑𝑒 ] [𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟 ] -[𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 ] × 100 (𝐵)
with Some data were also expressed as percentages of reduction of transporter activity using the following equation:

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

with A = percentage of transporter activity in the presence of a given concentration of carbamate pesticide, determined as described in equation (A) or equation (B). According to the US Food and Drug Administration (FDA), a threshold of 50% transporter activity reduction can be retained for fully validating the effect of a transporter inhibitor [START_REF] Giacomini | Membrane transporters in drug development[END_REF].

Trans-stimulation assays

Trans-stimulation corresponds to the stimulation of the transport of the labeled substrate in response to the presence of a chemical (the stimulating agent) on the opposite side of the transporting membrane [START_REF] Wright | Mechanism of cis-and trans-substrate interactions at the tetraethylammonium/H+ exchanger of rabbit renal brush-border membrane vesicles[END_REF], whereas, for cis-stimulation, the labeled substrate and the stimulating agent are present at the same side of the transporting membrane.

Because substrates are considered to trans-stimulate some SLC transporters, trans-stimulation studies permit to identify such substrates [START_REF] Sweet | Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient[END_REF][START_REF] Zhang | The interaction of ntetraalkylammonium compounds with a human organic cation transporter, hOCT1[END_REF]. Transstimulation assays were performed as previously described (Chedik et al., 2017a;[START_REF] Sayyed | Inhibition of organic anion transporter (OAT) activity by cigarette smoke condensate[END_REF]. Briefly, transporter-expressing HEK-293 cells were first preloaded with unlabeled reference substrates (2 mM TEA for OCT1, OCT2 and MATE2-K and 1 mM glutarate for OAT3) or 100 μM carbamate pesticides for 15 min (HEK-OAT3 cells) or 30 min (HEK-OCT1, HEK-OCT2 and HEK-MATE2-K cells) at 37°c in the transport assay medium [START_REF] Yang | admetSAR 2.0: web-service for prediction and optimization of chemical ADMET properties[END_REF]. For each carbamate pesticide, the prediction (P-gp substrate or non-substrate and P-gp inhibitor or non-inhibitor) was associated with a probability value, between 0.5 to 1.0 unit; the closer the score is to 1, the better the prediction (http://lmmd.ecust.edu.cn/admetsar2/). In silico admetSAR 2.0-based prediction of transport by P-gp was additionally performed for reference P-gp substrates (n=10) and non-substrates (n=10), as an internal validation control.

In silico prediction of P-gp-carbamate pesticides interactions

Prediction of in vivo modulation of drug transporter activity

In vivo modulation of transporter activities by carbamate pesticides was evaluated from in vitro data using the criteria defined by the US FDA guidance on in vitro drug interaction studies (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/vitrodrug-interaction-studies-cytochrome-p450-enzyme-and-transporter-mediated-druginteractions respectively. These ADI values were normalised to a subject of 70 kg body weight and by meal; for this purpose, we postulated that the oral ingestion of the pesticide dose occurs through two daily meals, via carbamate pesticides-contaminated food and drink. This resulted in oral doses of 1.75 mg (8.2 µmole) and 10.15 mg (53.9 µmole) for chlorpropham and propamocarb, respectively, with which pesticide I gut values were calculated, as described above.

Statistical analysis

Experimental data were routinely expressed as means ± SEM. They were statistically analysed through analysis of variance (ANOVA) followed by the Dunnett's or the Tukey's post-hoc test. The criterion of significance was p<0.05.

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

Results

Interactions of carbamate pesticides with ABC transporter activities

The carbamate pesticides aminocarb, carbofuran, chlorpropham and propamocarb were initially used at a non-toxic 100 μM concentration, in the range of those previously used in vitro [START_REF] Kawaratani | Influence of the carbamate fungicide benomyl on the gene expression and activity of aromatase in the human breast carcinoma cell line MCF-7[END_REF][START_REF] Yazdian | Evaluation of the expression of P-glycoprotein in propoxur-resistant Caco-2 cells[END_REF]. They failed to enhance cellular accumulation of rhodamine 123, a reference substrate for the R-site of P-gp (Jouan et al., 2016b), in P-gp overexpressing MCF7R cells (Figure 2A). In the same way, they did not alter cellular retention of Hoechst 33342, a reference substrate for the H-site of P-gp [START_REF] Martinez | Understanding polyspecificity within the substratebinding cavity of the human multidrug resistance P-glycoprotein[END_REF]. By contrast, the reference P-gp inhibitor verapamil enhanced cellular accumulation of rhodamine 123 and cellular retention of Hoechst 33342 in MCF7R cells (Figure 2A).

Taken together, such data indicate that aminocarb, carbofuran, chlorpropharm and propamocarb, unlike verapamil, did not inhibit transport activity of P-gp, whatever the considered active site (R or H) of P-gp. Such in vitro data were fully confirmed by in silico analysis using the admetSAR 2.0 web-tool, which predicts no inhibition of P-gp activity by these chemicals (Supplementary Table S2). In the same way, among a large set of 57 additional carbamate pesticides, 55 were predicted to not inhibit P-gp activity (Supplementary Table S2). Only alanycarb and furathiocarb were found to inhibit P-gp activity according to the in silico analysis, with, however, rather low probability values (0.6602 and 0.5935 for alanycarb and furathiocarb, respectively). Besides, P-gp was predicted to not transport aminocarb, carbofuran, chlorpropham and propamocarb, as well as the 57 additional carbamate pesticides investigated in the in silico study (Supplementary Table S2). The webtool admetSAR 2.0 was finally demonstrated to correctly in silico predict P-gp-mediated transport for various reference P-gp substrates and non-substrates, used here as positive and negative controls (Supplementary Table S3).

A c c e p t e d M a n u s c r i p t enhance cellular retention of the MRP substrate CDCF in HuH-7 cells, in contrast to the reference MRP inhibitor probenecid (Figure 2A). This suggests that MRP activity was not impaired by these agrochemicals. In the same way, aminocarb, carbofuran and propamocarb, unlike the reference BCRP inhibitor fumitremorgin C, did not enhance cellular retention of the BCRP substrate Hoechst 33342 in HEK-BCRP cells (Figure 2A); this indicates a lack of effect of these carbamate pesticides towards BCRP activity. By contrast, 100 µM chlorpropham significantly increased Hoechst 33342 retention in HEK-BCRP cells by a 1.5fold factor (Figure 2A), corresponding to a reduction of BCRP activity around 70%, greater than the threshold of 50%, retained by the US FDA for considering that a transporter inhibition may be validated [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. This inhibitory effect of chlorpropharm towards BCRP activity was concentration-dependent, with an IC 50 value of 53.2 µM (Figure 2B).

Interactions of carbamate pesticides with SLC transporters of organic anions

Aminocarb, carbofuran and propamocarb, used at 100 µM, did not inhibit uptake of the reference substrates DCF and 8-FcA in HEK-OATP1B1 and HEK-OATP1B3 cells (Figure 3).

Aminocarb and propamocarb also failed to modify E3S accumulation in HEK-OATP2B1 cells, whereas carbofuran slightly enhanced it (Figure 3). Chlorpropham did not alter accumulation of 8-FcA in HEK-OATP1B3 cells. By contrast, it diminished accumulation of DCF in HEK-OATP1B1 cells and that of E3S in HEK-OATP2B1 cells (Figure 3); such reductions of transporter activities were however rather modest, i.e., 100 µM chlorpropham decreased OATP1B1 and OATP2B1 activities by 37.2% and 24.9%, respectively, thus indicating that IC 50 values are higher than 100 µM.

Carbofuran, chlorpropham and propamocarb used at 100 µM failed to decrease uptake of the reference OAT substrate 6-CF in HEK-OAT1 cells, whereas aminocarb decreased it by A c c e p t e d M a n u s c r i p t a 1.36-fold factor (Figure 4A), corresponding to a slight reduction of OAT1 activity by 28.0%. Aminocarb, as well as carbofuran and propamocarb, similarly modestly reduced OAT3 activity, i.e., it was decreased by approximately 20-25% (Figure 4A). By contrast, chlorpropham markedly reduced 6-CF accumulation in HEK-OAT3 cells by a 4.64-fold factor (Figure 4A), reflecting a marked repression of OAT3 activity by 91.1%. Chlorpropham also hugely diminished OAT3-mediated-transport of the endogenous substrate E3S (Figure 4B).

The inhibition of OAT3 activity by chlorpropham was concentration-dependent (IC 50 = 5.0 µM) (Figure 4C). The carbamate pesticide however failed to trans-stimulate accumulation of the OAT3 substrate 6-CF in HEK-OAT3 cells (Figure 4D). By contrast, the reference OAT3 substrate glutarate trans-stimulated OAT3 activity (Figure 4D), which is fully consistent with the fact that OAT3 substrates can trans-stimulate OAT3 activity [START_REF] Sweet | Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient[END_REF].

Interactions of carbamate pesticides with SLC transporters of organic cations

Aminocarb and carbofuran failed to alter activity of OCT1, OCT2, MATE1 and MATE2-K (Figure 5A). Chlorpropharm was also without effect towards OCT1, OCT2 and MATE2-K and rather moderately decreased 4-DiASP accumulation in HEK-MATE1 cells by 33.4%

(Figure 5A). Propamocarb did not alter 4-DiASP accumulation in HEK-MATE1 cells, but reduced it in HEK-OCT1 (by a 2.3-fold factor) and HEK-OCT2 cell (by a 1.9-fold factor) (Figure 5A), corresponding to repressions of OCT1 and OCT2 activity by 69.5% and 57.2%, respectively. By contrast, propamocarb enhanced 4-DiASP uptake in HEK-MATE2-K cells (by a 2.4-fold factor) (Figure 5A). The inhibitory effects of propamocarb towards OCT1 and OCT2 activities were next found to be concentration-dependent, with IC 50 values around 40-50 µM; the cis-stimulating effect of the carbamate towards MATE2-K activity was also concentration-dependent (EC 50 = 5.6 µM) (Figure 5B). Propamocarb was next demonstrated to not trans-stimulate OCT1, OCT2 or MATE2-K activity, in contrast to the reference A c c e p t e d M a n u s c r i p t substrate TEA (Figure 6A). HEK-OCT1, HEK-OCT2 and HEK-MATE2-K cells finally failed to display significant increased accumulation of propamocarb when compared to parental HEK-wt cells (Figure 6B). By contrast, HEK-OCT1, HEK-OCT2 and HEK-MATE-2K exhibited higher accumulation of the reference OCT and MATE substrate TEA comparatively to HEK-wt cells (Figure 6B).

Prediction of in vivo modulation of drug transporter activities by chlorpropham and propamocarb

Evaluation of in vivo alteration of drug transporters by chlorpropham and propamocarb was performed from in vitro data using the criteria defined by the US FDA [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. Plasma concentrations of pesticides in environmentally-exposed humans, although very poorly characterised, are postulated to be in the picomolar to nanomolar range [START_REF] Seeger | Mixture effects of estrogenic pesticides at the human estrogen receptor alpha and beta[END_REF], and probably even less when considered their unbound fraction, which remains largely unknown. In agreement with this hypothesis, chlorpropham concentrations in 100 human blood samples collected from the general population in Beijing (China) were less than 3.8 nM [START_REF] Li | Background biomonitoring of residue levels of 137 pesticides in the blood plasma of the general population in Beijing[END_REF]. Available plasma human concentrations for other carbamate pesticides also correspond to values in the nM range or less [START_REF] Barr | Pesticide concentrations in maternal and umbilical cord sera and their relation to birth outcomes in a population of pregnant women and newborns in New Jersey[END_REF][START_REF] Petropoulou | Determination of carbofuran, carbaryl and their main metabolites in plasma samples of agricultural populations using gas chromatography-tandem mass spectrometry[END_REF][START_REF] Whyatt | Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns[END_REF]. We therefore retained the value of 3.8 nM as approximated I max,u for chlorpropham and also propamocarb. With this value, most likely over-estimated due to the absence of consideration of the unbound fraction, OCT1, OCT2 and MATE2-K activities were predicted to be not in vivo impaired by plasma concentrations of propamocarb, whereas OAT3 and BCRP were similarly not predicted to be in vivo inhibited by plasma levels of chlorpropham (Table 1). For calculating luminal gut concentrations (I gut ) of chlorpropham and propamocarb, we considered the admissible daily intake (ADI) of these two pesticides according to the European Union-Pesticides database, for estimating their oral dose for one meal and a 70 kg body weight (Table 2). Although the calculated ratio I gut /IC 50 A c c e p t e d M a n u s c r i p t reached notable levels (notably the value of 4.5 for propamocarb/OCT1), they did not fulfill the US FDA criteria for in vivo inhibiting intestinal BCRP and OCT1 (Table 2).

Discussion

The present study demonstrates that some carbamate pesticides inhibit drug transporter activities, thus adding such chemicals to the growing list of agrochemicals interacting with drug transporters. As already demonstrated for other classes of pesticides like organochlorine, pyrethroid and organophosphorus pesticides [START_REF] Gueniche | Implication of human drug transporters to toxicokinetics and toxicity of pesticides[END_REF], the inhibition of transporter activity by carbamates depends on the nature of the pesticide and of the transporter. The N-methylcarbamates aminocarb and carbofuran thus rather poorly interact with ABC and SLC drug transporters, whereas chlorpropham inhibits BCRP and OAT3

activities and propamocarb interacts with the organic cation transporters OCT1, OCT2 and MATE2-K. The basis for such differential interactions of carbamate pesticides with drug transporters is most likely due to the specific physicochemical features of each carbamate agrochemical, as already described for pyrethroid or organophosphorus pesticides [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]Chedik et al., 2017a). The reduced number of carbamate pesticides analysed in the present study, i.e., only four carbamate pesticides, unfortunately prevents to search the physicochemical parameters which may discriminate inhibitors from non-inhibitors among this pesticide class.

None of the four carbamate pesticides analysed in the present study was found to inhibit P-gp activity, whatever the considered active site of P-gp (R-or H-site). Similarly, the carbamate pesticides aldoxycarb, carbaryl, lannate and propoxur used at 100 or 250 µM failed to inhibit P-gp activity in P-gp-expressing cultured cells [START_REF] Bain | Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein[END_REF]. Taken together, such data suggest that no or little inhibitory effects towards P-gp can be expected for carbamate pesticides. Results from P-gp inhibition prediction with an in silico web-tool for a large set of carbamate pesticides fully argue in favor of this hypothesis. Indeed, only 2/61

A c c e p t e d M a n u s c r i p t carbamate pesticides, i.e., alanycarb and furathiocarb, were predicted to inhibit P-gp, with moreover rather low probability values. Carbamate pesticides were additionally predicted to be not substrates for P-gp. A P-gp-mediated efflux of carbamate pesticides at the apical pole of enterocytes can therefore likely be discarded, which fully agrees with the predicted high intestinal absorption of these agrochemicals (Chedik et al., 2017b). Transport of carbamate pesticides by P-gp expressed at the apical pole of capillary brain endothelial cells [START_REF] Cordon-Cardo | Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites[END_REF] can similarly most likely be ruled out; this may suggest a high passage of carbamate pesticides across the blood-brain barrier and agrees with their predicted high brain permeation (Chedik et al., 2017b). Besides P-gp activity, those of MRPs and BCRP were also not impacted by the carbamate pesticides used in the present study, excepted chlorpropham, which inhibited BCRP. The lack of inhibition of human BCRP by propamocarb does not agree with previous data reporting an inhibition of rabbit BCRP activity by this carbamate pesticide [START_REF] Halwachs | Assessment of ABCG2mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model[END_REF]. Such a discrepancy may be due to the use of different concentrations of propamocarb in the studies (100 µM in our present study and 265.5 µM in that of Halwach et al. (2016)). Alternatively, it may reflect species-dependent effects of propamocarb towards BCRP activity; such a concept of species-dependent inhibition of transporters is already established for at least P-gp [START_REF] Zolnerciks | Substrate-and species-dependent inhibition of P-glycoprotein-mediated transport: implications for predicting in vivo drug interactions[END_REF].

It is noteworthy that inhibition of transporters by chlorpropham and propamocarb is unlikely to occur in environmentally-exposed humans when applying the criteria of FDA for in vivo drug-drug interactions due to impairment of transporter activities. This reflects the fact that in vitro inhibition of drug transporters requires rather high concentrations of carbamate pesticides (IC 50 values are approximately in the 5-50 µM range), whereas their human plasma unbound concentrations, although very poorly characterised, may be postulated to be much lower, i.e., in the nM range or pM range. Luminal gut concentrations of chlorpropham and propamocarb, although in the 30-200 µM range for oral ingestion of the ADI dose, are also A c c e p t e d M a n u s c r i p t insufficient for inhibiting in vivo intestinal BCRP or OCT1. Other pesticides interacting with drug transporters in cultured cells, such as the pyrethoids allethrin and tetramethrin and some organophosphorus pesticides, have similarly been hypothesised to fail to alter transporter activities in environmentally-or occupationally-exposed humans [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]Chedik et al., 2017a;[START_REF] Gueniche | Implication of human drug transporters to toxicokinetics and toxicity of pesticides[END_REF]. It should however be kept in mind that humans are often exposed to mixtures of pesticides and/or other environmental pollutants like plasticizers, whose inhibitory effects towards drug transporters may add or synergize, as already demonstrated for inhibition of P-gp by pesticide combinations [START_REF] Pivcevic | Pesticides and their binary combinations as P-glycoprotein inhibitors in NIH 3T3/MDR1 cells[END_REF] or marine pollutants [START_REF] Nicklisch | Global marine pollutants inhibit Pglycoprotein: Environmental levels, inhibitory effects, and cocrystal structure[END_REF]. Exposure to such mixtures may consequently have to be considered for judging the in vivo relevance of transporter inhibition by pollutants, including carbamate pesticides [START_REF] Gueniche | Implication of human drug transporters to toxicokinetics and toxicity of pesticides[END_REF]. This assertion may be notably valuable for agricultural workers, often exposed to high concentrations of pesticides in an occupational manner [START_REF] Mostafalou | Pesticides: an update of human exposure and toxicity[END_REF]. Besides, human poisoning by carbamate pesticides, either accidentally or by self-ingestion, can result in relative high in vivo concentrations of these agrochemicals, reaching the µM range [START_REF] Michael | The pharmacokinetics and pharmacodynamics of severe aldicarb toxicity after overdose[END_REF][START_REF] Moriya | A fatal poisoning caused by methomyl and nicotine[END_REF] and thus putatively blocking transporter activities. The potential clinical consequences of such inhibitions of drug transporters are nevertheless most likely completely overshadowed by the severe cholinergic crisis characterizing carbamate insecticide poisoning [START_REF] Vale | Organophosphorus and carbamate insecticide poisoning[END_REF]. Carbamate poisoning also concerns domestic animals, such as cats and dogs [START_REF] De Siqueira | Poisoning of cats and dogs by the carbamate pesticides aldicarb and carbofuran[END_REF], for which inhibition of transporters by carbamates may consequently be hypothesized to also occur.

Propamocarb was able to cis-stimulate MATE2-K activity and such a stimulation of drug transporter activity, although not frequent, has also been reported to occur for OATP2B1 activity in response to organophosphorus pesticides [START_REF] Chedik | Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters[END_REF]. In the same way, some drugs have demonstrated to cis-stimulate activity of transporters; for example, the indomethacin enhanced activity of the ABC pump MRP2 [START_REF] Pedersen | Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2)[END_REF], whereas that of OATP2B1 is increased by rifampicin [START_REF] Vildhede | Hepatic uptake of atorvastatin: influence of variability in transporter expression on uptake clearance and drug-drug interactions[END_REF]. The molecular mechanism of such transporter cis-stimulations and the potential consequences, notably for intestinal absorption of xenobiotics, remain to be determined [START_REF] Ogura | Quercetin-3-rhamnoglucoside (rutin) stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1[END_REF].

Chloropropham failed to trans-stimulate activity of OAT3, whereas those of OCT1, OCT2 and MATE2-K were similarly not trans-stimulated by propamocarb. Because substrates are considered to trans-stimulate such transporters [START_REF] Sweet | Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient[END_REF][START_REF] Zhang | The interaction of ntetraalkylammonium compounds with a human organic cation transporter, hOCT1[END_REF], these data indicate that chlorpropham and propamocarb are likely not transported, or only poorly, by these transporters. The fact that HEK-OCT1, HEK-OCT2 and HEK-MATE2-K cells did not exhibit significant enhanced accumulation of propamocarb when compared to parental HEK-wt cells supports this conclusion. OAT3, expressed at the basolateral pole of proximal tubular cells [START_REF] Burckhardt | Drug transport by Organic Anion Transporters (OATs)[END_REF], is therefore unlikely to be implicated in the preferential accumulation of chlorpropham in the kidney and in its renal excretion, described in rats [START_REF] Fang | Metabolic studies of C-14-labelled propham and chlorpropham in female rat[END_REF]. In the same way, the renal transporters OCT2 and MATE2-K [START_REF] Motohashi | Organic cation transporter OCTs (SLC22) and MATEs (SLC47) in the human kidney[END_REF] are unlikely to participate to the major elimination of propamocarb by the kidney [START_REF] Kopp | Pharmacokinetics of propamocarb hydrochloride on single and repeated oral administration of 0.5 mg/kg in rats[END_REF], whereas intestinal OCT1 probably fails to contribute to the intestinal absorption of this pesticide.

In summary, the herbicide carbamate chlorpropham as well as the fungicide carbamate propamocarb were found to in vitro interact with some drug transporters. Chlorpropham inhibited BCRP and OAT3 activities, whereas propamocarb impaired those of OCT1, OCT2

and MATE-2K, without being transported. Chlorpropham and propamocarb concentrations required for interactions with transporters are however much higher than those expected in environmentally-exposed humans, making unlikely in vivo transporter activity alterations by these carbamate pesticides as single agents. The fact that chlorpropham and propamocarb No stimulation a In vivo inhibition of drug transporter activity can be predicted if the ratio I max,u /IC 50 ≥ 0.1 (and by analogy in vivo stimulation of drug transporter activity may be predicted if the ratio I max,u /EC 50 ≥ 0.1). b Defined as the maximum unbound plasma concentration. c From [START_REF] Li | Background biomonitoring of residue levels of 137 pesticides in the blood plasma of the general population in Beijing[END_REF].

  [𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑃𝑒𝑠𝑡𝑖𝑐𝑖𝑑𝑒 ] = cellular concentration of the reference substrate in the presence of a defined concentration of carbamate pesticide, [𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟 ] = cellular concentration of the reference substrate in the presence of the reference inhibitor and [𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 ] = cellular concentration of the reference substrate in control cells exposed neither to carbamate pesticide nor to the reference inhibitor.

  described above. After washing twice with PBS, cells were next re-incubated in transport assay medium containing 29 μM [ 14 C]-TEA (HEK-OCT1, HEK-OCT2 and HEK-MATE2-K cells) or 10 µM 6-CF (HEK-OAT3 cells) for 5 min at 37°C. After washing twice with PBS, cells were lysed in distilled water and intracellular accumulations of [ 14 C]-TEA and 6-CF were then determined by scintillation counting or spectrofluorimetry, respectively, and normalised to cellular protein content.

For

  , HEK-OCT2, HEK-MATE2-K and parental HEK-wt cells were incubated with 100 μM propamocarb or 29 μM [ 14 C]-TEA for 5 min at 37°C, in the transport assay medium defined above. Cells were next washed twice in ice-cold PBS and lysed in distilled water.Cellular accumulation of propamocarb was then determined by LC-MS/MS using the system described above; monitored ion transitions were at 189.1 > 144.1 m/z. [ 14 C]-TEA accumulation was determined by scintillation counting. Data were normalised to protein content. Accumulation of propamocarb and TEA in transporter-overexpressing HEK cells were finally expressed as fold-change comparatively to propamocarb or TEA accumulation found in control parental HEK-wt cells. Determination of kinetic parameters Half maximal inhibitory concentrations (IC 50 ) of chlorpropham towards BCRP activity and of propamocarb towards OCT1 and OCT2 activities and half maximal effective concentration (EC 50 ) of propamocarb toward MATE2-K activity were determined from nonlinear regression of concentration-response data based on the four parameter logistic function. They were calculated using GraphPad Prism software 8.3 (GraphPad Software, San Diego, CA, USA) through the following equations: (EC50-[I]))x Hill slope (𝐸) with A = percentage of transporter activity for a given concentration of carbamate pesticide determined as described in equation (A) (for propamocarb/OCT1/OCT2/MATE2-K) or in A c c e p t e d M a n u s c r i p t equation (B) (for chlorpropharm/BCRP), A max is the maximal percentage transporter activity, [I] is the carbamate pesticide concentration in the medium, and Hill slope is a coefficient describing the steepness of the curve.

  s c r i p t effects towards transporters may add on or synergize with those of other pesticides and/or chemicals contaminants, to which humans are often co-exposed in an environmental or occupational context, has however probably to be considered.

Figure 1 .

 1 Figure 1. Chemical structures of carbamate pesticides.

Figure 2 .

 2 Figure 2. Effects of carbamate pesticides on P-gp, MRP and BCRP activities.

Figure 3 .

 3 Figure 3. Effects of carbamate pesticides on OATP activities.

Figure 4 .

 4 Figure 4. Interactions of carbamate pesticides with OAT1 and OAT3.

Figure 5 .

 5 Figure 5. Effects of carbamate pesticides on OCT and MATE activities.

  Accumulation of reference substrates for OCT1 (TEA) or for OCT2, MATE1 and MATE2-K (4-DiASP) in HEK-OCT1, HEK-OCT2, HEK-MATE1 or HEK-MATE2-K cells was determined in the absence (control) or the presence of 100 µM carbamate pesticides or reference inhibitors (200 µM verapamil for OCT1 or 100 µM amitriptyline for OCT2, MATE1 or MATE2-K). Data are expressed as % of reference substrate accumulation in control cells; they are the means ± SEM of at least three independent assays. Dotted lines indicate the reference substrate levels in control cells. *, p < 0.05, **, p < 0.01 and ***, p < 0.001 when compared to control. (B) Effects of various concentrations of propamocarb on OCT1, OCT2 or MATE2-K activity were analysed through determining accumulation of TEA (OCT1 activity) or 4-DiASP (OCT2 or MATE2-K activity) in HEK-OCT1, HEK-OCT2 or HEK-MATE2-K cells. Data are expressed as % of transporter activity in control cells not exposed to propamocarb, arbitrarily set at 100%; they are the means ± SEM of at least three independent assays. IC 50 or EC 50 values of propamocarb are indicated at the top of the graphs.

Figure 6 .

 6 Figure 6. Trans-stimulating effect (A) and accumulation (B) of propamocarb in HEK-OCT1,

  IC 50 /maximum unbound plasma concentration of chemicals (I max,u ) ≥ 0.1[START_REF] Giacomini | Membrane transporters in drug development[END_REF]. For prediction of potential in vivo stimulation of transporter activity, the criteria defined by the FDA was adapted by replacing IC 50 by EC 50 . For intestinal P-gp and BCRP, they can be in vivo inhibited according to FDA if the ratio IC 50 /luminal gut concentration (I gut ,

	defined by the ratio dose orally ingested/250 mL) ≥ 10 (Giacomini et al., 2010). This criteria
	was also extended to intestinal OCT1 in the present study, because OCT1 is expressed at the
	apical membrane of enterocytes, like P-gp (Han et al., 2013). For defining the ingested oral
	dose of carbamate pesticides, we considered the admissible daily intake (ADI) of the
	pesticides,	defined	in	the	European	Union-Pesticides	database

). Briefly, transporters can be in vivo inhibited by xenobiotics if the ratio A c c e p t e d M a n u s c r i p t (https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public); ADI values are 0.05 mg/kg body weight and 0.29 mg/kg body weight for chlorpropham and propamocarb,

Table 1 :

 1 Prediction for in vivo transporter activity modulation by plasma concentrations of carbamate pesticides according to FDA criteria a

	Carbamate pesticide	Approximated I max,u (µM) b	Transporter	IC 50 /EC 50 (µM)	Ratio I max,u /

IC 50 or EC 50 Potential in vivo interaction with transporter

	Chlorpropham	0.0038 c	BCRP OAT3	53.2 (IC 50 ) 5.0 (IC 50 )	<0.001 <0.001	No inhibition No inhibition
			OCT1	48.1 (IC 50 )	<0.001	No inhibition
	Propamocarb	0.0038 c	OCT2	38.1 (IC 50 )	<0.001	No inhibition
			MATE2-K	4.6 (EC 50 )	<0.001

Table 2 :

 2 Prediction for in vivo transporter activity inhibition by luminal gut concentrations of carbamate pesticides according to FDA criteria a In vivo inhibition of intestinal P-gp activity (and by analogy intestinal OCT1 activity) can be predicted if the ratio I gut /IC 50 ≥ 10.

	Carbamate pesticide	Admissible daily intake (ADI) b (mg/kg body weight)	Oral dose c (µmole)	I gut (µM) d	Transporter	IC 50 (µM)	Ratio I gut /IC 50	Potential in vivo inhibition of transporter
	Chlorpropham	0.05	8.2	32.8	BCRP	53.2	0.6	No inhibition
	Propamocarb	0.29	53.9	215.6	OCT1	48.1	4.5	No inhibition

a b According to the European Union-Pesticides database. c Defined for one meal and a 70 kg body weight. d Luminal gut concentration, calculated as the ratio oral dose/250 mL.

Acknowledgements

The authors thanks Dr Y. Parmentier, Dr C. Denizot and Dr M. Le Vée for helpful support with HEK-293 cell clones overexpressing transporters and Technologie Servier (Orléans, France) for the gift of the LC-MS/MS system.

Disclosure statement

No potential conflict of interest was reported by the authors.