Barthélémy Brunier

Nida Sheibat-Othman

Mehdi Chniguir

Yves Chevalier

Elodie Bourgeat-Lami

Investigation of four different Laponite ® clays as stabilizers in Pickering emulsion polymerization

Keywords: Pickering emulsion polymerization, clay, Laponite, stabilization, adsorption

Clay-armored polymer particles were prepared by emulsion polymerization in the presence of Laponite platelets that adsorb at the surface of latex particles and act as stabilizers during the course of the polymerization. While Laponite ® RDS clay platelets are most often used, the choice of the type of clay still remains an open issue that is addressed in the present paper. Four different grades of Laponite were investigated as stabilizers in the emulsion polymerization of styrene. First, the adsorption isotherms of the clays, on preformed polystyrene particles, were determined by ICP-AES analysis of the residual clay in the aqueous phase. Adsorption of clay depended on the type of clay at low concentrations corresponding to adsorption as a monolayer.

Adsorption of clay particles as multilayers was observed for all the grades above a certain concentration under the considered ionic strength (mainly due to the initiator ionic species). The stabilization efficiency of these clays was investigated during the polymerization reaction (free of any other stabilizer). The clays did not have the same effect on stabilization, which was related to differences in their compositions and in their adsorption isotherms. The different grades led to different polymer particles sizes, and therefore to different polymerization reaction rates. Laponite RDS and S482 gave similar results, ensuring the best stabilization efficiency and the fastest reaction rate; the number of particles increased as the clay concentration increased.

Stabilization with Laponite XLS gave the same particles size and number as the latter two clays at low clay concentrations; but it reached an upper limit in the number of nucleated polymer particles at higher concentrations indicating a decrease of stabilization efficiency at high concentrations. Laponite JS did not ensure a sufficient stability of the polymer particles, as the polymerization results were comparable to a stabilizer-free polymerization system.

Introduction

Polymer nanocomposites allow combining polymer properties (e.g. stiffness, toughness) with those of inorganic materials (e.g. resistance to ignition) which enhances the end-use product properties (e.g. barrier properties, fire resistance or optical properties) while reducing their cost. [START_REF] Sill | Polymer-Nanoparticle Composites[END_REF] Among inorganic materials, Laponite ® 2 clay (BYK Additives Ltd) can be easily exfoliated in water as a colloidal suspension, making it particularly interesting for the stabilization of emulsions [START_REF] Ashby | Emulsions Stabilised by Laponite Clay Particles[END_REF] or latex particles. [START_REF] Bon | Pickering miniemulsion polymerization using Laponite clay as a stabilizer[END_REF][START_REF] Cauvin | Pickering stabilized miniemulsion polymerization: Preparation of clay armored latexes[END_REF][START_REF] Bourgeat-Lami | High Solids Content, Soap-Free, Film-Forming Latexes Stabilized by Laponite Clay Platelets[END_REF] Therefore, Laponite clay has been widely used to stabilize polymer particles during emulsion polymerization without any other stabilizing agent, which we refer to as Pickering emulsion polymerization in this work.

Pickering emulsion polymerization is a classical emulsion polymerization process, but where the conventional emulsifier is replaced by solid inorganic particles/platelets that adsorb at the surface of polymer particles during the course of the polymerization. It is a free radical polymerization (of styrene in the present case) initiated by a water-soluble initiator (potassium persulfate in the present case). The monomer is emulsified as coarse droplets, so that polymerization takes place in the aqueous phase and inside the monomer-swollen polymer particles after such particles have nucleated. It is to be distinguished from Pickering miniemulsion polymerization where a miniemulsion of monomer of sub-micron droplets is prepared such that the radicals generated in the aqueous phase are efficiently trapped by the monomer droplets; hence, the polymerization takes place in the monomer droplets and the final polymer particles correspond to the initial monomer droplets if coagulation is prevented.

The term Laponite is a brand that includes quite a large number of materials. [START_REF][END_REF] In the framework of clay chemistry, Laponite is a synthetic hectorite. [START_REF] Coelho | Argilas Especiais: Argilas Quimicamente Modificadas-Uma Revisão[END_REF] It is chemically composed of magnesium silicates (the neutral equivalent is talc) where part of the magnesium atoms has been substituted by monovalent lithium atoms. Such isomorphic substitution causes a departure from electrical stoichiometry that provides a negative surface charge to the platelets balanced by sodium cations.

The charge is related to the substitution rate of Mg by Li being in the 5-10 mol% range.

Physically, this clay is made of disc-shaped crystals (i.e. platelets) of approximately 25 nm diameter and 0.92 nm thickness. [START_REF] Fripiat | Thermodynamic and Microdynamic Behavior of Water in Clay Suspensions and Gels[END_REF] Its density is 2570 kg m -3 9 and it has a melting point of 900 °C. Each elementary particle is a crystal composed of about 1500 unit cells, and each cell is composed of six octahedrally coordinated atoms, either divalent magnesium or monovalent lithium, sandwiched between two layers of four tetrahedral silicon atoms. These groups are balanced by twenty oxygen atoms and four hydroxyl groups. In the powder form, the crystals share interlayer Na + ions forming stacks.

Though there are several grades of Laponite, many articles in the literature do not specify the studied grade, preventing a discussion with regards to the type of Laponite. Nevertheless, Laponite RD has been the most frequently used in emulsion or miniemulsion polymerizations or for the formation of gels. [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] Laponite RDS was found to be an efficient stabilizer of polymer particles produced by surfactant-free emulsion polymerization with [START_REF] Sheibat-Othman | A Kinetic Investigation of Surfactant-Free Emulsion Polymerization of Styrene Using Laponite Clay Platelets as Stabilizers[END_REF] or without addition of an auxiliary polar (macro)monomer. [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] Increasing the clay concentration increased the number of nucleated polymer particles, and hence decreased their final size. Moreover, Laponite RDS platelets adsorbed as multilayers on the surface of the polymer particles during the polymerization reaction as well as during adsorption experiments performed at room temperature on previously formed latex particles. [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] The objective of this work is to investigate the effect of different Laponite clay grades on the reaction rate and stabilization of polymer particles synthesized by surfactant-free emulsion polymerization. Four different grades were investigated: Laponite RDS, S482, XLS and JS. Such Laponite grades have been selected because they can be dispersed in water as individual nanoplatelets with no significant aggregation up to rather high concentrations, and present a long-term stability due to the stabilization of the rims by a peptizing agent. The character "S" in the grade name signifies that they are all sol-forming grades specifically designed to produce low viscosity dispersions in water through the addition of a so-called peptizing agent. The role of the peptizing agent is to avoid gel formation when dispersing the clay in water (more details are given below).

Materials and Methods

Materials

The monomer, styrene (Acros Organics, 99% extra pure, stabilized) and the initiator, potassium persulfate (KPS, Sigma-Aldrich, minimum 99%) were stored in a fridge until use. The four Laponite ® clays: RDS, XLS, JS and S482, were kindly donated by Rockwood additives (now BYK Additives Ltd). Deionized water of 18 MΩ cm resistivity was used throughout the work.

Laponite clays

In this work, three grades of Laponite were selected among the temporary sol-forming grades that are: Laponite RDS, XLS and JS. These clays can be well dispersed in water below 10 wt%. Also, Laponite S482 was selected as a permanent sol-forming grade. Fluid dispersions can be prepared up to 30 wt% concentration in water. All used clays are in powder form and contain a peptizing agent to prevent the formation of a thixotropic gel structure when dispersed in water.

The anionic peptizing agent adsorbs to the positively charged rims of the clays platelets. Charge reversal of the rims into negative charges avoids the electrostatic attraction between the rims and the faces. Tetrasodium pyrophosphate (Na 4 P 2 O 7 ) is used as peptizing agent for Laponite RDS, XLS and JS whereas (1-hydroxyethylidene)bisphosphonate (C 2 H 8 O 7 P 2.4 Na) (CAS # 7414-83-7) is used for the stabilization of Laponite S482. Laponite JS is a fluorohectorite that has its MgOH groups partly fluorinated (i.e. the OH groups are substituted by F atoms). The mean chemical compositions of these clays provided by the suppliers' product data sheet are presented in Ideal Laponite has the following chemical formula: [START_REF] Balnois | Probing the morphology of Laponite clay colloids by atomic force microscopy[END_REF][START_REF] Saunders | A Small-Angle X-Ray Scattering Study of the Structure of Aqueous Laponite Dispersions[END_REF] Na !

! Si ! Mg !!! Li ! 𝐹 ! O !" (OH) !!! !!
Though the chemical compositions given by the supplier do not fit the general chemical formula, the values of x and y have been tentatively estimated in Table 1 from the chemical compositions.

Besides, Laponite powder contains around 8 wt% water which should be either eliminated before weighing (by heating) or taken into consideration in the calculation of the concentration.

Laponite dispersion in water

Laponite platelets are hydrophilic. Their dispersion in water leads to pH = 8-9. The release of the Na + ions from the clay surface generates a strongly negative charge on their basal faces (700 elementary charges). A weakly positive charge present on the rim of the disks 4 causes destabilization and gel formation in the absence of specific stabilizer of the rims. [START_REF] Tawari | Electrical Double-Layer Effects on the Brownian Diffusivity and Aggregation Rate of Laponite Clay Particles[END_REF] The Laponite clays used in this work contain a peptizing agent: tetrasodium pyrophosphate (10 wt% based on clay) adsorbed onto the positively charged rims (edges); the tetravalent negatively charged pyrophosphate ions neutralize the rim charge and decrease electrostatic attractions between the positively charged rims and the negatively charged faces, and therefore avoid gelation. Reducing such interactions contributes to a longer stability of the dispersions in water. Laponite S482 is stabilized by the (1-hydroxyethylylidene)bisphosphonate that acts by the same mechanism as sodium pyrophosphate. Note that neutral low molar mass polyethylene oxide molecules have also been reported to delay Laponite aggregation by forming a steric barrier which prevents contact between platelets. [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] Dispersion of Laponite RDS in water has previously been investigated in order to determine the efficiency of dispersion and the time period before aging starts. [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF][START_REF] Shahin | Physicochemical Effects in Aging Aqueous Laponite Suspensions[END_REF][START_REF] Cummins | Gel: The Phases of Colloidal Laponite[END_REF][START_REF] Gupta | Time Temperature Superposition in Soft Glassy Materials[END_REF] TEM and cryo-TEM images indicated that the dispersion mainly contained isolated particles and only few stacks of two to six platelets, with an interlayer thickness of 1 nm.

Clay suspension aging

Aqueous Laponite suspensions undergo chemical aging, which is characterized by leaching of Mg !! ions taking place according to the following chemical reaction in acidic medium.

Si ! Mg !!! Li ! H ! O !" Na ! + 12 H ! + 8 H ! O → 𝑥 Na ! + 8 Si(OH) ! + 6 -𝑥 Mg !! + 𝑥 Li !
This reaction takes place in contact with CO 2 which dissolution in water generates an acidic medium (carbonic acid). It can be avoided by working under inert atmosphere. [START_REF] Fripiat | Thermodynamic and Microdynamic Behavior of Water in Clay Suspensions and Gels[END_REF] Conductometry indicated full dispersion in 1 hour and no aging (via the release of lithium and magnesium ions)

after 4 days at ambient temperature, which provides an estimate of the time period during which the non-aggregated dispersion can be safely used for polymerization.

Emulsion polymerization experiments

Ab initio semi-continuous polymerization experiments

A 1 L reactor was used with mechanical stirring at 400 rpm using a three blades Bohlender propeller. The polymerization reaction started by a batch period for polymer particles nucleation followed by a semi-continuous part for their growth. First, a known amount of clay was dispersed in 800 g of water for 30 min under stirring in the reactor at ambient temperature, degassed using nitrogen, and heated to 70 °C using a thermostated bath. Then, 40 g of styrene were added and the polymerization was initiated by adding 1.6 g of KPS dissolved in a small amount of degassed water. During the reaction, the stream of nitrogen was moved upwards off the reaction medium to maintain saturation of the gaseous atmosphere with nitrogen. At the end of the batch reaction, semi-continuous monomer addition was started where 160 g of monomer was added at a flow rate of 0.02 g s -1 . Samples were withdrawn at specific time intervals to measure the solids content (SC, i.e. mass fraction of solid) using a thermogravimetric balance and the particle size, which allowed calculating the particle number density. The solids content was used to calculate the amount of polymer and the monomer conversion (after subtracting the mass of clay and initiator). The coverage of the polymer particles surface by clay platelets was calculated as the ratio of the surface area of platelets' faces discs (of 25 nm diameter) to the polymer particles' area assuming spherical shape and full clay dispersion.

Surfactant-free polystyrene seed latex

The seed latex was synthesized for the adsorption analyses. It was prepared batch-wise using 4 g of KPS in 800 mL degassed water and 3 wt% of styrene (with respect to water). The final latex had a mean particle size of 300 nm and was concentrated under reduced pressure in a Rotavapor ® up to 20 wt% solids content. Such high concentration is required since the adsorption study using analyses by inductively coupled plasma -atomic emission spectroscopy (ICP-AES) involves diluting the latex with a dispersion of clay. Then, the latex was cleaned about 10 times with ion exchange resins (Dowex ® Marathon TM MR3) to eliminate free ionic species resulting from initiator decomposition and water-soluble species. The washing steps were repeated until a constant conductivity was obtained.

Characterizations

Particle size measurements

Particle mean size and size distribution were measured by dynamic light scattering (Zetasizer, Nano ZS, Malvern) at 25 °C at a fixed scattering angle of 173° after dilution in water. The z-

average hydrodynamic diameter (𝑑 ! , nm) is defined as 𝑑 ! = 𝑛 ! 𝑑 !" ! / 𝑛 ! 𝑑 !" !
, where 𝑛 ! is the number density of particles of diameter 𝑑 !" . The measured particle size was corrected to get the diameter of unswollen polymer particles with monomer but was not corrected with regards to the adsorbed platelets thickness. Indeed, it has previously been observed that the platelets laid flat on the polymer particles and therefore did not significantly contribute to the measured particle size, due to their small thickness (1 nm) compared to the particles diameter (of the order of 200 nm). 10

Zeta potential measurements

Zeta potentials of the Laponite platelets, dispersed at 1 g L -1 concentration in pure water, were measured by electrophoresis using a Nano ZS Zetasizer, (Malvern). The electrophoretic mobility (µ E ) was measured in a U-shaped sample holder equipped with two electrodes (Malvern folded capillary cells DTS 1070). It was converted into zeta potential (ζ) using the Henry equation

𝜇 ! = ! ! ! ! ! ! !
𝑓 𝜅𝑎 with the Smoluchowski approximation: 𝑓 𝜅𝑎 = 3 2, valid for ionic strength such that the Debye length (1/κ) is shorter than the particle size (κa > 1), where 𝜀 and 𝜀 ! are the permittivities of water and vacuum respectively, 𝜂 is the viscosity and κa is the ratio of particle radius to double layer thickness.

Inductively Coupled Plasma -Atomic Emission Spectroscopy (ICP-AES)

Adsorption of clay platelets on polystyrene latex was investigated by measuring the residual magnesium (Mg), silicon (Si) and lithium (Li) in water. A titration method was employed using at least two distinct wavelengths for each element: 279.8 nm, 280.3 nm and 285.2 nm for Mg; 252.9 nm, 255.6 nm and 288.2 nm for Si, and finally 610.4 nm and 670.8 nm for Li. First, a dispersion of clay in water was gently stirred for 24 h, then added to the surfactant-free seed described above and allowed to equilibrate for 12 h. Deionized water was added to reach the same solids content (5 wt%) for all samples, with different clay concentrations. Separation of polystyrene polymer particles from free clay platelets was ensured by two methods: 1) filtration through a syringe filter of 200 nm pore diameter or 2) centrifugation at 40 000 rpm during 20 min. To ensure that the separation method did not impact the results of adsorption; a test was done to validate that the particles did not pass through the filter while the clay did. In the centrifugation test, a test was also done to validate that clay platelets did not sediment while the particles did. [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] Each series of experiments was repeated four times.

Results and discussion

ζ potentials of the Laponite clay platelets

Electrophoresis measurements of ζ potential of clay platelets were performed in order to assess relevant data pertaining to the stabilization ability of clay platelets (as their elemental composition is not available). The ζ potential depends on the ionic strength of the aqueous medium. The adsorption isotherms of clay platelets on the surface of polymer particles were also found to depend on the ionic strength (by the comparison between cleaned latex using ion exchange resins and uncleaned latex). [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] The ionic species present in the aqueous phase during emulsion polymerization come from thermal decomposition of the KPS initiator, so that the ionic strength varies during the course of the polymerization. Pure water was however selected for the measurement of the ζ potential as the dispersion was more stable in this case.

The calculation of the ζ potential from the measured electrophoretic mobility, µ E , depends on the ionic strength and the shape of the particles. The Henry factor, 𝑓 𝜅𝑎 , in the Henry equation:

𝜇 ! = ! ! ! ! ! ! !
𝑓 𝜅𝑎 , assumes two limiting values for spherical particles depending on the ionic strength described by the width of the double electrical layer or the Debye length, κ -1 , with respect to the particle radius a: 𝑓 𝜅𝑎 = 1 for κa << 1 corresponding to low ionic strength, and 𝑓 𝜅𝑎 = 3 2 for κa >> 1, at high ionic strength. Though pure water was used as liquid medium, the ionic strength was fairly high because the Laponite platelets released ionic species in water upon dispersion. Indeed, the chemical compositions of the Laponite clays reveal an excess of sodium with respect to the substitution of lithium for magnesium x. The high ionic strength was revealed by a high electrical conductivity of the samples. The electrical conductivity measured during the electrophoresis experiments was between 0.1 and 0.4 mS cm -1 , which corresponded to an NaCl concentration of 0.01 M and a Debye length κ -1 = 3 nm, much shorter than the size of the clay platelets. Therefore, the Smoluchowki approximation where the thin double layer is not deformed by the electric field was valid and 𝑓 𝜅𝑎 was taken equal to 3 2.

The non-spherical shape of the Laponite clay platelets is another matter of concern. Such issue has been addressed both by theoretical calculations and experimental studies. [START_REF] Jiménez | The Electrokinetic Behavior of Charged Non-Spherical Colloids[END_REF] According to the calculations by Loewenberg [START_REF] Loewenberg | Unsteady Electrophoretic Motion of a Non-Spherical Colloidal Particle in an Oscillating Electric Field[END_REF] and comparative experimental studies on spherical and platelet-shaped particles, [START_REF] Rowlands | The Dynamic Mobility and Dielectric Response of Kaolinite Particles[END_REF][START_REF] Jiménez | Broadband Dielectric Spectra of Spheroidal Hematite Particles[END_REF] the application of the theoretical expressions valid for spherical particles to non-spherical particles is of acceptable accuracy as long as the surface conductivity of the particles remains low, which is the case here. Since such topic might be a matter of controversy, both the experimental electrophoretic mobilities and the calculated ζ potentials are reported in Table 2.

Table 2. Electrophoretic mobility and ζ potential of 1 g L -1 suspensions of Laponite in water.

Laponite RDS Laponite S482

Laponite XLS Laponite JS Electrophoretic mobility (m 2 V -1 s -1 ) -3.1×10 -8 -3.4×10 -8 -3.0×10 -8 -4.8×10 -8 ζ potential (mV) -40 -44 -38 -62

The ζ potentials of Laponite RDS, XLS and S482 were found to be similar in magnitude. This is not surprising owing to the close chemical compositions of Laponite RDS and XLS; the chemical composition of Laponite S482 is proprietary information of the manufacturer. Note that the ζ potential of the clay is reduced when measured in NaCl solution; for instance for Laponite RD at a concentration of 10 g L -1 in the presence of sodium pyrophosphate and NaCl (10 -3 M), the ζ potential is -29.5 mV. The ζ potential of the fluorohectorite Laponite JS is significantly higher than the three other Laponite grades. This is related to its electrical charge and the ionic adsorbed species, namely the pyrophosphate peptizing agent, and not to the substitution of F for OH that should not affect electrical charges. Thus, the substitution of lithium for magnesium is 50% higher for Laponite JS than for other grades (x = 0.75 against 0.50), which increases the electrical charge of platelets by the same amount. The amount of peptizing agent is also 50% higher in Laponite JS, causing larger negative charge. In summary, ζ potential measurements showed that the Laponite JS clay was different from the others, not only because it is a fluorohectorite, but also because it is more negatively charged. The later character is an important consideration with regards to its adsorption and the stabilization of latex particles.

Clay adsorption on polystyrene

Figure 1 shows the adsorption isotherm of Laponite clay platelets on surfactant-free polystyrene polymer particles. First of all, for all clay grades it can be noticed that the adsorption isotherms show two distinct regimes as a function of the clay concentration. For clay concentrations lower than 0.6 g L -1 (which corresponds to 0.35 g L -1 of residual clay in water), the clay platelets partitioned between the latex surface and bulk water. In this dilute regime, the adsorbed amount was lower than the amount allowing full coverage of the polymer surface. Above this clay concentration, adsorption rose almost vertically, showing that clay platelets in excess fully adsorbed onto the latex particles. In the concentrated regime, the adsorbed amount exceeded the limit of one dense monolayer. Such results strongly suggest that the clay was adsorbed as a monolayer for concentrations lower than 0.6 g L -1 but started forming multilayers for higher concentrations. With the used polymer seed with 𝑑 ! = 300 nm at SC = 5%, the surface area of polymer particles can be calculated (assuming spherical polymer particles) and compared to the surface area of adsorbed clay platelets to estimate the surface coverage. At a clay concentration of 0.6 g L -1 , the adsorbed amount of clay is 0.25 g L -1 , which gives a surface coverage of polymer particles of 40%. Full coverage can be attained (so multilayer formation can start) when the adsorbed amount of clay is 1.0 g L -1 , which corresponds to a total concentration of about

1.3 g L -1 .
The formation of multilayers is not due to the formation of aggregated stacks in water upon increasing the clay concentration. Indeed, cryo-TEM analysis of the clay suspensions before adsorption showed full clay dispersion as individual platelets. Moreover, a linear increase of the conductivity was observed when increasing the clay concentration in pure water, which indicated that the added clay was well dispersed. 10 Note that few measurements in the adsorption isotherms of Laponite S482 and XLS followed an unusual behavior. Thus, the concentration of residual clay in water could be as high as 1.2 g L -1

when the introduced clay concentration was at 3 g L -1 ; the formation of multilayers on the polymer surface started at higher concentration and the residual clay in water became similar as the other clays (at maximum 0.35 g L -1 ) leading to similar adsorption isotherms. This phenomenon might have been attributed to slow micro-gel formation that is concentrationdependent. However, according to the supplier and our observations, gel formation starts few

A B
days after the dispersion and the samples in this work were only 1 to 2 days old. Another explanation is that the samples were not at equilibrium. While adsorption can be assumed to be fast for Laponite RDS and Laponite JS thus ensuring fast equilibrium (~ 5 min for Laponite RDS), the adsorption would be slower for Laponite S482 and Laponite XLS. [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] Therefore, the data with residual clay concentrations higher than 0.5 g L -1 were discarded in fitting a theoretical model of adsorption isotherm to the experimental data.

A closer look at the dilute regime (Figure 1B), revealed significant differences between the adsorption isotherms of the clays. Laponite JS had the lowest adsorption to the particles surface, and Laponite RDS and S482 had similar first layer isotherms while the trend of the adsorption isotherm was less obvious for Laponite XLS. The noisy measurements of Laponite XLS can be due to a higher difficulty in fully dispersing this clay, especially at higher clay concentrations or to its non-equilibrium characteristics (which indicates slower adsorption). A Langmuir isotherm was nevertheless fitted to this part of the experimental data at low coverage. Note that the Langmuir isotherm assumes monolayer adsorption with no lateral interactions or steric hindrance between the adsorbed species: 22

! ! ! = ! ! ! !" !!! ! ! !" (1)
where 𝐶 !" (g L -1 ) is the equilibrium concentration in the liquid medium, 𝑞 (mg m -2 ) the surface density of adsorbed species, 𝑞 ! (mg m -2 ) the adsorbed amount at the plateau (maximum surface density for monolayer coverage) and 𝐾 ! the associated equilibrium constant (L mg -1 ). The Langmuir model was fitted to the adsorption isotherms in Figure 1B and the corresponding adsorption parameters are given in Table 3. The equilibrium constants K S are comparable for Laponite RDS and Laponite S482. A much lower value of K S was estimated for Laponite JS indicating lower binding strength of Laponite JS to the surface of polystyrene particles. The estimated 𝐾 ! parameter is lower for Laponite XLS, but this estimation should be considered with precaution due to the noisy measurements related to this clay. The adsorbed amount at full monolayer coverage q m was of similar magnitude for all Laponite clays, which was expected owing to their similar particle size. In order to describe multilayer adsorption, the Brunauer-Emmett-Teller (BET) isotherm was used. [START_REF] Brunauer | Adsorption of Gases in Multimolecular Layers[END_REF] This model, originally developed for adsorption from the gas phase, is an extension of the Langmuir theory under the additional hypothesis that molecules can also physically adsorb by binding to already adsorbed molecules, resulting in the formation of multilayers with no limit regarding the number of layers. Therefore, the model allows describing monolayer and multilayer adsorption behaviors:

! ! ! = !" !!! !!!!!" ( 2
)
where 𝑋 is the ratio of the partial pressure of the adsorbate to its saturation partial pressure at the system temperature and 𝑞 ! is the surface density of adsorbed species for a hypothetic monolayer at full coverage. 𝑐 is the BET constant defined as 𝑐 = exp

! ! !! ! ! ! !
where 𝐸 ! is the heat of adsorption for the first layer and 𝐸 ! is that for the higher layers. The BET model was applied to liquid phase adsorption by substituting the partial pressure of the adsorbate by its concentration in the liquid phase (𝑋 = 𝐶 !" 𝐶 ! ). [START_REF] Ebadi | What Is the Correct Form of BET Isotherm for Modeling Liquid Phase Adsorption?[END_REF][START_REF] Prestidge | Polymer and Particle Adsorption at the PDMS Droplet-Water Interface[END_REF] In this case, equation 2 has three degrees of freedom (𝑞 ! , 𝑐 and 𝐶 ! ). In order to estimate the adsorption energies corresponding to the different layers, Ebadi et al. [START_REF] Ebadi | What Is the Correct Form of BET Isotherm for Modeling Liquid Phase Adsorption?[END_REF] expressed the BET constant as the ratio of the equilibrium constants of the adsorption equilibria for the first layer and the higher layers:

𝑐 = ! ! ! !
, which led to the following equation:

! ! ! = ! ! ! !" !!! ! ! !" !!! ! ! !" !! ! ! !" (3)
where 𝐾 ! becomes the adsorption equilibrium constant of the 1 st layer and 𝐾 ! the adsorption equilibrium constant of upper layers. Based on this assumption, the model parameters were fitted to the data and are given in Table 3. It can be seen that 𝐾 ! is higher than 𝐾 ! , which indicates that the formation of multilayers takes place by means of stronger interactions in the first layer contacting polystyrene than for the upper layers (among clay platelets). 𝐾 ! takes the same value for all the studied Laponite clays. On the contrary, 𝐾 ! is much smaller for Laponite JS, which points the lower adsorption of Laponite JS on polystyrene.

Role of the clay in semi-continuous ab initio emulsion polymerization

Surfactant-free emulsion polymerization of styrene was carried out in the presence of increasing amounts of clay for each of the four Laponite grades by semi-continuous monomer addition in ab initio mode. The objective of these experiments was to investigate the effect of the clay concentration on the number density and stability of the nucleated particles.

Effect of Laponite RDS concentration

Investigation of Laponite RDS in ab initio semi-continuous emulsion polymerization was reported by Brunier et al (Figure 2). [START_REF] Brunier | Partitioning of Laponite Clay Platelets in Pickering Emulsion Polymerization[END_REF] The number of particles increased with increasing the clay concentration, but the increase slowed down at higher concentrations, although a clear leveling off was not observed. Increasing the clay concentration (for a fixed solids content of 20 wt%)

thus led to an increase in the reaction rate due to the formation of smaller particles. In addition, the clay platelets were quite efficient in ensuring particles' stability since the particles number remained constant until the end of the reaction, revealing that neither coagulation nor nucleation of new polymer particles took place. At the beginning of the polymerization, for clay concentrations higher than 0.5 g L -1 , the area of platelets was enough to cover the full surface area of particles and a significant excess of clay platelets was present. By the end of the reaction, as the particles grew, the amount of clay became insufficient to cover the full polymer particles' surface area, except for 10 g L -1 clay. The stabilization of polymer particles by adsorbed clay platelets is probably caused by the formation of a rigid coating around the polymer particles, which prevents the latex particles from close contact and coagulation. This is the main mechanism put forward for the stabilization of Pickering emulsions. [START_REF] Aveyard | Emulsions Stabilised Solely by Colloidal Particles[END_REF][START_REF] Chevalier | Emulsions Stabilized with Solid Nanoparticles: Pickering Emulsions[END_REF]

Effect of Laponite S482 concentration

A similar trend was observed for Laponite S482 (Figure 3). At the beginning of the reaction, the number of particles increased during the nucleation period, which lasted between 45 and 90 min. The higher the clay concentration, the longer was the nucleation period. The number of particles also increased with increasing clay concentration. At high concentrations, the increase in the particles size slowed down a little, without leveling off. This led to an increase in the reaction rate and to the formation of smaller particles when increasing the clay concentration (at the same solids content). The efficiency of this clay to stabilize the polymer particles could therefore be demonstrated as a small amount of clay (0.1 g L -1 ) was enough to reduce the particles size

A B C D
significantly compared to the clay-free experiment and the number of particles remained constant until the end of the reaction, with SC = 20%.

For clay concentrations higher than 0.5 g L -1 , the area of platelets was enough to cover the total surface of particles and a significant excess of clay platelets was present until 30 min of polymerization. As the particles grew, this amount became insufficient to cover the full polymer particles' surface. It can be noticed that the number of particles in some samples decreased during the very beginning of the reaction for high concentrations of clays. This suggests a coagulative nucleation mechanism. [START_REF] Ngai | Particle-Stabilized Emulsions and Colloids[END_REF] It is worth mentioning that this phenomenon was not observed for Laponite RDS.

Effect of Laponite XLS concentration

Figure 4 shows the results of ab initio emulsion polymerization using different concentrations of Laponite XLS. Laponite XLS behaved similarly as Laponite RDS and Laponite S482 up to 2 g L -1 (i.e. 0.05 % of clay based on monomer mass). Above 4 g L -1 , the influence of the clay concentration became small and the particles number density levelled off to around 2×10 [START_REF] Cummins | Gel: The Phases of Colloidal Laponite[END_REF] particles per liter. At this stage, the mean diameter of polymer particles was about 300 nm for a solids content of 20%. A similar levelling-off of the latex particle size was observed by Bon and coworkers [START_REF] Teixeira | Pickering Emulsion Polymerization Using Laponite Clay as Stabilizer to Prepare Armored "soft" Polymer Latexes[END_REF] during Pickering emulsion copolymerization of styrene and n-butyl acrylate using also Laponite XLS as stabilizer. It can also be noticed that the number of particles increased and then decreased during the very beginning of the reaction, which is again indicative of coagulative nucleation. This effect was more pronounced for Laponite XLS than for Laponite S482 and as a reminder, it was not observed for Laponite RDS.

Effect of Laponite JS concentration

Similarly to Laponite RDS and Laponite S482, increasing the amount of Laponite JS led to the formation of smaller particles (i.e., an increase in the number of particles) for the same solids content (Figure 5). However, the obtained latex particles were much bigger and the particles The nucleated number of particles was almost identical for the three grades Laponite RDS, S482

and XLS, for the two low concentrations (0.5 and 2 g L -1 ), (and much higher than for Laponite JS). This indicates similar surface charge for these grades. For higher concentrations, the number of particles continued to increase with increasing the clay concentration for Laponite RDS and S482 while it stayed constant for Laponite XLS. This might be due to the formation of some clay aggregates of Laponite XLS platelets at high concentrations, which reduces its stabilization efficiency. Another explanation would be that Laponite XLS adsorbs more slowly on the surface of particles, which is in agreement with the non-equilibrium characteristic of this clay observed in the adsorption isotherm measurements.

Laponite JS led to the generation of the lowest number of particles at all clay concentrations. This clay contains fluorine (4.8%), contrary to the other clays, and a higher amount of lithium (1.2% vs 0.8% for the other clays). It is therefore the most charged clay among the four studied ones in this work. This was confirmed by ζ potential measurements. Saunders et al. [START_REF] Saunders | A Small-Angle X-Ray Scattering Study of the Structure of Aqueous Laponite Dispersions[END_REF] also reported that clays with higher Li and F levels had a higher (more negative) basal surface charge and a lower (less positive) edge charge. [START_REF] Binks | Nanoparticle Silica-Stabilised Oil-in-Water Emulsions: Improving Emulsion Stability[END_REF][START_REF] Paunov | Adsorption of Charged Colloid Particles to Charged Liquid Surfaces[END_REF] The electrostatic repulsions may have a double effect: the adsorption of charged clay platelets increases the charge of the polymer particles and improves their stability against coagulation; but, electrostatic repulsions between the clay and the polymer particles, both negatively charged, may prevent the adsorption of the clay which hinders stabilization of polymer particles.

Such paradoxical role of electrostatic repulsions has already been reported in the case of oil/water Pickering emulsions (of liquid oil droplets instead of polystyrene particles). [START_REF] Binks | Nanoparticle Silica-Stabilised Oil-in-Water Emulsions: Improving Emulsion Stability[END_REF][START_REF] Paunov | Adsorption of Charged Colloid Particles to Charged Liquid Surfaces[END_REF] For example, silica particles have a very hydrophilic surface and are negatively charged at pH above the isoelectric point (iep = pH 2). As a consequence, silica particles are poor stabilizers of Pickering emulsions because they do not adsorb enough: the negative charge generated by the adsorption of few silica particles makes the oil droplets negatively charged, so that they repel other silica particles and prevent further adsorption. Every phenomenon that weakens such electrostatic repulsions allows a larger and faster adsorption of silica particles and improves the stabilization of oil droplets against coalescence. For instance, the addition of electrolytes (especially multivalent cations) increases the adsorption of silica particles and causes the aggregation of silica particles at the surface of oil droplets, thereby improving the stabilization of emulsions. [START_REF] Binks | Stability of Oil-in-Water Emulsions Stabilised by Silica Particles[END_REF] Similarly, lowering the pH closer to the isoelectric point of silica or the addition of polymers that cause bridging flocculation of the silica sol also weaken electrostatic repulsions. [START_REF] Hassander | The Mechanism of Emulsion Stabilization by Small Silica (Ludox) Particles[END_REF] Accordingly, it is presumed that the weak stability against coagulation taking place during emulsion polymerization in the presence of Laponite JS is caused by the weak adsorption of clay platelets due to the strong electrostatic repulsions between clay platelets and polystyrene particles partially covered by clay particles. Weak adsorption of Laponite JS has been observed at equilibrium in the concentration domain where monolayer adsorption takes place.

At higher concentrations, the adsorption isotherm showed that Laponite JS did adsorb at the surface of polymer particles; their adsorption should have generated enhanced electrostatic repulsions between polymer particles and prevented coagulation. However, during the course of emulsion polymerization, the rapidity of adsorption also becomes an important parameter, while the adsorption isotherm of this clay indicated a possible non-equilibrium characteristic, which suggested a slower adsorption. Indeed, efficient stabilization of nucleated polymer particles by clays requires that the rate of adsorption be faster than the rate of coagulation. Thus, a too large electrical charge of clay platelets may create electrostatic repulsions that make adsorption slower than coagulation, causing inefficient stabilization of the newly nucleated polymer particles within the time scale of coagulation. This involves non-equilibrium adsorption phenomena that may be studied using for instance Quartz Crystal Microbalance measurements.

Conclusions

Information about the ζ potential and adsorption isotherms was combined with the data of the different clay compositions, in order to elucidate Laponite stabilization behavior during

Pickering emulsion polymerization:

-The chemical compositions of Laponite RDS, Laponite XLS and Laponite JS are comparable, except for Laponite JS, which is a fluorohectorite (MgOH groups partly fluorinated, as the OH groups are substituted by F atoms). The composition of Laponite S482 is proprietary information of the manufacturer and it contains a different peptizing agent than the other clays.

-The ζ potential of Laponite JS was higher in magnitude than the other studied clays, due to a higher substitution of lithium for magnesium (50% higher than the other clays).

-The adsorption isotherm could be divided into two concentration regimes: at high clay concentrations, all clays followed a similar multilayer adsorption mechanism whereas at low clay concentration, the adsorption of the first layer of clay on the particle surface was larger for Laponite RDS and Laponite S482 than for Laponite XLS and Laponite JS. A non-equilibrium characteristic for Laponite XLS and Laponite JS was observed.

During the course of the polymerization:

-Laponite RDS and Laponite S482 were very similar in that increasing the clay concentration was found to increase the number of particles.

-Laponite JS was found the least efficient for stabilizing the latex particles, among the studied clays. At low clay concentrations, this can be explained by the lower adsorption (as shown by the adsorption isotherm), as a result of an increased repulsion between the clay and the polymer particles surface due to the higher charge of the clay. At higher concentrations, the adsorption isotherm showed that the clay adsorbed similarly as the other ones. However, a non-equilibrium characteristic was presumed in this isotherm, which suggested a slower adsorption of this clay than the other ones. The rapidity of adsorption is critical during the course of emulsion polymerization, and should be faster than the coagulation rate.

-Laponite XLS followed a similar behavior as Laponite RDS and Laponite S482 at low clay concentrations. This indicates that, for the three clays, individual platelets had the same surface charge (indeed the surface charge is better assessed under dilute conditions where the clay platelets do not interact with each other). But, a kind of saturation was observed for Laponite XLS at higher concentrations, and the nucleated number of particles reached a limit at a clay concentration of about 3 g L -1 . Increasing further the clay concentration did not affect the particles number. As the concentration of all clays on the particles surface was the same, this might be due to slower adsorption of this clay similarly to Laponite JS or to the formation of aggregates of Laponite XLS at high concentrations making it less efficient in stabilization.

Figure 1 .

 1 Figure 1. A: Adsorption isotherm of Laponite clay platelets on preformed polystyrene latex particles (with 5 wt% solids content and mean diameter 300 nm) measured using ICP-AES based on Si analysis and B: Zoom of the selected zone in Figure A with Langmuir fits (continuous line).

Figure 2 .

 2 Figure 2. Pickering ab initio semi-continuous emulsion polymerization of styrene stabilized by different amounts of Laponite RDS. Reprinted with permission from Langmuir, 2016, 32, 112-124, copyright 2016 American Chemical Society.

Figure 3 .

 3 Figure 3. Pickering ab initio semi-continuous emulsion polymerization of styrene stabilized by different amounts of Laponite S482.

Figure 4 .

 4 Figure 4. Pickering ab initio semi-continuous emulsion polymerization of styrene stabilized by

  almost 10 times lower than for the other clays, (i.e., close to the clay-free experiment), which led to lower reaction rates. The nucleation rate was also lower, which extended the nucleation period from 60 to 120 min. Laponite JS was therefore less efficient for stabilizing polystyrene particles in emulsion polymerization. A deeper comparison and analysis of the different clays is done in the next section.

Figure 5 . 5 .Figure 6

 556 Figure 5. Pickering ab initio semi-continuous emulsion polymerization of styrene stabilized by different amounts of Laponite JS.

Figure 6 .Figure 7 .

 67 Figure 6. Comparison of the different clay grades in Pickering ab initio semi-continuous emulsion polymerization of styrene for different amounts of Laponite: (A) 0.5 g L -1 , (B) 2.0 g L - 1 , (C) 3.0 g L -1 , and (D) 10.0 g L -1 .

Table

Table 1 .

 1 1. Chemical composition of the Laponite grades used in this study

	Chemical composition (%, dry basis)	Laponite RDS Laponite S482 Laponite XLS Laponite JS
	CAS #		53320-86-8		53320-86-8	64060-48-6
		SiO 2	54.5	54.5		50.2
	Components	MgO Li 2 O	26.0 0.8	26.0 0.8		22.2 1.2
		F	0	0		4.8
	Water (loss on ignition)	8.0	ns	8.2	8.7
	x		0.5		0.5	0.75
	y		0	0	0	2.4
	Surface area (m 2 g -1 )	330	370	330	300
	pH of a 2% suspension	9.7	9.8	9.7	10.0
	Peptizing	Na 2 O	5.6	ns	5.6	7.5
	agent	P 2 O 5	4.4	ns	4.1	5.4

* ns = non specified

Table 3 .

 3 Adsorption parameters of the Langmuir model (𝒒 𝒎 and 𝑲 𝑺 ) and BET model (𝒒 𝒎 , 𝑲 𝑺 and 𝑲 𝑳 ) for each Laponite grade investigated in this study

	Clay	𝒒 𝒎 (mg m -2 )	𝑲 𝑺 (L mg -1 )	𝑲 𝑳 (L mg -1 )
	Laponite RDS	1.1	212.8	3.1 10 -3
	Laponite S482	1.4	204.9	3.1 10 -3
	Laponite XLS	0.9	98.0	3.1 10 -3
	Laponite JS	0.2	31.6	3.1 10 -3

Acknowledgement

The support of the Agence Nationale pour la Recherche is gratefully acknowledged (PickEP project, grant n° ANR-12-JS09-0007-01).


Abeer M Radi

Abdelrahman Eman T Mohammed

Ibrahim Abushouk

Lotfi Aleya
email: lotfi.aleya@univ-fcomte.fr

Mohamed M Abdel-Daim

Abdelrahman Ibrahim Abushouk

Modulatory effects of sesame oil and ascorbic acid on abamectin-induced oxidative stress in rat liver and brain tissues

Keywords: abamectin, oxidative stress, sesame oil, ascorbic acid, liver and brain, rats

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pesticides are used worldwide to control pests, insects, and disease vectors in veterinary medicine and agriculture [START_REF] Jones | Pesticides and Insecticides: Development and Use Syrawood Publishing House[END_REF]. Abamectin (ABM) belongs to the avermectins family; 16-membered macrocyclic lactones with a disaccharide substituent at the carbon-13 position. It is generated from soil actinomycete fungi (Streptomyces avemitilis) and is a combination of 80% avermectin B1a plus 20% avermectin B1b. Abamectin is used as an anthelmintic agent in cattle and sheep to eliminate gastrointestinal nematodes, lung worms and nasal bots [START_REF] Campbell | Ivermectin and abamectin[END_REF].

Non-therapeutic exposures to ivermectin and other macrocyclic lactones may lead to toxic effects; only after oral ingestion of a large amount. Although the exact mechanisms remain unclear, macrocyclic lactones, when taken in large doses, may pass through the blood-brain barrier and produce GABA-mimetic toxic effects. In mammals, avermectin intoxication begins with hyperexcitability, incoordination, tremors, hypotension, and later develops into ataxia, coma, respiratory failure, and even death [START_REF] Yang | Acute human toxicity of macrocyclic lactones[END_REF].

Avermectin is poorly metabolized in mammals; 80-98% of the initially administered dose gets eliminated in stool [START_REF] Sun | Bioaccumulation and elimination of avermectin B1a in the earthworms (Eisenia fetida)[END_REF]. The highest levels of ABM were detected in the liver and fat (owing to its lipophilic nature), while the lowest was found in the brain [START_REF] Gonzalez Canga | The pharmacokinetics and metabolism of ivermectin in domestic animal species[END_REF]. Therefore, the detoxification of ABM may disturb the functions of hepatocytes. The biodistribution of ivermectin in the host depends on the efflux by P-glycoprotein (P-gp) and biotransformation by cytochromes P450 [START_REF] Alberich | Ivermectin exposure leads to up-regulation of detoxification genes in vitro and in vivo in mice[END_REF]. P-gp prevents brain toxicity by limiting the penetration of toxic compounds across the blood-brain barrier [START_REF] Roulet | MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoprotein substrate ivermectin[END_REF].

Besides, it contributes to the intestinal excretion of ivermectin [START_REF] Ballent | Modulation of the P-glycoproteinmediated intestinal secretion of ivermectin: in vitro and in vivo assessments[END_REF]. Hepatic drug metabolism is attained by the microsomal cytochrome P450 enzyme system that facilitates transformation into toxic intermediates, followed by reactive oxygen species (ROS) production, inflammatory cytokines release and lipid peroxidation [START_REF] Lu | Knockdown of cytochrome P450 2E1 inhibits oxidative stress and apoptosis in the cTnTR141W dilated cardiomyopathy transgenic mice[END_REF][START_REF] Maioli | The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes[END_REF].

These events initiate apoptosis and tissue inflammation through interaction with caspases and mitogen-activated protein kinases (MAPKs) [START_REF] Bayir | Bench-to-bedside review: Mitochondrial injury, oxidative stress and apoptosis-there is nothing more practical than a good theory[END_REF].

Oxidative stress is a key factor in avermectins-induced cytotoxicity [START_REF] Zhu | Avermectin induced liver injury in pigeon: mechanisms of apoptosis and oxidative stress[END_REF]. Therefore, the use of antioxidants to ameliorate its toxicity is a logical approach. Ascorbic acid is a watersoluble non-enzymatic antioxidant that defends the cellular compartments against reactive oxygen species (ROS) [START_REF] Jurczuk | Hepatic and renal concentrations of vitamins E and C in lead-and ethanol-exposed rats. An assessment of their involvement in the mechanisms of peroxidative damage[END_REF]. It is an essential element; must be supplied in diet because it cannot be synthesized in mammalian bodies. Ascorbic acid showed the ability to prevent lipid peroxidation and protect lipid membranes, proteins and DNA from oxidative harm [START_REF] Granger | Dietary vitamin C in human health[END_REF]. Moreover, it could antagonize the toxic effects of many xenobiotics (Abdel-Daim et al., 2019a;Abdel-Daim et al., 2019b;[START_REF] Özkan | The protective role of ascorbic acid (vitamin C) against chlorpyrifos-induced oxidative stress in Oreochromis niloticus[END_REF] and has been shown to renovate the antioxidant capacity of vitamin E [START_REF] Serbecic | Anti-oxidative vitamins prevent lipid-peroxidation and apoptosis in corneal endothelial cells[END_REF].

Sesame oil (SO) is extracted from Sesamum indicum seeds. These seeds contain many phytochemicals as flavonoids, phenolic acids, tannins, alkaloids, terpenoids, cephalin and lecithin and some minerals like iron, calcium, magnesium, copper, zinc, manganese and phosphorus [START_REF] Anilakumar | Nutritional, medicinal and industrial uses of sesame (Sesamum indicum L.) seeds-an overview[END_REF][START_REF] Sani | Phytochemicals and mineral elements composition of white Sesamum indicum L. seed oil[END_REF]. Several lignans, such as sesamolin, sesamin, and γ-tocopherol are potent polyphenolic antioxidants found in sesame seeds [START_REF] Rangkadilok | Variation of sesamin, sesamolin and tocopherols in sesame (Sesamum indicum L.) seeds and oil products in Thailand[END_REF]. For example, sesamin protects against oxidative stress and enhances hepatic drug detoxification [START_REF] Shuang | Sesamin protects the femoral head from osteonecrosis by inhibiting ROS-induced osteoblast apoptosis in rat model[END_REF]Zhang et al., 2016a). Over decades of research, sesame (oil) exhibited anti-inflammatory, antibacterial, hypolipidemic, antitumor [START_REF] Anilakumar | Nutritional, medicinal and industrial uses of sesame (Sesamum indicum L.) seeds-an overview[END_REF] and anti-allergic effects [START_REF] Jung | Changes in the Anti-Allergic Activities of Sesame by Bioconversion[END_REF].

The goals of this experiment were to explore the impact of acute exposure to the commercial formulation of ABM on the expression of metabolic cytochromes P450 2E1 (CYP-2E1), p38 MAPK and caspase-3 enzymes in the liver, as well as membrane p-glycoprotein efflux transporter and GABA-A signaling in rat brain. Further, we tested the preventive capacities of SO and/or AA against acute ABM toxicity. Biodiagnostic Company, Egypt. The kits for SGPT, SGOT, ALP enzymes, and direct bilirubin were supplied from Greiner Diagnostic GmbH-Bahlingen, Germany. The rest of used chemicals were of analytical grade. The chemical composition of sesame oil was analyzed using Trace GC-ISQ Mass Spectrometer (Thermo Scientific, Austin, TX, USA) as described in Supplementary file 1.

Materials and methods

Chemicals: Abamectin (Vertemic

Experimental design:

Thirty mature male albino rats (weighing 120 to 150 g), were purchased from the Egyptian Company for Biological Products and Vaccines. Animals were kept in stainless steel cages, fed rat chow and water ad libitum, and maintained at lab temperature of 25 ± 2 ᴼ C. All maintenance and care procedures were approved by the Research Ethical Committee at the Faculty of Veterinary Medicine, Beni-Suef University, Egypt.

After two weeks of acclimation, rats were randomly assigned to five groups (n = 6/group). The first group received distilled water (negative control). The second group (ABM) received a daily oral dose of ABM (2 mg/kg bwt, 1/5 LD50) for 5 days (LD50: 10 mg/kg) (Abdel-Daim and [START_REF] Abdellatief | Attenuating effects of caffeic acid phenethyl ester and betaine on abamectin-induced hepatotoxicity and nephrotoxicity[END_REF]. The third group (ABM+AA) received ABM plus a daily dose of AA (100 mg/kg bwt, orally) [START_REF] El-Ghoneimy | Synergistic protective effects of ceftriaxone and ascorbic acid against subacute deltamethrin-induced nephrotoxicity in rats[END_REF][START_REF] Seo | Protective effect of low dose of ascorbic acid on hepatobiliary function in hepatic ischemia/reperfusion in rats[END_REF], while the fourth group (ABM+SO) was given ABM plus a daily dose of SO (5 ml/kg bwt, orally) [START_REF] Saleem | Antioxidants and tumor necrosis factor alpha-inhibiting activity of sesame oil against doxorubicin-induced cardiotoxicity[END_REF]. The fifth group (Combination) received SO and AA with the same doses as before.

Groups (3 to 5) were given SO and/ or AA for 10 days before ABM exposure and 1 hour before ABM administration for 5 successive days. Figure 1 summarizes the experimental design and observed findings.

Blood sampling and tissue processing:

On the day following the last ABM dose, rats were anesthetized using isoflurane for blood withdrawal from the retro-orbital plexus, and then killed via cervical dislocation. The samples were centrifuged (at 3000 rpm for 15 min) to separate sera.

The liver and brain were excised, washed with saline, and then blotted over filter paper. The tissue was divided into three parts: the first (0.5 g) was homogenized in phosphate buffer saline (pH 7.4, 5 ml). Homogenates were later centrifuged at 3000 rpm for 15 min at 4 ˚C using a cooling, high-speed centrifuge; supernatants were collected and preserved at -80 ˚C until analysis of tissue oxidant and antioxidant indices. The second tissue part was preserved at -80 ˚C for molecular investigations. The third tissue part was prepared for histopathological examination.

Biochemical estimations:

The measurements of MDA, GSH concentrations and CAT activity were performed as per the methods described by Mihara and Uchiyama [START_REF] Mihara | Determination of malonaldehyde precursor in tissues by thiobarbituric acid test[END_REF], [START_REF] Beutler | Improved method for the determination of blood glutathione[END_REF] and Aebi [START_REF] Aebi | Catalase in vitro[END_REF], respectively. Serum samples were used to measure the activities of SGPT and SGOT [START_REF] Reitman | A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases[END_REF], ALP [START_REF] Tietz | A reference method for measurement of alkaline phosphatase activity in human serum[END_REF], and bilirubin (Tolman and Rej, 1999).

Detection of TNF-α by ELISA:

Tumor necrosis factor-α (TNF-α) levels were determined by ELISA technique as per the methods of [START_REF] Tietz | Clinical Guide to Laboratory Tests (ELISA)[END_REF][START_REF] Tietz | Clinical Guide to Laboratory Tests (ELISA)[END_REF]. ELISA kits were purchased from R&D system (MN, USA). Complementary DNA (cDNA) synthesis: The extracted RNA was reverse-transcribed into cDNA using high capacity cDNA reverse transcription kits (#K1621, Fermentas, USA) following the manufacturer's instructions.

Real-time quantitative PCR:

Real-time qPCR amplification and analysis were performed using an Applied Biosystem with software version 3.1 (StepOne™, USA) to measure the expression of mRNAs of target genes in the liver and brain, with B-actin as an internal reference (housekeeping gene). The isolated cDNA was amplified using SYBR Green Master Mix (Applied Biosystems) following the manufacturer's protocol. The primers used in the amplification are shown in Table 1 and were designed by Gene Runner Software (Hasting Software, Inc., Hasting, NY) from RNA sequences in the gene bank (based on published rat sequences). Data from realtime assays were analyzed using the v1.7 sequence detection software from PE Biosystems (Foster City, CA). Relative expression of the studied gene mRNA was calculated using the comparative Cycle threshold (Ct) method. All values were normalized to β-actin and reported as fold change over background levels detected in the treated groups. All these steps were performed according to the methods of Livak and Schmittgen [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Detection of p38 MAPK protein by Western Blot technique: p38 MAPK protein was

detected following the manufacturer's protocol (V3 Western Workflow™ Complete System, Bio-Rad® Hercules, CA). In brief, ice-cold radio immune precipitation assay (RIPA) buffer, along with phosphatase/protease inhibitors, were used for protein extraction from liver tissue homogenates. To visualize p38 MAPK protein, we used enhanced chemiluminescence (from ECL plus; Amersham, IL), followed by Molecular Analyst Software (Bio-Rad, Richmond, CA) for quantification. Protein levels were expressed in relation to β-actin.

Histopathological studies:

Sections from the liver and brain tissues (M1 motor cortex and hippocampus) were fixed in buffered formalin, then stained with Hematoxylin & Eosin to examine pathological findings under a light microscope.

Statistical analysis:

Data from the five experimental groups were summarized as means ± standard errors (SEM), and then transferred to a data sheet on the SPSS software (version 22, IBM Co., Armonk, NY). We used the ANOVA followed by Tukey's post-hoc test for experimental group comparison. P value ≤ 0.05 was accepted for significance.

Results

Sesame oil GC-MS analysis:

In the present study, 19 compounds have been identified in the SO sample by GC-MS analysis (Table 2). The major components were tetradecane,2,6,10trimethyl-(38.41%), octadecane (20.52%), 1-hexadecanol, 2-methyl-(9.14 %), octadecane, 6methyl-(5.72%), 1-tetradecanol (3.85%) and 9-octadecenoic acid (Z)-(3.70%). The mass spectra of all major components in the studied sample are shown in Supplementary file 1.

Serum biochemical analysis:

Oral administration of ABM was associated with significant (p ≤0.0001) elevations in serum levels of hepatocyte injury biomarkers (SGOT and SGPT) and biliary tract injury biomarkers (ALP and direct bilirubin), compared to the control group. Our results revealed the hepatoprotective effects of SO and AA, alone or in combination, as they significantly ameliorated the ABM-induced alterations in the four parameters. The obtained values in the ABM+SO and ABM+AA+SO combination groups were more frequently comparable to the control group, relative to the ABM+AA group (Table 3).

Hepatic oxidant/antioxidant markers:

Acute ABM exposure was associated with significant (p ≤0.05) increases in the hepatic tissue MDA concentration and CAT enzyme activity, as well as a significant drop in GSH level, compared to the control rats. Ascorbic acid and/or SO supplementation significantly (p ≤0.05) restored MDA, CAT and GSH to the normal control levels. There were no significant variations among the treated groups (ABM+AA, ABM+SO or Combination group) (Table 4).

Brain oxidant/antioxidant markers:

Our analysis showed a significant decrease (p ≤0.05) in MDA concentration and significant increases in GSH content and CAT activity in the brain tissues of rats, treated with SO and/or AA, in comparison with ABM-exposed rats. Both treatments, either alone or in combination, could restore MDA and GSH to normal levels; however, the effect of combined treatment was more pronounced than that of a single treatment (Table 4).

Hepatic tumor necrosis factor-α concentration:

Following ABM exposure, we observed significant elevations (p ≤0.05) in liver tissue TNF-α concentrations in comparison to control rats. Treatments of ABM-intoxicated rats with SO, AA, or their combination was associated with significant reductions in hepatic TNF-α in comparison to rats, exposed to ABM alone. There were no significant variations among the treated groups (ABM+SO, ABM+AA or Combination group); Figure 2.

Expression of CYP-2E1

, caspase-3, and p38 MAPK in the liver: Abamectin administration was associated with a significant boost in the hepatic tissue expression of CYP-2E1, caspase-3 and p38 MAPK proteins. In contrast, SO and/or AA supplementation ameliorated the ABM-induced increases in CYP-2E1, caspase-3, and p38 MAPK expression. There were no significant variations among the treated groups (ABM+SO, ABM+AA, or Combination groups).

Figure 3 show the observed changes in CYP-2E1, caspase-3, and p38 MAPK expression in rats' liver tissues. Figure 4 shows the western blotting analysis of p38 MAPK hepatic expression in relation to β-actin.

Expression of ABC efflux transporter (Abcb1a) and GABA-A receptor in the brain:

In this study, the expression of genes encoding the major ABC transporter (Abcb1a) was significantly increased following ABM exposure. This over-expression was significantly ameliorated in rats, treated with SO and AA, alone or in combination. Similarly, ABM exposure significantly upregulated GABA-A receptor expression in the brain of ABM-intoxicated rats (compared to normal controls), which was alleviated in rats treated with ABM plus SO, AA, or their combination (Figure 5).

Histopathological findings:

Liver tissue sections from the control group show normal hepatic lobules with granulated and radiating hepatocytic cords from the central vein. However, tissue sections from ABM-exposed rats display lymphocytic infiltration and hemorrhage in the portal area, sinusoidal dilatation, as well as cellular hydropic degeneration, nuclear displacement, and focal ballooning of hepatocytes around the central vein. Marked reductions in these pathological alterations were observed after administration of SO and AA or their combination.

Hepatic sections were nearly normal in the three treated groups except that those treated with SO and AA alone showed blood vessels congestion (Figure 6).

Cerebral cortex tissue sections from the control group show normal histological structure. In contrast, sections from ABM-intoxicated rats show dark stained nuclei, neuropil vaculation and hemorrhage. Rat groups, treated with SO or AA alone, showed marked reduction of hemorrhage, while the combination group sections show lightly-stained vesicular nuclei (Figure 7).

Hippocampal tissue sections from the control group show normal outer pleomorphic, middle pyramidal, and inner molecular layers. However, sections from ABM-intoxicated rats show pyknosis, dark stained pyramidal neurons and degenerative changes in the middle pyramidal layer. Interestingly, ABM-intoxicated rats treated with SO only showed few pyknotic pyramidal cells, while rats treated with AA and SO+AA combination show nearly normal pyramidal cells with euchromatic nuclei (Figure 8).

Discussion

Oxidative stress is a key player in ABM-induced toxicity. The hemeprotein cytochrome P450 multi-enzymatic complex act mainly as mono-oxygenases for the metabolism of many compounds. During the metabolism of toxic substrates by CYP2E1, more reactive and toxic products are formed with excess generation of ROS [START_REF] Danielson | The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans[END_REF]. These ROS further degrade the CYP hemeprotein to release iron, which catalyzes the Fenton's reaction, potentiating lipid peroxidation [START_REF] Caro | Oxidative stress, toxicology, and pharmacology of CYP2E1[END_REF]). In the present work, the detected overexpression of hepatic CYP2E1 gene and resulting ROS probably played a role in the observed hepatic injury in ABM-intoxicated rats as indicated by the significant increases in the hepatic lipid peroxidation indicator (MDA concentration) and depletion of GSH stores in the liver. In addition, the brain redox markers were altered following ABM exposure.

Reduced glutathione is the principal antioxidant that removes ROS, generated by CYP2E1 [START_REF] Chen | Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells[END_REF], thus GSH depletion (as observed in this study) leads to H2O2 accumulation, lipid peroxidation and cell damage. Catalase is a prominent endogenous antioxidant enzyme. The higher activity of hepatic CAT, noticed in the present study, may have been an adaptive response against H2O2 produced by ABM metabolism. In contrast, CAT activity was reduced in the brain of ABM-intoxicated rats. This result is parallel with that reported by Nasr et al. [START_REF] Nasr | Neuro and renal toxicity induced by chlorpyrifos and abamectin in rats[END_REF], which reflects the failure of the brain antioxidant capacity to overcome ABM-induced oxidative stress, probably due to the high oxidative metabolism in the brain [START_REF] Gandhi | Mechanism of oxidative stress in neurodegeneration[END_REF].

The link between ROS and TNF-α is complicated; ROS increase TNF-α release and TNF-α increases ROS production [START_REF] Blaser | TNF and ROS crosstalk in inflammation[END_REF]. Reactive oxygen species play as second messengers in the intracellular signal transduction pathways, including apoptosis. The p38 MAPK pathway is a series of serine/threonine kinases in mammalian cells, activated by excessive ROS generation (Di [START_REF] Lisa | beta(2)-Adrenoceptors, NADPH oxidase, ROS and p38 MAPK: another 'radical' road to heart failure?[END_REF] and inflammatory cytokines [START_REF] Segales | Regulation of Muscle Stem Cell Functions: A Focus on the p38 MAPK Signaling Pathway[END_REF]. It mediates inflammatory response in various cell types by up-regulating TNF-α, interleukin-1 and interleukin-8 [START_REF] Cuenda | p38 MAP-kinases pathway regulation, function and role in human diseases[END_REF]. In this study, ABM induced hepatocyte apoptosis, as confirmed by the increased caspase-3 expression in rat liver. These results are comparable to those detected in isolated rat hepatocytes [START_REF] Maioli | The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes[END_REF] and the pigeon liver [START_REF] Zhu | Avermectin induced liver injury in pigeon: mechanisms of apoptosis and oxidative stress[END_REF]. The increased caspase-3 expression detected in our study may be related to the increased TNF-α level, which is involved in the extrinsic apoptotic pathway [START_REF] Perez | TNF-alpha signals apoptosis through a bid-dependent conformational change in Bax that is inhibited by E1B 19K[END_REF]. Therefore, we suggest the possibility of ABM-induced apoptosis based on the extrinsic apoptotic pathway.

In addition, our results show that oral administration of ABM 1/5 LD50 (2 mg/kg bwt.) for 5 days significantly increased the serum activities of SGPT, SGOT and ALP in ABM-treated rats, compared to the control group. In parallel with prior investigations [START_REF] Hsu | Abamectin effects on aspartate aminotransferase and nitric oxide in rats[END_REF][START_REF] Khaldoun-Oularbi | Effect of subacute exposure to abamectin "insecticide" on liver rats (Rattus norvegicus)[END_REF], these findings indicate that ABM causes permeability alteration and blood leakage of intracellular enzymes from the damaged hepatocytes. Oxidative stress and inflammatory cytokines activate heme-oxygenase-1 that regulates the synthesis of bilirubin [START_REF] Yamamoto | Bilirubin oxidation provoked by nitric oxide radicals predicts the progression of acute cardiac allograft rejection[END_REF]. Direct bilirubin is actively excreted at the canalicular membrane after binding to transporter proteins [START_REF] Jedlitschky | ATPdependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2[END_REF]. Therefore, the damaged hepatocytes may be less able to produce the transporter proteins required for active transportation of direct bilirubin into the gall bladder, explaining the observed increase in its serum levels in the present study.

Because avermectins act as GABA-A receptor agonists in vertebrates, their safety in animals requires an intact blood brain barrier with integral P-gp. These are efflux transporters from the ATP-Binding Cassette (ABC) transporters superfamily [START_REF] Jones | The ABC transporter structure and mechanism: perspectives on recent research[END_REF], expressed within the capillary endothelial cells in the brain, placenta, and intestine [START_REF] Ballent | Modulation of the P-glycoproteinmediated intestinal secretion of ivermectin: in vitro and in vivo assessments[END_REF]. Pgp protects animals against the diffusion of avermectins into the brain, avoiding the consequent neurotoxicity [START_REF] Macdonald | Potential impact of ABCB1 (p-glycoprotein) polymorphisms on avermectin toxicity in humans[END_REF]. In rodent genome, P-gp is encoded as two genes known as MDR1a/Abcb1a and MDR1b /Abcb1b. Abcb1a is more similar to the human gene and is the main form at the blood-brain barrier and intestine [START_REF] Croop | The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues[END_REF]. In the current study, the expression of Abcb1a gene was significantly increased following ABM exposure. This finding is in accordance with a former study on ivermectin in murine hepatocytes [START_REF] Ménez | Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line[END_REF]. In contrast, earlier studies showed that avermectin induces neurotoxicity via either oxidative damage, apoptosis [START_REF] Li | Antioxidant response and histopathological changes in brain tissue of pigeon exposed to avermectin[END_REF] or down regulation of P-gp [START_REF] Sun | Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cells[END_REF].

The increased P-gp expression following ABM exposure in this study can be explained by several mechanisms. Reactive oxygen species increase the hepatic expression of P-gp [START_REF] Deng | 2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-kappa B pathway[END_REF] and brain endothelial cells in rats [START_REF] Hong | Up-regulation of P-glycoprotein expression by glutathione depletion-induced oxidative stress in rat brain microvessel endothelial cells[END_REF]. Moreover, p38 MAPK can activate NF-κB expression [START_REF] Saha | MAPK p38 regulates transcriptional activity of NF-kappaB in primary human astrocytes via acetylation of p65[END_REF], which activates MDR1 gene transcription [START_REF] Bentires-Alj | NF-kappaB transcription factor induces drug resistance through MDR1 expression in cancer cells[END_REF]. Additionally, previous investigations have shown that up-regulation of P-gp is stimulated by TNF-α in primary hepatocytes [START_REF] Hirsch-Ernst | Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures[END_REF] and the p38 MAPK pathway in rat prostate cancer cells [START_REF] Sauvant | Acidosis induces multi-drug resistance in rat prostate cancer cells (AT1) in vitro and in vivo by increasing the activity of the p-glycoprotein via activation of p38[END_REF]. In the present study, despite the detected overexpression of Abcb1a in the brain, the neurotoxic effect of ABM supports that it is an effective inhibitor of p-gp facilitated transport [START_REF] Lankas | Placental P-glycoprotein deficiency enhances susceptibility to chemically induced birth defects in mice[END_REF]. Therefore, ABM could increase Abcb1a expression as an adaptive response to oxidative stress. At the same time, ABM inhibits the P-gp efflux transport function, increasing its penetration into the CNS and facilitating the interaction with GABA receptors.

Another finding of this study is that ABM could significantly upregulate GABA-A receptor expression in the cerebral tissue of ABM-intoxicated rats. The mechanism of action of macrocyclic lactones-induced neurotoxicity (including ABM) in pests relies on their high affinity for glutamate-gated Cl channels in neuronal and muscular cells. In mammals, they bind to the receptor for the inhibitory neurotransmitter (GABA) and open the ionotropic GABA-A receptorgated Cl channels that are limited to the CNS [START_REF] Mccavera | Nematode ligand-gated chloride channels: an appraisal of their involvement in macrocyclic lactone resistance and prospects for developing molecular markers[END_REF]. Chloride ions then flow into the postsynaptic neuron in excess, causing hyperpolarization of the membrane potential and disrupting nerve signal transmission [START_REF] Novelli | Lethal effects of abamectin on the aquatic organisms Daphnia similis, Chironomus xanthus and Danio rerio[END_REF].

Ascorbic acid and SO are commonly used as dietary supplements. Oral SO and/or AA supplementation were able to inhibit the upregulation of CYP-2E1 expression in the liver of treated rats. Similarly, sesamin reduced the expression of CYP-2E1 in hepatocytes (Zhang et al., 2016b) and alleviated oxidative stress in acetaminophen or carbon tetrachloride-treated mouse liver [START_REF] Ma | Hepatoprotective properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway[END_REF]. Previous studies have demonstrated that AA could directly react with lipid peroxides, increase GSH and antioxidant enzyme levels, and prevent resulting apoptosis [START_REF] Santos | Oxidative stress in the hippocampus during experimental seizures can be ameliorated with the antioxidant ascorbic acid[END_REF][START_REF] Wang | Effect of ascorbic Acid and thiamine supplementation at different concentrations on lead toxicity in liver[END_REF]. Our results showed that antioxidants like SO and AA eliminated ROS and lipid peroxides in the liver and brain tissues. Additionally, the effect of combined treatment was more pronounced in alleviating the oxidative stress in the brain tissue than that of a single treatment. This may be attributed to the augmenting effect of both treatments.

Administration of SO and AA significantly alleviated ABM-induced apoptosis by inhibiting the hepatic expression of caspase-3, as well as p38 MAPK and TNF-α. The anti-apoptotic activities of SO components and AA have been described in earlier studies. Ascorbic acid inhibited p38 MAPK phosphorylation in vitro [START_REF] Carcamo | Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IkappaBalpha kinase beta[END_REF]. Similarly, Hou et al. reported that the inhibitory effect of sesamolin on caspase-3, p38 MAPK activation and ROS production could protect microglia against cell injury [START_REF] Hou | Protective effects of sesamin and sesamolin on murine BV-2 microglia cell line under hypoxia[END_REF]. Ma et al. found that CCl4-induced apoptosis was inhibited in liver by sesamin through reduction of hepatic TNF-α, Bak, Bax, Cyt.

C and caspase-3 expression levels [START_REF] Ma | Hepatoprotective properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway[END_REF].

Sesame oil, AA, and their combination could ameliorate the significant increases in serum SGPT, SGOT and ALP, caused by ABM. These changes were more pronounced in combination group compared with that of single treated groups. Previous studies have shown that AA ameliorated the elevated serum SGPT and SGOT levels in rat models of malathion hepatotoxicity [START_REF] Kalender | Malathion-induced hepatotoxicity in rats: the effects of vitamins C and E[END_REF] and organophosphate pesticide toxicity [START_REF] Ambali | Evaluation of subchronic chlorpyrifos poisoning on hematological and serum biochemical changes in mice and protective effect of vitamin C[END_REF]. Further, SO ameliorated subacute diazinon toxicity [START_REF] Taha | Synergistic ameliorative effects of sesame oil and alpha-lipoic acid against subacute diazinon toxicity in rats: hematological, biochemical, and antioxidant studies[END_REF] and protected brain cells against cypermethrin toxicity [START_REF] Hussien | Cypermethrin induced damage in genomic DNA and histopathological changes in brain and haematotoxicity in rats: the protective effect of sesame oil[END_REF]. Sesame oil significantly alleviated ABM-induced elevation serum direct bilirubin, as observed previously in diazinon intoxicated rats [START_REF] Elnaggar | Protective effect of some plant oils on diazinon induced hepatorenal toxicity in male rats[END_REF]. These effects may be explained in the light of GC-MS analysis findings; several compounds within the analyzed SO sample, such as octadecane and 9-octadecenoic acid have anti-inflammatory activities and have been shown to mitigate oxidative stress and inhibit the formation of arachidonic acid.

In this study, oral SO and/or AA supplementation counteracted the ABM-induced up-regulation of Abcb1a expression. Previous research has proven the ability of flavonoids to inhibit P-gp transporters via inhibition of ATPase activity [START_REF] Pulido | Interaction of enrofloxacin with breast cancer resistance protein (BCRP/ABCG2): influence of flavonoids and role in milk secretion in sheep[END_REF] or competitive inhibition [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF]. Thus, the phenolic sesamin, sesamol, and flavonoids in SO may be responsible for these effects concerning Abcb1a gene [START_REF] Anilakumar | Nutritional, medicinal and industrial uses of sesame (Sesamum indicum L.) seeds-an overview[END_REF]. Further, SO and AA alleviated ABM-induced overexpression of GABA-A receptor in rat brain. The brain is susceptible to oxidative injury due to its high oxygen consumption and a high content of polyunsaturated fatty acids [START_REF] Gandhi | Mechanism of oxidative stress in neurodegeneration[END_REF]. Redox agents can regulate the GABA-A receptors function [START_REF] Calero | Redox modulation of homomeric rho1 GABA receptors[END_REF]. In contrast, AA has been shown able to control the activity of glutamate and GABA receptors to protect neurons against glutamate excitotoxicity [START_REF] Calero | Allosteric modulation of retinal GABA receptors by ascorbic acid[END_REF]. Therefore, in the present study, the simultaneous administration of SO and AA could significantly modulate GABA-A receptor mRNA levels owing to their synergistic ROS-scavenging effect.

In conclusion, ABM induced oxidative stress and increases the expression of TNF-α, caspase-3 and p38 MAPK. Moreover, it upregulates the expression of drug detoxifying genes; the brain Abcb1a efflux transporter and the hepatic CYP-2E1 enzyme. However, pretreatments with SO and AA effectively ameliorated ABM-induced oxidative stress and apoptosis. Simultaneous administration of SO and AA was more efficient in protecting the rat liver and brain than single agent use.

GABA

Forward primer: 5'-TCGGGACCAACCCAACGTGC -3

Reverse primer: 5′-CGTGC TGGCCTGATTGACGCT -3

Abcb1a

Forward primer: 5-ACCAGCGGTCAGTGTGCT-3

Reverse primer: 5-CGGTTGTTTCCTACATTTGC-3

Cytochrome P450 2E1

Forward: 5-TTTGGATCCAATGGGTGATGTTGAG -3

Reverse: 5-TTTGAATTCCTCATTAGTAGCTTTTTTGAG-3

Caspase-3

Forward primer: 5'-TTC ATT ATT CAG GCC TGC CGA GG -3

Reverse primer: 5′-TTC TGA CAG GCC ATG TCA TCC TCA -3

Β-actin

Forward primer: 5'--GGTCGGTGTGAACGGATTTGG -3

Reverse primer: 5′-ATGTAGGCCATGAGGTCCACC-3

2. 6 .

 6 Quantitative analysis of gene expression of CYP-2E1, Caspase-3, GABA-A receptor and p-gp by real time PCR Total RNA extraction: Total RNA was extracted from the brain and liver tissue homogenates according to the manufacturer's instruction, using SV Total RNA Isolation System (Promega, Madison, WI, USA). The RNA concentrations and purity were measured with UV spectrophotometer.

Figure 1 :Figure 2 :

 12 Figure 1: Synthesis of the experimental design along with assessed molecular mechanisms. AA: Ascorbic acid, ABM: Abamectin, CAT: Catalase, CYP-2E1: Cytochrome P450 2E1, GSH: Reduced glutathione, MDA: Malondialdehyde, ROS: reactive oxygen species, SO: Sesame oil, TNF-α: Tumor necrosis factor-α

Figure 3 :

 3 Figure 3: Expression of cytochrome P450-2E1 (CYP-2E1), caspase-3, and p38 MAPK proteins in the liver of different experimental groups (in relation to β-actin). AA: Ascorbic acid, SO: Sesame oil. Columns represent means ± SEM (n=6). Columns with different superscript letters are significantly different at p ≤ 0.05.

Figure 4 :

 4 Figure 4: Western blotting analysis of p38 MAPK hepatic expression (in relation to β-actin) in different experimental groups.

Figure 5 :

 5 Figure 5: Relative expression of brain P-glycoprotein (Abcb1a) in different groups and GABA-A receptor in different groups. AA: Ascorbic acid, SO: Sesame oil. Columns represent means ± SEM (n=6). Columns with different superscript letters are significantly different at p ≤ 0.05.

Figure 6 :

 6 Figure 6: Photomicrographs of the hepatic sections stained with H&E. (A) Control group showed normal structure, (B) ABM group showed lymphocytic infiltration (*) and hydropic degeneration (curved arrow). The incite showed ballooning of hepatocytes (arrow). The treated groups with SO (C) and AA (D) showed congested bold vessels (thick arrow). (E) The combination group was nearly normal. Scale bar 50 um. Incite sale bar 20 um.

Figure 7 :

 7 Figure 7: Representative photomicrographs of cerebral cortices of experimental groups stained with H&E. (A) Control group showing normal histological structure. (B) ABM group showing dark stained nuclei (arrow), neuropil vacuolation (arrowhead) and hemorrhage (*). Rats treated with SO (C) and AA (D) showing marked reduction of hemorrhage and (E) Combination showing lightly stained vesicular nuclei (arrow). Scale bar 50 um.

Figure 8 :

 8 Figure 8: Representative photomicrographs of hippocampus (CA3) of experimental groups stained with H&E. (A) Control group showing outer pleomorphic layer (o), middle pyramidal layer (p) and inner molecular layer (M). (B) Abamectin group showed pyknosis, dark stained pyramidal neurons (arrow) and degenerative changes in the middle pyramidal layer. (C) treated with SO only few pyknotic pyramidal cells (arrow). (D) Rats treated with AA and (E) SO + AA combination showing nearly normal pyramidal cells with euchromatic nuclei (arrow). Scale bar 50 um.

  in serum SGPT, SGOT, ALP and direct bilirubin concentrations in different groupsValues are expressed as mean ± SEM (n = 6 per group). Values with different letters in a column are significantly different at level p < 0.0001. AA: Ascorbic acid, ABM: Abamectin, ALP: Alkaline phosphatase, SGPT: Serum glutamic pyruvic transaminase, SGOT: Serum glutamic oxaloacetic transaminase,

  ® , 1.8% EC) was bought from Syngenta Agro Services AG, Egypt. Sesame Oil was supplied by El-Captain Company (El-Obour City, Egypt) and AA (Ascorbin® 100% B.p) was obtained from Newvetrovit Company, Egypt. Commercial kits for malondialdehyde (MDA), reduced glutathione (GSH) and catalase (CAT) were purchased from

Table ( 1): The primer sequence of the studied rat genes Primer sequence

 (

Table 2 :

 2 The chemical composition of sesame oil sample by gas chromatography-mass spectrometry

	No.	RT	Name of the compound	Molecular	Molecular	Peak Area %
				Formula	weight
		3.36	Nonanol	C9H20O	144	0.40
		11.50	1-Tetradecanol	C14H30O	214	3.85
		13.30	Undecanal	C11H22O	170	0.49
		14.49	Dodecane	C12H26	170	0.59
		15.93	1-Hexadecanol, 2-methyl-	C17H36O	256	9.14
		16.25	Nonadecane	C19H40	268	2.64
		16.49	Tetradecane, 2,6,10-trimethyl-	C17H36	240	38.41
		18.19	12-Methyl-E,E-2,13-octadecadien-1	C19H36O	280	1.81
		18.68	Octadecane, 6-methyl-	C19H40	268	5.72
			Z-8-Methyl-9-tetradecenoic acid	C15H28O2	240	0.96

Table ( 4): Changes in MDA, GSH concentrations and catalase activity in the brain and liver of rats in different groups

 ( Values are expressed as mean ± SEM (n = 6 per group). Values with different letters in a column are significantly different at level p < 0.05. AA: Ascorbic acid, ABM: Abamectin, GSH: Reduced

			MDA		GSH		Catalase
		(n.mol/g tissue)	(mg/g tissue)		(U/g tissue)
	Control	Brain	62.27±2.82 a Liver	58.03±4.49 a Brain	68.58±3.92 a Liver	Brain	0.99±0.05 a Liver
	ABM Control	79.46±1.88 b 10.84±0.24 a 15.25±0.91 a	86.87±4.55 b 16±0.12 ac	125.6±6.03 b 16.24±0.12 a 0.634±0.01 a 2.09±0.10 b 0.236±0.01 a
	ABM+AA ABM	67.16±0.76 a 13.20±0.37 b 18.63±0.49 b	76.30±2.13 c 14.12±0.23 b	91.08±3.74 c 12.22±0.79 b 0.461±0.04 b 1.64±0.04 c 0.323±0.01 b
	ABM+SO ABM+AA	68.23±1.77 a 11.86±0.57 ab 15.19±0.32 a	70.72±3.41 ac 15.61±0.23 a	84.67±3.13 ac 15.79±0.06 a 0.624±0.01 a 1.43±0.03 c 0.303±0.01 bc
	ABM+AA+SO ABM+SO	68.83±0.49 a 11.51±0.52 ab 15.08±0.51 a	67.10±2.62 ac 15.79±0.14 a	80.21±1.94 ac 15.88±0.23 a 0.629±0.01 a 1.40±0.06 c 0.249±0.02 a
	ABM+AA+SO	10.99±0.43 a	15.24±0.49 a	16.68±0.07 c	16.64±0.29 a	0.630±0.01 a	0.222±0.01 a
	Glutathione, MDA: Malondialdehyde, SO: Sesame oil

Conflicts of