Jurkiewiez Bruno
email: bruno.jurkiewiez@univ-lyon1.fr

Alaa Koaik
email: alaa.koaik@gmail.com

Bel Sylvain
email: sylvain.bel@univ-lyon1.fr

Static behaviour up to failure of a full-scale GFRP-concrete hybrid footbridge

Keywords: GFRP pultruded profiles, Hybrid footbridge, bending tests, bonding / bolting interface. 1 Introduction

This paper presents an experimental investigation on the short-term behaviour up to failure of a full-scale GFRPconcrete hybrid footbridge. The connection between the slab and the GFRP profiles is a mixed bonding / bolting connection. The footbridge was first tested in the elastic domain under different configurations to mobilize the flexural and torsional stiffness. The numerous measurements show that the behaviour is in accordance with the regulatory requirements at SLS. The footbridge was finally loaded up to failure. The shear failure obtained in the GFRP section showed that the mixed connection used played its role perfectly and made it possible to fully exploit the bending capacity of the structure. In addition, the comparison with the results of the beam tests shows that the two-beam bridge can be analysed with sufficient precision as the juxtaposition of the two single-sided beams. This study confirms the important capacity of these hybrid GFRP-concrete structures in the construction of bridges or footbridges with classical engineer design tools.

fulfil standards' requirements for footbridges and are in good agreement with conventional analytical and numerical design methods. However, the ultimate static behaviour of this footbridge was not studied. In [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF], a similar and shorter footbridge was loaded up to failure. The failure is triggered by the shear failure at the webflange junction of one of GFRP profiles. The collapse was accompanied by high deflections which were a clear warning of the imminent failure.

In this context, the aim of the present study is to contribute to the current efforts to increase the knowledge and understanding of the instantaneous behaviour up to failure of hybrid structures with both bonding and bolting connection. To meet this objective, this study presents experimental investigations of the ultimate behaviour of two hybrid beams and a hybrid footbridge designed according the structural concept proposed by Gonilha et al. [START_REF] Gonilha | Creep response of GFRP-concrete hybrid structures: Application to a footbridge prototype[END_REF][START_REF] Gonilha | Dynamic response under pedestrian load of a GFRP-SFRSCC hybrid footbridge prototype: Experimental tests and numerical simulation[END_REF][START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF][START_REF] Gonilha | Static, dynamic and creep behaviour of a full-scale GFRP-SFRSCC hybrid footbridge[END_REF]. These structures consist of I-shaped GFRP pultruded profiles connected to a reinforced concrete slab. The two simply supported hybrid beams (named PB7 and PB8) with different mixed connections were first loaded up to failure. The results allowed designing a footbridge (BPB1) which was built, highly instrumented and tested under several loading configurations. This structure was first loaded in the elastic domain under 3 or 4 points bending, according to two different transversal bracing systems. The response of the structure, most notably between the two main girders, allows evaluating the influence of the bracing elements. Finally, the footbridge was loaded up to failure under 3 points bending. The experimental program is accompanied by an analytical study of deformations, stresses and deflections. The main results are presented and compared with test results in the elastic domain and close to failure.

Materials characteristics

Concrete

The concrete slabs were fabricated using Portland cement CEM I 52.5. The sand used was washed river sand and the coarse aggregate was 8 to 20 mm nominal size natural uncrushed gravel. The mix proportions were 1 (cement): 1.86 (sand): 2.33 (gravel) and a water-cement ratio of 0.47. For each concrete batch, five 100 mm edge cubes and three 160 mm x 320 mm cylinders were poured to evaluate compressive and tensile strengths (by splitting) respectively at different curing ages. The tests were performed according to NF EN 12390-3 [START_REF]Testing hardened concrete[END_REF] and NF P 18-408 [35], using a CONTROLAB machine with a maximum capacity of 1600 kN. For each hybrid beam, Table 1 presents the results of compression and tensile tests performed on concrete specimens the day the failure bending tests were done. The slabs of PB7 and PB8 were poured from the same batch. The difference of strength in the case of BPBP1 originated from the age of the concrete.

GFRP Profiles

The pultruded GFRP profile used in the experimental program was made of E-glass fibres embedded in a polyester matrix. The GFRP profiles contained both random continuous filament mat and E-glass roving and were produced by TOPGLASS. Burn-off tests previously performed on coupons taken from the profile showed heterogeneous distribution of the fibers [START_REF] Koaik | Experimental tests and analytical model of concrete-GFRP hybrid beams under flexure[END_REF]. The mass percentages of fibers in the flange and in the web were about 67% and 46% respectively. The Young's modulus and the tensile strength were measured by testing several rectangular prismatic coupon specimens extracted, in the pultrusion direction, from the flanges and web accordingly to the ISO 527-1 [START_REF]Determination of Tensile Properties -Part 1: General Principles[END_REF].

Five identical coupons were used for each test. In the flanges, the average longitudinal tensile strength and the average longitudinal elasticity modulus were 379.7 ± 36.5 MPa and 23.9 ± 2.6 GPa respectively. In the web, these properties were 395.3 ± 48.6 MPa and 25.0 ± 3.1 GPa respectively [START_REF] Alacheck | Experimental and finite element analysis of the long-term behaviour of GFRP-concrete hybrid beams fabricated using adhesive bonding[END_REF].

Adhesive

A two-component resin and hardener thixotropic epoxy adhesive (Sikadur® -31EF) was used to bond GFRP profiles to the concrete slabs as, according the product datasheet, this resin suits bonding concrete and GFRP in civil engineering applications. Experimental tests were performed 15 days after bonding to determine the main mechanical characteristic properties of the adhesive. The Young's modulus, the tensile strength and the ultimate strain were measured by testing 8 plates of bulk adhesive instrumented with strain gauges and prepared according to the ISO 527-2 [START_REF]Determination of Tensile Properties -Part 2: Test Conditions for Moulding and Extrusion Plastics[END_REF]. The specimens were tested on a Zwick machine at room temperature and at a crosshead speed of 1 mm.min -1 . All specimens were stored under ambient laboratory conditions until the testing date. The results showed behaviour almost linear elastic with an average ultimate stress of 13.4 ± 2.5 MPa, an elastic secant modulus of 6.7 ± 1.1 GPa and an ultimate strain of 0.18% ± 0.05% [START_REF] Alacheck | Bond strength's degradation of GFRP elements under aggressive exposure conditions[END_REF].

Bolts

In order to investigate the interface strength, three types of bolts were used: in PB7 class 6.8 bolts with a diameter of 12 mm, in PB8 class 8.8 bolts with a diameter of 10 mm, in BPB1 class 8.8 bolts with a diameter of 12 mm. Tensile and shear tests were conducted on each type of bolts, two specimens per type. The experimental results are presented in Table 2 and compared to the strength determined according to Eurocode 3 [START_REF]EN 1993-1-1. Eurocode 3: Design of steel structures -Part 1-1: General rules and rules for buildings[END_REF]. The EC3 strengthes were generally lower than the test result (between 6 and 33%) except for the shear strength of 10 8.8 bolts. This result may be explained by undesired parasite tension load applied on the bolts during the tests.

Steel reinforcement

The steel rebars in the concrete slabs are ribbed with guaranteed yield strength of 500 MPa. Tensile tests performed on 250 mm long pieces (5 specimens tested) according EN ISO 6892-1 showed that the real tensile strength reached about 561.0 ± 6.1 MPa.

Hybrid beams

Geometry and fabrication of the beams

Same concrete batches and same pultruded profiles were used to construct hybrid beams PB7 and PB8. These beams present the same geometry as shown in Figures 1 and2. The beams present a total length of 5000 mm and a simply supported span length of 4800 mm. The concrete slabs have a cross section of b × h = 600 mm × 70 mm. They were reinforced, in the tension zone (bottom of the slab), longitudinally with 4Ø7 mm steel bars and transversally with Ø7 mm/150 mm steel bar with a 20-mm cover. The cross-section was chosen based on design equations in order to benefit from the mechanical properties of each material (the high tensile strength of the GFRP and the high compressive strength of the concrete) and to delay the local/global buckling of the GFRP profile. The dimensions were then selected to obtain a neutral axis lying in the concrete slab. Five Precast slabs having the dimensions 840 mm x 600 mm x 70 mm, were used to cover the whole length. Spaces between precast slabs were cast in place with a similar concrete composition.

The pultruded I-profiles were 200 mm deep and 100 mm wide with a wall thickness of flanges and web of 10 mm. For all the used profiles, reinforced concrete jackets (concrete stiffeners) were executed between flanges at the support sections on both sides of the web, with a length of 200 mm, to avoid local web crushing failure.

The connection between the concrete slab and the GFRP profile was by bonding and bolting. The bonding is established by epoxy applied on contact zones between the profile and the slabs. The bolting however is done by bolts already tightened in predrilled holes in the profile top flange in cast in place zones. These zones are constituted from the spaces between the precast slabs and 90 mm x 90 mm reservations in precast slabs. PB7 and PB8 differ only in their bolts: PB7 was bolted with 6.8 12mm bolts, PB8 with 8.8 10 bolts. 36 bolts were used in each beam.

The following procedure was adopted for the preparation of both hybrid beams. The to-be-bonded surface of the hardened concrete slab was firstly sandblasted to remove all surface contaminants and to improve the roughness and the bond strength. Moreover, the surface roughness of the top flange of GFRP profiles was enhanced by means of fine sandpaper (grit P100). During the roughening process, special care was taken not to damage the fibres. After roughening, compressed air jet was used to blow up the dust and any stuck particles on the concrete slab or the profile. Then the top flange of the profile was cleaned with acetone to remove all grease and oil after which the surfaces were left to dry for few minutes. The two components of the epoxy adhesive were mixed according to the manufacturer's instructions and supplier's specifications (Sikadur® -31EF) until a homogeneous light grey paste was obtained. Once mixed, the adhesive was evenly distributed onto the treated area of the concrete slab and the top flange of the GFRP profile at the same time, paying attention to avoid air bubbles, and they were held together in position using clamping screws to insure a uniform thickness of 1 mm in the overlap area. Excess adhesive was chamfered on the extremities. Finally, the clamping screws were removed at least 48 hours after bonding, once the adhesive hardened. The holes, the reservations and the gaps between the precast slabs were concreted with the same concrete composition used for the slabs. After the preparation, the beams were stored under ambient laboratory conditions until testing day. Different steps of the beams construction are shown in Figure 3.

Testing set-up and instrumentation

Both beams were simply supported. Both supports allowed rotation by means of steel rollers fixed to hinges.

Beams were tested under 3 points bending, the hydraulic jack with a capacity of 500 kN was set at the mid-span of the beam. Steel plate was put between the loading point and the top concrete in order to minimize the local concentration of stresses. A displacement rate of 4 mm / min for the jack was used for the tests. The applied loads were measured by load cells (capacity: 500 kN) within the machine. The strains at different depths in the beam section were recorded using electrical resistance strain gauges positioned at mid-span and at a section (SN) located at 90 cm from the mid-span. Two types of gauges were used in this study: for concrete, (KC-70-120-A1-11) gauges and (KFG-10-120-C1-11) gauges for the other materials. The positions of these gauges in each beam are shown in Figure 4. The deflection in both sections was measured using a Linearly Variable Differential Transformer (LVDT) with a stroke of 100 mm and a precision of 0.01 mm. The applied load and the deflections at mid-span as well as the axial strains were registered in a PC using a Vishay data acquisition system with a rate of 2 Hertz. The test setup is illustrated in Figure 3c.

Results of bending tests

The behaviours of hybrid beams PB7 and PB8 are illustrated in Figure 5. They are very similar. Both beams showed an approximately linear behaviour up to a load of 50 kN after which a slight non-linearity is observed. This phase continued up to about 80 kN where the shear failure at the interface between the GFRP profile and the slab suddenly occurred. In the mid-span cross-section, for both beams, the maximum strains at failure in the top concrete layer and the bottom GFRP level were -0.31 % and +1.05 % respectively. There were no marks of concrete crushing at mid-span though the measures show high strains. Just before the shear failure, the interface was visibly intact and no cracks/separation was observed. This observation is verified by the strain diagram (Figure 5) that shows no significant slippage at the interface level before failure. A slight discrepancy may be noticed for the strains of the steel rebars which remained relatively small during the test (as they were close to the neutral axis): tensile stresses were noted for PB7 rebars while compression ones for that of PB8. This could be caused by casual imperfections when applying the formwork. In the cross-section SN, the strains were logically lower (-0.08 % and +0.65 % in the top fiber of the slab and in the bottom fiber of the profile respectively).

The inflexion of the force -displacement curves at about 50 kN can't be explained with certainty. However, it could be mentioned that the maximum strains in the concrete and in the GFRP beam did not exceed 0.11 % and 0.43 % respectively. In addition, as a shear failure at the interface occurred, it could be assumed that this inflexion in the behaviour of the beam at 50 kN could originate in the interface (microcracks in the bonding line, beginning of the bolt yielding…). This assertion should be confirmed by further investigations.

As a conclusion, the diameter and the strength of the bolts, which were the main differences in both beams, did not induce a significant effect on the behaviour of the beams. The fact that PB7 exhibited a failure load of 80.7 kN which is about 3% greater than that of PB8, could have been obtained for identical beams.

Hybrid footbridge

Conception, geometry and fabrication

Similar to composite steel-concrete structures, a composite footbridge can be designed as a twin-profile associated with a concrete slab via an interface connection system. The mechanical behavior of such structure shall then be considered in two directions. Along the longitudinal axis, the main beams and the concrete slab work together as a solidary mixed structure due to the connection. Thus this structure can be considered as a duplication of beams PB7 or PB8 presented earlier. Along the transverse direction, the slab can be considered supported on both beams with 2 cantilever spans. Considering that loads may be applied anywhere on the slab, it is necessary that the structure has a sufficient transverse flexural and torsional rigidity. This is obtained by the installation of regularly spaced transverse stiffeners (secondary girders) connecting the two main beams. This structural concept was adopted by Gonilha et al. [START_REF] Gonilha | Creep response of GFRP-concrete hybrid structures: Application to a footbridge prototype[END_REF][START_REF] Gonilha | Dynamic response under pedestrian load of a GFRP-SFRSCC hybrid footbridge prototype: Experimental tests and numerical simulation[END_REF][START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF][START_REF] Gonilha | Static, dynamic and creep behaviour of a full-scale GFRP-SFRSCC hybrid footbridge[END_REF]. As shown in Figures 6 and7, the footbridge consists of 2 I-GFRP beams identical to those used for PB7 and PB8 (see section 3) associated with a reinforced concrete slab of section 1200 mm x 70 mm. The beams are of a total length of 5000 mm and a simply supported span length of 4800 mm. The footbridge is designed as a duplication of PB7 / PB8 beams. The slab has the same steel density as PB7 / PB8 (at the bottom of the cross section, 8Ø7 mm longitudinal steel bars and Ø7 mm steel bar transversally spaced at 150 mm with a 20-mm cover). The same concrete stiffeners are applied at supports. The connection is also designed identically to that of PB7 / PB8 (bonding and bolting) but as PB7 and PB8 failed by shear at the interface, it was decided to reinforce the connection: the adhesive is identical (same adhesive and same thickness of 1 mm) but the bolts used are M12 8.8 bolts. The latter have a greater bearing capacity and stiffness than those of PB7 and PB8 (see Table 2). The bolts are paired in each reservation making a total of 72 bolts for the entire footbridge.

Similar to PB7/PB8, the slab is composed of five precast slabs having the dimensions 840 mm x 1200 mm x 70 mm and cast-in-place concrete areas are located between these 5 precast slabs. Slabs have 90 mm x 90 mm reservations for the bolts and tiny holes for the insertion of the clamping crews and slings.

Secondary girders (transverse braces), connecting the main I-profiles, consist of square GFRP tubes with 90 mm side length and 10 mm wall thickness. These elements are fixed to the web of both profiles at mid heights by GFRP angles and steel bolts (see Figures 8c and8d for connection details). Along the loading axis, these stiffeners are distributed as follows: a stiffener is located near each support and 2 intermediate stiffeners are located approximately at one-third of the span (Figure 6). In this study, the influence of the transverse stiffening density on the elastic response of the structure is analyzed: either the 4 stiffeners are present (complete bracing -Brace System # 1 named BS1), or only the extremity stiffeners are present (partially bracing -Brace System # 2 named BS2).

The main girders and stiffeners were first assembled and the slabs were prefabricated. Then, the surfaces to be bonded were prepared identically to PB7 / PB8 and the prefabricated slabs were bonded. Once the adhesive hardened, the bolts were tightened and the reservations were concreted with the same concrete as the prefabricated slabs. The footbridge was then kept in the open air until the day of testing. Figure 8 illustrates some of the construction steps. It may be noticed that no problem of shrinkage cracks were observed in the cast-inplace zones as these zones were short and hardly reinforced by several rebars.

Testing set-up and loading program

The loading configuration (jack, steel plate, supports, loading speed) was identical to that of the PB7 / PB8 beams. During the tests, in addition to the applied force, the vertical displacement was measured using (LVDT) sensors (stroke: 100 mmprecision: 0.01 mm) located at the center and at 1.5 m from the supports on each GFRP main profile. Interface slip between the slab and each profile was also measured by means of (LVDT) sensors (stroke: 10 mmprecision: 0.005 mm) at both extremities of the footbridge. The strains were measured by means of gauges in different sections of the footbridge and at different levels of each cross-section. Two types of gauges were used in this study: for concrete, (KC-70-120-A1-11) gauges and (KFG-10-120-C1-11) gauges for the other materials. Moreover, the strain of steel bolts along their axis at extremities was also measured by means of (KFG-5-120-D17-11) gauges. All the measurements were registered in a PC using a Vishay data acquisition system with a rate of 2 Hertz. In total, the device had 50 measurement channels. Figures 9 to 13 detail the testing configuration.

Elastic behavior

Before the connection with the concrete slabs, the double girder equipped with the stiffeners was tested in bending in the elastic domain in different configurations (Figures 14 and15). The results showed a behavior very close to that of an I-section alone tested under the same conditions, both for displacements and for deformations.

After the slab connection, the footbridge was first tested in the elastic domain under different configurations. The loading was first applied symmetrically, along the axis of the girders, under 3 and 4-point bending, with the bracing system No. 1 and the bracing system No. 2. The main test results are summarized in Figure 16 and Table 3. It may be noticed that the deflections and the strains in the west axis of the footbridge were slightly higher (less than 5%) than those in its east axis: the behavior of the footbridge exhibited a slight westeast asymmetry that may be explained by the 2 mains girders which did not press uniformly on the four supports. In addition, the longitudinal strains in the top and bottom fibers of the slab are about 4% lower that the strain in the axis of the Iprofiles: this result confirm that, in the case of wide slab, the stress and the strain are note uniform along the transverse axis. These observations generally did not depend on the cross-section (SC or SN), the loading system (3-pt or 4-pt bending) or the bracing system. Thus, the differences between the 2 bracing configurations are small which is logical because the loading is applied symmetrically along the axis of the beams.

The footbridge was then tested under 4-point diagonal bending: the loading was applied at 2 symmetrical points with respect to the mid-span, one located on the east beam on the northern side and the other on the west beam on the southern side (Figure 17). This test was carried out for the 2 bracing systems and then repeated by reversing the loading points (South-East and North-West). The main results are shown in Table 4, Figure 18 and Figure 19. Almost the same observations noticed in the previous paragraph can be made. It can be seen that the deflections are not influenced by the loading mode or the bracing system. On the other hand, the strains are higher on the side of the loading points in section SN. This is quite logical and indicates that the bridge undergoes a slight twist. These results also confirm that the two bracing systems used in this study (BS1 and BS2) have no significant influence on the overall elastic behavior.

In the last stage of the elastic tests, the bridge was subjected to centered flexure loading, that is by applying the loading along the axis of the slab and not along the axis of the main profiles under 3-point bending, only with the bracing system # 1 (see Figure 20). The most interesting results are summarized in Table 5 and are compared with the measurements obtained, for the same total load applied, in the case where the loading is applied in the axis of the main beams. It is first noted that the strains along the axis of the main girders are slightly affected by the loading at the center of the slab (differences of about 10% and less than 1% on the upper fiber of the slab and on the lower fiber of the profile respectively). On the other hand, the strains recorded in the central zone of the footbridge clearly show the bi-directional flexure of the slab in this zone. Indeed, tensile strains in the transverse steel (measurements 9 and 10) are significant whereas they did not show any deformation in earlier bending test.

Similarly, the transverse deformation of the upper fiber of the slab (measurement 7) indicates a shortening and therefore a compressive stress which reflects the fact that the slab is also bent along the transverse direction. It may be noticed that the bi-directional behavior of the slab is influenced by the bracing system as the latter prevents (more or less according the degree of bracing) the rotation of the GFRP profiles around their weak-axis and therefore the transversal strains of the RC slab.

These different measurements recorded in the elastic domain confirm that the footbridge operates in a predictable manner, analogous to what can be observed for mixed steel-concrete structures. These measurements provide an interesting database for in-depth modeling of the structure. In the case studied, the bracing system did not influence significantly the response of the footbridge as the twist / torsion action imposed to the structure was not high.

Testing up to failure

Once the elastic tests were completed, the footbridge was then completely unloaded, the bracing system # 1 (complete bracing) was put in place and the bridge was loaded under 3-point flexion (loading applied along the axis of the girders) monotonously increasing until failure. The same experimental setup was used. The main results are shown in Figures 21 to 30. They are very similar to those of [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF]. The deflection and strain diagrams of the slab and girders as a function of the force applied are almost linear until failure. This occurs at 218 kN and corresponds to the abrupt shear failure at the web-flange junction of the east profile (Figure 21). The maximum values of strain in the upper fiber of the slab, in the longitudinal rebars and in the lower fiber of the profile are respectively -0.22%, +0.13% and +0.94%, which indicates that the crushing of the concrete, the yielding of the steel or the tensile failure were not imminent (see Figures 22 to 24).

The load-strain diagrams at the center of the slab in sections SC and SN (Figures 24 to 26) show a similar and consistent evolution of strains in these areas of the footbridge. The measurements confirm that the transverse reinforcement is slightly solicited since the loading is applied along the axis of the main beams and thus induces no transverse flexure. The transversal deformations of the concrete in the upper and lower fibers are of opposite sign to the corresponding longitudinal deformations, which reflects Poisson's effect.

The deformation diagrams along the vertical axis (Navier diagrams) plotted along the axis of the main beams at sections SC and SN indicate that the assumption of conservation of the plane sections is quite acceptable approximation up to failure (Figures 27 and28).

In Figure 29, it is observed that the slip at the interface increases with the force applied in a non-linear manner and reaches maximum values between 25 and 55 m. These very low values confirm that slip can be neglected in calculations even in the case of a mixed connection bolting / bonding: the adhesive prevents any relative movement between the slab and the main girders. The small values obtained correspond to the eventual shear deformation of the adhesive, the flexibility of the bolts and their small movement in the holes. The strain measurements of the bolts located at the extremities along their vertical axis (see the position of the gauge in Figure 12) is given in Figure 30. The results show that two of the four bolts observed (bolts 2 and 3) were almost not solicited whereas the strain in bolts 1 and 4 remained low and evolved non-linearly with the applied load, in a similar manner to the slip curves. However, a clear relation linking bending of bolts and slip could not be proposed in this paper, other measures shall complement this analysis.

The behavior of the footbridge is compared to that of PB7 / PB8 single beams. It is necessary then to assume that the loading applied on the footbridge is distributed equally on the both symmetrical parts containing the girders. By doing this, it is considered that the total force applied on the footbridge is as twice as high that applied on the mono-sided footbridge equivalent to PB7 and PB8. It is first realized that the failure mode and load are different: PB7 / PB8 failed at a load of about 80 kN by shear failure at the interface whereas equivalent mono footbridge failed due to a shear of the GFRP profile at a load of 109kN (50% x 218kN: the failure load of the footbridge). Thus, strengthening the connection, in particular bolts, seems to have made it possible to better exploit the bearing capacity of the hybrid beams. Table 6 compares the main measurements of these two structures. It can be seen that up to 50 kN, the deflections and strains are very close. Starting from which, the differences get higher to reach a maximum at the failure of PB7/PB8, this change of behavior seems to be attributed to 2 different causes. On one hand, the strength and stiffness of the concrete at the time of the bending tests are slightly different since the concrete did not have the same age at both testing days (see Table 1). On the other hand, the stiffness of the footbridge bolts is greater than that of the PB7 / PB8 bolts, which means that the forces resisted by these bolts are probably higher in the footbridge than in the beams. Therefore, shear present in the adhesive joint is higher in the PB7 and PB8 which explains their failure mode. However, these results do not allow knowing with certainty if these two causes (strength -rigidity of the concrete and resistance / rigidity of the connection) have the same impact or whether one is more important than the other. It may also be noticed that the age of the adhesive in the beams PB7/PB8 at failure was about 40 days whereas the bonding in the footbridge had been made 400 days before the failure test.

Assuming a characteristic load defined in Eurocode 1 [START_REF]Eurocode 1: Actions on structures -Part 2: Traffic loads on bridges. European Committee for Standardization (CEN)[END_REF] for footbridge structures (5 kN/m 2 ), the maximum values of the bending moment and the shear force that should be sustained is equal to 17.3 kNm and 14.4 kN respectively for the span of the footbridge studied. Failure occurred for an applied load of 218 kN (in addition of the self-weight) which was at least 7.6 times greater than the resistance required by Eurocodes. This safety margin was higher but consistent with that noticed by Gonilha et al. [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF] who found a value of 4.2. Regarding the flexibility of the footbridge, the vertical deflection at failure was about 93.1 mm which corresponds to approximately L/51.6 (L being the span)about 10 times higher than the deflection limits defined for building floors in Eurocode [START_REF]Eurocode 1: Actions on structures -Part 2: Traffic loads on bridges. European Committee for Standardization (CEN)[END_REF] and more than 2.66 times higher than the deflection limit for pedestrian footbridges structures. It may be noticed that a safety factor of 2.35 was measured by Gonilha et al. [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF].

Analytical simulations

Elastic analysis

The elastic flexural response of a GFRP-concrete hybrid structure can be compared to that of steel-concrete composite beams in service limits states. Therefore, the elastic response may be predicted analytically using simple Timoshenko beam theory but several formulations are available according the reference axes used [START_REF] Correia | GFRP-concrete hybrid cross-sections for floors of buildings[END_REF][START_REF] Koaik | Experimental tests and analytical model of concrete-GFRP hybrid beams under flexure[END_REF][START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF]. In the case the axes are located at the neutral axis (Figure 31), the location of the neutral axis is calculated solving the following equation:

0 h 2 h A E z A E z 2 E b c p p p 1 p p 2 1 c c             (1)
In this equation, z 1 is the position of the neutral axis with respect of the top slab fiber, E p and E c are the longitudinal elastic modulus of the GFRP profile and the concrete slab respectively, h c and b c are the dimensions of the slab, A p and h p are the area and the height of the GFRP profile respectively. As the longitudinal elastic modulus of the flanges and the web were close to one to each other (see section 2.2), one can assume that E p was the average of both and was equal to 24.5 GPa.

The equation ( 1) is based on the following assumptions supported by the behavior of the hybrid beam and the footbridge shown in previous sections: the concrete in tension (cracks were observed) and the slip (not greater than 50 m) are neglected. The steel reinforcement may be also neglected as they were located close to the neutral axis (as shown in the following paragraphs).

The longitudinal stress (i.e axial stress) in each fiber of the composite cross-section can be calculated as follows:

z I M E E σ h p c c    (2) z I M σ h p   (3)
In these equations, z is the vertical position of the fiber with respect to the neutral axis, M is the applied bending moment and I h is the second moment of inertia of the whole composite cross-section around the neutral axis calculated as follows:

2 1 3 1 2 3                z h h A I z b E E I c p p p c p c h (4)
In this equation, I p is the second moment of inertia of the GFRP profile around its own centroid. The strain at each fiber can be deduced from stress using Hooke's law. Typical strain and stress diagrams are shown in Figure 31. In the present case, the structure was tested on a 3-point bending configuration over a simply supported span, thus the vertical deflection w(x) can be calculated as follows:

  hv p 2 2 h p Ω G 2 x F 4x 3L I E x F w(x)      (5)
where x denotes the longitudinal development of the structure, F is the total applied load, E p I h is the flexural stiffness of the composite cross-section, hv Ω is the shear area of the composite cross-section and G p is the inplane shear modulus of the GFRP profile. The latter, evaluated by means of three-point bending tests performed on a similar GFRP profile [START_REF] Alacheck | Experimental and finite element analysis of the long-term behaviour of GFRP-concrete hybrid beams fabricated using adhesive bonding[END_REF], is assumed to be equal to 2.5 GPa. For this type of hybrid structure, it may be admitted that only the web of the profile works efficiently, therefore in Eq. ( 5)

hv Ω may be substituted by the area of the webs A w .

Table 7 compares calculated values of vertical deflection and strains in two sections of the footbridge with those measured. The theoretical values were obtained considering the half footbridge. The relative errors were between 6.6% and 19.0% that confirms, as shown by Gonilha et al. [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF], this simple approach is able to predict, with an acceptable accuracy, the flexural response of hybrid GFRP-concrete structures for service conditions.

Failure analysis

In order to determine the resistance of the hybrid structure in bending, it is necessary to determine the ultimate bending moment and shear force that the structure is able to sustain. Actually, the instability phenomena were prevented in the footbridge by the bracing girders and therefore, the resistance is only limited by the crosssection strength, allowing a full exploitation of the materials' capacity.

The ultimate bending moment that the cross-section may sustain can be easily calculated assuming that the beam perceived a failure by crushing of concrete and the GFRP profile remains elastic as detailed by Koaik et al. [START_REF] Koaik | Experimental tests and analytical model of concrete-GFRP hybrid beams under flexure[END_REF].

The following assumptions were made: the concrete in tension, the slip and the steel reinforcement were neglected (for the same reasons as the elastic analysis) -E p = 24.5 GPathe concrete compression strength f c = 47.6 MPathe concrete ultimate compression strain  uc = 0.0035. Using this approach, the neutral axis is located 42.6 mm from the concrete top fiber, the ultimate bending moment and the theoretical failure load were equal to 390 kNm and 324 kN respectively. The latter value is logically greater than the measured failure load (218 kN) as no crushing of concrete was observed during the test and the maximum strain measured in the fiber of the concrete slab was about 0.0021.

Regarding the shear resistance, Gonilha et al. [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF] suggested to use a quadratic failure criterion initially proposed in [START_REF] Bank | Composites for construction: structural design with FRP materials[END_REF] and written as follows:

1 ) ( f (z) σ ) ( τ (z) τ 2 L tu, 2 LT u,                   z z (6)
where  and  are respectively the shear and axial stresses due to the applied load, z represents the vertical development of the cross-section,  u,LT is the in-plane shear strength of the GFRP, f tu,L is tensile strength of the GFRP for the longitudinal direction. As seen in section 2.2, f tu,L may be assumed to be equal to 379.7 MPa and 395.3 MPa in the flanges and in the web respectively.  u,LT was not measured but may assumed to be equal to 27.5 MPa as measured by Gonilha et al. for similar GFRP profiles [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF]. This value is closed to that mentioned in the technical data sheet of the supplier. In order to use this criterion, the shear and axial stresses were calculated assuming the materials remain in their elastic domain as indicated by the maximal strains measured during the test. The axial stress was determined using equations ( 2) and (3). The shear stress was calculated in each fiber located at z by the following equation:

(z) b I (z) S V τ(z) h h h  (7)
where V is the shear force acting in the cross-section, b(z) is the width the concrete slab, the width of the GFRP profile or its web thickness if the fiber is located in the concrete slab, in the profile top flange or in the web respectively. h S is the first moment of area of the upper part of the composite section with respect to the neutral axis.

The figure 32 shows the vertical distribution of the shear and axial stresses in the mid-span cross-section just before the failure. This graph confirms that the failure may be predicted at the junction between the flange and the web as it corresponds with the maximum computed value of equation ( 6) equal to 0.834. This value strongly depends on the in-plane shear strength of the GFRP  u,LT . For instance, equation ( 6) provides a value equal to 1 if  u,LT is 9% lower than the assumed value of 27.5 MPa.

Conclusions

In this study, results of bending tests under short-term quasi-static loading of beams and a hybrid compositeconcrete footbridge are presented. In these elements, the connection between the slab and the GFRP profiles is mixed bonding / bolting connection. The test results of the beams served to design the footbridge. The bridge is constructed, inspired by composite steel-concrete structures, as a double beam geometrically corresponding to the perfect juxtaposing of the 2 beams tested.

The footbridge was first tested in the elastic domain under different configurations (3 or 4-point bending, centered or eccentric loading) to mobilize the flexural and torsional stiffness. The numerous measurements recorded allowed to have a precise idea of the behavior in elastic phase. They show behavior consistent with expectations and in accordance with the regulatory requirements at SLS. These conclusions are in perfect agreement with those of Gonilha et al [START_REF] Gonilha | Structural behaviour of a GFRP-concrete hybrid footbridge prototype: Experimental tests and numerical and analytical simulations[END_REF].

The footbridge was then tested up to failure. The shear failure obtained in the GFRP section showed that the mixed connection used played its role perfectly and made it possible to fully exploit the bending capacity of the structure. In addition, the comparison with the results of the beam tests shows that the two-beam footbridge can be analyzed with sufficient precision considering only the half cross-section. On the other hand, the measurements recorded during these tests did not make it possible to determine with a sufficient certainty the part of the shear forces at the interface resisted by the bolts. This point will need to be studied in depth in future work

This study also confirms the important capacity of these hybrid GFRP-concrete structures in the construction of bridges or footbridges. The numerous experimental measurements carried out under different configurations are very useful to complement the experimental data already available in the literature and to develop and validate the computational models.

The analytical model developed in this study was able to predict the elastic response of the footbridge prototype with good accuracy. The shear failure observed at the web-flange junction could also be predicted using a failure

(5)

(5)

89 2 (Figure 1 :Figure 2 : 1 )Figure 3 :Figure 4 :

 212134 Figure 1: Geometry and loading of beams PB7 and PB8 -side and top views (dimensions are in mm -Passive reinforcements are not presented)

Figure 5 :

 5 Figure 5: Behaviour of PB7 and PB8

Figure 6 :

 6 Figure 6: Side (a) and top views (b) of the footbridge (dimensions are in mm) (Passive reinforcements and concrete stiffeners are not represented on these views)

Figure 7 : 32 Figure 8 :

 7328 Figure 7: Cross-sections of the footbridge (dimensions are in mm) (Secondary girders are not represented)

Figure 9 :Figure 10 :Figure 11 : 36 Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 17 :East

 91011361213141517 Figure 9: Location of the displacement sensors and the measurement locations (dimensions are in mm)

Figure 18 :Figure 19 :Figure 22 :Figure 23 :

 18192223 Figure 18: global response in case of diagonal loading

Figure 24 :Figure 25 :Figure 26 :Figure 27 :

 24252627 Figure 24: Behaviour of the footbridge at failure -Strains in SC in the central zone

Figure 28 :Figure 29 :

 2829 Figure 28: Behaviour of the footbridge at failure -Vertical diagram of strains in SN side east

Table 1 : Concrete compressive and tensile strengths of the different structures on the testing day Table 2: Strength of bolts

 1

			PB7	PB8	BPBP1
	Age (days)		96	440
	Compression (1) (MPa)	35.2 ± 3.1	47.6 ± 2.7
	Tensile (MPa)	3.4 ± 0.3	4.2 ± 0.4
	(1) The values represent the equivalent strengths on cylindrical specimens (0.8 x
	fc cube).
		Tensile strength f ub (MPa)	Shear strength (MPa)
						F u exp (kN) (1)
		Tests	EC3	Tests	EC3 (2)
		(max. / min.)		(max. / min.)
	 10 8.8 (PB8)	882.7 / 869.5	800	399.5 / 395.6	480	25.4
	 12 6.8 (PB7)	704.7 / 704.7	600	400.2 / 376.2	300	29.7
	 12 8.8 (BPBP1)	846.1 / 845.1	800	546.2 / 544.7	480	35.6
	(1) 0.5 x A s x f ub exp av
	(2) 0.6 x f ub in the case of 8.8 bolts and 0.5 x f ub in the case of 6.8 bolts (NF EN 20898-1)

Table 5 : Influence of the centered loading in the elastic domain (BS1 -3point bending)

 5

			Central	GFRP
			loading *	loading **
		West (1)	-218	-238
		Centre (5)	-289	-201
		East (3)	-186	-215
		West (2)	1235	1229
		East (4)	1167	1173
	Slab bottom fiber	Centre (6)	103	69
	Slab top fiber (transversal) Centre (7)	-67	32
	Slab bottom fiber (transversal) Centre (8)	59	-20
		West (12)	65	69
		Centre (11)	86	43
		North (9)	98	9
		South (10)

Table 4: Diagonal loading in the elastic domain

criterion based on stresses. Hence, the approach proposed in the paper is a reliable tool for the design of GFRPconcrete hybrid structures. However, the development of these structures can only be optimal if it is framed by regulatory texts that are still missing today.

Table 3: Influence of the bracing system in the elastic domain


Manon Le Goff

Dominique Lagadic-Gossmann

Remi Latour

Normand Podechard

Nathalie Grova

Fabienne Gauffre

Soizic Chevance

Agnès Burel

Brice M R Appenzeller

Lionel Ulmann

Odile Sergent

Eric Le Ferrec
email: ericleferrec@univ-rennes1.fr

PAHs increase the production of extracellular vesicles both in vitro in endothelial cells and in vivo in urines from rats

Keywords: aryl hydrocarbon receptor, AM: acetoxymethyl ester, Annexin V-PE: Annexin V-phycoerythrin, ARNT: aryl hydrocarbon receptor nuclear translocator, B[ cardiovascular diseases, CYP: cytochrome, DBA: dibenz[a, h]anthracene, DMSO: dimethyl sulfoxide, DTT: dithiothreitol, EVs: extracellular vesicles, FBS: fetal bovine serum, HMEC-1: Human microvascular endothelial cell-line 1, L-EVs: large extracellular vesicles, MVB: multivesicular bodies, NTA: nanoparticle tracking analysis, PAHs: polycyclic aromatic hydrocarbons, PBS: phosphatebuffered saline, PFA: paraformaldehyde, PYR: pyrene, RT-PCR: real-time quantitative PCR, SDS-PAGE: sodium dodecyl sulfate-polymerase gel electrophoresis, S-EVs: small extracellular vesicles, TEM: transmission electron microscopy, uEVs: urinary EVs, US-EPA: United States Environmental Protection Agency, WHO: World Health Organization, XREs: xenobiotic responsive elements, α-NF Polycyclic aromatic hydrocarbons, Aryl hydrocarbon receptor, Endothelial cells, Urine, Extracellular vesicles, Exosomes, Microvesicles, Large EVs, Small EVs Highlights

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

Introduction

Nowadays, there is broad consensus regarding the fact that our environmental health markedly depends on the impact of environmental contaminants to which humans are widely exposed [START_REF] Remoundou | Environmental Effects on Public Health: An Economic Perspective[END_REF] , especially via diet and ambient air. Most of these chemicals, such as heavy metals, persistent organic pollutants and biocides, come from anthropogenic activities. They are known to cause or worsen several diseases, like cardiovascular diseases (CVD), immune dysfunctions and cancers [START_REF] Ferronato | Waste Mismanagement in Developing Countries: A Review of Global Issues[END_REF] . Among these molecules, polycyclic aromatic hydrocarbons (PAHs) stand out since they are ubiquitous pollutants notably found in ambient air and diet. They are involved in the occurrence of human CVDs, and exhibit immunotoxic, mutagenic, and carcinogenic properties [START_REF] Bansal | Review of PAH contamination in food products and their health hazards[END_REF] . Most deleterious effects of PAHs are related to their binding to the aryl hydrocarbon receptor (AhR), a cytosolic ligandactivated transcription factor. PAH binding to this receptor triggers a canonical genomic pathway with the nuclear translocation of AhR followed by its interaction with aryl hydrocarbon receptor nuclear translocator (ARNT), thus allowing its binding to specific genomic elements termed xenobiotic responsive elements (XREs). XREs are found in the promoter of PAHs-responsive genes, such as cytochromes P-450 (CYP) 1A1 and 1B1, that are known carcinogen bioactivating enzymes.

AhR activation also triggers several non-genomic pathways, including intracellular calcium signaling and tyrosine kinase SRC activation [START_REF] Haarmann-Stemmann | Growth factors, cytokines and their receptors as downstream targets of arylhydrocarbon receptor (AhR) signaling pathways[END_REF][START_REF] Larigot | AhR signaling pathways and regulatory functions[END_REF] . The United States Environmental Protection Agency (US-EPA) as well as the World Health Organization (WHO) consider some of PAHs as priority pollutants due to their critical effects on human health [START_REF] Bruzzoniti | Determination of EPA's priority pollutant polycyclic aromatic hydrocarbons in drinking waters by solid phase extraction-HPLC[END_REF] . This has led to the development of research studies aiming at increasing the knowledge about these molecules and the discovery of biomarkers of exposure and/or effects.

During the last decade, extracellular vesicles (EVs) have drawn an increasing attention in the field of biomarkers of diseases or exposure to xenobiotics [START_REF] Benedikter | Extracellular vesicles released in response to respiratory exposures: implications for chronic disease[END_REF][START_REF] Conde-Vancells | Overview of extracellular microvesicles in drug metabolism[END_REF][START_REF] Rak | Extracellular Vesicles -Biomarkers and Effectors of the Cellular Interactome in Cancer[END_REF][START_REF] Soekmadji | The Emerging Role of Extracellular Vesicle-Mediated Drug Resistance in Cancers: Implications in Advanced Prostate Cancer[END_REF][START_REF] Wang | Plasma extracellular vesicle protein content for diagnosis and prognosis of global cardiovascular disease[END_REF] . Theoretically produced by all cells, EVs, including small extracellular vesicles (S-EVs or exosomes) and large extracellular vesicles (L-EVs or microvesicles), are delivery systems for multimolecular messages closely related to the nature and status of originating cells [START_REF] Bebelman | Biogenesis and function of extracellular vesicles in cancer[END_REF][START_REF] Maas | Extracellular Vesicles: Unique Intercellular Delivery Vehicles[END_REF] . These cellular nanostructures are differentiated by their size (S-EV: less than 200 nm, L-EV: less than 1000 nm), their biogenesis processes (S-EV: generated within endosomal compartments , L-EV: budding from the plasma membrane) and by their components (lipids, proteins, DNAs, mRNAs, miRNAs...) [START_REF] Hartjes | Extracellular Vesicle Quantification and Characterization: Common Methods and Emerging Approaches[END_REF] . EVs allow intercellular communication and consequently are involved in many biological processes such as cell maintenance and proliferation, tissue repair, angiogenesis... They have been detected within body fluids (i.e. blood, urine, lymph, saliva...) in healthy humans but also in patients [START_REF] Bayraktar | Cell-to-cell communication: microRNAs as hormones[END_REF][START_REF] Jaiswal | Intercellular Vesicular Transfer by Exosomes, Microparticles and Oncosomes -Implications for Cancer Biology and Treatments[END_REF] . In this latter case, EV production is exacerbated and their content altered. Such alterations notably occur during cancer, metabolic, neurodegenerative or CVDs [START_REF] Curtis | Endothelial microparticles: Sophisticated vesicles modulating vascular function[END_REF][START_REF] Martínez | Extracellular Vesicles in Metabolic Syndrome[END_REF] .

The EV overproduction is also observed during exposure to xenobiotics. Thus, some drugs or toxicants (like acetaminophen, alcohol, diclofenac or cigarette smoke) are able to increase in vitro and/or in vivo EV production, and to change their composition [START_REF] Bala | Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases[END_REF][START_REF] Benedikter | Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants[END_REF][START_REF] Cesselli | Extracellular Vesicles: How Drug and Pathology Interfere With Their Biogenesis and Function[END_REF][START_REF] Palomo | Abundance of Cytochromes in Hepatic Extracellular Vesicles Is Altered by Drugs Related With Drug-Induced Liver Injury[END_REF] . As a consequence, EVs could serve as biological markers of cell responses and possibly even susceptibility to toxicity. They also offer the possibility of detecting toxicity at the very early stages of development before irreversible effects occur.

Although the molecular mechanisms still remain to be fully clarified, there is evidence showing that an increase in intracellular calcium concentration ([Ca 2+ ]i), plasma membrane remodeling or oxidative stress are involved in the EV release by cells [START_REF] Pollet | Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?[END_REF][START_REF] Turturici | Extracellular membrane vesicles as a mechanism of cell-to-cell communication: advantages and disadvantages[END_REF] . Interestingly, we previously reported that PAHs and more specifically the prototypical PAH, benzo[a]pyrene (B[a]P), can trigger such cellular events. Indeed, our team previously demonstrated that cell exposure to various PAHs can increase the [Ca 2+ ]i, via an AhR-independent activation of the β2-adrenergic receptor [START_REF] Mayati | Induction of Intracellular Calcium Concentration by Environmental Benzo( a )pyrene Involves a β2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol 1,4,5-Trisphosphate Pathway in Endothelial Cells[END_REF] , and that membrane remodeling (characterized by an increase in fluidity and destabilization of lipid rafts), is a key mechanism in the B[a]P-associated toxicity [START_REF] Tekpli | Membrane remodeling, an early event in benzo[α]pyrene-induced apoptosis[END_REF] .

Exposures to xenobiotics or lifestyle factors, such as air pollutants 27 cigarette smoke [START_REF] Ryu | The Potential Roles of Extracellular Vesicles in Cigarette Smoke-Associated Diseases[END_REF] , alcohol [START_REF] Bala | Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases[END_REF] , or high fat diet [START_REF] Kranendonk | Extracellular vesicle markers in relation to obesity and metabolic complications in patients with manifest cardiovascular disease[END_REF] , would be able to modify EV trafficking, the amount produced and composition (e.g. proteins, miRNA), notably in biological fluids such as plasma or urine. In this context, circulating EVs might provide information about both exposure level and toxic responses of the organism. EVs in blood plasma of healthy humans mainly originate from platelets.

Comparatively, endothelial cells produce few EVs [START_REF] Iba | Role of extracellular vesicles in the development of sepsis-induced coagulopathy[END_REF] . However, while the level of EV production by these latter cells is low under physiological conditions, it can considerably increase in pathological situation [START_REF] Iba | Role of extracellular vesicles in the development of sepsis-induced coagulopathy[END_REF] . Thus, the amount of endothelium-derived EVs circulating in the blood stream has been shown to correlate with the severity of diverse diseases, eg. sepsis, stroke, atherosclerosis and metabolic syndrome [START_REF] Curtis | Endothelial microparticles: Sophisticated vesicles modulating vascular function[END_REF] . Endothelial cells are also particularly interesting to focus on since they also participate in the production of EVs detected in urine [START_REF] Barreiro | Urinary extracellular vesicles. A promising shortcut to novel biomarker discoveries[END_REF][START_REF] Lv | New insight into the role of extracellular vesicles in kidney disease[END_REF] . It is within this framework that our study was carried out.

In order to test whether EVs can serve as biomarkers of PAH exposure, the present study was designed to evaluate the effects of these pollutants on EV production. To this aim, two different approaches were applied: i) an in vitro approach, using the human endothelial cell line HMEC-1; and ii) an in vivo approach, using urine samples obtained from PAH-exposed rats. Urine provides a good alternative to blood in the field of disease biomarkers because this biological fluid can be collected noninvasively and in large amounts [START_REF] Pisitkun | Identification and proteomic profiling of exosomes in human urine[END_REF] . Our present data demonstrate that PAHs can increase EV production by endothelial cells. This overproduction observed in vitro concerns both S-EVs and L-EVs probably involving different molecular mechanisms. Indeed, it seems that only the increase of S-EVs would be AhR-dependent. Moreover, the PAH-related increase in EV production was also observed in vivo in urine samples from rats exposed to the 16 PAHs listed as "priority" compounds by US-EPA.

Materials and Methods

Chemicals and Reagents

Benzo

Cell Culture

The Human Microvascular Endothelial Cell line (HMEC-1) was obtained from the Center for Disease Control and Prevention (Atlanta, GA). Cells were routinely maintained in MCDB-131 medium containing hydrocortisone (1 µg/mL), epidermal growth factor (10 ng/mL), penicillin (50 unit/mL), streptomycin (50 unit/mL), L-glutamine (10 mM), and supplemented with 10% fetal bovine serum (FBS). At 90% confluence, before each treatment, cells were cultured overnight in serum-free medium. Chemicals were prepared as stock solutions in DMSO. The final concentration of vehicle did not exceed 0.2% (v/v); control cultures received the same concentration of DMSO. In all experiments using chemical inhibitors, cells were pre-treated 1 hour prior to and during exposure.

Animal experimentation

Long Evans rats (female of 180-200 g, Elevage Janvier, St Berthevin, France) were housed in plastic cages under controlled environment (12 h light/dark cycle, light on at 7 am, temperature of 22 ± 2°C and relative humidity of 40 ± 5 %). Food and water were available ad libitum. The water, food and oil were tested according to NF ISO 15302 to confirm that all these matrices were PAH-free down to a detection limit of 10 ng/L of water and 1 ng/g of fat. Rats were acclimatized to the animal facility for 2 weeks prior to experiment onset. The mix of PAHs was composed of the 16 compounds pointed out the US-EPA for their toxicity, and prepared in vegetable oil weekly (ISIO4, Lesieur, Neuilly-sur-Seine, France). Treatment of animals has been described in a previous study [START_REF] Grova | New insights into urine-based assessment of polycyclic aromatic hydrocarbon-exposure from a rat model: Identification of relevant metabolites and influence of elimination kinetics[END_REF]

Isolation of EVs from HMEC-1 cells

Cells were cultured using cell culture petri dishes of 151.9 cm 2 (CorningTM, Thermo Fisher Scientific, France). After PAH exposure, in order to isolate total EVs, L-EV or S-EV, the serum-free conditioned medium was first centrifuged at 3650 x g for 10 min to remove cells and cell debris and obtain the cell-cleared supernatants. Total EVs were pelleted by direct ultracentrifugation of the culture media at 100,000 x g for 1h45 at 4°C (Beckman Coulter Optima L-90K Series Ultracentrifuges, rotor Sw 28.1); the pellet was then resuspended in sterile phosphate-buffered saline (PBS), followed by another centrifugation at 100,000 x g for 1h45 min at 4°C. Total EV pellets were finally resuspended in sterile PBS.

L-EVs were recovered from cell-cleared supernatants following centrifugation at 10 000 × g for 30 min, followed by one washing step in PBS at 10 000 × g for 30 min, and then resuspended in sterile PBS. S-EVs were further isolated from L-EVs-depleted supernatants following filtration at 0.2 µm and a 100 000 x g ultracentrifugation step for 1h45 at 4°C, followed by one washing step in PBS at 100 000 x g for 1h45, and then resuspended in sterile PBS.

Isolation of urine EVs

About 9 mL of urine were first diluted half in PBS and centrifuged at 300 x g for 10 min, then 2 000

x g for 10 min to remove cells and cell debris. EVs were pelleted by ultracentrifugation at 100 000 × g for 2 h at 4°C (Beckman Coulter Optima L-90K Series Ultracentrifuges, rotor Sw 28.1). The pellet was next resuspended in sterile PBS containing dithiothreitol (DTT; 200 mg/mL) at 37°C for 10 minutes in order to dissociate of uromodulin aggregates [START_REF] Fernández-Llama | Tamm-Horsfall protein and urinary exosome isolation[END_REF][START_REF] Musante | A Simplified Method to Recover Urinary Vesicles for Clinical Applications and Sample Banking[END_REF] , and then resuspended in sterile PBS, followed by another centrifugation at 100 000 × g for 2h at 4°C. Total EV pellets were finally resuspended in sterile PBS.

Nanoparticle tracking analysis

EV samples were diluted in sterile PBS before nanoparticle tracking analysis (NTA). Size distribution and vesicle concentration were determined by NTA using a NanoSight LM14 (Malvern Instruments, Malvern, UK). A 405 nm laser beam is used to highlight the samples. Three 30 s captures per sample were recorded at 25°C. Videos were analysed using NanoSight NTA 3.1 software (Malvern, UK) using a detection threshold set to 3. The presented results correspond at least to an average from three videos for each of three independent samples of EVs, S-EVs and L-EVs. EV concentrations and distributions were then normalized to the cell count and expressed as number of EVs released per cell.

Electron microscopy

Transmission electron microscopy (TEM) was performed as described by Théry et al. [START_REF] Théry | Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids[END_REF] . Briefly, S-EVs and L-EVs were resuspended in 2% paraformaldehyde (PFA). Formvar-carbon coated copper grids (Agar Scientific, Stansted, UK) were placed on 5 µL S-EVs or L-EVs suspension for 20 min and washed with PBS. Vesicles were fixed in 1% glutaraldehyde in PBS for 5 minutes. After washing with distilled water, grids were placed on a drop of uranyl-oxalate for 5 min and of ice-cold 2% methyl cellulose/ 4% uranyl acetate (1/9; v/v) for 10 min. EVs were visualized using a JEOL JEM-1400 transmission electron microscope (JEOL, Japan).

Annexin V binding analysis

Staining by Annexin V-phycoerythrin (annexin V-PE, BD PharmingenTM) has been used to detect phosphatidylserine at the L-EV surfaces. An analysis window was defined using beads (Megamix, Stago) of diameters 0.5 μm, 0.9 μm and 3 μm. To determine the Annexin V positivity, L-EVs were incubated with Annexin V-PE, counting beads (Flow-Count™ Fluorospheres, Beckman) and buffer (14 mM NaCl, 10 mM Hepes, 2.5 mM calcium). Labelling in the absence of calcium was used as negative control. L-EVs stained with annexin V-PE were then analyzed by a BD FACSCanto™ III cytometer. Data have been exported using BD FACSDiva TM software.

Western Blotting

Total cellular, S-EV and L-EV protein amounts were estimated by a BCA Protein Assay Kit (Thermo Fisher Scientific, UK), using BSA as a standard. 5 µg proteins were separated by sodium dodecyl sulfate-polymerase gel electrophoresis (SDS-PAGE), and electrophoretically transferred onto nitrocellulose membranes (Merck Millipore). After blocking with a Tris-buffered saline solution supplemented with 2% bovine serum albumin for 2 h, membranes were hybridized with primary antibodies overnight at 4°C. Membranes were then incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h. Immunolabeled proteins were visualized by chemiluminescence using the LAS-3000 analyzer (Fujifilm) or Molecular Imager ChemiDoc TM XRS+ (Bio-Rad). Image processing was performed using Multi Gauge software (Fujifilm) or Image Lab TM software (Bio-Rad). For protein loading evaluation, a primary antibody against HSC70 was used.

Intracellular Calcium Concentration Measurements

Cells used for intracellular calcium concentration ([Ca 2+ ]i) measurements were grown on glass coverslips. The glass coverslips were sterilized in ethanol of increasing concentrations from 70 to 99%, and then coated with serum proteins using pure FBS prior to cell culture. Variations in [Ca 2+ ]i were analyzed in PAH-exposed HMEC-1 cells by real time fluorescence imaging, using Ca 2+ -sensitive probe Fura-2-AM, as previously reported [START_REF] Mayati | Induction of Intracellular Calcium Concentration by Environmental Benzo( a )pyrene Involves a β2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol 1,4,5-Trisphosphate Pathway in Endothelial Cells[END_REF][START_REF] Brinchmann | Lipophilic Chemicals from Diesel Exhaust Particles Trigger Calcium Response in Human Endothelial Cells via Aryl Hydrocarbon Receptor Non-Genomic Signalling[END_REF]

Apoptosis

Cells used for apoptosis analysis were cultivated in 6-well plates. Hoechst 33342/Sytox green staining was carried out to observe apoptotic cells by fluorescence microscopy. After PAH exposure, cells were stained with 50 µg/mL Hoechst 33342 and 93.5 nM Sytox green in the dark at 37°C for 30 min. More than 300 cells in randomly selected microscopic fields were analyzed and were scored under ZEISS Axio Scope A1 microscope. Cells with condensed or fragmented chromatin were counted as apoptotic.

RNA Isolation and Analysis

Total RNA was isolated from cells using the TRIzol® reagent (Invitrogen, Cergy Pontoise, France) and then reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). Real-time quantitative PCR (RT-PCR) was performed using SYBR Green PCR Master Mix on the CFX384 Touch TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).

The mRNA expressions were normalized by means of 18s mRNA levels. The 2 -∆∆ct method was used to express the relative expression of each selected gene. Primers were as follows: CYP1A1-forwards:

5'-CCCACAGCACAACAAGAGACA-3'; CYP1A1-reverse: 5'-CATCAGGGGTGAGAAACCGT-3'; CYP1B1forwards: 5'-AGCCAGGACACCCTTTCC-3'; CYP1B1-reverse: 5'-GAGTTGGACCAGGTTGTG-3'; 18Sforwards: 5'-CGCCGCTAGAGGTGAAATTC-3'; 18S-reverse: 5'-TTGGCAAATGCTTTCGCTC-3'.

Statistical analysis

All values were presented as means ± SEM from at least 3 independent experiments. Statistical analysis was performed using Student's t test and the one-way ANOVA with Dunnett's post hoc test (GraphPad Prism5, GraphPad Software, Inc, San Diego, California, USA). Significance was accepted at p < 0.05.

Results

B[a]P stimulates the production of EVs by endothelial HMEC-1 cells.

Endothelial cells constitute a well-known target for PAHs [START_REF] Mayati | Induction of Intracellular Calcium Concentration by Environmental Benzo( a )pyrene Involves a β2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol 1,4,5-Trisphosphate Pathway in Endothelial Cells[END_REF][START_REF] Brinchmann | Lipophilic components of diesel exhaust particles induce proinflammatory responses in human endothelial cells through AhR dependent pathway(s)[END_REF] , and are part of the cells mainly producing EVs under physiological conditions [START_REF] Iba | Role of extracellular vesicles in the development of sepsis-induced coagulopathy[END_REF] . We therefore decided to explore effects of B[a]P on EV production by the endothelial HMEC-1 cells using different concentrations and times of treatment. NTA revealed that B[a]P significantly stimulated EV production by HMEC-1 cells from 100 nM (Fig. 1A) and after 12h of treatment (Fig. 1B). After 24h of treatment with 100 nM B[a]P, observation by NTA of the size distribution profile of EVs (Fig. 1C) showed that they exhibited diameters ranging from 50 nm to 600 nm, and that B[a]P stimulated the production of the entire population of EVs (regardless of EV size).

B[a]P stimulates the production of both S-EVs and L-EVs by endothelial HMEC-1 cells

It is known that cells can release an heterogeneous population of vesicles with variable diameters and different biogenesis processes, including small EVs (i.e. exosomes) formed inside endosomal compartments and larger EVs (i.e. microvesicles) budding from the plasma membrane. To get more insight into the characterization of the EV overproduction in treated HMEC-1 cells, we next evaluated the B[a]P effect by looking at both small (diameter <200 nm) and large (diameter >200 nm) EVs, following differential ultracentrifugation and filtration through 0.22 µm filters. Thus, our experimental procedure allowed us to isolate two populations of EVs: the small EV population (S-EVs) corresponding to EVs with average diameter of 160.1 ± 12.6 nm, and the large EV population (L-EVs) corresponding to EVs with average diameter of 295.8 ± 13.2 nm. Whereas B[a]P exposure (100 nM, 24h) did not change EV diameters (Fig. S1), it nonetheless increased the production of both S-EVs and L-EVs (Fig. 2A and 2B respectively), to the same extent (by about 2 fold) as for total EVs (Fig. 1B).

S-EV and L-EV populations isolated from HMEC-1 cells correspond respectively to exosomes and microvesicles

In order to test whether S-EVs and L-EVs isolated under our experimental conditions correspond respectively to exosomes or microvesicles, we next examined their morphology and composition.

As expected, TEM images revealed structures with expected cup-shaped appearance and diameters (Fig. 3A). By western blotting (Fig. 3B), using total cell lysates as positive controls, we failed to detect any lamin A/C in isolated S-EVs and L-EVs, thus ruling out a possible contamination of our EV samples by nucleus material. In contrast, proteins traditionally observed in both exosomes and microvesicles such as caveolin-1, flotillin-1, β-actin, Tsg101 and HSC70 (used as loading control), were detected in both S-EVs and L-EVs. Finally, a protein described to be more specific to exosome, that is CD63, was only detected in S-EVs (Fig. 3B) and cell lysates. It is known that microvesicles expose phosphatidylserine on their outer leaflet of the membrane [START_REF] Head | Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function[END_REF][START_REF] Record | Extracellular vesicles: lipids as key components of their biogenesis and functions[END_REF] ; therefore we next used the fluorescently labeled annexin V to analyze its presence in L-EVs (Fig. 3C). By flow cytometry, we found that 69 ± 3.6 % of isolated L-EVs were annexin-V positive.

Altogether, these observations indicated that our technical procedure allowed the isolation of S-EVs and L-EVs corresponding respectively to exosome-and microvesicle-enriched populations produced by HMEC-1 cells.

B[a]P would modify the protein cargoes of EVs produced by HMEC-1

In parallel to the characterization of S-EVs et L-EVs produced by HMEC-1 cells, we also investigated whether the EV composition and characteristics studied were affected by B[a]P exposure. At first, we found that no significant modification was observed in presence of B[a]P when looking at EV morphologies (Fig. 3A) or annexin V labeling (Fig. 3C). To analyze the protein cargoes of EVs, 5 µg of total proteins were subjected to SDS-PAGE western blotting to compare protein composition of S-EV or L-EVs produced by cells in presence or not of B[a]P (Fig. 3B). Cell lysates were used as positive control. Among the studied proteins, the levels of caveolin-1, flotillin-1 or β-actin appeared to be increased in EVs produced upon B[a]P exposure, especially in S-EVs. The densitometric analysis thus revealed that caveolin-1 and flotillin-1 inductions were significant in the presence of this PAH (Fig. S2).

The increased production of S-EVs, but not L-EVs, upon B[a]P exposure would be AhRdependent and calcium-independent

PAHs are known to trigger numerous interconnected cellular phenomena, associated or not to AhR activation, among them the induction of xenobiotic-metabolism enzymes, intracellular calcium signaling, membrane remodeling or apoptotic signaling [START_REF] Larigot | AhR signaling pathways and regulatory functions[END_REF][START_REF] Holme | Combustion Particle-Induced Changes in Calcium Homeostasis: A Contributing Factor to Vascular Disease?[END_REF][START_REF] Tekpli | Role for membrane remodeling in cell death: Implication for health and disease[END_REF] . Interestingly, several of them are also implicated in the production of EVs by cells. Thus, upon cell activation or apoptosis, the increase of [Ca 2+ ]i underlies the initiation of cell signaling pathways, remodeling of cytoskeleton and the redistribution of membrane phospholipids associated to the intracellular processing for exosome and/or microvesicles release [START_REF] Yáñez-Mó | Biological properties of extracellular vesicles and their physiological functions[END_REF] . To get further insight into the possible mechanisms involved in the EV production upon B[a]P exposure, we decided to focus our attention on AhR activation, calcium signal and apoptosis. A and DBA were both capable of increasing the EV production by HMEC-1 cells in contrast to B[e]P and PYR which were ineffective (Fig. 4B). Note that, whatever the PAH tested, analysis of the size distribution profile of EVs (Fig. 4C-F) indicated that isolated EVs had diameters ranging from 50 to 600 nm and that, when PAHs exhibited an effect on EV production, this concerned the entire population of EVs (whatever the size of EVs) (Fig. 4E,4F). To determine if a higher concentration of PAHs with low affinity for AhR was able to stimulate EV production by HMEC-1 cells, we decided to expose cells to 1 µM of PYR for 24h. Even at this high concentration, PYR failed to modify the EV production by HMEC-1 cells (Fig. S4A). This result thus confirms our data with 100 nM, and is concordant with recent publication from our group [START_REF] Van Meteren | Polycyclic aromatic hydrocarbons can trigger a hepatocyte release of extracellular vesicles by various mechanisms of action depending on their affinity for aryl hydrocarbon receptor[END_REF] . Indeed, we have recently demonstrated that PYR was able to increase S-EV production by hepatocytes independently of AhR, and through a Constitutive Androstane Receptor (CAR)-dependent mechanism. Interestingly, unlike hepatocytes, mRNAs of CAR were not detected in HMEC-1 cells (Fig. S4B).

Only AhR ligands can increase EV production by

An AhR antagonist (alpha-naphthoflavone) inhibits the production of S-EVs but not that of L-EVs induced by B[a]P.

It is noteworthy that the PAH capacity to increase the mRNA levels of CYP1A1 and (FICZ, 100 nM, 24h), a potent high affinity ligand of AhR, further supported this finding. Indeed, FICZ was able to increase the production of S-EVs but not that of L-EVs (Fig. S5). Therefore, AhR activation would be involved in the B[a]P-induced production of S-EVs but not of L-EVs, thus suggesting differential mechanisms.

PAH capacities to induce intracellular calcium concentration do not overlap their capacities to induce EV production.

Knowing that calcium can be involved in EV production [START_REF] Martínez | Extracellular Vesicles in Metabolic Syndrome[END_REF][START_REF] Yáñez-Mó | Biological properties of extracellular vesicles and their physiological functions[END_REF] and that B[a]P can increase [Ca 2+ ]i in HMEC-1 cells [START_REF] Mayati | Induction of Intracellular Calcium Concentration by Environmental Benzo( a )pyrene Involves a β2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol 1,4,5-Trisphosphate Pathway in Endothelial Cells[END_REF] , we next decided to monitor the early [Ca 2+ ]i changes in response to our test PAHs. To do so, we used a real-time-fluorescence imaging method and the calcium-sensitive probe Fura2-AM. As previously described [START_REF] Mayati | Induction of Intracellular Calcium Concentration by Environmental Benzo( a )pyrene Involves a β2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol 1,4,5-Trisphosphate Pathway in Endothelial Cells[END_REF] , B[a]P (100 nM) was able to increase [Ca 2+ ]i in HMEC-1 cells (Fig. 6A). The maximum of increase occurred after about 20 min of exposure to B[a]P (Fig. S6). We then analyzed the effects of 100 nM of all other tested PAHs, on [Ca 2+ ]i in order

to determine the maximum level of [Ca 2+ ]i increase upon exposure to each molecule. Except for DBA, despite differences in terms of kinetics (Fig. S6), all PAHs were able to increase [Ca 2+ ]i in HMEC-1 cells, to the same extent as B[a]P (Fig. 6A). It is noteworthy that there was no correlation between PAH capacities to elevate [Ca 2+ ]i and those to increase EV production (Fig. 4B). Thus, B[e]P and PYR were found to induce [Ca 2+ ]i but failed to modify EV production by HMEC-1 cells; DBA stimulates EV production but did not modify [Ca 2+ ]i ; and B[a]P or B[a]A induced both [Ca 2+ ]i and EV production.

Therefore, the early calcium signal triggered by PAHs would likely not be involved in the increased EV production, whatever the type of EVs considered.

PAH capacities to induce apoptosis do not overlap their capacities to induce EV production.

As induction of CYP1A1 et 1B1 following AhR activation by PAHs could lead to apoptotic signaling [START_REF] Hardonnière | Environmental carcinogenesis and pH homeostasis: Not only a matter of dysregulated metabolism[END_REF] , we then decided to evaluate apoptosis following PAH exposures (100 nM, 24 h). This was performed using fluorescence microscopy and Hoechst/Sytox green staining (Fig. 6B). Consistent with literature, 100 nM B[a]P slightly (around 5%) but significantly increased the percentage of apoptotic cells. In contrast, the other tested PAHs, i.e. B[e]P, PYR, B[a]A and DBA, did not affect the apoptosis level.

As for [Ca 2+ ]i induction, we therefore noted that there was no correlation between PAH capacities to induce apoptosis and PAH capacities to increase EV production by HMEC-1 cells.

In vivo exposure to a mixture of PAHs increases urinary EV amount

EVs are widely found in all body fluids including blood, saliva or urine [START_REF] Yáñez-Mó | Biological properties of extracellular vesicles and their physiological functions[END_REF] . Urine appears as noninvasive source for information reflecting the physio-pathological state of urinary system but also of other physiological systems. Based upon the fact that endothelial cells are part of kidney intrinsic cells producing urinary EVs (uEVs) and that humans are widely exposed to PAH mixture, we finally decided to test the in vivo effect of a mixture of the 16 PAHs listed as "priority" compounds by US-EPA, on the amount of EVs in rat urine samples. Thus, we evaluated uEVs amounts in urines collected during 24 h from rats exposed through diet for 90 days to 0.8 mg/kg of PAH mixture in comparison to control rats receiving only vehicle (vegetal oil). After isolation, uEV amounts were analyzed by NTA. As shown in Fig. 7A, rat exposure to PAH mixture led to an increase of uEVs number (2.39.10 9 /mL vs 1.30.10 9 /mL) without any significant modification of renal functions, as determined by measuring urinary creatinine concentration and urinary volume (Fig. 7B andC).

Discussion

The growing interest for EVs originates (1)-from works demonstrating that EVs carry a plethora of bioactive molecules (i.e. nucleic acid, lipids, carbohydrates, proteins...) that play roles in cell-to-cell communication, and (2)-from works revealing the clinical interest of EVs as non-invasive diagnostic biomarkers in some human pathologies including cancer [START_REF] Nawaz | Mining Extracellular Vesicles for Clinically Relevant Noninvasive Diagnostic Biomarkers in Cancer[END_REF] . In this context, our study was performed in order to evaluate the capacity of PAHs to increase EV production both in vitro and in vivo with the aim of determining if these entities could represent potential biomarkers of exposure and/or effects of PAHs. Interestingly, only the increase of S-EV production was significantly counteracted by α-NF (AhR antagonist and inhibitor of CYP1), thus indicating that only the S-EV overproduction by B[a]P-treated HMEC-1 cells would be AhR dependent. These results therefore strengthen the fact that S-EV and L-EV biogenesis processes would differ, as previously proposed by other groups [START_REF] Abels | Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake[END_REF] It remains to determine the precise role for AhR in the S-EV production by HMEC-1 cells, notably in presence of B[a]P. Many hypotheses could be considered. Thus, a possible involvement of the sphingolipid pathway would be particularly interesting to evaluate since, in epithelial cells, several AhR ligands, including PAHs, are able to promote the generation of sphingosine-1-phosphate, a bioactive intermediate of sphingolipid metabolism [START_REF] Reiners | Aryl Hydrocarbon Receptor Regulation of Ceramide-induced Apoptosis in Murine Hepatoma 1c1c7 Cells: a function independent of aryl hydrocarbon receptor nuclear translocator[END_REF][START_REF] Wang | Aryl hydrocarbon receptor signaling promotes ORMDL3-dependent generation of sphingosine-1-phosphate by inhibiting sphingosine-1-phosphate lyase[END_REF] . Indeed, sphingolipids and the enzymes related to their biosynthesis have been associated to EV biogenesis and release, especially by shaping membrane curvature [START_REF] Verderio | Role of sphingolipids in the biogenesis and biological activity of extracellular vesicles[END_REF] . Another hypothesis might involve a change in the physicochemical characteristics of lipid raft microdomains. Indeed, these entities, that are detected in EV membranes [START_REF] Pollet | Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?[END_REF] , represent assemblies of proteins and lipids within the plasma membrane that are notably enriched in cholesterol and sphingolipids [START_REF] Head | Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function[END_REF] ; most interestingly, they are elements of the regulatory mechanisms associated to the vesicle formation and structure [START_REF] Pollet | Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?[END_REF] . As we have previously demonstrated that B[a]P alters the composition of the plasma membrane lipid microdomains notably through AhR activation [START_REF] Tekpli | Membrane remodeling, an early event in benzo[α]pyrene-induced apoptosis[END_REF] , it could be interesting to test a role for such a membrane remodeling in the production Nevertheless, considering averaged diameters (160 nm versus 300 nm for S-EVs and L-EVs respectively) and the corresponding averaged membrane surface area (0.08 µm 2 and 0.28 µm 2 for S-EVs and L-EVs respectively), it seems that L-EVs would not be a marginal population considering all EVs produced by HMEC-1. Since PAHs are environmental contaminants with numerous toxic effects on human cells, it could be interesting to determine if the observed L-EV overproduction is an attempt of exposed cells to remove B[a]P. Indeed, the microvesicle trafficking has been previously proposed as a biological process that helps cells to extrude potentially toxic xenobiotics [START_REF] Jorfi | The role of microvesicles in cancer progression and drug resistance[END_REF] , thus affording them a means of protection against an intracellular stress. This phenomenon is notably exploited by cancers cells as a mechanism of resistance. Indeed, microvesicles released from cancer cells, after drug treatment (i.e. doxorubicin), contain high levels of the chemotherapeutic agent [START_REF] Shedden | Expulsion of Small Molecules in Vesicles Shed by Cancer Cells: Association with Gene Expression and Chemosensitivity Profiles1,2[END_REF] . Regarding the mechanisms involved in L-EV biogenesis upon B[a]P exposure, they yet remain to be determined. However, it seems that neither early calcium signal triggered by PAHs, neither AhR would be involved in their production.

Regarding the effects, notably carcinogenic, of PAHs on human health, the monitoring of the populations likely exposed to these pollutants is currently performed through the measurement of urinary PAH metabolites (i.e. 1-hydroxy pyrene, 3-hydroxy B[a]P), or by evaluation of DNA-adducts in blood cells [START_REF] Srogi | Monitoring of environmental exposure to polycyclic aromatic hydrocarbons: a review[END_REF] . The use of these biomarkers is difficult since their presence does not necessarily reflect the level of PAH mixture humans are exposed to [START_REF] Srogi | Monitoring of environmental exposure to polycyclic aromatic hydrocarbons: a review[END_REF] . Moreover, these biomarkers are related to the capacity of cells to metabolize PAHs but not to the cellular reaction towards these pollutants.

Considering these aspects, it therefore seems that studying EVs in body fluids as urine would be a good alternative. Indeed, EV production can be correlated to cellular response, to disease, to drug exposure [START_REF] Martínez | Extracellular Vesicles in Metabolic Syndrome[END_REF] . Our present results indicate that B[a]P, B[a]A and DBA are each able to up-regulate production of EVs from endothelial cells. This induction effect was also observed in vivo upon exposure to a mixture of PAHs. Thus, our data show that rat exposure (90 days) to the mixture of 16 US-EPA PAHs, was capable of increasing the EV disposal from urine without influencing renal activity. These results therefore suggest that EVs in urine may have particular utility for monitoring exposure to toxicants and to detect the related early cellular response prior to end-organ damages.

Conclusion

Taken together, our data indicates that, i) PAH exposure increases in vitro the EV production by

  HMEC-1 cells. In order to test the possible involvement of AhR, we first decided to evaluate the potential of several PAHs with different AhR affinities, to increase EV production in HMEC-1 cells. To this aim, benzo[e]pyrene (B[e]P), pyrene (PYR), benz[a]anthracene (B[a]A) and dibenzo[a,h]anthracene (DBA) were chosen because they are part of the 16 U.S EPA priority pollutant PAH compounds; they are present in diet and possess different levels of affinity for AhR, therefore resulting in different potencies in terms of genomic AhR activation as compared to B[a]P (Fig. S3). The amount and size distribution of EVs produced by HMEC-1 cells were thus studied after 24 h of treatment with 100 nM of each PAH. Before focusing on EVs, we first evaluated the potential for AhR activation of our PAHs by studying the induction of known AhR-target genes, i.e. CYP1A1 and 1B1 (Fig. 4A). As expected, only B[a]A, B[a]P, and DBA (AhR ligands) induced CYP1A1 and 1B1 mRNA levels. The other PAHs tested (B[e]P and PYR), known to exhibit a very low affinity for AhR, failed to modify the expression of CYP1A1 and 1B1 in our cell model. Interestingly, like B[a]P, B[a]

  1B1 dovetail with their capacity to increase the EV production by HMEC-1 cells. These observations led us to hypothesize an implication of AhR in the increased EV production upon B[a]P exposure. To test this hypothesis, we next investigated the effects of α-NF (antagonist of AhR and inhibitor of CYP1) on the B[a]P-triggered induction of S-EVs or L-EVs. As shown in Fig.5, α-NF (10 µM) counteracted the up-regulation of S-EVs observed in presence of B[a]P (Fig.5A), while it failed to reduce the L-EV production (Fig.5B). Interestingly, treatment by 6-formylindolo[3,2-b]carbazole

  The present study highlights for the first time the capacity of PAHs to increase in vitro the EV production by endothelial HMEC-1 cells. Interestingly, only PAH ligands for AhR (i.e. B[a]P, B[a]A, DBA) led to this EV overproduction. Indeed, B[e]P and PYR, that are known weak AhR ligands, failed to modify such a production. Besides AhR activation, several PAHs can induce an intracellular calcium signaling[START_REF] Mayati | Induction of Intracellular Calcium Concentration by Environmental Benzo( a )pyrene Involves a β2-Adrenergic Receptor/Adenylyl Cyclase/Epac-1/Inositol 1,4,5-Trisphosphate Pathway in Endothelial Cells[END_REF] and trigger apoptosis[START_REF] Tekpli | Membrane remodeling, an early event in benzo[α]pyrene-induced apoptosis[END_REF] , two cellular phenomena previously related to EV biogenesis[START_REF] Pollet | Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?[END_REF][START_REF] Turturici | Extracellular membrane vesicles as a mechanism of cell-to-cell communication: advantages and disadvantages[END_REF] . Regarding influence of B[e]P, PYR, B[a]P, B[a]A, or DBA on intracellular calcium concentration, cell apoptosis and CYP1A1/1B1 mRNA levels, our results do support an AhR implication. Indeed, in contrast to the capacities to trigger an early [Ca 2+ ]i increase (as observed in presence of B[e]P, PYR, B[a]P, B[a]A) and apoptosis (as observed only in presence of B[a]P), the PAH capacity to induce mRNA levels of CY1A1 and 1B1 overlaps that to increase the EV production. These metabolism enzymes are well recognized as being under AhR control[START_REF] Larigot | AhR signaling pathways and regulatory functions[END_REF] and key actors in B[a]P induced apoptosis[START_REF] Tekpli | Membrane remodeling, an early event in benzo[α]pyrene-induced apoptosis[END_REF] , and, like EVs, are induced in our cell model only by strong AhR ligands.

  of S-EVs by Hmec-1 cells. Of note, the present results indicate that B[a]P exposure could modify EV protein cargoes, notably leading to an enrichment of caveolin-1 and flotillin-1 contents in S-EVs.Interestingly, caveolin-1 and flotillin-1 are well-known markers of lipid rafts. Although studies of greater extent (i.e. proteomics) are needed to conclude, besides stimulating EV production, it seems that PAHs would moreover be able to modify the messages carried by the vesicles by modifying the composition of EVs.Stimulating effects of PAHs, and more particularly B[a]P, towards EV production, concern the two main populations, namely S-EVs and L-EVs, as demonstrated by our EV characterization. As described in the literature, L-EV population (presently observed whatever the treatment applied), is relatively heterogonous as compared to the S-EV population. Looking only at their amount, L-EVs represent less than 20% of total EVs produced by HMEC-1 cells, whereas S-EVs represent more than 50%. Note that B[a]P treatment did not significantly affect this distribution (data not shown). Thus in terms of amount, S-EVs would represent the main type of EVs produced by HMEC-1 cells.

  endothelial cells and in vivo the release of EVs in urine, and that the stimulating effects of PAHs concern both S-EVs (or exosomes) and L-EVs (or microvesicles); ii) PAH exposure and more particularly, exposure to B[a]P, might influence the composition of the S-EVs produced by endothelial cells; and finally, iii) AhR would be involved in the PAH effects on the release of S-EVs, but not L-EVs. The inability of AhR weak ligands, like PYR or B[e]P, to increase the EV production in HMEC-1 cells, along with the inhibitory effects of α-NF, an AhR antagonist, towards the B[a]P effect on the S-EV production strongly support this assumption. In total, our results therefore point to EVs as possibly useful biomarkers of exposure for PAHs.

Figure 1 .

 1 Figure 1. B[a]P stimulates the extracellular vesicle release from endothelial HMEC-1 cells. Total

Figure 2 .

 2 Figure 2. B[a]P stimulates the production of both small vesicles (S-EVs) and large (L-EVs) vesicles

Figure 3 .

 3 Figure 3. Characterization of S-EV and L-EV populations isolated from endothelial HMEC-1 cells.

Figure 4 .F)

 4 Figure 4. Effects of PAHs on the EVs released from endothelial HMEC-1 cells. (A-F) HMEC-1 cells

Figure 5 :

 5 Figure 5: Involvement of AhR in the production of S-EVs, but not of L-EVs, produced upon B[a]P

Figure 6 .

 6 Figure 6. PAH effects on intracellular Ca 2+ concentrations ([Ca 2+ ]i) and on the apoptosis in HMEC-

Figure 7 .

 7 Figure 7. In vivo PAH exposure increases EV amount in urine. Rats received 0.8 mg/kg of a 16 PAH

  [e]pyrene (B[e]P), pyrene (PYR), benz[a]anthracene (B[a]A), benzo[a]pyrene (B[a]P),

	dibenz[a,h]anthracene	(DBA),	chrysene,	benzo[b]fluoranthene,	benzo[k]fluoranthene,
	benzo[g,h,i]perylene,	indeno[1,2,3-c,d]pyrene,	naphthalene,	fluorene,	acenaphthene,
	acenaphtylene, anthracene, phenanthrene, fluoranthene, alpha-naphthoflavone (α-NF), dimethyl
	sulfoxide (DMSO) and monoclonal antibody anti-β-actin were obtained from Sigma-Aldrich (St.
	Louis, MO, USA). Antibodies against CD63, lamin A/C and Hsc70 were from Santa Cruz Biotechnology
	(Santa Cruz, CA, USA), whereas Tsg101 was provided by Abcam and caveolin-1 and flotillin-1 by BD
	biosciences. All other chemicals used in this study were purchased from commercial sources at the
	highest purity available.

Acknowledgements

We first wish to thank Dr Carmen Martinez (UMR Inserm 1063, Angers, France) for valuable discussion on EVs at the beginning of this project. We are also very grateful to the MRiC platform (UMS BIOSIT, Rennes, France), notably Stéphanie Dutertre for training us on calcium measurements. This study was financially supported by the "Programme Environnement-Santé-Travail" of Anses with the funding from ITMO cancer in the context of the Cancer Plan 2014-2019 (EST-2016/1/31), and by the Cancéropôle Grand-Ouest/Région Bretagne (Concerto project). Manon Le Goff was a recipient of a PhD fellowship from the Collectivités Locales Mayennaises.