Dorra Trabelsi

Manel Zouari

Mohamed Kharrat

Mahe Dammak

Marielle Eyraud

Florence Vacandio

Structural, Micromechanical and Tribological Analyses of Electrodeposited Nickel-Graphite Coatings with Different Fractions of Graphite Microparticles

Keywords: Electrodeposition, Nickel, Micrographite, Composite, Friction, Wear

published or not. The documents may come

Introduction

In recent years, huge progresses have been made in the formulation of solid lubricant composite coatings for a large range as far as industrial applications are concerned. These composite coatings are intended to provide the highest level of performance in terms of self-lubricity and wear reduction as well as corrosion-resistance. Self-lubricating coatings have been the interest of many researchers [1]. They are used to control friction and wear, where conventional lubricants cannot afford the desired level of performance and durability under severe application conditions [2]. Many deposition procedures of composite coatings are used constantly such as plasma, physical vapor deposition (PVD), chemical vapor deposition (CVD), ion implantation and electrodeposition [3,4]. Among these procedures, electrodeposition is one of the most suitable one which can display a compromise between simplicity and economic concerns. It is considered as the major deposition technique that is industrially used for the co-deposition of both metal and particles into composite coatings. In fact, the problem of stable suspension of particles in the electrodeposition solution is generally overcome by the addition of suitable surfactant [5]. The latter also plays a fundamental role in the reduction of hydrogen embrittlement in the coatings, minimizing particles agglomeration and enhancing particles co-deposition [6]. Cetyltrymethyl ammonium bromide (CTAB) and sodium dedocyl sulfate (SDS) are frequently employed as surfactants [7,8]. Plated nickel (Ni) has been widely used as a coating matrix owing to its high brightness, hardness and corrosion resistance ability. Moreover, as illustrated by recent studies, the inclusion of fine lubricating particles (such as graphite, polytetrafluoroethylene (PTFE), molybdenum disulfide (MoS 2 ), etc. [9][10][11]) or hard particles (such as titanium carbide (TiC), alumina (Al 2 O 3 ), Zinc Oxide (ZnO), etc. [12,13]) into nickel matrix can enhance its mechanical and tribological properties [14]. For example, Goral et al. [15] have pointed out that the addition of aluminum oxide (Al 2 O 3 ) with a concentration of 80 g/L of Al 2 O 3 in the electrolytic bath exhibit a three times lower wear rate than the pure Ni coatings. Furthermore, Lekka et al. [16] have demonstrated that the presence of microand nano sized silicon carbides (SiC) particles in the Ni matrix leads to a decrease in the friction coefficient to 63 and 88% at 25°and 300 °C respectively. Several others researches have been focused on the study of Ni-graphite composite coatings, thanks to the economic and powerful characteristics of graphite particles [17,18]. Thus, many of them were interested in the study of the weight fraction of graphite influence in the properties of Ni-graphite composite coatings. Wu et al. [19] have characterized the graphite-Ni composite coatings obtained by automatic brush plating. They have found that the graphite content largely affects the roughness, the friction coefficient and the wear rate of Ni-graphite coatings. Whereas, He et al. [20] have focused on the study of the influence of graphite particles concentration on the surfaces morphology of Ni-graphite coatings obtained by electrodeposition from sulfamate bath. Further, Zhao et al. [9] have illustrated the influence of graphite concentration on the mechanical and tribological behaviors of Ni-graphite coatings elaborated by electrodeposition from sulfamate bath. They have found that the wear mechanism for these composite coatings is a delaminating wear, characterized by subsurface crack nucleation. Indeed, when the crack attains a critical length and propagates to the surface, delamination starts and the wear becomes catastrophic [21]. One of the possible reasons for this wear mechanism is the internal stress. Indeed, the addition of particles may change the state of internal stress due to the mismatch of lattice parameters, the distortions or the cracks between the particles [22]. The present study investigates the effect of graphite content on the structural, micromechanical and tribological properties of nickelgraphite coating obtained by electrodeposition from watts bath with specific parameters, extracted from previous study [23] and minimize the internal stress of coatings. These parameters have been used in order to enhance mechanical and tribological behaviors of Ni-graphite coatings.

Materials and Methods

Composite Coatings Preparation

The electrolyte used in the plating of Ni-graphite composite coatings was watts bath. The electrochemical bath components consisted of nickel sulfate (supplied by Sigma Aldrich) as Ni source, boric acid as buffer solution and nickel chloride (supplied by Sigma Aldrich) to prevent the anode from passivity. The bath composition and operating conditions are listed in Table 1. The weighted graphite particles in the range of 7-11 lm (supplied by Alfa Aesar) were used as a solid lubricant. Meanwhile, in order to enhance the electrostatic adsorption of suspended particles on the cathode surface, the surfactant such as sodium dodecyl sulfate (SDS) was added to the solution. All bath ingredients were agitated for 2 h by magnetic stirring. Subsequently, the sonication process had served for 30 min to provide homogeneous dispersion and to prevent the agglomeration of the particles. The operating conditions in terms of pH and current density were chosen from a previous research [23] in which the aim was to minimize the internal stresses. Actually, electroplating parameters such as current density, pH, bath temperature, and additive concentration strongly affect the internal stress [START_REF] Stein | A Practical Guide to Understanding, Measuring and Controlling Stress in Electroformed Metals[END_REF]. They directly affect the hydrogen codeposition, the overpotential of deposition and the lattice misfit, which are among the suggested theories responsible for internal stresses in the coatings [START_REF] Such | Nickel and Chromium Plating[END_REF]. Generally, increasing the current density leads to the increase in the internal stresses [START_REF] Lowenheim | Modern Electroplating[END_REF]. However, the effect of pH on the internal stresses depends on the bath composition. For example, it has been recommended that pH should be less than 5 for watts bath [START_REF] Lowenheim | Modern Electroplating[END_REF] while, internal stresses attain the minimum at pH = 4 and increase below 4 in sulfamate bath [START_REF] Barrett | Nickel Plating from the Sulfamate bath[END_REF]. The temperature of the bath also affects the internal stresses: the rise in the temperature of the electroplating bath leads to the decrease of internal stresses in most cases [START_REF] Dini | The Materials Science of Coatings and Substrates[END_REF].

Carbon sheet was used as anode and cathode was made using mild steel E24 (0.17% C, 1.4% Mn, 0.045% S, 0.045% P and 98.34% Fe) with the dimensions of 30 9 20 9 15 mm 3 as a substrate. The active surface area was 450 mm 2 and the remaining surfaces were hidden with adhesive tape. Before electrodeposition, the active surface area of substrate was mechanically polished with abrasive paper, degreased by NaOH solution with pH = 11 and etched by means of 10% H 2 SO 4 for 1 min. After each preparation step, the substrate was rinsed with distilled water so as to completely remove the residuals of each stage.

Characterization Techniques

Atomic absorption technique was used to evaluate the graphite phase content into the Ni composite coating for each of the considered concentration of graphite particles in the electrochemical bath (0 g/l, 1 g/l, 5 g/l, 10 g/l, and 20 g/l). The mass of the deposits was determined by a balance of precision equal to 0.01 mg at the first step. Subsequently the coated substrate was put in a concentrated nitric acid (HNO 3 ). This solvent dissolved the nickel without attacking the steel according to the norm NF A 91,101 [START_REF] Laroche | contro ˆle des reve ˆtements e ´lectrolytiques et chimiques[END_REF]. After complete dissolution of Ni, (i.e when the residual mass was equal to the mass of the substrate before electrodeposition), the steel substrate was picked up and the obtained solution (HNO 3 ? Nickel) was heated up to 100 °C until HNO 3 got fully evaporated. Thereafter, distilled water was added to the solution that was heated to 100 °C in order to rinse the sample from residual acid. After its full evaporation, 50 ml of distilled water was added. Then, the obtained solution was filtered to remove the graphite particles. Finally, it was examined by atomic absorption to know the weight percentage of nickel in this solution. Therefore, the weight percentage of graphite was also deduced. SEM and XRD were used to investigate the coating's surface morphologies as well as the wear traces. Cross sections of substrates were prepared and analyzed by optical microscope to determine the thickness of the coatings. The roughness of various coatings and wear volume were investigated by tactile profilometer. The surface roughness parameter Ra was used for analysis and was measured over a tracing length of 12 mm. Micro hardness of various coatings were measured by Vickers indentation at a load of 1000 g.

The friction and wear test were carried out using a linear reciprocating tribometer designed in our laboratory (Fig. 1). A ball on plane contact configuration was utilized. High chromium steel (100Cr6) was the material of the counter body, having a diameter of 15 mm and a surface roughness Ra of 0.02. Preceding each friction test, the coating surface as well as the ball surface were rinsed with ethanol. The apparatus allowed contact between the steel ball and the coating surface under a constant normal load. Tangential cyclic motion was then applied to the coated specimen. A load cell located between the coated specimen and its holder allowed to measure the tangential force. The output of this load cell was continually stored using a data acquisition system. Normal load, tangential motion amplitude and frequency were adjusted to 6 N, 7.5 mm and 1 Hz, respectively. The wear tests were performed at room temperature (25 °C) and with a relative humidity of 55 ± 5%. Following each friction test, wear track on the coating surface was observed by SEM and worn surface of the spherical counter body was observed by optical microscope (OM). The wear volume was measured on the basis of wear track profile developed by a tactile profilometer over a tracing length of 4 mm in a direction perpendicular to the sliding direction (Fig. 2).

Graphite Weight Fractions

The weight percentage of graphite particles in the Ni composite coating as a function of their concentration in the electrolytic bath is shown in Fig. 3. The incorporation of graphite particles into the Ni matrix can be explained by the codeposition mechanism of inert particles from electrolyte bath. According to Wasekar et al. [START_REF] Wasekar | [END_REF] and in analogy to Guglielmi's simplified model for composite electrodeposition [31], during first stage of addition to the metal ions, there is the adsorption of Ni on the surface of graphite particles. In the second step, migration of graphite particles with ionic cloud to the cathode surface through diffusion layer takes place. Here these particles get adsorbed thereby loosing hydrated shell by electron transfer reaction. Finally, the Ni atoms get incorporated into crystal lattice surrounding the graphite particles. Thus, a graphite particle gets encapsulated in Ni matrix. A higher concentration of graphite particles in plating bath enhances the adsorption rate leading to a higher volume percentage of the codeposited graphite particles [14]. However, the capturing capacity of the growing metal does not increase simultaneously with the increase of graphite concentration [32] and when the amount of graphite particles exceed the maximum amount of saturation state, an elastic collision of particles inside the electrolyte take place, leading to particles agglomeration and thereafter a shielding effect for particles codeposition can take place [33]. Therefore in this study, the amount of particles is almost constant after exceeding a boundary concentration of particles in the electrolytic bath (10 g/l). This result is similar to that found by Wang et al. [22] for Al 2 O 3 -Cu(Sn) composite and Ghorbani et al. [34] for graphite-brass composite coatings. The XRD diagram of pure nickel and Ni-graphite composite layers which contain 23 wt% of graphite particles are presented in Fig. 5. As can be seen, the pure Ni pattern displays typical peaks of Nickel having a face centered cubic structure (FCC) and exhibits a strong (200) reflection peak that reveals the preferred orientation. In fact, the growth of pure nickel coatings is carried out by nucleation on the substrate surface followed by columnar growth of grains (perpendicular to the substrate surface) in (200) preferred crystallographic direction. These grains end up at the coating surface, leading to pyramidal structure [35]. The addition of graphite microparticles results in the change of the predominant reflection peak to (111) which, in turn, is the most intense peak in the XRD pattern from standard randomly oriented nickel [36]. This denotes that Ni-graphite composite coatings have more random grain structure, and subsequently, a rougher surface texture. A similar observation has been revealed by different authors in their studies of Ni composite coating. Srivastava et al. [37] inferred that the change of preferred orientation with the incorporation of nano SiC particles is attributed to the change of the surface morphology of Ni grains from dense spherical smooth surface to rough nodular surface.

Morphologies

Cross sections have been prepared on the considered coatings and observed by OM (Fig. 6). The thickness seems to be constant (about 50 lm) for the different coatings (Table 2). A slight decrease can be observed for coatings containing 42% graphite (40 lm). It seems that, at high concentration of particles, the graphite particles play the role of physical barriers to grain growth and decelerate the growth rate; therefore, thinner composite coating can be seen. This result has also been found by Borkar et al. [38] while studying the effect of Al 2 O 3 content on the microstructure of nickel composite coatings.

Surface Roughness

The evolution of surface roughness Ra with graphite content in the coating is presented in Fig. 7. It can be seen that the particle content in the coating strongly influences the surface roughness. The values of Ra are in the range of 0.8 lm for pure Ni coating to 2.4 lm for composite coating containing 42 wt% graphite. This increase of surface roughness with the increase of wt% of particles seems to be caused by the random oriented texture (as explained in the previous paragraph) and more collision of particles when their amount increases in the bath. Ghorbani et al. also found similar results for the surface roughness of electrodeposited bronze-graphite [39].

Micromechanical and Tribological Behaviors

of Ni-Graphite Coatings

Microhardness

Figure 8 illustrates the effect of weight fraction of graphite in the composite coating on the microhardness of the considered coatings. The microhardness presents a strong drop between 0 and 10 wt% of graphite. Subsequently, the surface microhardness of Ni composite coatings displays a quasi-linear decrease as the graphite particle's amount in the Ni matrix increases. This decrease is attributed to the sliding and soft nature of graphite particles. Hence, they are easily deformed under a contact stress: the increase of graphite fraction in the composite coatings leads to the decrease in the capability of anti-plastic deformation that brings in lower microhardness of the composite coatings [40].

Friction Coefficient

This section highlights the evolution of friction coefficient of Ni composite coatings, filled with different weight fractions of graphite particles, during 2000 sliding cycles as illustrated in Fig. 9. For all coatings, the friction coefficient increases with the number of sliding cycles during the running-in stage and stabilizes afterwards to a steady state value. Figure 10 presents the effect of the graphite weight fraction in the Ni coatings, on the stabilized value of the coefficient of friction (COF) after 2000 sliding cycles. COF for Ni-graphite composite coatings are much reduced compared to the one for pure nickel coating. Hence, the COF value reaches 0.15 for the coating containing the highest amount of graphite used in the present work (42 wt%). Indeed, an easy slippage takes place between the layers of graphite due to its hexagonal structure. Accordingly, an adherence of graphite particles to the worn surface as well as formation of solid self-lubricant layer is shown on the wear track. Thereupon, the thickness of this layer increases with the increase of graphite content and the metal-metal contact is changed into the graphite filmmetal contact [40]. By an EDX technique, Tang et al. [18] has proved the formation of tribofilm composed of mainly graphite, on the worn surface of Ni-graphite coating deposited by supersonic atmospheric plasma spraying system during the reciprocating sliding friction process. Furthermore, the rate of COF decrease is obviously lowered beyond 38 wt% graphite. For this graphite content, the performances of the tribofilm are quite saturated, and no more additional graphite particles are required to reduce frictional interactions between the first contacting bodies, which accords well with the results found by Zhao et al. [9] while, Nickchi et al. [41] found that friction coefficient decreases with the increase in the micro graphite content in the bronze layer, whereas, friction coefficient increases with the increase in nano graphite content in the bronze layer.

Wear Behavior

Figure 11 reveals the wear volume loss of Ni composite coatings versus different graphite fractions. It can be noted that the wear volume decreases upto 38 wt% of graphite particles and then it increases slightly. The initial reduction of wear volume loss as the graphite wt% increases can be explained by the ability of graphite particles to form a layer of soft wear debris (third body) which covers the wear track on the coated sample. It also forms a thin transfer film mainly composed of graphite that strongly adheres to the spherical counterface, resulting in a lower friction between the contacting solids. When the graphite wt% exceeds 38%, the particles smash the continuity and uniformity of the matrix and then result in weakness and fragility of composite coatings. Thus, the wear volume loss increases immensely for coatings with graphite amount equal to 42 wt%. Hence, 38 wt% of graphite corresponds to the optimal graphite concentration that provides the best condition to promote the solid lubricant layer stability and to maintain the Ni matrix integrity.

Figure 12 illustrates the SEM micrographs of worn surfaces of Ni-graphite composite coatings after 2000 sliding cycles. As noted for the pure Ni coatings (Fig. 12a), the wear track is deep with some plastic grooves, clear adhesive spalling pits and, indeed, some areas of massive peelings. The addition of 10 wt% of graphite is still insufficient to improve the wear resistance of Ni composite coatings (Fig. 12b) and the same wear mechanisms as for the pure Ni coatings are found. Thus, the amount of lubricating particles is not enough to form a continuous third body layer [40]. Therefore, the addition of 10 wt% of graphite particles does not have a significant effect on the wear process. The incorporation of 23 wt% of graphite solid lubricant particles in Ni coating (Fig. 12c) leads to a smoother wear scar with fewer and smaller adhesive craters. This can be allocated to the formation of a third-body layer on the whole surface contact during the sliding process. Nonetheless, some grooves are clearly visible, indicating that more severe plastic deformation has taken place. Figure 12d illustrates the micrographs of worn surface of Ni coatings containing 38 wt% of graphite particles. As shown, the wear scar is smooth and straight with little scratches. It seems that with 38 wt% fraction of graphite added to the Ni coating, a self-lubricating Nigraphite is obtained, and the wear resistance of coating is greatly enhanced. With the high graphite fraction (42 wt%), a wide wear track with some plastic grooves and spalling pits reappears (Fig. 12e). This result can be accounted for by the huge decrease of microhardness of the corresponding coating. Eventually, the loose particles are easily carried out. These snatch particles act as abrasive particles in the contact zones, which, in turn, are considered to harm the transfer film and bring severe abrasive wear [42]. It can be clearly seen that the use of specific operating conditions during the coating process (a current density of 4.8 A/dm 2 , a pH of 2 and a coating thickness of 50 lm), like those suggested by Gu ¨ler et al. [23], largely affects the wear behavior of the Ni-graphite coating. In fact, these specific conditions, which are found to minimize the level of internal stresses, have discarded the catastrophic delaminating wear mechanism found by Zhao et al. [9] for electrodeposited Ni-graphite composite coatings.

Optical micrographs of steel ball worn surfaces, after 2000 sliding cycles, are shown in Fig. 13. Abrasive striations are obvious on the worn surface of the counterface rubbing against pure Nickel (Fig. 13a). Figure 13b exhibits the formation of a discontinuous transfer film on the worn surface of the steel ball in case of coating containing 10 wt% of graphite. Besides, abrasive striations are still visible. Hence, the addition of graphite particles in coatings leads to the development of a transfer film on the counterface causing a decrease of COF and wear volume. The increase in %wt of graphite particles in coatings leads to an expansion of the transfer film to cover most of the worn surface of the counterface for Ni-23 wt% of graphite coating (Fig. 13c) and to totally cover the worn surface of the counterface for Ni-38 wt% of graphite coating (Fig. 13d). This result confirms that the addition of graphite with optimal value results in making a self-lubricating coating. After the addition of 42 wt% of graphite to the coating (Fig. 13e), a discontinuous transfer film reappears on the counterface and a denuded zone is observed in the center of the transfer film, confirming that at this concentration, the detachment of particles easily take place and they act as abrasive particles in the contact zone.

Conclusion

In this study, Ni-graphite composite coatings were electrodeposited from typical watts bath with specific operating conditions which was found to minimize the level of internal stresses. Different weight fractions of graphite microparticles were considered. The microstructures and morphologies of the obtained coatings were explored using atomic absorption technique, SEM, OM, tactile profilometry and XRD. Concerning the micromechanical and tribological behaviors of Ni-graphite coatings, they were analyzed with microhardness tester and linear reciprocation ball on plane tribometer. The following conclusions could be drawn:

• The weight percentage of particles in coatings increases as the concentration of graphite particles in the electrolytic bath increases. • The coating surface roughness increases with the weight fraction of graphite. • The coating's microhardness depends on the graphite particle amount. Indeed, the rise of this amount exhibits a notable decrease of microhardness, which is attributed to the sliding ability and soft nature of graphite particles. • The stabilized COF value decreases as the graphite content in the Ni matrix increases. This behavior is ascribed to the development of solid self-lubricant layer on the wear track as well as transfer film on the counterface allowing an easy slippage between the layers of graphite due to its hexagonal structure. • The wear volume loss of Ni-graphite coatings decreases with the increase of the graphite fraction upto 38 wt% at which a continuous transfer film is formed on the steel counterface. Exceeding this optimal fraction, a discontinuous transfer film reappears and the volume loss re-increases. • The optimal graphite concentration of 38 wt% provides the best condition to promote the solid lubricant layer stability and to maintain the Ni matrix integrity.

Fig. 1

 1 Fig. 1 Reciprocating friction and wear apparatus

3. 1 . 2 Microstructures

 12 SEM micrographs exhibiting surface morphologies of pure Ni and Ni-graphite composite coatings filled with different weight fractions of graphite solid lubricant (10, 23, 38, 42 wt%) are shown in Fig. 4. The micrograph of pure Ni coating reveals a regular pyramidal structure with uniform grain size and smooth surface compared to the surface of Ni matrix filled with graphite particles. Indeed, it seems that the graphite particles with irregular shapes marked in the SEM micrographs as a light phase, are homogeneously dispersed into the Ni matrix.

Fig. 3

 3 Fig. 3 Weight fraction of graphite particles in coatings versus graphite concentration in electrolytic bath

Fig. 4 Fig. 5

 45 Fig. 4 SEM micrographs of Ni-graphite composite coatings with different weight fraction of graphite a 0%, b 10%, c 23%, d 38%, e 42%

Fig. 6 Table 2 Fig. 7 Fig. 8

 6278 Fig. 6 Cross section of Ni-graphite composite coatings filled with different weight fraction of graphite: a 0%, b 10%, c 23%, d 38%, e 42%

Fig. 9

 9 Fig. 9 Friction coefficient trends of Ni-graphite composite coatings filled with different weight fraction of graphite

Fig. 10

 10 Fig. 10 Evolution of stabilized value of friction coefficient versus different weight fraction of graphite in Ni coatings

Fig. 12

 12 Fig. 12 SEM micrographs of wear track after 2000 sliding cycles of Ni composite coatings filled with different weight fraction of graphite: a pure, b 10%, c 23%, d 38%, e 42%

Fig. 13

 13 Fig. 13 Optical micrographs of the counterbody worn surface after 2000 sliding cycles against Ni composite coatings filled with different weight fraction of graphite: a pure, b 10%, c 23%, d 38%, e 42%

Table 1

 1 Bath composition and operating conditions

	Bath composition (g/l)

Acknowledgements The authors gratefully acknowledge the help and support of Professor Mongi Feki from the Laboratory of Materials Engineering and Environment (National Engineering School of Sfax; Sfax; Tunisia).


Bertrand Pourrut

Séverine Jean

Jérôme Silvestre

Eric Pinelli
email: pinelli@ensat.fr

Lead-induced DNA damage in Vicia faba root cells: Potential involvement of oxidative stress

Keywords:

Genotoxic effects of lead (0-20 M) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10 M. In addition, at this concentration, DNA damage time-dependently increased until 12 h. Then, a decrease in DNA damages was recorded. The significant induction of micronucleus formation also reinforced the genotoxic character of this metal. Direct interaction of lead with DNA was also evaluated with the a-cellular comet assay. The data showed that DNA breakages were not associated with a direct effect of lead on DNA. In order to investigate the relationship between lead genotoxicity and oxidative stress, V. faba were exposed to lead in the presence or absence of the antioxidant Vitamin E, or the NADPH-oxidase inhibitor dephenylene iodonium (DPI). The total inhibition of the genotoxic effects of lead (DNA breakage and micronucleus formation) by these compounds reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead. These results highlight, for the first time in vivo and in whole-plant roots, the relationship between ROS, DNA strand-breaks and chromosome aberrations induced by lead.

Introduction

Lead (Pb) is one of the most useful metals, and it is also one of the most toxic [START_REF] Shotyk | Biogeochemistry and cycling of lead[END_REF]. The widespread use of lead has caused global contamination of air, water and soil. Extensive research has found that lead can cause neurological, haematological, gastrointestinal, reproductive, circulatory, and immunological pathologies [START_REF] Patrick | Lead toxicity part II: the role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity[END_REF]. Moreover, the International Agency for Research on Cancer has listed inorganic lead compounds as possibly carcinogenic (Group-2B) chemicals [START_REF] Iarc | Monographs on the Evaluation of the Carcinogenic Risk to Human-Genetic and Related Effects: An Updating of Selected[END_REF][START_REF] Pulido | Metal-induced apoptosis: mechanisms[END_REF]. In plants, lead has been described to influence various morphological, physiological and biochemical processes. Pb is known to strongly inhibit plant growth, root elongation, seed germination, seedling development, transpiration, chlorophyll production, lamellar organisation in chloroplasts and cell division [START_REF] Wierzbicka | How lead loses its toxicity to plant[END_REF][START_REF] Patra | Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance[END_REF]. However, only limited data are available on the effects of Pb on plant genetic material. It has been observed that Pb promoted single strand-breaks in lupine roots [START_REF] Rucinska | Genotoxicity of lead in lupin root cells as evaluated by the comet assay[END_REF]. In a recent study, Gichner et al. [START_REF] Gichner | Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants[END_REF] demonstrated that large concentrations of lead induced DNA strand-breaks. In Vicia faba roots, lead shortened the mitotic stage and prolonged the interphase, thus prolonging the cell cycle [START_REF] Patra | Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance[END_REF]. At low concentrations (below 10 M) lead increased the mitotic index, but decreased it at higher concentrations. Pb also dose-dependently increased the micronucleus frequency [START_REF] Patra | Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance[END_REF][START_REF] Shahid | Lead-induced genotoxicity to Vicia faba L. roots in relation with metal cell uptake and initial speciation[END_REF].

Lead genotoxicity mechanisms are complex and not yet well understood. In vitro studies demonstrated that lead interacts with protein and nucleic acids, particularly at sulfhydryl groups and the phosphate backbone [START_REF] Gerbert | Toxicity, mutagenicity and teratogenicity of lead[END_REF][START_REF] Tajmir-Riahi | The effects of Cu and Pb on the solution structure of calf thymus DNA: DNA condensation and denaturation studied by Fourier transform IR difference spectroscopy[END_REF]. Lead decreases the fidelity of DNA polymerases and disturbs DNA and RNA synthases. Pb may also act as co-carcinogen by synergistically potentiating the genotoxicity of UV light and alkylating carcinogens [START_REF] Hartwig | Indirect mechanism of leadinduced genotoxicity in cultured mammalian cells[END_REF]. In addition, ascorbic acid can reduce sister-chromatid exchange caused by lead, implying that free radicals may play a role in genotoxicity of this metal [START_REF] Dhir | Relative efficiency of Phyllantus emblica fruit extract and ascorbic acid in modifying lead and aluminium-induced sister chromatid exchanges in mouse bone marrow[END_REF]. Using a plasmid-relaxation assay, Yang et al. [START_REF] Yang | Singlet oxygen is the major participating in the induction of DNA strand Breakage and 8-hydroxydeoxyguanosine adduct by lead acetate[END_REF] demonstrated that high concentrations of lead promote DNA strand-breakage and the formation of 8-hydroxydeoxyguanosine (8-OHdG) adducts in DNA.

In previous work, we demonstrated that an early step in the lead-induced oxidative burst in V. faba roots is strongly associated with activation of NADPH-oxidase and production of O 2

•- [START_REF] Pourrut | Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots[END_REF]. It is assumed that lead induces DNA damage in mammalian cells through excessive production of ROS and aldehydes, such as malondialdehyde (MDA) [START_REF] Xu | Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice[END_REF]. However, in plants, DNA damageinduction mediated by lead remains unclear. The aim of this work was to investigate the role of ROS in lead-induced genotoxicity. For this purpose, DNA damage was studied in lead-treated plants, in the presence or absence of Vitamin E, a powerful scavenger of ROS and a soluble lipid by-product of oxidative stress [START_REF] Wang | The location and function of vitamin E in membranes (review)[END_REF] and DPI, an NADPH oxidase inhibitor [START_REF] Pourrut | Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots[END_REF]. Pb-induced DNA alterations in V. faba root cells were evaluated by use of (i) the comet assay, which allowed the detection of DNA strand-breaks and (ii) the V. faba micronucleus test (MN), which revealed chromosome aberrations. Direct interactions between lead and DNA were also evaluated with the a-cellular comet assay.

Materials and methods

Materials

The V. faba L. cultivar "aguadulce" (Tezier, France) was used for this study. Dry seeds were soaked for 24 h in de-ionized water. The seed coats were removed and placed on moistened filter paper in a germination chamber under optimal conditions of germination, i.e. in the dark at 22 ± 1 • C and 100% humidity. After 3 days, when the primary roots were about 2-3 cm in length, the seedlings were transferred to a PVC tank (3 plants per tank) containing continuously aerated Hoagland nutrient solution. Four days were necessary to obtain secondary roots of suitable length (1-2 cm) for the different tests. For experiments, roots were exposed to different concentrations of Pb(II) (Pb(NO3)2; Sigma-Aldrich chemical), in the presence or absence of Vitamin E or DPI (Sigma-Aldrich chemical). All plants were grown and exposed under controlled conditions during a 16-h photoperiod at 70% relative humidity and day/night temperatures of 24/22 ± 1 • C. Light was supplied by 600-W Osram Nav-T Super High Pressure Sodium Lamps providing a minimum photosynthetic photon-flux density of 500 mol m -2 s -1 at the top of the plant [START_REF] Pourrut | Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots[END_REF].

Cellular comet assay

Single-cell gel electrophoresis (SCGE) was performed according to Gichner et al. [START_REF] Gichner | Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves[END_REF] with some modifications. For dose-response experiments, V. faba roots were treated at 20 • C in the dark, with different Pb(NO3)2 concentrations (1, 5, 10, and 20 M) for 8 h. For kinetic experiments, roots were exposed to 10 M Pb(NO3)2 for 1, 4, 8, 12, 16 and 20 h. To evaluate the implication of ROS in lead-induced genotoxicity, some plants were co-incubated with Vitamin E (1 and 10 M) or DPI (0.1 and 1 M), an inhibitor of the oxidative burst induced by lead in V. faba root tips [START_REF] Pourrut | Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots[END_REF]. In order to compare results obtained with those from the normalized micronucleus test, plants were incubated with lead (10 M) for 6 h. Some plants were also pretreated with Vitamin E (1 or 10 M) or DPI (0.1 or 1 M) for 1 h before exposure to lead, while others were only treated during 7 h with Vitamin E or DPI at the same concentrations as previously described. In each experiment, negative controls were placed in Hoagland's solution without lead for the same exposure times, while positive controls were incubated with 35 M H2O2 for 1 h. It is noted that maleic hydrazine (MH) cannot be used as a positive control in the comet assay [START_REF] Gichner | Differential genotoxicity of ethyl methanesulphonate, N-ethyl-Nnitrosourea and maleic hydrazide in tobacco seedling based on data of the Comet assay and two recombination assays[END_REF]. After treatment of the seedlings, excised roots were rinsed in water and placed in a 60mm petri dish kept on ice and spread with 1.5 mL of cold PBS buffer (NaCl 130 mM, Na2HPO4 7 mM, NaH2PO4 3 mM, EDTA 50 mM, pH 7). The roots were gently sliced with a fresh razor blade. The plate was kept tilted in the ice so that the isolated root nuclei would collect in the buffer. The suspension with released nuclei was cleaned of debris by filtering through a 20-m nylon cloth. All operations were conducted under inactinic red light to avoid light-induced damage.

Then comet-assay slides were prepared according to Gichner et al. [START_REF] Gichner | Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves[END_REF]. For each slide, 50 randomly chosen nuclei were analyzed with an Olympus fluorescence microscope with an excitation filter of 510-560 nm and a barrier filter of 590 nm. A computerized image-analysis system (Komet version 5.1, Andor Technology) was employed. The percentage DNA in tail was used as the primary measure of DNA damage according to Hartmann et al. [START_REF] Hartmann | Recommendations for conducting the in vivo alkaline Comet assay[END_REF]. Three slides were evaluated per treatment and each treatment was repeated at least three times. From the repeated experiments, the averaged median value for the percent DNA in tail was calculated for each treatment group from the DNA per cent value of each slide [START_REF] Lovell | Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro comet studies[END_REF].

A-cellular comet assay

SCGE slides with nuclei from untreated root cells were prepared as outlined above and the slides were immersed in solutions of 1 M NaCl, 10 mM Trizma containing 1, 5, 10 or 20 M Pb(NO3)2 for 1 h at 4 • C. Negative control slides were immersed in the same solution without lead, while positive controls were incubated in the same solution with 8 M H2O2. After the treatment period, the slides were rinsed three times for 5 min by immersion in cold 400 mM Tris buffer, then electrophoresed and analyzed as described earlier.

Micronucleus test

The V. faba MN test was carried out according to Ma et al. [START_REF] Ma | The improved Allium/Vicia root tip assay for clastogenicity of environmental pollutants[END_REF]. Exposure time was 30 h for the negative control, and 6 h for Pb(NO3)2-treated groups, followed by a 24-h recovery period. The test concentrations for Pb(NO3)2 were 1, 5, 10 and 20 M. To evaluate the potential role of oxidative burst in lead-induced MN formation, V. faba roots were pre-treated with 10 M Vitamin E or 1 M DPI, 1 h before treatment with lead. The direct effects of these two substances were evaluated by exposure of V. faba roots to Vitamin E or DPI during 7 h, followed by a 24-h recovery period. For each experiment, five seeds were used per treatment. Maleic hydrazine (MH; 40 M) was used as a positive control. Aerated Hoagland's solution was used as a negative control. After treatment, root tips were fixed in Carnoy's solution (glacial acetic acid/ethanol 1:3) at 4 • C for one night, and transferred to 70% ethanol for storage. Root tips were then hydrolyzed in 1 N HCl at 60 • C for 5-7 min. Five slides were prepared for each of the five seeds. After staining the root tips with 1% acetoorcein, the interphase cells were scored for micronucleus frequency under a 1000× magnification. Five thousand cells per tip were counted. In order to avoid underestimation of micronuclei due to impaired cell-proliferation rate [START_REF] Ma | The improved Allium/Vicia root tip assay for clastogenicity of environmental pollutants[END_REF], the MN test was performed only on root tips with a mitotic index superior to 2%.

Analysis of lead content

Lead content was analyzed as previously described [START_REF] Pourrut | Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots[END_REF]. V. faba plants roots were exposed to 10 M of lead nitrate in Hoagland solution during different time periods (0, 4, 8, 12, 16 and 20 h). Roots were collected and rapidly washed in distilled water. Lead bound to the rhizoderm was removed as follows: fragments were shaken with 40 mL of 0.001 M HCl during 3 min, and 360 mL of 1 M HCl were added to yield a final concentration of 0.01 M HCl. After shaking for another 5 min, roots were washed in distilled water, oven-dried at 80 • C for 48 h, and then weighed. Roots were mineralized in a 1:1 mixture of 65% HNO3 and 30% H2O2 at 80 • C during 6 h. After filtration, lead concentrations were determined with an IRIS Intrepid II XDL ICP-OES. The accuracy of the analytical procedure was verified with a reference material: Virginia tobacco leaves (CTA-VTL-2, polish certified reference material; ICHTJ).

Statistical analysis

For each experiment, statistical analysis was performed with a one-way ANOVA and the multiple comparison method of Tukey [START_REF] Keppel | Design and Analysis: A Researcher's Hand Book[END_REF].

Results

DNA-damage analysis by use of the cellular comet assay

Fig. 1 illustrates the DNA-damaging effect of lead applied for 8 h on V. faba roots. The positive control (PC) was obtained by incubation of roots with 35 M of H 2 O 2 . Under these conditions, H 2 O 2 induced 76 ± 2.5% DNA in tail. Cellular comet-assay data clearly demonstrate a significant increase in DNA fragmentation correlated with increasing concentrations of lead (1-20 M). The percentage DNA in tail increased from 23 ± 1.2% (negative control) to 64 ± 2.1% for 20 M lead treatment (P < 0.01). Under these experimental conditions, the maximum effect was observed after exposure to 10 M of lead.

Evaluation of chromosome aberrations

Chromosome aberrations analyzed with the micronucleus (MN) test are shown in Fig. 2. It is worth noting that 20 M of lead induced a blackening of the root tips and a loss of mitosis. Under these conditions, micronuclei were not quantified. Results in Fig. 2A illustrate that 1, 5 and 10 M Pb(NO 3 ) 2 had no significant effect on cell mitosis. Data shown in Fig. 2B demonstrate that lead dosedependently induced MN formation in V. faba root tips. The MN induction became significant with 5 M of lead (7.2 ± 0.15‰). The maximum effect was observed with 10 M (8.2 ± 0.3‰). This MN value was slightly higher than the positive control obtained with MH (6.45 ± 0.21‰).

Kinetics of lead uptake and lead-induced DNA damage in V. faba roots

To investigate the relationship between DNA fragmentation and Pb concentration in the roots, the kinetics of lead accumulation (10 M) were analyzed (Fig. 3). The concentration of 10 M lead was chosen because it induced the maximum genotoxic effect on V. faba roots without blackening of root tips. Data reveal that the entry of lead into the roots was very fast between 0 and 4 h (11.25 g g -1 dry weight h -1 ), and reached 46.5 ± 6.5 g g -1 dry weight. Then, lead uptake decreased to 2.53 g g -1 dry weight h -1 and became constant. Under these experimental conditions the maximum lead content was reached at 20 h (90.2 ± 6.9 g g -1 dry weight).

As exposure time is fixed in the normalized MN test, it cannot be used for the kinetic exposure experiment. So, the comet assay was used to evaluate the kinetics of DNA-damage induction by 10 M lead nitrate (Fig. 4). Results show that, after 4 h of exposure, DNA strand-breaks increased of about 14% in comparison with the control. After 4 h of incubation with lead, a strong and significant increase in DNA strand-breaks was recorded, with a maximum effect at 12 h (72 ± 1.7% DNA in tail). After this time, DNA strand damage decreased until 20 h (58.3 ± 3.5%).

Analysis of DNA damage with the a-cellular comet assay

In order to evaluate the potential direct effects of lead on V. faba root-cell DNA, the a-cellular comet assay was performed for 1 h. Treatment longer than 2 h cannot be applied in the a-cellular comet assay as even control nuclei become severely damaged, resulting in a strong increase in the percentage DNA in the tail [START_REF] Gichner | Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves[END_REF]. Results obtained with the a-cellular comet assay reveal that 8 M H 2 O 2 induced 83 ± 3.1% DNA in tail after 1 h of incubation (data not shown). Under the same experimental conditions, whatever the concentration, lead did not directly induce DNA damage (28 ± 1.7% for the control vs. 30.2 ± 1.5% for 20 M lead).

Effects of Vitamin E and DPI on lead-induced DNA fragmentation and micronucleus formation

To identify the potential role of ROS in lead-induced genotoxic effects, V. faba roots were pre-incubated with vitamin E or DPI during 1 h before exposure to lead. The results (Fig. 5) indicate that lead-induced DNA fragmentation (59.3 ± 1.8% in tail) was dosedependently inhibited by vitamin E: with 1 M, a reduction of 90%in DNA fragmentation was observed, while 10 M Vitamin E totally abolished it. Similarly, at the two test concentrations used, DPI totally inhibited lead-induced DNA breakdown. Under these conditions, the percentages of DNA in tail were similar to those in the negative control. Treatments of roots with Vitamin E or DPI without exposure to lead did not influence root-cell DNA fragmentation.

The effects of vitamin E and DPI were also evaluated in the MN test (Fig. 6). Results show that 10 M vitamin E alone significantly decreased the MN frequency (0.5 ± 0.1‰) in comparison with the control (1.95 ± 0.2‰), without modification of the cell mitosis. Vitamin E completely inhibited the effect of lead (10 M) on MN induction (0.9 ± 0.2‰). Under the same conditions, DPI (1 M) alone had no effect on the basal level of MN, while it significantly inhibited the effects of lead on MN induction (1 ± 0.1‰). These data clearly illustrate the potential role of oxidative stress in lead-induced MN formation.

Discussion

Even though lead toxicity in plants is well known [START_REF] Pourrut | Lead uptake, toxicity and detoxification in plants[END_REF], only few studies have examined its genotoxic effects. Relevant in vivo studies on DNA damage by lead in plant systems are lacking. Gichner et al. [START_REF] Gichner | Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants[END_REF] showed that lead could induce somatic mutations and DNA strand-breaks in tobacco plants. In a recent study we observed that the genotoxicity of lead was associated with its chemical speciation [START_REF] Shahid | Lead-induced genotoxicity to Vicia faba L. roots in relation with metal cell uptake and initial speciation[END_REF]. In the present work, data of the cellular comet assay illustrate the DNA-damaging effect of lead (Fig. 1). These results indicate that the DNA damage observed in V. faba roots are correlated with the external lead concentration, as previously described with high lead concentrations [START_REF] Rucinska | Genotoxicity of lead in lupin root cells as evaluated by the comet assay[END_REF][START_REF] Gichner | Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants[END_REF]. The maximum effect was observed with 10 M of lead. The alkaline comet assay reliably detects singlestrand (SS) and double-strand (DS) DNA breaks, even at very low damage levels. SS and DS DNA breaks result from numerous reaction types: base-and nucleotide-excision repair, direct scission of the DNA backbone by chemical or radical attack, scission following the binding of an intercalating agent, the action of endonuclease or topoisomerase, the presence of alkali-labile DNA adducts or alkalilabile sites [START_REF] Horvathova | The nature and origin of single-strand breaks determined with the comet assay[END_REF]. Chromosome aberrations analyzed by the MN test indicate that lead dose-dependently induced MN formation in V. faba root tips (Fig. 2B). The maximum effect was obtained at 10 M (8.2 ± 0.3‰) as previously observed using the cellular comet assay. These data also illustrate the genotoxic potential of lead [START_REF] Steinkellner | Genotoxic effects of heavy metals: comparative investigation with plant bioassays[END_REF]. The V. faba MN test is a very sensitive and useful method that allows detection of both clastogenic and aneugenic effects [START_REF] Sabti | Fish micronuclei for assessing genotoxicity in water[END_REF][START_REF] Hajjouji | Assessment of the genotoxicity of olive mill waste water (OMWW) with the Vicia faba micronucleus test[END_REF]. Micronuclei are the result of chromosome breaks (or mitotic anomalies) that require a passage through mitosis to be recognisable. The molecular mechanism of MN induction is not yet clearly understood. Under our experimental conditions, the same kind of dose-dependent response observed in the two tests (comet assay and MN test) suggests a close link between lead-induced DNA damage and MN formation.

To determine the potential relationship between lead concentration in roots and genotoxicity, lead (10 M) uptake into the roots was analyzed as a function of time (Fig. 3). Our data illustrate the fast uptake and accumulation of lead into V. faba roots during the first hours of treatment. The kinetics of lead accumulation in V. faba roots are in agreement with those found by Nedelkoska and Doran [START_REF] Nedelkoska | Characteristics of heavy metal uptake by plant species with potential for phytoremediation and phytomining[END_REF] in several plants treated with Cd, Ni or Cu. Lead accumulation in plant roots has been demonstrated by many authors [START_REF] Patra | Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance[END_REF]. It has been reported that lead effects are directly related to exposure dose and duration [START_REF] Rucinska | Genotoxicity of lead in lupin root cells as evaluated by the comet assay[END_REF][START_REF] Steinkellner | Genotoxic effects of heavy metals: comparative investigation with plant bioassays[END_REF]. However, while lead was still taken up after 12 h of culture, a decrease in DNA damage was observed (Figs. 3 and4). If these results seem directly correlated to induction of DNA-repair systems, this could also be associated with a rapid and efficient system of lead detoxification in plants [START_REF] Wierzbicka | Lead in the apoplast of Allium cepa L. root tips: ultrastructural studies[END_REF]. This system includes chelation by organic or amino acids [START_REF] Wierzbicka | Comparison of the toxicity and distribution of cadmium and lead in plant cells[END_REF] or by phytochelatins [START_REF] Piechalak | Accumulation and detoxification of lead ions in legumes[END_REF] and sequestration in vacuole [START_REF] Piechalak | Enhancing phytoremediative ability of Pisum sativum by EDTA application[END_REF] or plasma-tubule [START_REF] Wierzbicka | Lead in the apoplast of Allium cepa L. root tips: ultrastructural studies[END_REF]. Although it has been observed that Pb detoxification was quickly activated [START_REF] Wierzbicka | Lead in the apoplast of Allium cepa L. root tips: ultrastructural studies[END_REF][START_REF] Piechalak | Accumulation and detoxification of lead ions in legumes[END_REF], our results indicate that these detoxification and repair systems became efficient only after 12 h of exposure to lead.

In order to determine pathways involved in lead genotoxicity, the direct effect of lead on DNA was assessed with the a-cellular comet assay. Results reveal that lead did not directly induce DNA damage after 1 h of incubation (data not shown). These results confirm those of Valverde et al. [START_REF] Valverde | Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA-metal interaction?[END_REF], who did not find DNA damage due to direct DNA-lead interaction in different mouse-cell types. These data are also in agreement with those of Hartwig [START_REF] Hartwig | Carcinogenicity of metal compounds: possible role of DNA repair inhibition[END_REF], who proposed that direct genotoxic effects of metals are rather weak and/or restricted to high concentrations (more than 1 mM). Our results suggest that lead genotoxicity was mediated by another indirect pathway. In view of the lead-induced ROS production in plant roots [START_REF] Pourrut | Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots[END_REF][START_REF] Przymusi Ński | Organospecific responses of lupin seedlings to lead localization of hydrogen peroxide and peroxidase activity[END_REF] and according to Rucinska et al. [START_REF] Rucinska | Genotoxicity of lead in lupin root cells as evaluated by the comet assay[END_REF], the induction of an indirect mechanism, such as oxidative stress is suggested. It appears likely that many of the genotoxic effects of lead in mammalian cells are mediated by ROS and/or lipid-soluble by-products of oxidative stress such as MDA.

To identify the potential role of ROS in the induction of DNA alterations, V. faba plant roots were incubated with two concentrations of Vitamin E or DPI, during 1 h before exposure to lead. Fig. 5 results suggest the implication of ROS in lead-induced DNA strandbreaks. Vitamin E and the NADPH oxidase-inhibitor DPI completely abolished the increase in the percentage DNA in tail after lead treatment. At the same time, neither Vitamin E nor DPI influenced lead accumulation kinetics in V. faba plant roots (data not shown). Vitamin E is a fat-soluble vitamin that protects cells against ROS [START_REF] Sattler | Vitamin E is essential for seed longevity and preventing lipid peroxidation during germination[END_REF]. Recently, Collin et al. [START_REF] Collin | Vitamin E is essential for the tolerance of Arabidopsis thaliana to metal-induced oxidative stress[END_REF] demonstrated that Vitamin E is essential for the tolerance of Arabidopsis thaliana to metal-induced oxidative stress. In a previous work, we highlighted the fundamental role of NADPH oxidase in lead-induced ROS production in V. faba root tips [START_REF] Pourrut | Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots[END_REF]. In these plant roots, ROS production was totally inhibited by 0.1 or 1 M DPI, a well-known inhibitor of NADPH oxidase. In the present work, data obtained with DPI and Vitamin E illustrate a relationship between oxidative stress and lead-induced DNA strand-breakage. In in vitro system, it has been demonstrated that singlet oxygen is the major species participating in the induction of DNA strand-breakage and 8-hydroxydeoxyguanosine adduct induction by lead [START_REF] Yang | Singlet oxygen is the major participating in the induction of DNA strand Breakage and 8-hydroxydeoxyguanosine adduct by lead acetate[END_REF]. In addition, the • OH radical is considered to be the ultimate reactive oxygen species that interacts with DNA and promotes genetic damage [START_REF] Halliwell | Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts[END_REF]. This radical attacks DNA on the sugar residue and induces DNA fragmentation, base loss, and strand-breaks with a terminal sugar residue fragment [START_REF] Rhaese | Chemical analysis of DNA alterations: I. Base liberation and backbone breakage of DNA and oligodeoxyadenylic acid induced by hydrogen peroxide and hydroxylamine[END_REF]. It also produces thymine glycol, 7,8-dihydro-8-oxoguanine or 2,6diamino-4-hydroxy-5-formamido-pyrimidine.

Because lead can promote oxidative stress, DNA strand-breaks and chromosome aberrations, the MN test was also conducted in the presence of Vitamin E or DPI. Fig. 6 shows that these compounds completely inhibited the effect of lead on MN formation. These data strongly support the role of oxidative stress in lead-induced MN formation in V. faba root tips. According to Figs. 5 and6, genotoxic effects of lead were rapid to appear in V. faba root tips. Six hours of exposure were sufficient to increase both DNA fragmentation (48% DNA in tail) and micronucleus formation. These results underline the role of DNA fragmentation in MN formation induced by lead. Overall, these data highlight for the first time in a plant a possible relationship between lead, oxidative stress, DNA fragmentation and micronucleus formation. However, genotoxic processes are very complex, and epigenetic effects could also be involved in lead-induced MN formation [START_REF] Thier | Interaction of metal salts with cytoskeletal motor protein systems[END_REF]. Indeed, lead is able to interfere with the spindle apparatus of dividing cells and promotes genotoxic effects. In this work, we cannot totally exclude this hypothesis.

In conclusion, these data strongly suggest that, in whole plant roots, lead genotoxicity could be mediated by ROS production. The use of Vitamin E and DPI helps to establish a link between ROS production, DNA fragmentation and probably micronucleus formation. Although metallic salts are effective mitotic poisons due to their affinity for thiol groups, the total inhibition of lead-induced MN formation in the presence of Vitamin E or DPI strongly suggests the implication of ROS in MN induction. If our results underline the role of oxidative stress in DNA strand-breakage, we cannot rule out that lead genotoxicity and especially MN formation are almost exclusively mediated by ROS. In this work, it was also impossible to discern the precise nature of the radical that directly or indirectly interacts with DNA. Lipid-oxidation products quenched by Vitamin E could be also associated with DNA-adduct formation and breakages. Other research is still necessary to fully understand the complete genotoxic mechanism.

Fig. 1 .

 1 Fig. 1. (A) Effects of lead on V. faba root-cell DNA: undamaged (left) and damaged (right) nuclei (×400). (B) Dose-effect of lead on DNA strand-breakage assessed with the comet assay in whole V. faba roots. The negative control (NC) was realized by incubation of roots in Hoagland's solution without lead for the same exposure duration (8 h). The positive control (PC) was realized by incubation of roots with 35 M H2O2 in Hoagland's solution during 1 h. Each treatment was repeated five times. Each value represents the mean ± SE of three separate experiments. *Values significantly different from the control (P < 0.01).

Fig. 2 .Fig. 3 .

 23 Fig. 2. Mitotic index (A) and micronucleus frequency (B) values in V. faba roots exposed to different concentrations of lead (1, 5, and 10 M). NC: negative control, PC: positive control. Each value represents the mean ± SE of five seeds in three separate experiments. *Values significantly different from the control (P < 0.01).

Fig. 4 .

 4 Fig. 4. Time-dependent DNA strand-breakage induced by lead nitrate in whole plant roots. V. faba roots were incubated with ( ) or without (᭹) 10 M lead nitrate in Hoagland's solution. Each value represents the mean ± SE of five plants in three separate experiments. *Values significantly different from the control (P < 0.01).

Fig. 5 .

 5 Fig. 5. Effect of the ROS scavenger Vitamin E or the NADPH-oxidase inhibitor DPI on lead-induced DNA strand-breakage in V. faba whole plant roots. V. faba plant roots were incubated with Vitamin E (1 and 10 M) or DPI (0.1 and 1 M) in culture medium 1 h before addition of lead nitrate (10 M). NC: negative control, PC: positive control. Each value represents the mean ± SE of five plants in three separate experiments. *Values significantly different from lead treated roots (P < 0.01).

Fig. 6 .

 6 Fig. 6. Mitotic index (A) and micronucleus frequency (B) values in V. faba roots exposed to lead nitrate (10 M) in the presence or absence of Vitamin E (10 M) or DPI (1 M). NC: negative control, PC: positive control. Each value represents the mean ± SE of five seeds in three separate experiments. *Values significantly different from the control (P < 0.01).

Conflict of interest statement

The authors declare that there are no conflicts of interest.