Pb low doses induced genotoxicity in Lactuca sativa plants

Accepted Manuscript Pb low doses induced genotoxicity in Lactuca sativa plants S. Silva, P. Silva, H. Oliveira, I. Gaivão, M. Matos, O. Pinto-Carnide, C. Santos PII: S0981-9428(16)30493-4 DOI: 10.1016/j.plaphy.2016.12.026 Reference: PLAPHY 4766 To appear in: Plant Physiology and Biochemistry Received Date: 6 September 2016 Revised Date: 26 December 2016 Accepted Date: 26 December 2016 Please cite this article as: S. Silva, P. Silva, H. Oliveira, I. Gaivão, M. Matos, O. Pinto-Carnide, C. Santos, Pb low doses induced genotoxicity in Lactuca sativa plants, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2016.12.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Pb low doses induced genotoxicity in Lactuca sativa plants Silva S1*, Silva P2, Oliveira H3, Gaivão I4, Matos M2, 5, Pinto-Carnide O6, Santos C7 1 Department of Chemistry, QOPNA and CESAM, University of Aveiro, 3810-193 2 RI PT Aveiro, Portugal. Department of Genetics and Biotechnology (DGB), University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal. Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal. 4 Animal and Veterinary Research Centre (CECAV) and Department of Genetics and SC 3 5 Biosystems & Integrative Sciences Institute (BioISI), Faculty of Sciences, University of Lisboa, Lisboa, Portugal. 6 M AN U Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal. Centre for the Research and Technology of Agro-Environmental and Biological TE D Sciences (CITAB) & Department of Biology and Environment, University of Trás-osMontes e Alto Douro, Apartado 1013, 5001-801 Vila Real, Portugal. 7 Department of Biology & GreenUP-CitabUP, Faculty of Sciences, University of Porto, + AC C EP Rua Campo Alegre s/n, 4169-007 Porto, Portugal. Corresponding author E-mail: [email protected] Tel: + 351 234 370 766 fax: + 351 234 372 587 ACCEPTED MANUSCRIPT Abstract Soil and water contamination by lead (Pb) remains a topic of great concern, particularly regarding crop production. The admissible Pb values in irrigation water in several countries range from ≈0.1 to ≈5 mg L-1. In order to evaluate putative effects of Pb RI PT within legal doses on crops growth, we exposed Lactuca sativa seeds and seedlings to increasing doses of Pb(NO3)2 up to 20 mg L-1. The OECD parameter seed germination and seedling/plant growth were not affected by any of the Pb-concentrations used. SC However, for doses higher than 5 mg L-1 significant DNA damage was detected: Comet assay detected DNA fragmentation at ≥ 5 mg L-1 and presence of micronuclei (MN) M AN U were detected for 20 mg L-1. Also, cell cycle impairment was observed for doses as low as 0.05 mg L-1 and 0.5 mg L-1 (mostly G2 arrest). Our data show that for the low doses of Pb used, the OECD endpoints were not able to detect toxicity, while more sensitive endpoints (related with DNA damage and mitotic/interphase disorders) identified TE D genotoxic and cytostatic effects. Furthermore, the nature of the genotoxic effect was dependent on the concentration. Finally, we recommend that MN test and the comet AC C Keywords: EP assay should be included as sensitive endpoints in (eco)toxicological assays. Cell cycle; comet assay; metal toxicity; micronucleus; phytotoxicity. 1. Introduction The presence of heavy metals in the environment occurs naturally or as a consequence of human actions. Since the industrial revolution, the levels of heavy metals as contaminants in the environment increased, often reaching values that became toxic for living organisms (Nagajyoti et al. 2010; Agency for Toxic Substances and Disease ACCEPTED MANUSCRIPT Registry, 2007). Soil, water and atmospheric contamination by lead (Pb) is a worldwide problem and humans are commonly exposed to Pb above naturally-occurring levels (Agency for Toxic Substances and Disease Registry, 2007). Being a toxic metal, Pb is a environmentaly persistent and multi-target pollutant (Shahid et al. 2013; Kumar and RI PT Kumari, 2015) and is known to have no function either in animals or plants. Furthermore, based on the frequency of occurrence, toxicity and human exposure potential, Pb ranks second after arsenic (Agency for Toxic Substances and Disease SC Registry, 2015). Therefore, Pb remediation is a matter of discussion and concern worldwide. M AN U Most plant species are susceptible to Pb exposure with effects at morphological, physiological and/or biochemical levels (Pourrut et al. 2011b). There are, however, several plant species that are Pb hyper accumulators (accumulating ≥1000 ppm) and are recommended for soil/water remediation (Kumar and Kumari, 2015). Pb reaches plants TE D mostly by soil (where it accumulates mostly due to atmospheric depositions) and by water. According to the Agency for Toxic Substances and Disease Registry (2015), industrial Pb release to aquatic systems contributes to the observed high levels of metal EP detected in drinking and irrigation water. Also, Pb concentration in surface water depends on contamination sources, water physicochemical properties and Pb content in AC C soil. According to the US Environmental Protection Agency (EPA), Pb values in surface/ground water usually range from 5 to 30 µg L-1, but higher levels (< 890 µg L-1) may be detected (EPA, 1986). Pb content in drinking/tap water is usually ˂5 µg L-1 but higher levels (100 µg L-1) have also been found (World Health Organization, 2011). For irrigation water, the recommended values differ with country/organization: for example, Food and Agriculture Organization (FAO, http://www.fao.org/docrep/003/t0234e/t0234e06.htm) and EPA (2012) recommend 5 ACCEPTED MANUSCRIPT mg L–1, whereas Korea, Israel, Cyprus, Greece and Italy recommend 0.1 mg L-1 (Jeong, 2016), and Canada allows a maximum of 0.2 mg L–1 (http://www.env.gov.bc.ca/wat/wq/BCguidelines/lead/lead.html). Roots are the first organ of contact and the main system of entrance and accumulation RI PT of Pb in plants (Pourrut et al. 2011b), being therefore expected that roots are also the primary organ to evidence sensitivity to Pb. As the metal reacts with important biomolecules, a large number of metabolic pathways may be disrupted (Patra et al. SC 2004) leading to diverse phytotoxic symptoms. Nevertheless, Pb induced toxicity depends on the species, the plant stage of development and the exposure conditions, M AN U including Pb concentration and speciation, exposure period, pH, soil or mineral solution and organic composition (Patra et al. 2004; Lamb et al. 2010; Pourrut et al. 2011a; Capelo et al. 2012). Pb can delay or even inhibit germination, as described for wheat (Yang et al. 2010; TE D Lamhamdi et al. 2011; Wang et al. 2011) and rice (Mishra et al. 1999), but the most commonly reported physiological effect concerns plant growth imbalances. Shoot and/or root growth inhibition was detected in Vicia faba (Wang et al. 2010a), Sesbania EP grandiflora (Malar et al. 2014), Lactuca sativa (Capelo et al. 2012), Hordeum vulgare and Lycopersicon esculentum (Cheyns et al. 2012), Hordeum vulgare (Varun et al. AC C 2011), Cynara cardunculus (Burak Batir et al. 2016), Triticum aestivum (Lamhamdi et al. 2011) and Pisum sativum (Rodriguez et al. 2015). Nevertheless, most of these alterations were observed at moderate to extremely high Pb concentrations (> 100 ppm). Under more environmentally relevant concentrations, Pb effects on plant growth (length and/or biomass) is species dependent (Lamb et al. 2010; Lamhamdi et al. 2011; Capelo et al. 2012; Ali et al. 2014; Bharwana et al. 2014; Rodriguez et al. 2015; Burak Batir et al. 2016; Silva et al. 2016b). Taking all together, it seems that parameters related with ACCEPTED MANUSCRIPT growth are not the most sensitive and adequate endpoints to assess Pb toxicity. Therefore, alternative and more sensitive biomarkers are required to accurately assess and predict Pb phytoxicity. Photosynthesis related endpoints have been proposed as sensitive biomarkers, such as CO2 assimilation rate and ribulose bisphosphate RI PT carboxylase oxygenase activity (Rodriguez et al. 2015; Silva et al. 2016b). On the other hand, genotoxicity induced by Pb has been reported even under low concentrations (Pourrut et al. 2011a, b; Shahid et al. 2011; Batir et al. 2016). Several parameters seem SC promising for the determination of Pb genotoxicity in plants, including micronucleus (MN) frequency, and the DNA damage assessed by the comet assay (Pourrut et al. and are relatively easy to perform. M AN U 2011a; Shahid et al. 2011), since these techniques have demonstrated high sensitivity The aim of the present work was to analyze the effects of environmentally relevant Pb concentrations in irrigation water on growth, cell cycle dynamics and DNA stability of TE D the economically important crop L. sativa. The cultivar Reine de Mai has demonstrated to be a reliable model for toxicological/ISO assays and we have previously demonstrated that plants of this cultivar accumulated Pb (67.3 mg gDW-1 in roots) when EP exposed to 125 mg L-1 Pb, whereas at low doses, as 12.5 mg L-1, no significant alterations were detected between control and exposed roots (Capelo et al. 2012). L. AC C sativa plants of the cultivar Reine de Mai were grown hydroponically in the presence of low Pb concentrations and several endpoints were used to assess Pb toxicity: germination, growth, micronuclei (MN) frequency, DNA damage (by comet assay), ploidy stability and cell cycle analyses. At the end we aim to propose the most sensitive biomarkers for assessing Pb phytotoxicity. 2. Material and Methods ACCEPTED MANUSCRIPT 2.1 Germination Lactuca sativa (cultivar Reine de Mai) seeds were surface disinfected with sodium hypochlorite 10% (v/v), rinsed in water and germinated for 7 days in Petri dishes containing filter paper (25 seeds per dish). Seeds were grown on modified Hoagland’s RI PT nutrient solution with the following composition: 57.52 mg L–1 NH4H2PO4; 2.86 mg L–1 H3BO3; 656.4 mg L–1 Ca(NO3)2; 0.04 mg L–1 CuSO4.5H2O; 5.32 mg L–1 Fe-tartrate; 120.38 mg L–1 MgSO4; 1.81 mg L–1 MnCl2.4H2O; 0.016 mg L–1 MoO3; 606.6 mg L–1 SC KNO3; 0.11 mg L–1 ZnSO4.7H2O. Different concentrations of Pb: 0, 0.05, 0.5, 5, 10 and 20 mg L-1 Pb(NO3)2 corresponding to 0.031, 0.31, 3.1, 6.25 and 12.5 mg L–1 Pb were M AN U used in the assay. To each dish, 9 mL of the nutritive solution (pH 5.8) supplemented with Pb were added, and germination took place in the dark at 24 °C. After 7 days, the germination rate, plantlet growth (root and aerial portion) and biomass were assessed. For germination rate determination 4 dishes were used and for growth evaluation three TE D to six plantlets from four different dishes were used. 2.2 Hydroponic culture EP Lactuca sativa (cultivar Reine de Mai) plants (2 weeks old) purchased from “Viveiros Litoral” (Aveiro, Portugal) were washed and grown for a further 28 days on modified AC C Hoagland’s nutrient solution described in 2.1 section. Nutritive solution was supplemented with different concentrations of Pb(NO3)2: 0, 0.05, 0.5, 5, 10, 20 mg L–1 (Pb concentrations described in 2.1 section). These concentrations were chosen in order to include the maximum recommended values for Pb in irrigation waters (EPA, 2012). Plants were grown in a climate chamber at 24 oC, under light intensity of 200 µmol m–2 s–1 with 70% humidity and 16/8h photoperiod (Silva et al. 2016b). The nutrient solution was continuously aerated and renewed every 3 days. The pH was maintained at 5.8 ACCEPTED MANUSCRIPT throughout the assay. All measurements were performed at the end of the exposure period. After the exposure period, root length, root biomass and root water content (WC) were assessed. For that, were used six different plants from each treatment. The RI PT WC was calculated as follows: (fresh weight-dry weight)/ fresh weight *100. 2.2.1 Cell cycle analysis For flow cytometry (FCM) studies, nuclear suspensions of root apices (6 pools of 5-8 SC fresh apices from three different replicates) were obtained by chopping the roots in Woody Plant Buffer (Loureiro et al. 2007), and processed according to Silva et al. M AN U (2012). After addition of RNAse (e.g., Loureiro et al. 2007; Dolezel and Bartos, 2005) and 50 µL propidium iodide (PI), approximately 3000 nuclei were analyzed with a Coulter EPICS-XL flow cytometer (Coulter Electronics, USA), with an argon-ion laser (15mW, 488 nm). The percentage of nuclei in each phase of the cell cycle (G0/G1, S and USA). TE D G2 phases) were analyzed using the FlowJo software (Tree Star Inc., Ashland, Oregon, For mitotic phase analysis, root tips were fixed in Carnoy’s solution (3:1 methanol: EP acetic acid) and stored at 4 ºC. The apices were hydrolyzed in 1 N HCl at 70 ºC, washed AC C in water and stained with PI (Silva et al. 2016a). The mitotic index and the number of cells in each mitotic phase and in cytokinesis were determined with a fluorescence microscope Nikon Epics 80i (Nikon, Japan). For that, in each condition, 2-3 tips from three replicates were assessed and from each tip 1000 cells were analyzed. The percentage of cells in mitosis (in 1000 cells), corresponding to the mitotic index, and the percentage of cells in each phase of mitosis and cytokinesis (of the total number of cells in mitosis+ cytokinesis) were determined. ACCEPTED MANUSCRIPT 2.2.2 Genotoxicity 2.2.2.1 Micronucleus test MN assessment was performed according to Silva et al. (2016a) and similarly to the described in section 2.2.1 for mitotic phase analysis. For each condition 2-3 tips from RI PT three replicates were assessed for MN frequency. From each tip 1000 cells were analyzed under a 40x magnification. SC 2.2.2.2 Comet assay DNA damage assessed by the comet assay was conducted according to Gichner et al. M AN U (2004). Fresh tissues of roots and leaves were gently sliced with a razor blade and nucleoids collected in 0.4 M Tris buffer (pH 7.5). Fifty µL of the nucleoids suspension were mixed in 50 µL 1% low melting point agarose at 40 ºC and spread on a slide covered with dried normal point melting agarose. Nuclei were unwound by placing the TE D slides in alkaline electrophoresis buffer for 15 min, prior to electrophoresis at 0.74 V cm-1 for 30 min. The slides were then rinsed with 0.4 M Tris buffer and dried overnight. After staining with ethidium bromide, nucleoids were analyzed with a fluorescence EP microscope (Nikon Eclipse 80i) using filters of 510-560 nm (excitation) and 590 nm (barrier). Images were captured with NIS Elements F 3.00, SP7 software. The comets AC C were classified using CaspLab v1.2.2 program, scoring 300-400 nucleoids for each condition (from 3 replicates) under a 40x magnification. 2.2.2.3 Analysis of DNA content and ploidy stability For nuclear DNA content (nDNA) and ploidy level analysis, leaf and root fresh nuclei were prepared as described for the cell cycle analysis (2.2.1 section) but Vicia faba L. cv. Inovec (with 2C = 26.90 pg DNA (Dolezel and Bartos, 2005), provided by J. ACCEPTED MANUSCRIPT Dolezel, Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic) was used as internal standard as described previously (Silva et al. 2012, for recommendation procedures and review see Dolezel and Bartos, 2005). Ploidy was analyzed using the FlowJo software (Tree Star RI PT Inc., Ashland, Oregon, USA). The nuclear genome size of the samples was calculated as follows (Dolezel and Bartos, 2005): Sample 2C nuclear DNA content (pg)=((lettuce genotype G0/G1 peak mean)/(V. faba G0/G1 peak mean)) * 26.90. SC To score for possible clastogenic damages the full peak coefficient of variation (FPCV) M AN U of the lettuce G0/G1peaks was analyzed according to Rodriguez et al. (2011). 2.3 Statistical analysis Values are presented as mean ± standard deviation. The comparison between treatments was made using One Way ANOVA test. When data was statistically different, ANOVA TE D test was followed by a Holms Sidak Comparison Test (p<0.05). 3 Results EP 3.1 Germination and Growth After seven days under germination conditions, seeds’ germination rates and seedlings AC C length and biomass were not impaired by Pb exposure (p>0.05, Fig. 1). Also, Pb exposure (≤20 mg L-1) did not delay germination, nor induced morphological changes in seedlings compared to the control (data not shown). After 28 d exposure, root length and biomass were not significantly (p>0.05) affected by Pb exposure (Fig. 2A, B). Also, Pb exposure did not induce imbalances in root WC (Fig. 2C). 3.2 Cell cycle analysis ACCEPTED MANUSCRIPT The FCM histogram of control tip roots showed the typical diploid profile with a dominant G0/G1 peak (Fig. 3B, C). Nevertheless, the cell cycle dynamics of the different populations was affected by the concentration of Pb (Fig. 3A). Roots exposed to 0.05 mg L-1 Pb(NO3)2 showed a decrease of subpopulation of cells in G0/G1 phase (≈14%), RI PT while the percentage of cells in S (≈100%) and G2 (≈634%) phases increased (Fig. 3C). Similarly, roots exposed to 0.5 mg L-1 Pb(NO3)2 presented a higher number of nuclei in G2 phase (217% higher than the control). SC Pb did not impair the mitotic index, but at 20 mg L-1 a trend to decrease was detected (Fig. 4A). Concerning the mitotic phases, roots exposed to 0.05 mg L-1 Pb(NO3)2 detected in the cytokinesis (Fig. 4B). 3.3 Genotoxicity M AN U showed a significant decline of cells in anaphase (Fig. 4B). No significant changes were TE D Pb concentrations above 0.05 mg L-1 induced genotoxicity (Fig. 5, 6) in lettuce roots. Concerning MN (Fig. 5), control roots presented on average 0.1 ± 0.25 ‰ micronucleated cells. All Pb-treated roots showed more cells with MN than control EP roots, although this increase was only significant for 20 mg L-1 Pb(NO3)2 (3.5 ± 1.3 ‰ cells, corresponding to ≈2,700 % increase). AC C By the comet assay two groups may be observed with distinct responses (Fig. 6): a) control and 0.05 mg L-1 Pb(NO3)2; b) 5, 10, 20 mg L-1 Pb(NO3)2. Overall, DNA damage (measured by the tail length) was statistically evident when plants were exposed to ≥ 5 mg L-1 Pb(NO3)2, although comets could be detected even at lower doses (0.5 mg L-1, p>0.05) (Fig. 6C). Tail DNA % (Fig. 6A) and Tail Moment (Fig. 6B) presented similar profiles, being higher than control in plants exposed to 5 and 10 mg L-1 Pb(NO3)2 (≈850 % and ≈6,000 % higher than control for Tail DNA and Tail moment, respectively). ACCEPTED MANUSCRIPT Exposed roots showed similar diploid histograms and no occurrence of aneuploidy/polyploidy was detected. The nuclear DNA content (pg nDNA) (Table 1) of control plants ranged between 6.21 ± 0.02 pg/2C and no differences (p>0.05) were detected between treatments (Table 1). The values of FPCV obtained in all treatments RI PT were similar to the control (p>0.05). 4. Discussion SC Germination tests are recognized by OECD guidelines for the assessment of the toxicity of chemicals/contaminants (OECD, 1984), but the sensitivity of these tests is under M AN U discussion and several authors do not consider the germination rate as a sensitive endpoint (Lamb et al. 2010; Silva et al. 2016a). In the present work, Pb did not alter lettuce germination rates or seedling growth. Similarly, lettuce exposed to Pb and other metals (Cu, Zn and Cd) did not show disturbance in germination rates [0.1-1000 µM TE D (0.033 - 331 mg L-1) Pb(NO3)2] (Lamb et al. 2010). On the other hand, maize showed a decrease of germination and seedling growth (length, fresh and dry weight) under low doses of Pb (ranging from 0.01 to 1 mg L-1 Pb) (Ahmad et al. 2011). A similar EP impairment was described in Triticum aestivum [0.3-3 mM (18.7 – 623 mg L-1 Pb)] AC C (Lamhamdi et al. 2011) and in Brassica campestris, where Pb inhibited radicle emergence (Singh et al. 2011). Pb can also stimulate germinattion as described by Islam et al (2007) in Elshotzia argyi exposed to low-moderate doses of Pb [25-400 µM (5-80 mg L-1)], despite the negative effect on plant growth. Impairments on plant growth after exposure to high Pb doses have been reported for several species and it seems that above 100 ppm Pb (inclusively), most of the species show susceptibility concerning length and/or biomass growth (Wang et al. 2010a; Lamhamdi et al. 2011; Varun et al. 2011; Capelo et al. 2012; Cheyns et al. 2012; Yan ACCEPTED MANUSCRIPT and Tam 2013; Ali et al. 2014; Malar et al. 2014; Batir et al. 2016). At lower concentrations, as reported in this work, some species did not show growth alterations as is the case of Vicia faba (Wang et al. 2010a, b) and T. aestivum (Lamhamdi et al. 2011), while other species, such as cabbage, showed growth stimulation (Paschalidis et RI PT al. 2013). In other species, such as Nicotiana tabacum (Alkhatib et al. 2012) or Zea mays ( Wierzbicka 1999; Ahmad et al. 2011) growth was negatively affected by Pb exposure. Concerning lettuce, Lamb and colleagues (2010) reported root growth SC reduction above 10 µM Pb2+ (2 mg L-1) and an EC50 value for roots of about 180 µM (36 mg L-1), whereas Capelo et al. (2012) detected growth (shoot and roots) M AN U impairments only under 125 mg L-1 Pb2+ and Silva et al. (2016b) did not find shoot growth alterations in lettuce exposed to 0.03 - 12.5 mg L-1 Pb2+. Furthermore, using the same cultivar as used in this work, Capelo et al. (2012) did not detect accumulation of Pb in plants (roots and shoots) exposed to 12.5 mg L–1 Pb for 15 days, which can TE D indicate that at lower concentrations Pb may have predominantly an indirect action. As conclusion, Pb effects on growth are strongly dependent on plant species/variety and it seems that lettuce presents some tolerance to this metal, as demonstrated by Monteiro et EP al (2012) for this cultivar under cadmium exposure (Monteiro et al. 2012). AC C Despite no significant alterations were observed in root growth, cell cycle dynamics was significantly affected, particularly the interphase progression. At the mitotic phase, alterations were detected in anaphase (decrease) at the lowest concentration. Alterations in anaphase were also reported by Liu et al. (1994), which observed anaphase bridges in Allium cepa roots. Despite imbalances on mitotic phase, no significant modifications on mitotic index values were observed. Similarly, in Vicia faba Pb did not induce alterations on mitotic index [1 – 10 µM (0.2 – 2 mg L-1)] (Pourrut et al. 2011a; Shahid et al. 2011). Nevertheless, several authors reported an antimitotic effect of Pb in ACCEPTED MANUSCRIPT different plant species, but again mostly in higher doses (Patra et al. 2004; Pourrut et al. 2013; Jiang et al. 2014). At the interphase, variations occurred under 0.05 and 0.5 mg L–1 Pb(NO3)2, increasing the number of cells in S and G2 phases and a decrease of cells in G0/G1. These changes suggest that even at low and admissible doses, Pb induces an RI PT arrest at G2/M checkpoint rather than an increment of cell proliferation. This hypothesis is supported by the absence of length and biomass enhancements. For higher doses, Pb shortened the mitotic stage and prolonged the interphase in V. faba (Qun and Xiao, SC 1995) and also induced an arrest of cells at G2/M in P. sativum (Rodriguez, 2011). Concerning Pb cytotoxicity, our data show that cell cycle is not linearly dependent on M AN U the Pb doses, as the lowest concentration was more cytotoxic than the more concentrated ones. Cell cycle disturbance induced by Pb is thought to be a consequence of Pb binding to cell wall and membranes, increasing their rigidity, and/or due to disturbance of microtubules organization (Pourrut et al. 2013; Jiang et al. 2014). For TE D example, microtubules depolymerizations, loss of microtubules transverse orientation and cell death was induced under low doses of Pb (0.1-40 mg L-1) in Cymodosa nodose (Malea et al. 2014). These mechanisms of action are however not sufficient to explain EP the observed effects of low doses of Pb in lettuce, since no alterations were detected at cytokinesis. Despite the mechanisms by which Pb induces cell cycle imbalances, those AC C alterations may result in abnormal cells (Liu et al. 1994; Pourrut et al. 2013; Jiang et al. 2014) and even in cell death (Malea et al. 2014). The MN test has been reported to be a reliable and sensitive biomarker and is recommended by OECD as a robust test for the effects of chemicals in animal cells (OECD, 2010) and for assessment of environmental contamination (Ma et al. 1995; Castiglione et al. 2011). MN test detects genotoxic damage in interphase cells as result of aneugenic (whole chromosome) or clastogenic (chromosome breakage) damage and ACCEPTED MANUSCRIPT require passage through the mitosis to be recognizable. On the other hand, the comet assay detects single-strand (SS) (repairable) and double-strand (DS) (unrepairable) DNA breaks at low level of damage (Horvathova et al. 1998). Therefore, these biomarkers provide distinct and complementary information and both seem to be RI PT sensitive and reliable methods to evaluate Pb toxicity. Nevertheless, the use of these methods to assess Pb genotoxicity remains limited in plants, and even less when used in combination. Lead induced genotoxicity was observed in V. faba by MN test and comet SC assay (Pourrut et al. 2011a; Shahid et al. 2011), in A. cepa using MN test (Liu et al. 1994) and the comet assay (Jiang et al. 2014) and in Lupinus luteus by comet assay M AN U (Rucinska et al. 2004). DNA damage associated to Pb exposure was also detected using other molecular techniques, such as random amplified polymorphic DNA (RAPD) and microsatellite instability profiles (eg. Cenkci et al. 2010; Rodriguez et al. 2013; Malar et al. 2014; Batir et al. 2016). Genotoxicity induced by low (and legal) doses of Pb was TE D evident in lettuce roots, as proven by the increase in the number of micronucleated cells, and the increase of DNA fragmentation. Also, it was found that DNA damage increased, as expected, with Pb concentration in a dose-dependent manner. However, EP the type of damage differed as 5 and 10 mg L-1 induced SS/DS DNA breaks, whereas at 20 mg L-1 Pb induced more aneugenic/clastogenic damage. This last fact not only is AC C demonstrated by the increase of MN, but is in accordance with the observed decrease of the tail moment at the highest concentration, which is a consequence of tail length reduction. The tail length reduction indicates a change of the nature of DNA damage. This behavior was also observed in lupin roots, which presented elevated number of nuclei with high tail lengths and moments values under 150 mg L-1 Pb, whereas at higher concentrations (350 mg L-1 Pb) the number decreased, in an apparent hormetic effect (Rucinska et al. 2004). Similar hormetic effect on the tail length was shown in ACCEPTED MANUSCRIPT lettuce roots exposed to Cd (Monteiro et al. 2012). Taking all together, the nature of the Pb-induced DNA damage (and probably other metals as Cd) seems to be concentration dependent: a) lower concentrations induce the formation of short, rapidly migrating DNA fragments; b) higher doses induce the formation of longer/heavier DNA fragments RI PT with reduced electrophoretic DNA migration. The increase of tail length and MN occurrence in root cells of plants exposed to 20 mg L-1 Pb was not supported by any alteration in growth or in surviving rates, suggesting that these surviving root cells had dose, Pb did not induce ploidy alterations. SC insufficient DNA repair systems for blocking cell cycle. Nevertheless, and for the same M AN U Although genotoxicity seems to be a consequence of Pb exposure, little is known about the Pb induced genotoxic mechanisms on plants (Pourrut et al. 2013). The observed SS and/or DS DNA breakes could be a result of ROS formation or Pb direct interaction with DNA. It was proposed that at low doses, Pb-induced genotoxicity is mostly TE D mediated by indirect mechanisms, as an increase of ROS levels (Pourrut et al. 2011a). This last hypothesis is supported by the Pb-induced oxidative stress reported for different plants and animals (Lamhamdi et al. 2011; Dai et al. 2012; Liu et al. 2012; EP Shahid et al. 2013; Bharwana et al. 2014; Alya et al. 2015) and, in the case of lettuce, by the absence of significant Pb accumulation under Pb low concentrations (Capelo et al. AC C 2012). In conclusion we demonstrate that low doses of Pb despite not inducing apparent toxicity to seed germination and seedling/plant growth (both parameters recommended by OCDE 208), showed significant toxicity when chosen endpoints are related with DNA damage and mitotic/interfase disorders. These genotoxic effects address interesting questions regarding the low doses used here, namely the potential accumulation of mutations that may occur when crops are irrigated with waters ACCEPTED MANUSCRIPT containing some of the Pb levels reported here. Finally, we also demonstrate that the inclusion of MN and comet assay should be recommended as more sensitive endpoints to (eco)toxicological assays, in complement to more classical ones as germination and RI PT growth. 5. Contributions SS was responsible for plant exposure and for cytological analysis and gave assistance SC in the comet assay, helped and provided material for flow cytometry analyses and wrote the manuscript; SP performed the germination test, was involved in plant exposure, M AN U performed the comet assay and helped in flow cytometry analyses; OH performed the flow cytometry analysis an collaborated in manuscript correction; GI assisted the comet assay and collaborated in manuscript correction, Matos M. supervised the germination tests and collaborated in manuscript correction; P-CO collaborated in manuscript TE D correction; SC contributed in designing the study, corrected the manuscript and gave valuable suggestions to improve the final document. EP 6. Acknowledgments Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino AC C Superior (FCT/MCTES) supported S. Silva (SFRH/BPD⁄74299⁄2010) and H. Oliveira (SFRH/BPD/111736/2015) grants from the financing program QREN–POPH/FSE – Tipologia 4.1 – Formação Avançada. Thanks are due, for the financial support to CESAM (UID/AMB/50017) and UI QOPNA (FCT UID/QUI/00062/2013), to FCT/MEC through national founds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. ACCEPTED MANUSCRIPT RI PT 7. References Agency for Toxic Substances & Disease Registry (ATSDR) (2007) Toxicological profile for Lead. U.S. department of health and human services, Atlanta, Georgia. 2015 priority list of SC Agency for Toxic Substances and Disease Registry (ATSDR) (2015) Summary data for hazardous substances. M AN U https://www.atsdr.cdc.gov/spl/#modalIdString_myTable2015 at 27-07-2016 Ahmad, M.S., Ashraf, M., Tabassam, Q., Hussain, M., Firdous, H., 2011. Lead (Pb)induced regulation of growth, photosynthesis, and mineral nutrition in maize (Zea mays L.) plants at early growth stages. 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Different letters correspond to significant differences (p < 0.05) between treatments. FPCV 0 5.38 ± 1.95 ab 6.21 ± 0.02 ab 4.47 ± 0.48 ab 6.26 ± 0.02 ab 0.5 4.30 ± 0.89 ab 6.21 ± 0.02 ab 5 3.51 ± 0.63 b 6.27 ± 0.01 b 10 5.56 ± 0.93 ab 6.12 ± 0.02 ab 20 6.68 ± 2.08 a 6.10 ± 0.04 a EP Pb(NO3)2 (mg L-1) AC C 0.05 nDNA content (pg) M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D Figures EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C Figure 1. Germination rate (A), seedling biomass (B) and seedling shoot and root lengths (C) of seeds exposed to different Pb(NO3)2 concentrations for 5 days. No differences were found (p≥0.05). ACCEPTED MANUSCRIPT 6 A 4 3 2 RI PT Root biomass (g) 5 1 50 0 B 30 SC Root lenght (cm) 40 20 M AN U 10 120 00 C 80 60 40 20 0 0 0.05 TE D Root WC (%) 100 0.5 5 10 Pb(NO 3 ) 2 (mgL -1 ) 20 EP Figure 2. Root biomass (A), root length (B) and root water content (C) of plants exposed during 28 days to different AC C Pb(NO3)2 concentrations. Values correspond to average ± standard deviation. No differences were found (p≥0.05). RI PT ACCEPTED MANUSCRIPT Figure 3. Cell cycle profile of root tips analysed by flow cytometry. Percent of cells in each phase (A) and representative histograms of cell cycle profile (B, C): A) control; B) roots roots exposed to 0.05 mg L-1 Pb(NO3)2. SC Values correspond to average ± standard deviation. In each phase different letters correspond to significant M AN U differences (p < 0.05) between treatments. 12 A 8 6 TE D Mitotic index (%) 10 4 2 120 0 60 * AC C % 80 B EP 100 Prophase Metaphase Anaphase Telophase Cytokinesis 40 20 0 0 0.05 0.5 10 20 Pb(NO3)2 (mg L-1) Figure 4. Mitotic index (A) and the % of the total number of cells in mitosis + cytokinesis that are in each mitotic phase (B) of roots grown in the presence of Pb(NO3)2. Values correspond to average ± standard deviation. * ACCEPTED MANUSCRIPT correspond to significant differences (p < 0.05) between treatments and control. The absence of letters in A means no RI PT statistical differences (p ≥ 0.05) 6 b 5 3 ab 2 ab SC MN (‰) MN (‰) 4 ab ab 1 0 0 0.05 0.5 5 10 Pb(NO3)2 (mgL -1) 20 M AN U a Figure 5. Number of cells (‰) with micronucleus in roots exposed to Pb(NO3)2. Values correspond to average ± AC C EP TE D standard deviation. Different letters correspond to significant differences (p < 0.05) between treatments. ACCEPTED MANUSCRIPT 70 A 60 b b ab 40 ab RI PT 30 20 10 a a 0 300 0 b B 250 b ab 150 ab 100 50 a a 500 00 b C b 400 M AN U Tail Moment 200 SC Tail DNA % 50 300 a 200 100 a a 0 0.05 0 5 10 EP 0.5 TE D Tail lenght b 20 Pb(NO3)2 (mg L-1) AC C Figure 6. DNA damage assessed by the comet assay in roots exposed to Pb(NO3)2: Tail DNA% (A), Tail Moment (B) and Tail length (C). Values correspond to average ± standard deviation. Different letters correspond to significant differences (p < 0.05) between treatments. ACCEPTED MANUSCRIPT Exposure to low doses of Pb induced genotoxicity in lettuce • Cell cycle was impaired after exposure to low doses of Pb • MN and comet assay were sensitive and valuable endpoints to assess Pb phytotoxicity • The nature of DNA damage was Pb-concentration dependent AC C EP TE D M AN U SC RI PT •