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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Cytotoxic and genotoxic effects of metal(oid)s bioactivated in rocket leaves (Eruca vesicaria subsp. sativa Miller)
Myriam Villatoro-Pulido a, Rafael Font b, Sara Obregn-Cano c, Rafael Moreno-Rojas d, Manuel ngel Amaro-Lpez d, Jauoad Anter e, Andrs Muoz-Serrano e, Antonio De Haro Bailn c, ngeles Alonso-Moraga e, Mercedes Del Ro-Celestino b,
a

Department of Plant Breeding and Crop Biotechnology, Center IFAPA Alameda del Obispo s/n, Apartado 3092, 14080 Crdoba, Spain Department of Plant Breeding and Crop Biotechnology, Center IFAPA La Mojonera, Camino San Nicols, 1, 04745 La Mojonera, Almera, Spain Department of Genetics and Plant Breeding, Institute of Sustainable Agriculture (IAS), CSIC, Apartado 4084, Crdoba, Spain d Department of Bromatology, Campus of Rabanales, University of Crdoba, 14071 Crdoba, Spain e Department of Genetics, Campus of Rabanales, University of Crdoba, 14071 Crdoba, Spain
b c

h i g h l i g h t s
 Rocket accessions were used to test genotoxic and cytotoxic effects of metal(oid)s.  Three efcient metal excluding accessions of rocket were identied.  Metal and isothiocyanates were the modulator agents of their biological activity.  SMART and cytotoxicity tests, offer an effective rst-step screening to assess toxicity.

a r t i c l e

i n f o

a b s t r a c t
Rocket is an important source of essential elements. However, it may also accumulate toxic elements such as metal(oids). The objectives of the present work were (i) to study the uptake of arsenic, lead, cadmium and zinc in rocket grown in contaminated soils, (ii) to establish the genotoxic and cytotoxic activities of this vegetable material, and (iii) to study the modulator role of the glucosinolate and metal contents in the genotoxic/cytotoxic activities. Lead, cadmium and zinc leaf concentrations in our study were over the concentrations allowed by the statutory limit set for metal(oid) contents in vegetables. The accessions were non genotoxic at the different concentrations studied, although one of the accessions showed the highest mutation rates doubling those of negative control. The cytotoxicity assays with HL60 human leukaemia cells showed that the tumouricide activities of rocket leaves decreased with the increasing of metal(oid) concentrations and also with the decreasing of glucosinolate concentrations in their tissues. An interaction between metal(oid)s and glucosinolate degradation products contained in rocket leaves is suggested as the main modulator agents of the biological activity of the plants grown in metal-contaminated soils. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 9 April 2013 Received in revised form 29 August 2013 Accepted 20 September 2013 Available online 22 October 2013 Keywords: Arsenic Lead Cadmium Glucosinolate Toxicity In vivo

1. Introduction Some members of the Brassicaceae family have shown to accumulate from moderate to high levels of metal(oid)s (Ebbs and Kochian, 1997). Rocket, that belongs to Brassicaceae family, may accumulate considerable amounts of essential elements (P, K, Mg, Ca, Fe, Mg, Cu, Zn and Na) (Bozokalfa et al., 2009; Villatoro-Pulido et al., 2012b), but also toxic elements such as arsenic (As) (Stilwell et al., 2006) and other heavy metals like lead (Pb), chromium (Cr)
Corresponding author. Tel.: +34 671632003; fax: +34 950156453.
E-mail address: mercedes.rio.celestino@juntadeandalucia.es (M. Del Ro-Celestino). 0045-6535/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.09.071

and cadmium (Cd) (Saleh, 2001; Euzbio Filho et al., 1998; Stilwell et al., 2006; Al-Qurainy et al., 2010). Rocket (Eruca vesicaria subsp. sativa Miller) has been identied as a vegetable with potentially anti-cancer activity due to their content in isothiocyanates that are glucosinolates (GLs) hydrolysis products (Fenwick et al., 1983; Mithen, 2001). There is a great concern about metal(oid)s as they can accumulate in some agricultural species and enter the human food chain through the consumption of vegetable edible parts being potential carcinogens (Kabata-Pendias and Mukherjee, 2007). Studies of genotoxicity can help to evaluate the risk/safety of healthy food products. The somatic mutation and recombination test (SMART) in proliferating wing imaginal discs of Drosophila

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melanogaster is a eukaryotic assay based on the loss of heterozygosity for two genetic markers affecting the phenotype of wing hairs in the adult (Graf et al., 1984). This wing spot test is a versatile and reliable system to test complex mixtures for genotoxicity. It was shown to be suitable to carry out both genotoxicity and antigenotoxicity assays, thanks to the capabilities of treated larvae to bioactivate metabolites either as a single compound or as complex mixtures depending on the form by which they are consumed (Graf et al., 1998; Anter et al., 2011; Tasset-Cuevas et al., 2013). In the case of As, a well-known genotoxin and carcinogen, Rizki et al. (2006) concluded that inorganic As was non-genotoxic in the SMART test for D. melanogaster at the tested concentrations. There is limited data on the genotoxicity of heavy metals (Pb, Cd and Zn) in rocket. Only two studies have been carried out in rocket seedlings (Al-Qurainy, 2010; Al-Qurainy et al., 2010). These studies concluded that high concentrations of Pb and Cd damaged the seedlings of E. sativa and were potentially genotoxic. HL60 human leukaemia cells have been used in cytotoxicity assays in order to determine the tumouricide activity of the plant (Anazetti et al., 2003). There is little information about the in vitro activity of rocket. Cytotoxicity against melanoma cells (Khoobchandani et al., 2011) and antigenotoxicity on human hepatoma (HepG2) cells towards benzo(a)pyrene induced toxicity (Lamy et al., 2008) have been reported. However no studies have been carried out to demonstrate the presence or absence of in vitro activity of rocket leaves with accumulation of toxic elements. The main objectives of this work are (i) to study the uptake of As, Pb, Cd and Zn in rocket leaves and (ii) to establish the in vivo genotoxic and in vitro cytotoxic activities of this vegetable material (iii) to study the modulator role of the GL and metal contents in the genotoxic/cytotoxic activities.

2.2. Sample preparation The plants (Pex-1, Pex-11, Pex-14 and Pex-17 accessions) were harvested once they were ready for human consumption (40 d). They were thoroughly washed with tap water, rinsed with deionised water (0.2% non phosphate detergent solution) and weighed to assess their biomass. Finally, samples were freeze-dried at 80 C (1.8 101 mbars) for 3 d, in a Telstar freeze-drier equipment (mod. Lioalfa 680). 2.3. Determination of arsenic and heavy metal in soils and plants The metal extractable fraction in soil was estimated with a diethylenetriamine pentaacetic acid (DTPA) extraction, buffered at pH 7.3 (Lindsay and Norvell, 1978). The As concentration was determined by the ow injection-hydride generation-atomic absorption spectrometry (FIAHGAAS) (Muoz et al., 2000), Pb and Cd were determined by atomic absorption spectrometry (AAS) with graphite chamber (Perkin Elmer Analyst 600 with an autosampler AS 800) (Guzman et al., 1994) and Zn was determined using ame atomic absorption spectroscopy (Perkin Elmer 1100B). The accuracy and precision of the analytical methods was assessed carrying out analyses of the Community Bureau of Reference (reference sample CMR 279, sea lettuce) (Griepink and Muntau, 1988). The values obtained for the reference sample by FIAHGAAS and AAS were concordant with the certied values (data not showed). 2.4. Metal transfer factor coefcients In order to nd out what proportion of the total soil metal concentration was available and transferred to plant aerial parts, the transfer coefcient (TC) was calculated. This is dened as the ratio of metal concentration in the plant to the total metal concentration in the soil (Adriano, 2001). 2.5. Determination of glucosinolate content in rocket leaves

2. Material and methods 2.1. Plant material and greenhouse experiments Four rocket accessions were used based on their total content of GLs. Seeds of Eruca vesicaria subsp. sativa Pex-1, Pex-11, Pex-14 and Pex-17 were obtained from Botanischer Garten der Universitat of Karlsruhe, Germany; Dipartimento di Scienze Botaniche of Palermo, Italy; Facult des Sciences Agronomiques of Gembloux, Belgium; and from commercial sources (Cobham, Surrey, U.K.). Pots were placed in the greenhouse under natural light, with a temperature of 27/18 C (day/night) and a relative humidity of 50%/70% (day/night). Seeds were germinated in Petri dishes for 48 h and when the plants reached an adequate height (812 cm), they were transferred to plastic pots containing 3 kg of contaminated soil in order to study the uptake and accumulation of As, Pb, Cd and Zn. The contaminated soil was obtained from the experimental area El Vicario (latitude N 37260 2100 , longitude W 06130 0000 ), within the Green Corridor, close to the pyrite mine of Aznalcllar (Seville, Spain) (Santos et al., 2002). The soil was classied as Typic Haploxeralf (USDA-SSS, 1999). One week before planting, soil was mixed with commercial potting mixture (1:1 vol). The commercial potting mixture was used as the control. The soil was a sandy loam soil (sand 50%, silt 33% and clay 17%) which chemical characteristics were pH = 6.0, organic C = 35%, N = 0.3% and organic matter = 60%. In order to study the As, Pb, Cd and Zn accumulation, a complete random design was used for 40 d of exposure. Controls with non contaminated soil were also included. All treatments were replicated 10 times. Freeze-dried rocket young leaves (100 mg) were ground in a Janke and Kunkel (A10 mill, IKA-Labortechnik). The our was heated at 75 C to inactivate myrosinase (15 min, 2.5 mL of 70% aqueous methanol). Glucosinolates were analysed by liquid chromatography with ultraviolet photometric detection (for further details see Villatoro-Pulido et al., 2012a). The total GL content was computed as the sum of all the individual GLs present in the sample. The chromatograms were compared to the desulfo-glucosinolate proles provided by three certied reference materials recommended by the U.E. (CRMs 366, 190 and 367) and using a pure standard GLS for the glucosativin (Wathelet et al., 1991). 2.6. Genotoxicity assays 2.6.1. Drosophila melanogaster strains Two Drosophila Strains were used:  mwh/mwh, carrying the recessive mutation mwh (multiple wing hairs) that produces multiple tricomas per cell.  r3/In (3LR) TM3, ri pp sep bx34e es BdS, where the r3 (are) marker is a homozygous recessive lethal mutation that produces deformed tricomas but it is viable in homozygous somatic cells once larvae start developing. 2.6.2. Treatments The genotoxicity assays were carried out following the method described by Graf et al. (1984). Treatment vials contained 0.85 g of Drosophila Instant Medium (Formula 424, Carolina Biological

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Supply, Burlington, NC) wetted with 4 mL of different prepared concentrations of lyophilized vegetable samples. The accessions PEX-1, PEX-11, PEX-14 and PEX-17 were assayed at 0.675, 1.25, 2.5 and 5 mg mL1 concentrations. One hundred larvae were used for each accession and treatment. The negative controls were prepared with the medium and water; and positive controls with medium and hydrogen peroxide (0.12 mM) as oxidative genotoxicant (Romero-Jimnez et al., 2005). After emergence, adult ies were collected and stored in a 70% ethanol solution. See Villatoro-Pulido et al. (2012a) for detailed maintenance, crosses and treatments. 2.6.3. Wing analysis Twenty pairs of wings of each control and concentration of wild type transheterozygous marker ies (mwh/r3 genotype) were removed and mounted on slides using Faures solution. Female and male wings were mounted separately. Both dorsal and ventral surfaces of the wings were screened under a photonic microscope with the 400 magnication. Wing hair mutations (spots) were scored among a total of 24 400 monotricoma cells per wing. 2.7. In vitro cytotoxicity assays 2.7.1. Cell culture and incubation conditions Cells were grown at a density of 105 cells mL1 before beginning the assay in 2 mL well plates (For further detailed information see Anter et al., 2011). 2.7.2. Cell viability assay HL60 cells were placed in 12 well culture plates (1 105 cells mL1) and treated for 72 h with different concentrations (0.5, 1, 1.5 and 2 mg mL1) of the lyophilized plants whereas the negative controls had only culture medium. The cell viability was assessed using the trypan blue (Fluka, 93 595) exclusion assay, which was added to the cell culture with a volume ratio of 1:1. The number of living cells was counted using a Neubauer chamber under an inverted microscope (Motic, AE30/31) at 100 magnications. A growth curve was established and IC50 values (concentration of tested compound causing 50% inhibition of cell growth) were estimated. 2.8. Data evaluation and statistical analysis The values are expressed as mean standard deviation (S.D.). Differences between metal(oid) concentrations in soils and leaves, and glucosinolate content in leaves were assessed by mean comparison tests (t-test and Tukeys multiple comparison test). Data normality was tested prior to analysis and, when necessary, variables were logarithmically transformed. SPSS Version 10.0 software was used to perform all statistical analyses (SPSS, 2000). Wing hair spots were grouped into three different categories: S, a small single spot corresponding to one or two cells exhibiting the mwh phenotype; L, a large single spot with three or more cells showing mwh or r3 phenotypes; T, a twin spot corresponding to two juxtaposing clones, one showing the mwh phenotype and other the r3 phenotype. Small and large spots can be originated by somatic point mutation, chromosome aberration as well as somatic recombination whilst twin spots are produced exclusively by somatic recombination between the r3 locus and the centromere. The total number of spots was also evaluated. A multi-decision procedure was applied to determine whether a result is positive, inconclusive or negative (Frei and Wrgler, 1988). The frequencies of each type of mutant clone per wing were compared to the concurrent negative control and the signicance was given at <5% level. All inconclusive and positive results were analysed with the non-parametric U-test of Mann, Whitney and

Wilcoxon (a = b = 0.05, one sided) according with Frei and Wrgler (1995). Growth curves for the cell viability assay were established and plotted as survival percentage with respect to the control growing at 72 h. Each experiment was repeated in triplicate and IC50 values were estimated. 3. Results 3.1. As, Cd, Pb and Zn content in soil Arsenic concentration was always above potentially phytotoxic levels in the contaminated soils (Table 1), and exceeded both the proposed statutory maximum allowable value (<20 mg kg1) and the proposed intervention value (>50 mg kg1) suggested by the Andalusian regulations for agricultural soils (BOJA, 1999). Concentrations of the other elements were below intervention values for Andalusian soils: >200 mg kg1 (Pb), >3 mg kg1 (Cd) and > 300 mg kg1 (Zn). In the case of the control soil, element concentrations were lower than the reference values established by Andalusian regulations for agricultural soils, and were also signicantly lower than the concentrations found in the contaminated soil. The diethylene triamine pentaacetic acid (DPTA) extractable element concentrations of the soils are shown in Table 1. Mean concentration values of total and DPTA-extractable As (67.7 and 0.69 mg kg1), Pb (105 and 16.3 mg kg1), Cd (0.4 and 0.2 mg kg1) and Zn (100 and 21.1 mg kg1) in contaminated soil were signicantly higher than in the control soil. Plant-available metals were very low (Table 1) and element concentrations in contaminated soils not exceeded the intervention values for soils by the Andalusian environmental authority (BOJA, 1999). 3.2. Accumulation of As, Pb, Cd and Zn by rocket plants After 40 d of exposure to control and contaminated soils, all the plants looked quite healthy and did not show any phytotoxicity symptoms except a slight necrosis at the edge and tip of the leaves in a few plants. This indicated that these accessions were relatively tolerant to metal(oid)s. The rocket accessions varied widely in their abilities to accumulate metal(oid)s in the leaves (Table 2). Commercial accession (PEX-17) was the most efcient in As, Pb, Cd and Zn accumulation (1.96 mg kg1 As, 30.9 mg kg1 Pb, 2.9 mg kg1 Cd and 165 mg kg1 Zn) with around 35 times higher concentrations than those accessions grown in the non-contaminated soils. The metal transfer factor coefcient (TC) has been used to describe a plants ability to concentrate the contaminant with respect to the medium. PEX-17 accession showed the highest TC for all the elements. Transfer coefcients for all accessions were <1 for As (0.0050.03) and Pb (0.010.28), indicating low mobility from soil to aerial plant parts. For Cd, TC values were always >1 (2.27.25) with the highest value for PEX-17 accession grown in contaminated soil (7.25). In the case of Zn, TC values were only >1 for PEX-17 accession grown in contaminated soil (1.64), while PEX1, PEX-11 and PEX-14 accessions showed TC values <1 (0.40.9). 3.3. Glucosinolate composition of the rocket accessions Among the aliphatic GLs, glucoerucin (GER) and glucoraphanin (GRA) were the most abundant (Table 3), while glucosativin (4-mercaptobutylglucosinolate) was the predominant indole GL. The accessions showed a mean total content of GLs ranging from 20.7 to 45.63 l moles of GLs g1 of dry weight (dw) in contaminated soil (PEX-17 and PEX-11 respectively). The Tukeys multiple

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Table 1 Total and DPTA extractable concentrations of As and heavy metals content (mean standard deviation) in non-contaminated (control) and contaminated soils (for each treatment n = 3). Soil Concentrations of toxic metals (mg kg1) As Total DPTA Maximuma Toxic range for plantsb Control Contaminated Control Contaminated 9.1 0.1 67.7 0.1** 0.12 0.0 0.69 0.80* 20 1550 Pb 41.8 0.1 105.1 0.5* 5.9 0.2 16.3 0.3* 200 100400 Cd 0.2 0.0 0.4 0.0* 0.01 0.0 0.2 0.0* 23 38 Zn 60 0.9 100.4 1.4* 40 1.7 21.1 13.4 300 70400

Means were compared between the control and contaminated soils for As, Pb, Cd and Zn within each total and DPTA extractable concentrations. Signicantly different at P < 0.05. ** Signicantly different at P < 0.01. a Maximum allowed concentrations for agricultural soils according to the Andalusian regulations (BOJA, 1999). b Total concentrations in soil considered as phytotoxic potentially toxic for plants (Kabata-Pendias and Pendias, 1992).
*

Table 2 Concentrations of As, Pb, Cd and Zn (mean standard deviation) in leaf rocket grown in control and contaminated soils (expressed as mg kg1). Accessions Metal(oid)s in soil As Control Contaminated Pb Control Contaminated Cd Control Contaminated Zn Control Contaminated PEX-1 0.34 0.00 0.40 0.03 5.52 0.12 2.67 0.12 0.84 0.04 0.92 0.00 58 1.10 51 0.78 PEX-11 0.28 0.06 0.36 0.07 2.67 0.12 2.60 0.12 0.94 0.02 0.96 0.00 63 0.90 51 0.34 PEX-14 0.31 0.02 0.40 0.10 7.41 0.09 1.70 0.08 0.68 0.02 0.92 0.01 31 0.81 48 1.12 PEX-17 0.30 0.05 1.96 0.20*,b 1.70 0.04 30.89 1.96* 0.44 0.00 2.90 0.12* 48 1.02 165 0.91* Maximum allowed in vegetables (mg kg1)a 10

0.5

60

a Maximum allowed concentrations for metal(oid) contents in vegetables (ANZFZ, 1998; Codex Alimentarius Commission (FAO/WHO), 2001; Commission Regulation, 2008). b Means were compared between the control and contaminated soils for As, Pb, Cd and Zn within each accession. * Signicantly different at P < 0.05.

comparison test (P < 0.05) showed statistically signicant differences (P < 0.05) among accessions grown in the contaminated soil for the traits compared. PEX-14 and PEX-17 commercial accessions showed lower mean values of GRA (7.9 and 3.0 l moles of GLs g1 dw, respectively) and total GLs (22.3 and 20.7 l moles of GLs g1 dw, respectively) in plants grown in contaminated soil. The PEX-1 and PEX-11 accessions showed the highest content of GRA (>24 l moles g1 dw) and total GLs (>40 l moles g1 dw) in plants grown in contaminated soil. No signicant differences were found between GL concentrations in accessions grown in control and contaminated soils.

3.4. Genotoxicity assays of rocket Hydrogen peroxide exhibited a signicant total mutation rate which triplicates the control rate (Table 4), implying that the accuracy of the genotoxicity assay was ensured (Romero-Jimnez et al., 2005). None of the accessions were genotoxic at the concentrations assayed. The highest mutation rate (0.3 spots/wing) was scored for the 5 mg mL1 concentration of PEX-17 accession, followed by 1.25 mg mL1 concentration of PEX-1 and PEX-17 accessions, all of them are non-signicant rates although doubling the water control mutation rate. The lowest concentration assayed of PEX-14

Table 3 Glucosinolate composition (mean standard deviation) in rocket accessions grown in control and contaminated soils. Glucosinolates Glucoraphanin Soil Accessions PEX-1 PEX-11 PEX-14 PEX-17 Control 9.4 6.2 7.3 2.3 4.2 1.6 2.2 0.3 Contaminated 24.11 2.1ab 25.0 2.0a 7.9 1.0b 3.0 0.5b Glucosativin Soil Control 12.1 0.5 18.7 4.2 11.3 3.9 10.2 3.1 Contaminated 14.78 1.3a 17.0 1.3a 12.5 08a 12.0 1.5a Glucoerucin Soil Control 0.8 0.1 1.7 0.3 2.6 0.5 4.5 1.3 Contaminated 0.77 0.1b 1.3 0.1b 1.3 0.1b 4.9 0.7a Othersa Soil Control 0.6 0.2 1.0 0.4 0.9 0.4 0.8 0.3 Contaminated 0.95 0.3b 2.4 0.3a 0.6 0.1b 0.8 0.0b Total GLs Soil Control 23.0 5.4 28.6 2.6 19.0 5.3 17.7 4.5 Contaminated 40.6 3.2a 45.63 3.1a 22.3 2.0b 20.7 2.6b

a Others included: aliphatic GLs (glucoiberverin, gluconapin, progoitrin, gluconapoleiferin and glucobrassicanapin), aromatic GLs (gluconasturtiin), indole GLs (4-hidroxyglucobrassicin, 4-metoxyglucobrassicin and neoglucobrassicin). b Means for each individual and total GLs were compared among accessions grown in the contaminated soil using Tukeys multiple comparison test (P < 0.05). Means followed by a common letter are not signicantly different from each other at P < 0.05.

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Table 4 Genotoxicity in the Drosophila SMART test of the treatments with rocket. Compounds Frequency of spots per wing (number of spots) and diagnosisa Number of wings H2O H2O2 (0.12 M) Rocket (mg mL1) PEX-1 [0.675] [1.250] [2.500] [5.000] PEX-11 [0.625] [1.250] [2.500] [5.000] PEX-14 [0.625] [1.250] [2.500] [5.000] PEX-17 [0.675] [1.250] [2.500] [5.000] 40 40 Small spots (12 cells) m = 2 0.100 (4) 0.350 (14) + Large spots (more than two cells) m = 5 0.025 (1) 0.000 (0) Twin spots m = 5 0.000 (0) 0.025 (1) Total spots m = 2 0.125 (5) 0.375 (15) +

40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40

0.100 0.250 0.125 0.125 0.125 0.100 0.075 0.075 0.075 0.150 0.125 0.125 0.125 0.225 0.100 0.225

(4) (10) (5) (5) (5) (4) (3) (3) (3) (6) (5) (5) (5) (9) (4) (9)

0.075 0.025 0.050 0.000 0.025 0.025 0.100 0.025 0.000 0.050 0.075 0.075 0.050 0.025 0.050 0.050

(3) (1) (2) (0) (1) (1) (4) (1) (0) (2) (3) (3) (2) (1) (2) (2)

0.000 0.000 0.000 0.000 0.075 0.025 0.025 0.025 0.000 0.000 0.000 0.025 0.025 0.025 0.050 0.025

(0) (0) (0) (0) (3) (1) (1) (1) (0) (0) (0) (1) (1) (1) (2) (1) i

0.175 0.275 0.175 0.125 0.225 0.150 0.200 0.125 0.075 0.200 0.200 0.200 0.200 0.275 0.200 0.300

(7) (11) (7) (5) (9) (6) (8) (5) (3) (8) (8) (8) (8) (11) (8) (12)

a Statistical diagnoses according to Frei and Wrgler (1988): + (positive), - (negative) and i (inconclusive). Signicance levels a = b = 0.05. The inconclusive and positive data were evaluated by the non-parametric U test of Mann, Whitney and Wilcoxon according to Frei and Wrgler (1995).

showed a mutation rate (0.075) even lower than water control (0.125) although statistically non-signicant. 3.5. Cytotoxicity assays of rocket

with the PEX-17 accession. The lethal doses 50 were 0.75, 0.75 and 1.5 mg mL1 for PEX-1, PEX-11 and PEX-14 accessions, respectively. 4. Discussion

Different trends and inhibitory concentrations (IC50) were observed depending on the plant material used (Fig. 1). Relative growth of tumour cells decreased in the three experiments with PEX-1, PEX-11 and PEX-14 accessions, while IC50 was not reached

Contamination of soils by metal(oid)s is a worldwide problem that involves a great risk for the environment, wildlife and human health, mostly caused by their entry in the food chain.

Fig. 1. Effects of rocket extracts from accessions (PEX-1, PEX-11, PEX-14 and PEX-17) grown in metal-contaminated soils on viability of HL-60 cells. Cell viability was assessed by trypan blue exclusion assay. Data are expressed as percentages of control (mean standard deviation values from three independent experiments).

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In this research, all elements showed a low extractability in the contaminated soil due to the previous chemical treatments (soil amendments with calcium carbonate and ferric oxides) used by regional authorities to x metals in the soil of Aznalcllar (Junta De Andaluca, 2001) (Table 1). These results are in concordance with other authors (Del Ro et al., 2002; Madejn et al., 2007) who concluded in their studies that elements showed low mobility in the soil of Aznalcllar. The results of the present work showed that only PEX-17 accession accumulated signicantly higher concentration of As, Pb, Cd and Zn in the leaves compared to the other accessions in contaminated soils. Differential metal accumulation by species of Brassicaceace family has been previously reported (Sharma et al., 2010). According to the metal transfer factor coefcient, PEX-17 accession showed signicantly higher (P < 0.05) ability to concentrate the contaminant than other accessions. Our ndings are in agreement with previous studies, which reported similar TC for Cd ratio (3.66) (Abe et al., 2008) to ours, although coefcients lower than our values (0.510.85) have also been reported (Fotiadis et al., 2008). With regard to Zn, TC for PEX-17 (1.64) was somewhat higher (0.81) than those found by Terzano et al. (2008). Higher TC values for As and Pb than ours have been reported, with TC = 2.06 for As (Stilwell et al., 2006) and TC = 2.745.80 for Pb (Euzbio Filho et al., 1998). Our results suggest that PEX-17 accession showed limited accumulation of these elements (As and Pb) in their leaves. In general, the results indicated that none of the accessions were identied as hyperaccumulator because they accumulated < 100 mg Cd kg1, <1000 mg kg1 for As, Pb and <10 000 mg kg1 for Zn (Baker and Brooks, 1989; Zhou and Song, 2004). Based on TC values, PEX-1, PEX-11 and PEX-14 accessions could have potential for phytostabilization uses due to all three accessions excluded metal(oid)s from their leaf tissues. From the human and animal consumption scope, Pb, Cd and Zn concentrations in PEX-17 accession in our study were over the concentrations allowed by the statutory limit set for metal(oid) content in vegetables (3, 0.5 and 60 mg kg1 dw, respectively). With regard to As concentration (1.96 mg kg1 dw), it was below the maximum content allowed in vegetables (10 mg kg1 dw) (ANZFZ, 1998; Codex Alimentarius Commission (FAO/WHO), 2001; Commission Regulation, 2008). In relation to the total GL and GRA contents of rocket leaves, this study has revealed concentrations higher (PEX-1 and PEX-11 accessions grown in contaminated soil) than those found in other studies (Bennet et al., 2006; Kim and Ishii, 2006). This fact is of great importance for their contribution to the chemopreventive activity of cruciferous. The GL concentration of rocket plants grown in contaminated soils in this study were unaffected by soil metal(oid) concentrations. However, a remarkable trend was observed towards a higher total GL and GRA contents, especially evident for the PEX-1 and PEX-11 accessions (Table 3). With respect to the genotoxicity assays, no genotoxic effects were observed in the four accessions when compared to the water negative control. Nevertheless, a remarkable trend in PEX-17 accession to genotoxicity was found by doubling the control spot/wing rate of total genotoxic damage in the highest concentration assayed. Regarding the chemical elements characterized in the present research i.e. metal(oid)s and glucosinolate prole, the tandem high metal-low glucosinolate combined variable was only fullled by PEX-17 accession. Although PEX-14 accession exhibited low glucosinolate content, its value of metal content and TC index was similar to the other studied accessions.PEX-1 and PEX-11 showed high GL concentration in leaves containing low levels of metal(oid)s (Tables 2 and 3)). Therefore, our data partially supported the trade-off hypothesis proposed previously (Martens

and Boyd, 1994), which stating that when plants accumulate metals it would induce less GLs providing an adaptive advantage against biotic stress to accumulator plants (Asad et al., 2013). Consequently the metal content was the variable that could be the cause of the high mutation rates observed in the somatic mutation in vivo system of Drosophila melanogaster for PEX-17 accession. Metals are all potential carcinogens; being effective mutagens and have been extensively described (Finney and OHalloran, 2003; Donma and Donma, 2005; Mishra et al., 2010). Previous works have also showed different genotoxic effects on plants grown in contaminated soils. Soils containing Pb higher concentrations than ours (10 000 mg kg1) resulted non genotoxic in a Nicotiana plant model (Mouchet et al., 2008) but, contrarily, Pb was genotoxic on the micronucleous test in Vicia faba system (Shahid et al., 2011). Treatments of Zn (350 mg kg1) on roots of Phaseolus vulgaris induced changes in the RAPD proles (Cenkci et al., 2009), and Al-Shami et al. (2012) determined genotoxic effects of Zn when tested at 50 mg L1 concentration in the comet assay of Chironomus kiiensis. Similar to the Pb, detecting genotoxicity for cadmium strongly depends on the tester system rather than on the used concentrations. Monteiro et al. (2010) could show DNA clastogenic damage of Cd (10 and 100 lM) in Lactuca sativa but neither in Thlaspi caerulescens nor Thlaspi arvense, independently of their accumulator or non-accumulator ability. In the case of arsenic, a well-known genotoxin and carcinogen, Rizki et al. (2006) concluded that inorganic arsenic was non-genotoxic in the SMART test for Drosophila melanogaster. Villatoro-Pulido et al. (2009) found genotoxic effects by the metal-contaminated aerial parts of Raphanus using the SMART test. Our results, not reaching the signicant level but doubling the control rate, are nearer the ndings by the last authors, concluding that the relative genetic damage observed in somatic proliferative cells of Drosophila treated with the PEX-17 accession can be due to the presence of As, Pb, Cd and Zn elements. Similar results are observed in the cytotoxicity assays where IC50 was reached in all the accessions except the PEX-17 accession with the lowest concentration of glucosinolates. In a previous study Valeriana wallichii and Xanthium strumarium were tested for heavy metal content and cytotoxicity on brine shrimps. They showed cytotoxicity values of 1.930 lg mL1 and 4.973 lg mL1 respectively, which are much lower than the values obtained in this study, probably due to our higher concentrations of Zn (165 mg kg1) (Khuda et al., 2012). Our results are supported by evidence from experimental studies in mammals, where methylated metabolites of arsenic are more genotoxic than inorganic arsenic since Drosophila is unable to biotransform arsenic to methylated forms (Rizki et al., 2006). Drosophila by itself can only detect methylated forms of As, and needs a previous bioactivation in the plant matrix of rocket to produce high mutation rates. Cadmium induces many genotoxic events like DNA strand breaks, sister chromatid exchanges and chromosomal aberrations in human cells (Donma and Donma, 2005); on the other hand lead is considered a potential mutagen by inducing direct DNA damage, clastogenicity and inhibition of DNA synthesis, or interfering with DNA repair (Donma and Donma, 2005). Zinc is a trace element which, when in excess is deleterious because it may displace other trace metals leading to protein dysfunction (Finney and OHalloran, 2003). The genotoxicity of zinc has been studied in different shortterm mutagenicity assays with controversial results (Thompson et al., 1989). Zinc acetate resulted mutagenic in two of the assays, L5178Y mouse lymphoma assay and in the in vitro cytogenetic assay with Chinese hamster ovary cells, althougth it did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. The zinc acetate showed also to be, neither mutagenic nor toxic in

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M. Villatoro-Pulido et al. / Chemosphere 93 (2013) 25542561 Anazetti, M.C., Melo, P.S., Duran, N., Haun, M., 2003. Comparative cytotoxicity of dimethylamide-crotonin in the promyelocytic leukemia cell line (HL60) and human peripheral blood mononuclear cells. Toxicology 188, 261274. Anter, J., Fernndez-Bdmar, Z., Villatoro-Pulido, M., Demyda-Peyrasa, S., MorenoMilln, M., Alonso-Moraga, A., Muoz-Serrano, A., Luque de Castro, M.D., 2011. A pilot study on the DNA-protective, cytotoxic, and apoptosis-inducing properties of olive-leaf extracts. Mutat. Res. 723, 165170. ANZFZ, 1998. Australian Food Standard Code. Australia New Zealand Food Authority, Camberra. 41. Asad, S.A., Young, S., West, H., 2013. Effect of nickel and cadmium on glucosinolate production in Thlaspi caerulescens. Pal. J. Bot. 45 (S1), 495500. Baker, A.J.M., Brooks, R.R., 1989. Terrestrial higher plants which hyperaccumulate metallic elements: a review of their distribution, ecology, and phytochemistry. Biorecovery 1, 81126. Bennet, R.N., Rosa, E.A.S., Mellon, F.A., Kroon, P.A., 2006. Ontogenic proling of glucosinolates, avonoids, and other secondary metabolites in Eruca sativa (salad rocket), Diplotaxis erucoides (wall rocket), Diplotaxis tenuifolia (wild rocket), and Bunias orientalis (Turkish rocket). J. Agric. Food Chem. 54, 4005 4015. BOJA (Boletn Ocial de la Junta de Andaluca), vol. 5, Seville, 12th January, 1999. pp. 408409. Bozokalfa, M.K., Yagmur, B., Ilbi, H., Esiyok, D., Kavak, S., 2009. Genetic variability for mineral concentration of Eruca sativa L. and Diplotaxis tenuifolia L. accessions. Crop Breed. Appl. Biotechnol. 9, 372381. erci, I.H., Konuk, M., Bozdag , A., 2009. Toxic chemicalsCenkci, S., Yildiz, M., Cig induced genotoxicity detected by random amplied polymorphic DNA (RAPD) in bean (Phaseolus vulgaris L.) seedlings. Chemosphere 76, 900906. Codex Alimentarius Commission (FAO/WHO), 2001. Food additives and contaminants. Joint FAO/WHO Food Standards Programme, ALINORM 01/12A. pp. 1289. Commission Regulation (EC) No. 629/2008 of 2 July, 2008. Amending Regulation (EC) No. 1881/2006 Setting Maximum Levels for Certain Contaminants in Foodstuffs (1), Ofcial Journal of the European Union, 2008. Del Ro, M., Font, R., Almela, C., Velez, D., Montoro, R.De., Haro Bailn, A., 2002. Heavy metals and arsenic uptake by wild vegetation in the Guadiamar river area after the toxic spill of the Aznalcllar mine. J. Biotechnol. 98, 125137. Donma, O., Donma, M., 2005. Cadmium, lead and phytochemicals. Med. Hypotheses. 65, 699702. Ebbs, S.D., Kochian, L.V., 1997. Toxicity of zinc and copper to Brassica species: implications for phytoremediation. J. Environ. Qual. 26, 776781. Euzbio Filho, O., Scalize, F.E., Ferreira, M.E., Pessa da Cruz, M.C., 1998. Dry matter and heavy metal contents in Eruca sativa L. fertilized with urban waste vermicompost. Proceedings of the 16th World Congress of Soil Science. 2026 August; Montpellier, France. <http://natres.psu.ac.th/Link/SoilCongress/en/ symt40.htm>. Fenwick, G.R., Heaney, R.K., Mullin, W.J., 1983. Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr. 18, 123201. Finney, L.A., OHalloran, T.V., 2003. Transition metal speciation in the cell: insights from the chemistry of metal ion receptors. Science 300, 931936. Fotiadis, E.A., Vardavakis, E., Lolas, P.C., 2008. Assessment of remediation potential of certain weed and crop species in Cd contaminated soils. In: Proceedings of the International conference Protection and Restoration of the Environment IX, 29 June3 July 2008, Kefalonia, Greece. Frei, H., Wrgler, F.E., 1988. Statistical methods to decide whether mutagenicity test data from Drosophila assays indicate a positive, negative, or inconclusive result. Mutat. Res. 203, 297308. Frei, H., Wrgler, F.E., 1995. Optimal experimental design and sample size for the statistical evaluation of data from somatic mutation and recombination tests (SMART) in Drosophila. Mutat. Res. 334, 247258. Graf, U., Abraham, S.K., Guzmn-Rincn, J., Wrgler, F.E., 1998. Antigenotoxicity studies in Drosophila melanogaster. Mutat. Res. 402, 203209. Graf, U., Wrgler, F.E., Katz, A.J., Frei, H., Juon, H., Hall, C.B., Kale, P.G., 1984. Somatic mutation and recombination test in Drosophila melanogaster. Environ. Mutagen. 6, 153188. Griepink, B., Muntau, H., 1988. The certication of the contents (mass fractions) of As, Cd, Cu, Pb, Se and Zn in a sea lettuce (Ulva lactuca). CRM 279. Report no EUR 11185 EN, Luxembourg: Commission of the European, Communities, 1988. Guzman, G., Alcantara, E., Barron, V., Torrent, J., 1994. Phytoavailability of phosphate adsorbed on ferrihydrite, hematite, and goethite. Plant Soil. 159, 219225. Junta de Andaluca, 2001. Corredor Verde del Guadiamar, Corredor Verde del Guadiamar (Eds.), Consejera de Medio Ambiente de la Junta de Andaluca, Junta de Andaluca, Sevilla, Spain. pp. 170. Kabata-Pendias, A., Mukherjee, A.B., 2007. Trace Elements from Soil to Human. Springer-Verlag, Berlin. p. 550. Kabata-Pendias, A., Pendias, H., 1992. Trace Elements in Soils and Plants. CRC Press, Florida. p. 365. Khoobchandani, M., Ganesh, N., Gabbanini, S., Valgimigli, L., Srivastava, M.M., 2011. Phytochemical potential of Eruca sativa for inhibition of melanoma tumor growth. Fitoterapia 82, 647653. Khuda, F., Iqbal, Z., Zakiullah, Khan, A., Nasir, F., Muhammad, N., Khan, J.A., Khan, M.S., 2012. Metal analysis, phytotoxic, insecticidal and cytotoxic activities of selected medicinal plants of Khyber Pakhtunkhwa. Pak. J. Pharm. Sci. 25 (1), 51 58.

the Salmonella mutation assay. Nevertheless, when bonded to an organic ligand like 2,4-pentanedione the authors could observe mutagenic results (Thompson et al., 1989). The results of genotoxicity testing in Drosophila using metal contaminated rocket are partially in agreement with the genotoxicity observed for heavy metals in the above mentioned works as the accession with significant accumulation of metals is the only one in the border line of genotoxicity. A palliative factor that could account for the non-genotoxic effects of treatments with metal contaminated rocket could be the GL content of the plant. Many research articles deal with GLs and generally emphasize the toxicity and antinutritive properties in animals (Mawson et al., 1994). Much less has been written about the potentially benecial activities of GLs and their enzymatic hydrolysis products (Mithen, 2001). In human nutrition, GLs are important for their avor, and their degradation products (isothiocyanates) can act as antioxidant, antimutagenic and antiproliferative compounds, but also as potent agents with biocidal potential (bactericide, fungicide, herbicide and pesticide) (Vig et al., 2009). They have also shown antigenotoxic effects in mice and Salmonella systems and an inhibitory activity against leukaemia HL60 cells among others (Vig et al., 2009). Our results t in the main corpus of healthy and chemopreventive data, as the accession with the lowest glucosinolate contents (PEX-17) shows the higher mutation rates, suggesting that GL contents in rocket contaminated with metal(oid)s would help to block the formation of endogenous and exogenous carcinogens and, consequently, would prevent initiation of the carcinogenesis process. In fact, the PEX-17 accession is unable to reduce the proliferation of HL60 cells, but on the contrary, it accelerates the cell viability up to 150% respect to the control (Fig. 1). 5. Conclusions In summary, the results obtained in the present assay from rocket plants grown in contaminated soils revealed, (a) negative relationships between the metal(oid) uptake and the total GL and GRA contents, (b) positive relationships between the metal(oid) contents and the mean total mutation rates as well as between the GL content and cytotoxicity and nally, (c) a negative relationship between the metal(oid) content and the cytotoxic potential. An interaction between metal(oid)s and the GL degradation products contained in the rocket leaves is suggested as the main modulator agents of the biological activity of the plants grown in soils contaminated with metal(oid)s. Acknowledgements The authors thank to the Department for Innovation, Science and Business of the Regional Autonomous Government of Andalusia for funding the Project P06-AGR-02230. We would also like to thank Nicholas Davies and Juan Carlos Villatoro for their help in grammatical revision of the manuscript. References
Abe, T., Fukami, M., Ogasawara, W., 2008. Cadmium accumulation in the shoots and roots of 93 weed species. J. Soil Sci. Plant Nutr. 54, 566573. Adriano, D.C., 2001. Trace Elements in Terrestrial Environments: Biochemistry, Bioavailability and Risks of Metals. Springer-Verlag, New York. Al-Shami, S.A., Md Rawi, C.S., Mohd Nor, S.A., Ahmad, A.H., Mohd Ishadi, N.A., Dieng, H., 2012. Genotoxicity in Chironomus kiiensis (Chironomidae: diptera) after exposure to polluted sediments from rivers of north peninsular Malaysia: implication for ecotoxicological monitoring. River Res. Appl., 14671535. Al-Qurainy, F., 2010. Application of inter simple sequence repeat (ISSR marker) to detect genotoxic effect of heavy metals on Eruca sativa (L.). African J. Biotechnol. 9, 467474. Al-Qurainy, F., Alameri, A.A., Khan, S., 2010. RAPD prole for the assessment of genotoxicity on a medicinal plant; Eruca sativa. J. Med. Plant. Res. 4, 579586.

Author's personal copy

M. Villatoro-Pulido et al. / Chemosphere 93 (2013) 25542561 Kim, S.J.Ishii, G., 2006. Glucosinolate proles in the seeds, leaves and roots of rocket salad (Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and puried 4-methoxyglucobrassicin. Soil Sci. Plant Nutr. 52, 394400. Lamy, E., Schrder, J., Paulus, S., Brenk, P., Stahl, T., Mersch-Sundermann, V., 2008. Antigenotoxic properties of Eruca sativa (rocket plant), erucin and erysolin in human hepatoma (HepG2) cells towards benzo(a)pyrene and their mode of action. Food. Chem. Toxicol. 46, 24152421. Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci. Soc. Amer. J. 42, 421428. Madejn, P., Murillo, J.M., Maran, T., Lepp, N.W., 2007. Factors affecting accumulation of thallium and other trace elements in two wild Brassicaceae spontaneously growing on soils contaminated by tailings dam waste. Chemosphere 67, 2028. Martens, S.N., Boyd, R.S., 1994. The ecological signicance of nickel hyperaccumulation: a plant chemical defense. Oecologia 98, 379384. Mawson, R., Heaney, R.K., Zdunczyk, Z., Kozlowska, H., 1994. Rapeseed mealglucosinolates and their antinutritional effects Part 4. Goitrogenicity and internal organs abnormalities in animals. Mol. Nutr. Food Res. 38, 178191. Mishra, S., Dwivedi, S.P., Singh, R.B.A., 2010. Review on epigenetic effect of heavy metal carcinogens on human health. Open Nutr. J. 3, 188193. Mithen, R., 2001. Glucosinolates-biochemistry, genetics and biological activity. Plant Growth Reg. 34, 91103. Monteiro, M.S., Rodriguez, E., Loureiro, J., Mann, R.M., Soares, A.M., Santos, C., 2010. Flow cytometric assessment of Cd genotoxicity in three plants with different metal accumulation and detoxication capacities. Ecotox. Environ. Safety 73, 12311237. Mouchet, F., Cren, S., Deydier, E., Guilet, R., Gauthier, L., 2008. Preliminary study of lead (Pb) immobilization by meat and bone meal combustion residues (MBMCR) in soil: assessment of Pb toxicity (phytotoxicity and genotoxicity) using the tobacco model (Nicotiana tabacum var. xanthi Dulieu).. Biometals 21, 443458. Muoz, O., Devesa, V., Suer, M.A., Vlez, D., Montoro, R., Urieta, I., Macho, M.L., Jaln Principio del formulario, M., 2000. Total and inorganic arsenic in fresh and processed sh products. J. Agric. Food Chem. 48, 43694376. Rizki, M., Kossatz, E., Velzquez, A., Creus, A., Farina, M., Fortaner, S., Sabbioni, E., Marcos, R., 2006. Metabolism of arsenic in Drosophila melanogaster and the genotoxicity of dimethylarsinic acid in the Drosophila wing spot test. Environ. Mol. Mutagen. 47, 162168. Romero-Jimnez, M., Campos-Snchez, J., Analla, M., Muoz-Serrano, A., AlonsoMoraga, A., 2005. Genotoxicity and antigenotoxicity of some traditional medicinal herbs. Mutat. Res. 585, 147155. Saleh, A.A.H., 2001. Effect of Cd and Pb on growth, certain antioxidant enzymes activity, protein prole and accumulation of Cd, Pb and Fe in Raphanus sativus and Eruca sativa seedlings. Proc. of the First International Conference (Egyptian British Biological Society, EBB Soc). Egypt. J. Biol. 3, 131139. Santos, A., Alonso, E., Callejn, M., Jimnez, J.C., 2002. Heavy metal content and speciation in groundwater of the Guadiamar river basin. Chemosphere 48, 279285.

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Shahid, M., Pinelli, E., Pourrut, B., Silvestre, J., Dumat, C., 2011. Pb-induced genotoxicity to Vicia faba L. roots in relation with metal cell uptake and initial speciation. Ecotox. Environ. Safety 74, 7884. Sharma, A., Sainger, M., Dwivedi, S., Srivastava, S., Tripathi, R.D., Singh, R.P., 2010. Genotypic variation in Brassica juncea (L.) Czern. cultivars in growth, nitrate assimilation, antioxidant responses and phytoremediation potential during cadmium stress. J. Environ. Biol. 31, 773780. SPSS, 2000. SPSS for Windows, Version 10.0. (19891999) SPSS Inc., Chicago. Stilwell, D.E., Musante, C.L., Sawhney, B.L., 2006. Copper, Chromium and Arsenic in soil and plants near coated and uncoated CCA wood. Proc. Ann. Int. Conf. Soils, Sediments, Water Energy 11, 147163. Tasset-Cuevas, I., Fernndez-Bedmar, Z., Lozano-Baena, M.D., Campos-Snchez, J., De Haro-Bailn, A., Muoz-Serrano, A., Alonso-Moraga, A., 2013. Protective Effect of Borage Seed Oil and Gamma Linolenic Acid on DNA. In Vivo and In Vitro Studies. PLoS ONE 8 (2), e56986. http://dx.doi.org/10.1371/ journal.pone.0056986. Terzano, R., Al Chami, Z., Vekemans, B., Miano, T., Ruggiero, P., 2008. Zinc distribution and speciation within rocket plants (Eruca vesicaria L. Cavalieri) Ggrown on a polluted soil amended with compost as determined by XRF microtomography and micro-XANES. J. Agric. Food Chem. 56, 32223231. Thompson, E.D., McDermott, J.A., Zerkle, T.B., Skare, J.A., Evans, B.L., Cody, D.B., 1989. Genotoxicity of zinc in 4 short-term mutagenicity assays. Mutat. Res. 223, 267 272. USDA-SSS, 1999. Soil Taxonomy. Agriculture handbook nr. 436 2nd ed. Soil Survey Staff SSS, Natural Resources Conservation Service NRCS, United States Department of Agriculture USDA, U.S. Government Printing Ofce, Washington, DC 20402. Vig, A.P., Rampal, G., Thind, T.S., Arora, S., 2009. Bio-protective effects of glucosinolates A review. LWT-Food Sci. Technol. 42, 15611572. Villatoro-Pulido, M., Font, R., De Haro-Bravo, M.I., Romero-Jimnez, M., Anter, J., De Haro Bailn, A., Alonso-Moraga, A., Del Ro-Celestino, M., 2009. Modulation of genotoxicity and cytotoxicity by radish grown in metal-contaminated soils. Mutagenesis 24, 5157. Villatoro-Pulido, M., Font, R., Saha, S., Obregn-Cano, S., Anter, J., Muoz-Serrano, A., De Haro Bailn, A., Alonso-Moraga, A., Del Ro-Celestino, M., 2012a. In vivo biological activity of rocket extracts (Eruca vesicaria subsp. sativa (Miller) Thell) and sulforaphane. Food Chem. Toxicol. 50, 13841392. Villatoro-Pulido, M., Moreno Rojas, R., Muoz-Serrano, A., Cardeosa, V., Amaro Lpez, M.., Font, R., Del Ro-Celestino, M., 2012b. Characterization and prediction by near-infrared reectance of mineral composition of rocket (Eruca vesicaria subsp. sativa and Eruca vesicaria subsp. vesicaria). J. Sci. Food Agric. 92 (7), 13311340. Wathelet, J.P., Wagstaffe, P., Boenke, A., 1991. The certication of the total glucosinolate and sulphur contents of three rapeseeds (colza), CRMs 190, 366 and 367. Fresenius J. Anal. Chem. 347, 396399. Zhou, Q.X., Song, Y.F., 2004. Principles and Methods of Contaminated Soil Remediation. Science Press, Beijing, China.