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Mutagenesis Advance Access originally published online on September 23, 2008
Mutagenesis 2009 24(1):51-57; doi:10.1093/mutage/gen051
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Modulation of genotoxicity and cytotoxicity by radish grown in metal-contaminated soils

Myriam Villatoro-Pulido, Rafael Font, Maria Isabel De Haro-Bravo1, Magdalena Romero-Jiménez2, Jaouad Anter2, Antonio De Haro Bailón1, Ángeles Alonso-Moraga2,* and Mercedes Del Río-Celestino1

Departamento de Mejora y Biotecnologia de Cultivos Centro IFAPA Alameda del Obispo s/n, Apartado 3092, 14080 Córdoba, Spain 1Departamento de Mejora Genética Vegetal Instituto de Agricultura Sostenible (CSIC), Apartado 4084, 14080 Córdoba, Spain 2Departamento de Genética, C-5 Campus de Rabanales, Universidad de Córdoba, 14071 Córdoba, Spain

Members of the Brassicaceae family are known for their anticarcinogenic and genetic material protective effects. However, many of the species of this family accumulate high amounts of metals, which is an undesirable feature. Radish (Raphanus sativus L.) has shown to accumulate metals in roots to a higher extent than others members of Brassicaceae. The main objectives of this work are (i) to study the distribution of the accumulated As, Pb and Cd in radish plants and (ii) to establish the genotoxic, antigenotoxic and cytotoxic activities of the root and shoot of this vegetable. Results indicate that (i) the shoots of radish accumulate higher concentrations of metal(oid)s than roots; (ii) the shoots were genotoxic at the different concentrations studied, with the root showing such genotoxic effect only at the highest concentration assayed; (iii) the antigenotoxic potential of radish is reduced in plants with high metal content and (iv) the tumouricide activities of the radish plants were negatively correlated to their metal(oid) contents. An interaction between metal(oid)s and the isotyocianates (hydrolysis products of the glucosinolates) contained in the radish is suggested as the main modulator agents of the genotoxic activity of the plants grown in contaminated soils with metal(oid)s.


   Introduction
Top
 Introduction
Material and methods
 Results
 Discussion
 Funding
 References
 
Several studies have indicated that vegetables, particularly leafy crops, grown in heavy metals contaminated soils have higher concentrations of heavy metals than those grown in uncontaminated soil (1Go). A major pathway of soil contamination is through atmospheric deposition of heavy metals from point sources such as metaliferous mining, smelting, agricultural and industrial activities (2Go). In addition, foliar uptake of atmospheric heavy metals emissions has also been identified as an important pathway of metal contamination in vegetable crops (3Go).

Elements such as Pb, Cd and As are not eliminated and can accumulate in human vital organs producing progressive toxicity. Arsenic (As) is one of the most important global environmental toxicants. Chronic arsenic poisoning can cause serious health effects including cancers, melanosis, hyperkeratosis, restrictive lung disease, peripheral vascular disease, gangrene, diabetes mellitus, hypertension and ischaemic heart disease (4Go–7Go). Lead and cadmium are among the most abundant heavy metals and are particularly toxic. Cadmium can impair renal function, and some studies indicate a neoplastic effect (8Go). Lead is a well-known physiological and neurological toxic affecting many biochemical processes and almost every organ and system in the human body (9Go).

Typical uncontaminated agricultural soils contain 1–20 mg/kg of As in the soil (10Go), 2–300 mg/kg of Pb in the soil and 0.01–2 mg/kg of Cd in the soil (11Go). Generally, in unpolluted environments, ordinary crops do not accumulate enough arsenic to be toxic to humans. However, in arsenic contaminated soil, the uptake of arsenic by the plant tissue is significantly elevated, particularly in vegetables and edible crops (12Go). Therefore, there is, concern regarding accumulation of As in agricultural crops and vegetables grown in arsenic-affected areas.

Some members of the Brassicaceae family have been shown to accumulate from moderate to high levels of Pb, Cr, Cd, Ni, Zn and Cu (13Go). Carbonell-Barrachina et al. (14Go) also reported that radish (Raphanus sativus L.) plants grown on higher soil concentrations of As accumulated high As concentration in roots and shoots.

Besides the logical cautions about the use of contaminated soils, there is a great concern about As and heavy metal pollution in Spain due to an environmental accident in a pyrite mine located in the city of Aznalcóllar, Sevilla (Southern Spain) (15Go,16Go). Arsenic, lead and cadmium from these soils may accumulate in any of the agricultural species being grown in them and enter the human food chain through their edible parts.

This pollutants are all potential carcinogens and therefore they are dangerous when present in human diet. Studies of genotoxicity and antigenotoxicity can help to evaluate the risk/safety and effectiveness of healthy food products (17Go). The somatic mutation and recombination test (SMART) in wings of Drosophila melanogaster is a well-known eukaryotic assay based on the loss of heterozygosity for two genetic markers affecting the phenotype of wing hairs (18Go). This wing spot test is a versatile and reliable system to test complex mixtures for geno/antigenotoxicity. 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 single compound or as complex mixtures depending on the form on which they are up taken (19Go,20Go). A wide variety of compounds and complex mixtures have been assayed with this test, such as food additives, beverages and insecticides (21Go,22Go).

In the case of arsenic, a well-known genotoxin and carcinogen, Rizki et al. (23Go) concluded that inorganic arsenic was non-genotoxic in the SMART test for D.melanogaster. However, there have been no studies to test complex mixtures such as edible vegetables grown in contaminated soils that contain metals.

HL60 human leukaemia cells have been used in cytotoxicity assays in order to determine the tumouricide activity of the plant. The HL60 line was isolated from peripheral blood leukocytes of a 36-year-old Caucasian patient suffering from promyelocytic leukaemia (24Go). This cell line has been studied intensely for many years in order to clarify the mechanisms that induce the differentiation of normal cells into tumoural cells, with a view to control this proliferation in living organisms (25Go). In this way, the induction of differentiation and apoptosis in tumoural cells would be an efficient anticancer therapy strategy.

The main objectives of this work are (i) to study the uptake and distribution of As, Pb and Cd in radish plants and (ii) to establish the genotoxic, antigenotoxic and cytotoxic activities of this vegetable's roots and shoots.


   Material and methods
Top
 Introduction
 Material and methods
Results
 Discussion
 Funding
 References
 
Plant material and greenhouse experiments
The plant species studied was the variety of radish namely Middle East Giant of R.sativus L.

Pots were placed in the greenhouse under natural light, 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 had reached adequate height (8–12 cm), they were transferred to plastic pots containing 3 kg of contaminated soil in order to study the uptake and accumulation of As, Pb and Cd.

The contaminated soil was obtained from the experimental area ‘El Vicario’ (37° 26'21''N, 6°13'00''W) within the Green Corridor, close to the pyrite mine of Aznalcóllar (26Go). The soil was classified as Typic Haploxeralf. One week before planting, soil was mixed with commercial potting mixture (1:1 vol). The commercial potting mixture was used as control. The soil was a sandy loam soil (sand 50%, silt 33% and clay 17%) which chemical characteristics were pH = 6.0, Corg = 35%, NT = 0.3% and organic matter = 60%.

In order to study the As, Pb and Cd accumulation, a complete random design was used for 40 days of exposure. Controls with unpolluted soil were also included. All treatments were replicated 10 times.

Sample preparation and chemical analysis
The plants were separated into shoots and edible parts, washed with tap water, rinsed several times with distilled water and weighed to assess their biomass.

The arsenic concentration was determined by FIA-HG-AAS (27Go), and heavy metal contents (Pb and Cd) were determined by AAS with graphite chamber (Perkin Elmer Analyst 600 with an autosampler AS 800) (28Go). The accuracy and precision of the analytical methods was assessed by carrying out analyses of the Community Bureau of Reference reference sample CMR 279 (sea lettuce) (29Go). The values obtained for the reference sample by FIA-HG-AAS and AAS were concordant with the certified values (data not showed).

Genotoxicity assays
Strains Two Drosophila strains were used containing genetics markers on the left arm of chromosome 3: mwh/mwh, carrying the wing cell marker multiple wing hairs (mwh) and flr3/In(3LR)TM3, ri pp sep bx34e es BdS (flr3/TM3, BdS abbreviated). This wing cell marker flare (flr3) is a zygotic recessive lethal, which is maintained in the strain over the balancer chromosome TM3. More detailed genetic information is provided by Lindsley and Zimm (30Go).

Treatment procedure
Crosses Virgin females with the genotype flr3/TM3, BdS were mated to mwh/mwh males. An optimal design requires 300 females and 150 males. Flies are allowed to mate for 3 days in order to obtain an optimal production of hybrid eggs on the fourth day after mating.

Treatments Genotoxicity tests were carried out as described by Graf et al. (18Go). Hybrid eggs were collected over an 8-h period. After 72 ± 4 h later, the emergent larvae were washed from remaining feeding medium using a 20% sodium chloride solution and transferred to treatment vials. These vials contained 0.85 g of Drosophila Instant Medium (Formula 4-24, Carolina Biological Supply, Burlington, NC), and different concentrations of lyophilized vegetable samples wetted with distilled water. The negative controls were prepared with medium and water and positive controls with medium and hydrogen peroxide as oxidative genotoxin (22Go). Antigenotoxicity tests were performed by mixing the mutagen (hydrogen peroxide) with the lyophilized samples in appropriate concentrations. Larvae were fed until pupation (~48 h) at 25 ± 1°C. After emergence, adult flies were collected and stored in a 70% ethanol solution.

Wing scoring Twenty pairs of wings of each control and concentrations of transheterozygous marker flies (mwh flr+/mwh+ flr3) were removed and mounted on slides using Faure’s solution. Female and male wings were mounted separately. Both dorsal and ventral surfaces of the wings were analysed under a photonic microscope with the x400 magnification. Wing hair mutations (spots) were scored among a total of 24 000 monotricoma cells per wing. In the positive control and genotoxic single treatments, balancer wings (mwh/TM3, BdS) were also mounted.

In vitro cytotoxicity assays
Cell culture and incubation conditions The human leukaemia cell line HL60 (promyelocytic cells) was supplied by Dr José M. Villalba-Montoro (Department of Cell Biology, University of Cordoba, Spain). HL-60 myeloid leukaemia cells were grown in RPMI-1640 medium (Invitrogen, Verviers, Belgium) supplemented with the antibiotics penicillin, streptomycin and amphotericin (commercial mixture, A5955, antibiotic–antimycotic solution 100x stabilized, Sigma, St. Louis, MO, USA), L-glutamine (G7513, Sigma) and heat-inactivated foetal bovine serum (S01805, Linus), in a humidified atmosphere containing 5% CO2 at 37°C (Shel Lab, Cornelius, OR, USA) (31Go). Cultures were passed every 2–3 days to maintain logarithmic growth. Cells were grown at a density of 105 cells/ml before beginning the assay in 2-ml well plates. The HL-60 cells of the assays were incubated with increasing concentrations of filters from lyophilized plants whereas the negative controls had only culture medium. At least three independent repetitions of the assays were carried out to calculate means for statistical analysis.

Survival assay
Cell viability was determined by the trypan blue dye (T8154, Sigma) exclusion test. Cells were counted by adding an aliquot of 10 µl of the culture to 10 µl of the trypan blue dye. The mix of cells and dye were put on a Neubauer chamber and counted under a light inverted microscope (AE30/31, Motic). Aliquots were taken at 24, 36 48, 60 and 72 h of incubation. After each incubation period, a growth curve was established and IC50 values (concentration of tested compound causing 50% inhibition of cell growth) were estimated. Curves are expressed as survival percentage with respect to controls at 72 h of growth.

Data evaluation and statistical analysis
Student t-test [applied to data of metal(oids) contents of soil and plants grown in control and contaminated soils] was used to detect differences between soils and plants in their concentration of the two heavy metals (Pb and Cd) and As. The test allowed us to see how and where the plants concentrate the pollutants analysed. SPSS Version 10.0 software (32Go) was used to perform all statistical analyses.

For each plant, we calculated the shoot/root metal concentration quotient (MS/MR) as a measure to assess the metal(oid) uptake strategy of plants.

Wing spot data are broken down into three different categories: small single spots (S) consisting of 1 or 2 mwh or flr3 cells, large single spots (L) with three or more mwh or flr3 cells and twin spots (T) with mwh and flr3 cells. The total number of spots was evaluated.

For evaluation of the genotoxic effects, the frequency of spots in the treated assay was compared to negative controls, using distilled water. The statistical significance of spots frequency per wing was evaluated using a multi-decision procedure to determine whether a result was positive, negative or inconclusive based on two alternative hypotheses (33Go).

In the balancer-heterozygous genotype (mwh/TM3, BdS), mwh spots are produced mainly by somatic point mutation and chromosome aberrations since mitotic recombination between the balancer chromosome and its structurally normal homologue is a lethal event. To quantify the recombinogenic activity of the mutagenic samples, the frequency of mwh clones on the marker transheterozygous wings (mwh single spots plus twin spots) was compared with the frequency of mwh spots on the balancer transheterozygous wings. The difference in mwh clone frequency is a direct measure of the proportion of recombination (20Go).

The percentage of inhibition of mutagenic events by lyophilized samples was calculated from the control-corrected frequencies of total spots, as proposed by Abraham (34Go): [inhibition = (genotoxin alone-sample plus genotoxin) x 100/genotoxin alone].


   Results
Top
 Introduction
 Material and methods
 Results
Discussion
 Funding
 References
 
Availability and accumulation of metal(oids)
Soil analysis Table I shows concentrations of heavy metals (Pb and Cd) and arsenic of the contaminated soils used in this work. The mean concentration of total and diethylene triamine pentaacetic acid (DPTA) extractable arsenic in the contaminated soils (75 and 4 mg/kg, respectively) were significantly higher than control soil and higher than the upper limit of the range from normal soils, as shown in Table I (10Go,11Go). Mean concentration values of total and DPTA-extractable Pb (83 and 14 mg/kg) and Cd (0.4 and 0.08 mg/kg) in contaminated soil were significantly higher than control soil, although both Pb and Cd concentrations in contaminated soils were within the range found in normal soils (10Go,11Go). Available data in the literature show that the values of As concentration in contaminated soils (<20 mg/kg) can be considered toxic for plant growth (2Go). Plant-available metal was very low (Table I) and was within the ranges of normal soils.


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  		Table I. Total and extractable in DTPA concentrations (mean ± standard error, n = 6) of heavy metals and arsenic (mg/kg dry matter) of the sampling site

 
Table I shows concentrations of heavy metals (Pb and Cd) and arsenic of the contaminated soils used in this work. The mean concentration of As in the contaminated soils (75 mg/kg) was higher than the upper limit of the range from normal soils; as shown in Table I (10Go,11Go). Mean concentration values of Pb (83 mg/kg) and Cd (0.4 mg/kg) in contaminated soils were within the range found in normal soils. Available data in the literature show that the values of As concentration in contaminated soils (<20 mg/kg) can be considered toxic for plant growth (2Go). Plant-available metal was very low (Table I) and was within the ranges of normal soils.

Accumulation and distribution of Pb, Cd and As by radish plants
The metal(oid)s concentrations from plants grown in contaminated and uncontaminated soils are shown in Table II. Due to the low bioavailability of metals in the soils used (Table I), the accumulation of metals in the tissues of the plants studied was low (Table II). Significant differences were found for Pb, Cd and As concentrations in roots and shoots from radish plants from contaminated and uncontaminated sites (Table II).


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  		Table II. Pb, Cd and As concentrations (mg/kg dry weight) in shoots and roots of radish grown in contaminated and non-contaminated soils (mean ± standard deviation)

 
When radish plants were grown in contaminated soil, a little portion of As, Pb and Cd remained in the roots, with a major portion of the element translocating to the shoots (Table II). Radish plants accumulated significantly higher concentration of metal(oid)s in shoots as compared to the roots. The behaviour of the radish plants with respect to metal(oid)s was characterized in all the cases by MS/MR (shoot/root metal concentration quotient > > 1).

Genotoxicity assays of R.sativus L.
The SMART was applied to discern the genotoxicity and possible antigenotoxicity of radish plants grown in standard and metal-contaminated soils. The proliferative imaginal discs of the wing in Drosophila larvae gave the expected results for internal water-negative control and concurrent hydrogen peroxide-positive control. Hydrogen peroxide exhibited a total mutation rate (0.225 mutant clones per wing) which duplicates the control rate, implying that the accuracy of the genotoxicity and antigenotoxicity assays was ensured (22Go).

Table III shows the results obtained with the genotoxicity assays. Data are expressed as small single, large single, twin and total spots per wing scored in 40 transheterozygous wings for each assayed concentration. The non-metal-treated roots were not genotoxic for any of the concentrations (0.125 total spots per wing on average). The metal-treated roots showed genotoxic results but only at the highest concentration (5 mg/ml), and the metal-treated shoot parts were genotoxic for all the assayed concentrations (5, 2.5 and 0.625 mg/ml).


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  		Table III. Genotoxicity in the Drosophila SMART by radish

 
In order to evaluate the recombinogenic potency of mutagenic concentrations, we also looked at additional information on the spots per wing scored in balancer wings (Serrate phenotype) where no recombinogenicity is accounted (Table III). Values of recombinogenicity with respect to the total induced clones ranged from 16 to 50%, with the aerial part reaching the highest values (50%) at the two highest concentrations. It is remarkable that the hydrogen peroxide used as oxidative genotoxin gave a recombinogenic activity lower (44%) than the shoot of metal-treated radish.

All the samples tested for genotoxicity were tested in parallel for antigenotoxicity against the oxidative mutagen hydrogen peroxide in the Drosophila wing spot test. Hydrogen peroxide is a well-known mutagen in D.melanogaster. Studies published by Romero-Jiménez et al. (22Go) and Allen and Tresini (35Go) have described >200 effects of hydrogen peroxide on >100 genes, including those of stress. The results of the antimutagenic effects against hydrogen peroxide are shown in Table IV. The only sample that had genotoxic effect was the treated root at concentrations of 0.625 mg/ml. As expected, the antigenotoxic effects of non-contaminated roots were higher than those of roots grown in contaminated soils.


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  		Table IV. Antigenotoxicity in the Drosophila SMART by radish in combined treatments with the genotoxin hydrogen peroxide

 
For root of plants that grew in non-contaminated soils, the percentage of inhibition increased with the doses (0.11, 0.22 and 0.33 for the doses 0.625, 2.5 and 5 mg/ml, respectively, which means an average inhibition rate of 0.22). These results agree with data (data not shown, 36Go) from other species of the same family, in which a strong antimutagenic activity against hydrogen peroxide has been detected. Results obtained from the samples that grew in contaminated soil were diverse: the highest concentrations were capable of inhibiting the damage induced by hydrogen peroxide (0.11 inhibition average for treated root). Nevertheless, the lower concentrations of treated roots could not inhibit the effect, but increased the mutagenic activity of hydrogen peroxide.

Cytotoxicity assays
Figure 1a, b and c show the results of cytotoxicity assays performed using lyophilized radish material against exponential growing of HL-60 cancer cells. The curves express the survival percentage with respect to controls growing after 72 h of treatment. The relative growth of the tumour cells decreased as concentration increased in the three experiments. Nevertheless, shapes and IC50s were different for each case. The lethal dose 50 were reached at a concentration of 0.65 mg/ml for the control sample (Figure 1a) whereas this dose was reached at a concentration of 5 mg/ml with sample of contaminated roots (Figure 1b). This value was 15 times higher than the dose needed to inhibit tumour growth by control roots. As stated above, metals are related to cancer promotion, and the moderately high content of As, Cd and Pb in the treated roots could explain the high inhibitory concentrations needed. The IC50 was not reached with the samples from contaminated shoots. Higher concentrations of the samples were tested (data not shown) and the IC50 was never reached. In this case, two main factors could have influenced this unhealthy result: the high amount of metals incorporated and the lack of glucosinolates in the aerial part of the plant. It is needed to say that the radish shoot is not normally used as a food, but it is the root that is the edible part.


Figure 1
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  		Fig. 1. Relative growing of HL60 tumour cells at 72 h of treatments with non-metal-treated root (a), metal-treated root (b) and metal-treated shoot (c) samples.

 

   Discussion
Top
 Introduction
 Material and methods
 Results
 Discussion
Funding
 References
 
The accumulation in the tissues of the plants studied was low (17, 20 and 5% for Pb, Cd and As, respectively) (Table II) due to the previous chemical treatments (soil amendments with calcium carbonate and ferric oxides) used by regional authorities to fix metals in the soil of Aznalcóllar (37Go) (Table I).

The concentrations of Pb, Cd and As in shoots of radish (1.6, 0.9 and 3.9 mg/kg dw, respectively) were higher than those found in roots (edible part). These results contrast with those from Marchiol et al. (38Go), which reported a higher concentration of Pb and Cd in the root system of radish as compared to the shoots. Tlustos et al. (39Go) found that the distribution of arsenic among plants was affected by the rate of As in soil. Plants grown on lower As soil accumulated more As in the leaves than in the roots, whereas those grown on higher As soil had more As in the roots than in the leaves. Carbonell-Barrachina et al. (14Go) also reported that radish plants grown in soils with higher concentrations of As had a higher concentration of As in the roots than in the shoots.

From the perspective of human and animal consumption, the Pb, Cd and As concentrations in radish plants in our study were below the maximum concentrations allowed by the statutory limit set for metal(oid)s content in vegetables (3, 0.5 and 10 mg/kg dw, respectively) (40Go,41Go).

The levels of As, Pb and Cd found in radish plants in this experiment could have been higher considered the low bioavailability of As, Pb and Cd in the soils used. Further research is needed using higher As, Pb and Cd content in soils to determine the plant ability to accumulate these elements in their edible parts.

The results of genotoxicity observed for contaminated shoots and roots can be due to the presence of metal(oid)s that are related to DNA damage and have a negative influence on the protective role of radish. Nevertheless, although carcinogenicity of metals(oid)s is not always related to genotoxicity results, we found a clear relation between metal content and genotoxicity in our study .

In the case of arsenic, a well-known genotoxin and carcinogen, Rizki et al. (23Go) concluded that inorganic arsenic is non-genotoxic in the SMART test for Drosophila. Recent evidence from experimental studies in mammals indicates that methylated metabolites of arsenic are more genotoxic than inorganic arsenic. These authors (23Go) hypothesized that inorganic arsenic is non-genotoxic in Drosophila because they are unable to biotransform arsenic to methylated forms. The absence of biomethylation in Drosophila could explain the lack of genotoxicity for inorganic arsenic and the genotoxicity of methylated arsenic in the SMART wing spot assay found by these authors. Our experiment design solves this limitation of Drosophila. Radish grown in contaminated soils incorporate and bioactivate arsenic inorganic species in bioavailable molecules that results mutagenic for Drosophila. Our results open a new way to test complex single and complex compounds in Drosophila by feeding larvae not with the inactive molecule but with the molecules that are already bioactivated by plants in the same way and concentrations that would be consumed by humans.

Cadmium, a potent immunotoxic metal, induces DNA strand breaks, sister chromatid exchanges and chromosomal aberrations in human cells; 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 (42Go). The genotoxicity observed in Drosophila fed by contaminated radish could be due to the sum of the three mutagenic activities of As, Cd and Pb which are bioavailable and bioactivated.

The percentage of inhibition (Table IV) calculated by the algorithm of Abraham (34Go) for antigenotoxicity assays gives the reduction ability of mutagenic effect of the plant assayed against hydrogen peroxide. The synergism observed between hydrogen peroxide and metal(oid)s can activate several genes which codify stress enzymes that detoxify the effects of hydrogen peroxide, but only at the highest concentrations (43Go). The glucosinolate and isothyocyanate content of Raphanus could behave as desmutagens by counteracting the effect of the reactive oxygen species (ROS) generated by hydrogen peroxide and metals, but only when a certain threshold concentration is reached.

When assayed using model systems in which both intragenic and multilocus mutations can readily be detected, arsenic is, indeed, found to be a strong, dose-dependent mutagen which induces mostly multilocus deletions. Furthermore, the roles of oxygen and nitrogen reactive species in mediating the genotoxic response are presented in a systematic and logical fashion in support of a working model. The data from the study of Hei and Filipic (44Go) suggest that antioxidants may be a useful interventional treatment in reducing the deleterious effects of arsenic. Cd leads to the enhanced production of ROS and exerts its effects on cellular structure and mechanisms, and Pb may also generate ROS and cause oxidative damage to DNA. Pb can be substituted for Zn in several proteins which function as transcriptional regulators, e.g. Zn finger (42Go). Nevertheless, a negative synergism between the two types of doses of ROS (those produced by metals and those produced by hydrogen peroxide) is observed in our experiences, probably due to stress genes that become activated.

The cytotoxic activity of radish can be explained by the high content in glucosinolates and isothiocyanates in the root part of the plant (45Go). Evidence supporting the relation between metal(oid)s and cancer has been previously reported (5Go,8Go). In the case of the As, much of the evidence suggests that As and most of its derivates are cancer promoters rather than carcinogens in animal studies (46Go). The mechanisms of metal-induced carcinogenesis may involve induction of lipid peroxidation and an increase in the levels of free radical within the cells, following Pb or Cd exposure, suggesting that the induction of genotoxicity and carcinogenicity is achieved by indirect interactions (e.g. oxidative stress) of these metals with DNA (42Go). Cd affects cell proliferation and differentiation. This metal interferes with antioxidant defence mechanisms and stimulates the production of ROS, which may act as signalling molecules in the induction of gene expression (46Go) and suppression of apoptosis (42Go). The inhibition of DNA repair processes by Cd represents a mechanism by which the genotoxicity of other agents is enhanced and may contribute to the tumour initiation by this metal (47Go). Lead is considered as another potential human carcinogen. Cruciferous vegetables are also known to concentrate Cd and Pb, and these metals are considered to be potential human carcinogens (42Go).

The absence of genotoxicity of the non-contaminated root and the genotoxicity of contaminated shoot and root has been demonstrated. The antigenotoxic effects of non-contaminated roots are higher than the effects of the shoots grown in contaminated soil. All the samples showed tumouricide activity but with different rates of inhibition. The non-treated plants had the highest anti-proliferative activity. The glucosinolates and its hydrolysis products could be the principal modulator agents of the antigenotoxic and cytotoxic activities of the plants grown in soils contaminated with metals.

It has already been mentioned that governmental agencies have set limits for Pb and Cd concentrations above which horticultural crops of the family Brassicaceae is considered unsuitable for human consumption (40Go). This regulation sets the maximum limit for Pb and Cd in vegetables at 0.30 and 0.20 mg/kg wet weight, respectively, and 1 mg/kg wet weight for As (41Go). For edible part of radish analysed in the present study, total Pb, Cd and As concentrations were 0.02, 0.04 and 0.12 mg/kg wet weight (90% of water content) which did not exceed limits.

Also, the European Environment and Health Information System of the World Health Organization (WHO) has established regulatory guidelines regarding dietary Pb, Cd and As intake. It recommends a provisional tolerable weekly intake (PTWI) of 25, 7 and 15 µg/kg body wt of total Pb, Cd and As, respectively (48Go). To estimate the ‘degree’ of Pb, Cd and As intake through radish, our results were interpreted in terms of the WHO PTWI. Using the means of radish, weekly consumption of the Spanish population of 183 g (49Go), mean Pb, Cd and As concentrations in radish and human body weight (70 kg), weekly intake calculated were 5.20 x 10–2, 14.9 x 10–2 and 31.5 x 10–2 µg/kg body wt for Pb, Cd and As, respectively.

On the basis of the results obtained in this work, we can conclude that it is necessary to perform more studies to review the criteria used to set the maximum limits allowed by law regarding different metals. This statement is based on the fact that in spite of the estimated weekly intake of total Pb, Cd and As does not exceed the safety limits allowed by the WHO current legislation, this consumption of metals caused more genotoxic effects and less citotoxicity than the radish cultivated in non-contaminated soil. Both, SMART and cytotoxicity tests, offer a rapid and cost-effective first-pass screening capable to assess toxicity when conventional toxicology data are limited or lacking.


   Funding
Top
 Introduction
 Material and methods
 Results
 Discussion
 Funding
References
 
Consejería de Agricultura y Pesca (Junta de Andalucía, Spain) (C03-070).


   Acknowledgments
 
The authors thank Gloria Fernández (IAS-CSIC, Córdoba) for technical assistance in the analysis of plants.

Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +34 957 218674; Fax: +34 957 212072; Email: ge1almoa{at}uco.es


   References
Top
 Introduction
 Material and methods
 Results
 Discussion
 Funding
 References
 

    1. Guttormsen G, Singh BR, Jeng AS. Cadmium concentrations in vegetable crops grown in a sandy soil as affected by Cd levels in fertiliser and soil pH. Fert. Res. (1995) 41:27–32.[CrossRef]

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    3. Salim R, Al-Subu MM, Douleh A, Chenavier L, Hagemeyer J. Effects of root and foliar treatments on carrot plants with lead and cadmium on the growth, uptake and the distribution of metals in treated plants. J. Environ. Sci. Health Part A (1992) 27:1739–1758.

    4. Chen CJ, Chiou HY, Chiang MH, Lin TM, Tai TY. Dose-response relationship between ischemic heart disease mortality and long-term arsenic exposure. Arterioscler. Tromb. Vasc. Biol. (1996) 16:504–510.[Abstract/Free Full Text]

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Received on May 16, 2008; revised on August 1, 2008; accepted on August 22, 2008.


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