Investigation of DNA changes in wheat ( Triticum aestivum L . ) induced by cadmium using random amplified polymorphic DNA ( RAPD ) analysis

In recent years, many plants have been used as bioindicators or living indices and several experiments have been conducted to evaluate the genotoxic effects of environmental pollution on plant species. Plants such as Arabidopsis and barley have been used as biological indicators by several researchers and have been able to identify DNA changes using molecular markers. This study was conducted on wheat as an important cereal that provides human daily food and has an important role in human health. The purpose of study was to evaluate the effect of different concentrations of cadmium on amounts of the soluble proteins, investigation of growth and possible changes to the structure of DNA using random amplified polymorphic DNA (RAPD) marker. In the present study, wheat seedlings were used for detection of genotoxic effects of cadmium contamination in the range of 0 to 120 mgl -1 . The limiting effects of cadmium on length of root and plant height and total soluble proteins in root were quite evident by increase of cadmium concentration. Change in DNA was observed using RAPD marker as there was change in number of bands, present or absent of bands at the range of above 5 mgl -1


INTRODUCTION
In the last several decades, cadmium (Cd) has been used extensively as materials and/or intermediates in the chemical industry and agriculture for electroplating, metallurgy, painting, plastic production, etc., and is being released into the biosphere, and largely found in the water and soil (Baryla et al., 2001;Liu et al., 2005;Shahrtash et al., 2010).Through food chain, Cd accumulation could pose a direct threat to human health and cause diseases such as hypertension, angiopathy, kidney and bone function decay (Liu et al., 2005).The toxic and genotoxic effects of cadmium have been demonstrated in different plant and animal species and it has been shown that cadmium caused change in DNA and cellular structures (Conte et al., 1998;Liu et al., 2005;Shahrtash et al., 2010).However, the molecular mechanism responsible for the genotoxicity of cadmium remains unclear.It has been suggested that it may involve direct interaction of Cd 2+ with DNA through the binding of Cd at G, A and T bases (Valverde et al., 2001;Hossain and Huq, 2002;Anastassopoulou, 2003).Furthermore, recent studies indicate that Cd acts as a mutagen primarily by direct inhibition of an essential DNA mismatch repair, resulting in a high level of genetic instability (Andrea, 1994;Jin et al., 2003).Cadmium cellular toxicity and genotoxicity may also be mediated indirectly; cells under oxidative stress display various dysfunctions due to lesions caused by reactive oxygen species (ROS, e.g.O 2 − , H 2 O 2 and OH − ) to lipids, proteins and DNA (Waisberg et al., 2003;Joseph, 2009).Advantages of measuring effects of genotoxic chemicals directly on DNA are mainly related to the sensitivity and short response time.Recently, advances in molecular biology have led to the development of a number of selective and sensitive assays for DNA analysis in ecogenotoxicology.DNA based techniques [RFLP, QTL, random amplified polymorphic DNA (RAPD), AFLP, SSR and VNTR] are used to evaluate the variation at the DNA sequence level.RAPD of these techniques can be used to detect genotoxicity and differences in RAPD profiles can clearly be shown when comparing DNA fingerprints from untreated and treated individuals to genotoxic agents (Savva, 1996;Conte et al., 1998;Theodorakis and Bickham, 2001;Enan, 2006;Atienzar and Jha, 2006;Liu et al., 2007;Cenkci et al., 2009).The aim of this study was to detect DNA damage induced by Cd using the RAPD technique.Detection of the genotoxic effect involves comparison of RAPD profiles of the root tip DNA generated by control and treated wheat seedlings.

Plant material, growth and total soluble protein level test of seedlings
Seeds of Cascogen wheat cultivar provided by Seed and Plant Improvement Institute of Mashhad, were sterilized by using incubation in 5% (w/v) sodium hypochlorite, soaked in distilled water, incubated for three days in a dark growth chamber at temperature of 18 ± 1°C and germinated to primary roots of 1 cm long.Uniformly 100 germinated seeds were selected and transferred to Petri pots (length 18 cm, width 10 cm and height 7 cm) containing 0.5 kg sandy soil of 1 to 3 mm (washed with 1 N HCl) with 200 ml of distilled water or test solution.The growth inhibition test was performed with the above wheat plantlets exposed to 0, 5, 10, 15, 20, 30, 60, 90, 120 mgl -1 Cd (in the form of CdCl 2. H 2 O with purity of 99.9% (Merk, Germany) for nine days, respectively.Petri pots were incubated in a growth chamber at temperature of 20 ± 1°C and a 16-h-day-8-h-night photoperiod.After nine days of incubation, the root and shoot length was measured using a ruler and total soluble protein level of root-tips in plantlets were measured with Bradford method (1976) by using nanodrop (Thermo scientific nanodrop 2000 Spectophotometry, U.S.A).The data were expressed as mean ± HSD.Inhibitory rate (IR, %) of the above indices was calculated using the following formula: IR = (1-x/y) × 100, where x and y are the average values detected in the control and each sample treated, respectively.Three replicate were set up for each treatment and comparison experiments were carried out as duplicate.The differences between mean values of treated plantlets and the controls were statistically investigated using HSD tests.

DNA extraction and RAPD procedures
After nine days of growth, approximately 1 cm root tips of seedlings were collected, ground in liquid nitrogen, and total genomic DNA was extracted using DNA purification kit (Germany, 5 prime).Purified DNA concentration and integrity of total genomic DNA in each sample were estimated by nanodrop (Thermo scientific nanodrop 2000 Spectophotometry., U.S.A).The PCR amplification was carried out with 15 10-base pair random primers (Bioneercompany) and genomic DNA as the template (Table 1).PCR were performed in a reaction mixture of 25 μl containing approximately 75 ng of the genomic DNA dissolving in sterile TE, 10X PCR buffer (2/5 μl), 2 mM MgCl 2 , 0.2 mM of each dNTP, 2 μl of 10 μM primer and 1 U Taq DNA polymerase.Amplifications were performed in a DNA thermocycler (Biometra.,U.S.A) programmed for 4 min at 95°C (initial denaturing step), 35 consecutive cycles each consisting of 4 min at 94°C (denaturing), 1 min at 34°C (annealing), 2 min at 72°C (extension), and followed by 1 cycle for 10 min at 72°C (final extension step).Negative controls with water, without any template DNA, were always included to monitor contamination.PCR reaction products were mixed with one-sixth volume of gel loading buffer (Fermentas, Germany), and then separated on 1.1% agarose gel using Tris-Borate-EDTA (TBE) buffer and GeneRuler DNA ladder (Fermentas, Germany).DNA bands were stained with ethidium bromide, visualized and photographed under UV light.The size and intensity of each amplification product was automatically estimated using the UV-Transilluminator image analyzer system.

RAPD profile and data analysis
All amplifications were repeated twice in order to confirm the reproducible amplification of scored fragments.Only reproducible and clear amplification bands were scored for the construction of the data matrix.Polymorphism observed in RAPD profiles included disappearance and/or appearance and variation in band intensity when comparison with untreated control treatments were evaluated (Liu et al., 2005;Cenkci et al., 2009).Change in soluble protein level and growth rate values were calculated as a percentage of their control (Liu et al., 2005).

Statistical analysis and computations
The growth index experiments and total soluble protein content was calculated according to randomized complete block designed with three replicates.Raw data were imported to Microsoft Excel 2010 program for calculation.The SPSS (Version 17.1) program was used for analysis of variance and comparison of the means was performed by HSD's method at P< 0.05.

Effect of Cd on root and shoot growth in wheat seedlings
Cadmium treatment showed toxic effect on seedling growth and root growth of wheat seedlings in all treatment as compared to the control (Figure 1).Seedling and root length of wheat were significantly (p<0.05)decreased at 10, 15, 20, 30, 60, 90, 120 mgl -1 of cadmium treatment as compared to untreated seedlings.The inhibitory rate of root and shoot growth is given in Table 2. Toxicity effect of cadmium on plant has been previously described in few studies (Shafiq et al., 2008;Liu et al., 2005).

Effect of Cd on total soluble protein content of root tips of wheat seedlings
The data for total soluble protein level in root tips of seedlings are shown in Table 3.Total soluble protein levels in seedlings decreased at 5, 10, 15, 20, 30, 60, 90, and 120 mgl -1 Cd, but significant (P<0.05)reduction was obtained at concentrations above 10 mgl -1 Cd, and inhibited substantially respectively when compared with control plantlets.Results show that there was a negative correlation between Cd concentration and total soluble protein content in wheat seedlings, with a correlation coefficient of (r 2 ) 0.825.

DNA extraction
Since extracted genomic DNA was in varied concentration, it is needed to equal the concentration of DNA for our experiment.So genomic DNA concentration was equaled by using nanodrop and separated on agarose gel.Nanodroping and electrophoresis data showed that purity grade of DNA is suitable for RAPD.The integrity of the genomic DNA extracted is shown in Figure 2.

RAPD profile analysis
Aimed at verifying the genetic effect of Cd tested, the RAPD analysis was performed on DNA extracted from the roots.Fifteen (15) 10mer oligonucleotide primers of 70 to 75% GC content were utilized for screening the wheat genome for changes, whilst only 13 primers generated specific and stable results.The total number of bands was 80 for untreated control treatments.All 13 primers produced same RAPD profiles for the untreated roots seedlings.On the other hand, RAPD profiles showed substantial differences between untreated control and treated seedlings with apparent changes (disappearance and/or appearance) in the number and size of amplified DNA fragments for different primers.The changes in RAPD profiles were summarized for wheat roots of treated seedlings in comparison to their controls (Tables 4 and 5).The maximum change in RAPD profiles (disappearance b and/or appearance a) was obtained in 120, 90, 60, 20, 15 and 30 mgl -1 of Cd concentration, respectively, when compared with control plantlets.Also, maximum change in band intensity (decrease d or increase c) was obtained in 90, 120, 60, 15, 20 and 30 mgl -1 of Cd, as compared to untreated seedling.In this experiment, change in band intensity (decrease and increase) greatly occurred in high Cd concentrations.For Az.1 primer, disappearances of bands were more evident in 120, 30 and 20 mgl -1 concentration of Cd (Figure 3).With Az.2 and Az.4 primers, no change was observed in RAPD profiles (Figure 3).With Az.3 primer, new bands appeared in 20, 30, 60, 90 and 120 mgl -1 of Cd concentration (Figure 3).For Az.6, Az.5 and Az.13 primers, disappearance of bands occurred in 5 mgl -1 of Cd concentration (Figure 3).Disappearance of bands was also detected in 15, 20, 60,90 and 120 mgl -1 with Az.7 primer (Figure 3).Disappearance and decrease in band was observed in 15, 20 and 30 mgl -1 , and increase in band intensity was detected in 60, 90 and 120 mgl -1 (Figure 3).With Az.12 primer, band intensity was only an appearance of DNA change (Table 5) (Figure not shown).
Appearance of new bands in low molecular weight was obtained in Az.13 profiles above 15 mgl -1 Cd (Table 5)     in Tables 4 and 5.In Tables 4 and 5, data shows that there is a direct relationship between Cd concentration and band changes, thus suggesting nonrandom interaction between DNA and Cd.

Comparison of RAPD profiles, root growth and soluble protein content of roots in barley seedlings under cadmium stress
In Figure 4, the genomic template stability, a qualitative measure reflecting changes in RAPD patterns, was used to compare the modifications in RAPD profiles with reductions in root and shoot growth and soluble protein content of root tips in wheat seedlings.Following exposure to increasing Cd, root and shoot growth and soluble protein content of root tips in wheat seedlings decreased gradually.In contrast, the genomic template stability decreased after exposure to Cd too, but stabilized basically after 15 mgl -1 Cd of exposure.The plateau effect was ascribed to multiple changes in RAPD profiles (for example appearance, disappearance of new bands), which tend to counterbalance each other.In other words, the disappeared band was compensated by the low frequency of newly appearing bands at 15 mgl -1 Cd concentration for primers used in this experiment.

DISCUSSION
In the present study, the application of RAPD marker analysis is reported for an assessment of cadmium genotoxicity in wheat.This technique has been used by many researchers for determination of genotoxic effect of heavy metal (Conte et al., 1998;Atienzar et al., 2002;Liu et al., 2005;Gupta and Sarin, 2009;Al-Qurainy, 2009;Liu et al., 2009;Cenkci et al., 2009;Taspinar et al., 2009;Shahrtash et al., 2010;Sameer and Qari 2010;Al-Qurainy et al., 2010).The concentration of Cd used in the present study induced some changes in DNA template which was detected by RAPD marker.Cadmium is neither an essential microelement for the growth of plants and animals, nor does it participate in the processes of cell metabolism.However, it is readily taken up probably through zinc transporter proteins (Suzuki, 2005;Fusconi et al., 2007), and it is toxic even at very low concentrations.Cadmium interacts with the root cell wall and, at the cellular level, it induces numerous physiological and metabolic disturbances (Liu et al., 2004;Suzuki, 2005;Fusconi et al., 2007).Cadmium enhances the production of reactive oxygen species that may damage major classes of macromolecules in plant cells, causing DNA strand breaks and increases membrane lipid peroxidation (Andrea, 1994;Fusconi et al., 2007).Metal ions can directly bind to DNA or indirectly through hydrogen bonding of the coordinating water molecules surrounding the metal ions.Metal binding to the bases usually disrupts base pair hydrogen bonding and destabilizes the double helix.The binding of metals to DNA and RNA also influences indirectly the sugar conformation.As a result of this change in sugar puckering, the two helical conformations of A-DNA and B-DNA or RNA are characterized by the orientation of the bases with respect to the axis of the double helix (Valverde et al., 2001;Hossain and Huq, 2002;Anastassopoulou, 2003;Oliveira et al., 2008).This may be the reason why some metal ions influence DNA synthesis and the replication process.Like other metal ions, cadmium has direct interaction with DNA (Fuente et al., 2004;Oliveira et al., 2008).In this study, DNA damage was shown by RAPD profiles via disappearance or appearance and alteration in intensity (decrease or increase) of bands.Bands intensity in some primers under different concentration of cadmium showed some changes (increase or decrease).Changes observed in the DNA profiles such as modifications in band intensity and loss of bands may be due to the changes in oligonucleotide priming sites leading to genomic rearrangements, and less likely to point mutations or DNA damage in the primer binding sites which can block or reduce the efficiency of DNA polymerization in the PCR reaction (Atienzar et al., 2002;Liu et al., 2005;Gupta and Sarin, 2009;Gao et al., 2010).Appearance of new PCR products occurred because some oligonucleotide priming sites could become accessible oligonucleotide primers after structural change or because some changes in DNA sequence have occurred due to mutations (resulting in new annealing events), and/or large deletions (bringing two pre-existing annealing sites closer), and/or homologous recombination (juxtaposing two sequences that match the sequence of the primer) (Atienzar et al., 1999;Liu et 2005).Trends in disappearing of RAPD bands in profiles in all primers were not similar.However, the number in high concentration was greater than in low concentration.
Disappearance of bands are likely to be due to changes in oligonucleotide priming sites, originating from rearrangements and less likely from point mutations and DNA damage in the primer binding sites (Liu et al., 2005;Enan, 2006;Liu et al., 2009).The disappearance of a normal RAPD product may be related to the events such as DNA damage (example, single and double strand breaks, modified bases, a basic sites, oxidized bases, bulky adducts, DNA-protein cross-links), point mutations and/or complex chromosomal rearrangements induced by genotoxins (Atienzar et al., 1999(Atienzar et al., , 2000)).The low concentration of Cd (5 and 10 mgl -1 ) can also induce mutation and produce polymorphism in RAPD profiles.It was found that three informative 10-mer primers, Az.5, Az.12 and Az.13, may have great potential for detecting cadmium induced genomic alterations in low concentration.In Tables 4 and 5, data shows that there is a relative relationship between Cd concentration and RAPD profiles polymorphism, suggesting a nonrandom interaction between DNA and Cd.
Our finding on comparison between 'untreated' and 'treated' genomes show that RAPD analysis can be used for evaluation of toxicity on plants caused by environmental pollutants.On the basis of these considerations, we could suggest that RAPD technique is a powerful tool for measuring qualitative and quantitative genotoxic activities produced by cadmium.This method can be applied to a wide range of bioindicator organisms and may become a universal methodology to identify target genes for specific genotoxic agents .
In conclusion, the heavy metal such as Cd, can damage the seedlings of wheat and cause mutations even at low concentration.Since, wheat is immensely used as an important plant that provides human daily food and has an important role in human health; therefore, biomarkers are necessary for detection of heavy metals pollution in this species and its growing area to make human health free of hazardous materials.Thus, RAPD fingerprints appear to be stable in detection of genotoxic doses of those plants which are growing in heavy metal polluted soils.

Figure 1 .
Figure 1.Effect of Cd on wheat seedling length after nine days of treatment.

Figure 2 .
Figure 2. Genomic DNA of about 100 ng extracted by using DNA purification kit (Germany, 5 PRIME) in root-tips of wheat.
appearance of new bands; b, disappearance of normal bands; a + b, polymorphic bands.(Figurenot shown).The number of disappearance of RAPD bands was more at two Cd concentrations (90 and 120 mgl -1 ) and appearance of new bands was greater at three Cd concentrations (15, 90 and 120 mgl -1 ) for Az.14 primer.Absent of bands was particularly obvious for wheat exposed to 60, 90 and 120 mgl -1 Cd for primer Az.15 (Table 4) (Figure not shown).Decrease or increase in bands intensity generated by different primers is given

Figure 3 .
Figure 3. RAPD profiles of genomic DNA from root-tips of wheat seedlings exposed to varying Cd concentrations.

Figure 4 .
Figure 4. Comparison of root growth, plant height, total soluble protein level and genomic DNA template stability in root tips of wheat seedlings exposed to different Cd concentrations.

Table 1 .
Sequences and temperature (°C) of the 15 primers used in this experiment.

Table 2 .
Effect of Cd on root and shoot length of wheat seedlings after 9 days of treatment.

Table 3 .
Effect of Cd on total soluble protein content of root tips seedlings after 9 days of treatment.

Table 5 .
Changes of total bands intensity in control, and polymorphic bands and varied bands in Cd-contaminated wheat seedlings.