Environ Sci Pollut Res DOI 10.1007/s11356-014-3932-2
RESEARCH ARTICLE
Evaluation of arsenic trioxide genotoxicity in wheat seedlings using oxidative system and RAPD assays Ozkan Aksakal & Nevzat Esim
Received: 21 July 2014 / Accepted: 1 December 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Arsenic is a metalloid that is toxic to living organisms. It is known that high concentration of arsenic causes toxic damage to cells and tissues of plants. While the toxic effect of arsenic is known, limited efforts have been made to study its genotoxic effect on the crops. In the present study, effects of arsenic trioxide (As2O3) on seed germination, root length, reactive oxygen species (ROS), lipid peroxidation (malondialdehyde (MDA)), and activities of antioxidant enzymes, as well as DNA in wheat seedlings were investigated. Seedlings were exposed to different (10 to 40 mg/L) As2O3 concentrations for 7 days. Seed germination and root elongation decreased with increase of As2O3 concentration. The values of hydrogen peroxide (H2O2), superoxide anion (O2·−), and MDA contents significantly increased by As2O3 concentrations. The highest values for H2O2, O2·−, and MDA were obtained in 40 mg/L treated wheat seedling. A significant increase of peroxidase (POX) and catalase (CAT) activity in seedlings were observed with increased concentration of As2O3, then decreased when reaching a value of 40 mg/L, whereas the activities of superoxide dismutase (SOD) were gradually enhanced with increasing As2O3 concentration. Alterations of DNA in wheat seedlings were detected using randomly amplified polymorphic DNA (RAPD) technique. The changes occurring in RAPD profiles of seedlings following As2O3 treatment included loss of normal bands and appearance of new bands in comparison to that of control seedlings. The results of our study showed that As2O3 induced DNA damage in a dose-dependent meaner, and the root cells of wheat studied showed a defense Responsible editor: Philippe Garrigues O. Aksakal (*) Department of Biology, Science Faculty, Atatürk University, 25240 Erzurum, Turkey e-mail: [email protected] N. Esim Vocational Training School, Bingöl University, Bingöl, Turkey
against As2O3-induced oxidative stress by enhancing their antioxidant activities. Keywords As2O3 . Catalase . MDA . Peroxidase . RAPD . Superoxide dismutase
Introduction Arsenic is one of the most carcinogenic elements, frequently found in groundwater. High concentrations of arsenic exist in groundwater as a consequence of volcanic activity, erosion of mother rock, or forest fires, as well as from anthropogenic sources such as arsenic-based pesticides or herbicides, metal processing, coal burning, and geothermal discharge (Cao et al. 2009a; Dho et al. 2010). Millions of people in different parts of the world are chronically exposed to arsenic through drinking water. Long-term exposure to inorganic arsenic compounds through water can lead to various diseases such as hyperpigmentation, birth defects, cardiovascular disease, disorders of the central nervous system, and skin, lung, and kidney cancers (Mandal and Suziki 2002). Arsenic is classified as class A carcinogen according to the International Agency for Research of Cancer. The World Health Organization (WHO) has recommend 10 ppb as the maximum permissible arsenic level for drinking water (WHO 2001). Inorganic arsenic forms used previously as pesticides, plant defoliants, and herbicides may accumulate in agricultural soils and plants (Saha and Ashraf 2007). Many plant species including cereals grown in arsenic-contaminated fields accumulate substantial amounts of arsenic in their edible parts that may pose health risks (Sinha et al. 2010). The toxic effects of arsenic inhibit crop growth and development. The phytotoxicity of arsenic is affected considerably by the chemical form and concentration of arsenic in the soil, the tolerance of the genotype species to the metalloid, and the environmental
Environ Sci Pollut Res
condition governing growth (Patra et al. 2004). Plants readily take up inorganic arsenite and arsenate, the chemical forms most commonly detected in the environment, which is greatly influenced by soil texture. These decrease some macronutrient and micronutrient concentrations in plant tissues, with most notable effects on plant phosphorus status (Patra et al. 2004; Dho et al. 2010). As arsenic has a chemical structure similarity to phosphorus, it participates in many cell reactions. Specific organo-arsenical compounds have been detected in some organisms where these compounds are reported to replace phosphorus in the phosphate groups of DNA (Pavlik et al. 2010). Both arsenite and arsenate disturb photosynthetic activity through reducing chlorophyll levels in leaves (Sinha et al. 2010). Furthermore, both arsenic forms cause some morphological changes (e.g., wilting, browning, and dehydration), growth inhibition, disruption of enzyme metabolism, and energy flow. Various authors previously reported that exposure to high concentration of arsenic induce the production of reactive oxygen species (ROS) in plants, which damage cell membrane, DNA, protein, and lipid (Mandal and Suziki 2002; Cao et al. 2009b; Sinha et al. 2010). To minimize the harmful effects of ROS, plants have evolved an effective scavenging system composed of antioxidant molecules and antioxidant enzymes (Lin et al. 2008; Moreno-Jimenez et al. 2009). However, arsenic causes induction of chromosome breaks or exchanges, formation of apurinic/apyrimidinic sites, DNA and oxidative base damage, DNA protein cross-links, chromosomal aberrations, and interaction with spindle function during mitosis or meiosis inducing chromosome segregational errors. The comet assay, micronucleus or chromosome aberration assays have been used to evaluate the genotoxic effects of toxic chemicals on plants (Yi et al. 2007; Yi and Si 2007). The advantages of these methods evaluating the direct effects of genotoxic substances on DNA are their sensitivity and short response time (Xue-mei et al. 2006; Aksakal 2013). The recent advances in molecular biology have led to the development of several PCR-based techniques, which can be used for DNA analysis in the field of genotoxicology (Liu et al. 2007). The random amplified polymorphic DNA (RAPD) method is a PCR-based technique that amplifies random DNA fragments with the use of single short primers of arbitrary nucleotide sequence under low annealing conditions. The technique has been extensively used for species classification, genetic mapping, and phylogeny (Zhiyia and Haowen 2004). Also, its use in surveying genomic DNA for evidence of various types of DNA damage and mutation shows that RAPD may potentially form the basis of novel biomarker assay to detect DNA damage and mutational events in cells of bacteria, plants, invertebrate, and vertebrate animals (Aksakal 2013). Plant bioassays, which are more sensitive and simpler than most methods used to detect the genotoxic effects of toxic
chemicals, have been demonstrated to be efficient tests for monitoring genotoxicity of environmental pollutants (Radic et al. 2009). Wheat, a member of the Graminae family, is one of the plant species used for determining the potential genotoxicity of toxic chemical substances. In the present study, we focused on phytotoxicity and genotoxicity of As2O3 in wheat. For this purpose, we screened genomic DNA alterations in wheat root cells exposed to various concentrations of As2O3 by using RAPD. In addition to this, we addressed ROS, MDA, and the activities of the antioxidative enzymes to evaluate a possible interference of As2O3 with the oxidative system of wheat. Based on the data obtained, the results of this study will provide significant knowledge on mechanism of As2O3 in cereals and eventually can lead to crops.
Materials and methods Chemicals and growth conditions Four different concentrations (10, 20, 30, and 40 mg/L) of As2O3 were used for genotoxicite assays (Yi et al. 2007). All the test solutions were prepared using distilled water. Wheat seeds were sterilized with 75 % ethanol for 2 min, followed by 10 % sodium hypochloride for 10 min and washed thoroughly with distilled water for five times. The seeds were germinated to primary roots of 2–4-mm length in a sterile glass jar containing three layers of Whatman paper for 48 h at 25± 2 °C in dark. Fifteen germinated seeds were selected and transferred to sterile petri plates, and they were treated with different (10, 20, 30, and 40 mg/L) As2O3 concentrations. Plant seedlings were exposed to As2O3 for 7 days in the growth chamber under the same conditions. After 5 days of incubation, oxidative system parameters of wheat seedlings were determined. In these experiments, the seedlings that were merely acquired from the germinated seeds were used. To obtain the enzyme extracts, 0.5-g seedling tissues were homogenized in ice-cold 0.2 M potassium phosphate buffer (pH 7.8). The homogenate was centrifuged for 15 min at 12.000×g at 4 °C and the supernatant was obtained and stored at −20 °C for determination the rate of oxidative system parameters. In addition to this, after 7 days, root length and germination rate were measured in the wheat seedlings. ROS and lipid peroxidation Following He et al. (2005), hydrogen peroxide (H2O2) levels were measured by monitoring the absorbance at 410 nm of the titanium-peroxide complex. Absorbance values were calibrated to a standard curve generated with known concentrations of H2O2. Superoxide anion (O2·−) content was measured as described by Elstner and Heupel (1976). The absorbance
Environ Sci Pollut Res
was monitored at 530 nm after 3 ml of n-butyl alcohol was added to the mixture. Sodium nitrite was used as a standard solution to calculate the production rate of superoxide. MDA content, indicator of lipid peroxidation, was determined according to the method of Heath and Packer (1968). Leaves were weighed, and homogenates containing 10 % trichloroacetic acid (TCA) and 0.65 % 2-thiobarbituric acid (TBA) were heated at 95 °C for 60 s and then cooled to room temperature and centrifuged at 10,000×g for 10 min. The absorbance of the supernatant was read at 532 and 600 nm against a reagent blank. MDA content was expressed as nanomoles per gram (nmol g−1) FW. Antioxidant enzymes activity Total SOD activity was estimated according to the modified method of Agarwal and Pandey (2004). One unit of enzyme activity was taken as that amount of enzyme, which reduced the absorbance reading to 50 % in comparison with tubes lacking enzyme. CAT activity was measured according to the method of Havir and McHale (1987). One unit of CAT activity was defined as the amount of enzyme that used 1 μmol H2O2 min−1. POX activity was measured according to Ye et al. (2003). One unit of POX activity was defined as the amount of enzyme that caused an increase in absorbance of 0.01 min−1. Statistical analysis All data presented are mean values. Each value was presented as the mean±SE with a minimum of six experiments. Means were compared by one-way analysis of variance and Duncan’s multiple range tests at 5 % level of significance. Genomic DNA isolation DNA was extracted by using the procedure reported by Aksakal et al. (2013) with minor modifications. Approximately 10–15-mg fresh sample was snap-frozen in liquid nitrogen in 2-ml Eppendorf tubes. DNA extraction buffer [100 mM Tris–HCl (pH 8.0); 50 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0); 500 mM NaCl; 2 % SDS (w/ v); 2 % 2-mercaptoethanol (v/v); 1 % PVP (w/v)] was added and the whole mixed well. The mixture was incubated at 65 °C in a water bath for 40 min with intermittent shaking at 5-min intervals. It was centrifuged at 12,000×g for 15 min at 4 °C; the supernatant was transferred into a new 1.5-ml tube and mixed with an equal volume of phenol/chloroform/ isoamylalcohol (25:24:1), and centrifuged. The supernatant was collected and mixed with 1/10 volume 10 % CTAB-0.7 M NaCl in a new tube. After centrifugation (12,000×g for 15 min), the supernatant was collected and an equal volume of chloroform/isoamylalcohol (24:1) was added and mixed
gently. The DNA was precipitated by the addition of 0.6 volume of cold isopropanol, left at −20 °C for 10 min. The DNA was pelleted by centrifugation (12,000×g for 15 min), and the isopropanol was poured off; the DNA was allowed to air-dry before being dissolved in 100 μl of TE buffer. RAPD procedures Samples were screened for RAPD variation using standard tenbase primers supplied by Operon Technologies Inc. (Alameda, CA, USA). Thirty microliters of reaction cocktail was prepared as follows: 10× buffer 3.0 μl, dNTPs (10 mM) 1.2 μl, magnesium chloride (25 mM) 1.2 μl, primer (5 μM) 2.0 μl, Taq polymerase (5 units) 0.4 μl, water 19.2 μl sample DNA 3.0 μl (100 ng/μl). Forty oligonucleotide primers were screened, and among them, 16 primers were selected and used for further studies. Sequences (5′→3′) from primer 1 to 16 utilized are CAGCACCCAC (OPA-13), TGCCGAGCTG (OPA-2), GAATCGGCCA (OPH-18), AAGGCTCACC (OPY-6), AGGCAGAGCA (OPY-8), GGGCCAATGT (OPY-16), GTCCACACGG (OPB-8), CTGGACGTCA (OPW-7), GGCGGATAAG (OPW-5), AGTCGCCCTT (OPY15), GTCC TGGGTT (OPW-17), CTGATGCGTG (OPW-11), TTCAGG GCAC (OPW-18), AATCGGGCTG (OPA-4), CTGACCAG CC (OPH-19), and GGGTCTCGGT (OPY-13), respectively. The thermal cycle was as follows: 2 min at 95 °C; 2 cycles of 30 s at 95 °C, 1 min at 37 °C, 2 min at 72 °C; 2 cycles of 30 s at 95 °C, 1 min at 35 °C, 2 min at 72 °C; 41 cycles of 30 s at 94 °C, 1 min at 35 °C, 2 min at 72 °C, followed by a final 5min extension at 72 °C then brought down to 4 °C. Electrophoresis The PCR products (27 μl) were mixed with 6× gel loading buffer (3 μl) and loaded onto an agarose (1.5 % w/v) gel electrophoresis in 0.5× Tris-Borate-EDTA (TBE) buffer at 70 V for 150 min. Amplification products separated by gel was stained in ethidium bromide solution (2 μl Etbr/100 ml 1× TBE buffer) for 40 min. The amplified DNA product was detected by using the Bio Doc Image Analysis System with Uvisoft analysis package (Cambridge Electronic Design Ltd., Cambridge, UK). RAPD patterns were evaluated by using computer software TotalLab TL120. Genomic template stability (GTS, %) was calculated as follows: a GT S ¼ 1− 100 n where a is the average number of polymorphic bands detected in each treated sample and n is the number of total
Environ Sci Pollut Res 50 45 40 H2O2 Content (ng.g-1 FW)
bands in the control. Polymorphism in RAPD profiles included disappearance of a normal band and appearance of a new band in comparison to control. The average was calculated for each experimental group exposed to different As2O3 treatments. To compare the sensitivity of each parameter, changes in these values were calculated as a percentage of their control (set to 100 %).
Results
35
b
b
10
20
ab
a
c
30 25 20 15 10 5 0 C
Effect of As2O3 on seed germination and root length
40
10 O2.- Concentration (nmol.g-1 FW)
Dose-dependent germination inhibition of wheat seeds after 7 days of As2O3 treatment was observed (Table 1). Seed germination was inhibited more than 50 % in wheat seeds when applied 30 and 40 mg/l As 2 O 3 concentrations. Moreover, root lengths decreased with increase of As2O3 concentration compared with the control seedlings (Table 1) and these increases were statistically significant (p
RESEARCH ARTICLE
Evaluation of arsenic trioxide genotoxicity in wheat seedlings using oxidative system and RAPD assays Ozkan Aksakal & Nevzat Esim
Received: 21 July 2014 / Accepted: 1 December 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Arsenic is a metalloid that is toxic to living organisms. It is known that high concentration of arsenic causes toxic damage to cells and tissues of plants. While the toxic effect of arsenic is known, limited efforts have been made to study its genotoxic effect on the crops. In the present study, effects of arsenic trioxide (As2O3) on seed germination, root length, reactive oxygen species (ROS), lipid peroxidation (malondialdehyde (MDA)), and activities of antioxidant enzymes, as well as DNA in wheat seedlings were investigated. Seedlings were exposed to different (10 to 40 mg/L) As2O3 concentrations for 7 days. Seed germination and root elongation decreased with increase of As2O3 concentration. The values of hydrogen peroxide (H2O2), superoxide anion (O2·−), and MDA contents significantly increased by As2O3 concentrations. The highest values for H2O2, O2·−, and MDA were obtained in 40 mg/L treated wheat seedling. A significant increase of peroxidase (POX) and catalase (CAT) activity in seedlings were observed with increased concentration of As2O3, then decreased when reaching a value of 40 mg/L, whereas the activities of superoxide dismutase (SOD) were gradually enhanced with increasing As2O3 concentration. Alterations of DNA in wheat seedlings were detected using randomly amplified polymorphic DNA (RAPD) technique. The changes occurring in RAPD profiles of seedlings following As2O3 treatment included loss of normal bands and appearance of new bands in comparison to that of control seedlings. The results of our study showed that As2O3 induced DNA damage in a dose-dependent meaner, and the root cells of wheat studied showed a defense Responsible editor: Philippe Garrigues O. Aksakal (*) Department of Biology, Science Faculty, Atatürk University, 25240 Erzurum, Turkey e-mail: [email protected] N. Esim Vocational Training School, Bingöl University, Bingöl, Turkey
against As2O3-induced oxidative stress by enhancing their antioxidant activities. Keywords As2O3 . Catalase . MDA . Peroxidase . RAPD . Superoxide dismutase
Introduction Arsenic is one of the most carcinogenic elements, frequently found in groundwater. High concentrations of arsenic exist in groundwater as a consequence of volcanic activity, erosion of mother rock, or forest fires, as well as from anthropogenic sources such as arsenic-based pesticides or herbicides, metal processing, coal burning, and geothermal discharge (Cao et al. 2009a; Dho et al. 2010). Millions of people in different parts of the world are chronically exposed to arsenic through drinking water. Long-term exposure to inorganic arsenic compounds through water can lead to various diseases such as hyperpigmentation, birth defects, cardiovascular disease, disorders of the central nervous system, and skin, lung, and kidney cancers (Mandal and Suziki 2002). Arsenic is classified as class A carcinogen according to the International Agency for Research of Cancer. The World Health Organization (WHO) has recommend 10 ppb as the maximum permissible arsenic level for drinking water (WHO 2001). Inorganic arsenic forms used previously as pesticides, plant defoliants, and herbicides may accumulate in agricultural soils and plants (Saha and Ashraf 2007). Many plant species including cereals grown in arsenic-contaminated fields accumulate substantial amounts of arsenic in their edible parts that may pose health risks (Sinha et al. 2010). The toxic effects of arsenic inhibit crop growth and development. The phytotoxicity of arsenic is affected considerably by the chemical form and concentration of arsenic in the soil, the tolerance of the genotype species to the metalloid, and the environmental
Environ Sci Pollut Res
condition governing growth (Patra et al. 2004). Plants readily take up inorganic arsenite and arsenate, the chemical forms most commonly detected in the environment, which is greatly influenced by soil texture. These decrease some macronutrient and micronutrient concentrations in plant tissues, with most notable effects on plant phosphorus status (Patra et al. 2004; Dho et al. 2010). As arsenic has a chemical structure similarity to phosphorus, it participates in many cell reactions. Specific organo-arsenical compounds have been detected in some organisms where these compounds are reported to replace phosphorus in the phosphate groups of DNA (Pavlik et al. 2010). Both arsenite and arsenate disturb photosynthetic activity through reducing chlorophyll levels in leaves (Sinha et al. 2010). Furthermore, both arsenic forms cause some morphological changes (e.g., wilting, browning, and dehydration), growth inhibition, disruption of enzyme metabolism, and energy flow. Various authors previously reported that exposure to high concentration of arsenic induce the production of reactive oxygen species (ROS) in plants, which damage cell membrane, DNA, protein, and lipid (Mandal and Suziki 2002; Cao et al. 2009b; Sinha et al. 2010). To minimize the harmful effects of ROS, plants have evolved an effective scavenging system composed of antioxidant molecules and antioxidant enzymes (Lin et al. 2008; Moreno-Jimenez et al. 2009). However, arsenic causes induction of chromosome breaks or exchanges, formation of apurinic/apyrimidinic sites, DNA and oxidative base damage, DNA protein cross-links, chromosomal aberrations, and interaction with spindle function during mitosis or meiosis inducing chromosome segregational errors. The comet assay, micronucleus or chromosome aberration assays have been used to evaluate the genotoxic effects of toxic chemicals on plants (Yi et al. 2007; Yi and Si 2007). The advantages of these methods evaluating the direct effects of genotoxic substances on DNA are their sensitivity and short response time (Xue-mei et al. 2006; Aksakal 2013). The recent advances in molecular biology have led to the development of several PCR-based techniques, which can be used for DNA analysis in the field of genotoxicology (Liu et al. 2007). The random amplified polymorphic DNA (RAPD) method is a PCR-based technique that amplifies random DNA fragments with the use of single short primers of arbitrary nucleotide sequence under low annealing conditions. The technique has been extensively used for species classification, genetic mapping, and phylogeny (Zhiyia and Haowen 2004). Also, its use in surveying genomic DNA for evidence of various types of DNA damage and mutation shows that RAPD may potentially form the basis of novel biomarker assay to detect DNA damage and mutational events in cells of bacteria, plants, invertebrate, and vertebrate animals (Aksakal 2013). Plant bioassays, which are more sensitive and simpler than most methods used to detect the genotoxic effects of toxic
chemicals, have been demonstrated to be efficient tests for monitoring genotoxicity of environmental pollutants (Radic et al. 2009). Wheat, a member of the Graminae family, is one of the plant species used for determining the potential genotoxicity of toxic chemical substances. In the present study, we focused on phytotoxicity and genotoxicity of As2O3 in wheat. For this purpose, we screened genomic DNA alterations in wheat root cells exposed to various concentrations of As2O3 by using RAPD. In addition to this, we addressed ROS, MDA, and the activities of the antioxidative enzymes to evaluate a possible interference of As2O3 with the oxidative system of wheat. Based on the data obtained, the results of this study will provide significant knowledge on mechanism of As2O3 in cereals and eventually can lead to crops.
Materials and methods Chemicals and growth conditions Four different concentrations (10, 20, 30, and 40 mg/L) of As2O3 were used for genotoxicite assays (Yi et al. 2007). All the test solutions were prepared using distilled water. Wheat seeds were sterilized with 75 % ethanol for 2 min, followed by 10 % sodium hypochloride for 10 min and washed thoroughly with distilled water for five times. The seeds were germinated to primary roots of 2–4-mm length in a sterile glass jar containing three layers of Whatman paper for 48 h at 25± 2 °C in dark. Fifteen germinated seeds were selected and transferred to sterile petri plates, and they were treated with different (10, 20, 30, and 40 mg/L) As2O3 concentrations. Plant seedlings were exposed to As2O3 for 7 days in the growth chamber under the same conditions. After 5 days of incubation, oxidative system parameters of wheat seedlings were determined. In these experiments, the seedlings that were merely acquired from the germinated seeds were used. To obtain the enzyme extracts, 0.5-g seedling tissues were homogenized in ice-cold 0.2 M potassium phosphate buffer (pH 7.8). The homogenate was centrifuged for 15 min at 12.000×g at 4 °C and the supernatant was obtained and stored at −20 °C for determination the rate of oxidative system parameters. In addition to this, after 7 days, root length and germination rate were measured in the wheat seedlings. ROS and lipid peroxidation Following He et al. (2005), hydrogen peroxide (H2O2) levels were measured by monitoring the absorbance at 410 nm of the titanium-peroxide complex. Absorbance values were calibrated to a standard curve generated with known concentrations of H2O2. Superoxide anion (O2·−) content was measured as described by Elstner and Heupel (1976). The absorbance
Environ Sci Pollut Res
was monitored at 530 nm after 3 ml of n-butyl alcohol was added to the mixture. Sodium nitrite was used as a standard solution to calculate the production rate of superoxide. MDA content, indicator of lipid peroxidation, was determined according to the method of Heath and Packer (1968). Leaves were weighed, and homogenates containing 10 % trichloroacetic acid (TCA) and 0.65 % 2-thiobarbituric acid (TBA) were heated at 95 °C for 60 s and then cooled to room temperature and centrifuged at 10,000×g for 10 min. The absorbance of the supernatant was read at 532 and 600 nm against a reagent blank. MDA content was expressed as nanomoles per gram (nmol g−1) FW. Antioxidant enzymes activity Total SOD activity was estimated according to the modified method of Agarwal and Pandey (2004). One unit of enzyme activity was taken as that amount of enzyme, which reduced the absorbance reading to 50 % in comparison with tubes lacking enzyme. CAT activity was measured according to the method of Havir and McHale (1987). One unit of CAT activity was defined as the amount of enzyme that used 1 μmol H2O2 min−1. POX activity was measured according to Ye et al. (2003). One unit of POX activity was defined as the amount of enzyme that caused an increase in absorbance of 0.01 min−1. Statistical analysis All data presented are mean values. Each value was presented as the mean±SE with a minimum of six experiments. Means were compared by one-way analysis of variance and Duncan’s multiple range tests at 5 % level of significance. Genomic DNA isolation DNA was extracted by using the procedure reported by Aksakal et al. (2013) with minor modifications. Approximately 10–15-mg fresh sample was snap-frozen in liquid nitrogen in 2-ml Eppendorf tubes. DNA extraction buffer [100 mM Tris–HCl (pH 8.0); 50 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0); 500 mM NaCl; 2 % SDS (w/ v); 2 % 2-mercaptoethanol (v/v); 1 % PVP (w/v)] was added and the whole mixed well. The mixture was incubated at 65 °C in a water bath for 40 min with intermittent shaking at 5-min intervals. It was centrifuged at 12,000×g for 15 min at 4 °C; the supernatant was transferred into a new 1.5-ml tube and mixed with an equal volume of phenol/chloroform/ isoamylalcohol (25:24:1), and centrifuged. The supernatant was collected and mixed with 1/10 volume 10 % CTAB-0.7 M NaCl in a new tube. After centrifugation (12,000×g for 15 min), the supernatant was collected and an equal volume of chloroform/isoamylalcohol (24:1) was added and mixed
gently. The DNA was precipitated by the addition of 0.6 volume of cold isopropanol, left at −20 °C for 10 min. The DNA was pelleted by centrifugation (12,000×g for 15 min), and the isopropanol was poured off; the DNA was allowed to air-dry before being dissolved in 100 μl of TE buffer. RAPD procedures Samples were screened for RAPD variation using standard tenbase primers supplied by Operon Technologies Inc. (Alameda, CA, USA). Thirty microliters of reaction cocktail was prepared as follows: 10× buffer 3.0 μl, dNTPs (10 mM) 1.2 μl, magnesium chloride (25 mM) 1.2 μl, primer (5 μM) 2.0 μl, Taq polymerase (5 units) 0.4 μl, water 19.2 μl sample DNA 3.0 μl (100 ng/μl). Forty oligonucleotide primers were screened, and among them, 16 primers were selected and used for further studies. Sequences (5′→3′) from primer 1 to 16 utilized are CAGCACCCAC (OPA-13), TGCCGAGCTG (OPA-2), GAATCGGCCA (OPH-18), AAGGCTCACC (OPY-6), AGGCAGAGCA (OPY-8), GGGCCAATGT (OPY-16), GTCCACACGG (OPB-8), CTGGACGTCA (OPW-7), GGCGGATAAG (OPW-5), AGTCGCCCTT (OPY15), GTCC TGGGTT (OPW-17), CTGATGCGTG (OPW-11), TTCAGG GCAC (OPW-18), AATCGGGCTG (OPA-4), CTGACCAG CC (OPH-19), and GGGTCTCGGT (OPY-13), respectively. The thermal cycle was as follows: 2 min at 95 °C; 2 cycles of 30 s at 95 °C, 1 min at 37 °C, 2 min at 72 °C; 2 cycles of 30 s at 95 °C, 1 min at 35 °C, 2 min at 72 °C; 41 cycles of 30 s at 94 °C, 1 min at 35 °C, 2 min at 72 °C, followed by a final 5min extension at 72 °C then brought down to 4 °C. Electrophoresis The PCR products (27 μl) were mixed with 6× gel loading buffer (3 μl) and loaded onto an agarose (1.5 % w/v) gel electrophoresis in 0.5× Tris-Borate-EDTA (TBE) buffer at 70 V for 150 min. Amplification products separated by gel was stained in ethidium bromide solution (2 μl Etbr/100 ml 1× TBE buffer) for 40 min. The amplified DNA product was detected by using the Bio Doc Image Analysis System with Uvisoft analysis package (Cambridge Electronic Design Ltd., Cambridge, UK). RAPD patterns were evaluated by using computer software TotalLab TL120. Genomic template stability (GTS, %) was calculated as follows: a GT S ¼ 1− 100 n where a is the average number of polymorphic bands detected in each treated sample and n is the number of total
Environ Sci Pollut Res 50 45 40 H2O2 Content (ng.g-1 FW)
bands in the control. Polymorphism in RAPD profiles included disappearance of a normal band and appearance of a new band in comparison to control. The average was calculated for each experimental group exposed to different As2O3 treatments. To compare the sensitivity of each parameter, changes in these values were calculated as a percentage of their control (set to 100 %).
Results
35
b
b
10
20
ab
a
c
30 25 20 15 10 5 0 C
Effect of As2O3 on seed germination and root length
40
10 O2.- Concentration (nmol.g-1 FW)
Dose-dependent germination inhibition of wheat seeds after 7 days of As2O3 treatment was observed (Table 1). Seed germination was inhibited more than 50 % in wheat seeds when applied 30 and 40 mg/l As 2 O 3 concentrations. Moreover, root lengths decreased with increase of As2O3 concentration compared with the control seedlings (Table 1) and these increases were statistically significant (p
