Plant Cell Rep (2011) 30:1713–1721 DOI 10.1007/s00299-011-1079-x
ORIGINAL PAPER
Copper-induced oxidative damage, antioxidant response and genotoxicity in Lycopersicum esculentum Mill. and Cucumis sativus L. ¨ zlem Darcansoy I˙s¸ eri • Didem Aksoy Ko¨rpe • O Erkan Yurtcu • Feride Iffet Sahin • Mehmet Haberal
Received: 17 January 2011 / Revised: 6 April 2011 / Accepted: 22 April 2011 / Published online: 10 May 2011 Ó Springer-Verlag 2011
Abstract Adequate copper (Cu2?) concentrations are required for plants; however, at higher concentrations it can also cause multiple toxic effects. In the present study, lipid peroxidation, hydrogen peroxide levels as well as ascorbate peroxidase (APX: EC 1/11/1/11) and catalase (CAT: EC 1.11.1.6) activities were determined in Lycopersicum esculentum Mill. and Cucumis sativus L. seedlings after 7-day exposure to copper sulfate. In addition, DNA damage in these two crops was assessed by measuring micronucleus (MN) frequency and tail moments (TM) as determined by Comet assay. Inhibitory copper concentrations (EC50: 30 and 5.5 ppm for L. esculentum and C. sativus, respectively) were determined according to dose-dependent root inhibition curves, and EC50 and 29EC50 were applied. Malondialdehyde (MDA) and H2O2 levels significantly increased in all groups studied. CAT activity increased in
treatment groups of C. sativus. APX activity increased in L. esculentum seedlings due to 29EC50 treatment. Reductions in mitotic indices (MI) represented Cu2?dependent root growth inhibition in all treatment groups studied. According to TMs and MN frequencies, copper exposure induced significant DNA damage (p \ 0.05) in all study groups, whereas the DNA damage induced was dose dependent in C. sativus roots. In conclusion, Cu2?induced oxidative damage, elevations in H2O2 levels and alterations in APX and CAT activities, as well as significant DNA damage in nuclei of both study groups. To our knowledge, this is the first comparative and comprehensive study demonstrating the effects of copper on two different plant species at relevant cytotoxic concentrations at both biochemical and genotoxicity levels with multiple end points. Keywords Cucumber
Communicated by J. Zou. ¨ . D. ˙Is¸ eri (&) D. A. Ko¨rpe F. I. Sahin M. Haberal O Institute of Transplantation and Gene Sciences, Baskent University, 06980 Kazan, Ankara, Turkey e-mail:
[email protected] E. Yurtcu Department of Medical Biology, Faculty of Medicine, Baskent University, Ankara, Turkey F. I. Sahin Department of Medical Genetics, Faculty of Medicine, Baskent University, Ankara, Turkey M. Haberal Department of General Surgery, Faculty of Medicine, Baskent University, Ankara, Turkey
Copper Comet Micronucleus Tomato
Introduction Heavy metals are stable soil contaminants that accumulate due to mining, and industrial and agricultural processes. Soil contaminated with heavy metals including copper is one of the concerns of agriculture (e.g., due to use of copper-based fungicides, bactericides and fertilizers) (Alloway 1995). Adequate copper (Cu2?) concentrations are required for plants, since it acts as a cofactor of enzymes required for normal growth and development (Marschner 1995). However, copper, besides being a micronutrient, can also cause multiple toxic effects in plants in high concentrations. Copper binds to soil particulate organic matter and humic substances, so its accumulated levels are localized in the soil (Herna´ndez et al. 2006; Sayen et al. 2009). In
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addition, toxic levels of copper are released to aquatic environments due to industrial processes. When accumulated in plant tissues at toxic levels, copper was shown to induce oxidative damage by generating reactive oxygen species (ROS) such as singlet oxygen, hydrogen peroxide and hydroxyl radical, which can damage biological molecules (Aust et al. 1985; De Vos et al. 1989; Weckx and Clijsters 1996). For example, free radicals damage DNA by abstractions and addition reactions leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases, and membranes by binding to sulfhydryl groups of membrane proteins and inducing lipid peroxidation (Halliwell and Gutteridge 1984; Dizdaroglu et al. 2002). Copper accumulation can cause alterations in vital processes of plants such as mineral nutrition, photosynthesis, enzyme activities and biosynthesis of various macromolecules including chlorophyll, leading to inhibition of seed germination, growth and development (Phalsson 1989; Lidon et al. 1993; Doncheva et al. 1996). Copper-induced growth inhibition, oxidative damage and antioxidant response have been studied in different plant species. For example, 20–50 lM Cu2? has been shown to reduce the growth of rice seedlings, causing changes in antioxidant enzyme activity levels, hydrogen peroxide and cell wall peroxidases (Chen et al. 2000). Inhibitory effects of 0.01–10 lM Cu2? were investigated in maize grown in nutrient solution in terms of growth, mineral and chlorophyll contents, and enzyme activities (Mocquot et al. 1996). In a study conducted on soil, tomato growth was reduced with soil Cu2? levels above 150 mg/ kg with a soil pH below 6.5, and soil Cu2? levels above 330 mg/kg with a soil pH above 6.5 (Rhoads et al. 1989). More recently, the genotoxic effects of 1.5 and 3 ppm copper sulfate were studied in a model plant, Allium cepa (Yıldız et al. 2009). In addition, copper chloride was shown to have cytogenetic effects on Helianthus annuus L. and Vicia hirsuta L. root tip cells at a concentration range of 10–100 ppm (I˙nceer and Beyazog˘lu 2000; I˙nceer et al. 2003). Copper concentration may vary in different types of soils at a range of 1–120 ppm. For example, copper level can be below 6 ppm in mineral soils, and below 30 ppm in organic soils. Crop species have differing requirement, tolerance and toxicity thresholds for Cu2? in soil and tissues (reviewed in Padmavathiamma and Li 2007). Toxicological levels of ecopollutants should be well defined for species of economical importance and human consumption. In addition, species may show variable antioxidant response and elimination ability to toxic levels of copper. In accordance, depending on their response, oxidative damage to tissues and cytogenetic and genotoxic effects may vary. The aim of the present study was to determine the inhibitory effects of copper sulfate and comparatively
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evaluate copper-induced cytotoxicity and genotoxicity on Lycopersicon esculentum Mill. (cv. H2274) and Cucumis sativus (cv. Beith Alpha) seedlings in terms of oxidative damage, hydrogen peroxide levels, ascorbate peroxidase (APX: EC 1.11.1.11) and catalase (CAT: EC 1.11.1.6) activity levels, mitotic index, micronucleus frequency and DNA tail moments measurements of Comet assays.
Materials and methods Plant materials and treatments Lycopersicon esculentum Mill. commercial cv. H2274 (MayAgro Seed Corporation, Turkey) and Cucumis sativus commercial cv. Beith Alpha (MayAgro Seed Corporation, Turkey) seeds were used in experiments. After surface sterilization, seeds were left to germinate for 2 days between two layers of moist filter paper in the dark. Germination was defined by the presence of a radicle at least 2 mm long. A total of 15–20 germinated seeds were transferred to sterile strength Hogland’s nutrient solution (Sigma, USA) at 22–25°C, with 50 ± 5% relative humidity and a photoperiod of 16-/8-h (day/night) in growth chamber. To determine the effective copper sulfate (CuSO45H2O) concentrations, seedlings were grown for 7 days in Hoagland’s solutions containing different copper concentrations (0–500 ppm) and root growth was measured. Effective copper concentrations (EC50) for tomato and cucumber were calculated from logarithmic trend lines. Treatments for biochemical and genotoxic studies were performed at EC50 (30 and 5.5 ppm, for L. esculentum and C. sativus, respectively) and 29EC50 (60 and 11 ppm, for L. esculentum and C. sativus, respectively) concentrations for 7 days. Determination of lipid peroxidation Lipid peroxidation was assayed by determining the amount of malondialdehyde (MDA), a product of lipid peroxidation (Ohkawa et al. 1979). In brief, samples were homogenized with mortar and pestle in liquid nitrogen, and the homogenates were suspended in tricholoroacetic acid (TCA; Merck). Samples were centrifuged at 12,000 rpm for 15 min and the supernatant was mixed with a 1:1 volume of thiobarbituric acid (TBA; Sigma) in TCA. Following a 96°C, 25 min incubation, samples were cooled to room temperature and centrifuged at 10,000 rpm for 5 min. Absorbance of the supernatant was recorded at 532 and 600 nm against TBA in TCA. After subtracting the non-specific absorbance at 600 nm, MDA concentration was calculated by its extinction coefficient of 155 mM-1 cm-1.
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Hydrogen peroxide contents Samples were homogenized by mortar and pestle in liquid nitrogen, and the homogenates were suspended in 100-mM potassium phosphate buffer (pH 6.8). The slightly modified method of Bernt and Bergmeyer (1974) reported by (Celikkol Akcay et al. 2010) was used to determine the H2O2 content. Homogenates were centrifuged at 18,000g for 20 min at 4°C and the supernatant was mixed with peroxidase reagent containing peroxidase (Sigma) and o-dianisidine (Sigma) in potassium phosphate buffer. After incubation at 30°C, the reaction was terminated with perchloric acid (Sigma) and the samples were centrifuged at 5,000g for 10 min. Absorbance of the supernatant was measured at 436 nm, and H2O2 content was calculated using its extinction coefficient of 39.4 mM-1 cm-1.
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were performed in 45% acetic acid. The slides were immediately examined under light microscope (Nikon Eclips 500, Japan) with 4009 magnification. Three replicates were performed for each treatment and scoring was made from at least five root tips for each replicate. A minimum of 1,000 cells were counted for each treatment. The mitotic index (MI; %) was calculated as: (number of mitotic cells/total number of cells) 9 100. Micronuclei (MN) were blindly scored in accordance with the acceptable quality criteria reported by Tolbert et al. (1992) and expressed as percent frequency. Alkaline Comet assay (alkaline single cell gel electrophoresis; SCGE)
For determination of APX activity (Wang et al. 1991), samples were homogenized with mortar and pestle in liquid nitrogen, and the homogenates were suspended in Tris– HCl buffer containing polyvinylpolypyrrolidone (Sigma), ascorbate (Sigma) and EDTA (Applichem, USA). After centrifugation at 12,000 rpm for 20 min, at 4°C, the total protein amount in supernatants was determined according to the Bradford method (Bradford 1976). 100 lg of total protein was added to the assay solution (potassium phosphate buffer with ascorbate) and reaction was initiated by the addition of H2O2. Decrease in the absorbance of ascorbate was recorded at 290 nm for 3 min against the assay solution (e = 2.8 mM-1 cm-1). For determination of CAT activity (Chance and Mahly 1995), samples were homogenized with mortar and pestle in liquid nitrogen, and the homogenates were suspended in suspension buffer. After centrifugation at 12,000 rpm for 20 min, at 4°C, total protein amount in supernatants was determined according to the Bradford method; 100 lg of total protein was added to potassium phosphate buffer and reaction was initiated by the addition of H2O2. Decrease in the absorbance of H2O2 was recorded at 240 nm for 3 min against the assay solution (e = 39.4 mM-1 cm-1).
For the determination of the genotoxic effects of copper treatments on seedlings, alkaline SCGE was performed as previously described (Gichner et al. 2004) with modifications. Nuclei from roots of the seedlings were isolated by chopping root tips in 250 lL of ice-cold 0.2 M Tris, pH 7.5, containing 4 mM MgCl2 and 0.5% w/v Triton X-100 (Yıldız et al. 2009) on a watch glass on ice. Nuclear suspension (25 lL) was mixed with 25 lL of 1% (w/v) low melting point agarose (LMPA; Aldrich) and added on to slides coated with 1% (w/v) normal melting point agarose (NMPA; Aldrich). Coverslips were placed and slides were incubated on ice packs until the solidification of the agarose. Coverslips were removed and 50 lL of 0.5% (w/v) LMPA was added on to the slides. Slides were incubated in electrophoresis buffer (300 mM NaOH, 1 mM EDTA disodium salt; pH [13) for 20 min in the dark and electrophoresis was run at 24 V (300 mA) for 30 min. After neutralization (0.4 M Tris; pH 7.5), slides were stained with 2 lg/mL EtBr and observed under florescence microscope (Nicon, Eclipse 600, Japan). A computerized image analysis system (Comet Assay IV; Perceptive Instruments, UK) was employed. Olive tail moment (TM) was used as the end point measure of DNA damage. A minimum of three SCGE slides were prepared for each treatment replicate and, in total, a minimum of 150 nuclei were analyzed per treatment.
Feulgen staining of root tips
Statistical analysis
Root tips were fixed in ethanol and glacial acetic acid (3:1) solution for 24 h and stored in 70% ethanol at 4°C until use. After rinsing, the root tips in dH2O were incubated in 1 N HCl at 60°C for 15 and 8 min for tomato and cucumber, respectively, and subsequently in 1 N HCl for 2 min at room temperature. Roots were washed with dH2O twice and then stained with Feulgen for 1.5–2 h in the dark. Root tips were transferred to dH2O and squash preparations
All data are expressed as mean ± standard error of the means (SEM) of at least four replicate experimental setups. All statistical analyses were performed using SPSS 11.5 Software (SPSS Inc., USA). The mean differences between the control and copper-treated groups of two cultivars were statistically evaluated by one-way ANOVA analysis at the 0.05 level, and post hoc Tukey analyses were performed to identify significant differences.
Determination of APX and CAT activities
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(a) 10
(b) 40
9 8 7 6 5 4 3 2 1 0
root length (mm)
root length (mm)
Fig. 1 Dose-dependent inhibition of a L. esculentum, and b C. sativus seedling root growth by copper treatment
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Results
Dose-dependent growth inhibition of L. esculentum and C. sativus roots after 7 days of copper treatment was observed (Fig. 1). To investigate stress response under similar toxic concentrations, EC50 was calculated from the logarithmic trend lines of root growth curves; 30 and 5.5 ppm EC50 were calculated for L. esculentum and C. sativus, respectively. Copper treatment induced lipid peroxidation To assess oxidative damage to membranes, lipid peroxidation levels of the seedlings were determined in terms of the final product of lipid peroxidation, malondialdehyde (MDA) amounts (Fig. 2). Copper treatment induced dose-dependent oxidative damage in L. esculentum and C. sativus. MDA levels in L. esculentum seedlings were significantly increased in EC50 (24.20 ± 2.62 nmol/gFW) and 29EC50 (38.57 ± 3.25 nmol/gFW) treatment groups (2.6- and 4.2fold, respectively) when compared with untreated seedlings (9.17 ± 0.88 nmol/gFW). Likewise, EC50 (3.75 ± 0.15 nmol/gFW) and 29EC50 (8.14 ± 0.95 nmol/gFW) copper treatment caused 2.2- and 4.9-fold increases in MDA levels of C. sativus relative to the untreated group (1.67 ± 0.13 nmol/gFW). Elevations in hydrogen peroxide content Increase in hydrogen peroxide levels in cells is an indication of increased oxidative stress. H2O2 contents of L. esculentum and C. sativus roots increased due to copper treatments. In roots of L. esculentum, the levels significantly increased in EC50 (84.66 ± 3.98 nmol/gFW) and 29EC50 (97.26 ± 4.57 nmol/gFW) treatment groups (4- and 4.6-fold, respectively) when compared with untreated seedlings (21.29 ± 2.26 nmol/gFW) (Fig. 3). Similarly, EC50 (39.10 ± 4.72 nmol/gFW) and 29EC50 (85.26 ± 10.28 nmol/gFW) copper treatment caused 4.7- and 10.3-fold increases in MDA
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MDA (nmole/gFW)
Growth inhibition of roots and determination of effective copper concentrations (EC50)
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Cu (ppm)
Cu (ppm)
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L.esculentum
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C.sativus
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** *
** *
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*
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0 NT
EC50
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Fig. 2 MDA levels. * and ** represent significant difference between copper treatment groups of L. esculentum with p \ 0.001 (i.e., * control vs. other treatments, ** EC50 vs. 29EC50), and 9 and 99 represent significant difference between copper treatment groups of C. sativus with p \ 0.001 (i.e., 9 control vs. other treatments, 99 EC50 vs. 29EC50)
levels of C. sativus relative to the untreated group (8.29 ± 1.83 nmol/gFW). Alterations in antioxidant enzyme activity levels Changes in catalase and ascorbate peroxidase activities in L. esculentum and C. sativus seedlings were measured for the assessment of changes in enzymatic antioxidant defenses after copper treatments (Fig. 4). In the tomato seedlings, after EC50 and 29EC50 copper treatment, there were not any significant changes (Fig. 4a). However, APX activity in 29EC50 copper treatment group (12.83 ± 0.39 lmolASA/ min/mg protein) significantly increased when compared with the untreated group (2.18 ± 0.77 lmolASA/min/mg protein) (Fig. 4b). On the contrary, APX levels did not significantly change due to copper treatment in cucumber seedlings. On the other hand, EC50 and 29EC50 copper treatments caused significant increases in catalase activity levels (307.91 ± 28.03 and 405.17 ± 42.35 nmolH2O2/ min/mg protein, respectively) in cucumber seedlings when compared with control (113.93 ± 14.07 nmolH2O2/min/mg protein) (Fig. 4a). Conclusively, enhanced enzymatic antioxidant response, as increased CAT and APX activity, was demonstrated in C. sativus and L. esculentum roots, respectively.
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* L.esculentum
H2O2 (nmole/gFW)
100
×× ×
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Table 1 The MI and MN frequencies in root tips of copper-treated tomato and cucumber seedlings Treatment
*
MN (% ±SEM)
16.51 ± 2.11a
0.22 ± 0.02b
L. esculentum 60
NT
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40
* 20
EC50
CAT activity (nmoleH2O2/min/mg protein)
5.73 ± 0.71
0.82 ± 0.14
29EC50
2.62 ± 0.04a
1.18 ± 0.14b
17.21 ± 0.58c
0.30 ± 0.02e
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L.esculentum
400
C.sativus
** *
7.26 ± 0.23c,d 5.02 ± 0.29c,d
0.73 ± 0.13f 1.08 ± 0.10e,f
a Significant difference in MI frequencies between copper treatment groups of tomato with p \ 0.009 b
Significant difference in MN frequencies between copper treatment groups of tomato with p \ 0.021 c, d Significant difference in MI frequencies between copper treatment groups of cucumber with p \ 0.001 (i.e., c for control vs. other treatments, d for 5.5 ppm vs. 11 ppm) e, f Significant difference in MN frequencies between copper treatment groups of cucumber with p \ 0.009 (i.e., e for control vs. other treatments, f for 5.5 ppm vs. 11 ppm)
** *
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2xEC50
Fig. 3 Hydrogen peroxide content of roots. * Represent significant difference between control and copper treatment groups of tomato with p \ 0.001, and 9 and 99 represent significant difference between copper treatment groups of cucumber with p \ 0.023 (i.e., 9 control vs. other treatments, 99 EC50 vs. 29EC50)
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APX activity (µmoleAsA/min/mg protein)
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C. sativus
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MI (% ±SEM)
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2xEC50
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L.esculentum
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C.sativus
** *
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roots of tomato and cucumber, respectively, where the increments were statistically insignificant in EC50 treatment groups. MN frequency was significantly higher in 29EC50 treatment group of cucumber when compared with EC50. Copper treatment caused growth reduction in the roots of L. esculentum and C. sativus roots, as represented by reduced MIs, together with genotoxic damage induced as demonstrated by increments in MN frequencies.
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Assessment of DNA damage by Comet assay **
*
2 0 NT
EC50
2xEC50
Fig. 4 a Catalase activity levels in roots of copper-treated tomato and cucumber seedlings. * and ** represent significant difference between copper treatment groups of cucumber with p \ 0.001 (i.e., * control versus other treatments, ** EC50 vs. 29EC50); b ascorbate peroxidase activity levels in roots of copper-treated tomato and cucumber seedlings. * and ** represent significant difference between copper treatment groups of tomato with p \ 0.001 (i.e., * control vs. 29EC50, ** EC50vs. 29EC50)
Alterations in mitotic index (MI) and micronucleus (MN) frequencies Results obtained from Feulgen staining of root tips are summarized in Table 1. MI of L. esculentum roots significantly decreased due to EC50 and 29EC50 Cu2? treatments. EC50 and 29EC50 Cu2? treatments also caused dosedependent reductions in MIs of C. sativus root tips. MN frequency significantly increased in 29EC50 Cu2?-treated
The image analysis of Comet assay measures the amount of DNA in the head and tail and the length of the tail, based on the principle that migration of nuclei with intact and damaged DNA on agarose differs. Both TM value and percentage of tail DNA obtained by Comet assay represent DNA damage (Gichner et al. 2004). Results of the Comet assay as expressed TM values (Fig. 5) demonstrated copper-induced genotoxic damage in the root nuclei of L. esculentum and C. sativus. Root nuclei of control groups had 1.92 ± 1.01 and 5.35 ± 0.94 lm TM for L. esculentum and C. sativus, respectively, where these values increased to 10.99 ± 2.35 and 18.94 ± 4.24 lm (5.7- and 3.5-fold, respectively) in EC50 Cu2?-applied groups, respectively. The TM values increased by 6.5- and 6.3-fold (12.43 ± 1.81 and 33.83 ± 2.72 lm) for 29EC50 Cu2? treatments of L. esculentum and C. Sativus seedlings, respectively. According to Fig. 5, copper treatments induced significant dose-dependent increases in average tail moments of C. Sativus, i.e., doubling the effective
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40 35
TM (µm)
30
×× ×
L. esculentum C. sativus
25
×× ×
20 15
*
*
10
× 5
*
0 NT
EC50
2xEC50
Fig. 5 a The mean tail moments (TM) in roots of copper-treated tomato and cucumber seedlings obtained by alkaline Comet assay. * Represent significant difference between control and copper treatment groups of tomato with p \ 0.018, and 9 and 99 represent significant difference between copper treatment groups of cucumber with p \ 0.001 (i.e., 9 control vs. other treatments, 99 EC vs. 29EC50)
copper concentrations caused approximately doubling of the tail moment. On the other hand, TM values obtained by EC50 and 29EC50 copper treatments of tomato seedlings were statistically indifferent from each other.
Discussion Toxicological levels of ecopollutants should be well defined for species of economical importance and human consumption. In addition, species may show variable antioxidant response and elimination ability to toxic levels of copper (Mocquot et al. 1996; Weckx and Clijsters 1996; Chen et al. 2000). Accordingly, depending on their response, oxidative damage to tissues, and cytogenetic and genotoxic effects may vary (I˙nceer and Beyazog˘lu 2000; ˙Inceer et al. 2003; Yildiz et al. 2009). So, when studying different species, species variations in response to test materials should be considered. Like other eukaryotic systems, the effective concentration 50 (EC50), the concentration of a toxic substance at which the half growth is attained relative to control, has become a useful parameter for selecting the test concentrations in ecotoxicological studies of plants (Yildiz and Arikan 2008). However, if the EC50 value is chosen as the highest concentration, the observed cytotoxic and genotoxic effects may be sublethal (Yildiz and Arikan 2008). Concordantly, in the present study, EC50 and 29EC50 were selected to determine the effects of copper on L. esculentum and C. sativus seedlings. EC50 for tomato was calculated as 30 ppm (Fig. 1a), and 30 and 60 ppm copper was applied. EC50 for cucumber was considerably lower, i.e., calculated as 5.5 ppm (Fig. 1b), and 5.5 and 11 ppm copper was applied. Previously, EC50 value was found to be 1.5 ppm for A. cepa (Yildiz et al. 2009), 30–40 lM (* 4–6.4 ppm) copper sulfate was
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reported to cause *50% reduction in the root growth of rice (Chen et al. 2000), 30 ppm copper chloride caused *60% reduction in root growth of eggplant (Ko¨rpe and Aras 2011), and significant decrease of root biomass was reported with 10 lM Cu2? (*1.5 ppm) in maize (Mocquot et al. 1996), whereas EC50 (442 ppm) for radish was considerably higher (Sun et al. 2010). Accordingly, the sensitivity of cucumber was close to rice and higher than that of onion and maize, whereas tomato was less sensitive to copper and the sensitivity was close to the eggplant. Variations in the rate of Cu-influx, detoxification through the metal chelators such as phytochelatins, metallothioneins, organic acid, amino acids and enzymatic quenching of ROS contribute to copper toxicity of plants (reviewed in Ducic and Polle 2005). Two of the parameters to assess oxidative stress are lipid peroxidation and H2O2 accumulation. MDA formation is used as the general indicator of the extent of lipid peroxidation resulting from oxidative stress. MDA levels (Fig. 2) in L. esculentum seedlings significantly increased in the EC50 and 29EC50-treated plants when compared with the untreated group. Likewise, EC50 and 29EC50 copper treatment increases the MDA levels of C. sativus relative to the untreated group. According to fold changes, copperinduced lipid peroxidations were similar in two species at physiologically relevant toxic concentrations. Increases in H2O2 levels also demonstrated copper-induced oxidative stress in seedlings of L. esculentum and C. Sativus (Fig. 3). Though the increments by EC50 copper treatment were similar, 29EC50 copper treatment caused higher accumulation of hydrogen peroxide in cucumber than tomato. H2O2 can be generated by SOD, NADPH oxidase, xanthine oxidase, amine oxidase and a cell wall peroxidase (reviewed in Neill et al. 2002). As reported by Zhang et al. (2010), NADPH oxidase can use cytosolic NADPH to produce O2-, which quickly dismutates to H2O2 via SOD. This was supported by the increased activity of plasma membrane NADPH oxidase in Elsholtzia haichowensis roots, where activities of most of the enzymes increased significantly due to copper treatment compared to the control (Zhang et al. 2008). Furthermore, accomplishment of steady-state levels of H2O2 in the presence of copper, even with increased levels of ROS scavenging enzymes, may also be dependent on other detoxification systems (i.e., metal chelators), as well as the time course of the experimental setup (Wang et al. 2004). Catalase and ascorbate peroxidase are among enzymatic ROS scavenging mechanisms in plants. APX, GPX and CAT detoxify hydrogen peroxide. APX requires ascorbate and GSH regeneration system, the ascorbate–glutathion cycle (reviewed by Mittler 2002). The steady-state level of ROS in the different cellular compartments is determined by the interplay between multiple ROS-producing pathways
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and ROS scavenging mechanisms, and upon exposure to various abiotic stresses, ROS scavenging enzymes are induced to decrease the concentration of toxic intracellular ROS levels (reviewed by Apel and Hirt 2004). Since the extent of oxidative stress in a cell is determined by the amounts of superoxide, hydrogen peroxide and hydroxyl radicals, the balance of SOD, APX and CAT activities is important for the suppression of toxic ROS levels in a cell. APX has a higher affinity for H2O2 (lM range) than CAT (mM range) and ascorbate–glutathione cycle is found in all compartments of the cells where CAT is only present in peroxisomes (Mittler 2002). Conclusively, APX plays a more crucial role in controlling the ROS level, although CAT is indispensible when high levels of ROS are produced (Willekens et al. 1996) and, therefore, might act as a buffer zone to control the overall level of ROS that reaches different cellular compartments during stress. It is also supported by the remarkably higher APX activity than CAT activity (i.e., units were expressed as lmol ASC min-1 mg-1 protein and nmol H2O2 min-1 mg-1 protein, respectively; Fig. 4) in all groups studied. Considering changes in CAT activity of tomato, copper treatment did not induce any changes in the levels (Fig. 4a). On the other hand, APX activity in 29EC50 copper treatment group significantly increased when compared to untreated group (Fig. 4b). On the contrary, APX levels did not significantly change due to copper treatment in cucumber seedlings, where significantly elevated levels of CAT activity were observed in both copper-treated groups of cucumber. Firstly, untreated cucumber samples had considerably higher APX activity than that of tomato, which was probably sufficient to eliminate the initial ROS levels in cells. Secondly, considering elevations in hydrogen peroxide level, though intrinsic levels in tomato seedlings were higher when compared with cucumber seedlings, fold changes in hydrogen peroxide levels were higher especially in 29EC50 treatment group of cucumber in comparison to tomato, so plants might have needed extra CAT to deal with increased ROS levels. Previously, enzymes involved in copper-induced oxidative damage were reported to be APX in bean (Cuypers et al. 1999), CAT and peroxidase in wheat (Singh et al. 2007), SOD, APX and guaiacol peroxidase in Brassica juncea (Wang et al. 2004), and SOD, APX, GR and peroxidase in rice (Chen et al. 2000). Regulation of the enzymatic response may also differ during exposure times as evidenced in Brassica juncea (Wang et al. 2004). The induction of these enzymes may depend on intracellular signaling and diverse molecular networks in different plants, such as mitogen-activated protein kinase cascade (Xiong and Zhu 2002; Yeh et al. 2003). Moreover, the control and regulation of biological processes by ROS, such as growth and development, cell cycle, programmed cell death and hormone signaling have recently emerged in addition to their role in
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biotic and abiotic stress responses (reviewed by Mittler et al. 2004). Changes in gene expression and protein that are induced under these conditions may vary depending on the species and intracellular distributions of H2O2. Mitotic index, considered as a parameter of cellular division frequency and inhibition of mitosis, may be used to estimate cytotoxicity of substances (Yıldız et al. 2009). Reductions in mitotic indices (Table 1) indicated dosedependent inhibition of root growth due to copper exposure. MN results from chromosomal fragments or whole chromosome lagging during cell division (Heddle et al. 1991; Fenech et al. 1999) and was accepted as one of the cytogenetic end points in toxicity studies including heavy ¨ nyayar et al. 2006; Seth et al. 2008). According to metals (U Table 1, 29EC50 Cu2? treatments caused an increase in MN frequency in root tips of tomato and cucumber, respectively. Though the increments in EC50 were statistically insignificant, there were two- to fourfold increases in MN frequencies for both tomato and cucumber. Another quantitative parameter to measure DNA damage induced by various agents in plants is the Comet assay (Gichner 2003; Gichner et al. 2004; Yildiz et al. 2009). TM values significantly increased in root nuclei of all copper-treated groups. Interestingly, doubling the copper concentration resulted in significant increase (approximately doubled) in DNA damage in the root nuclei of cucumber, in concordance with the decreases in MI and increments in MN as well as increments of the H2O2 content (Fig. 3). So, copper-induced genotoxicity seems to be dose dependent in cucumber. When the ROS generated in cells exceed the capacity of the antioxidant systems, in addition to the unspecific oxidation of proteins and membrane lipids, DNA damage may also occur depending on the ROS produced (Mittler 2002). ROS-induced DNA damage may be directly caused by DNA strand breaks. Alternatively, metals themselves or ROS may irreversibly bind to/damage proteins involved in DNA replication, repair and transcription, which in turn leads to damage at the nucleic acid level as previously proposed for Cd genotoxicity (Lin et al. 2007; Seth et al. 2008). Moreover, DNA–protein crosslinks may cause heavy metal-induced DNA damage (Tuteja et al. 2001). Considering increments in MN frequencies and TM values, DNA damage observed in the root nuclei of tomato and cucumber might have been induced by direct or indirect acting of Cu2? on nucleic acids, and these deleterious effects may show species variation depending on the antioxidant systems and differences in other molecules of cells. In conclusion, in the present study, (a) dose-dependent growth inhibition of root growth (b) Cu2?-induced oxidative damage, (c) increased ROS as demonstrated by elevations in H2O2 levels, (d) enhanced enzymatic antioxidant response, i.e., APX and CAT, and (e) Cu2?-induced
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genotoxic damage to root nuclei were demonstrated in two different biological systems, L. esculentum and C. sativus. Cu2? concentrations were selected based on the dosedependent root growth inhibition. Heavy metal tolerance may show variations among species as well illustrated in the present study, i.e., EC50 for tomato was 30 ppm and 5.5 ppm for cucumber. So, relevant toxic concentrations should be applied to study heavy metal-induced damage in relation to their requirements and tolerance. Cu2? exposure caused significant DNA damage in the nuclei of both study groups as demonstrated by MN and Comet assays. Though Cu2?-induced DNA damage seems to be correlated to ROS produced in cells, possible direct effects of copper should also be considered (as indicated previously). There are several reports on the deleterious effects of Cu2? on different plants (Mocquot et al. 1996; Chen et al. 2000; ˙Inceer and Beyazog˘lu 2000; ˙Inceer et al. 2003; Yildiz et al. 2009). However, to our knowledge, this is the first comparative and comprehensive study demonstrating the effects of Cu2? on two different plant species at relevant cytotoxic concentrations at both biochemical and genotoxicity levels with multiple end points. Acknowledgments This study was approved by the Baskent University Institutional Review Board (Project No: DA09/31) and supported by the Baskent University Research Fund.
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