Environ Sci Pollut Res DOI 10.1007/s11356-015-4501-z


Nitric oxide mitigates arsenic-induced oxidative stress and genotoxicity in Vicia faba L. Pratiksha Shukla 1 & A. K. Singh 1

Received: 20 October 2014 / Accepted: 6 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract The protective effects of nitric oxide (NO) against arsenic (As)-induced structural disturbances in Vicia faba have been investigated. As treatment (0.25, 0.50, and 1 mM) resulted in a declined growth of V. faba seedlings. Arsenic treatment stimulates the activity of SOD and CAT while the activities of APX and GST content were decreased. The oxidative stress markers such as superoxide radical, hydrogen peroxide and malondialdehyde (lipid peroxidation) contents were enhanced by As. Overall results revealed that significant accumulation of As suppressed growth, photosynthesis, antioxidant enzymes (SOD, CAT, APX, and GST activity), mitotic index, and induction of different chromosomal abnormalities, hence led to oxidative stress. The concentration of SNP (0.02 mM) was very effective in counteracting the adverse effect of As toxicity. These abnormalities use partially or fully reversed by a simultaneous application of As and NO donor and sodium nitroprusside and has an ameliorating effect against As-induced oxidative stress and genotoxicity in V. faba roots.

Keywords Arsenic stress . Antioxidant system . Chromosomal abnormalities . Mitotic index . Nitric oxide . Oxidative stress

Responsible editor: Philippe Garrigues * Pratiksha Shukla [email protected] 1

Department of Botany, Genotoxic Lab, Udai Pratap Autonomous College, Varanasi 221002, India

Introduction Toxic metal contamination in soil has become a serious problem in crop production worldwide (Sarma and Mondal 2011). Arsenic (As) is one of the most harmful toxic metals which is widespread in nature. The potential sources of As in soil and water include oxidation of pyrite and use of phosphate fertilizers, herbicides, and insecticides, etc. (Srivastva et al. 2009). Being a nonessential element for the plant growth, As contamination in the soil negatively affects growth, development, and crop yield (W-X Li et al. 2006). However, As availability in the soil and plant uptake depends greatly upon its form in the soil. In the aerobic soils, arsenate dominates, whereas arsenite dominates in the anaerobic conditions and is highly toxic to plants (E.Smith et al. 1998). Meharg and Hartley-Whitaker (2002) demonstrated that arsenate disrupts phosphate metabolism, whereas arsenite disrupts cellular functions through interference with function of enzymes and proteins and causes death. Plants grown in soil containing high As concentration show visible injury such as chlorosis and growth inhibition (Stoeva et al. 2005). Under a variety of environmental stresses (abiotic, xenobiotic, heavy metal, and herbicide), reactive oxygen species (ROS) such as singlet oxygen (1O2), superoxide radical (O·-2), hydrogen peroxide (H2O2), and hydroxyl radical (·OH) are generated, which consequently promotes the oxidative stress (Yadav 2010). ROS are highly toxic to plant cell, and in absence of any protective mechanism, they can react with proteins, lipids, DNA, and inactivate antioxidant defense system (Yadav et al. 2005a, b). To avoid heavy metal toxicity, plants have a well-developed ROS scavenging system that serves as a defensive role, reduces the ROS-induced injury, and helps in resistance toward heavy metals (P.L.Gratäo et al. 2005). Plants have well-developed enzymatic and nonenzymatic scavenging systems to quench ROS (Vranová et al. 2002). So, the ROS is

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always generated at a controlled balance under unstressed conditions. When plants are subjected to adverse conditions, the scavenging system may lose its function, and the balance between producing and quenching ROS can be disturbed, resulting in accumulation of ROS, which in general causes lipid peroxidation, protein modification, breakage of DNA strands, chlorophyll decay, ion leakage, and cell death (Scandalios 1993). Nitric oxide (NO) is signaling bioactive molecules. The high reactivity and free diffusion across membranes make NO ideal for a transient signal molecule between adjacent cells and within cells. NO supplied exogenously provides resistance against stress induced by heavy metals like cadmium (N.V. Laspina et al. 2005a, b), copper (C.C. Yu et al. 2005), and aluminum (Wang and Yang. 2005). The defensive role of NO relates to its antioxidant ability to quench ROS and protect the cells against abiotic stress (Wang and Yang. 2005). Earlier, As is reported to cause oxidative stress leading to cellular damage in the sensitive plant species like mung bean, pea, and red clover (N. Stoeva et al. 2005). However, no information is available regarding the role of NO, a signaling and messenger molecule, in regulating the As-induced stress. Further, information is lacking whether exogenous NO can alleviate the As-induced oxidative stress by quenching ROS and thus prevent cellular damage. With this background in mind, a study was conducted to elucidate the role of NO in regulating As-induced oxidative stress and genotoxicity in Vicia faba. Treatment with sodium nitroprusside (SNP) as a donor of NO increases the rate of photosynthesis, chlorophyll content, transpiration rate, and stomata conductance in cucumber seedlings (Fan et al. 2007). NO has been found to enhance chlorophyll content in potato, lettuce, Arabidopsis thaliana L., and maize leaves (Graziano et al. 2002). Depending on the concentration of NO, the plant tissue, and the type of stress, some author considered NO to be a stressinducing agent (Leshem et al. 1997), while others have reported its protective role (Hsu and Kao 2004). The genetic evidence is limited to define the functions of NO in signal transduction events related to the cell cycle control in higher plants (Besson-Bard et al. 2008; Pagnussat et al. 2002; CorreaAragunde et al. 2004, 2006; Lozano-Juste et al. 2011; He Y.K et al. 2004; Ma W.et al. 2010). Through modulating NO levels in alfalfa cell cultures by exogenous treatment with NO donors or scavengers, it was revealed that NO is required for auxin-mediated activation of cell division and embryogenic cell formation (Otvos et al. 2005). V. faba L. is a widely grown vegetable in different parts of India and world. It is rich in mineral and vitamins and thus a popular vegetable. There exists a possibility that if V. faba was grown in As-polluted soil, then it could too have adverse effects as discussed above. Therefore, it becomes necessary to investigate the rate of As accumulation and its effects on physiological and biochemical processes in V. faba. It possesses many

advantages that make them ideal for use by scientists in the field of environmental mutagenesis for screening and monitoring of genotoxic agents according to the standard protocol for the plant assays established by the International Program on assays established by the International Program on Chemical Safety (IPCS) and the World Health Organization (WHO) (Soliman and Ghoneam 2004). V. faba is well known as an excellent model plant and useful biomarker for the detection of heavy metal pollution (Duan et al. 2000). In the present study, the alleviating potential of NO against As-induced oxidative stress and genotoxicity in V. faba has been examined especially on the basis of germination rate, seedling height, chlorophyll content, antioxidant enzyme, lipid peroxidation, reactive oxygen species, mitotic index, and chromosomal aberration assay.

Materials and methods Plant material and tested chemicals For the present investigation, the seeds of V. faba L. variety, EC 349608, were obtained from IARI Pusa Samastipur, Bihar. Treatment of arsenic used in the form of sodium arsenate (molecular weight 312.01) of technical grade (purity = 98.5 %) was purchased from Loba-Chemei, Mumbai, India. All the chemicals used in the study for enzymatic and biochemical estimations were of technical grade and procured from Merck Ltd., India; Loba-Chemie Pvt., Ltd., India. Sodium nitroprusside (as NO donor) was obtained from Qualigens Company batch no. NL727263 OIS. Fixative and stains used in the present experiment were procured from Loba Chemic Merck Specialties Pvt. Ltd. India. Plant growth and treatment Seeds of V. faba L. (EC 349608) were obtained from IARI Pusa, Samastipur, and Bihar. The seeds were surface-sterilized with 0.1 % (w/v) sodium hypochlorite solution and then washed with distilled water three times and soaked in distilled water for 12 h. Then, the seeds were divided into two groups. One half of the seeds were treated with concentrations of sodium arsenate(0.25, 0.50, 1 mM) for 6 h, and other half were pretreated with SNP (0.02 mM) for 12 h followed by intertreatment washing for 1 h and finally treated with concentrations of sodium arsenate (0.25, 0.50, 1 mM) for 6 h. After the treatment of sodium arsenate, the seeds were subjected for 4-h washing under running distilled water. Some seeds were also treated with distilled water with or without SNP to be used as control. The treated seeds were germinated on wet filter papers in petriplates with regular moistening with distilled water, and about 50 % seeds were also sown in the field for plant growth. Used concentration of SNP (0.02 mM) was determined on the basis of literatures present.

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Determination of As content For the dermination of As content, dried root and shoot sample (50 mg) from control and treated seedlings was digested in mixed acid (HNO3/HCLO4; 85:15, v/v) until transparent solution was obtained. The volume of digested sample was maintained up to 30 ml with double distilled water. The content of As in digested sample was determined by using inductively coupled argon plasma-atomic emission spectroscopy (ICAP-AES). Determination of nitric oxide For sample preparation, 600-mg tissue was ground in 3 ml of cooled buffer (0.1 M sodium acetate buffer, pH 6.0, 1 M NaCl, and 1 % (w/v) ascorbic acid). Homogenate was centrifuged at 10,000×g for 20 min at 4 °C. Supernatant was collected, and immediately, NO was quantified in cleared solution by using spectrophotometer measuring the conversion of oxy-hemoglobin to methemoglobin. Assay of NO was done by the hemoglobin-trapping technique, based on conversion of ferrous form of hemoglobin (oxyhemoglobin or HbO2) into ferric form; methemoglobin (metHb), by nitric oxide (Murphy and Noack 1994), was used for the detection of NO in tissue homogenate. Oxyhemoglobin was prepared from bovine-crystallized hemoglobin as follows. Sodium dithionite was added to the buffer (1–2 mg in 1 ml of sodium acetate buffer), followed by 1 ml of hemoglobin (10−6 equivalents ml−1). Continuous O2 was provided to this flask. The conversion of metHb to HbO2 was monitored by the change of color from brown to purple as the metHb was reduced by the sodium dithionite to HbO2 (deoxyhemoglobin) and then from purple to bright red. This HbO2 was stored on ice in dim light for NO determination. For the measurement of nitric oxide production, 10 μM HbO2 from stock was added to the above assay sample buffer. After 2 min, the assay solution was collected, and the conversion of HbO2 to metHb was quantified by taking its absorbance at 401 and 421 nm. Determination of growth and photosynthetic pigments The seed plated in petriplates was allowed to grow at room temperature in laboratory condition by constant moistening with double distilled water. After 72 h, the data for germination and root length were taken. The germination percentages of seeds were determined by radical formation basis, and the root lengths of the germinated seeds were measured with a millimetric rular as described by Fiskesjo (1985). Seedling height was measured by a ruler on 10th day after planting the seeds by the rular (Singh 1982). The 20 mg fresh leaves from each sample were crushed in 80 % acetone, and the pigments were extracted and centrifuged. The absorbance of extract was read at 663, 646, and 470 nm by using UV-visible

spectrophotometer. The amount of chlorophyll a, chlorophyll b, and carotenoids was calculated following the method of Lichtenthaler (1987). Determination of SOD, CAT, APX, and GST activities The superoxide dismutase (SOD; EC activity was estimated according to the method of Giannopolitis and Reis (1977). Fresh leaves (50 mg) of each sample were thoroughly homogenized in EDTA—phosphate buffer (pH 7.8) under ice cool condition using mortar and pestle and centrifuged at 15, 000g for 20 min at 4 °C, and supernatant was used as enzyme extract. Reaction mixture (3 ml) contained 50 mM potassium phosphate buffer (pH 7.8), 1.3 mM riboflavin, 0.1 mM EDTA, 13 mM methionine, 63 mM nitroblue tetrazolium (NBT), 0.05 M sodium carbonate (pH 10.2), and enzyme extract (0.1 ml). The reaction mixtures were illuminated for 20 min under white light intensity of 100 mmol photons m−2 s−1. The photoreduction of NBT (formation of purple formazone) was recorded spectrophotometrically at 560 nm and compared with blank samples having no enzyme extract. One unit (U) of SOD activity is defined as the amount of enzyme required to cause 50 % inhibition in reduction of NBT. For the estimation of catalase (CAT; EC and ascorbate peroxidase (APX; EC activity, fresh leaves (50 mg) of each sample were crushed in 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, with the addition of 1 mM ascorbic acid in case of APX. Homogenate was centrifuged at 15,000g for 20 min at 4 °C, and supernatant was used as enzyme. CAT activity was assayed as decrease in absorbance at 240 nm due to the dissociation of H2O2, and the activity of enzyme was calculated by using an extinction coefficient of 39.4 mM−1 cm −1(Aebi 1984). Reaction mixture (2 ml) contained 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 10 mM H2O2, and 0.2 ml enzyme extract. One unit (U) of enzyme activity is defined as 1 nmol H2O2 dissociated min−1. The APX activity was determined according to the method of Nakano and Asada (1981). Reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H2O2, and enzyme extract. The decrease in absorbance for 1 min was measured at 290 nm. The enzyme activity was calculated by using an extinction coefficient of 2.8 mM−1 cm−1. One unit (U) of enzyme activity is defined as 1 nmol ascorbate oxidized min−1. The glutathione S-transferase (GST, EC activity was measured following the method of Habig et al. (1974) using 1chloro-2,4-dinitrobenzene (CDNB) as substrate. For enzyme extraction, fresh leaves (50 mg) from each sample were homogenized in 100 mM potassium phosphate buffer (pH 6.25) and centrifuged. Enzyme assay was carried out in 2-ml reaction mixture containing 100 mM potassium phosphate buffer (pH 6.25), 0.75 mM CDNB, 30 mM GSH, and 0.2 ml enzyme

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extract. The increase in absorbance due to the formation of conjugates between GSH and CDNB was monitored at 340 nm. Enzyme activity was calculated by using an extinction coefficient 9.6 mM−1 cm−1. One unit (U) of enzyme activity is defined as 1 nmol of CDNB conjugates formed min−1. Determination of superoxide radical and hydrogen peroxide Superoxide radical (SOR; O2−) in each sample was determined following the method of Elstner and Heupel (1976). This assay is based on formation of NO2− from hydroxylamine in the presence of O2−. Fresh leaves (50 mg) were crushed in 65 mM potassium phosphate buffer (pH 7.8) and centrifuged at 10,000g for 10 min at 4 °C. The reaction mixture consisted of 65 mM potassium phosphate buffer (pH 7.8), 10 mM hydroxylamine hydrochloride, and leaf extract and then incubated for 20 min at 25 °C. After this, 17 mM sulfanilamide and 7 mM naphthylethylene diamine dihydrochloride were mixed to the incubated reaction mixture. After 15 min of reaction, diethyl ether was mixed to the same reaction mixture gently and centrifuged at 2000g for 5 min. The absorbance of the colored aqueous phase was recorded at 530 nm. A standard curve was prepared with NaNO−2 and used to calculate the production of O2−. For the estimation of H2O2, fresh leaf samples (50 mg) from control and treated seedlings were crushed in 0.1 % (w/v) trichloroacetic acid and centrifuged at 10,000g for 10 min at 4 °C (Velikova et al. 2000). The reaction mixture (2 ml) contained tissue extract (0.5 ml), 10 mM potassium phosphate buffer (pH 7.0), and 1 M KI solution. Absorbance of reaction mixture was recorded at 390 nm. The hydrogen peroxide concentration was calculated by using a standard curve prepared with H2O2.

Percentage Amelioration ¼

Determination of lipid peroxidation Lipid peroxidation was determined by measuring malondialdehyde (MDA) content following the method of Heath and Packer (1968). Fresh fully expanded young leaves (500 mg) were homogenized with 2.5 ml of 0.1 % trichloroacetic acid (TCA) solution. The extract was centrifuged at 10, 000 rpm for 10 min. Four milliliters of 20 % TCA containing 0.5 % thiobarbituric acid (TBA) was added to every 1 ml of the aliquot. After properly treating the mixture, it was centrifuged for 15 min at 10,000 rpm, and the absorbance of the supernatant was read at 532 nm. An extinction coefficient of 155/mM/cm was used for calculating MDA content. Cytogenetic assay When the primary roots became 3–5 cm long, the root tips were cut off to promote the growth of lateral roots. The tips of lateral roots (after 2–5-cm growth) were randomly sampled and fixed in Carnoy’s solution (6 ethyl alcohol/3 chloroform/1 acetic acid). These root tips were preserved in 70 % alcohol at 4 °C temperature for cytological studies. For the study of mitotic index and chromosomal aberrations, the temporary slides were prepared by hydrolyzing the root tips in 1 N HCl at 60 °C, removing the acid by thorough washing and 5 % ferrous alum and then staining with 0.5 % hematoxylin (Chauhan et al. 1998). Data evaluation and statistical analysis Percentage amelioration of toxicity by SNP was calculated according to Waters et al. (1990) and Rao and Tiwari (2006) by using the following formula:

ðArsenic treated groups  SNP pretreated with As groupsÞ  100 ðArsenic treated groups  ControlÞ

The results were statistically analyzed by analysis of variance (ANOVA). Duncan’s multiple range test was applied for mean separation for significant differences among treatments at P

Nitric oxide mitigates arsenic-induced oxidative stress and genotoxicity in Vicia faba L.

The protective effects of nitric oxide (NO) against arsenic (As)-induced structural disturbances in Vicia faba have been investigated. As treatment (0...
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