ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
ROLE OF METAL SPECIATION IN LEAD-INDUCED OXIDATIVE STRESS
TO Vicia faba ROOTS1
© 2015 M. Shahid*, C. Dumat**, ***, B. Pourrut****, G. Abbas*, N. Shahid*, E. Pinelli**, ***
*Department of Environmental Sciences, COMSATS Institute of Information Technology, Vehari, Pakistan
**Université de Toulouse, INP-ENSAT, Castanet-Tolosan, France ***UMR 5245 CNRS-INP-UPS, EcoLab (Laboratoire d'écologie fonctionnelle), Castanet-Tolosan, France ****LGCgE, Equipe Sols et Environnement, ISA, Lille Cedex, France Received December 2, 2014
Chemical speciation of metals in soil/solution plays an important role in determining their biogeochemical behavior in soil—plant system. The current study evaluated the influence of applied form of Pb (metal speciation) on its toxicity to metal sensitive Vicia faba L. roots. Lead was applied to young V. faba seedlings alone or chelated by organic ligands (citric acid and ethylenediaminetetraacetic acid). Plants were exposed to all treatments for 1, 4, 8, 12, and 24 h in nutrient solution, and contents of H2O2 and thiobarbituric-acid-reac-tive substances (TBARS) production were analyzed in V. faba roots. The results showed that Pb toxicity to V. faba roots depended on its applied chemical form and duration of exposure. Lead alone caused two burst of lipid peroxidation and H2O2 induction at 1 h and 12 h. Addition of EDTA dose-dependently inhibited Pb-induced H2O2 and TBARS production, indicating a protective role of this chelator against Pb toxicity during the first 24 h. In contrast, citric acid did not show significant effects on Pb-induced H2O2 and TBARS production, but delayed the induction of these effects. This study suggested that Pb toxicity to V. faba roots varies with Pb speciation in growth medium.
Keywords: Vicia faba — lead — organic ligands — oxidative stress
DOI: 10.7868/S0015330315040156
INTRODUCTION
Environmental contamination by heavy metal pollution is a pervasive global problem, which has threatened the safety and quality of environment [1]. According to the U.S. Geological Survey, the worldwide mine production ofheavy metals has increased during the last decade due to their increased use in industry [2]. Many thousands of places on earth contaminated with heavy metals, including Europe, Asia, and North America [3]. According to the European Environmental Agency (EEA), pollutions in Europe are subject to the order of 250000 places.
Among the heavy metals, lead (Pb) is a non-essential, widespread and potentially toxic metal that presents a severe environmental concern and health risks to living organisms [4]. Lead is used widely in various industrial processes due to its useful physico-chemical properties, which led to a significantly enhanced con-
1 This text was submitted by the authors in English.
Abbreviations'. CA — citric acid; ROS — reactive oxygen species; TBARS — thiobarbituric-acid-reactive substances. Corresponding author. Dr. Muhammad Shahid. Department of Environmental Sciences, COMSATS Institute of Information Technology, \ehari-61100, Pakistan; e-mail. muhammadshahid@ ciitvehari.edu.pk
centration of this metal in environment [5, 6]. Lead does not play any known necessary function in living metabolism, and is reported to cause a number of toxic effects to metabolic reactions in living organisms [7, 8].
Lead-induced toxicity to plants is often a result of interference of uptake and transport of nutrients and water, altered nitrogen metabolism, reduced plant growth, photosynthesis and respiration and disorder of the photosynthetic apparatus in chloroplasts [7, 9]. Lead toxicity to plants also causes invisible symptoms of injury like leaf roll, necrosis and chlorosis of leaves and reduced length and browning of roots [7, 10]. Lead-induced excess production of reactive oxygen species (ROS) is the most common and initial biochemical effect in plant under Pb toxicity [1, 11, 12]. Enhanced generation of these ROS deteriorates the cell redox status and results in oxidative stress [2, 13]. Lead-induced oxidative stress causes several dysfunctions to plant cells due to lesions between ROS and bio-molecules [2, 14]. Overproduction of ROS as a result of Pb toxicity may lead to cell death due to oxidative reactions such as membrane lipid peroxidation, RNA and DNA damage, enzyme inhibition, protein oxidation, and malondialdehyde (MDA) generation [2]. Lead is well known to cause lipid peroxidation via increased
Experimental design and composition of all the treatments
Treatment Visual Minteq input Visual Minteq output, %
Notation Composition Pb-chelated Pb-free
Control Hoagland solution (HS) - -
Pb HS + 5 ^M Pb 0 85
Pb-EDTA-a HS + 5 ^M Pb + 2.25 ^M EDTA 40 51
Pb-EDTA-b HS + 5 ^M Pb + 10 ^M EDTA 99 1
Pb-CA-a HS + 5 ^M Pb + 550 ^M CA 25 64
Pb-CA-b HS + 5 ^M Pb + 1000 ^M CA 40 51
Pb-chelated and Pb-free data were calculated using Visual Minteq, v. 2.60.
ROS generation in plants [12]. Lipid peroxidation occurs when ROS such as H2O2 or HO' cause oxidation lipids in cell membranes. The process of lipid peroxidation most commonly occurs in polyunsaturated fatty acids [14]. The evaluations ofROS production and lipid peroxidation are commonly used plant bioassays to predict metal toxicity in risk assessment studies [13, 14].
Despite considerable progress in recent years regarding toxic effects of Pb to plants, little is known about physiological responses of Pb with respect to its chemical speciation and bioavailability [15, 16]. Recent literature indicates that the potential toxicity of Pb varies with its chemical speciation [17]. Different forms of Pb vary greatly regarding their toxic effects to plants and other living organisms [2, 3]. Therefore, it is of great importance to know the effect of applied chemical form of Pb on its toxicity and physiological responses of plants. Organic chelating agents have been reported to modify the metal speciation by com-plexing the Pb through their binding groups [9]. Consequently, these organic ligands could modify physiological responses of plants to heavy metal [12, 18]. However, most of the studies carried out using organic ligands employed plant-hyperaccumulator with objectives of metal solubilization in soil, uptake by plants or translocation to aerial parts. There is very rare data regarding plant physiological responses to heavy metals under organic ligand application. Thus, the objective of this study was to evaluate the effect of metal speciation on Pb toxicity to V. faba plants in term of ROS production and lipid peroxidation.
MATERIALS AND METHODS
Experimental conditions and treatments. The broad bean (Vicia faba L., cv. Primabel; type aguadulce; Fa-baceae) plants were used in this study. V. faba plants are commonly used as bioindicator for determining the toxicity of heavy metals [19]. The high use of V. faba plants as model plant of metal toxicity is due to its important biomass, fast growth and high sensitivity to metals [17].
Dry seeds of V. faba were soaked in deionized water for 6—8 h. The seeds were allowed to germinate by putting them between two layers of moist cotton in a germination chamber at 100% relative humidity and 22°C. After a germination period of one week, uniform and healthy seedlings of V. faba were transferred to plastic tubs containing nutrient solution ("Sigma") for vegetative growth. Nutrient solution used for vegetative growth contained 5 mM KNO3, 5 mM Ca(NO3)2, 2 mM KH2PO4, 1.5 mM MgSO4, 9.11 |M MnSO4, 1.53 |M ZnSO4, 24.05 |M H3BO3, 0.235 |M CuSO4, 0.1 |M Na2MoO4, and 268.6 |M Fe [20]. V. faba seedlings were grown under growth chamber conditions: 70% relative humidity, 24/22°C day/night temperatures and 16-h photoperiod.
Three-week-old plants were exposed to different treatments of Pb (table). Lead was exposed to V. faba plants at 5 ^M as lead nitrate {Pb(NO3)2} alone or in combination with ethylene diamine tetraacetic acid (EDTA) and citric acid (CA). Both organic ligands were added to nutrient solution at two levels, i.e., EDTA at 2.25 and 10 |M whereas CA at 550 and 1000 |M. The highest applied levels of CA (1000 |M) and EDTA (10 |M) were also exposed to plants alone as controls to check their individual effects on ROS production and lipid peroxidation. All treatments were applied for five time intervals, i.e., 1, 4, 8, 12, and 24 h in order to evaluate the early steps of Pb toxicity to V. faba plants. Pb(NO3)2 in 5 |M concentration was chosen because, according to Pourrut et al. [21], at this concentration Pb causes genotoxicity in V. faba seedlings while remaining a representative of the pollution levels often found in the environment. The applied level of KH2PO4 in all treatments was lowered to 0.2 mM in order to reduce Pb precipitation as phosphate in nutrient solution [17]. The computer software speciation model Visual Minteq (v. 2.60) was employed to determine the chemical speciation of Pb in nutrient solution under applied levels of CA and EDTA. Visual Minteq calculations showed that application of 2.25 and 10 |M EDTA chelated, respectively, 40 and 99% of Pb, whereas 550 and 1000 |M of CA chelated, respectively, 25 and 40% of Pb in nutrient solution (table).
Lipid peroxidation assay. Lipid peroxidation was evaluated by calculating the thiobarbituric-acid-reac-tive substances (TBARS) as described by Shahid et al. [18]. Each sample was homogenized at 4°C in liquid nitrogen using a solution of hydro-alcoholic (80/20, v/v). The homogenate was then incubated at 95°C with the thiobarbituric acid in the presence of butyl hydroxytol-uene (HTB) to avoid any oxidation of the mixture. After centrifugation, spectrophotometer was used to determine the absorbance of mixture assay at 532 nm. The TBARS contents were determined using 155/(mM cm) extinction coefficient.
Evaluation of H2O2. The measurement ofH2O2 contents was carried out in accordance to Shahid et al. [18]. All samples were homogenized in liquid nitrogen using 5 mL trichloroacetic
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