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Modulation of antioxidant defense system after long term arsenic exposure in Zantedeschia aethiopica and Anemopsis californica
Ecotoxicology and Environmental Safety 94 (2013) 67–72
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Modulation of antioxidant defense system after long term arsenic exposure in Zantedeschia aethiopica and Anemopsis californica Carmen Lizette Del-Toro-Sánchez a, Florentina Zurita a, Melesio Gutiérrez-Lomelí a, Brenda Solis-Sánchez a, Laura Wence-Chávez a, Araceli Rodríguez-Sahagún a, Osvaldo A. Castellanos-Hernández a, Gabriela Vázquez-Armenta b, Fernando Siller-López b,n a
Centro Universitario de la Ciénega, Universidad de Guadalajara, Av. Universidad 1115, Ocotlán, Jalisco, Mexico Departamento de Fisiología. Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada, 950, Guadalajara, Jalisco 44340, Mexico
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art ic l e i nf o
a b s t r a c t
Article history: Received 25 November 2012 Received in revised form 1 April 2013 Accepted 26 April 2013 Available online 17 May 2013
Zantedeschia aethiopica (calla lily) and Anemopsis californica (yerba mansa) are plant species capable of accumulating arsenic (As) and therefore proposed as phytoremediation for removal of As from drinking water. The effects of a continuous 6 month As exposure (347 11 μg/L) from local contaminated groundwater on the antioxidant response of Z. aethiopica and A. californica were evaluated in leaves and stems of the plants bimonthly in a subsurface flow constructed wetland. As increased the activities of the antioxidant enzymes ascorbate peroxidase, glutathione reductase and catalase where higher levels were observed in Z. aethiopica than A. californica. No significant differences were detected on lipid peroxidation levels or antioxidant capacity evaluated by ORAC and DPPH assays or total phenol contents in any part of the plant, although in general the leaves of both plants showed the best antioxidant defense against the metal. In conclusion, Z. aethiopica and A. californica were able to cope to As through induction of a more sensitive enzymatic antioxidant response mechanism. & 2013 Elsevier Inc. All rights reserved.
Keywords: Plants Zantedeschia aethiopica Anemopsis californica Arsenic Phytoremediation Antioxidant response
1. Introduction Chronic exposure to As is a significant worldwide environmental health concern (Agency for Toxic Substances and Disease Registry, 2007). The general population may be exposed to As in air, drinking water and food. Of these, food is usually the largest source of As. However, in some parts of the world, such as some regions of Mexico, the primary route of exposure is through drinking water that has been contaminated by anthropometric or natural geologic sources of As. Chronic exposure to low levels of As is associated with the development of various dermal effects (e.g., hyperpigmentation, hyperkeratosis), peripheral neuropathy, heart and kidney diseases and an increased risk of skin, bladder and lung cancer (Agency for Toxic Substances and Disease Registry, 2007). In México, As contamination of groundwater is of increasing concern as 75 percent of drinking water comes from groundwater sources (Armienta and Segovia, 2008). Remediation of As-contaminated soil and water is necessary for protecting both human life and agricultural production. Phytoremediation, which makes use of plants to remove, detoxify and/or
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Corresponding author. E-mail addresses: [email protected], [email protected] (F. Siller-López).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.04.024
sequester toxic pollutants, has been considered an environmentally friendly and cost-effective alternative to physical or chemical remediation methods (Salt et al., 1995). Plants have developed a number of mechanisms to control the homeostasis of essential elements and to cope with the stress induced by toxic elements (Vazquez et al., 2009). A result of the environmental metal stress is an increased production of reactive oxygen species (ROS) (Lyubenova and Schroder, 2011). Oxidation and damage of key plant macromolecules like proteins, lipids and DNA may result from sustained exposure to ROS and are observed as an increase in the amount of carbonylated proteins, lipid peroxidation and DNA oxidation. In response, plants have evolved several mechanisms to control the level of reactive oxygen molecules and to efficiently repair the oxidative damage of affected biomolecules. The defense against the oxidative levels is achieved by nonenzymatic and enzymatic antioxidant systems. Examples of the former include ascorbate, glutathione, phytochelatins, tocopherol, polyphenols, etc., that can act as metal chelators, provide reducing equivalents or directly scavenge molecular species of active oxygen. As with the chemical antioxidants, a network of antioxidant enzymes protects cells against oxidative stress through diverse mechanisms. Representative enzymes are peroxidases (e.g., ascorbate peroxidase (APX), peroxiredoxin), catalases (CAT), superoxide dismutase), thioredoxin and members
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Table 1 Enzymatic protocols. APX (EC.1.11.1.11) Extraction buffer 100 mM phosphate buffer (pH 6.0), 0.1% (v/v) Triton X-100, 2 mM CaCl2, 1 mM sodium ascorbate Reaction buffer 50 mM phosphate buffer (pH 7.0), 1 mM sodium ascorbate (AsA) Volume/well 185 a Extract volume 10 Substrate [stock H2O2 [1 mM] solution] Substrate 5 μL [0.025 mM] volume [final] OD 290 nm (AsA) Recording time 5 min Extinction 2.8 mM/cm coefficient Units nmol AsA min/mg/prot Reference Murshed et al. (2008) with modifications
a
GSR (EC.1.8.1.7)
CAT (EC.1.11.1.6)
100 mM Hepes buffer (pH 8.0), 0.1% (v/v) Triton X-100, 1 mM EDTA 50 mM Hepes buffer (pH 8.0), 0.5 mM EDTA, 0.25 mM NADPH 150 40 GSSG [40 mM]
100 mM phosphate buffer (pH 7.0), 0.1% (v/v) Triton X-100, 1 mM EDTA 50 mM phosphate buffer (pH 7.3) 165 5 H2O2 [2 M]
10 μL [2.0 mM]
20 μL [200 mM]
340 nm (NADPH) 5 min 6.2 mMcm
240 nm (H2O2) 5 min 40 mMcm
nmol NADPH min/mg/prot Murshed et al. (2008) with modifications
nmol H2O2 min/mg/ prot Chance and Maehly (1955) with modifications
Previous to substrate addition an absorbance reading was made at the indicated OD for 3 min at 25 1C to determine nonspecific degradation.
of the glutathione system like glutathione reductase (GSR) and glutathione S-transferases (Gill and Tuteja, 2010). Antioxidant systems and their significance for the acclimation of plants to air pollution and climatic stresses have been reviewed frequently with emphasis on the responses of leaves, although less attention has been paid to other parts of the plant (Schutzendubel and Polle, 2002); since in general, tolerant plants exposed to water contaminated with metals take up the metal by the cell roots where it is transported to the aerial tissues to be sequestered in the cell vacuoles (Finnegan and Chen, 2012). Several species of plants have been studied over the years in search of those able to cope and extract more effectively metals for phytoremediation purposes. Particularly, the construction of artificial wetlands is being considered as a low-cost option for As remotion from drinking water in less-developed nations (Bundschuh et al., 2010). In a preliminary study, Zantedeschia aethiopica and Anemopsis californica, two non-edible plants commonly found in the occidental region of México that shows a vigorous growth in constructed wetlands, were tested in a batch-fed subsurface flow constructed wetland (SSFCW) for phytoremediation of As contaminated groundwater with efficient As removing results. The mean As mass removal efficiency were 12.4 percent and 4.8 percent for Z. aethiopica and A. californica, respectively, with the peak on As removing at 2 months after exposure to As contaminated groundwater (Zurita et al., 2012). These results were similar to other studies where the As removal was observed in planted cells on SSFCW ( 15 percent) (Rahman et al., 2011). Therefore, the aim of this research was to assess the role of the antioxidant systems in leaves and stems of Z. aethiopica and A. californica in the ability to resist the stress induced by As.
2. Material and methods 2.1. Plant material and growth conditions Groundwater exposure to As in the organisms under study and As quantification by an inductively coupled plasma atomic emission spectrometer were performed previously and described in Zurita et al., (2012). Briefly, Z. aethiopica and A. californica, obtained from Ocotlán, Jalisco, México, were exposed during 6 months to control As-free bottled water or local As-contaminated groundwater in a SSFCW. Eight plastic cells (58 cm 22 cm 16 cm; L W H), were used as small-scale SSFCW. All the cells were fed with As-free bottled water during the first month of experimentation in order to allow plant establishment. Six liters of the corresponding water were fed to each of the cells under batch operation with a
hydraulic retention time of 5 days. Four cells were planted with three young plants of Z. aethiopica (ca. 20 cm-height) contained in each cell and four were planted with three young plants of A. californica (ca. 10 cm-height). Two of the four cells in each set were fed with As-free bottled water and two were fed with As-contaminated groundwater. As time weighted average concentration on groundwater water was: 34711.3 μg/L; range: 25–125 μg/L (median: 28 μg/L, min: 22 μg/L, max: 123 μg/L). Pools of sections of leaves and stems of both plant species were collected at 2, 4, and 6 months (n¼2 plant samples/exposure group/time point), grounded in an ice-cooled mortar with liquid nitrogen and stored without oxygen at −80 1C until further analysis. The antioxidant activities evaluated in the present study were a continuation of the assays described in Zurita et al., 2012. A Synergy four multi-mode microplate reader (Biotek, Winooski, VT) was used for all absorbance and fluorescence measurements using UV- or fluorescent-microplate wells.
2.2. Assays of antioxidant enzyme activities Ground frozen material (0.25 g) was homogenized at 4 1C in a Powergen 700 homogenizer (Fisher Scientific, Waltham, MA) with 1 mL of the corresponding extraction buffer for each enzyme assay (Table 1). The homogenate, filtered through four layers of muslin cloth, was centrifuged at 12,000g for 15 min at 4 1C and the supernatant (enzyme extract) was analyzed immediately for enzyme activities in triplicate. Kinetic readings of blanks (no enzyme extract) were used to correct for enzyme activities. Simplified version of the antioxidant enzyme protocols are shown in Table 1. Protein concentration was determined on enzyme extracts by the fluorescent assay with the compound o-phthaldialdehyde (Held, 2006), using bovine serum albumin (BSA) as the standard.
2.3. Oxidative stress and nonenzymatic antioxidant activity evaluation Thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, was used as the oxidative stress marker and nonenzymatic antioxidants were evaluated by measuring total phenols and total antioxidant capacity determined with two radical scavenging methods: oxygen radical absorptive capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays.
2.3.1. Estimation of lipid peroxidation The level of lipid peroxidation was estimated following the method of Heath and Packer (1968) and Ohkawa et al., (1979). Approximately 0.1 g of frozen samples was homogenized with 1.0 mL of 0.1 percent trichloroacetic acid (TCA), and centrifuged at 10,000g for 15 min at room temperature. An amount of 0.25 mL of the supernatant taken in a separate test tube was added with 1.0 mL of 0.5 percent thiobarbituric acid (TBA) made in 20 percent TCA. The mixture was heated at 95 1C for 30 min, cooled quickly in an ice-bath and centrifuged at 5000g for 5 min. The absorbance of malondialdehyde (MDA), one of the final decomposition products of lipid peroxidation that reacts with TBA, was recorded at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm. Results were expressed as thiobarbituric acid-reacting substances (TBARS) in nmol MDA g−1 fresh weight (FW) using the extinction coefficient of 155 Mm-1cm-1.
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2.3.2. Total phenolic content Stems and leaves extracts were obtained by the following method: 0.5 g of sample was dissolved in 0.5 mL of methanol. The solution was centrifuged at 12,000g for 15 min at 4 1C. The supernatant (methanolic extract) was used for the quantification of total phenols and determination of the total antioxidant capacity. Estimation of total phenols was determined according to the method of Prior et al., (2005) with minor modifications. The method consisted of mixing 20 mL of methanolic extract with 100 mL of 0.1 N Folin–Ciocalteu reagent on a 96-well microplate. After 5 min, 80 mL of 10 percent (w/v) Na2CO3 were added at the mixture. The absorbance was measured after 40 min at 765 nm. The amount of total phenolic content was calculated as gallic acid equivalents (GAE) using gallic acid as the standard and were expressed as mg GAE g−1 FW. 2.3.3. Determination of nonenzymatic antioxidant activity 2.3.3.1. ORAC assay. The ORAC assay was carried out using the method of Huang et al., (2002) and performed essentially as described by Held (2005). The reaction was carried out in 75 mM phosphate buffer (pH 7.4). An amount of 150 μL of sodium fluorescein (4 10−3 μM) and 25 μL of extracts (3 mg/mL) were added in the well of the microplate. The mixture was preincubated for 10 min at 37 1C and reactions were initiated by the addition of 25 μL of AAPH (2,2′-Azobis(2-amidinopropane) dihydrochloride) solution. The fluorescence was then monitored kinetically with data taken every minute during 60 min. The final results were expressed as μmol Trolox equivalents (TE) g−1 FW using a Trolox standard curve. 2.3.3.2. DPPH assay. A 96-well microplate method was used to measure the scavenging activity toward the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical according to the method of Kim et al., (2002) with some modifications. DPPH was dissolved in methanol to 1 mM. This stock solution was prepared daily. An amount of 70 mL of extract (1:100) was added with 130 mL stock solution. The absorbance of the reaction mixture was measured after 30 min at 515 nm. The final results were calculated by using a Trolox standard curve (0–100 mM) expressed as μmol TE g−1 FW. 2.4. Data analysis A one-way analysis of the variance (ANOVA) was employed to compare the biochemical changes and significant differences were considered at p o0.05. All the parameters evaluated are presented as the mean7 SD of three replicates per sample. Statistical analysis was performed using a StatGraphics software package.
3. Results and discussion As affects plant metabolism through diverse mechanisms: competing with phosphate in phosphate-dependent mechanisms, binding to reactive cellular dithiols and by the production of ROS (Finnegan and Chen, 2012). Plants make changes to cope with the toxicity associated to As, mainly by adjustments to antioxidant metabolites and numerous antioxidant and stress-related enzymes. In our study, plant antioxidant enzymes were more prone to response to the As exposure than the nonenzymatic antioxidants evaluated in the SWFC system. No visible symptoms of inhibition of plant growth or death tissue sections were observed by As exposure on both plant species. 3.1. Antioxidant enzyme activities
Fig. 1. Enzyme activity of (A) APX, (B) GSR and (C) CAT in leaves and stems of control (Ctr) and arsenic (As) exposed Zantedeschia aethiopica and Anemopsis californica at 2, 4 and 6 months. Values are the mean 7SD of at least three replicates.
Baseline activity of APX in leaves and stems of Z. aethiopica was higher than those of A. californica (Fig. 1A). Upon treatment with As, an apparent decrease on APX activity was observed at 2 months on leaves and stems of both plants, however at the end of the 6 months of As exposure, APX activity was significantly higher in leaves of Z. aethiopica (260 percent) and stems of A. californica (150 percent) compared to their respective controls (Fig. 1A). Regarding GSR, a trend on increasing GSR activity was observed in Z. aethiopica for the length of As exposure (Fig. 1B); however, only leaves showed a significant augment after 6 months of As treatment (335 percent of control values). Similar significant results were observed in leaves of A. californica at the end of the 6 months of As exposure (322 percent increase compared to control) (Fig. 1B). In A. californica, leaves also showed a higher basal level of GSR activity than stems.
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Fig. 2. Lipid peroxidation levels (TBARS) (A) and total phenols (B), ORAC (C) and DPPH (D) antioxidant levels in leaves and stems of control (Ctr) and arsenic (As) exposed Z. aethiopica and A. californica at 2, 4 and 6 months. Values are the mean 7 SD of at least three replicates.
For CAT assays, baseline CAT activity in leaves of Z. aethiopica was higher than A. californica (Fig. 1C), and leaves showed a higher basal activity than stems in Z. aethiopica. After 6 months of As exposure, a significant increase in CAT activity was observed in leaves of both plants compared to controls (363 percent for Z. aethiopica and 475 percent for A. californica). As observed, after 6 months of As exposure the enzymatic stimulation of APX, CAT and GSR, key players for the H2O2 metabolism and cellular thiol–redox balance in plants, suggest the mechanism of As toxicity that induces the response required to tolerate the chronic oxidative stress of As, which corresponds to a similar general response of antioxidant modulation described in different types of plants and sources of oxidative stress (Khan et al., 2009; Murugan and Harish, 2007; Srivastava et al., 2005). In general, in our study, leaves have a higher enzymatic antioxidant system than stems, and the response of Z. aethiopica to As exposure was more marked than in A. californica, which demonstrates a higher ability to cope with As toxicity in that structure and particular plant. As previously described in Ashyperaccumulators plants, the aerial tissues tend to have higher concentrations of As compared to the root (Finnegan and Chen, 2012); and it has been shown that the accumulation of As led to a significant increase in the level of ROS and hence, the up regulation on the antioxidant system to help combat the As toxicity (Srivastava et al., 2011).
3.2. Lipid peroxidation A significant difference was observed in basal TBARS content of leaves of A. californica compared to their stems and to the leaves and stems of Z. aethiopica. Z. aethiopica and A. californica showed no visual toxic symptoms when exposed to As stress and no effects were observed on TBARS lipid peroxidation, a sign of As toxicity (Fig 2A). TBARS levels, an indicator of the reaction of ROS with the polyunsaturated fatty acids of lipid membranes, demonstrated that the plants under study did not suffer lipid damage even after the prolonged 6 months of exposure. Srivastava et al., (2005) reported similar decreased TBARS levels on As tolerant Pteris vittata fern as results of the antioxidant defence. Also, Lokhande et al., (2011) showed comparable results on malondialdehyde concentration, another marker of lipid peroxidation, in Sesuvium portulacastrum exposed to As during 30 days where no association with the high presence of the heavy metal was reported. 3.3. Non-enzymatic antioxidant activity In general, similar basal levels of the antioxidant markers under study were found in leaves and stems when inter- and intra-plant comparisons were analyzed. Only in Z. aethiopica, control ORAC levels were higher at 2 months of growth on the SSFCW (Fig. 2C) and DPPH activity was lower in leaves than stems of the plant
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(Fig. 2D). No differences were observed in any of the nonenzymatic antioxidant levels of total phenol content, ORAC or DPPH activity upon exposure to As either in Z. aethiopica or A. californica (Fig. 2B–D). As described by several authors, the enzymatic and nonenzymatic antioxidant response mounted by plants in As detoxification is dependent on the concentration and exposure duration (Duman et al., 2010; Khan et al., 2009). In this study, the predominance on enzymatic antioxidant response over nonenzymatic could be explained by these factors, as observed by Cao et al., (2004) who reported a predominant role of enzymatic antioxidants in P. vittata to low As exposure (≤20 mg/kg) whereas nonenzymatic antioxidants were more critical at high As exposure (50–200 mg/kg). We hypothesized that the prevalence for the As-induced enzymatic over non-enzymatic antioxidant response in leaves that we observed in our study could be associated to a number of events that might occur on the metabolism of As in plants as has been reported by several authors. Most plants retain much of the As burden in the root (Finnegan and Chen, 2012) and a small fraction is preferentially transported to the leaves where it is sequestered and accumulated in the vacuole (Pickering et al., 2000). Complexation of As with phytochelatin or GSH followed by sequestration into the vacuole is an effective mechanism to tolerate As and maintain ROS under controlled balance. However, when As exposure is prolonged, the steady-state level of ROS concentration is chronically increased leading to oxidative modification of cellular constituents causing redox imbalance (Lushchak, 2011). Regulation of cellular antioxidant potential remains critical for survival and involves the sensing of reactive species and transduction of the signal to express antioxidant enzymes and stress proteins via the reversible oxidation of specific cysteine residues of key proteins, such as Rap2.4a (Shaikhali et al., 2008). The abundant titers of the low molecular mass antioxidants GSH and ascorbic acid support the redox homeostasis and are considered as crucial intracellular defense against ROS induced oxidative damage (Gill and Tuteja, 2010). However, even moderate oxidation of the GSH pool by redox imbalances might be sufficient to activate Rap2.4a-dependent gene expression (Shaikhali et al., 2008). In the present study, the increased activities of the enzymatic antioxidants APX, GSR and CAT and the minor but not significant changes on TBARS levels, sign of oxidative toxicity, or the antioxidant capacity evaluated by ORAC, DPPH assays or phenol content, might be a consequence of the fine sensing redox mechanism of signal transduction that up-regulate the enzymes involved in antioxidant defense even when no effect on the oxidant status and antioxidant capacity were observed after As exposure. More studies are needed for defining mechanistic relationships of the implicated antioxidant defense in As tolerance of Z. aethiopica and A. californica.
4. Conclusion A progressive antioxidative response was mediated by Z. aethiopica and A. californica to tolerate the chronic groundwater exposure to As in a SSFCW system. In general, a significant higher antioxidant enzymatic response was observed after 6 months of As exposure, and was more evident in leaves than stems of every plant. This effect could be related as a sign of recovery and augmentated response to tolerate a continuous chronic metal toxic stress. Also, As exposure evoked a more sensitive enzymatic antioxidant response than the observed by nonenzymatic biochemical elements (i.e., total phenols, total antioxidant capacity-ORAC, DPPH-). These plants appear to be candidates for application in As removal; however, further explorations will be
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necessary for the total characterization of the antioxidant response involved.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Modulation of antioxidant defense system after long term arsenic exposure in Zantedeschia aethiopica and Anemopsis californica Carmen Lizette Del-Toro-Sánchez a, Florentina Zurita a, Melesio Gutiérrez-Lomelí a, Brenda Solis-Sánchez a, Laura Wence-Chávez a, Araceli Rodríguez-Sahagún a, Osvaldo A. Castellanos-Hernández a, Gabriela Vázquez-Armenta b, Fernando Siller-López b,n a
Centro Universitario de la Ciénega, Universidad de Guadalajara, Av. Universidad 1115, Ocotlán, Jalisco, Mexico Departamento de Fisiología. Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada, 950, Guadalajara, Jalisco 44340, Mexico
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art ic l e i nf o
a b s t r a c t
Article history: Received 25 November 2012 Received in revised form 1 April 2013 Accepted 26 April 2013 Available online 17 May 2013
Zantedeschia aethiopica (calla lily) and Anemopsis californica (yerba mansa) are plant species capable of accumulating arsenic (As) and therefore proposed as phytoremediation for removal of As from drinking water. The effects of a continuous 6 month As exposure (347 11 μg/L) from local contaminated groundwater on the antioxidant response of Z. aethiopica and A. californica were evaluated in leaves and stems of the plants bimonthly in a subsurface flow constructed wetland. As increased the activities of the antioxidant enzymes ascorbate peroxidase, glutathione reductase and catalase where higher levels were observed in Z. aethiopica than A. californica. No significant differences were detected on lipid peroxidation levels or antioxidant capacity evaluated by ORAC and DPPH assays or total phenol contents in any part of the plant, although in general the leaves of both plants showed the best antioxidant defense against the metal. In conclusion, Z. aethiopica and A. californica were able to cope to As through induction of a more sensitive enzymatic antioxidant response mechanism. & 2013 Elsevier Inc. All rights reserved.
Keywords: Plants Zantedeschia aethiopica Anemopsis californica Arsenic Phytoremediation Antioxidant response
1. Introduction Chronic exposure to As is a significant worldwide environmental health concern (Agency for Toxic Substances and Disease Registry, 2007). The general population may be exposed to As in air, drinking water and food. Of these, food is usually the largest source of As. However, in some parts of the world, such as some regions of Mexico, the primary route of exposure is through drinking water that has been contaminated by anthropometric or natural geologic sources of As. Chronic exposure to low levels of As is associated with the development of various dermal effects (e.g., hyperpigmentation, hyperkeratosis), peripheral neuropathy, heart and kidney diseases and an increased risk of skin, bladder and lung cancer (Agency for Toxic Substances and Disease Registry, 2007). In México, As contamination of groundwater is of increasing concern as 75 percent of drinking water comes from groundwater sources (Armienta and Segovia, 2008). Remediation of As-contaminated soil and water is necessary for protecting both human life and agricultural production. Phytoremediation, which makes use of plants to remove, detoxify and/or
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Corresponding author. E-mail addresses: [email protected], [email protected] (F. Siller-López).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.04.024
sequester toxic pollutants, has been considered an environmentally friendly and cost-effective alternative to physical or chemical remediation methods (Salt et al., 1995). Plants have developed a number of mechanisms to control the homeostasis of essential elements and to cope with the stress induced by toxic elements (Vazquez et al., 2009). A result of the environmental metal stress is an increased production of reactive oxygen species (ROS) (Lyubenova and Schroder, 2011). Oxidation and damage of key plant macromolecules like proteins, lipids and DNA may result from sustained exposure to ROS and are observed as an increase in the amount of carbonylated proteins, lipid peroxidation and DNA oxidation. In response, plants have evolved several mechanisms to control the level of reactive oxygen molecules and to efficiently repair the oxidative damage of affected biomolecules. The defense against the oxidative levels is achieved by nonenzymatic and enzymatic antioxidant systems. Examples of the former include ascorbate, glutathione, phytochelatins, tocopherol, polyphenols, etc., that can act as metal chelators, provide reducing equivalents or directly scavenge molecular species of active oxygen. As with the chemical antioxidants, a network of antioxidant enzymes protects cells against oxidative stress through diverse mechanisms. Representative enzymes are peroxidases (e.g., ascorbate peroxidase (APX), peroxiredoxin), catalases (CAT), superoxide dismutase), thioredoxin and members
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Table 1 Enzymatic protocols. APX (EC.1.11.1.11) Extraction buffer 100 mM phosphate buffer (pH 6.0), 0.1% (v/v) Triton X-100, 2 mM CaCl2, 1 mM sodium ascorbate Reaction buffer 50 mM phosphate buffer (pH 7.0), 1 mM sodium ascorbate (AsA) Volume/well 185 a Extract volume 10 Substrate [stock H2O2 [1 mM] solution] Substrate 5 μL [0.025 mM] volume [final] OD 290 nm (AsA) Recording time 5 min Extinction 2.8 mM/cm coefficient Units nmol AsA min/mg/prot Reference Murshed et al. (2008) with modifications
a
GSR (EC.1.8.1.7)
CAT (EC.1.11.1.6)
100 mM Hepes buffer (pH 8.0), 0.1% (v/v) Triton X-100, 1 mM EDTA 50 mM Hepes buffer (pH 8.0), 0.5 mM EDTA, 0.25 mM NADPH 150 40 GSSG [40 mM]
100 mM phosphate buffer (pH 7.0), 0.1% (v/v) Triton X-100, 1 mM EDTA 50 mM phosphate buffer (pH 7.3) 165 5 H2O2 [2 M]
10 μL [2.0 mM]
20 μL [200 mM]
340 nm (NADPH) 5 min 6.2 mMcm
240 nm (H2O2) 5 min 40 mMcm
nmol NADPH min/mg/prot Murshed et al. (2008) with modifications
nmol H2O2 min/mg/ prot Chance and Maehly (1955) with modifications
Previous to substrate addition an absorbance reading was made at the indicated OD for 3 min at 25 1C to determine nonspecific degradation.
of the glutathione system like glutathione reductase (GSR) and glutathione S-transferases (Gill and Tuteja, 2010). Antioxidant systems and their significance for the acclimation of plants to air pollution and climatic stresses have been reviewed frequently with emphasis on the responses of leaves, although less attention has been paid to other parts of the plant (Schutzendubel and Polle, 2002); since in general, tolerant plants exposed to water contaminated with metals take up the metal by the cell roots where it is transported to the aerial tissues to be sequestered in the cell vacuoles (Finnegan and Chen, 2012). Several species of plants have been studied over the years in search of those able to cope and extract more effectively metals for phytoremediation purposes. Particularly, the construction of artificial wetlands is being considered as a low-cost option for As remotion from drinking water in less-developed nations (Bundschuh et al., 2010). In a preliminary study, Zantedeschia aethiopica and Anemopsis californica, two non-edible plants commonly found in the occidental region of México that shows a vigorous growth in constructed wetlands, were tested in a batch-fed subsurface flow constructed wetland (SSFCW) for phytoremediation of As contaminated groundwater with efficient As removing results. The mean As mass removal efficiency were 12.4 percent and 4.8 percent for Z. aethiopica and A. californica, respectively, with the peak on As removing at 2 months after exposure to As contaminated groundwater (Zurita et al., 2012). These results were similar to other studies where the As removal was observed in planted cells on SSFCW ( 15 percent) (Rahman et al., 2011). Therefore, the aim of this research was to assess the role of the antioxidant systems in leaves and stems of Z. aethiopica and A. californica in the ability to resist the stress induced by As.
2. Material and methods 2.1. Plant material and growth conditions Groundwater exposure to As in the organisms under study and As quantification by an inductively coupled plasma atomic emission spectrometer were performed previously and described in Zurita et al., (2012). Briefly, Z. aethiopica and A. californica, obtained from Ocotlán, Jalisco, México, were exposed during 6 months to control As-free bottled water or local As-contaminated groundwater in a SSFCW. Eight plastic cells (58 cm 22 cm 16 cm; L W H), were used as small-scale SSFCW. All the cells were fed with As-free bottled water during the first month of experimentation in order to allow plant establishment. Six liters of the corresponding water were fed to each of the cells under batch operation with a
hydraulic retention time of 5 days. Four cells were planted with three young plants of Z. aethiopica (ca. 20 cm-height) contained in each cell and four were planted with three young plants of A. californica (ca. 10 cm-height). Two of the four cells in each set were fed with As-free bottled water and two were fed with As-contaminated groundwater. As time weighted average concentration on groundwater water was: 34711.3 μg/L; range: 25–125 μg/L (median: 28 μg/L, min: 22 μg/L, max: 123 μg/L). Pools of sections of leaves and stems of both plant species were collected at 2, 4, and 6 months (n¼2 plant samples/exposure group/time point), grounded in an ice-cooled mortar with liquid nitrogen and stored without oxygen at −80 1C until further analysis. The antioxidant activities evaluated in the present study were a continuation of the assays described in Zurita et al., 2012. A Synergy four multi-mode microplate reader (Biotek, Winooski, VT) was used for all absorbance and fluorescence measurements using UV- or fluorescent-microplate wells.
2.2. Assays of antioxidant enzyme activities Ground frozen material (0.25 g) was homogenized at 4 1C in a Powergen 700 homogenizer (Fisher Scientific, Waltham, MA) with 1 mL of the corresponding extraction buffer for each enzyme assay (Table 1). The homogenate, filtered through four layers of muslin cloth, was centrifuged at 12,000g for 15 min at 4 1C and the supernatant (enzyme extract) was analyzed immediately for enzyme activities in triplicate. Kinetic readings of blanks (no enzyme extract) were used to correct for enzyme activities. Simplified version of the antioxidant enzyme protocols are shown in Table 1. Protein concentration was determined on enzyme extracts by the fluorescent assay with the compound o-phthaldialdehyde (Held, 2006), using bovine serum albumin (BSA) as the standard.
2.3. Oxidative stress and nonenzymatic antioxidant activity evaluation Thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, was used as the oxidative stress marker and nonenzymatic antioxidants were evaluated by measuring total phenols and total antioxidant capacity determined with two radical scavenging methods: oxygen radical absorptive capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays.
2.3.1. Estimation of lipid peroxidation The level of lipid peroxidation was estimated following the method of Heath and Packer (1968) and Ohkawa et al., (1979). Approximately 0.1 g of frozen samples was homogenized with 1.0 mL of 0.1 percent trichloroacetic acid (TCA), and centrifuged at 10,000g for 15 min at room temperature. An amount of 0.25 mL of the supernatant taken in a separate test tube was added with 1.0 mL of 0.5 percent thiobarbituric acid (TBA) made in 20 percent TCA. The mixture was heated at 95 1C for 30 min, cooled quickly in an ice-bath and centrifuged at 5000g for 5 min. The absorbance of malondialdehyde (MDA), one of the final decomposition products of lipid peroxidation that reacts with TBA, was recorded at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm. Results were expressed as thiobarbituric acid-reacting substances (TBARS) in nmol MDA g−1 fresh weight (FW) using the extinction coefficient of 155 Mm-1cm-1.
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2.3.2. Total phenolic content Stems and leaves extracts were obtained by the following method: 0.5 g of sample was dissolved in 0.5 mL of methanol. The solution was centrifuged at 12,000g for 15 min at 4 1C. The supernatant (methanolic extract) was used for the quantification of total phenols and determination of the total antioxidant capacity. Estimation of total phenols was determined according to the method of Prior et al., (2005) with minor modifications. The method consisted of mixing 20 mL of methanolic extract with 100 mL of 0.1 N Folin–Ciocalteu reagent on a 96-well microplate. After 5 min, 80 mL of 10 percent (w/v) Na2CO3 were added at the mixture. The absorbance was measured after 40 min at 765 nm. The amount of total phenolic content was calculated as gallic acid equivalents (GAE) using gallic acid as the standard and were expressed as mg GAE g−1 FW. 2.3.3. Determination of nonenzymatic antioxidant activity 2.3.3.1. ORAC assay. The ORAC assay was carried out using the method of Huang et al., (2002) and performed essentially as described by Held (2005). The reaction was carried out in 75 mM phosphate buffer (pH 7.4). An amount of 150 μL of sodium fluorescein (4 10−3 μM) and 25 μL of extracts (3 mg/mL) were added in the well of the microplate. The mixture was preincubated for 10 min at 37 1C and reactions were initiated by the addition of 25 μL of AAPH (2,2′-Azobis(2-amidinopropane) dihydrochloride) solution. The fluorescence was then monitored kinetically with data taken every minute during 60 min. The final results were expressed as μmol Trolox equivalents (TE) g−1 FW using a Trolox standard curve. 2.3.3.2. DPPH assay. A 96-well microplate method was used to measure the scavenging activity toward the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical according to the method of Kim et al., (2002) with some modifications. DPPH was dissolved in methanol to 1 mM. This stock solution was prepared daily. An amount of 70 mL of extract (1:100) was added with 130 mL stock solution. The absorbance of the reaction mixture was measured after 30 min at 515 nm. The final results were calculated by using a Trolox standard curve (0–100 mM) expressed as μmol TE g−1 FW. 2.4. Data analysis A one-way analysis of the variance (ANOVA) was employed to compare the biochemical changes and significant differences were considered at p o0.05. All the parameters evaluated are presented as the mean7 SD of three replicates per sample. Statistical analysis was performed using a StatGraphics software package.
3. Results and discussion As affects plant metabolism through diverse mechanisms: competing with phosphate in phosphate-dependent mechanisms, binding to reactive cellular dithiols and by the production of ROS (Finnegan and Chen, 2012). Plants make changes to cope with the toxicity associated to As, mainly by adjustments to antioxidant metabolites and numerous antioxidant and stress-related enzymes. In our study, plant antioxidant enzymes were more prone to response to the As exposure than the nonenzymatic antioxidants evaluated in the SWFC system. No visible symptoms of inhibition of plant growth or death tissue sections were observed by As exposure on both plant species. 3.1. Antioxidant enzyme activities
Fig. 1. Enzyme activity of (A) APX, (B) GSR and (C) CAT in leaves and stems of control (Ctr) and arsenic (As) exposed Zantedeschia aethiopica and Anemopsis californica at 2, 4 and 6 months. Values are the mean 7SD of at least three replicates.
Baseline activity of APX in leaves and stems of Z. aethiopica was higher than those of A. californica (Fig. 1A). Upon treatment with As, an apparent decrease on APX activity was observed at 2 months on leaves and stems of both plants, however at the end of the 6 months of As exposure, APX activity was significantly higher in leaves of Z. aethiopica (260 percent) and stems of A. californica (150 percent) compared to their respective controls (Fig. 1A). Regarding GSR, a trend on increasing GSR activity was observed in Z. aethiopica for the length of As exposure (Fig. 1B); however, only leaves showed a significant augment after 6 months of As treatment (335 percent of control values). Similar significant results were observed in leaves of A. californica at the end of the 6 months of As exposure (322 percent increase compared to control) (Fig. 1B). In A. californica, leaves also showed a higher basal level of GSR activity than stems.
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Fig. 2. Lipid peroxidation levels (TBARS) (A) and total phenols (B), ORAC (C) and DPPH (D) antioxidant levels in leaves and stems of control (Ctr) and arsenic (As) exposed Z. aethiopica and A. californica at 2, 4 and 6 months. Values are the mean 7 SD of at least three replicates.
For CAT assays, baseline CAT activity in leaves of Z. aethiopica was higher than A. californica (Fig. 1C), and leaves showed a higher basal activity than stems in Z. aethiopica. After 6 months of As exposure, a significant increase in CAT activity was observed in leaves of both plants compared to controls (363 percent for Z. aethiopica and 475 percent for A. californica). As observed, after 6 months of As exposure the enzymatic stimulation of APX, CAT and GSR, key players for the H2O2 metabolism and cellular thiol–redox balance in plants, suggest the mechanism of As toxicity that induces the response required to tolerate the chronic oxidative stress of As, which corresponds to a similar general response of antioxidant modulation described in different types of plants and sources of oxidative stress (Khan et al., 2009; Murugan and Harish, 2007; Srivastava et al., 2005). In general, in our study, leaves have a higher enzymatic antioxidant system than stems, and the response of Z. aethiopica to As exposure was more marked than in A. californica, which demonstrates a higher ability to cope with As toxicity in that structure and particular plant. As previously described in Ashyperaccumulators plants, the aerial tissues tend to have higher concentrations of As compared to the root (Finnegan and Chen, 2012); and it has been shown that the accumulation of As led to a significant increase in the level of ROS and hence, the up regulation on the antioxidant system to help combat the As toxicity (Srivastava et al., 2011).
3.2. Lipid peroxidation A significant difference was observed in basal TBARS content of leaves of A. californica compared to their stems and to the leaves and stems of Z. aethiopica. Z. aethiopica and A. californica showed no visual toxic symptoms when exposed to As stress and no effects were observed on TBARS lipid peroxidation, a sign of As toxicity (Fig 2A). TBARS levels, an indicator of the reaction of ROS with the polyunsaturated fatty acids of lipid membranes, demonstrated that the plants under study did not suffer lipid damage even after the prolonged 6 months of exposure. Srivastava et al., (2005) reported similar decreased TBARS levels on As tolerant Pteris vittata fern as results of the antioxidant defence. Also, Lokhande et al., (2011) showed comparable results on malondialdehyde concentration, another marker of lipid peroxidation, in Sesuvium portulacastrum exposed to As during 30 days where no association with the high presence of the heavy metal was reported. 3.3. Non-enzymatic antioxidant activity In general, similar basal levels of the antioxidant markers under study were found in leaves and stems when inter- and intra-plant comparisons were analyzed. Only in Z. aethiopica, control ORAC levels were higher at 2 months of growth on the SSFCW (Fig. 2C) and DPPH activity was lower in leaves than stems of the plant
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(Fig. 2D). No differences were observed in any of the nonenzymatic antioxidant levels of total phenol content, ORAC or DPPH activity upon exposure to As either in Z. aethiopica or A. californica (Fig. 2B–D). As described by several authors, the enzymatic and nonenzymatic antioxidant response mounted by plants in As detoxification is dependent on the concentration and exposure duration (Duman et al., 2010; Khan et al., 2009). In this study, the predominance on enzymatic antioxidant response over nonenzymatic could be explained by these factors, as observed by Cao et al., (2004) who reported a predominant role of enzymatic antioxidants in P. vittata to low As exposure (≤20 mg/kg) whereas nonenzymatic antioxidants were more critical at high As exposure (50–200 mg/kg). We hypothesized that the prevalence for the As-induced enzymatic over non-enzymatic antioxidant response in leaves that we observed in our study could be associated to a number of events that might occur on the metabolism of As in plants as has been reported by several authors. Most plants retain much of the As burden in the root (Finnegan and Chen, 2012) and a small fraction is preferentially transported to the leaves where it is sequestered and accumulated in the vacuole (Pickering et al., 2000). Complexation of As with phytochelatin or GSH followed by sequestration into the vacuole is an effective mechanism to tolerate As and maintain ROS under controlled balance. However, when As exposure is prolonged, the steady-state level of ROS concentration is chronically increased leading to oxidative modification of cellular constituents causing redox imbalance (Lushchak, 2011). Regulation of cellular antioxidant potential remains critical for survival and involves the sensing of reactive species and transduction of the signal to express antioxidant enzymes and stress proteins via the reversible oxidation of specific cysteine residues of key proteins, such as Rap2.4a (Shaikhali et al., 2008). The abundant titers of the low molecular mass antioxidants GSH and ascorbic acid support the redox homeostasis and are considered as crucial intracellular defense against ROS induced oxidative damage (Gill and Tuteja, 2010). However, even moderate oxidation of the GSH pool by redox imbalances might be sufficient to activate Rap2.4a-dependent gene expression (Shaikhali et al., 2008). In the present study, the increased activities of the enzymatic antioxidants APX, GSR and CAT and the minor but not significant changes on TBARS levels, sign of oxidative toxicity, or the antioxidant capacity evaluated by ORAC, DPPH assays or phenol content, might be a consequence of the fine sensing redox mechanism of signal transduction that up-regulate the enzymes involved in antioxidant defense even when no effect on the oxidant status and antioxidant capacity were observed after As exposure. More studies are needed for defining mechanistic relationships of the implicated antioxidant defense in As tolerance of Z. aethiopica and A. californica.
4. Conclusion A progressive antioxidative response was mediated by Z. aethiopica and A. californica to tolerate the chronic groundwater exposure to As in a SSFCW system. In general, a significant higher antioxidant enzymatic response was observed after 6 months of As exposure, and was more evident in leaves than stems of every plant. This effect could be related as a sign of recovery and augmentated response to tolerate a continuous chronic metal toxic stress. Also, As exposure evoked a more sensitive enzymatic antioxidant response than the observed by nonenzymatic biochemical elements (i.e., total phenols, total antioxidant capacity-ORAC, DPPH-). These plants appear to be candidates for application in As removal; however, further explorations will be
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necessary for the total characterization of the antioxidant response involved.
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