Relationship between copper-and zinc-induced oxidative stress and ...

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ORIGINAL ARTICLE. B. N. Tripathi Ж J. P. Gaur. Relationship between copper- and zinc-induced oxidative stress and proline accumulation in Scenedesmus sp.
Planta (2004) 219: 397–404 DOI 10.1007/s00425-004-1237-2

O R I GI N A L A R T IC L E

B. N. Tripathi Æ J. P. Gaur

Relationship between copper- and zinc-induced oxidative stress and proline accumulation in Scenedesmus sp.

Received: 3 November 2003 / Accepted: 5 February 2004 / Published online: 11 March 2004  Springer-Verlag 2004

Abstract A 4-h exposure of Scenedesmus sp. to Cu or Zn enhanced intracellular levels of both test metals and proline. The level of intracellular proline increased markedly up to 10 lM Cu, but higher concentrations were inhibitory. However, intracellular proline consistently increased with increasing concentration of Zn in the medium. Cu and Zn induced oxidative stress in the test alga by increasing lipid peroxidation and membrane permeability, and by reducing SH content. Pretreatment of the test alga with 1 mM proline for 30 min completely alleviated Cu-induced lipid peroxidation, minimized K+ efflux and also reduced depletion of the SH pool. But proline pretreatment could only slightly reduce Zn-induced oxidative stress. Interestingly, proline pretreatment increased the level of Cu (25–54%) and Zn (19– 49%) inside the cells. It did not affect the activities of superoxide dismutase, ascorbate peroxidase or catalase, but improved glutathione reductase activity under Cu and Zn stress. A comparison of the effects of proline pretreatment on lipid peroxidation by Cu, Zn, methyl viologen and ultraviolet-B radiation suggests that proline protects cells from metal-induced oxidative stress by scavenging reactive oxygen species rather than by chelating metal ions. Pretreatment of cells with a known antioxidant (ascorbate) and a hydroxyl radical scavenger (sodium benzoate) considerably reduced metal-induced lipid peroxidation and proline accumulation. However, sodium benzoate had a very mild effect on Zninduced lipid peroxidation and proline accumulation. The present study demonstrates that proline possibly acts by detoxifying reactive oxygen species, mainly hydroxyl radicals, rather than by improving the antioxidant defense system under metal stress. Keywords Antioxidant Æ Metal toxicity Æ Oxidative stress Æ Proline Æ Scenedesmus B. N. Tripathi Æ J. P. Gaur (&) Laboratory of Algal Biology, Department of Botany, Banaras Hindu University, 221005 Varanasi, India E-mail: [email protected]

Abbreviations APOX: Ascorbate peroxidase Æ CAT: Catalase Æ GR: Glutathione reductase Æ MDA: Malondialdehyde Æ MV: Methyl viologen Æ ROS: Reactive oxygen species Æ SH: Sulphydryl Æ SOD: Superoxide dismutase Æ UV-B: Ultraviolet-B radiation

Introduction Reactive oxygen species (ROS), such as, superoxide, hydrogen peroxide and the hydroxyl radical, are formed by the leakage of electrons onto molecular oxygen from the electron transport activities of chloroplasts, mitochondria and the plasma membrane in plant cells during normal metabolism (Foyer et al. 1994). Elevated levels of heavy metals and various other environmental stresses accelerate the formation of ROS. Redox-active metals (e.g., Cu and Fe) catalyze the formation of hydroxyl radicals by directly participating in the Haber– Weiss reaction, and metals without redox capacity (e.g., Cd, Pb, Hg, Zn) inactivate the cellular antioxidant pool and disrupt the metabolic balance, eventually enhancing the load of ROS (Stohs and Bagchi 1995; Briat 2002). To cope with the harmful effects of ROS, plants cells are equipped with a well-developed antioxidant defense system comprising enzymes, namely, superoxide dismutase (SOD), ascorbate peroxidase (APOX), catalase (CAT), and glutathione reductase (GR), as well as nonenzymatic compounds, such as, ascorbate, thiols, tocopherols, etc. (Mallick and Mohn 2000). Proline accumulation often occurs in a variety of plants in the presence of elevated levels of metals, but a consensus has yet to emerge on its role in combating metal stress. Some researchers assume that proline accumulation is a mere symptom of damage, and is not at all involved in protection against metal stress (Lutts et al. 1996). Others believe that proline does protect cells from metal stress (Farago and Mullen 1979; Smirnoff and Cumbes 1989; Wu et al. 1995; Mehta and Gaur 1999; Siripornadulsil et al. 2002). Proline may protect

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plants from osmotic stress and enzymes from denaturation, help in regulating cytosolic pH and the NAD(P)+/NAD(P)H ratio, serve as a source of carbon and nitrogen, scavenge toxic ROS, etc. (Sharmila and Pardha Saradhi 2002). It has been proposed that proline might protect plants from metal toxicity by chelating heavy metals in the cytoplasm (Farago and Mullen 1979). In fact, proline has been found to protect glucose6-phosphate dehydrogenase and nitrate reductase from metal toxicity in vitro due to reduction of free metal ion activity (Sharma et al. 1998). But the latter authors believe that intracellular accumulation of proline does not effectively contribute to metal detoxification by chelation. Wu et al. (1995, 1998) suggested that proline protects algal cells from metal toxicity by reducing metal uptake. Mehta and Gaur (1999) and Siripornadulsil et al. (2002) showed that proline protects algal cells from metal-induced oxidative stress. However, it is not certain as yet if proline-mediated mitigation of metal-induced oxidative stress is by direct involvement of proline in the detoxification of free radicals, or by improvement of the antioxidant defense machinery already existing in the cell. The present study examines the relationship between Cu- and Zn-induced oxidative stress and proline accumulation in Scenedesmus sp., and discusses the possible mechanisms through which proline offers protection against metal-induced oxidative stress. Cu and Zn were used as the test metals because the former is redox-active whereas the latter is not.

Materials and methods Test organism and culture conditions The freshwater green alga Scenedesmus sp. (obtained from Dr. J.W. Rijstenbil, Netherlands Institute of Ecology, Yerseke, The Netherlands) was cultivated in 4-times diluted BG 11 culture medium (Hughes et al. 1958). The pH of the culture medium was adjusted to 7. The cultures received 72 lmol photons m 2 s 1 photosynthetically active radiation (PAR) in a 12:12 h light and dark cycle at 27±1C. The cultures were hand-shaken 3–4 times every day. All the experiments were conducted in triplicate with cultures in the exponential phase of growth.

Metal uptake and proline accumulation An exponentially growing culture of Scenedesmus sp. was supplemented with the stock solution of Cu or Zn, prepared from their analytical grade salts CuCl2.2H2O and ZnCl2, respectively, so as to give the selected concentrations of each test metal (2.5, 5, 10, 20, and 40 lM Cu and 5, 10, 25, 50, and 100 lM Zn) in the medium. Preliminary experiments showed approximately 50% inhibition of the growth of the test organism at 10 and 25 lM of Cu and Zn, respectively. Metal uptake by the test alga was measured by determining the intracellular metal content. For this purpose, algal suspension (20 ml, 109 cells ml 1) treated with different concentrations of the test metals for 4 h was taken and the cells were harvested by centrifugation. Sufficient accumulation of test metals occurred within 4 h to elicit toxic effects on the test alga, and therefore this time period was selected for all the experiments. After washing in

10 ml of 2 mM EDTA for 10 min to remove the metal ions adhering to the cell surface, the pellet was transferred to 5 ml of digestion mixture containing HNO3 (70%), H2O2 (30%) and deionized water in 1:1:3 ratio (Bates et al. 1982). Digestion was performed on a hotplate at 80C until the solution became colourless. The residue was dissolved in 2% (v/v) HNO3 and the final volume adjusted to 5 ml. The digested samples were analyzed for metal content with an atomic absorption spectrophotometer (Perkin Elmer, model 2380). For the determination of Cu- and Zn-induced proline accumulation, a 50-ml algal suspension (109 cells ml 1) was treated with different concentrations of the test metals for 4 h. Thereafter, cells were harvested for measurement of proline content by the method of Bates et al. (1973). The harvested cells were resuspended in 10 ml of 3% sulfosalicylic acid and disrupted with an ultrasonicator (Vibronics, Mumbai, India) for 10 min at 300 mA. The extract was then separated from cell debris by centrifugation at 5,000 rpm for 20 min. Free proline in the supernatant was treated with acid-ninhydrin at 80C for 1 h. The reaction was terminated in an ice bath and absorbance of the coloured complex was recorded at 520 nm. The standard curve for proline was prepared by dissolving proline in 3% (w/v) sulfosalicylic acid and covering the concentration range 0.1–5.0 lg ml 1.

Relationship between intracellular proline and metal-induced oxidative damage Experiments were conducted to explore the relationship between proline accumulation and metal-induced oxidative stress. For this purpose, the test alga was pretreated with different concentrations of proline for 30 min under the conditions described earlier. Thereafter, cells were harvested and then exposed to different concentrations of Cu and Zn. The role of proline in mitigating metal-induced oxidative stress was evaluated by using prolinepretreated cells. These cells were subjected to metal stress for 4 h, and subsequently lipid peroxidation, membrane integrity, and SH content were measured. Lipid peroxidation was estimated by measuring the formation of malondialdehyde (MDA) with TBA (2-thiobarbituric acid) according to De Vos et al. (1989). Algal cells were harvested and the pellet was transferred to 4 ml of 0.25% (w/v) TBA in 10% (w/v) trichloroacetic acid (TCA) and heated at 95C for 30 min. After cooling in ice, the mixture was centrifuged at 5,000 rpm for 10 min. MDA was determined by subtracting absorbance of the supernatant at 600 nm from that at 532 nm and using an absorbance coefficient of 155 mM cm 1 (Kwon et al. 1965). The integrity of the plasma membrane, in terms of K+ efflux, in metal-treated as well as the control cells was determined by the method used by Mehta and Gaur (1999). After 4 h exposure to different concentrations of the test metals, the cells were harvested, and CsCl2 (0.1%, w/v) was added to the filtrate before measuring its K+ content by atomic absorption spectrophotometry. Total SH groups of the metal-treated and the control cells were assayed as per Sedlak and Lindsay (1968). For the assay, cells were harvested from a 3-ml cell suspension (1.5·106 cells ml 1) and resuspended in 2 ml of 1.0 mM Tris–HCl at pH 8.2. Thereafter, a 0.5 ml of 5% SDS and 0.1 ml of 10 mM 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB) in absolute methanol were added. The mixture was vortexed for 5 min and the homogenate was incubated at room temperature for 20 min with intermittent stirring, and nucleic acids and proteins were precipitated by adding 3 ml cold methanol. The mixture was centrifuged at 8,000 rpm for 10 min and total SH concentration was determined in the supernatant by measuring absorbance at 412 nm, using glutathione as the standard. The effect of proline pretreatment on the uptake of Cu and Zn was also studied to test whether the ameliorative effect of proline on metal-induced oxidative stress was direct or due to inhibition of metal uptake.

399 Relationship between intracellular proline and activities of antioxidant enzymes in Scenedesmus sp. exposed to test metals Experiments were carried out to understand whether proline acts directly on ROS for alleviating metal-induced oxidative stress or enhances the activities of antioxidant enzymes of the test alga. Hence, the effect of proline pretreatment on Cu- and Zn-induced changes in the activities of the antioxidant enzymes SOD, APOX, CAT, and GR was studied. For the determination of enzyme activity, 50 ml exponentially growing dense algal suspension (cell density, 109 cells ml 1) from metal-treated and control cultures was centrifuged. The cell pellet was mixed with an extraction mixture comprising 50 mM phosphate buffer (pH 7.0), 1 mM EDTA, 0.05% (v/v) Triton X-100, 2% (w/v) polyvinylpyrrolidone and 1 mM ascorbic acid, and sonicated. The homogenate was centrifuged at 16,000 rpm for 25 min and the resulting supernatant, hereafter referred to as the enzyme extract, was kept ice cold and used for the measurement of SOD, APOX, CAT and GR activities. The SOD assay was based on that of Beauchamp and Fridovich (1971) as suggested by Schickler and Capsi (1999); it measures inhibition of photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm. The reaction mixture contained 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 75 mM NBT, 16.7 mM riboflavin and 50 ll of the enzyme extract in a final volume of 3 ml. Tubes containing the mixture were incubated under a fluorescent lamp for 10 min. An illuminated blank without enzyme gave the maximum reduction of NBT, and therefore the maximum absorbance at 560 nm. SOD activity is presented as absorbance of the blank minus absorbance of the sample. The APOX activity of the test alga was determined according to the method given by Asada (1984). 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid and the enzyme extract (total volume, 1 ml) were added to a cuvette. The peroxidase reaction was started by the addition of 20 ll of 5 mM H2O2 and the decrease in the absorbance was recorded at 290 nm after 10– 20 s. Correction was made for the non-enzymatic oxidation of ascorbate by H2O2 (about 1 nmol min 1), and for ascorbate oxidase activity, if any. Under these conditions, a decrease of 0.01 absorbance units corresponds to 3.6 mM ascorbate oxidized. The method given by Clairbone (1985) was followed for the determination of CAT activity of the test alga. The assay mixture contained 3.125 mM H2O2 in 50 mM phosphate buffer (pH 7.0) and 200 ll of enzyme extract in a total volume of 3 ml. CAT activity was estimated by recording decrease in absorbance of H2O2 at 240 nm. The extinction coefficient for CAT was 0.039 mmol cm 1 (Aebi 1984). The GR activity of the test alga was determined as per Schaedle and Bassham (1977). A 1-ml reaction mixture containing 0.05 M Tris–HCl (pH 7.5), 0.15 mM NADPH, 0.5 mM oxidised glutathione (GSSG) and 3 mM MgCl2 was mixed with the enzyme extract (50–200 ll) to achieve a rate of NADPH oxidation between 0.05 and 0.08 absorbance units/min at 340 nm.

The role of proline as a free-radical scavenger or metal-ion chelator An effort was made to ascertain whether proline acts as a freeradical scavenger or as a metal chelator. In this context, the protective role of proline against Cu- and Zn-induced oxidative stress was compared with that against two different elicitors of oxidative stress, ultraviolet-B radiation (UV-B) and methyl viologen (MV). UV-B and MV cannot chelate with proline, and therefore protection offered against oxidative stress by UV-B and MV was dependent on the free-radical detoxification by proline rather than on chelation. To verify this, proline-mediated protection from oxidative stress was compared considering levels of Cu, Zn, MV and UV-B that produced almost similar extents of lipid peroxidation. An exponentially growing culture of Scenedesmus sp. was treated with the selected concentrations (0.1, 0.5, 1.0, 2.5, 5.0, and 10 lM) of MV in the culture medium. MDA content and proline accumulation were measured after exposure of the test alga to MV for 4 h.

UV-B radiation was provided by a UV-B lamp (No. 3-4408; Fotodyne Inc.) at a maximum output of 310 nm. The UV-B dose (12.9 MW m 2 nm 1) was selected on the basis of the latitude of the authors’ place of work and the mean percentage depletion of the ozone layer calculated by Smith et al. (1980). The above dose was obtained by adjusting the distance between the UV-B source and the algal suspension (10 ml), kept in an open glass petri dish. The algal suspension was stirred continuously with the help of a magnetic stirrer to allow uniform exposure. Proline accumulation and lipid peroxidation were studied in the test alga exposed to the selected dose of UV-B for 5, 10, 20, 30, 45, and 60 min.

Effect of ascorbate and sodium benzoate on metal-induced proline accumulation After collecting substantial evidence on the relationship between intracellular proline and metal-induced oxidative stress, one more experiment was conducted to strengthen the above observations. The test alga was pretreated with ascorbate, a well-known antioxidant (Noctor and Foyer 1998), and sodium benzoate, a scavenger of hydroxyl radicals (Luna et al. 1994), and thereafter exposed to test metals. It was speculated that if algal cells already had sufficient antioxidant or free-radical scavenger, there would be no need to accumulate some other antioxidant or free-radical scavenger, like proline. The algal suspension was pretreated with different concentrations of ascorbate (5, 10, 25, 50, and 100 lM) or sodium benzoate (5, 10, 25, 50, and 100 lM) for 30 min following the method already described for proline pretreatment. After pretreatment, the algal cells were exposed to different concentrations of Cu and Zn for 4 h and lipid peroxidation and proline accumulation were measured. All the data are presented on a per cell basis, and hence the cell number was counted with a hemocytometer during each experiment.

Statistical analyses The effect of proline pretreatment on metal-induced changes under different parameters was statistically analyzed using two-way ANOVA. All other data were analyzed by one-way ANOVA.

Results The intracellular levels of Cu and Zn in Scenedesmus sp. in relation to their concentration in the culture suspension are shown in Fig. 1. The intracellular levels of both the metals increased following increase in their concentration in the medium. The intracellular level of Cu remained higher than that of Zn. The intracellular level of proline significantly increased with a rise in concentration of Cu (F5,12=640.4, P