Journal of Applied Phycology 15: 1319, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Cadmium-induced changes in antioxidant enzymes from the marine alga Nannochloropsis oculata
Mi Young Lee 1,* and Hyun Woung Shin 2
Department of Life Science, Soonchunhyang University, Asan PO Box 97, Chungnam, 336-600, Korea; Marine Research & Development Institute, Soonchunhyang University, Asan PO Box 97, Chungnam, 336-600, Korea; *Author for correspondence (e-mail: miyoung@sch.ac.kr; phone: +82-041-530-1355; fax: +82-041-530-1355)
2 1

Received 21 May 2002; accepted in revised form 11 November 2002

Key words: Antioxidant enzymes, Cadmium, Nannochloropsis oculata, Oxidative stress Abstract Cadmium-induced oxidative stress symptoms such as lipid peroxidation and H 2O 2 production were examined in the marine alga Nannochloropsis oculata. Changes in antioxidant enzyme levels and isozyme patterns were also examined. Increasing concentrations of Cd produced growth inhibition. Among the responses to added Cd, the H 2O 2 content and malonyldialdehyde accumulation increased signicantly, indicating a state of oxidative stress. In the case of ascorbate peroxidase activity the increase was about 2.5 times and a marked induction of the isozyme APX2 contributed to this increase. Guaiacol peroxidase activity increased about 4-fold, this being due mainly to the isozyme GPX3. Catalase activity increased slightly, whereas superoxide dismutase and glutathione reductase activity decreased markedly. Alterations of antioxidant enzyme levels and isozyme pattern changes in Cd-treated alga suggest that they might be involved in the heavy metal tolerance in this alga. Abbreviations: APX ascorbate peroxidase, CAT catalase, GPX guaiacol peroxidase, GR glutathione reductase, ROS reactive oxygen species, SOD superoxide dismutase Introduction Heavy metals constitute an environmental pollutant with high toxicity to most animal and plants. If a particular metal concentration is not tolerated, growth is inhibited and the organism eventually dies (Payne and Price 1999). Among the features inuencing the survival capacity of cells are the exclusion of the toxic metal outside the plasma membrane and biosynthesis of metal-binding ligands in phytochelatin (Ahner and Morel 1995, 1999; Pistocchi et al. 2000). Heavy metals may produce oxidative stress possibly by generating free radicals and reactive oxygen species (ROS). ROS includes superoxide radical (O 2 ), hydrogen peroxide (H 2O 2) and the hydroxyl radical (OH). The importance of antioxidant enzymes is generally emphasized in preventing oxidative stresses by scavenging ROS (Dawes 2000). The antioxidant system comprises several enzymes such as ascorbate peroxidase(APX), guaiacol peroxidase(GPX), catalase(CAT), superoxide dismutase (SOD) and glutathione reductase (GR). Superoxide radicals generated are converted to H 2O 2 by the action of SOD. The accumulation of H 2O 2 is prevented in the cell by CAT, GPX, or by the ascorbate-glutathione cycle where APX reduces it to H 2O (Donahue et al. 1997). Among these, the GPX level was changed in response to various physical, chemical and biological stresses as previously reported (Lee 1997, 2002). Changes in GPX activity and isozyme proles have been suggested as indicators for environmental stresses (Lee et al. 2000; Mullet 2000). The involvement of antioxidants in the response to Cd is unclear, because Cd is not a transition metal like copper and iron, which may induce oxidative stress via a Fenton-type reaction (Robinson 1989). The

XPS 0110534TX (JAPH) product element JAPH871 Grafikon

14 question as to how Cd acts at the cellular level and how plants and marine organisms defend themselves against this pollutant is receiving increasing attention. Increased productions of extra- and intracellular metal-ligands in phytoplankton exposed to cadmium has been reported (Pistocchi et al. 2000). Moreover, cadmium inhibited epoxidation of diatoxanthin to diadinoxanthin in the xanthophyll cycle of the marine diatom (Bertrand et al. 2001). However, there is in general less information on Cd-induced oxidative stress and antioxidant enzyme systems in marine algae. Nannochloropsis oculata was chosen as a model system, because marine ecosystems are particularly vulnerable to pollution by heavy metals such as cadmium, and algae are often recommended as biological monitoring organisms in heavy metal-polluted areas (Whitton 1984; Whitton et al. 1989). The aim of this study was to quantify the effects of Cd-induced enhancements on lipid peroxidation, H 2O 2 content, antioxidant enzyme levels and isozyme patterns. Determination of H 2O 2 and malonyldialdehyde content The H 2O 2 and malonyldialdehyde content were measured under various illumination conditions. The light intensity conditions included 12:12 light-dark cycle, 16:8 light-dark cycle and continuous illuminations of 50 mol photon m 2 s 1. Algal material (1 g), treated with 10 M Cd for 4 days under various illumination conditions, was homogenized in an ice bath with 0.1% (w/v) trichloroacetic acid (TCA). The supernatant after centrifugation was mixed with 50 mM phosphate buffer (pH 7.0) and 1 M KI. The absorbance of the supernatant was read at 390 nm, and H 2O 2 content was obtained from a standard curve for H 2O 2. Lipid peroxidation level was determined in terms of 2-thiobarbituric acid (TBA) reactive metabolite, chiey malonyldialdehyde (MDA). Alga was extracted with 0.25% TBA in 10% TCA, and the extract was quickly cooled after heating. After centrifugation at 10,000 g for 15 min, the absorbance of the supernatant was measured at 532 nm. The level of lipid peroxidation is expressed as mol of MDA formed using an extinction coefficient of 155 mM cm 1. Preparation of enzyme extracts A unialgal strain of Nannochloropsis oculata was obtained from the Korea Marine Microalgae Culture Collection (KMC 064) and cultured in the f/2 medium of Guillard and Ryther (1962). Erlenmeyer asks containing 100 mL medium were inoculated with 25 mL of a mother culture. The alga was grown at room temperature with shaking at 100 rpm under a 12:12 light-dark cycle of 50 mol photon m 2 s 1 illumination. Effect of Cd on growth yield The effects of cadmium on growth yield were investigated in f/2 medium under a 12:12 light-dark cycle of 50 mol photon m 2 s 1 illumination. For this, a 10-day old culture was further cultured for 4 days in similar medium, but with various concentrations of CdCl 2. Growth yield was measured daily as cell density (Leupold 1988). Algal material treated with 10 M Cd for 4 days was powdered in liquid nitrogen. This powdered was further homogenized in 50 mM sodium phosphate buffer (pH 6.0) containing sea sand. The extract was centrifuged at 10,000 g for 30 min and the supernatant was used for antioxidant enzyme analysis. Antioxidant enzyme assays Catalase (CAT) activity was determined spectrophotometrically by measuring the decrease of absorbance at 240 nm due to H 2O 2 decomposition (Rao et al. 1996). Guaiacol peroxidase (GPX) activity was determined with guaiacol as a substrate. The reaction was initiated by the addition of H 2O 2 and the increase in absorbance at 470 nm was measured (Lee 2002). Ascorbate peroxidase (APX) activity was measured with ascorbate as a substrate, and the decrease in absorbance at 290 nm was measured (Mittler and Zilinskas 1993). Superoxide dismutase (SOD) activity was determined using the xanthine/xanthine oxidase/nitroblue tetrazolium system. Inhibition of cytochrome c reduction by SOD was measured by the reduction

Materials and methods Algal culture

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Table 1. Effect of Cd concentration on growth yield of N. oculata. Mean S.E. of three separate experiments. (Ten-day old culture in f/2 medium and 12:12 light-dark cycle with 50 mol photon m 2 s 1 transferred to text medium for 4 days.) CdCl 2 (M) 0 5 10 15 20 Relative Growth (%) 100 90 85 70 50

5.3 3.1 4.5 6.4

of nitroblue tetrazolium (Vitoria et al. 2001). Glutathione reductase (GR) activity was determined by measuring the reduction of oxidized glutathione at 340 nm. The reduction of oxidized glutathione was measured by NADPH oxidation (Vitoria et al. 2001). All determinations are expressed as the mean S.E. of three separate experiments. Native- polyacrylamide gel electrophoresis and activity staining of antioxidant enzymes

containing 10 mg methylthiazolyl tetrazolium, 6 mg phenazine methosulfate and 15 mg MgCl 2. The enzyme bands were seen as pale zones on a dark blue background (Asada et al. 1974). For staining of APX isozymes (Mittler and Zilinskas 1993), the gel was incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 4 mM ascorbate and 2 mM H 2O 2 for 20 min. The gel was washed with 50 mM potassium phosphate buffer (pH 7.0) and soaked in a solution of 50 mM potassium phosphate buffer (pH 7.8) containing 28 mM N,N,N ,N -tetramethylene-ethylenediamine and 2.45 mM nitroblue tetrazolium. The APX activity was observed as an achromatic band on a purple-blue background. GR activity was detected by incubating the gel in a solution of 0.25 M Tris-HCl buffer (pH 7.8) containing 0.24 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0.4 mM NADPH, 0.34 mM 2,6-dichlorophenolindophenol and 3.6 mM oxidized glutathione in darkness for 1 h (Kang et al. 1999)

Results Equal amounts of protein extracts from alga were subjected to native PAGE using discontinuous buffer system of Laemmli (1970). Antioxidant enzymes were separated on the 10% separating polyacrylamide gel with a 5% stacking gel at 100 V for 5 h at 4 C. After electrophoretic separation of antioxidant enzymes, the gels were stained for individual enzyme activity. Staining of CAT activity was performed as described by Woodbury et al. (1971). The gel was incubated in 3.27 mM H 2O 2 for 25 min, rinsed in water, and soaked in a solution of 1% ferric chloridepotassium ferricyanide (III). Staining of GPX isozyme was performed by incubating the gel in 50 mM sodium acetate buffer (pH 5.0) containing 2.4 mM 3-amino-9-ethyl carbazole and 5% N,N-dimethylformamide for 4 h. Thereafter, the reaction was stopped by a brief washing in deionized water (Lee 2002). Staining of SOD isozymes was performed by incubating the gel in 50 mM Tris-HCl buffer (pH 8.5) Effects of Cd on growth yield, H 2O 2 content and lipid peroxidation There was a signicant inhibition of growth yield with increasing Cd concentration (Table 1). The reduction was about 15% with 10 M Cd, and 50% growth with 20 M Cd. The H 2O 2 content and lipid peroxidation level in terms of malonyldialdehyde (MDA) accumulation in the alga were measured under various illumination conditions. The illumination conditions included 12:12 light-dark cycle, 16:8 lightdark cycle and continuous illumination of 50 mol photon m 2 s 1. As shown in Table 2, about 30% increase in H 2O 2 content occurred after Cd application under 12:12 light-dark cycle. The level of MDA, one of the major TBA reactive metabolites, also increased in Cd-treated alga about 33%. Increasing the light length in the light-dark cycle to 18 h also

Table 2. Effects of Cd on the H 2O 2 content and malonyldialdehyde accumulation in N. oculata under various illumination conditions. Mean S.E. of three separate experiments. (Ten-day old culture in f/2 medium and 12:12 light-dark cycle with 50 mol photon m 2 s 1 was grown for 4 days under similar conditions, but with 10 M Cd) Control Cd-treated alga 12 h:12 h 4.25 0.17 0.12 0.003

16 h:8 h 4.32 0.11 0.13 0.004

24 h:0 5.2 0.21 0.24 0.002

H 2O 2 (mol g 1 d. wt) malonyldialdehyde (mol g 1 d. wt)

3.19 0.15 0.09 0.006

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Table 3. Comparison of activities of various antioxidant enzymes in N. oculata exposed to Cd. Relative activity refers to comparison with the control. Mean S.E. of three separate experiments. (Tenday old culture in f/2 medium and 12:12 light-dark cycle with 50 mol photon m 2 s 1 was grown for 4 days under similar conditions, but with 10 M Cd) Antioxidant enzyme guaiacol peroxidase ascorbate peroxidase catalase superoxide dismutase glutathione reductase Relative activity (%) 400 5.3 250 6.1 115 10.5 85 5.2 75 4.7

* Ten day old N. oculata cultured in f/2 medium under a 12:12 light-dark cycle of 50 mol photon m 2 s 1 illumination was further cultured in the same medium, but containing 10 M Cd for 4 days. All determinations are expressed as the mean S. E. of three separate experiments. The values presented here have been shown relative to the enzyme activity without treatment of Cd, which was given a value of 100.

Figure 1. Changes in the isozyme patterns of ascorbate peroxidase (APX) from N. oculata grown for 4 days in medium containing various concentrations of CdCl 2. Lane 1: control. Lane 2: CdCl 2treated alga.

resulted in the notable enhancement of H 2O 2 and MDA content following Cd treatment. Especially, about 63% and 66% increase of H 2O 2 and MDA content, respectively, occurred by Cd treatment under continuous illumination. Effects of Cd on activities and isozyme patterns of APX and GPX Table 3 showed the activities of several antioxidant enzymes such as GPX, APX, CAT, SOD and GR following Cd application. Upon exposure to 10 M Cd, about 2.5 fold increase of total APX activity occurred. The zymogram of native-PAGE revealed multiple forms of APX, named APX1, APX2 and APX3 (Figure 1); APX3 was prominent and APX1 was very faint in the control. Interestingly, APX2 isozyme was signicantly induced after Cd treatment possibly by biosynthesis of APX2 isozyme. These results suggest that APX2 isozyme might have played an important role in Cd tolerance. There was about a 4-fold enhancement of total GPX activity compared to the control (Table 3). When Cd was tested on in vitro total GPX activity in the enzyme extracts, no signicant alteration of en-

Figure 2. Changes in the isozyme patterns of guaiacol peroxidase (GPX) from N. oculata grown for 4 days in medium containing various concentrations of CdCl 2. Lane 1: control. Lane 2: CdCl 2treated alga.

zyme activity was observed (data not shown), suggesting that Cd had no effect on GPX activity itself in vitro. The changes of GPX isozyme patterns in response to Cd were also analyzed by means of activity staining on the native-PAGE gel (Figure 2). The levels of GPX3 increased notably as revealed by en-

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Figure 3. Changes in patterns of catalase from N. oculata grown for 4 days in medium containing various concentrations of CdCl 2. Lane 1: control. Lane 2: CdCl 2-treated alga.

hanced band intensity. The result indicated that farmigrating GPX3 isozyme contributed prominently to the total GPX activity enhancement according to the GPX isozyme proles. Effects of Cd on the activities of CAT, SOD and GR and their isozyme patterns CAT activity slightly increased after Cd treatment (Table 3). (CAT dismutates H 2O 2 into H 2O and O 2, whereas GPX decomposes H 2O 2 by peroxidation of co-substrates such as phenolic compounds.) In order to examine whether CAT isozyme proles might be affected by Cd, algal extract was subjected to nativePAGE and stained for CAT activity (Figure 3). There was only a single enzyme band for CAT activity in the control and Cd-treated alga. There was about a 15% decrease in SOD activity in response to Cd (Table 3). As shown in Figure 4, SOD in N. oculata existed as three isozymes, and SOD isozyme proles did not change after Cd treatment. GR, which catalyses the NADPH-dependent reduction of oxidized glutathione, showed about 25% decrease in its activity (Table 3). The alga had ve isozymes of GR in the control (Figure 5). The intensities of four pre-existing isozymes (GR1, GR2,

Figure 4. Changes in isozyme patterns of superoxide dismutase from N. oculata grown for 4 days in medium containing various concentrations of CdCl 2. Lane 1: control. Lane 2: CdCl 2-treated alga.

Figure 5. Changes in isozyme patterns of glutathione reductase from N. oculata grown for 4 days in medium containing various concentrations of CdCl 2. Lane 1: control. Lane 2: CdCl 2-treated alga.

18 GR3,GR4) remained unaffected by Cd, whereas isozyme GR5 disappeared. levels of APX, GPX and CAT might be concomitantly involved in the removal of H 2O 2 formed by Cd-induced oxidative stress. SOD activity decreased after Cd application, in contrast to APX, GPX and CAT. Cd-induced reduction of SOD has been attributed to an inactivation of the enzyme by H 2O 2 produced in different compartments, where SOD catalyses the disproportionation of superoxide radicals (Vitoria et al. 2001). GR activity, another enzyme of the ascorbateglutathione cycle, remained 25% lower than the control. It is likely that Cd also inhibits the total GR activities through inactivation of GR5 isozyme. Taken together, these results suggest that enhanced APX2, GPX3 and CAT activities may functioning concurrently to remove H 2O 2 when GR and SOD become inactivated in the Cd-treated alga.

Discussion Many studies have been performed on the enhancement of plant tolerance to oxidative stress by modifying the plant antioxidant defense system (Anderson et al. 1995; Vitoria et al. 2001). In addition, it is believed that the alterations in antioxidant enzymes may be due to the synthesis of new isozymes or enhancement of pre-existing enzyme levels for the metabolism of ROS (Kang et al. 1999). However, detailed information concerning activation of the antioxidant system for the detoxication of metal ions in marine algae is scarce, although algae are recommended as biological monitors for heavy metal pollution (Whitton et al. 1989). In the present study enhancements of lipid peroxidation and H 2O 2 content after Cd treatment (Table 2) suggest that cadmium caused oxidative damage, possibly by generating reactive oxygen species (ROS). ROS are generated by cellular exposure to heavy metals, and all of these ROS are extremely reactive and rapidly disrupt normal cell metabolism (Dawes 2000). Cd-induced oxidative stress is believed to increase in the light, so Cd toxicity in terms of H 2O 2 and MDA content was also compared in the light versus dark. Increasing the light period in the light-dark cycle from 12 to 24 h resulted in a marked enhancement of H 2O 2 and MDA contents following Cd treatment. The enhancement of lipid peroxide and H 2O 2 are indicators of ROS generation, showing a state of oxidative stress. The severe oxidative stress shown under continuous illumination may have been due to the increase of Cd toxicity in the light. The markedly elevated levels of APX and GPX following Cd treatment indicated the protective role of these enzymes against Cd-induced oxidative stress (Table 3). Isozyme APX2 and isozyme GPX3 contributed chiey to the enhancement of total APX and GPX activity, respectively (Figures 1 and 2). APX was reported to be involved in H 2O 2 removal through the ascorbate-glutathione cycle in the chloroplast (Donahue et al. 1997). Changes of GPX activity and GPX isozyme patterns under environmental stresses have been suggested as indicators for biotic or abiotic stresses (Lee 1997). CAT activity slightly increased in response to Cd. CAT and peroxidase are two major systems for the enzymatic removal of H 2O 2. Markedly elevated

Acknowledgements This work was supported by grants for National Hazard Prevention Research (00-J-01-B-26) in Korea. We thank Dr M. Sidharthan for the algal culture, and Hye Lin Park, Byoung Sul Min, Joong Ki Min and Yoon Kyoung Kim for technical assistance.

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