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Catalase- and Superoxide Dismutase-induced Morphological Changes and Growth. Inhibition in the Red Tide Phytoplankton Chattonella marina. Tatsuya ODA ...

Biosci. Biotech. Biochem., 59 (11), 2044-2048, 1995

Catalase- and Superoxide Dismutase-induced Morphological Changes and Growth Inhibition in the Red Tide Phytoplankton Chattonella marina Tatsuya ODA, Junko MORITOMI, Ienobu KAWANO, Shiho HAMAGUCHI, Atsushi ISHIMATSU,* and Tsuyoshi MURAMATSU

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Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan * Nomo Fisheries Station, Nagasaki University, Nomozaki, Nagasaki 851-05, Japan Received April 14, 1995 Chattonella is one of the most toxic red tide phytoplankton and causes severe damage to fish farming. Recent studies demonstrated that Chattonella sp. generates superoxide and hydroxyl radicals, which may be responsible for the toxicity of this plankton. However, little is known about the mechanism of the production of oxygen radicals by Chattonella, and the role of oxygen radicals in Chattonella themselves is also unclear. In this study, we found that superoxide dismutase (SOD) and catalase inhibited the growth of Chattonella marina concomitant with their morphological changes. In the presence of these enzymes, the shape of vegetative C. marina cells changed from spindle to round. Furthermore, the generation of oxygen radicals by C. marina depended on the growth phase; the rate of superoxide and hydrogen peroxide generation was the highest during exponentially growing phase and subsequently decreased to one-fifth of the maximal level in the stationary growth phase. These results suggest that oxygen radicals generated by C. marina play an essential role in their own survival, especially in cell division.

There has recently been a global increase in the fre- little is known about the mechanisms of oxygen radicalquency, magnitude, and geographical extent of red tides. generation by Chattonella, and the significant role or conThis increase seems to be closely correlated with the degree tribution of oxygen radicals to Chattonella themselves is of coastal pollution or the use of coastal waters for aqua- still unclear. culture. 1 ,Z) In Japan, red tides often appear in the Seto Thus, this study was undertaken to gain insight into the Inland Sea, and in the coastal areas of Kyushu during the mechanism of oxygen-radical generation by C. marina and summer. Several species of plankton have been identified as to ascertain possible roles of the oxygen radicals for the the caustative organisms of red tide. 3 •4 ) Chattonella marina Chattonella themselves. (Raphidophyceae) is one of the most frequently appearing noxious red tide phytoplankton and is highly toxic to fish, Materials and Methods especially to the yellowtail, Seriola quinqueradiata. Materials. Cu, Zn-superoxide dismutase (SOD) (3800 units/mg of Although the precise toxic mechanism responsible for protein, from bovine erythrocyte), catalase (5900 units/mg of protein), fish death caused by Chattonella remains unclear, recent cytochrome c (from horse heart), and horseradish peroxidase (100 units/mg of protein) were purchased from Wako Pure Chemical Industry, Co., Ltd., studies 5 -7) demonstrated that a decrease in oxygen partial Osaka, Japan. The inactive form of SOD was prepared according to the pressure of arterial blood is the earliest physiological dis- method described previously. 24 - 26) Briefly, 10,000 units/ml of SOD in turbance observed in fish after exposure to Chattonella. 25mM sodium phosphate buffer (pH 7.5) containing 20mM H 2 0 2 was In addition, physiological and histological studies of fish incubated for 1h at 37°C followed by dialysis against Erd-Schreiber (ESM) medium. Activity of SOD was assayed by the cytochrome exposed to Chattonella suggested that the blockade of respi- cmodified reduction assay using hypoxanthine and xanthine oxidase as an ratory water flow through the gill lamellae caused by exces- O 2-generating system as described by McCord and Fridovich. 27 ) Catalase sive mucus interferes with oz transfer, resulting in asphyx- activity was assayed by the method described by Beers and Sizer 28 ) in ia. 8 11) Furthermore, recent studies found that Chattonella which the disappearance of H 2 0 2 is followed spectrophotometrically at sp. produces oxygen radicals such as 2 , HzO z , and, OH 240 nm. Inactive catalase was obtained by·heat treatment (121 °C, 15 min) of catalase solution (50,000 units/ml in ESM medium). Other chemicals radicals as secondary products, using several methods in- were of the highest grade commercially available. cluding electron spin resonance (ESR) spectroscopy with the spin traps 5,5-dimethyl-l-pyrroline-N-oxide and N-tPlankton culture. C. marina was generously provided by Kagoshima Prefectural Fisheries Experimental Station, Japan, and was cultured at butyl-~-phenylnitrone, and luminol-enhanced chemilumin8.2) under 3000 Ix illumination with a cycle of escence response. 1Z 17) Since harmful effects of oxygen 26°e in ESM medium (pH 12 h light and 12 h dark. 29 ) ESM medium was prepared according to the radicals generated in various biological systems have been composition reported originally without soil extract, i.e., 120mg NaN0 , 3 well documented, 18 - 22) these results suggest that oxygen 5mg K 2 HP0 4 , 0.1 mg vitamin B1 , 0.01 mg vitamin B12 , 0.001 mg biotin, radicals generated by Chattonella may responsible for gill 0.26 mg EDTA-Fe 3 +,0.33 mg EDTA-Mn l +, and 1g tris(hydroxymethyl)tissue injury which eventually causes fish death. It has been aminomethane were dissolved in 1 liter of seawater and the pH was adshown that higher plant cells also produce superoxide anion justed to 8.2, followed by autoclaving (121 °e, 15 min).4 Under these conditions, a maximum plankton concentration of 5 x 10 cells/mt was rouand H zO/ 3 ) in addition to animal cells such as phagocytic tinely attained. Unless otherwise noted, exponentially growing plankton cells in which oxygen radicals produced by these cells are was used throughout the experiments. All cultivation was done using involved in the killing of invading microbes. 19l However, sterilized instruments. Cells were counted with a hemocytometer.

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Abbreviations: ESM medium, Erd-Schreiber modified medium; SOD, superoxide dismutase; ESR, electron spin resonance.

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Growth Inhibition of Chattonella by Radical Scavengers

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Measurement of superoxide anion. Generation of superoxide anion (0;) from C. marina was measured spectroscopically on the basis of SOD-inhibitable reduction of cytochrome c. After the addition of cytochrome c (final concentration, 50 flM) to the plankton suspension in ESM medium, reduction of cytochrome c during the first I min of incubation was measured as an increase in the difference absorbance between 550 and 540 nm with a spectrophotometer (Beckman DU-40, Beckman Instruments, Inc., Fullerton, CA) in the presence or absence of 100 units/ml of SOD at 26°C. The reading was converted to nmol of cytochrome c reduced using a molar absorbance coefficient of 19.1 mM - ! em - ! 30) after subtracting the 0 time value in the presence of 100 units/ml of SOD as a background value. Measurement of H 2 0 2 • Detection of H 2 0 2 in the C. marina suspension was done by the scopoletin method 3 !) at 26°C. After the addition of scopoletin (final concentration, 1 flM) and horseradish peroxidase (final concentration, 20 units/ml) to the plankton suspension in ESM medium, a decrease in fluorescence intensity during the first I min of incubation was measured with a fluorescence spectrophotometer (Hitachi Model 650-60) at an excitation wavelength of 350 and an emission wavelength of 460 nm in the presence or absence of 500 units/ml catalase. The catalase-inhibitable decrease of fluorescence intensity was considered to reflect actual H 2 0 Z ' The concentration of HzO z was estimated by using a standard curve of HzO z in cell-free ESM medium. Under the assay conditions, the decrease of fluorescence intensity was proportional to the concentration of HzO z'

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Effects of superoxide dismutase and catalase on the growth of C. marina To ascertain whether or not the oxygen radicals produced by C. marina are essential for the plankton themselves, we examined the effects of superoxide dismutase (SOD) and catalase on the growth of C. marina. After the addition lil o.15-,---------------------, -

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Relationship between the generation of oxygen radicals by C. marina and plankton growth We examined the relationship between the production of oxygen radicals and the growth phase of C. marina. After the inoculation of C. marina cells into ESM medium at the initial cell-concentration of 800-1000 cells/ml, the growth was measured by counting of living cells at 3-day intervals until the growth reached the stationary phase. Motile cells were considered to be viable cells. Generation of superoxide anion (0 2) and hydrogen peroxide (HzO z) by C. marina was measured at 3-day intervals at the same time. As shown in Fig. 1, the highest rates of generation of O 2 and HzO z were observed during the exponential growth phase with a cell density of about 10,000cells/ml. After the cell population reached stationary phase on the 6th day with a cell density of about 30,000 cells/ml, the production of both O 2 and HzO z gradually decreased and remained at a low level during the stationary phase. Thus, the generation of oxygen radicals by C. marina appears to be related to the metabolic potential of the organisms. C. marina may have specific metabolic or enzymatic systems that are responsible for the production of oxygen radicals. Although the details of the mechanism of the production of oxygen radicals by C. marina is still unclear, our previous observations have demonstrated that the amount of HzO z increased 10- to 15-fold after the disruption of plankton cells by sonication as compared to the HzO z level detected in intact plankton cell suspensions. 15) These results suggest that C. marina has a compartment in which HzO z accumulated at high concentrations and from which a small amount of HzO z is gradually released from intact cells during normal growth. At exponential growth phase, the highest level of H 2 0 Z was also observed in disrupted plankton cell suspensions, suggesting that the intracellular level of HzO z may be correlated to the growth phase of plankton as well as the release of HzO z (Fig. 2). However, we cannot exclude the possibility that sonication treatment acts on C. marina as a physical stimulation and induces the

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Fig. 1. Production of Superoxide (A) and Hydrogen Peroxide (B) by Chattonella marina during the Growth (C). A sample of exponentially growing plankton cells was inoculated into 50 ml of ESM medium with a final cell density of 1000 1500 cells/ml and cultured under the conditions described in Materials and Methods. Viable cell count and measurement of superoxide and hydrogen peroxide produced by Chaltonclla marina were done at 3-day intervals until the growth reached the stationary phases. The generation of 0; and H 2 0 2 from C. marina during the first I min of incubation was measured as described in Materials and Methods.

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