Profiles of antioxidant gene expression and physiological changes by thermal and hypoosmotic stresses in black porgy (Acanthopagrus schlegeli)

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Abstract

We determined oxidative stress by measuring the expression and activity of 3 antioxidant enzymes [Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPX)] in black porgy exposed to thermal (20 °C  30 °C) and hypoosmotic (35 psu→10 psu and 0 psu) stresses. The expression and activity of antioxidant enzymes were significantly higher after exposure to 30 °C, 10 psu, and 0 psu. Furthermore, we measured H2O2 and lipid peroxidation (LPO) levels. As a result, H2O2 and LPO levels were significantly increased after exposure to thermal (20 °C  30 °C) and hypoosmotic stress (35 psu→10 psu and 0 psu) stress. These results indicate that thermal and hypoosmotic stress induces oxidative stress in black porgy. Additionally, we investigated the changes due to thermal and hypoosmotic stress by measuring plasma cortisol and ion (Na+ and Cl) levels. Plasma cortisol levels increased at 30 °C and at 10 psu and then decreased at 0 psu. However, plasma Na+ and Cl levels did not change after exposure to thermal stress (30 °C), and decreased at 10 psu and 0 psu. In conclusion, thermal and hypoosmotic environments increase oxidative stress, thereby these results may be indicators of oxidative stress in black porgy.

Introduction

Stress factors in fish can be divided into 2 groups: physical factors, such as salinity, temperature, culture density, temperature, dissolved oxygen, and chemical factors (Beckmann et al., 1990). Physical factors, such as salinity and temperature change, affect growth, reproduction, metabolism, osmoregulation, and immune function, causing negative effects under physiological conditions, such as a disturbance in growth and reproduction (Ackerman et al., 2000). Furthermore, stress induced by changes in salinity has been associated with enhanced reactive oxygen species (ROS) generation, which may seriously affect immune function and lead to oxidative stress (Paital & Chainy, 2010, Shin et al., in press).

ROS, including superoxide (O2), hydrogen peroxide (H2O2), hydroxyl radicals (OH), and singlet oxygen (1O2), are produced naturally during oxidative metabolism (Roch, 1999). Overproduction of ROS in response to environmental stress can lead to increased lipid peroxidation (LPO) and may affect cell viability by causing membrane damage and enzyme inactivity (Nordberg and Arnér, 2001). Subsequently, cell senescence and apoptosis and oxidation of nucleic acids and proteins may be accelerated. The resultant DNA damage may provoke a variety of physiological disorders such as accelerated aging, reduced disease resistance, and reduced reproductive ability (Kim & Phyllis, 1998, Pandey et al., 2003).

Complex antioxidant defense systems maintain homeostasis and protect aerobic organisms against ROS and the subsequent damage of oxidative stress. Antioxidants may be enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST), as well as compounds such as metallothionein, quercetin, vitamin C, and vitamin E (α-tocopherol) (McFarland et al., 1999, Jayaraj et al., 2007). Antioxidant defense systems are found in the livers and kidneys of marine organisms (Basha Siraj and Rani Usha, 2003). Both SOD and CAT directly scavenge ROS. SOD removes O2 through the process of dismutation to O2 and H2O2 (2O2 + 2H+  H2O2 + O2; Kashiwagi et al., 1997). The H2O2 produced by Cu/Zn-SOD is sequentially reduced to H2O and O2 by CAT (Kashiwagi et al., 1997). CAT is an oxidoreductase that breaks down 2 molecules of H2O2 into 2 molecules of H2O and O2 (2H2O2  2H2O + O2), thereby counteracting the toxicity of H2O2 (Kashiwagi et al., 1997).

Black porgy (Acanthopagrus schlegeli) is an euryhaline teleost that moves from coastal waters to near-shore shallow areas during the transition from larvae to juveniles and lives in coastal waters near land or in estuaries and in 18–21 °C water (Kinoshita and Tanaka, 1990). In summer, water temperature is increased, and then stress and death of the fish occurs than in any other season (Collazos et al., 1995). Therefore, we investigated the expression and activity of antioxidant enzymes and the changes in the H2O2, and LPO levels in black porgy to understand the oxidative stress induced by thermal and hypoosmotic stress. In addition, we analyzed changes in Na+, Cl, and cortisol levels to provide basic data about the physiological responses and the index of stress induced by thermal and hypoosmotic stresses in black porgy.

Section snippets

Experimental fish and conditions

The study was performed with 1-year-old black porgy (Acanthopagrus schlegeli, Sparidae) (n = 60, 14.3 ± 0.4 cm, 51.0 ± 6.0 g) reared in three 220-L circulating filter tanks in the laboratory. Before the experiment, water temperature and photoperiod were 20 ± 1 °C and 12L:12D, respectively.

Temperature changes

Fish were reared in seawater in the two circulating filter tanks (40 L) with automatic temperature regulation systems (JS-WBP-170RP; Johnsam Co., Seoul, Korea) and allowed to acclimatize to the conditions for 24 h. The

Expression of antioxidant enzymes induced by thermal and hypoosmotic stress

Cu/Zn-SOD and CAT mRNA expressions significantly increased by about 5-fold, and GPX mRNA significantly increased by about 3.8-fold in the 30 °C group as compared to those in the control group (20 °C) (Fig. 1). Cu/Zn-SOD and CAT mRNA expressions significantly increased by about 17.5-fold and 13-fold, respectively, and GPX mRNA significantly increased by about 7.3-fold at 10 psu, and then decreased at 0 psu (Fig. 2).

Activity of antioxidant enzymes induced by thermal and hypoosmotic stress

Cu/Zn-SOD and CAT activities significantly increased by about 2-fold, and GPX

Discussion

This study was performed to understand the oxidative stress induced in the body by rapid environmental changes, such as the transfer of black porgy to high water temperatures or hypoosmotic environments. We measured the expression and activity of antioxidant enzymes (Cu/Zn-SOD, CAT and GPX) as well as an index of oxidative stress (H2O2 and LPO) to investigate the oxidative stress induced by high water temperatures and hypoosmotic environments. In addition, we investigated the physiological

Acknowledgements

This research was supported by the MKE (The Ministry of Knowledge Economy), Korea, under the ITRC (Information Technology Research Center) support program supervised by the NIPA (National IT Industry Promotion Agency (NIPA-2009-C1090-0903-0007).

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