Anti-oxidative responses of zebrafish (Danio rerio) gill, liver and brain tissues upon acute cold shock

Abstract

The present study seeks to detect oxidative damage and to compare anti-oxidative responses among liver, gills and brain of adult zebrafish that were cooled from 28 °C (control) to 12 °C (treatment) for 0–24 h. The lipid peroxidation of liver, gill and brain tissues significantly increased at 1 h after transfer, but reactive oxygen species in the treatment group increased significantly after 24 h as compared to the control. The fish were found to develop a cascading anti-oxidative mechanism beginning with an increase in Cu/Zn-SOD levels, followed by increased CAT and GPx mRNA expressions in the three tissue types. Both smtB and mt2 mRNAs increased in the hepatic and brain tissues following 1 h of cold stress, but only smtB exhibited a significant increase in the gills at 1 h and 6 h after transfer to 12 °C. Furthermore, cellular apoptosis in the brain was not evident after cold shock, but liver and gills showed cellular apoptosis at 1–3 h, with another peak in the liver at 6 h after cold shock. The results suggest that the cold shock induced oxidative stress, and the enzymatic (SOD, GPx and CAT) and non-enzymatic (mt-2 and smt-B) mRNA expressions all play a role in the resulting anti-oxidation within 1–6 h of cold shock. A functional comparison showed that the brain had the most powerful antioxidant defense system of the three tissue types since it had the highest smtB mRNA expression and a lower level of cell apoptosis than the liver and gills after exposure to cold stress.

Introduction

Temperature is an important environmental factor for the health of poikilothermic animals, such as fish, influencing physiological functions from the molecular to whole body scale, along with the structures of communities and ecosystems. Cold shock can occur under natural conditions, such as thermocline temperature variation, rapid changes in solar heat, abnormal water movements, rapid precipitation and rapid changes in seasonal temperatures. In recent years, most related studies have been more concerned with the potential impact of global warming and have thus focused on stress responses to sudden water temperature increases. However, cold shock can be characterized as a physiological cascade of primary, secondary and tertiary responses, respectively including neuroendocrine responses, metabolic response and developmental change (Donaldson et al., 2008). A small number of papers have investigated the oxidative stress induced by cold shock. In fact, seasonal variations cause environmental temperature changes which may be reflected in free radical metabolism over the course of the year.

Antioxidant responses can be induced by short-term temperature shocks which result in higher (Lushchak and Bagnyukova, 2006a, Bagnyukova et al., 2007) or lower body temperatures (Malek et al., 2004, Lushchak and Bagnyukova, 2006b) in fish. Acute low temperatures can also induce DNA damage, lipid peroxidation and changes in osmolarity following oxidative stress in white shrimp (Qiu et al., 2011). The present study used zebrafish (Danio rerio) as an animal model since our previous study found that only mt2 (metallothionein 2) mRNA appeared in zebrafish larvae but not smtB (similar as metallothionein B) upon exposure to low temperatures. However, both genes show transcript signals in the brains of adult zebrafish after exposure to low temperatures or acid environments with in situ hybridization assay (manuscript in preparation). We supposed that the mRNA expression of the two genes might differ between larval and adult fish, but the physiological functions of mt2 and smtB were clearer in larvae than in adult fish. Thus, we seek to compare the physiological function of both genes in mature zebrafish.

As in many other vertebrates, environmental stress can significantly increase ROS in fish, causing injury to cells. Organisms use antioxidant defense systems to minimize damage from oxidative stress. Thus, the levels of oxidative stress could be estimated through different ways: direct assessment of ROS overproduction, assessment of LPO products, or determination of changes in antioxidant mechanisms (enzymatic and non-enzymatic) (Cazenave et al., 2006). Such systems include antioxidant molecules, such as glutathione (GSH), vitamins C and E, and carotenoids (Alvarez et al., 2005); antioxidant enzymes such as SOD, CAT, and GPx (Valavanidis et al., 2006); SOD, which catabolizes the dismutation of superoxide radicals to molecular oxygen and hydrogen peroxide; CAT, which degrades hydrogen peroxide into molecular oxygen and water; and GPx, which reduces hydrogen and organic peroxides to water and corresponding alcohols (Castro et al., 2012). In addition, several studies have found that antioxidant non-enzyme genes include metallothionein (Chen and Maret, 2001, Choi et al., 2007). Thus, the present study investigates the expression of antioxidant genes including the Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx), as indicators of anti-oxidants to determine the anti-oxidative functions of mt2 and smtB in zebrafish upon exposure to low temperatures.

Metallothioneins (MTs) are a small family of thiol-rich metals binding protein devoid of enzymatic activities. Previous studies have reported that MTs exhibit antioxidant activity and, together with GSH, are important in the regulation of redox homeostasis and protection against superoxide and hydroxyl radical (HO) (Nzengue et al., 2012). This means that MTs protect organisms from cell injury caused by ROS. In addition, zebrafish (D. rerio) features many types of MT, and their various expressions differ among tissues or in terms of physiological function. For example, D. rerio mt1 is more prevalent than mt2 in the brain, gills and liver upon Cd^2 + exposure (Bourdineaud et al., 2006), and produces mt2 signals in zebrafish, but not smtB signals upon Cd^2 + exposure and cold shock (Wu et al., 2008). Thus, it will compare the smtB and mt2 mRNA expression among liver, gill and brain tissues of zebrafish upon exposure to cold shock.

It is also known that other environmental factors such as pollution and heavy metals cause oxidative stress in aquatic organisms (Migliarini et al., 2005, Barillet et al., 2010). Oxidative stress induced by Cd^2 + can result in DNA damage. The oxidation of DNA bases can be due to HO. radical attacks on the pyrimidines aromatic cycles. Lipid peroxidation (LPO) is also a source of endogenous DNA damage via malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) which can react with DNA bases. Reactive oxygen species (ROS) reaction with proteins can also produce new radicals that can react with DNA. Taken together, these constitute examples of DNA damage induction by lipids and protein oxidation products. When this damage occurs on a great scale and the cell may no longer recover, it induces apoptosis. In addition, oxidative stress produces indirect genotoxic effects such as ROS production-induced oxidative DNA damage and lipid oxidation, thus producing short-term cell apoptosis. However, during short term stress, antioxidant molecules, antioxidant enzymes, and proteins produce an antioxidant defense system to protect the organism. Both ROS and LPO are known to be reduced (scavenged) by the activity of antioxidant enzymes. Otherwise, it will induce cellular apoptosis. The present study addresses the effects of a cold shock (short-term experiment) in three organs (gill, liver and brain) of zebrafish. More specifically, several stress biomarkers were measured including 1) enzymatic anti-oxidants (Cu/Zn-SOD, GPx and CAT), 2) non-enzymatic anti-oxidants (mt2 and smtB) and 3) oxidative damage parameters (ROS and LPO levels). The levels of cell apoptosis were also investigated to confirm the capacity of the anti-oxidative mechanism in zebrafish after acute cold shock.