Bioaccumulation, Oxidative Stress, Immune Responses in Channa Argus Exposure to Waterborne Hexavalent Chromium

Hexavalent chromium (Cr (VI)) is a very common and harmful heavy metal pollutant in the world. However, the responses of Cr (VI) to aquatic environmental toxicants have not been well described. In this study, we evaluated the waterborne Cr (VI) [ 0 mg/L (C1), 0.5 mg/L (C2), 1 mg/L (C3), 2 mg/L (C4)] exposed chronically to Channa argus. After 14 and 28 days, we measured biochemical parameters, Cr (VI) accumulation, antioxidant activity and immune response in the serum, liver and gill, respectively. Our results shown that Cr (VI) accumulation will occur in certain tissues as the time of exposure in water prolongs, and the biochemical parameters in the serum will increase. Antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) gradually decreased with the increasing time of Cr (VI) concentration, while the malondialdehyde concentration increases. In addition, in the immune response, cortisol, aminotransferase (ALT), aspartate aminotransferases (AST), alkaline phosphatase (AKP), immunoglobulin M increase, but lysozyme decrease. These results indicated that exposure to waterborne Cr (VI) can exert adverse effects in C. argus by inducing Cr (VI) accumulation, immune responses and oxidative stress. et al. Astaxanthin enhances hematology, antioxidant and immunological parameters, immune-related gene expression, and disease resistance against in Channa argus.


Introduction
Due to human activities, the content of heavy metals in the environment has increased gradually, and the deterioration of environmental quality has seriously affected human life and health. At present, environmental heavy metals have become a worldwide environmental problem. Heavy metal pollution is mainly manifested in water pollution. Because of the importance of sh in the aquatic ecosystem, sh are typically used for environmental monitoring. Chromium is a key micronutrient for maintaining physiological homeostasis. There are two main forms of Cr in the aquatic environment: trivalent chromium (Cr (III)) and hexavalent chromium (Cr (VI)). Cr (III) is an important trace element. Cr (VI) is an important environmental pollutant released from domestic and industrial wastewater, which harms animal health and causes malformations, allergic reactions, and damages the liver and kidneys as well as animal cancer [1]. Cr (VI) is more active on living cells than Cr (III), and it easily penetrates cell membranes. Therefore Cr (VI) causes greater damage to cells [2]. Exposure to Cr (VI) may lead to metabolic conversion of normal growth and development to detoxi ed energy chromium toxicity and inhibit the growth of sh [3]. It is well known that Cr (VI) and other toxic heavy metals will accumulate in the speci c tissues of a variety of sh through exposure to heavy metals in water and in the diet, and continue to exert toxic effects leading to sub-health and even death of sh [4][5].
Aquatic animal blood factors have been widely used as indicators of physiological and pathological changes in toxicology and environmental research, aiming to assess the impact of toxin exposure [6].
Because sh blood has a close relationship with the external environment through the circulatory system [7]. Therefore, sh blood parameters are a good indicator to re ect water environmental toxicity. Shaheen et al. (2012) also reported that since blood is highly sensitive to environmental changes and therefore can be used as a reliable parameter to measure environmental toxicity, and Cr (VI) has negative effects on aquatic animal blood and biochemical factors [8].
Cr exposure in water can lead to accumulation of speci c tissues in aquatilia, in uence the normal metabolic enzyme activity, cause the reactive oxygen species (ROS) and tissues precipitation, peroxidation of lipid and protein, and later cause oxidant stress. Aquatic lives contain an active antioxidation system with various protective enzymes preventing or limiting tissue damaged by inhibiting the generation of ROS [9]. The anti-oxidation system of sh is an important element in the decomposition of H2O2, including antioxidant enzymes such as catalase (CAT) and glutathione peroxidase (Gsh-Px), which are recognized as antioxidant damage and response. An indicator of irritating response [10]. The production of lipid peroxide and malondialdehyde (MDA) can be also reduced by Gsh-Px [11]. Therefore, the effects of chromium on sh exposure can be studied by the use of antioxidant enzymes. Besides, it has been proposed that selenium exposure can in uence oxidant stress along with immune function [12]. Trace elements in water can affect the immune indices of sh [13]. Lysozyme (LZM) which is important is a natural immune defense molecule. In a non-speci c immune response, not only does LZM dissolve bacteria, but also activates the complement system and is important in preventing microbial invasion [14]. Immunoglobulin M (IgM) is a large molecule involved in a speci c immune response [15]. Researches have revealed that the levels of LZM and IgM are convenient parameters to monitoring the possible effects of environmental impacts on sh immune responses [16][17]. In addition, serum cortisol (COR) is a key parameter to measure the physiological state of exposure to pollutants. Therefore, they can be used as indicators of stress response.
Channa argus is a carnivorous freshwater sh, commonly known as black sh and mullet [13]. Because of its high nutritional value, strong environmental adaptability and fast growth, it is one of the main sh species cultured in freshwater ponds in China, North Korea, Japan, India and Russia widely [16]. It's an expensive edible sh, but few knows about the Cr (VI) effects. Thus, the purpose of this study is to make an investigation of the Cr (VI) accumulation in C. argus tissue and its adverse effects on Cr (VI) antioxidant activity, oxidant stress, serum biochemistry and tissue accumulation.

Experimental Fish and Design
C. argus (13.42 ± 0.35 g) were collected from a commercial sh farm. During the acclimation period, C. argus were cultured in 100 L polyethylene tanks with aerated water and fed commercial carp diet twice daily at 1-2% BW for 14 days.
After acclimation, 240 C. argus were randomly assigned to four groups (5 tanks in each group, 12 sh in each tank). The Cr (VI) concentrations in blue plastic boxes were 0, 0.5, 1.0, and 2.0mg/L, respectively. Cr (VI) solution was prepared from potassium dichromate solution (CAS:7778-50-9, MDL: MFCD00011367, Macklin Biochemical Co., Ltd, Shanghai, China). Commercial feed (Table 1) of C. argus (Dry matter: crude protein 48.1%, crude lipid 11.3%, ash 12.3%, carbohydrate 20.1%, gross energy, 19.3 kJ/g and protein: energy ratio, 26.0 mg/kJ, Alpha Feed Co., Ltd Shenzhen, China) was used as experimental feed. The tanks were placed under experimental conditions (temperature: 22.3 ± 1.2°C; pH: 7.6 ± 0.2; ammonia: less than 0.3 mg/L; nitrites: less than 0.04 mg/L and dissolved oxygen: 5.82 ± 0.22 mg/L) and in photoperiod (14 h light/10 h dark photoperiod). Feed the sh twice a day (08:00 and 16:00) at 3% body weight for 28 days. A siphon was used to remove debris from the bottom of the tank every day and half of the water in each tank was changed every two days. The sh for research were performed according to the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee, Heilongjiang Bayi Agricultural University. The concentration of Cr (VI) were approximately equal to or higher than that present in the environment.

Sample Collection
The sh were put on a 24-hour fast before the samples were collected. On days 14 and 28, sh were anesthetized in buffered Methane-Sulfonate-222 (MS-222). Blood samples were taken from the tail vein, and centrifugation was performed at 3000 RPM for 10 min and serum was collected, which was stored at − 20°C for future research. Tissue samples were taken from liver, intestine, gill. All tissues were rapidly frozen in liquid nitrogen and stored at -80°C for future research.

Chromium Analysis
On days 14 and 28, in order to observe Cr concentration, 3 tissues (liver, intestine, gill) were taken from each group. All tissues were dried in an oven at 120°C before analysis and the dried samples were colddigested with 20 mL of concentrated nitric acid (65% HNO 3 ). The digested samples were collected and centrifuged at 14,000 ×g for 20 min at 4°C and the puri ed liquid was used for determining Cr by atomic absorption spectrometer AA-6300 (Shimadzu, Japan).

Statistical Analysis
The results of the research are expressed as mean ± S.D. One-way analysis of variance (ANOVA) was used to determine the signi cant differences. In order to compare the mean values (P < 0.05), Duncan's multiple range test was used, which had signi cant differences. SPSS statistics 19.0 software (IBM, USA) was used for analysis.

Chromium Accumulation
As Fig. 1 shown, after 14 and 28 days of Cr (VI) exposure, the accumulation levels in each tissue of C. argus was liver > gill > intestine. Compared with the 0 mg/L group, the accumulation of Cr (VI) in gill, intestine and liver were signi cantly increased (P < 0.05) in all groups exposed to Cr (VI).

Antioxidant Parameters in Liver and Gill
The liver and gill antioxidant coe cients of C. argus in response to Cr (VI) are shown in Fig. 2. As shown in the above results, compared with 0 mg/L, the activity of CAT, Gsh-Px and SOD in liver and gill signi cantly reduced (P < 0.05) when exposed to 2 mg/L Cr (VI) at the 28 day. On the contrary, MDA content in liver dramatically increased (P < 0.05) when exposed to 2 mg/L Cr (VI), and MDA content in gill approached the peak level at 2 mg/L on the 28 day.

Serum Immunological Parameters
Changes in serum immunological parameters after 28 days of C. argus exposure to Cr (VI) are shown in Fig. 3 and Fig. 4. Compared with 0 mg/L group, LZM activity was obviously decreased (P < 0.05) when it was exposed to 0.5-2mg/L Cr (VI) in the 14 day and the 28 day, and was lower than the other groups when it was exposed to 2mg/L Cr (VI) in the 28 day. However, the activity of AST, AKP, LDH and COR content were different. In dose-dependent effect, it signi cantly increased (P < 0.05) when it was exposed to 1-2mg/L Cr (VI) on the days 14 and 28. On day 28 after Cr (VI) exposure, IgM concentration and ALT activity were signi cantly higher than others when they were exposed to 2mg/L Cr (VI).

Discussion
Heavy metals are known to be a toxic pollutant that has always been existed, which may cause harmful effects on sh in aquatic ecosystems. The exposure of heavy metal toxic substances will have toxic effects on aquatic animals, but such toxic effects will vary with the biological conditions of the animals, the physical and chemical properties of the water, and the types, types and exposure time of toxic substances [20]. We also know that the type of sh, age and metabolic activity, the concentration of metals in food and sediments, as well as the temperature and salinity of environmental factors, may all affect the degree of heavy metal accumulation in their tissues [21]. Fish are usually at the top of the aquatic food chain, and exposure to heavy metals from bioaccumulation in sh can be considered as an assessment of metal pollution in the aquatic environment [22].In addition, the accumulation of Cr (VI) in sh may lead to potential health risks for those who consume them [23]. Therefore, it is necessary to explore the accumulation and puri cation of water. Cr (VI) in sh tissues, to evaluate the toxic effects of Cr (VI) as antioxidants in sh systems and relative eating risks. And the route of ingestion affects accumulation in aquatic animals, whether through respiration or through digestive organs.
In this study, through exposure to heavy metals in water, the highest accumulation of Cr (VI) in the tissue was observed in the liver of C. argus. In general, the accumulation of sh in the liver is exposed to a much greater amounts of tested metals than other organs. This is attributed to the higher tendency of the elements to be related to the sulfur Al-sulfhydryl groups in carboxylate, amino, nitrogen and metallothionein. Vutukuru et al. (2007) reported that there is a large accumulation of chromium in the liver of major carp in India, and Labeo rohita is exposed to Cr (VI), leading to metabolic processes and detoxi cation [24]. Intestines and gills are important in ingestion. The content of heavy metals and other toxic substances in sh [25]. Intestinal tract is very crucial for the nutrients digestion and absorption in sh, and the integrity of its organizational structure is crucial for the growth and development of sh. In general, the ingestion of metals in the intestine is mainly largely diet-related, because it will ingest metals in the case of water transmission. The high accumulation of metals in intestines may be related to enterohepatic circulation that bile could excrete metals into the intestines [24]. This research showed that the bioaccumulation of Cr (VI) aqueous exposed to different tissues is as follows: liver > gills > intestine.
Because of the interpenetration between sh blood and the external environment, blood parameters have proven to be sensitive and reliable indexes of toxic metal exposure for aquatic animals [26][27][28][29].
The SOD, CAT, Gsh-Px and MDA evaluation are generally known as the important indicators to detect the antioxidant capacity of aquatilia [30]. Antioxidant enzymes such as SOD, CAT, and Gsh-Px are important in the cellular defense against xenobiotic exposure [31]. It is known that SOD can catalyze the search for superoxide anion radicals and can quickly turn oxygen-free radicals into hydrogen peroxide molecules, so as to balance the metabolism of free radicals and protect the cells [32][33]. Both CAT and Gsh-Px have the ability to eliminate H 2 O 2 and transform it into H 2 O and O 2 , thereby helping to remove H 2 O 2 and reduce tissue damage [34]. All of the enzymes mentioned can defense against the stress of exposure to toxins in the organism. Their proper function and activity are essential to prevent any cell damage or death [35].
The secondary product MDA is produced by lipids peroxidation, indicating that oxidant cell damage caused by binding to free protein amino acids and then crosslinking within and between protein molecules [36][37]. So, an increased MDA level is the main mechanism for oxidative damage of cells. Therefore, MDA accumulation is the sign of the risk of oxidant cell damages [38]. The results above indicated, compared with 0 mg/L, the CAT activity, Gsh-Px and the SOD levels in liver and gill markedly decreased (P < 0.05) when they were exposed to 2 mg/L Cr (VI) on the 28 day. Exposure of Cr (VI) in water can cause massive generation of free radicals and reduce the antioxidant capacity of aquatilia. Opposite trend, in liver and gill, the level of MDA increased signi cantly (P < 0.05) when exposed to 2 mg/L Cr (VI) compared with 0 mg/L, in the 28 day. The level of MDA which exposed to 2 mg/L Cr (VI) is the highest, especially in the liver at 28 day.
Because of the interpenetration between sh blood and the external environment, blood parameters have proven to be sensitive and reliable indexes of toxic metal exposure for aquatic animals [39][40][41]. In order to determine the degree of liver and gill toxicity caused by Cr (VI) exposure, the levels of several enzymes were estimated, including ALT, AST and LDH. ALT and AST are important transaminases in sh liver, and AST is an indicator of liver tissue. The serum biochemical parameters of injury are therefore considered to be important indicators for the diagnosis of liver diseases in aquatic animals [42][43]. Compared with the control, they were greatly improved at 28 day. This increase may be due to damage to liver cells and the leakage of these enzymes from damaged cells [44]. COR is synthesized by the adrenal cortex and is for regulating glucose metabolism and stress response. Meanwhile, the serum COR level is related to the stress process [45]. Srious metal pollution can cause abnormal COR metabolism of sh [46]. Previous studies have shown that the accumulation of Cr (VI) tends to lead to immunosuppression, thus increasing sensitivity of sh to diseases [47]. AKP is an important indicator of liver disease diagnosis. When the liver is damaged, liver cells overproduce AKP. LZM is an essential protective immune factor, hydrolyzing the peptidoglycan structure to remove all kinds of bacteria [48]. IgM is a large molecule involved in the speci c immune response and is an important index to measure immune response [49]. This study shows that as the concentration of Cr (VI) increases, the level of LZM in the serum decreases signi cantly. It is suggested that immunosuppression may be caused because of in ammation by heavy metal stress [49]. At the same time, with the increase of lead concentration, the content of COR and AKP in this research increased signi cantly, which indicated that the lead accumulation caused an increase in the degree of stress. This could be due to the damage of the liver tissue structure caused by the accumulation of Cr (VI), and the COR and AKP in the serum also increase. However, IgM shows an upward trend with the prolonged exposure time in water. Some reports indicate that exposure to heavy metals and other toxic substances can cause changes in the body's immune system parameters [17]. Besides, higher levels of LZM and IgM have been observed in diseased sh [50]. Some studies have revealed that the environmental pressure has a negative impact on the immunity system, leading to increased sensitivity to diseases [51]. As the Cr (VI) content of water increases, the IgM level of C. argus also increases.

Conclusion
This research indicated that the waterborne Cr (VI) exposure to C. argus leads to the signi cant Cr (VI) accumulation in organs. Besides, Cr (VI) exposure in water changed antioxidant parameters (MDA, CAT, SOD and Gsh-Px) and immune coe cients (LZM, IgM, ALT, AST, AKP, COR, LDH) expression. These results suggest that the Cr (VI) exposure has a negative effect C. argus.