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Effects of dietary chromium exposure to rockfish, Sebastes schlegelii are ameliorated by ascorbic acid
Ecotoxicology and Environmental Safety 139 (2017) 109–115
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Effects of dietary chromium exposure to rockfish, Sebastes schlegelii are ameliorated by ascorbic acid Jun-Hwan Kima, Ju-Chan Kangb, a b
MARK
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West Sea Fisheries Research Institute, National Institute of Fisheries Science, Incheon 22383, South Korea Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, South Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Sebastes schlegelii Chromium Ascorbic acid Hematological parameters Accumulation
Juvenile rockfish Sebastes schlegelii (mean length 10.8 ± 1.4 cm, and mean weight 31.7 ± 3.6 g) were exposed for 4 weeks with the different levels of dietary chromium (Cr6+) at 0, 120 and 240 mg/L and ascorbic acids (AsA) at 100, 200 and 400 mg/L. Significant accumulation occurred in specific tissues and hematological parameters were altered: red blood cell count, hematocrit, and hemoglobin increased; plasma components were altered including calcium, glucose, cholesterol, total protein, glutamic oxalate transaminase, and glutamic pyruvate transaminase. However, magnesium and alkaline phosphatase concentrations were unchanged. Ascorbic acids reduced both chromium uptake into tissues and altered hematological parameters.
1. Introduction While chromium (Cr) can be a highly toxic metal, it is also a critical nutrient for aquatic animals. In aquatic environment, Cr exists in two main forms: trivalent chromium [Cr(III)] and hexavalent chromium [Cr(VI)]. Oxidized state, Cr(VI), is highly toxic due to its ability to be absorbed more easily in biological system through anion-exchange carriers than is Cr(III) (Dayan and Paine, 2001; Salnikow and Zhitkovich, 2008). Hematological factors in aquatic animals have been widely considered as useful indicators of physiological and pathological changes in toxicological and environmental research seeking to evaluate influences of exposure to toxins (Adhikari et al., 2004). Fish blood parameters are good indicators of toxicity of aquatic environment owing to the close relationship between circulatory system and external environment. Moreover, blood parameters can be affected by various factors such as xenobiotic type, target species, and exposure concentration (Chandrasekara and Pathiratne, 2005). Fish physiological status, e.g. hematological factors, is known to be affected by metallic stress. For example, Cr(VI) exposure was shown to significantly affect hematological factors of Indian major carp, Labeo rohita (Vutukuru, 2005). Shaheen and Akhtar (2012) also reported blood can be a reliable parameter for measuring environmental toxicity because it is highly susceptible to alterations in the environment, and Cr(VI) has a negative effect on hematological and biochemical factors in aquatic animals. In addition, Mazon et al. (2002) reported that hematological and physiological alterations in fish can suggest the homeostatic status in fish
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exposed to environmental pollution. Ascorbic acid (AsA) is a critical nutrient required for the proper metabolic function and immunity of aquatic animals, in addition to their growth and development (Wang et al., 2003). Moreover, AsA supplementation is known to aid in detoxification against contamination by various toxic metals (Kim and Kang, 2015a). AsA is a strong antioxidant, and its supplementation is considerably effective to attenuate Cr(VI) toxicity caused by reactive oxygen species formed during Cr(VI) reduction (Poljsak et al., 2005). AsA supplementation reduced metal retention in tissues caused by metal exposure (Kadrabova et al., 1992). AsA supplementation is also highly effective to attenuate metal-induced alterations in hematological and serum biochemical factors (Yousef, 2004). Ambali et al. (2007) reported that vitamin C has a protective function that can ameliorate damage to hematological factors of mice caused by chlorpyrifos. Fish biomarkers have been considered useful tools in the following risk assessment procedures: effect, exposure and hazard assessment, risk characterization or classification, and monitoring aquatic ecosystems (Oost et al., 2003). Information on toxic effects of Cr(VI) on bioaccumulation and hematological factors, and detoxification effects of AsA for exposure of aquatic animals to Cr is limited. Given that rockfish, Sebastes schlegelii, is a widely consumed species in South Korea, research on toxic effects of Cr on S. schlegelii and ameliorative effects of AsA may be useful for identifying potential bioindicators for Cr toxicity in marine environment.
Corresponding author. E-mail address: [email protected] (J.-C. Kang).
http://dx.doi.org/10.1016/j.ecoenv.2017.01.029 Received 28 October 2016; Received in revised form 9 January 2017; Accepted 17 January 2017 0147-6513/ © 2017 Published by Elsevier Inc.
Ecotoxicology and Environmental Safety 139 (2017) 109–115
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2.2. Feed ingredients and diet formulation
Table 1 The chemical components of seawater and experimental condition used in the experiments. Item
Value
Temperature (°C) pH Salinity (‰) Dissolved oxygen (mg/L) Chemical oxygen demand (mg/L) Ammonia (µg/L) Nitrite (µg/L) Nitrate (µg/L)
19.0 ± 1.0 8.1 ± 0.5 33.2 ± 0.5 7.3 ± 0.4 1.31 ± 0.3 11.0 ± 0.6 1.6 ± 0.3 9.41 ± 1.2
Formulation of diets is shown in Table 2. Potassium dichromate and ascorbic acid were obtained from Sigma Chemical Co., Ltd. All diets contained 33% casein, 23% fish meal, 20% wheat flour, 5% corn starch, 2% vitamin premix (vitamin C-free), and 2% mineral premix. 10% fish oil was added to meet the essential fatty acid (EFA) requirements of S. schlegelii. Chromium premix was made up of 2 g chromium with 98 g cellulose. Chromium premix was added at different concentrations in diets for supplementation of different dietary chromium concentrations of 0, 120, and 240 mg/kg diet. Ascorbic acid premix was made up of 2 g ascorbic acids with 98 g cellulose. Three isonitrogenous and isolipidic diets were formulated supplementation of different dietary ascorbic acid concentrations of 100, 200 and 400 mg/kg. All ingredients were blended thoroughly. At last, water was added into the mixture to produce stiff dough. Then the dough was pelleted by experimental feed mill, and dried for 24 h at room temperature. After processing, all diets were packed and kept at −20 °C until use. The actual chromium and AsA concentrations are shown in Table 3. For determination of total chromium concentrations in diets, ICP-MS measurements were performed using an ELAN 6600DRC ICP-MS instrument with argon gas (Perkin-Elmer). Total chromium concentrations were determined by external calibration. ICP multi-element standard solution VI (Merck) was used to develop a standard curve. The chromium bioaccumulation in diet samples was expressed as mg/kg dry wt. To determine of total ascorbic acid concentrations in diets, HPLC measurements were performed using an Agilent 1200 series. The ascorbic acid content in diet samples was expressed mg/kg dry wt.
2. Materials and methods 2.1. Experimental fish and conditions Juvenile S. schlegelii were obtained from a local fish farm in Tongyeong, Korea. Fish were acclimatized for 2 weeks under laboratory conditions. During the acclimation period, fish were fed a Cr-free diet twice daily and maintained on a 12-h:12-h light/dark cycle and constant condition at all times (Table 1). After acclimatization, 90 fish (body length, 10.8 ± 1.4 cm; body weight, 31.7 ± 3.6 g) were randomly selected for this study. Dietary chromium exposure took place in 500 L circular tanks with 5 fish per treatment group. Dietary chromium and ascorbic acid concentrations were 0, 120, and 240 mg/kg and 100, 200 and 400 mg/kg (Table 2), and fish were fed each chromium concentration at a rate of 2% body weight daily (as two 1% meals per day). Chromium concentration of 240 mg/kg seems to be unrealistic. However, the chromium concentration in the coast near the Incheon North Harbor, Korea reached 214 ppm (Um et al., 2003). Ascorbic acid requirement in S. schlegelii for growth and development is 65 mg/kg based on Standard Manual of Black Rockfish Culture (NFRDI, 2003). However, there is no research about the proper ascorbic acid requirement for detoxification. At the end of each period (at 2 and 4 weeks), fish were anesthetized in buffered 3-aminobenzoic acid ethyl ester methanesulfonate (Sigma Chemical, St. Louis, MO). Anesthetization concentration and time were different to fish species and size. In this study, anesthetization was conducted at 20 ppm concentration for 5 min.
2.3. Chromium accumulation Tissue samples of liver, kidney, spleen, intestine, gill, and muscle of S. schlegelii were removed using clean techniques and freeze-dried to measure the dry weight. The freeze-drying samples were digested by the wet digestion method (Arain et al., 2008). The dried samples were digested in 65%(v/v) HNO3, and re-dried at 120 °C on a hot plate. The procedure was repeated until total digestion. The entirely digested samples were diluted in 2%(v/v) HNO3. The samples were filtered through a 0.2 µm membrane filter (Advantec MFS, Ins., CA, USA) under pressure. For determination of total Cr concentrations, the digested and extracted solutions were analyzed by ICP-MS. The ICP-MS measure-
Table 2 Formulation of the experimental diet (% dry matter). Ingredient (%)
a
Casein Fish mealb Wheat flourc Fish oild Cellulosea Corn starchc Vitamin Premix (Vitamin C-free)e Mineral Premixf Chromium Premixg Ascorbic acid Premixh
Concentration (mg/kg) M0V1
M0V2
M0V3
M1V1
M1V2
M1V3
M2V1
M2V2
M2V3
33.0 23.0 20.0 10.0 4.5 5.0 2.0 2.0 0.0 0.5
33.0 23.0 20.0 10.0 4.0 5.0 2.0 2.0 0.0 1.0
33.0 23.0 20.0 10.0 3.0 5.0 2.0 2.0 0.0 2.0
33.0 23.0 20.0 10.0 3.9 5.0 2.0 2.0 0.6 0.5
33.0 23.0 20.0 10.0 3.4 5.0 2.0 2.0 0.6 1.0
33.0 23.0 20.0 10.0 2.4 5.0 2.0 2.0 0.6 2.0
33.0 23.0 20.0 10.0 3.3 5.0 2.0 2.0 1.2 0.5
33.0 23.0 20.0 10.0 2.8 5.0 2.0 2.0 1.2 1.0
33.0 23.0 20.0 10.0 1.8 5.0 2.0 2.0 1.2 2.0
(M0: Pb 0 mg/kg, M1: Pb 120 mg/kg, M2: Pb 240 mg/kg, V1: AsA 100 mg/kg, V2: AsA 200 mg/kg, V3: AsA 400 mg/kg) a United States Biochemical (Cleveland, OH). b Suhyup Feed Co., Ltd., Gyeong Nam Province, Korea. c Young Nam Flour Mills Co., Pusan, Korea. d Sigma Chemical Co., St. Louis, MO. e Vitamin Premix (vitamin C-free) (mg/kg diet): dl-calcium pantothenate, 400; choine chloride 200; inositol, 20; menadione, 2; nicotinamide, 60; pyridoxine·HCl, 44; riboflavin, 36; thiamine mononitrate, 120, dl-a-tocopherol acetate, 60; retinyl acetate, 20000IU; biotin, 0.04; folic acid, 6; vitamin B12, 0.04; cholecalcifero, 4000IU. f Mineral Premix (mg/kg diet): Al, 1.2; Ca, 5000; Cl, 100; Cu, 5.1; Co, 9.9; Na, 1280; Mg, 520; P, 5000; K, 4300; Zn, 27; Fe, 40; I, 4.6; Se, 0.2; Mn, 9.1. g Chromium Premix (mg/kg diet): 20,000 mg Pb/ kg diet. h Ascorbic acid Premix (mg/kg diet): 20,000 mg ascorbic acid/ kg diet.
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Table 3 Analyzed dietary concentration (mg/kg) from each source. Diets (mg/kg) Concentrations
M0V1
M0V2
M0V3
M1V1
M1V2
M1V3
M2V1
M2V2
M2V3
Cr concentrations Actual Cr concentrations AsA concentrations Actual AsA concentrations
0 4.3 100 83.4
0 5.2 200 174.6
0 3.7 400 367.3
120 131.2 100 88.1
120 124.5 200 168.2
120 128.3 400 359.7
240 246.6 100 79.9
240 249.7 200 171.2
240 244.7 400 363.6
(M0: Pb 0 mg/kg, M1: Pb 120 mg/kg, M2: Pb 240 mg/kg, V1: AsA 100 mg/kg, V2: AsA 200 mg/kg, V3: AsA 400 mg/kg)
200 and 400 mg/kg AsA at 240 mg/kg Cr exposure was considerably lower than the fish receiving diet supplemented with 100 mg/kg AsA. At 4 weeks, Cr bioaccumulation in the liver of fish receiving diets supplemented with 200 and 400 mg/kg AsA at 120- and 240-mg/kg Cr exposure was substantially lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. Cr accumulation in the spleen was also notably increased in the fish exposed to greater than 120-mg/kg Cr at 2 and 4 weeks. At 2 weeks, AsA supplementation had no effect on Cr accumulation. However, 400 mg/kg AsA supplementation at 240-mg/kg Cr exposure at 4 weeks considerably affected Cr accumulation compared to the dietary supplementation at 100 and 200 mg/kg AsA. For the intestinal tissue, significant accumulation was observed when Cr exposure was greater than 120 mg/kg. At 2 weeks, the 200 and 400 mg/kg AsA supplementation at 240 mg/kg Cr exposure notably influenced retention of Cr accumulation, compared to the 100 mg/kg AsA. At 4 weeks, the concentrations of bioaccumulation in the intestine of fish receiving diets supplemented with 200 and 400 mg/kg AsA at 120 and 240 mg/kg Cr exposure were substantially lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. In gill tissue, substantial accumulation was observed in fish exposited to greater than 120 mg/kg dietary Cr. At 2 weeks, the concentrations of bioaccumulation in gill tissue of fish receiving diets supplemented with 200 and 400 mg/kg AsA and exposed to 240 mg/kg Cr were substantially lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. At 4 weeks, the concentrations of bioaccumulation of those receiving diets supplemented with 200 and 400 mg/kg AsA at 120- and 240-mg/kg Cr exposure were considerably lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. In muscle tissue, there was no considerable Cr accumulation except for the group receiving diets supplemented with 100 mg/kg AsA at 240mg/kg Cr exposure at 2 weeks. At 4 weeks, the concentration of bioaccumulation in fish receiving a diet supplemented with 400 mg/kg AsA at 240 mg/kg Cr exposure was considerably lower than the accumulation in fish receiving diets supplemented with 100 and 200 mg/kg AsA. At 4 weeks of dietary Cr exposure, depending on the AsA supplementation, the profile of tissue Cr accumulation was in the following order: kidney > liver > spleen > intestine > gill > muscle.
ments were performed using an ELAN 6600DRC ICP-MS instrument with argon gas (Perkin-Elmer). Total Cr concentrations were determined by external calibration. ICP multi-element standard solution VI (Merck) was used for the standard curve. The Cr bioaccumulation in tissue samples was expressed as µg/g dry wt. 2.4. Blood samples and hematological assay Blood samples were collected within 35–40 s through the caudal vein of the fish in 1-ml disposable heparinized syringes. The blood samples were kept at 4 °C until the blood parameters were completely studied. The total red blood cell (RBC), hemoglobin (Hb), concentration and hematocrit (Ht) value were determined immediately. Total RBC counts were counted using an optical microscope with hemo-cytometer (Improved Neubauer, Germany) after diluted by Hendrick's dilution solution. The Hb concentration was determined using the Cyanmethemoglobin technique (Asan Pharm. co., Ltd). The Ht value was determined by the microhematocrit centrifugation technique. The blood samples were centrifuged to separate plasma from blood samples at 3000g for 5 min at 4 °C. The serum samples were analyzed for inorganic substances, organic substances, and enzyme activity using a clinical kit (Asan Pharm. Co., Ltd). Calcium and magnesium were analyzed by the o-cresolphthalein-complexone technique and xylidyl blue technique. Glucose and total protein were analyzed by the GOD/ POD technique and biuret technique. Glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were analyzed by the Kind-king technique and alkaline phosphatase (ALP) was analyzed using clinic al kit (Asan Pharm. Co., Ltd). 2.5. Statistical analysis The experiment was conducted in exposure periods for 4 weeks and performed in triplicate. Statistical analyses were performed using the SPSS/PC+ statistical package (SPSS Inc, Chicago, IL, USA). Significant differences between groups were identified using one-way ANOVA and Duncan's test for multiple comparisons or Student's t-test for two groups (Duncan, 1955). The significance level was set at P < 0.05. 3. Results 3.1. Chromium accumulation
3.2. Hematological factors Cr accumulation in the kidney, liver, spleen, intestine, gill, and muscle of S. schlegelii exposed to various dietary Cr concentrations depended on AsA supplementation (Fig. 1). We observed the highest Cr accumulation in the kidney. This significant accumulation in the kidney was observed in fish given dietary Cr greater than 120 mg/kg at 2 weeks and 4 weeks. Furthermore, for the 240 mg/kg Cr exposure at 2 and 4 weeks, Cr bioaccumulation in kidney was considerably lower in the fish receiving a diet supplemented with 400 mg/kg AsA than in the fish receiving diets supplemented with 100 and 200 mg/kg AsA. In liver tissue, Cr accumulation significantly increased at Cr exposure greater than 120 mg/kg, at 2 and 4 weeks. At 2 weeks, Cr bioaccumulation in the liver of fish receiving diets supplemented with
The RBC count, hematocrit value, and hemoglobin concentration of S. schlegelii exposed to dietary Cr all depended on AsA supplementation (Fig. 2). In S. schlegelii, a significant decrease in RBC count at 2 weeks was observed in 120 mg/kg Cr exposure and dietary supplementation with 100 and 200 mg/kg AsA, and in 240 mg/kg Cr and dietary supplementation with 400 mg/kg AsA. At 4 weeks, the RBC values were notably reduced by the dietary Cr exposure over 120 mg/kg. In 240 mg/kg Cr, the reduction in dietary supplementation with 400 mg/ kg AsA was significantly less than in dietary supplementation with 100 and 200 mg/kg AsA. The hematocrit of S. schlegelii was significantly decreased in 120 mg/kg Cr at 2 weeks. At 4 weeks, a substantial 111
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Fig. 1. Cr accumulation of rockfish, Sebastes schlegelii exposed to different concentrations of dietary chromium and ascorbic acid for 4 weeks. Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by Duncan's multiple range test.
cantly lower than those in 120 mg/kg Cr in fish receiving diets supplemented with 100 and 200 mg/kg AsA and 240 mg/kg Cr in fish receiving a diet supplemented with 100 mg/kg. The hematological factors of S. schlegelii were significantly reduced by the dietary Cr exposure, and the high concentrations of AsA supplementation were highly effective to attenuate Cr exposure. The alterations in plasma components of S. schlegelii by the dietary Cr exposure depended on the AsA supplementation (Fig. 3). In the inorganic components such as calcium and magnesium, calcium was reduced at 240 mg/kg Cr with 100 mg/kg AsA at 2 weeks, and 100 and
reduction was observed in 120 mg/kg Cr and dietary supplementation with 100 and 200 mg/kg AsA and in 240 mg/kg Cr and dietary supplementation with 400 mg/kg AsA. In the hemoglobin of S. schlegelii, the concentration at 2 weeks was notably decreased at Cr exposure greater than 120 mg/kg Cr in fish receiving a diet supplemented with 100 mg/kg AsA and in those exposed to a dietary concentration of 240 mg/kg Cr and receiving diets supplemented with 200 and 400 mg/ kg. At 4 weeks, the concentrations in 120 mg/kg Cr in fish receiving a diet supplemented with 400 mg/kg AsA and 240 mg/kg Cr in fish receiving diets supplemented with 200 and 400 mg/kg were signifi-
Fig. 2. Changes of RBC count, Hematocrit and Hemoglobin in rockfish, Sebastes schlegelii exposed to different concentrations of dietary chromium and ascorbic acid for 4 weeks. Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by Duncan's multiple range test.
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Fig. 3. Changes of plasma parameters in rockfish, Sebastes schlegelii exposed to different concentrations of dietary chromium and ascorbic acid for 4 weeks. Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by Duncan's multiple range test.
nase (GPT) of S. schlegelii were notably increased by the dietary Cr exposure, whereas no change was observed in the ALP. The GOT of S. schlegelii was notably increased with 120 mg/kg Cr at 2 weeks. At 4 weeks, a significant increase in the GOT was observed in 120 mg/kg supplemented with 100 and 200 mg/kg AsA and in 240 mg/kg supplemented with 400 mg/kg AsA. The GPT of S. schlegelii was substantially increased in 120 mg/kg Cr supplemented with 100 and 200 mg/kg AsA at 2 weeks. At 4 weeks, a notable increase was observed in 120 mg/kg Cr supplemented with 100 mg/kg AsA and in 240 mg/kg Cr supplemented with 200 and 400 mg/kg AsA. However, there was no alteration in the ALP of S. schlegelii due to dietary Cr when exposed with AsA supplementation. The plasma components of S. schlegelii were significantly altered by dietary Cr exposure, and the high concentrations of AsA supplementation were highly effective to attenuate alterations due
200 mg/kg AsA at 4 weeks. Magnesium was not changed by the dietary Cr exposure. Of the organic components, such as glucose, cholesterol, and total protein, the glucose and cholesterol concentrations were significantly increased by dietary Cr exposure, whereas total protein was notably decreased. Glucose was substantially increased at 240 mg/ kg Cr and dietary supplementation with 100 mg/kg AsA at 2 weeks and at 120 mg/kg Cr exposure and dietary supplementation with 100, 200, and 400 mg/kg AsA at 4 weeks. Cholesterol was significantly increased at concentration of 240 mg/kg Cr supplemented with 100 mg/kg AsA at 2 weeks and 120 mg/kg Cr supplemented with 100 mg/kg AsA. A significant decrease in total protein was observed in the 240 mg/kg Cr supplemented with 100 and 200 mg/kg AsA at 2 weeks and 100, 200, and 400 mg/kg AsA at 4 weeks. In the enzyme components, the glutamic oxalate transaminase (GOT) and glutamic pyruvate transami-
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liorate the reduction in the hematological factors associated with dietary Cr exposure. Vani et al. (2011) reported significant reductions in the toxicity of deltamethrin to the major carp, Catla catla by AsA. Hounkpatin et al. (2012) also reported that high concentrations of AsA supplementation were effective to attenuate effects on hematological factors caused by cadmium and mercury exposure. In plasma, calcium and magnesium are major constituents that function as the ion regulators for homeostasis, and can be affected by metal-induced toxicity (Rogers et al., 2005). The calcium level of S. schlegelii was decreased by dietary Cr exposure, whereas no significant change was observed in the magnesium of S. schlegelii. In plasma, the glucose and cholesterol level of S. schlegelii were raised through dietary Cr exposure, whereas total protein decreased. Plasma glucose is commonly elevated by gluconeogenesis and can be a good parameter to monitor environmental toxicity (Saravanan et al., 2011). Al-Akel and Shamsi (1996) reported that Cr(VI) exposure in carp, Cyprinus carpio L., resulted in significant alterations in carbohydrate metabolism and blood factors, which caused the breakdown of tissue glycogen necessary to meet the energy demand in the muscle for the anaerobic stress by impairing oxygen supply to tissue. Cholesterol is a major structural component of cell membranes and precursor of steroid hormones and an increase in cholesterol can be a reliable indicator to assess metalinduced toxicity (Oner et al., 2008). Plasma protein can be significantly changed under stress situations (Gopal et al., 1997). In the present study, GOT and GPT of S. schlegelii were notably increased by dietary Cr exposure, but there was no alteration in the ALP. These enzyme components have been considered as a critical indicator for evaluating liver damage caused by toxic substance exposure (Oost et al., 2003). Dietary Cr exposure affected significant alterations in the plasma components of S. schlegelii. The high concentrations of AsA supplementation were highly effective for alleviating the alterations in the plasma components due to dietary Cr exposure. Other authors have reported the protective effects of AsA on hematological factors of animals exposed to toxicants (Yousef, 2004; Hounkpatin et al., 2012). Vani et al. (2011) also reported the ameliorating effect of dietary AsA supplementation in the serum components of Catla catla exposed to deltamethrin. In conclusion, dietary Cr exposure in S. schlegelii results in a significant Cr accumulation in all tissues, although the accumulation concentrations in each tissue vary due to metabolic differences. High concentrations of AsA supplementation were highly effective for alleviating Cr accumulation associated with dietary Cr exposure. Hematological factors and plasma components were significantly affected by dietary Cr exposure, and high concentrations of AsA supplementation were highly effective for attenuating the alterations in the factors. High concentrations of AsA supplementation effectively attenuate Cr-induced bioaccumulation and other effects.
to Cr exposure. 4. Discussion Metal levels in aquatic ecosystems have increased by industrial and agricultural activities, and the metal exposure induces metal accumulation in fish tissues (Uysal et al., 2008). The accumulation and dispersion patterns for metal exposure depend on the exposure vectors (water, feed, and sediment), exposure concentration and period, and absorption ability of each tissue (Leonard et al., 2014). Among chromium, hexavalent chromium (Cr VI) can be much toxic to fish due to its ability to enter cells via anion transport than trivalent chromium (Cr III) (Kim and Kang, 2016b). In this study, the highest Cr accumulation was observed in the kidney of S. schlegelii by dietary Cr exposure. Farag et al. (2006) suggested that kidney is a major target tissue of Cr exposure. Kidney plays a role in excreting xenobiotics and toxicants, as well as having a detoxification function (Bodo et al., 2003), which causes a significant accumulation in kidney tissue. Similar to the kidney, a significant accumulation was observed in the liver of S. schlegelii exposed to dietary Cr. Webb and Wood (2000) also reported a notable accumulation in the liver of four marine teleosts and two marine elasmobranchs exposed to silver. A significant accumulation in the liver of fish and other vertebrates exposed to metals is induced by the metallothioneins in the liver, which binds to metals, allowing the liver to accumulate higher doses of metals than other tissues (Atli and Canli, 2003). In another study, a significant accumulation in the liver and kidney of S. schlegelii was observed through dietary Cr exposure (Kim and Kang, 2016a). Jezierska and Witeska (2006) reported that the kidney and liver in fish are the tissues with the greatest accumulation through metal exposure. Notable Cr accumulation was also observed in the spleen of S. schlegelii. Generally, the metabolically active tissues such as kidney, liver, and spleen have much greater accumulation than other tissues (Karaytug et al., 2007). Metal absorption in aquatic animals occurs via different pathways including through direct uptake from water by gills, and ingestion from food by the intestines (Oost et al., 2003); metals accumulation in fish is generally through diet (Hall et al., 1997). In this study, the higher accumulation of the intestine in S. schlegelii than that of the gill may be caused by dietary exposure. A significant accumulation in the intestine of S. schlegelii may be due to direct metal uptake by dietary exposure. The lowest accumulation was observed in the muscle of S. schlegelii exposed to dietary Cr. The relative accumulation of S. schlegelii exposed to dietary Cr was: kidney > liver > spleen > intestine > gill > muscle. In this study, the high concentrations of AsA supplementation were highly effective at alleviating Cr accumulation of all tissues in S. schlegelii with dietary Cr exposure. AsA supplementation to various animals helps reduce metal retention in tissues due to metal exposure (Kadrabova et al., 1992; Grosicki, 2004). In fish, Kumar et al. (2009) reported AsA supplementation was highly effective at reducing cadmium accumulation in the liver and kidney of catfish, Clarias batrachus, exposed to cadmium. The results of this study demonstrate that the high concentrations of AsA supplementation should be effective to attenuate accumulation in aquatic animals with Cr exposure. The hematological factors such as RBC count, hematocrit, and hemoglobin have been considered as sensitive and reliable indicators to evaluate fish physiological status under toxicant exposure. Authors reported anemia and significant decreases in hematocrits, hemoglobin, and erythrocyte counts in fish exposed to toxic substances (Benifey and Biron, 2000; Jee et al., 2004). Vutukuru (2003) also reported hematological alterations of the Indian major carp, Labeo rohita, exposed to Cr. In this study, dietary Cr exposure induced a substantial reduction in RBC count, hematocrit, and hemoglobin of S. schlegelii. The changes in the hematological factors may be due to osmotic alterations induced by metal toxicity causing hemodilution and red blood cell fragility, in addition to impaired hematopoietic tissues (Shah, 2006). The high concentrations of AsA supplementation were highly effective to ame-
Acknowledgment This study was supported by the project the Environmental-friendly Aquaculture Technology using biofloc (R2017016) of the National Institue of Fisheries Science, Incheon, South Korea. References Adhikari, S., Sarkar, B., Chatterjee, A., Mahapatra, C.T., Ayyappan, S., 2004. Effects of cypermethrin and carbofuran on certain hematological parameters and prediction of their recovery in a freshwater teleost, Labeo rohita (Hamilton). Ecotoxicol. Environ. Saf. 58, 220–226. Al-Akel, A.S., Shamsi, M.J.K., 1996. Hexavalent chromium: toxicity and impact on carbohydrate metabolism and haematological parameters of carp (Cyprinus carpio L.) from Saudi Arabia. Aquat. Sci. 58, 24–30. Ambali, S., Akanbi, D., Igbokwe, N., Shittu, M., Kawu, M., Ayo, J., 2007. Evaluation of subchronic chlorpyrifos poisoning on hematological and serum biochemical changes in mice and protective effect of vitamin C. J. Toxicol. Sci. 32, 111–120. Arain, M.B., Kazi, T.G., Jamali, M.K., Afridi, H.I., Jalbani, N., 2008. Total dissolved and bioavailable metals in water and sediment samples and their accumulation in
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Oreochromis mossambicus of polluted Manchar Lake. Chemosphere 70, 1845–1856. Atli, G., Canli, M., 2003. Natural occurrence of metallothionein-like proteins in the liver of fish Oreochromis niloticus and effects of cadmium, lead, copper, zinc, and iron exposures on their profiles. Bull. Environ. Contam. Toxicol. 70, 619–627. Benifey, T.J., Biron, M., 2000. Acute stress response in triploid rainbow trout (Onchrhynchus mykiss) and brook trout (Salvelinus fontinalis). Aquaculture 184, 167–176. Bodo, A., Bakos, E., Szeri, F., Varadi, A., Sarkadi, B., 2003. The role of multidrug transporters in drug availability, metabolism and toxicity. Toxicol. Lett. 140–141, 133–143. Chandrasekara, H.U., Pathiratne, A., 2005. Influence of low concentrations of Trichlorfon on haematological parameter and brain acetylcholinesterase activity in common carp, Cyprinus carpio L. Aquac. Res. 36, 144–149. Dayan, A.D., Paine, A.J., 2001. Mechanisms of chromium toxicity, carcinogenicity and allergenicity: review of the literature from 1985 to 2000. Hum. Exp. Toxicol. 20, 439–451. Duncan, D.B., 1955. Multiple range and multiple f tests. Biometrics 11, 1–42. Farag, A.M., May, T., Marty, G.D., Easton, M., Harper, D.D., Little, E.E., Cleveland, L., 2006. The effect of chronic chromium exposure on the health of Chinook salmon (Oncorhynchus tshawytscha). Aquat. Toxicol. 76, 246–257. Gopal, V., Parvathy, S., Balasubramanian, P.R., 1997. Effect of heavy metals on the blood protein biochemistry of the fish Cyprinus carpio and its use as a bio-indicator of pollution stress. Environ. Monit. Assess. 48, 117–124. Grosicki, A., 2004. Influence of vitamin C on cadmium absorption and distribution in rats. J. Trace Elem. Med. Biol. 18, 183–187. Hall, B.D., Bodaly, R.A., Fudge, R.J.P., Rudd, J.W.M., Rosenberg, D.M., 1997. Food as the dominant pathway of methyl mercury uptake by fish. Water, Air, Soil Pollut. 100, 13–24. Hounkpatin, A.S.Y., Johnson, R.C., Guedenon, P., Domingo, E., Alimba, C.G., Boko, M., Edorh, P.A., 2012. Protective effects of vitamin C on hematological parameters in intoxicated wistar rats with cadmium, mercury and combined cadmium and mercury. Int. Res. J. Biol. Sci. 1, 76–81. Jee, J.H., Kim, S.G., Kang, J.C., 2004. Effects of phenanthrene on growth and basic physiological functions of the olive flounder, Paralichthys olivaceus. J. Exp. Mar. Biol. Ecol. 304, 123–136. Jezierska, B., Witeska, M., 2006. The metal uptake and accumulation in fish living in polluted waters. Soil Water Pollut. Monit., Prot. Remediat. 69, 107–114. Kadrabova, J., Madaric, A., Ginter, E., 1992. The effect of ascorbic acid on cadmium accumulation in guinea pig tissues. Experientia 48, 989–991. Karaytug, S., Erdem, C., Cicik, B., 2007. Accumulation of cadmium in the gill, liver, kidney, spleen, muscle and brain tissues of Cyprinus carpio. Ekoloji 16, 16–22. Kim, J.H., Kang, J.C., 2015a. Influence of dietary ascorbic acid on the immune responses of juvenile Korean rockfish Sebastes schlegelii. J. Aquat. Anim. Health 27, 178–184. Kim, J.H., Kang, J.C., 2016a. The chromium accumulation and its physiological effects in juvenile rockfish, Sebastes schlegelii, exposed to different levels of dietary chromium (Cr6+) concentrations. Environ. Toxicol. Pharmacol. 41, 152–158. Kim, J.H., Kang, J.C., 2016b. Oxidative stress, neurotoxicity, and metallothionein (MT) gene expression in juvenile rock fish Sebastes schlegelii under the different levels of dietary chromium (Cr6+) exposure. Ecotoxicol. Environ. Saf. 125, 78–84. Kumar, P., Prasad, Y., Patra, A.K., Ranjan, R., Swarup, D., Patra, R.C., Pal, S., 2009. Ascorbic acid, garlic extract and taurine alleviate cadmium-induced oxidative stress in freshwater catfish (Clarias batrachus). Sci. Total Environ. 407, 5024–5030. Leonard, E.M., Banerjee, U., D’Silva, J.J., Wood, C.M., 2014. Chronic nickel bioaccumulation and sub-cellular fractionation in two freshwater teleost's, the round
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of dietary chromium exposure to rockfish, Sebastes schlegelii are ameliorated by ascorbic acid Jun-Hwan Kima, Ju-Chan Kangb, a b
MARK
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West Sea Fisheries Research Institute, National Institute of Fisheries Science, Incheon 22383, South Korea Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, South Korea
A R T I C L E I N F O
A B S T R A C T
Keywords: Sebastes schlegelii Chromium Ascorbic acid Hematological parameters Accumulation
Juvenile rockfish Sebastes schlegelii (mean length 10.8 ± 1.4 cm, and mean weight 31.7 ± 3.6 g) were exposed for 4 weeks with the different levels of dietary chromium (Cr6+) at 0, 120 and 240 mg/L and ascorbic acids (AsA) at 100, 200 and 400 mg/L. Significant accumulation occurred in specific tissues and hematological parameters were altered: red blood cell count, hematocrit, and hemoglobin increased; plasma components were altered including calcium, glucose, cholesterol, total protein, glutamic oxalate transaminase, and glutamic pyruvate transaminase. However, magnesium and alkaline phosphatase concentrations were unchanged. Ascorbic acids reduced both chromium uptake into tissues and altered hematological parameters.
1. Introduction While chromium (Cr) can be a highly toxic metal, it is also a critical nutrient for aquatic animals. In aquatic environment, Cr exists in two main forms: trivalent chromium [Cr(III)] and hexavalent chromium [Cr(VI)]. Oxidized state, Cr(VI), is highly toxic due to its ability to be absorbed more easily in biological system through anion-exchange carriers than is Cr(III) (Dayan and Paine, 2001; Salnikow and Zhitkovich, 2008). Hematological factors in aquatic animals have been widely considered as useful indicators of physiological and pathological changes in toxicological and environmental research seeking to evaluate influences of exposure to toxins (Adhikari et al., 2004). Fish blood parameters are good indicators of toxicity of aquatic environment owing to the close relationship between circulatory system and external environment. Moreover, blood parameters can be affected by various factors such as xenobiotic type, target species, and exposure concentration (Chandrasekara and Pathiratne, 2005). Fish physiological status, e.g. hematological factors, is known to be affected by metallic stress. For example, Cr(VI) exposure was shown to significantly affect hematological factors of Indian major carp, Labeo rohita (Vutukuru, 2005). Shaheen and Akhtar (2012) also reported blood can be a reliable parameter for measuring environmental toxicity because it is highly susceptible to alterations in the environment, and Cr(VI) has a negative effect on hematological and biochemical factors in aquatic animals. In addition, Mazon et al. (2002) reported that hematological and physiological alterations in fish can suggest the homeostatic status in fish
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exposed to environmental pollution. Ascorbic acid (AsA) is a critical nutrient required for the proper metabolic function and immunity of aquatic animals, in addition to their growth and development (Wang et al., 2003). Moreover, AsA supplementation is known to aid in detoxification against contamination by various toxic metals (Kim and Kang, 2015a). AsA is a strong antioxidant, and its supplementation is considerably effective to attenuate Cr(VI) toxicity caused by reactive oxygen species formed during Cr(VI) reduction (Poljsak et al., 2005). AsA supplementation reduced metal retention in tissues caused by metal exposure (Kadrabova et al., 1992). AsA supplementation is also highly effective to attenuate metal-induced alterations in hematological and serum biochemical factors (Yousef, 2004). Ambali et al. (2007) reported that vitamin C has a protective function that can ameliorate damage to hematological factors of mice caused by chlorpyrifos. Fish biomarkers have been considered useful tools in the following risk assessment procedures: effect, exposure and hazard assessment, risk characterization or classification, and monitoring aquatic ecosystems (Oost et al., 2003). Information on toxic effects of Cr(VI) on bioaccumulation and hematological factors, and detoxification effects of AsA for exposure of aquatic animals to Cr is limited. Given that rockfish, Sebastes schlegelii, is a widely consumed species in South Korea, research on toxic effects of Cr on S. schlegelii and ameliorative effects of AsA may be useful for identifying potential bioindicators for Cr toxicity in marine environment.
Corresponding author. E-mail address: [email protected] (J.-C. Kang).
http://dx.doi.org/10.1016/j.ecoenv.2017.01.029 Received 28 October 2016; Received in revised form 9 January 2017; Accepted 17 January 2017 0147-6513/ © 2017 Published by Elsevier Inc.
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2.2. Feed ingredients and diet formulation
Table 1 The chemical components of seawater and experimental condition used in the experiments. Item
Value
Temperature (°C) pH Salinity (‰) Dissolved oxygen (mg/L) Chemical oxygen demand (mg/L) Ammonia (µg/L) Nitrite (µg/L) Nitrate (µg/L)
19.0 ± 1.0 8.1 ± 0.5 33.2 ± 0.5 7.3 ± 0.4 1.31 ± 0.3 11.0 ± 0.6 1.6 ± 0.3 9.41 ± 1.2
Formulation of diets is shown in Table 2. Potassium dichromate and ascorbic acid were obtained from Sigma Chemical Co., Ltd. All diets contained 33% casein, 23% fish meal, 20% wheat flour, 5% corn starch, 2% vitamin premix (vitamin C-free), and 2% mineral premix. 10% fish oil was added to meet the essential fatty acid (EFA) requirements of S. schlegelii. Chromium premix was made up of 2 g chromium with 98 g cellulose. Chromium premix was added at different concentrations in diets for supplementation of different dietary chromium concentrations of 0, 120, and 240 mg/kg diet. Ascorbic acid premix was made up of 2 g ascorbic acids with 98 g cellulose. Three isonitrogenous and isolipidic diets were formulated supplementation of different dietary ascorbic acid concentrations of 100, 200 and 400 mg/kg. All ingredients were blended thoroughly. At last, water was added into the mixture to produce stiff dough. Then the dough was pelleted by experimental feed mill, and dried for 24 h at room temperature. After processing, all diets were packed and kept at −20 °C until use. The actual chromium and AsA concentrations are shown in Table 3. For determination of total chromium concentrations in diets, ICP-MS measurements were performed using an ELAN 6600DRC ICP-MS instrument with argon gas (Perkin-Elmer). Total chromium concentrations were determined by external calibration. ICP multi-element standard solution VI (Merck) was used to develop a standard curve. The chromium bioaccumulation in diet samples was expressed as mg/kg dry wt. To determine of total ascorbic acid concentrations in diets, HPLC measurements were performed using an Agilent 1200 series. The ascorbic acid content in diet samples was expressed mg/kg dry wt.
2. Materials and methods 2.1. Experimental fish and conditions Juvenile S. schlegelii were obtained from a local fish farm in Tongyeong, Korea. Fish were acclimatized for 2 weeks under laboratory conditions. During the acclimation period, fish were fed a Cr-free diet twice daily and maintained on a 12-h:12-h light/dark cycle and constant condition at all times (Table 1). After acclimatization, 90 fish (body length, 10.8 ± 1.4 cm; body weight, 31.7 ± 3.6 g) were randomly selected for this study. Dietary chromium exposure took place in 500 L circular tanks with 5 fish per treatment group. Dietary chromium and ascorbic acid concentrations were 0, 120, and 240 mg/kg and 100, 200 and 400 mg/kg (Table 2), and fish were fed each chromium concentration at a rate of 2% body weight daily (as two 1% meals per day). Chromium concentration of 240 mg/kg seems to be unrealistic. However, the chromium concentration in the coast near the Incheon North Harbor, Korea reached 214 ppm (Um et al., 2003). Ascorbic acid requirement in S. schlegelii for growth and development is 65 mg/kg based on Standard Manual of Black Rockfish Culture (NFRDI, 2003). However, there is no research about the proper ascorbic acid requirement for detoxification. At the end of each period (at 2 and 4 weeks), fish were anesthetized in buffered 3-aminobenzoic acid ethyl ester methanesulfonate (Sigma Chemical, St. Louis, MO). Anesthetization concentration and time were different to fish species and size. In this study, anesthetization was conducted at 20 ppm concentration for 5 min.
2.3. Chromium accumulation Tissue samples of liver, kidney, spleen, intestine, gill, and muscle of S. schlegelii were removed using clean techniques and freeze-dried to measure the dry weight. The freeze-drying samples were digested by the wet digestion method (Arain et al., 2008). The dried samples were digested in 65%(v/v) HNO3, and re-dried at 120 °C on a hot plate. The procedure was repeated until total digestion. The entirely digested samples were diluted in 2%(v/v) HNO3. The samples were filtered through a 0.2 µm membrane filter (Advantec MFS, Ins., CA, USA) under pressure. For determination of total Cr concentrations, the digested and extracted solutions were analyzed by ICP-MS. The ICP-MS measure-
Table 2 Formulation of the experimental diet (% dry matter). Ingredient (%)
a
Casein Fish mealb Wheat flourc Fish oild Cellulosea Corn starchc Vitamin Premix (Vitamin C-free)e Mineral Premixf Chromium Premixg Ascorbic acid Premixh
Concentration (mg/kg) M0V1
M0V2
M0V3
M1V1
M1V2
M1V3
M2V1
M2V2
M2V3
33.0 23.0 20.0 10.0 4.5 5.0 2.0 2.0 0.0 0.5
33.0 23.0 20.0 10.0 4.0 5.0 2.0 2.0 0.0 1.0
33.0 23.0 20.0 10.0 3.0 5.0 2.0 2.0 0.0 2.0
33.0 23.0 20.0 10.0 3.9 5.0 2.0 2.0 0.6 0.5
33.0 23.0 20.0 10.0 3.4 5.0 2.0 2.0 0.6 1.0
33.0 23.0 20.0 10.0 2.4 5.0 2.0 2.0 0.6 2.0
33.0 23.0 20.0 10.0 3.3 5.0 2.0 2.0 1.2 0.5
33.0 23.0 20.0 10.0 2.8 5.0 2.0 2.0 1.2 1.0
33.0 23.0 20.0 10.0 1.8 5.0 2.0 2.0 1.2 2.0
(M0: Pb 0 mg/kg, M1: Pb 120 mg/kg, M2: Pb 240 mg/kg, V1: AsA 100 mg/kg, V2: AsA 200 mg/kg, V3: AsA 400 mg/kg) a United States Biochemical (Cleveland, OH). b Suhyup Feed Co., Ltd., Gyeong Nam Province, Korea. c Young Nam Flour Mills Co., Pusan, Korea. d Sigma Chemical Co., St. Louis, MO. e Vitamin Premix (vitamin C-free) (mg/kg diet): dl-calcium pantothenate, 400; choine chloride 200; inositol, 20; menadione, 2; nicotinamide, 60; pyridoxine·HCl, 44; riboflavin, 36; thiamine mononitrate, 120, dl-a-tocopherol acetate, 60; retinyl acetate, 20000IU; biotin, 0.04; folic acid, 6; vitamin B12, 0.04; cholecalcifero, 4000IU. f Mineral Premix (mg/kg diet): Al, 1.2; Ca, 5000; Cl, 100; Cu, 5.1; Co, 9.9; Na, 1280; Mg, 520; P, 5000; K, 4300; Zn, 27; Fe, 40; I, 4.6; Se, 0.2; Mn, 9.1. g Chromium Premix (mg/kg diet): 20,000 mg Pb/ kg diet. h Ascorbic acid Premix (mg/kg diet): 20,000 mg ascorbic acid/ kg diet.
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Table 3 Analyzed dietary concentration (mg/kg) from each source. Diets (mg/kg) Concentrations
M0V1
M0V2
M0V3
M1V1
M1V2
M1V3
M2V1
M2V2
M2V3
Cr concentrations Actual Cr concentrations AsA concentrations Actual AsA concentrations
0 4.3 100 83.4
0 5.2 200 174.6
0 3.7 400 367.3
120 131.2 100 88.1
120 124.5 200 168.2
120 128.3 400 359.7
240 246.6 100 79.9
240 249.7 200 171.2
240 244.7 400 363.6
(M0: Pb 0 mg/kg, M1: Pb 120 mg/kg, M2: Pb 240 mg/kg, V1: AsA 100 mg/kg, V2: AsA 200 mg/kg, V3: AsA 400 mg/kg)
200 and 400 mg/kg AsA at 240 mg/kg Cr exposure was considerably lower than the fish receiving diet supplemented with 100 mg/kg AsA. At 4 weeks, Cr bioaccumulation in the liver of fish receiving diets supplemented with 200 and 400 mg/kg AsA at 120- and 240-mg/kg Cr exposure was substantially lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. Cr accumulation in the spleen was also notably increased in the fish exposed to greater than 120-mg/kg Cr at 2 and 4 weeks. At 2 weeks, AsA supplementation had no effect on Cr accumulation. However, 400 mg/kg AsA supplementation at 240-mg/kg Cr exposure at 4 weeks considerably affected Cr accumulation compared to the dietary supplementation at 100 and 200 mg/kg AsA. For the intestinal tissue, significant accumulation was observed when Cr exposure was greater than 120 mg/kg. At 2 weeks, the 200 and 400 mg/kg AsA supplementation at 240 mg/kg Cr exposure notably influenced retention of Cr accumulation, compared to the 100 mg/kg AsA. At 4 weeks, the concentrations of bioaccumulation in the intestine of fish receiving diets supplemented with 200 and 400 mg/kg AsA at 120 and 240 mg/kg Cr exposure were substantially lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. In gill tissue, substantial accumulation was observed in fish exposited to greater than 120 mg/kg dietary Cr. At 2 weeks, the concentrations of bioaccumulation in gill tissue of fish receiving diets supplemented with 200 and 400 mg/kg AsA and exposed to 240 mg/kg Cr were substantially lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. At 4 weeks, the concentrations of bioaccumulation of those receiving diets supplemented with 200 and 400 mg/kg AsA at 120- and 240-mg/kg Cr exposure were considerably lower than the accumulation in fish receiving a diet supplemented with 100 mg/kg AsA. In muscle tissue, there was no considerable Cr accumulation except for the group receiving diets supplemented with 100 mg/kg AsA at 240mg/kg Cr exposure at 2 weeks. At 4 weeks, the concentration of bioaccumulation in fish receiving a diet supplemented with 400 mg/kg AsA at 240 mg/kg Cr exposure was considerably lower than the accumulation in fish receiving diets supplemented with 100 and 200 mg/kg AsA. At 4 weeks of dietary Cr exposure, depending on the AsA supplementation, the profile of tissue Cr accumulation was in the following order: kidney > liver > spleen > intestine > gill > muscle.
ments were performed using an ELAN 6600DRC ICP-MS instrument with argon gas (Perkin-Elmer). Total Cr concentrations were determined by external calibration. ICP multi-element standard solution VI (Merck) was used for the standard curve. The Cr bioaccumulation in tissue samples was expressed as µg/g dry wt. 2.4. Blood samples and hematological assay Blood samples were collected within 35–40 s through the caudal vein of the fish in 1-ml disposable heparinized syringes. The blood samples were kept at 4 °C until the blood parameters were completely studied. The total red blood cell (RBC), hemoglobin (Hb), concentration and hematocrit (Ht) value were determined immediately. Total RBC counts were counted using an optical microscope with hemo-cytometer (Improved Neubauer, Germany) after diluted by Hendrick's dilution solution. The Hb concentration was determined using the Cyanmethemoglobin technique (Asan Pharm. co., Ltd). The Ht value was determined by the microhematocrit centrifugation technique. The blood samples were centrifuged to separate plasma from blood samples at 3000g for 5 min at 4 °C. The serum samples were analyzed for inorganic substances, organic substances, and enzyme activity using a clinical kit (Asan Pharm. Co., Ltd). Calcium and magnesium were analyzed by the o-cresolphthalein-complexone technique and xylidyl blue technique. Glucose and total protein were analyzed by the GOD/ POD technique and biuret technique. Glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were analyzed by the Kind-king technique and alkaline phosphatase (ALP) was analyzed using clinic al kit (Asan Pharm. Co., Ltd). 2.5. Statistical analysis The experiment was conducted in exposure periods for 4 weeks and performed in triplicate. Statistical analyses were performed using the SPSS/PC+ statistical package (SPSS Inc, Chicago, IL, USA). Significant differences between groups were identified using one-way ANOVA and Duncan's test for multiple comparisons or Student's t-test for two groups (Duncan, 1955). The significance level was set at P < 0.05. 3. Results 3.1. Chromium accumulation
3.2. Hematological factors Cr accumulation in the kidney, liver, spleen, intestine, gill, and muscle of S. schlegelii exposed to various dietary Cr concentrations depended on AsA supplementation (Fig. 1). We observed the highest Cr accumulation in the kidney. This significant accumulation in the kidney was observed in fish given dietary Cr greater than 120 mg/kg at 2 weeks and 4 weeks. Furthermore, for the 240 mg/kg Cr exposure at 2 and 4 weeks, Cr bioaccumulation in kidney was considerably lower in the fish receiving a diet supplemented with 400 mg/kg AsA than in the fish receiving diets supplemented with 100 and 200 mg/kg AsA. In liver tissue, Cr accumulation significantly increased at Cr exposure greater than 120 mg/kg, at 2 and 4 weeks. At 2 weeks, Cr bioaccumulation in the liver of fish receiving diets supplemented with
The RBC count, hematocrit value, and hemoglobin concentration of S. schlegelii exposed to dietary Cr all depended on AsA supplementation (Fig. 2). In S. schlegelii, a significant decrease in RBC count at 2 weeks was observed in 120 mg/kg Cr exposure and dietary supplementation with 100 and 200 mg/kg AsA, and in 240 mg/kg Cr and dietary supplementation with 400 mg/kg AsA. At 4 weeks, the RBC values were notably reduced by the dietary Cr exposure over 120 mg/kg. In 240 mg/kg Cr, the reduction in dietary supplementation with 400 mg/ kg AsA was significantly less than in dietary supplementation with 100 and 200 mg/kg AsA. The hematocrit of S. schlegelii was significantly decreased in 120 mg/kg Cr at 2 weeks. At 4 weeks, a substantial 111
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Fig. 1. Cr accumulation of rockfish, Sebastes schlegelii exposed to different concentrations of dietary chromium and ascorbic acid for 4 weeks. Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by Duncan's multiple range test.
cantly lower than those in 120 mg/kg Cr in fish receiving diets supplemented with 100 and 200 mg/kg AsA and 240 mg/kg Cr in fish receiving a diet supplemented with 100 mg/kg. The hematological factors of S. schlegelii were significantly reduced by the dietary Cr exposure, and the high concentrations of AsA supplementation were highly effective to attenuate Cr exposure. The alterations in plasma components of S. schlegelii by the dietary Cr exposure depended on the AsA supplementation (Fig. 3). In the inorganic components such as calcium and magnesium, calcium was reduced at 240 mg/kg Cr with 100 mg/kg AsA at 2 weeks, and 100 and
reduction was observed in 120 mg/kg Cr and dietary supplementation with 100 and 200 mg/kg AsA and in 240 mg/kg Cr and dietary supplementation with 400 mg/kg AsA. In the hemoglobin of S. schlegelii, the concentration at 2 weeks was notably decreased at Cr exposure greater than 120 mg/kg Cr in fish receiving a diet supplemented with 100 mg/kg AsA and in those exposed to a dietary concentration of 240 mg/kg Cr and receiving diets supplemented with 200 and 400 mg/ kg. At 4 weeks, the concentrations in 120 mg/kg Cr in fish receiving a diet supplemented with 400 mg/kg AsA and 240 mg/kg Cr in fish receiving diets supplemented with 200 and 400 mg/kg were signifi-
Fig. 2. Changes of RBC count, Hematocrit and Hemoglobin in rockfish, Sebastes schlegelii exposed to different concentrations of dietary chromium and ascorbic acid for 4 weeks. Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by Duncan's multiple range test.
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Fig. 3. Changes of plasma parameters in rockfish, Sebastes schlegelii exposed to different concentrations of dietary chromium and ascorbic acid for 4 weeks. Values with different superscript are significantly different in 2 and 4 weeks (P < 0.05) as determined by Duncan's multiple range test.
nase (GPT) of S. schlegelii were notably increased by the dietary Cr exposure, whereas no change was observed in the ALP. The GOT of S. schlegelii was notably increased with 120 mg/kg Cr at 2 weeks. At 4 weeks, a significant increase in the GOT was observed in 120 mg/kg supplemented with 100 and 200 mg/kg AsA and in 240 mg/kg supplemented with 400 mg/kg AsA. The GPT of S. schlegelii was substantially increased in 120 mg/kg Cr supplemented with 100 and 200 mg/kg AsA at 2 weeks. At 4 weeks, a notable increase was observed in 120 mg/kg Cr supplemented with 100 mg/kg AsA and in 240 mg/kg Cr supplemented with 200 and 400 mg/kg AsA. However, there was no alteration in the ALP of S. schlegelii due to dietary Cr when exposed with AsA supplementation. The plasma components of S. schlegelii were significantly altered by dietary Cr exposure, and the high concentrations of AsA supplementation were highly effective to attenuate alterations due
200 mg/kg AsA at 4 weeks. Magnesium was not changed by the dietary Cr exposure. Of the organic components, such as glucose, cholesterol, and total protein, the glucose and cholesterol concentrations were significantly increased by dietary Cr exposure, whereas total protein was notably decreased. Glucose was substantially increased at 240 mg/ kg Cr and dietary supplementation with 100 mg/kg AsA at 2 weeks and at 120 mg/kg Cr exposure and dietary supplementation with 100, 200, and 400 mg/kg AsA at 4 weeks. Cholesterol was significantly increased at concentration of 240 mg/kg Cr supplemented with 100 mg/kg AsA at 2 weeks and 120 mg/kg Cr supplemented with 100 mg/kg AsA. A significant decrease in total protein was observed in the 240 mg/kg Cr supplemented with 100 and 200 mg/kg AsA at 2 weeks and 100, 200, and 400 mg/kg AsA at 4 weeks. In the enzyme components, the glutamic oxalate transaminase (GOT) and glutamic pyruvate transami-
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liorate the reduction in the hematological factors associated with dietary Cr exposure. Vani et al. (2011) reported significant reductions in the toxicity of deltamethrin to the major carp, Catla catla by AsA. Hounkpatin et al. (2012) also reported that high concentrations of AsA supplementation were effective to attenuate effects on hematological factors caused by cadmium and mercury exposure. In plasma, calcium and magnesium are major constituents that function as the ion regulators for homeostasis, and can be affected by metal-induced toxicity (Rogers et al., 2005). The calcium level of S. schlegelii was decreased by dietary Cr exposure, whereas no significant change was observed in the magnesium of S. schlegelii. In plasma, the glucose and cholesterol level of S. schlegelii were raised through dietary Cr exposure, whereas total protein decreased. Plasma glucose is commonly elevated by gluconeogenesis and can be a good parameter to monitor environmental toxicity (Saravanan et al., 2011). Al-Akel and Shamsi (1996) reported that Cr(VI) exposure in carp, Cyprinus carpio L., resulted in significant alterations in carbohydrate metabolism and blood factors, which caused the breakdown of tissue glycogen necessary to meet the energy demand in the muscle for the anaerobic stress by impairing oxygen supply to tissue. Cholesterol is a major structural component of cell membranes and precursor of steroid hormones and an increase in cholesterol can be a reliable indicator to assess metalinduced toxicity (Oner et al., 2008). Plasma protein can be significantly changed under stress situations (Gopal et al., 1997). In the present study, GOT and GPT of S. schlegelii were notably increased by dietary Cr exposure, but there was no alteration in the ALP. These enzyme components have been considered as a critical indicator for evaluating liver damage caused by toxic substance exposure (Oost et al., 2003). Dietary Cr exposure affected significant alterations in the plasma components of S. schlegelii. The high concentrations of AsA supplementation were highly effective for alleviating the alterations in the plasma components due to dietary Cr exposure. Other authors have reported the protective effects of AsA on hematological factors of animals exposed to toxicants (Yousef, 2004; Hounkpatin et al., 2012). Vani et al. (2011) also reported the ameliorating effect of dietary AsA supplementation in the serum components of Catla catla exposed to deltamethrin. In conclusion, dietary Cr exposure in S. schlegelii results in a significant Cr accumulation in all tissues, although the accumulation concentrations in each tissue vary due to metabolic differences. High concentrations of AsA supplementation were highly effective for alleviating Cr accumulation associated with dietary Cr exposure. Hematological factors and plasma components were significantly affected by dietary Cr exposure, and high concentrations of AsA supplementation were highly effective for attenuating the alterations in the factors. High concentrations of AsA supplementation effectively attenuate Cr-induced bioaccumulation and other effects.
to Cr exposure. 4. Discussion Metal levels in aquatic ecosystems have increased by industrial and agricultural activities, and the metal exposure induces metal accumulation in fish tissues (Uysal et al., 2008). The accumulation and dispersion patterns for metal exposure depend on the exposure vectors (water, feed, and sediment), exposure concentration and period, and absorption ability of each tissue (Leonard et al., 2014). Among chromium, hexavalent chromium (Cr VI) can be much toxic to fish due to its ability to enter cells via anion transport than trivalent chromium (Cr III) (Kim and Kang, 2016b). In this study, the highest Cr accumulation was observed in the kidney of S. schlegelii by dietary Cr exposure. Farag et al. (2006) suggested that kidney is a major target tissue of Cr exposure. Kidney plays a role in excreting xenobiotics and toxicants, as well as having a detoxification function (Bodo et al., 2003), which causes a significant accumulation in kidney tissue. Similar to the kidney, a significant accumulation was observed in the liver of S. schlegelii exposed to dietary Cr. Webb and Wood (2000) also reported a notable accumulation in the liver of four marine teleosts and two marine elasmobranchs exposed to silver. A significant accumulation in the liver of fish and other vertebrates exposed to metals is induced by the metallothioneins in the liver, which binds to metals, allowing the liver to accumulate higher doses of metals than other tissues (Atli and Canli, 2003). In another study, a significant accumulation in the liver and kidney of S. schlegelii was observed through dietary Cr exposure (Kim and Kang, 2016a). Jezierska and Witeska (2006) reported that the kidney and liver in fish are the tissues with the greatest accumulation through metal exposure. Notable Cr accumulation was also observed in the spleen of S. schlegelii. Generally, the metabolically active tissues such as kidney, liver, and spleen have much greater accumulation than other tissues (Karaytug et al., 2007). Metal absorption in aquatic animals occurs via different pathways including through direct uptake from water by gills, and ingestion from food by the intestines (Oost et al., 2003); metals accumulation in fish is generally through diet (Hall et al., 1997). In this study, the higher accumulation of the intestine in S. schlegelii than that of the gill may be caused by dietary exposure. A significant accumulation in the intestine of S. schlegelii may be due to direct metal uptake by dietary exposure. The lowest accumulation was observed in the muscle of S. schlegelii exposed to dietary Cr. The relative accumulation of S. schlegelii exposed to dietary Cr was: kidney > liver > spleen > intestine > gill > muscle. In this study, the high concentrations of AsA supplementation were highly effective at alleviating Cr accumulation of all tissues in S. schlegelii with dietary Cr exposure. AsA supplementation to various animals helps reduce metal retention in tissues due to metal exposure (Kadrabova et al., 1992; Grosicki, 2004). In fish, Kumar et al. (2009) reported AsA supplementation was highly effective at reducing cadmium accumulation in the liver and kidney of catfish, Clarias batrachus, exposed to cadmium. The results of this study demonstrate that the high concentrations of AsA supplementation should be effective to attenuate accumulation in aquatic animals with Cr exposure. The hematological factors such as RBC count, hematocrit, and hemoglobin have been considered as sensitive and reliable indicators to evaluate fish physiological status under toxicant exposure. Authors reported anemia and significant decreases in hematocrits, hemoglobin, and erythrocyte counts in fish exposed to toxic substances (Benifey and Biron, 2000; Jee et al., 2004). Vutukuru (2003) also reported hematological alterations of the Indian major carp, Labeo rohita, exposed to Cr. In this study, dietary Cr exposure induced a substantial reduction in RBC count, hematocrit, and hemoglobin of S. schlegelii. The changes in the hematological factors may be due to osmotic alterations induced by metal toxicity causing hemodilution and red blood cell fragility, in addition to impaired hematopoietic tissues (Shah, 2006). The high concentrations of AsA supplementation were highly effective to ame-
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