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Effects of ethoxyquin on the blood composition of turbot, Scophthalmus maximus L.
Comparative Biochemistry and Physiology Part C 127 (2000) 1 – 9
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Effects of ethoxyquin on the blood composition of turbot, Scophthalmus maximus L. Tapati Bose Saxena a, Karl Erik Zachariassen a,*, Leif Jørgensen b a
Laboratory of Ecophysiology and Toxicology, Department of Zoology, Norwegian Uni6ersity of Science and Technology (NTNU), 7491 Trondheim, Norway b SINTEF Fisheries and Aquaculture, 7465 Trondheim, Norway Received 12 March 1999; received in revised form 11 February 2000; accepted 20 March 2000
Abstract Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (EQ) is a synthetic antioxidant used for preventing rancidity in animal foodstuffs. Three groups of ten fish were given a diet containing respectively 75 (control group with the commercial food), 200 and 400 ppm EQ for 16 days. The control group had a plasma osmolality and chloride concentration within the normal range of marine teleosts, but sodium concentrations of only about 110 mM, indicating the presence in the plasma of substantial amounts of another cation. Fish given food with 400 ppm EQ displayed a 70 mM increase in the plasma concentration of sodium. This indicates that EQ has disturbed the iono-regulatory mechanisms, probably by reducing the ATP production or inhibiting directly the Na/K-ATPase in the gills. The large increase in plasma sodium concentration was not accompanied by any significant increase in plasma osmolality, indicating that at least a part of the sodium added to the plasma is made osmotically inactive. In spite of the elevated plasma sodium concentration, the sodium content of erythrocytes of the 400-ppm EQ fish was reduced to half, while the content of calcium was unaffected. The transmembrane energy gradient of sodium in the EQ exposed turbot obviously increased, allowing them to use a sodium coupled antiport system to keep the cellular calcium content low when the Ca-ATPases is blocked. A mechanism of this kind is also likely to be important to turbot that experience hypoxia under natural conditions. The 400-ppm group also displayed a substantial increase in liver weight, but the physiological significance of this effect is not clear. The leucocyte counts indicated the absence of obvious immunological effects. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ethoxyquin; Turbot; Scophthalmus maximus; Osmoregulation; Plasma osmolality; Sodium; Chloride; Calcium; Haematocrit
1. Introduction Ethoxyquin (EQ) is a synthetic antioxidant used as a preservative for fish, poultry and dairy food products. It is used on large scale as an antioxidant for preventing oxidation of fishmeal, * Corresponding author. Tel.: + 47-73596299; fax: + 4773591309. E-mail address: [email protected] (K.E. Zachariassen).
and in some fishmeal products the content of EQ is as high as 400 ppm. EQ has been shown to have significant toxic effects on poultry, swine, rats and mice. The toxic effects seem in part to be due to its capacity to inhibit energy metabolism and ATP production. Hernandez et al. (1993) demonstrated that EQ induces an inhibition of those renal secretory mechanisms that depend on metabolic energy, and Reyes et al. (1995) demonstrated that EQ inhibits renal ATPases and electron transport in
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the mitochondrial respiratory chain. EQ is highly nephrotoxic (Hard and Neal, 1992; Manson et al., 1992), and scanning electron microscopy of the kidney of EQ-treated rats has shown calcification and stone formation (Emazawa et al., 1989). EQ causes an increase in liver weight and hepatic cell proliferation in broilers, cockerels and swine (Bailey et al., 1996; Dibner et al., 1996). It affects the gut associated immune system in broiler and swine (Dibner et al., 1996). In catfish microsomes, 100 ppm dietary EQ completely inhibited lipid peroxidation activity (Eun et al., 1993). EQ is accumulated in the tissue of mice (Kim, 1991), and Skaare and Nafstad (1979) reported that EQ is accumulated for several days in the renal cortex of rats given a single dose of the substance. Although EQ is commonly mixed with fish food products, very little work has been done to study its possible effects on the physiology of fish. The purpose of the present experiment was to assess the effects of EQ on blood parameters of turbot. The main parameters focused on were plasma osmolality and the concentrations of sodium and calcium in plasma and red blood cells. In marine teleost fish the plasma osmolality and sodium concentration are only one third of the corresponding values in the ambient seawater, and the gradients are maintained by the Na/K-ATPase in the chloride cells in the gills. If these mechanisms are disturbed, the plasma osmolality and sodium concentrations will increase, and several investigators have used these parameters to monitor the physiological status of fish under stress (Blackburn and Clarke, 1987; Finstad et al., 1988). Na/K-ATPase also maintains the intracellular concentration of sodium at a substantially lower level than in the extracellular fluid, thus providing animal cells with a high transmembrane electrochemical potential difference or energy gradient of sodium (Ganong, 1987). A substantial fraction (30 – 70%) of the ATP produced in the mitochondria is spent to maintain the sodium gradient (Florey, 1966; Ganong, 1987), which in turn, is the energy source of a number of vital cellular processes. The high sodium gradient depends on a number of factors, such as an adequate supply of oxygen, an intact metabolic system for the production of ATP, an intact Na/K-ATPase and a low membrane permeability of sodium (Ganong, 1987). Consequently, the sodium gradient is sensitive to any disturbance of the metabolism or other factors influencing the Na/K-ATPase activity
(Børseth et al., 1995). It is technically rather complicated to determine the sodium gradient precisely, but since the total tissue content of sodium appears to be well correlated with the sodium gradient (Aunaas and Zachariassen, 1994), the total tissue or intracellular content of sodium may be used as an index for the sodium gradient. Through a sodium coupled antiport system, the energy gradient of sodium is also important for cellular calcium regulation. The concentration of free intracellular calcium is kept below 10 − 7 M (Ganong, 1987), and there is substantial evidence indicating that an elevation of cellular free calcium is highly cytotoxic (Kretsinger, 1990; Orhenius et al., 1991). The low cellular calcium content is maintained also by a membrane bound calcium ATPase. Lymphocytes form the basis of the immuno defence system of animals, and antibody producing cells are assumed to derive from the lymphocytes (Fa¨nge, 1992). Several investigators have reported a reduction in the number of lymphocytes in fish exposed to toxic chemicals (Brown et al., 1998; Fo¨rlin et al., 1995; Schwaiger et al., 1996). Therefore also differential counts of white blood cells were included in this study. Also plasma chloride concentration and osmolality, haematocrit, and liver weight were measured to monitor the physiological status of the fish.
2. Materials and methods The experiments were conducted at Brattøra Research Center, Trondheim. Thirty turbot, Scophthalmus maximus L., with a length of 26.179 2.14 cm (Mean 9 S.D.) and a body mass of 352.269 79.28 g (Mean 9 S.D.) were used in the experiment. The fish were first acclimated over a period of 2 months in a 1 m3 fiber glass tank with 700 l recirculated and aerated 35‰ sea water at 8–10°C and under a light regime of 12 h light:12 h darkness. During this period they were fed three times a week with Nor Aqua protein mix pellets (diameter 10 mm with approximately 100 ppm EQ). The amount of food corresponded to 1% of their body mass at each feeding. One week before the exposure experiment, the fish were divided into three equal groups and placed in three tanks. Physical factors such as salinity, temperature, light conditions and gas ten-
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sions in the three tanks were the same as under the acclimation period and they were kept constant throughout the experiment. The experiments were carried out with special food pellets with different concentrations of ethoxyquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) (EQ). The pellets were made from fishmeal, which was obtained from Ryttervik Fabrikker ASA, Egersund, Norway. According to the Norwegian Herring Oil and Meal Industry Research Institute, the fishmeal contained 75 ppm EQ when it came from the factory. Pellets (diameter 5 mm) with three different concentrations of EQ were prepared. One was the original meal with 75 ppm EQ, a second had 200 ppm and the third had 400 ppm of EQ. The EQ was manufactured by Sigma Chemical Co, USA. The three groups of fish were fed three times a week with the respective types of food, 1% of their body mass each time over a period of 16 days. After the 16-day exposure, blood samples were collected. To reduce stress, the fish were dip netted one at a time. To avoid possible effects of anaesthetics, anaesthetics were not used when the blood samples were taken. The fish were kept on a dissecting tray with their head covered with a wet cloth. In this way they remained without any visible sign of stress for 5 – 10 min. Within a few seconds, blood was drawn from the caudal vessels into a heparinized syringe. Only traces of heparin solution were used. Care was taken to avoid air bubbles in the blood samples since this could lead to a change in the cell volume (Fugelli, 1967). The blood cells were separated from plasma by centrifugation for 20 min at 2000× g. Both the plasma and the red blood cell (RBC) fractions were immediately transferred to a deep freezer and kept frozen until analysis. For measuring haematocrit blood was transferred to heparinized capillary tubes and centrifuged in a Compur M 1100 microcentrifuge. After blood sampling, the fish were killed with a blow to the head and weighed and measured. The liver was then dissected out and weighed. Plasma osmolality was measured on 10 ml plasma samples on a Wescor 5500 Vapour Pressure Osmometer. Plasma chloride concentration was determined on 10-ml plasma samples with a Radiometer CMT 10 chloride titrator. Sodium and calcium were measured on a Perkin Elmer atomic absorption spectrophotome-
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ter (AAS). For analysis of calcium, the plasma samples were diluted by a factor of 50 with a 0.1% solution of lanthanum oxide (La2O3). For the analysis of sodium, the samples were diluted with deionized water by another factor of 50. The concentrations were measured in relation to standard solutions with known concentrations within the linear range of the AAS instrument. For determination of intracellular ions, thawed cell fractions were transferred to platinum crucibles and weighed to determine their wet mass (WM). They were then dried to constant weight at 80°C and their dry mass (DM) determined. The relative water content of the cell samples was calculated as (WM-DM)/WM × 100. The dried cells were then ashed at 550°C for about 10 h. The ashes were dissolved in one to two droplets of concentrated HNO3 solution and diluted with 0.1% lanthanum oxide solution to a total volume of 5 ml. Since the cellular water content may vary during the experiments, the intracellular solutes were calculated both as molal concentration and as solute content. The latter values were calculated in relation to cellular dry mass to correct for variations in cell sample size. The values were corrected for the contents of ions present in the extracellular fraction of the packed blood cell column, assuming that the extracellular fluid fraction was 3.3% of the total water content of the cell fraction (Fugelli and Zachariassen, 1976). To carry out differential counts of white blood cells (WBCs) a droplet of non-heparinized blood was drawn into a film on a glass slide. The film was air dried and fixed in absolute methanol for 5 min. The fixed blood films were then stained with May–Grunnwald Giemsa’s stain, and the WBC were counted as described by Burrows and Fletcher (1987), Quentel and Obach (1992). Mean 9S.E. are given throughout. Student’s two tailed t-test (paired design) was used to test for statistical significance of differences between the groups (Zar, 1984). A level of P= 0.05 was considered significant.
3. Results None of the fish died during the experiment. During the first 3 days the fish in all three groups were equally active in feeding, but from the fourth day the fish of the two high EQ groups showed reduced feeding activity. All three groups of fish
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consumed all their food, except at the last feeding, when fish in the 400-ppm group consumed food corresponding to only 0.6% of their body mass. Fig. 1a shows that the mean plasma sodium concentration of the control group was 106 mM. The value increased to about 150 mM in the 200-ppm group and to 180 mM in the 400-ppm group. The mean plasma chloride concentration of the control group was 136 mM (Fig. 1b), and in both the two high EQ groups there was a moderate concentration increase to 147 mM. The mean plasma osmolality values were 325 mOsm in the control group and about 335 mOsm in both the high EQ groups (Fig. 1c), but the differences were not statistically significant. Fig. 1d shows that in the 400-ppm group, there was a statistically significant increase in mean plasma calcium concentration compared to the control group.
Fig. 2a shows that both the concentration and content of sodium in the RBCs in the EQ exposed fish were reduced to about half the values of the low EQ control fish, whereas the intracellular concentrations and contents of calcium were not affected. Table 1 shows that there was a substantial and statistically significant increase in the relative liver mass of the fish exposed to 400 ppm EQ. Both the two high EQ fish groups and the control group had haematocrit values of about 15% (Table 1). The relative water content of the RBCs dropped in the 200-ppm EQ group compared with the control groups (PB 0.05, t= 2.2, n= 20), while the water content of the 400-ppm group was not significantly different from that in the control group (P B 0.10). There was no statistically significant effect of EQ on any of the leucocyte counts (Table 2).
Fig. 1. Plasma concentration of sodium (a) and chloride (b), plasma osmolality (c) and plasma concentration of calcium (d) in groups of turbot (S. maximus) given different doses of ethoxyquin in the food. * P B0.05, *** P B0.001. No asterisk — not significantly different from control value.
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Fig. 2. Intracellular contents and concentrations of sodium (a) and calcium (b) in three groups of turbot (S. maximus) given different doses of ethoxyquin in the food. ** PB 0.002. No asterisk — not significantly different from control value.
4. Discussion The turbot used in the present study were all from the same stock and had been reared under identical conditions. Factors such as temperature, salinity, light conditions and oxygen supply were the same in the three tanks throughout the experiments, and except for the concentration of EQ in the food there was no known factor that differed
among the groups. Hence, the changes seen in the physiological parameters of the fish given the two highest concentrations of EQ in the food are likely to be caused by the higher levels of EQ. The blood plasma concentration of chloride in the control fish agrees well with the plasma osmolality in that chloride made up a little less than half the osmotic activity in the plasma. Both values are also in agreement with the values ob-
Table 1 Liver mass, haematocrit and erythrocyte water content of turbot (S. maximus) exposed to different contents of dietary ethoxyquin
Percentage of liver mass to body mass Haematocrit (%) Water content of the RBC (%)
Ethoxyquin content 75 ppm
200 ppm
400 ppm
1.036 90.175 16.0 92.0 76.4 93.6
1.2 90.22* 14.5 92.4* 73.0 94.2*
1.225 9 0.2** 15.6 9 2.3* 80.5 9 4.5*
* P\0.05 and; ** P= 0.05, statistical significance of difference from control.
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served in other marine teleosts (Schmidt-Nielsen, 1997). The plasma concentrations of sodium in the control turbot were substantially lower than the about 140 mM that appears to be normal for marine teleost fish (Schmidt-Nielsen, 1997). The values were also lower than what should be expected from the observed osmolality values. We are not aware that similarly low of sodium/osmolality ratios have previously been reported from teleosts, and therefore the methods were controlled by corresponding measurements on trout plasma. These measurements gave values of 139.3910.6 mM sodium and 313.6 9 7.1 mOsm, i.e. values within the normal ranges of marine teleosts, and hence, the methods seem to be reliable. The low sodium concentrations suggest that another osmotically active cation is present in the plasma of the control fish at quite high concentrations, i.e. about 30 mmolal. Solutes like potassium, magnesium, ammonium and ninhydrin positive substances (NPS) were all present in the plasma at concentrations below 5 mM, and the identity of the cation is so far unknown. Feeding the turbot with high EQ food gave rise to a strong increase in plasma sodium concentration. The increase suggests that EQ has an inhibitory effect on the ionoregulatory mechanisms. The disturbance is likely to be due to effects on the chloride cells, either through an inhibition of the ATP producing metabolic processes or through a direct inhibition of the Na/K-ATPase. The plasma sodium concentration of the turbot given higher dietary EQ contents was considerably higher than the about 150 mM, which is normal in marine teleosts (Schmidt-Nielsen, 1997). The increase in the plasma concentration of sodium was far greater than the corresponding increases in plasma osmolality, and in the 400ppm EQ group the sodium concentration was Table 2 Differential counts (%) of leucocytes in turbot (S. maximus) exposed to different contents of ethoxyquin in the food Types of leucocytes
75 ppm
200 ppm
400 ppm
Lymphocytes Thrombocytes Monocytes Granulocytes (neutrophilic)
37.1 9 2.2 52.894.0 3.991.6 5.99 3.4
40.79 2.4 50.8 9 2.4 4.19 2.8 5.19 2.5
39.4 9 2.8 54.69 3.4 3.1 9 1.6 3.7 9 1.5
actually higher than expected from the osmolality values. The discrepancy between the changes in plasma sodium concentration and plasma osmolality may have several causes. One possible cause is that the unknown cation, which seems to be present in significant concentrations only in the control fish, was removed from the plasma as the plasma sodium level increased. The cation may have been removed by metabolization or transferred to other fluid compartments. However, as judged from the concentration of this cation in the control fish, the removal of this ion cannot compensate osmotically for the entire 70 mmolal increase in plasma sodium concentration. Another possibility is that the sodium flowing into the plasma is made osmotically inactive by binding to other plasma molecules, most likely to proteins or other macromolecules. A factor of this kind would also explain how plasma sodium in the exposed fish can occur at such high concentrations relatively to the osmolality. None of these two factors alone can explain all the observed effects on solute concentrations and osmolality, and hence the response to EQ intake is most likely to involve a combination of the two. Although the precise features of these factors are not known, some speculations may be made about their nature and adaptive significance. In nature, turbot are likely to experience episodes with substantial salt stress. Turbot live dug down in soft sediments, waiting for prey or hiding from predators. When threatened by a predator, they do not try to escape, but remain hidden on the bottom (Kalmijn, 1971). Sharks and rays are important predators on turbot, and since these fishes use electrosensitive organs to locate their prey, the turbot are likely to respond by bradycardia and arrest of water pumping over the gills to avoid electrodetection (Kalmijn, 1971, 1974). The bradycardia will reduce the oxygen supply to the tissues and inhibit metabolism. The subsequent reduction of the ATP production will in turn reduce the sodium extrusion over the gills, and a net influx of sodium from the seawater would follow. During prolonged bradycardia, the plasma concentration of sodium may increase substantially. Against this background it would be an advantage for turbot to possess mechanisms preventing plasma sodium activity from rising to intolerable levels. The low sodium concentrations in the blood plasma of the unstressed turbot,
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increases the capacity of the fish to receive a great sodium influx before toxically high levels are reached. Also a sodium binding plasma protein activated during salt stress would have such a function. In the 400-ppm EQ group, there is also a 30% increase in the plasma concentration of calcium. The 10 mM calcium in the seawater is substantially higher than the concentration in the blood plasma, and together with the electrical potential between the blood plasma and seawater, this gradient causes calcium to diffuse passively into the fish, and active extrusion of calcium is required to prevent an increase in plasma calcium concentration. The observed increase in plasma calcium concentration in the EQ exposed fish indicates that EQ also affected the active regulation of calcium, probably by reducing the metabolic production of ATP that provides energy for calcium extrusion. The fish also display a surprising capacity to defend their cellular concentrations of sodium and calcium during toxic stress. Although EQ is likely to have inhibited the metabolic production of ATP (Reyes et al., 1995) and thus the ATPases that transport sodium and calcium out of the cells (Ganong, 1987), the intracellular concentrations and contents of sodium and calcium in the 400ppm group did not increase. The 400-ppm group actually displayed an unexpected reduction in both concentration and content of intracellular sodium, implying that sodium must have been transferred from cellular to extracellular fluid compartments. This change in the transmembrane distribution of sodium indicates that even the transmembrane energy gradient of sodium has increased in the EQ exposed fish. The paradoxical increase in the sodium energy gradient raises the question of what is the energy source of the elevation of the sodium gradient. One possibility is that the energy input is related to the disappearance from the plasma of the unidentified cation present in the control fish. If the intracellular concentration of the unidentified cation in the control fish is low, the cation may have moved into cellular compartments in an antiport exchange with cellular sodium. Sodium is known to participate in exchange mechanisms of this kind with a number of organic and inorganic substances (Ganong, 1987). Since the sodium gradient is an important energy source for various cellular processes, it is important to the turbot to
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maintain it even when the fish are under various types of stress. Through another antiport exchange mechanisms the sodium gradient is important for the maintenance of low cellular levels of calcium. This antiport is probably particularly important when there is a shortage of ATP and the calcium extrusion through the calcium ATPase is out of function, and the capacity of turbot to elevate their sodium gradient in situations when the metabolic production of ATP is inhibited may be an adaptation to prevent toxic cell killing by calcium. An adaptation of this kind is likely to be important to turbot exposed to hypoxic stress in nature, but the same mechanism may offer protection also during toxically induced ATP shortage. The haematocrit values were rather low (about 16%), but they agree with the values of 16.7% reported by Quentel and Obach (1992) for turbot with a mean body mass of 250 g. There was no significant change in the haematocrit in any of the high EQ fed groups. This agrees well with the fact that the relative water content of the RBCs remained unaffected in the 400-ppm EQ group, but not so well with the reduced relative water content of the RBCs of the 200-ppm EQ group. The fact that the WBC counts did not reveal any change in the numbers of lymphocytes indicates that EQ did not affect the immuno defence system of the fish. The EQ induced increase in liver weight in the 400-ppm group is in agreement with the results of Bailey et al. (1996), who found that EQ induced an increase in the liver weight of cockerels. The increase may be due to an increased hepatic cell proliferation as observed by Dibner et al. (1996) or by an increased cellular water content. In conclusion, the present study indicates that turbot possess mechanisms that protect them when they are exposed to toxic agents like EQ, and these mechanisms are probably also important to turbot under natural conditions. However, under natural conditions hypoxic stress is only transient, and the protective mechanisms are likely to provide only temporary protection. Although the results suggest that turbot are able to handle the EQ-induced stress, exposure to high contents of EQ in the food over longer periods may have more serious effects. Due to their different life strategies, other fish species such as salmon are unlikely to possess the protective mechanisms of turbot, and may thus be more vulnerable than turbot to EQ in the food.
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Acknowledgements The authors are grateful to the Ryttervik fabrikker a.s., Egersund, Norway, for providing fishmeal for the experiment. Svein Ramstad of SINTEF Applied Chemistry is thanked for assistance in making the food pellets. Thanks are also due to Jørund S. Larsen for technical assistance. References Aunaas, T., Zachariassen, K.E., 1994. Physiological biomarkers and the Trondheim biomonitoring system. In: Kramer, K.J.M. (Ed.), Biomonitoring of Coastal Waters and Estuaries. CRC Press, Boca Raton, FL. Bailey, C.A., Srinivasan, L.J., McGeachin, R.B., 1996. The effect of ethoxyquin on tissue peroxidation and immune status of single comb white leghorn cockerels. Poultry Sci. 75, 1109–1112. Blackburn, J., Clarke, W.C., 1987. Revised procedure for the 24 hour seawater challenge test to measure seawater adaptability of juvenile salmonids, Can. Tech. Rep. Fish. Aquat. Sci. 1515. Børseth, J.F., Aunaas, T., Denstad, J.-P., Nordtug, T., Olsen, A.J., Schmid, R., Skjærvø, G., Zachariassen, K.E., 1995. Transmembrane sodium energy gradient and calcium content in the adductor muscle of Mytilus edulis L. in relation to the toxicity of oil and organic chemicals. Aquat. Toxicol. 31, 263– 276. Brown, S.B., Delorme, P.D., Evans, R.E., Lockhart, W.L., Muir, D.C.G., Ward, F.J., 1998. Biochemical and histological responses in rainbow trout (Oncorhynchus mykiss) exposed to 2,3,4,7,8-pentachlorodibenzofuran. Environ. Toxicol. Chem. 17, 915–921. Burrows, A.S., Fletcher, T.C., 1987. Blood leucocytes of the turbot, Scophthalmus maximus (L). Aquaculture 67, 214–215. Dibner, J.J., Atwell, C.A., Kitchell, M.L., Shermer, W.D., Ivey, F.J., 1996. Feeding of oxidized fats to broilers and swine: effects on enterocyte turnover, hepatocyte proliferation and the gut associated lymphoid tissue. Anim. Feed Sci. Technol. 62, 1– 13. Emazawa, T., Toyoda, K., Shinoda, K., Okamiya, H., Furukawa, F., Imaida, K., Takahashi, M., Hayashi, Y., 1989. Analysis of renal calcification and stone formation in rats treated with ethoxyquin. Bull. Natl. Inst. Hyg. Sci. (Tokyo) 107, 68–72. Eun, J.B., Hearnsberger, J.O., Kim, J.M., 1993. Antioxidants, activators and inhibitors affect the enzymatic lipid peroxidation system of catfish muscle microsomes. J. Food Sci. 58, 71–74.
Fa¨nge, R., 1992. Fish blood cells. In: Hoar, W.S., Randall, D.J., Farrell, A.P. (Eds.), Fish Physiology. Academic Press, New York. Finstad, B., Staurnes, M., Reite, O.B., 1988. Effect of low temperature on sea-water tolerance in rainbow trout, Salmo gairdneri. Aquaculture 72, 319 – 328. Florey, E., 1966. An Introduction to General and Comparative Physiology. W.B. Saunders, Philadelphia, PA. Fo¨rlin, L., Andersson, T., Balk, L., Larsson, A., 1995. Biochemical and physiological effects in fish exposed to bleached Kraft mill effluents. Ecotoxicol. Environ. Saf. 30, 164 – 170. Fugelli, K., 1967. Regulation of cell volume in flounders (Pleuronectes flesus) erythrocytes accompanying a decrease in plasma osmolality. Comp. Biochem. Physiol. 22, 253 – 260. Fugelli, K., Zachariassen, K.E., 1976. The distribution of taurine, gamma-aminobutyric acid and inorganic ions between plasma and erythrocytes in flounder (Platichthys flesus) at different plasma osmolalities. Comp. Biochem. Physiol. 55A, 173 – 177. Ganong, W.F., 1987. Review of Medical Physiology, thirteenth edn. Appleton & Lange, East Norwalk. Hard, G.C., Neal, G.E., 1992. Sequential study of the chronic nephrotoxicity induced by dietary administration of ethoxyquin in Fischer 344 rats. Fundam. Appl. Toxicol. 18, 278 – 287. Hernandez, M.E., Reyes, J.L., Gomez-Lojero, C., Sayavedra, M.S., Melendez, E., 1993. Inhibition of the renal uptake of p-aminohippurate and tetraethylammonium by the antioxidant ethoxyquin in the rat. Food Chem. Toxicol. 31, 363 – 367. Kalmijn, A.J., 1971. The electric sense of sharks and rays. J. Exp. Biol. 55, 371 – 383. Kalmijn, A.J., 1974. The detection of electric fields from inanimate and animate sources other than electric organs. In: Fessard, A. (Ed.), Electroreceptors and Other Specialized Receptors in Lower Vertebrates. Handbook of Sensory Physiology, vol. III/3. Springer, Berlin. Kim, H.L., 1991. Accumulation of ethoxyquin in the tissue. J. Toxicol. Environ. Health 33, 229 – 236. Kretsinger, R.H., 1990. Why cells must export calcium. In: Liss, A.R. (Ed.), Intracellular Calcium Regulation. Alan R. Liss, New York. Manson, M.M., Green, J.A., Wright, B.J., Carthew, P., 1992. Degree of ethoxyquin-induced nephrotoxicity in rat is dependent on age and sex. Arch. Toxicology 66, 51 – 56. Orhenius, S., McConkey, D.J., Bellomo, G., Nicotera, P., 1991. Role of Ca2 + in toxic cell killing. Trends Pharmacol. Sci. 10, 281 – 285. Quentel, C., Obach, A., 1992. The cellular composition of the blood and haematopoietic organs of turbot, Scophthalmus maximus L. J. Fish Biol. 41, 709 – 716.
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Reyes, J.L., Hernandez, M.E., Melendez, E., Gomez-Lojero, C., 1995. Inhibitory effect of the antioxidant ethoxyquin on electron transport in the mitochondrial respiratory chain. Biochem. Pharm. 49, 283–289. Schmidt-Nielsen, K., 1997. Animal Physiology. Cambridge University Press, Cambridge. Schwaiger, J., Fent, K., Strecher, H., Ferling, H., Negele, R.D., 1996. Effects of sublethal concentrations of
.
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triphenyltinacetate on rainbow trout (Oncorhynchus mykiss). Arch. Environ. Contam. Toxicol. 30, 327 – 334. Skaare, J.U., Nafstad, I., 1979. The distribution of 14C-ethoxyquin in rats. Acta Pharm. Toxicol. 44, 303 – 307. Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice Hall, Englewood Cliffs, NJ.
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Effects of ethoxyquin on the blood composition of turbot, Scophthalmus maximus L. Tapati Bose Saxena a, Karl Erik Zachariassen a,*, Leif Jørgensen b a
Laboratory of Ecophysiology and Toxicology, Department of Zoology, Norwegian Uni6ersity of Science and Technology (NTNU), 7491 Trondheim, Norway b SINTEF Fisheries and Aquaculture, 7465 Trondheim, Norway Received 12 March 1999; received in revised form 11 February 2000; accepted 20 March 2000
Abstract Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (EQ) is a synthetic antioxidant used for preventing rancidity in animal foodstuffs. Three groups of ten fish were given a diet containing respectively 75 (control group with the commercial food), 200 and 400 ppm EQ for 16 days. The control group had a plasma osmolality and chloride concentration within the normal range of marine teleosts, but sodium concentrations of only about 110 mM, indicating the presence in the plasma of substantial amounts of another cation. Fish given food with 400 ppm EQ displayed a 70 mM increase in the plasma concentration of sodium. This indicates that EQ has disturbed the iono-regulatory mechanisms, probably by reducing the ATP production or inhibiting directly the Na/K-ATPase in the gills. The large increase in plasma sodium concentration was not accompanied by any significant increase in plasma osmolality, indicating that at least a part of the sodium added to the plasma is made osmotically inactive. In spite of the elevated plasma sodium concentration, the sodium content of erythrocytes of the 400-ppm EQ fish was reduced to half, while the content of calcium was unaffected. The transmembrane energy gradient of sodium in the EQ exposed turbot obviously increased, allowing them to use a sodium coupled antiport system to keep the cellular calcium content low when the Ca-ATPases is blocked. A mechanism of this kind is also likely to be important to turbot that experience hypoxia under natural conditions. The 400-ppm group also displayed a substantial increase in liver weight, but the physiological significance of this effect is not clear. The leucocyte counts indicated the absence of obvious immunological effects. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ethoxyquin; Turbot; Scophthalmus maximus; Osmoregulation; Plasma osmolality; Sodium; Chloride; Calcium; Haematocrit
1. Introduction Ethoxyquin (EQ) is a synthetic antioxidant used as a preservative for fish, poultry and dairy food products. It is used on large scale as an antioxidant for preventing oxidation of fishmeal, * Corresponding author. Tel.: + 47-73596299; fax: + 4773591309. E-mail address: [email protected] (K.E. Zachariassen).
and in some fishmeal products the content of EQ is as high as 400 ppm. EQ has been shown to have significant toxic effects on poultry, swine, rats and mice. The toxic effects seem in part to be due to its capacity to inhibit energy metabolism and ATP production. Hernandez et al. (1993) demonstrated that EQ induces an inhibition of those renal secretory mechanisms that depend on metabolic energy, and Reyes et al. (1995) demonstrated that EQ inhibits renal ATPases and electron transport in
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the mitochondrial respiratory chain. EQ is highly nephrotoxic (Hard and Neal, 1992; Manson et al., 1992), and scanning electron microscopy of the kidney of EQ-treated rats has shown calcification and stone formation (Emazawa et al., 1989). EQ causes an increase in liver weight and hepatic cell proliferation in broilers, cockerels and swine (Bailey et al., 1996; Dibner et al., 1996). It affects the gut associated immune system in broiler and swine (Dibner et al., 1996). In catfish microsomes, 100 ppm dietary EQ completely inhibited lipid peroxidation activity (Eun et al., 1993). EQ is accumulated in the tissue of mice (Kim, 1991), and Skaare and Nafstad (1979) reported that EQ is accumulated for several days in the renal cortex of rats given a single dose of the substance. Although EQ is commonly mixed with fish food products, very little work has been done to study its possible effects on the physiology of fish. The purpose of the present experiment was to assess the effects of EQ on blood parameters of turbot. The main parameters focused on were plasma osmolality and the concentrations of sodium and calcium in plasma and red blood cells. In marine teleost fish the plasma osmolality and sodium concentration are only one third of the corresponding values in the ambient seawater, and the gradients are maintained by the Na/K-ATPase in the chloride cells in the gills. If these mechanisms are disturbed, the plasma osmolality and sodium concentrations will increase, and several investigators have used these parameters to monitor the physiological status of fish under stress (Blackburn and Clarke, 1987; Finstad et al., 1988). Na/K-ATPase also maintains the intracellular concentration of sodium at a substantially lower level than in the extracellular fluid, thus providing animal cells with a high transmembrane electrochemical potential difference or energy gradient of sodium (Ganong, 1987). A substantial fraction (30 – 70%) of the ATP produced in the mitochondria is spent to maintain the sodium gradient (Florey, 1966; Ganong, 1987), which in turn, is the energy source of a number of vital cellular processes. The high sodium gradient depends on a number of factors, such as an adequate supply of oxygen, an intact metabolic system for the production of ATP, an intact Na/K-ATPase and a low membrane permeability of sodium (Ganong, 1987). Consequently, the sodium gradient is sensitive to any disturbance of the metabolism or other factors influencing the Na/K-ATPase activity
(Børseth et al., 1995). It is technically rather complicated to determine the sodium gradient precisely, but since the total tissue content of sodium appears to be well correlated with the sodium gradient (Aunaas and Zachariassen, 1994), the total tissue or intracellular content of sodium may be used as an index for the sodium gradient. Through a sodium coupled antiport system, the energy gradient of sodium is also important for cellular calcium regulation. The concentration of free intracellular calcium is kept below 10 − 7 M (Ganong, 1987), and there is substantial evidence indicating that an elevation of cellular free calcium is highly cytotoxic (Kretsinger, 1990; Orhenius et al., 1991). The low cellular calcium content is maintained also by a membrane bound calcium ATPase. Lymphocytes form the basis of the immuno defence system of animals, and antibody producing cells are assumed to derive from the lymphocytes (Fa¨nge, 1992). Several investigators have reported a reduction in the number of lymphocytes in fish exposed to toxic chemicals (Brown et al., 1998; Fo¨rlin et al., 1995; Schwaiger et al., 1996). Therefore also differential counts of white blood cells were included in this study. Also plasma chloride concentration and osmolality, haematocrit, and liver weight were measured to monitor the physiological status of the fish.
2. Materials and methods The experiments were conducted at Brattøra Research Center, Trondheim. Thirty turbot, Scophthalmus maximus L., with a length of 26.179 2.14 cm (Mean 9 S.D.) and a body mass of 352.269 79.28 g (Mean 9 S.D.) were used in the experiment. The fish were first acclimated over a period of 2 months in a 1 m3 fiber glass tank with 700 l recirculated and aerated 35‰ sea water at 8–10°C and under a light regime of 12 h light:12 h darkness. During this period they were fed three times a week with Nor Aqua protein mix pellets (diameter 10 mm with approximately 100 ppm EQ). The amount of food corresponded to 1% of their body mass at each feeding. One week before the exposure experiment, the fish were divided into three equal groups and placed in three tanks. Physical factors such as salinity, temperature, light conditions and gas ten-
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sions in the three tanks were the same as under the acclimation period and they were kept constant throughout the experiment. The experiments were carried out with special food pellets with different concentrations of ethoxyquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) (EQ). The pellets were made from fishmeal, which was obtained from Ryttervik Fabrikker ASA, Egersund, Norway. According to the Norwegian Herring Oil and Meal Industry Research Institute, the fishmeal contained 75 ppm EQ when it came from the factory. Pellets (diameter 5 mm) with three different concentrations of EQ were prepared. One was the original meal with 75 ppm EQ, a second had 200 ppm and the third had 400 ppm of EQ. The EQ was manufactured by Sigma Chemical Co, USA. The three groups of fish were fed three times a week with the respective types of food, 1% of their body mass each time over a period of 16 days. After the 16-day exposure, blood samples were collected. To reduce stress, the fish were dip netted one at a time. To avoid possible effects of anaesthetics, anaesthetics were not used when the blood samples were taken. The fish were kept on a dissecting tray with their head covered with a wet cloth. In this way they remained without any visible sign of stress for 5 – 10 min. Within a few seconds, blood was drawn from the caudal vessels into a heparinized syringe. Only traces of heparin solution were used. Care was taken to avoid air bubbles in the blood samples since this could lead to a change in the cell volume (Fugelli, 1967). The blood cells were separated from plasma by centrifugation for 20 min at 2000× g. Both the plasma and the red blood cell (RBC) fractions were immediately transferred to a deep freezer and kept frozen until analysis. For measuring haematocrit blood was transferred to heparinized capillary tubes and centrifuged in a Compur M 1100 microcentrifuge. After blood sampling, the fish were killed with a blow to the head and weighed and measured. The liver was then dissected out and weighed. Plasma osmolality was measured on 10 ml plasma samples on a Wescor 5500 Vapour Pressure Osmometer. Plasma chloride concentration was determined on 10-ml plasma samples with a Radiometer CMT 10 chloride titrator. Sodium and calcium were measured on a Perkin Elmer atomic absorption spectrophotome-
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ter (AAS). For analysis of calcium, the plasma samples were diluted by a factor of 50 with a 0.1% solution of lanthanum oxide (La2O3). For the analysis of sodium, the samples were diluted with deionized water by another factor of 50. The concentrations were measured in relation to standard solutions with known concentrations within the linear range of the AAS instrument. For determination of intracellular ions, thawed cell fractions were transferred to platinum crucibles and weighed to determine their wet mass (WM). They were then dried to constant weight at 80°C and their dry mass (DM) determined. The relative water content of the cell samples was calculated as (WM-DM)/WM × 100. The dried cells were then ashed at 550°C for about 10 h. The ashes were dissolved in one to two droplets of concentrated HNO3 solution and diluted with 0.1% lanthanum oxide solution to a total volume of 5 ml. Since the cellular water content may vary during the experiments, the intracellular solutes were calculated both as molal concentration and as solute content. The latter values were calculated in relation to cellular dry mass to correct for variations in cell sample size. The values were corrected for the contents of ions present in the extracellular fraction of the packed blood cell column, assuming that the extracellular fluid fraction was 3.3% of the total water content of the cell fraction (Fugelli and Zachariassen, 1976). To carry out differential counts of white blood cells (WBCs) a droplet of non-heparinized blood was drawn into a film on a glass slide. The film was air dried and fixed in absolute methanol for 5 min. The fixed blood films were then stained with May–Grunnwald Giemsa’s stain, and the WBC were counted as described by Burrows and Fletcher (1987), Quentel and Obach (1992). Mean 9S.E. are given throughout. Student’s two tailed t-test (paired design) was used to test for statistical significance of differences between the groups (Zar, 1984). A level of P= 0.05 was considered significant.
3. Results None of the fish died during the experiment. During the first 3 days the fish in all three groups were equally active in feeding, but from the fourth day the fish of the two high EQ groups showed reduced feeding activity. All three groups of fish
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consumed all their food, except at the last feeding, when fish in the 400-ppm group consumed food corresponding to only 0.6% of their body mass. Fig. 1a shows that the mean plasma sodium concentration of the control group was 106 mM. The value increased to about 150 mM in the 200-ppm group and to 180 mM in the 400-ppm group. The mean plasma chloride concentration of the control group was 136 mM (Fig. 1b), and in both the two high EQ groups there was a moderate concentration increase to 147 mM. The mean plasma osmolality values were 325 mOsm in the control group and about 335 mOsm in both the high EQ groups (Fig. 1c), but the differences were not statistically significant. Fig. 1d shows that in the 400-ppm group, there was a statistically significant increase in mean plasma calcium concentration compared to the control group.
Fig. 2a shows that both the concentration and content of sodium in the RBCs in the EQ exposed fish were reduced to about half the values of the low EQ control fish, whereas the intracellular concentrations and contents of calcium were not affected. Table 1 shows that there was a substantial and statistically significant increase in the relative liver mass of the fish exposed to 400 ppm EQ. Both the two high EQ fish groups and the control group had haematocrit values of about 15% (Table 1). The relative water content of the RBCs dropped in the 200-ppm EQ group compared with the control groups (PB 0.05, t= 2.2, n= 20), while the water content of the 400-ppm group was not significantly different from that in the control group (P B 0.10). There was no statistically significant effect of EQ on any of the leucocyte counts (Table 2).
Fig. 1. Plasma concentration of sodium (a) and chloride (b), plasma osmolality (c) and plasma concentration of calcium (d) in groups of turbot (S. maximus) given different doses of ethoxyquin in the food. * P B0.05, *** P B0.001. No asterisk — not significantly different from control value.
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Fig. 2. Intracellular contents and concentrations of sodium (a) and calcium (b) in three groups of turbot (S. maximus) given different doses of ethoxyquin in the food. ** PB 0.002. No asterisk — not significantly different from control value.
4. Discussion The turbot used in the present study were all from the same stock and had been reared under identical conditions. Factors such as temperature, salinity, light conditions and oxygen supply were the same in the three tanks throughout the experiments, and except for the concentration of EQ in the food there was no known factor that differed
among the groups. Hence, the changes seen in the physiological parameters of the fish given the two highest concentrations of EQ in the food are likely to be caused by the higher levels of EQ. The blood plasma concentration of chloride in the control fish agrees well with the plasma osmolality in that chloride made up a little less than half the osmotic activity in the plasma. Both values are also in agreement with the values ob-
Table 1 Liver mass, haematocrit and erythrocyte water content of turbot (S. maximus) exposed to different contents of dietary ethoxyquin
Percentage of liver mass to body mass Haematocrit (%) Water content of the RBC (%)
Ethoxyquin content 75 ppm
200 ppm
400 ppm
1.036 90.175 16.0 92.0 76.4 93.6
1.2 90.22* 14.5 92.4* 73.0 94.2*
1.225 9 0.2** 15.6 9 2.3* 80.5 9 4.5*
* P\0.05 and; ** P= 0.05, statistical significance of difference from control.
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served in other marine teleosts (Schmidt-Nielsen, 1997). The plasma concentrations of sodium in the control turbot were substantially lower than the about 140 mM that appears to be normal for marine teleost fish (Schmidt-Nielsen, 1997). The values were also lower than what should be expected from the observed osmolality values. We are not aware that similarly low of sodium/osmolality ratios have previously been reported from teleosts, and therefore the methods were controlled by corresponding measurements on trout plasma. These measurements gave values of 139.3910.6 mM sodium and 313.6 9 7.1 mOsm, i.e. values within the normal ranges of marine teleosts, and hence, the methods seem to be reliable. The low sodium concentrations suggest that another osmotically active cation is present in the plasma of the control fish at quite high concentrations, i.e. about 30 mmolal. Solutes like potassium, magnesium, ammonium and ninhydrin positive substances (NPS) were all present in the plasma at concentrations below 5 mM, and the identity of the cation is so far unknown. Feeding the turbot with high EQ food gave rise to a strong increase in plasma sodium concentration. The increase suggests that EQ has an inhibitory effect on the ionoregulatory mechanisms. The disturbance is likely to be due to effects on the chloride cells, either through an inhibition of the ATP producing metabolic processes or through a direct inhibition of the Na/K-ATPase. The plasma sodium concentration of the turbot given higher dietary EQ contents was considerably higher than the about 150 mM, which is normal in marine teleosts (Schmidt-Nielsen, 1997). The increase in the plasma concentration of sodium was far greater than the corresponding increases in plasma osmolality, and in the 400ppm EQ group the sodium concentration was Table 2 Differential counts (%) of leucocytes in turbot (S. maximus) exposed to different contents of ethoxyquin in the food Types of leucocytes
75 ppm
200 ppm
400 ppm
Lymphocytes Thrombocytes Monocytes Granulocytes (neutrophilic)
37.1 9 2.2 52.894.0 3.991.6 5.99 3.4
40.79 2.4 50.8 9 2.4 4.19 2.8 5.19 2.5
39.4 9 2.8 54.69 3.4 3.1 9 1.6 3.7 9 1.5
actually higher than expected from the osmolality values. The discrepancy between the changes in plasma sodium concentration and plasma osmolality may have several causes. One possible cause is that the unknown cation, which seems to be present in significant concentrations only in the control fish, was removed from the plasma as the plasma sodium level increased. The cation may have been removed by metabolization or transferred to other fluid compartments. However, as judged from the concentration of this cation in the control fish, the removal of this ion cannot compensate osmotically for the entire 70 mmolal increase in plasma sodium concentration. Another possibility is that the sodium flowing into the plasma is made osmotically inactive by binding to other plasma molecules, most likely to proteins or other macromolecules. A factor of this kind would also explain how plasma sodium in the exposed fish can occur at such high concentrations relatively to the osmolality. None of these two factors alone can explain all the observed effects on solute concentrations and osmolality, and hence the response to EQ intake is most likely to involve a combination of the two. Although the precise features of these factors are not known, some speculations may be made about their nature and adaptive significance. In nature, turbot are likely to experience episodes with substantial salt stress. Turbot live dug down in soft sediments, waiting for prey or hiding from predators. When threatened by a predator, they do not try to escape, but remain hidden on the bottom (Kalmijn, 1971). Sharks and rays are important predators on turbot, and since these fishes use electrosensitive organs to locate their prey, the turbot are likely to respond by bradycardia and arrest of water pumping over the gills to avoid electrodetection (Kalmijn, 1971, 1974). The bradycardia will reduce the oxygen supply to the tissues and inhibit metabolism. The subsequent reduction of the ATP production will in turn reduce the sodium extrusion over the gills, and a net influx of sodium from the seawater would follow. During prolonged bradycardia, the plasma concentration of sodium may increase substantially. Against this background it would be an advantage for turbot to possess mechanisms preventing plasma sodium activity from rising to intolerable levels. The low sodium concentrations in the blood plasma of the unstressed turbot,
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increases the capacity of the fish to receive a great sodium influx before toxically high levels are reached. Also a sodium binding plasma protein activated during salt stress would have such a function. In the 400-ppm EQ group, there is also a 30% increase in the plasma concentration of calcium. The 10 mM calcium in the seawater is substantially higher than the concentration in the blood plasma, and together with the electrical potential between the blood plasma and seawater, this gradient causes calcium to diffuse passively into the fish, and active extrusion of calcium is required to prevent an increase in plasma calcium concentration. The observed increase in plasma calcium concentration in the EQ exposed fish indicates that EQ also affected the active regulation of calcium, probably by reducing the metabolic production of ATP that provides energy for calcium extrusion. The fish also display a surprising capacity to defend their cellular concentrations of sodium and calcium during toxic stress. Although EQ is likely to have inhibited the metabolic production of ATP (Reyes et al., 1995) and thus the ATPases that transport sodium and calcium out of the cells (Ganong, 1987), the intracellular concentrations and contents of sodium and calcium in the 400ppm group did not increase. The 400-ppm group actually displayed an unexpected reduction in both concentration and content of intracellular sodium, implying that sodium must have been transferred from cellular to extracellular fluid compartments. This change in the transmembrane distribution of sodium indicates that even the transmembrane energy gradient of sodium has increased in the EQ exposed fish. The paradoxical increase in the sodium energy gradient raises the question of what is the energy source of the elevation of the sodium gradient. One possibility is that the energy input is related to the disappearance from the plasma of the unidentified cation present in the control fish. If the intracellular concentration of the unidentified cation in the control fish is low, the cation may have moved into cellular compartments in an antiport exchange with cellular sodium. Sodium is known to participate in exchange mechanisms of this kind with a number of organic and inorganic substances (Ganong, 1987). Since the sodium gradient is an important energy source for various cellular processes, it is important to the turbot to
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maintain it even when the fish are under various types of stress. Through another antiport exchange mechanisms the sodium gradient is important for the maintenance of low cellular levels of calcium. This antiport is probably particularly important when there is a shortage of ATP and the calcium extrusion through the calcium ATPase is out of function, and the capacity of turbot to elevate their sodium gradient in situations when the metabolic production of ATP is inhibited may be an adaptation to prevent toxic cell killing by calcium. An adaptation of this kind is likely to be important to turbot exposed to hypoxic stress in nature, but the same mechanism may offer protection also during toxically induced ATP shortage. The haematocrit values were rather low (about 16%), but they agree with the values of 16.7% reported by Quentel and Obach (1992) for turbot with a mean body mass of 250 g. There was no significant change in the haematocrit in any of the high EQ fed groups. This agrees well with the fact that the relative water content of the RBCs remained unaffected in the 400-ppm EQ group, but not so well with the reduced relative water content of the RBCs of the 200-ppm EQ group. The fact that the WBC counts did not reveal any change in the numbers of lymphocytes indicates that EQ did not affect the immuno defence system of the fish. The EQ induced increase in liver weight in the 400-ppm group is in agreement with the results of Bailey et al. (1996), who found that EQ induced an increase in the liver weight of cockerels. The increase may be due to an increased hepatic cell proliferation as observed by Dibner et al. (1996) or by an increased cellular water content. In conclusion, the present study indicates that turbot possess mechanisms that protect them when they are exposed to toxic agents like EQ, and these mechanisms are probably also important to turbot under natural conditions. However, under natural conditions hypoxic stress is only transient, and the protective mechanisms are likely to provide only temporary protection. Although the results suggest that turbot are able to handle the EQ-induced stress, exposure to high contents of EQ in the food over longer periods may have more serious effects. Due to their different life strategies, other fish species such as salmon are unlikely to possess the protective mechanisms of turbot, and may thus be more vulnerable than turbot to EQ in the food.
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Acknowledgements The authors are grateful to the Ryttervik fabrikker a.s., Egersund, Norway, for providing fishmeal for the experiment. Svein Ramstad of SINTEF Applied Chemistry is thanked for assistance in making the food pellets. Thanks are also due to Jørund S. Larsen for technical assistance. References Aunaas, T., Zachariassen, K.E., 1994. Physiological biomarkers and the Trondheim biomonitoring system. In: Kramer, K.J.M. (Ed.), Biomonitoring of Coastal Waters and Estuaries. CRC Press, Boca Raton, FL. Bailey, C.A., Srinivasan, L.J., McGeachin, R.B., 1996. The effect of ethoxyquin on tissue peroxidation and immune status of single comb white leghorn cockerels. Poultry Sci. 75, 1109–1112. Blackburn, J., Clarke, W.C., 1987. Revised procedure for the 24 hour seawater challenge test to measure seawater adaptability of juvenile salmonids, Can. Tech. Rep. Fish. Aquat. Sci. 1515. Børseth, J.F., Aunaas, T., Denstad, J.-P., Nordtug, T., Olsen, A.J., Schmid, R., Skjærvø, G., Zachariassen, K.E., 1995. Transmembrane sodium energy gradient and calcium content in the adductor muscle of Mytilus edulis L. in relation to the toxicity of oil and organic chemicals. Aquat. Toxicol. 31, 263– 276. Brown, S.B., Delorme, P.D., Evans, R.E., Lockhart, W.L., Muir, D.C.G., Ward, F.J., 1998. Biochemical and histological responses in rainbow trout (Oncorhynchus mykiss) exposed to 2,3,4,7,8-pentachlorodibenzofuran. Environ. Toxicol. Chem. 17, 915–921. Burrows, A.S., Fletcher, T.C., 1987. Blood leucocytes of the turbot, Scophthalmus maximus (L). Aquaculture 67, 214–215. Dibner, J.J., Atwell, C.A., Kitchell, M.L., Shermer, W.D., Ivey, F.J., 1996. Feeding of oxidized fats to broilers and swine: effects on enterocyte turnover, hepatocyte proliferation and the gut associated lymphoid tissue. Anim. Feed Sci. Technol. 62, 1– 13. Emazawa, T., Toyoda, K., Shinoda, K., Okamiya, H., Furukawa, F., Imaida, K., Takahashi, M., Hayashi, Y., 1989. Analysis of renal calcification and stone formation in rats treated with ethoxyquin. Bull. Natl. Inst. Hyg. Sci. (Tokyo) 107, 68–72. Eun, J.B., Hearnsberger, J.O., Kim, J.M., 1993. Antioxidants, activators and inhibitors affect the enzymatic lipid peroxidation system of catfish muscle microsomes. J. Food Sci. 58, 71–74.
Fa¨nge, R., 1992. Fish blood cells. In: Hoar, W.S., Randall, D.J., Farrell, A.P. (Eds.), Fish Physiology. Academic Press, New York. Finstad, B., Staurnes, M., Reite, O.B., 1988. Effect of low temperature on sea-water tolerance in rainbow trout, Salmo gairdneri. Aquaculture 72, 319 – 328. Florey, E., 1966. An Introduction to General and Comparative Physiology. W.B. Saunders, Philadelphia, PA. Fo¨rlin, L., Andersson, T., Balk, L., Larsson, A., 1995. Biochemical and physiological effects in fish exposed to bleached Kraft mill effluents. Ecotoxicol. Environ. Saf. 30, 164 – 170. Fugelli, K., 1967. Regulation of cell volume in flounders (Pleuronectes flesus) erythrocytes accompanying a decrease in plasma osmolality. Comp. Biochem. Physiol. 22, 253 – 260. Fugelli, K., Zachariassen, K.E., 1976. The distribution of taurine, gamma-aminobutyric acid and inorganic ions between plasma and erythrocytes in flounder (Platichthys flesus) at different plasma osmolalities. Comp. Biochem. Physiol. 55A, 173 – 177. Ganong, W.F., 1987. Review of Medical Physiology, thirteenth edn. Appleton & Lange, East Norwalk. Hard, G.C., Neal, G.E., 1992. Sequential study of the chronic nephrotoxicity induced by dietary administration of ethoxyquin in Fischer 344 rats. Fundam. Appl. Toxicol. 18, 278 – 287. Hernandez, M.E., Reyes, J.L., Gomez-Lojero, C., Sayavedra, M.S., Melendez, E., 1993. Inhibition of the renal uptake of p-aminohippurate and tetraethylammonium by the antioxidant ethoxyquin in the rat. Food Chem. Toxicol. 31, 363 – 367. Kalmijn, A.J., 1971. The electric sense of sharks and rays. J. Exp. Biol. 55, 371 – 383. Kalmijn, A.J., 1974. The detection of electric fields from inanimate and animate sources other than electric organs. In: Fessard, A. (Ed.), Electroreceptors and Other Specialized Receptors in Lower Vertebrates. Handbook of Sensory Physiology, vol. III/3. Springer, Berlin. Kim, H.L., 1991. Accumulation of ethoxyquin in the tissue. J. Toxicol. Environ. Health 33, 229 – 236. Kretsinger, R.H., 1990. Why cells must export calcium. In: Liss, A.R. (Ed.), Intracellular Calcium Regulation. Alan R. Liss, New York. Manson, M.M., Green, J.A., Wright, B.J., Carthew, P., 1992. Degree of ethoxyquin-induced nephrotoxicity in rat is dependent on age and sex. Arch. Toxicology 66, 51 – 56. Orhenius, S., McConkey, D.J., Bellomo, G., Nicotera, P., 1991. Role of Ca2 + in toxic cell killing. Trends Pharmacol. Sci. 10, 281 – 285. Quentel, C., Obach, A., 1992. The cellular composition of the blood and haematopoietic organs of turbot, Scophthalmus maximus L. J. Fish Biol. 41, 709 – 716.
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Reyes, J.L., Hernandez, M.E., Melendez, E., Gomez-Lojero, C., 1995. Inhibitory effect of the antioxidant ethoxyquin on electron transport in the mitochondrial respiratory chain. Biochem. Pharm. 49, 283–289. Schmidt-Nielsen, K., 1997. Animal Physiology. Cambridge University Press, Cambridge. Schwaiger, J., Fent, K., Strecher, H., Ferling, H., Negele, R.D., 1996. Effects of sublethal concentrations of
.
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triphenyltinacetate on rainbow trout (Oncorhynchus mykiss). Arch. Environ. Contam. Toxicol. 30, 327 – 334. Skaare, J.U., Nafstad, I., 1979. The distribution of 14C-ethoxyquin in rats. Acta Pharm. Toxicol. 44, 303 – 307. Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice Hall, Englewood Cliffs, NJ.