Biotransformation and glutathione homeostasis in rainbow trout Exposed to chemical and physical stress

Marine Environmental Research Vol. 42, No. 1-4, pp. 323-321, 0141-1136(95)00077-l

1996 Copyright 0 1996 Elswier Science Ltd Printed in Great Britain. All rights reserved 0141-1136/96/515.00+0.00

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Biotransformation and Glutathione Homeostasis in Rainbow Trout Exposed to Chemical and Physical Stress Pirjo Lindstrijm-Seppti,” Sashwati Roy a Sirpa Huuskonen,’ Katri Tossavainen,ab Ossi Ritolaub & Eine Marina “Department of Physiology, University of Kuopio, P.O.B. 1627, FIN-7021 1 Kuopio, Finland bDepartment of Applied Zoology and Veterinary Medicine, University of Kuopio, P.O.B. 1627, FIN-7021 1 Kuopio, Finland

ABSTRACT Glutathione, together with biotransformation and antioxidative enzymes, plays an important role in protecting the cells against damage caused by free radicals, peroxides, oxidizing metabolites and xenobiotics introduced by environmental stress. Pollution of the environment causes many long-term physiological effects on fish. Furthermore, in mammals exercise increases oxidative stress (increase of reactive oxygen species) which overloads the defence mechanisms. Induction of the glutathione system could show whether physical stress also affects defence mechanisms offish. In the present series of studies we have investigated the biotransformation and glutathione homeostasis of rainbow trout exposed to chemical and physical stress. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne hexachlorobenzene (HCB; 2 pgllitre) together with duck-weed (Lemna minor) using dtrerent combinations of fish, plants and HCB. In another experiment, fish were held in different water flows (none, medium, high). Hepatic monooxygenase (EROD) and conjugation (GST) enzyme activities, as well as antioxidant systems (GPX, GR, tGSH) were measured. In the HCB-exposed-fish-plant group EROD activities in fish were suppressed compared with the uncontaminated group. It is probable that through digested plants the fish could have been exposed to possible plant-derived HCB metabolites or secondary metabolites. Fish of different physical activity showed slightly elevated EROD activities three weeks from the beginning of the experiment. After the following three-week period EROD activities had decreased to the starting level. Three weeks from the beginning GST activities were at their highest level. The elevation of mono-oxygenase activities may have caused increased production of reactive intermediates which were further metabolized through activated glutathione system. Elevated total glutathione contents in studied tissues showed the potency of these fish to resist oxidative stress. Copyright 0 1996 Elsevier Science Ltd 323

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Metabolism of redox cycling xenobiotics in aquatic organisms is very similar to that of mammals (Winston, 1991). Aquatic organisms contain the same major antioxidant enzymes as mammals, however, quantitative differences among various fish species exist. Fish are known to regulate hepatic glutathione (GSH) levels by in situ hepatic GSH biosynthesis, although no labile hepatic cytosolic GSH pool has been found (Gallagher ef al,, 19923). The responses of glutathione metabolism of fish are supposed to resemble the mammalian system. Hexachlorobenzene (HCB), a persistent environmental organochlorine compound, has contaminated the environment mainly through agricultural processes where it was extensively used as a fungicide. The release of HCB into the environment also occurs as a byproduct of various industrial and combustion processes. HCB is an environmentally persistent organochlorine compound that has great potential to bioaccumulate in the aquatic food chain. The possible ecotoxicological effects of such accumulation on the aquatic biota and human health implications, following consumption of HCB contaminated food of aquatic origin, are of concern. HCB has been reported as a ‘mixed-type’ inducer of cytochrome P-450 in mammals (Link0 er al., 1986), whereas in juvenile rainbow trout, HCB exposure did not induce hepatic microsomal cytochrome P-450 and ethoxyresorufin-0-deethylase (EROD) activity (Tyle et al., 1991). Uptake and metabolism of HCB have also been studied in plants (Roy et al., 1995). The present series of studies were aimed to investigate: (i) the fish-plant interactions and responses of biotransformation and antioxidant enzymes in rainbow trout (Oncorhynchus mykiss) following exposure to HCB in the presence of the aquatic plant Lemna minor; and (ii) the effect of physical activity on biotransformation and glutathione metabolism in rainbow trout. Juvenile rainbow trout (8-10 g fresh wt/litre) were transferred to glass aquaria (32 fish in each) containing 2 pg/litre HCB dissolved in dechlorinated and filtered tap water, and L. minor (N 0.50 g fresh wt/litre) or HCB alone without plants. Control sets of fish and plants were simultaneously maintained. All experiments were carried out at 10 f 1°C water temperature and 12 h-12 h light-dark cycle. Fish samples (n = 8) were collected from the pool of 32 fish after 1,2, 7 and 14 d of exposure. The fish consumed most of the L. minor by the end of the experiment (14 d). In another set of experiments hatcheryraised immature rainbow trout (total 138 fish) were held in tanks where the water flow was 0 (none), 0.64 (medium) or 1.48 (high) body length/s. The samples (n = 18-20) were taken 0, 3 and 6 weeks after a one-week adaptation period. Hepatic mono-oxygenase (7-ethoxyresorufin 0-deethylase, EROD), conjugation (glutathione S-tranferase, GST) and antioxidant (glutathione peroxidase, GPX; glutathione reductase, GR) enzyme activities, as well as the amount of glutathione (total glutathione, tGSH) were measured (Roy et al., 1995). Enzyme activities were measured from the liver and the amount of glutathione from the liver, heart, red and white muscle, as well as from the blood. Statistical significance of changes were tested with one-way analysis of variance using Duncan’s test (P-C 0.05). Exposure to plants in uncontaminated water did not cause any major changes in the hepatic microsomal mono-oxygenase, conjugation or antioxidant enzyme activities in fish (Fig. l(A-D)). However, hepatic EROD activities of 0. mykiss were significantly inhibited after 2 and 14 days of exposure to HCB in fish that consumed L. minor contaminated with HCB (Fig. l(A)). No significant changes in the activities of EROD and AHH were

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Fig. 1. Response of hepatic microsomal: glutathione S-transferase @mol/min/mg (D) glutathione reductase (mnol/min/mg exposed to hexachlorobenzene for 1, 2, Difference of HCB-exposed

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(A) ethoxyresorufin-0-deethylase (pmol/min/mg prot); (B) prot); (C) glutathione peroxidase (mnol/min/mg prot); and prot) activities in rainbow trout (Oncorhynchus mykiss) 7 and 14 d in the presence of Lemna minor (mean, n = 8). groups with their controls, P < 0.05 (Duncan).

observed in fish that were exposed to HCB in the absence of plants (not illustrated). The consumption of HCB-contaminated plants significantly increased the activities of the antioxidant enzymes GPX and GR in fish liver after 14 days of exposure (Fig. l(C and D)). No major metabolites (e.g. pentachlorophenol) of HCB were detected in plants by GC-MS analysis. Such observations suggest that interactions between fish and plants in a HCB-contaminated environment may affect hepatic biotransformation and antioxidant enzymes in fish. Significant inhibition of hepatic EROD activities in fish exposed to HCB in the presence of plants might have occurred as a result of: (i) overloading of HCB that went into the fish through the gastric route, e.g. consumption of HCB-loaded plants; (ii) presence of toxic metabolites (e.g. PCP) in plants as a result of biotransformation of HCB; and/or (iii) induction of secondary stress metabolites, such as phenols, quinones, flavonoids, etc. The possibility of altered bioavailability due to the loading of HCB in plant material could be one explanation. However, the presence of HCB metabolites (e.g. PCP) in plants was not observed. Studies have shown that the exposure of plants to stressors usually induces specific secondary metabolites (e.g. alkaloids, terpenoids and phenylpropanoids) (Rhodes, 1994). It is possible that inhibition of hepatic mono-oxygenase activities in fish might have resulted owing to HCB-induced accumulation of secondary stress metabolites in the exposed plants. The effects of plant stress or secondary metabolites on the cytochrome P-450 system in fish are not yet properly defined. However, the aromatic hydrocarbon-responsive [Ah] gene battery of mammals has been reported to respond to phytoalexin (a group of plant stress metabolites) induced cytotoxicity (Nebert et al., 1990).

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As a protective response, to encounter oxidative stress caused by oxygenated metabolites of plant stress metabolites, the enzymatic (GPX and GR) antioxidant defence status may have been boosted in fish exposed to HCB in the presence of plants. In the other set of experiments, EROD activities showed increased values three weeks from the start especially in groups that were in medium and high water flow (Fig. 2(A)). Three weeks later EROD activities had decreased to the same level as at the start of the experiment. This could reflect some induction of mono-oxygenase activities caused by physical stress which later, e.g. because of adaptation, decreases. Three weeks from the start, GST activities were at their highest level (Fig. 2(B)). The group with no water flow clearly showed the highest values. Six weeks from the start, GST activities were still elevated in all groups. There are reports indicating the depletion of glutathione levels after pesticide exposure (Ghosh et al., 1992). However, channel catfish, exposed to chlorothalonil, had increased gill GSH concentrations (Gallagher et al., 1992a). It has also been shown that the length of exposure affects the amount of GSH; three days of exposure of fish to No. 2 fuel oil decreased GSH levels, however, after 21 days of exposure GSH was elevated (Steadman et al., 1991). In the present experiment elevated glutathione contents in the liver, heart, red and white muscle and blood (Fig. 2(C-G)) show the potency of rainbow trout to cope with oxidative stress under physical stress. In summary, significant inhibition in liver mono-oxygenase enzyme activities and increase in the antioxidant enzymes were observed in fish which consumed L. minor contaminated with HCB. Such observations suggest that interactions between fish and plants

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Fig. 2. (A) Hepatic EROD (pmol/min/mg prot.) and (B) GST (nmol/min/mg prot.) activities and

the amount of total glutathione (pmol/g wet weight) in (C) liver, (D) heart, (E) red and (F) white muscle as well as in (G) the blood of rainbow trout at the beginning and after 3 and 6 weeks of exposure to low (none), medium and high water flow (n = 18-20). *Difference of experimental groups with O-point, P< 0.05 (Duncan).

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in a HCB-contaminated environment may affect fish hepatic biotransformation and antioxidant enzymes. On the other hand, concurrent elevation of mono-oxygenase and glutathione activities in physical stress may indicate the increased production of reactive intermediates which are further metabolized through activated glutathione system.

ACKNOWLEDGEMENTS Authors would like to thank Mr Arvo Tuvikene, M.Sc., MS Tiina Soininen, MS Riitta Venllainen and MS Katalin Urban for technical assistance during the experiments and analyses.

REFERENCES Gallagher, E. P., Canada, A. T. & DiGiulio, R. T. (1992). Aquat. Toxicol., 23, 155-168. Gallagher, E. P., Hasspieler, B. M. & DiGiulio, R. T. (1992). Biochem. Pharmacol., 43, 2209-2215. Ghosh, P., Ghosh, S., Bose, S. & Bhattacharya, S. (1992). Sci. Total Environ., l-2 Suppl. Linko, P., Yeowell, T. A., Gasiewicz, T. A. & Goldstein, J. A. (1986). .I. Biochem. Toxicol., 1, 95-107. Nebert, D. W., Petersen, D. D. & Fomace, A. J. (1990). Environ. Hlth Perspect., 88, 13-25. Rhodes, M. J. C. (1994). Plant Molec. Biol., 24, l-20. Roy, S., LindstrBm-SeppH, R., Huuskonen, S. & Hgnninen, 0. (1995). Chemosphere, 30, 1789-1798. Steadman, B. L., Farag, A. M. & Bergman, H. L. (1991). Environ. Toxicol. Chem., 10, 365-374. Tyle, H., Egsmose, M. & Harrit, H. (1991). Comp. Biochem. Physiol., lOOC, 161-164. Winston, G. W. (1991). Comp. Biochem. Physiol., lOOC, 173-176.