Fish Physiology and Biochemistry vol. 8 no. I pp 11-17 (1990) Kugler Publications, Amsterdam/Berkeley
Oxidation of glutathione during hydroperoxide metabolism in isolated hepatocytes of rainbow trout (Salmo gairdneri) J. Gordon Bell and Colin B. Cowey2 1 NERC Unit of Aquatic Biochemistry, University of Stirling, Stirling FK9 4LA, Scotland Keywords: glutathione, hydroperoxide, hepatocyte, rainbow trout, lipid peroxidation
Abstract Freshly isolated rainbow trout hepatocytes were exposed to tert-butyl hydroperoxide (BuOOH), a substrate for glutathione peroxidase. BuOOH at a concentration approximately equimolar (1 mM) with intracellular reduced glutathione (GSH) caused a reversible increase in intracellular glutathione disulphide (GSSG) but did not compromise cell viability or damage membrane lipids. BuOOH at 10 mM caused a large irreversible increase in intracellular GSSG followed by efflux into the medium. Considerable leakage of lactate dehydrogenase and loss of highly unsaturated fatty acids, particularly docosahexaenoic acid also occurred. Dependence of hydroperoxide removal on flux through the hexose monophosphate pathway was suggested by the increased release of 14CO 2 from [1-1 4C] glucose from hepatocytes incubated with BuOOH.
Introduction In biological systems hydroperoxides can arise not only via the metabolism of various xenobiotics but also as a result of normal oxygen metabolism (Halliwell and Gutteridge 1985). Vertebrate cells possess a multi-level defence mechanism which prevents pathogenesis arising from free radical initiated lipid peroxidation. The selenium containing enzyme glutathione peroxidase (GSHPx) (EC1.11.1.9) has a prominent role in this process (Simmons and Jamall 1988). GSHPx catalyses the reduction of H 20 2 and various organic hydroperoxides with the simultaneous oxidation of glutathione (GSH) to glutathione disulphide (GSSG) (Ursini and Bindoli 1987). GSHPx activity has been identified in salmonid fish tissues (Bell et al. 1985, 1986). GSSG is reduced to GSH by the NADPH dependent en2
zyme glutathione reductase (ECI.6.4.2) thus maintaining a high GSH:GSSG ratio. The damaging effects of lipid peroxidation to cellular components are related to the degree of membrane lipid unsaturation (Tien and Aust 1982) and increasing unsaturation renders membrane fatty acids more susceptible to peroxidative damage (May and McCay 1968). Phosphatides from fish tissues are particularly rich in fatty acids of the (n-3) series especially the highly unsaturated species, docosahexaenoic acid (Henderson and Tocher 1987) which shows the highest rate of peroxidation in mammalian systems in vitro (Tien and Aust 1982). The presence of a highly efficient system for the removal of lipid hydroperoxides is therefore particularly important in fish. GSHPx activity in salmonid tissue homogenates has been demonstrated with various hydroperoxide
Present address: Department of Nutritional Sciences, University of Guelph, Ontario NIG 2W1, Canada
12 substrates (Bell et al. 1985). In this study the role of GSHPx as a component of a complex antioxidant system was investigated in an intact cell system so that the consequences of its action could be related to the overall metabolism of the cell. Isolated rainbow trout hepatocytes were challenged with t-butyl hydroperoxide (BuOOH) to observe the capacity of the GSHPx/GSSG reductase system and to assess the effect of oxidative challenge on cellular glutathione status and membrane integrity.
Materials and methods Fish Rainbow trout (200-800 g) were obtained from Almondbank Trout Farm, Perth, Scotland, and maintained in outdoor fibreglass tanks supplied with fresh water from the Oban town supply and given a commercial pelleted feed. Reagents D-[1- 14 C] and D-[6-14C] glucose and L-[U-14 C] lactate were obtained from Amersham International, U.K. Glutathione reductase type III, oxidised and reduced glutathione, t-butyl hydroperoxide and collagenase type IV were obtained from Sigma Chemical Company Ltd., U.K. 2-vinyl pyridine was obtained from Aldrich, U.K. All other reagents were of the highest grade available. Preparationof hepatocytes The procedure described by Moon et al. (1985) was used to isolate hepatocytes. The perfusion medium was a Modified Hanks medium containing 6 mM NaHCO 3. After final washing the hepatocytes were resuspended in Modified Hanks medium containing 1 mM CaC1 2 and 0.2 mM alanine, cysteine and glutamine (incubation medium). Hepatocyte viability was checked periodically by measuring gluconeogenesis from L-[U-1 4C] lactate as described by Walton and Cowey (1979). During incubations leakage of intracellular lactate dehydrogenase into the medium was measured as described
by Lush et al. (1969) to assess membrane integrity (Moon et al. 1985). Hepatocyte incubation Washed hepatocytes were resuspended in incubation medium at a concentration of approximately 100 mg fresh weight/ml of medium and placed in 25 ml 'Reacti-Flasks' which had been pre-treated with 'Sigmacote' to prevent cell adhesion. The cells were maintained in suspension by rotating the flasks using a 'Roto-torque' rotator (Cole-Parker Instruments, Illinois, USA). Incubations were carried out in air at 20°C. All experiments were started between 11.00 and 12.00 a.m. to avoid changes in cellular glutathione levels due to circadian rhythm. Glutathione measurement Total glutathione and GSSG were measured by the method of Griffith (1980) with the following adaptations. At various time intervals 1.0 ml samples of hepatocytes were removed from the flasks and centrifuged in a microcentrifuge (12000 x g for 1 min). 0.6 ml of the supernatant (medium) was removed and deproteinized with 0.3 ml 5°% (w/v) sulphosalicylic acid. The cell pellet was homogenized in 9 volumes of 2% (w/v) sulphosalicylic acid. Both were then recentrifuged (12000 x g for 1 min) to remove precipitated protein. The two supernatant fractions were treated as follows: (a) Intracellular glutathione: 150 1 of the homogenized cell supernatant was placed in each of two vials containing 10 /1l 3% (w/v) triethanolamine. One vial was stored on ice until assayed for total intracellular GSH equivalents. 3 1 of 2-vinyl pyridine was added to the second vial, the contents mixed and incubated at room temperature for 45 minutes prior to assay of GSSG. (b) Extracellular glutathione: 300 l of deproteinized medium supernatant was placed in each of 2 vials containing 20 Itl 3% triethanolamine. One vial was stored on ice until assayed for total extracellular GSH equivalents. 3 tl of 2-vinyl pyridine was added to the second vial, the contents mixed and incubated at room temperature for 45 minutes prior to the assay of GSSG. Total GSH equivalents and
13 H 2SO 4. Sixty minutes after acidification the filter paper wicks were removed and along with two cup washings (distilled H 20) were placed in scintillation vials containing 10 ml 'Instagel' scintillant (Packard, U.K.). The samples were counted in an LKB 'Rackbeta' No. 1219 scintillation counter. Counting efficiency was determined using an external standard.
E 0 0
Fatty acid analysis
-
time(min)
Fig. 1. Time course of GSSG content (nmol/g cells) of control C) and 10 mM BuOOH · , 1 mM BuOOH, ( (· A ) treated hepatocytes. Results are means + SEM from ( 3-5 different experiments.
GSSG were assayed by an enzymatic recycling procedure in which GSH is sequentially oxidised by 5,5' dithiobis-(2-nitrobenzoic acid) (DTNB) and reduced by NADPH in the presence of GSSG reductase as described by Tietze (1979) and adapted by Griffith (1980). A standard curve was produced with known amounts of GSSG. Each standard cuvette contained triethanolamine at a concentration equivalent to adding 100 tl of sample. Glucose metabolism One ml samples of hepatocyte suspension were placed in 25 ml 'Reacti-Flasks' containing 0.9 ml incubation medium. The flasks were incubated at 20°C for 15 minutes on a 'Roto-torque' rotator after which 0.1 ml (0.1 ItCi) of either [1- 14C] or [6-14 C] glucose was added to the flasks. The isotope had been diluted in cold glucose making the final flask concentration 3 mM. Zero time flasks had 1.0 ml of 5 M H 2SO4 added before isotope addition. The flasks were then stoppered but aeration was provided via a 19G syringe needle which also supported a plastic cup containing 5 cm 2 of filter paper soaked in 0.3 ml of 20% KOH to act as a CO 2 trap. The incubations were terminated at 30, 60, 90 and 180 minutes by addition of 1.0 ml of 5M
Approximately 100 mg of hepatocytes were homogenised in 4 ml chloroform:methanol (2:1, v:v) containing 0.00125% butlated hydroxytoluene. Water was added (0.2 volumes) and the contents extracted on a vortex mixer. The lower chloroform layer was removed, blown to dryness under N 2 and methyl esters of fatty acids prepared by acid catalysed transmethylation (Christie 1982), then purified by TLC. Fatty acid methyl esters were resolved and measured in a Carlo Erba HRGC 41 gas chromatograph equipped with an FFAP capillary column (50 m x 0.22 mm i.d.) and using hydrogen as carrier gas (Cowey et al. 1984). Identification was by comparison with known standards and by reference to published data (Ackman and Eaton, 1978). Results Control hepatocytes incubated up to 3 h have a relatively constant intracellular GSSG level of around 10 nmol/g representing between 1 and 2% of total cellular glutathione (see Figs. 1 and 2a). When BuOOH is added to the incubation medium intracellular GSSG increases indicating activity of glutathione peroxidase. BuOOH added at 1 mM concentration causes an initial rise in GSSG which is reduced to normal levels after about 1 hour. BuOOH added at 10 mM causes a large initial rise of intracellular GSSG which is gradually reduced to control levels after 90 minutes (Fig. 1). GSSG as a 70 of total glutathione increases to around 60% and remains at this level throughout the incubation period indicating that GSSG reductase is unable to restore the normal GSH:GSSG ratio when hydroperoxide is present at this concentration (Fig. 2a). Total intracellular GSH remains fairly constant
14 aN
0 0 0 9
A
E DI
a
c .0 c~
0
time(min)
Fig. 2a. Time course of percentage total GSH equivalents as GSSG in control (· ), 1 mM BuOOH ( [ ) and 10 mM BuOOH ( * ) treated hepatocytes. Results are means + SEM from 3-5 different experiments.
tlm®(min)
Fig. 3. Time course of GSSG release into the extracellular medium from control ( *), 1 mM BuOOH ( ) and 10 mM BuOOH ( ) treated hepatocytes. Results are means + SEM from 3-5 different experiments.
X aR o E
e e oI
aI 0
tim(min)
Fig. 4. Time course of extracellular lactate dehydrogenase (LDH) activity in control (· *), 1 mM BuOOH ( B) tlme(min)
Fig. 2b. Time course of total intracellular GSH equivalents (~Imol/g cells) of control ( *), 1 mM BuOOH, (D[ B), and 10 mM BuOOH ( A) treated hepatocytes. Results are means ± SEM from 3-5 different experiments.
(around 1.2 /Amol/g) in control cells during incubation and addition of 1 mM BuOOH causes no significant alterations. Addition of 10 mM BuOOH causes a rapid and irreversible loss of GSH (Fig. 2b). BuOOH at both concentrations causes an increased efflux of GSSG into the medium (Fig. 3). However, only 307o of GSSG formed intracellular-
and 10 mM BuOOH (A
A
) treated hepatocytes. Results are
means ± SEM from 3-5 different experiments.
ly on addition of 10 mM BuOOH appears in the medium, the majority is probably reacting with protein free thiols to produce glutathione-protein mixed disulphides. Leakage of lactate dehydrogenase into the medium during the period of incubation showed little change in control and 1 mM BuOOH treated cells but those incubated with 10 mM BuOOH showed a steady accumulation of this enzyme in the medium after I h incubation (Fig. 4). Analysis of cellular total lipid fatty acid composition showed no change during incubation in control
15 40
m2 o
o
B.uOOH
30
20
10
O
300
0
10
time(mln)
Fig. 5 Time course of docosahexaenoic acid (DHA) content of contr, ol ( *), 1 mM BuOOH ( 0) and 10 mM BuOC)H (A A) treated hepatocytes. Results are means SEM from 3-5 different experiments.
Fig. 5. The graph gives values for DHA as % by weight of total cell lipid. In molar terms initial DHA concentrations in control, 1 mM and 10 mM BuOOH treated cells were 48.2 + 2.42, 43.9 ± 4.11 and 45.7 4.47 tmol DHA/g wet weight of hepatocytes respectively. In control and 1 mM BuOOH treated cells there was no significant loss of DHA over the 3h incubation period, but in hepatocytes treated with 10 mM BuOOH DHA concentration fell to 32.2 3.04 tmol/g after 3h. Fig. 6 indicates that utilisation of [1-14C] glucose to 14CO 2 by hepatocytes was increased as a result of increased intracellular GSSG concentration. Cells treated with 10 mM BuOOH showed increased [1-14 C] glucose utilisation between 30 and 60 minutes but thereafter glucose utilisation tended to plateau suggesting that the metabolic pathways had been disrupted by oxidative damage (result not shown). Metabolism of [6-14C] glucose was not sigHIlcanILIy
altered In
UuJr
treated
cells.
Discussion
time(min)
Fig. 6. Time course of 14 CO2 release from either [1- 1 4C] or [6-14C] glucose by control and 1 mM BuOOH stimulated hepatocytes. [1-14 C] glucose; control (o o), [1-14 C] glucose; BuOOH ( *), [6-14C] glucose; control ( ), [6- 14 C] glucose; BuOOH ( -). Results are means + SEM from 3-5 different experiments.
and I mM BuOOH treated hepatocytes. However, those cells treated with 10 mM BuOOH showed decreased levels of highly unsaturated fatty acids (HUFA) particularly docosahexaenoic acid (DHA) with increased levels of monoenes and saturates. The effect on DHA levels versus time is shown in
In this study we have investigated the metabolism of BuOOH, a recognised substrate for rainbow trout GSHPx (Bell et al. 1984) in isolated hepatocytes. BuOOH at a concentration of 1 mM caused a reversible increase in cellular GSSG which did not enhance leakage of lactate dehydrogenase or damage the HUFA component of cellular lipids. Ten mM BuOOH, however, caused an irreversible oxidation of GSH which resulted in extensive leakage of lactate dehydrogenase and loss of DHA. Accumulation of intracellular GSSG was followed by efflux into the extracellular medium, a phenomenon also observed in rat hepatocytes (Sies and Summer 1975; Eklow et al. 1984) and human erythrocytes (Srivastava et al. 1974). In trout cells challenged with 10 mM BuOOH only 30% of intracellular GSSG appeared extracellularly, the remainder presumably forming mixed disulphides by reacting with free thiol groups on cellular proteins. It has been proposed that formation of proteinglutathione mixed disulphides may be an important regulatory mechanism in rat hepatocytes (Isaacs and Binkley 1977a, b) although excessive oxidation
16 of protein thiols could obviously result in indiscriminate enzyme deactivation causing a breakdown in metabolic pathways and a collapse of cellular integrity. A relatively small increase in intracellular GSSG resulting from addition of 1 mM BuOOH, caused an increased release of 14 CO 2 from the metabolism of [1- 14 C] glucose. This demonstrates the dependence of hydroperoxide removal on increased flux through the hexose monophosphate pathway. This dependence is linked to the ability of the hexose monophosphate pathway to supply NADPH, the limiting substrate in the reduction of GSSG to GSH via GSSG reductase (Sies et al. 1972). Stimulation of the hexose monophosphate pathway in mammals, and presumably also in fish, occurs by activation of glucose-6-phosphate dehydrogenase via formation of enzyme-glutathione mixed disulphides (Keeling et al. 1982). Accumulation of lipid peroxides within cell and organelle membranes results in reduced membrane fluidity, uncontrolled ion permeability and inactivation of membrane bound enzymes (Halliwell 1987). Incubation of trout hepatocytes with 10 mM BuOOH clearly overloads the cellular capacity to metabolise the hydroperoxide by the GSHPx/GSSG reductase couple. The reversal of the normal GSH:GSSG ratio will result in indiscriminate formation of mixed disulphides in cellular proteins resulting in their deactivation and ultimately in cell death. Furthermore, unmetabolised BuOOH will decompose to yield unstable free radical products capable of attacking membrane HUFA and thereby reducing cell integrity. These experiments demonstrate the activity of GSHPx in an intact cell system from rainbow trout and further elucidate its role in integrating with other enzymes and metabolic pathways to reduce intracellular hydroperoxides. Obviously, concentrations of endogenous hydroperoxides are not likely to reach a level of 10 mM normally. However instances which can compromise cellular hydroperoxide metabolism are not unknown and these could allow an accumulation of potentially damaging hydroperoxides, e.g. deficiencies in selenium, riboflavin or essential amino acids or loss of superoxide dismutase activity could reduce the ability to metabolise hydroperoxides thus predisposing the likeli-
hood of oxidative damage. In normal cells, however, the presence of hydroperoxide levels up to an equimolar concentration with respect to GSH, while causing a transient increase in GSSG, would appear to cause no significant damage to the fatty acid membrane components of rainbow trout hepatocytes. References cited Ackman, R.G. and Eaton, C.A. 1978. Some contemporary applications of open-tubular gas-liquid chromatography in analyses of methyl esters of longer-chain fatty acids. Fette Seifen Anstr. Mittel. 80: 21-37. Bell, J.G., Cowey, C.B. and Youngson, A. 1984. Rainbow trout liver microsomal lipid peroxidation; the effect of purified glutathione peroxidase, glutathione S-transferase and other factors. Biochim. Biophys. Acta. 795: 91-99. Bell, J.G., Cowey, C.B., Adron, J.W. and Shanks, A.M. 1985. Some effect of vitamin E and selenium deprivation on tissue enzyme levels and indices of tisue peroxidation in rainbow trout (Salmo gairdneri). Br. J. Nutr. 53: 149-157. Bell, J.G., Pirie, B.J.S., Adron, J.W. and Cowey, C.B. 1986. Some effects of selenium deficiency on glutathione peroxidase (EC1.11.19) activity and tissue pathology in rainbow trout (Salmo gairdneri). Br. J. Nutr. 55: 305-311. Christie, W.W. 1982. Lipid Analyses, 2nd edition. Pergamon Press, Oxford. Cowey, C.B., Degener, E., Tacon, A.G.J., Youngson, A. and Bell, J.G. 1984. The effect of vitamin E and oxidised fish oil on the nutrition of rainbow trout (Salmo gairdneri)grown at natural, varying water temperatures. Br. J. Nutr. 51: 443451. Eklow, L., Moldens, P. and Orrenius, S. 1984. Oxidation of glutathione during hydroperoxide metabolism. A study using hepatocytes and the glutathione reductase inhibitor 1,3-bis (2-chloroethyl)-nitrosourea. Eur. J. Biochem. 138: 459-463. Griffith, O.W. 1980. Determination of glutathione and glutathione disulphide using glutathione reductase and 2-vinyl pyridine. Anal. Biochem. 106: 207-212. Halliwell, B. and Gutteridge, J.M.C. 1985. Free radicals in biology and medicine. Oxford University Press, U.K. Halliwell, B. 1987. Free radicals and metal ions in health and disease. Proc. Nutr. Soc. 46: 13-26. Henderson, R.J. and Tocher, D.R. 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26: 281-347. Isaacs, J.T. and Binkley, F. 1977a. Glutathione dependent control of protein disulphide-sulphydryl content by subcellular fractions of hepatic tissue. Biochim. Biophys. Acta. 497: 192-204. Isaacs, J.T. and Binkley, F. 1977b. Cyclic AMP-dependent control of the rat hepatic glutathione disulphide-sulphydryl ratio. Biochim. Biophys. Acta. 298: 29-38.
17 Keeling, P.L., Smith, L.L. and Aldridge, W.N. 1982. The formation of mixed disulphides in rat lung following paraquat administration; correlation with changes in intermediary metabolism. Biochim. Biophys. Acta. 716: 249-257. Lush, I.E., Cowey, C.B. and Knox, D. 1969. The lactate dehydrogenase isozymes of 12 species of flatfish (Heterosomata). J. Exp. Zool. 171: 105-118. May, H.E. and McCay, P.B. 1968. Reduced triphosphopyridine nucleotide oxidase-catalysed alterations of membrane phospholipids. 11. Enzyme properties and stoichiometry. J. Biol. Chem. 243: 2296-2305. Moon, T.W., Walsh, P.J. and Mommsen, T.P. 1985. Fish hepatocytes: A model metabolic system. Can. J. Fish. Aquat. Sci. 42: 1772-1782. Sies, H., Gerstenecker, C., Menzel, H. and Flohe, L. 1972. Oxidation in the NADP system and release of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides. FEBS Lett. 27: 171-175. Sies, H. and Summer, K.-H. 1975. Hydroperoxide metabolizing
systems in rat liver. Eur. J. Biochem. 57: 503-512. Simmons, T.W. and Jamall, I.S. 1988. Significance of alterations in hepatic antioxidant enzymes; primacy of glutathione peroxidase. Biochem. J. 251: 913-917. Srivastava, S.K., Yogesh, C.A. and Beutler, E. 1974. Useful agents for the study of glutathione metabolism in erythrocytes. Biochem. J. 139: 289-295. Tien, M. and Aust, S.D. 1982. Rabbit liver microsomal lipid peroxidation; the effect of lipid on the rate of peroxidation. Biochim. Biophys. Acta. 712: 1-9. Tietze, F. 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidised glutathione; applications to mammalian blood and other tissues. Anal. Biochem. 27: 507-522. Ursini, F. and Bindoli, A. 1987. The role of selenium peroxidases in the protection against oxidative damage of membranes. Chem. Phys. Lipids 44: 255-276. Walton, M.J. and Cowey, C.B. 1979. Gluconeogenesis by isolated hepatocytes from rainbow trout Salmo gairdneri. Comp. Biochem. Physiol. 62B: 75-79.
Oxidation of glutathione during hydroperoxide metabolism in isolated hepatocytes of rainbow trout (Salmo gairdneri) J. Gordon Bell and Colin B. Cowey2 1 NERC Unit of Aquatic Biochemistry, University of Stirling, Stirling FK9 4LA, Scotland Keywords: glutathione, hydroperoxide, hepatocyte, rainbow trout, lipid peroxidation
Abstract Freshly isolated rainbow trout hepatocytes were exposed to tert-butyl hydroperoxide (BuOOH), a substrate for glutathione peroxidase. BuOOH at a concentration approximately equimolar (1 mM) with intracellular reduced glutathione (GSH) caused a reversible increase in intracellular glutathione disulphide (GSSG) but did not compromise cell viability or damage membrane lipids. BuOOH at 10 mM caused a large irreversible increase in intracellular GSSG followed by efflux into the medium. Considerable leakage of lactate dehydrogenase and loss of highly unsaturated fatty acids, particularly docosahexaenoic acid also occurred. Dependence of hydroperoxide removal on flux through the hexose monophosphate pathway was suggested by the increased release of 14CO 2 from [1-1 4C] glucose from hepatocytes incubated with BuOOH.
Introduction In biological systems hydroperoxides can arise not only via the metabolism of various xenobiotics but also as a result of normal oxygen metabolism (Halliwell and Gutteridge 1985). Vertebrate cells possess a multi-level defence mechanism which prevents pathogenesis arising from free radical initiated lipid peroxidation. The selenium containing enzyme glutathione peroxidase (GSHPx) (EC1.11.1.9) has a prominent role in this process (Simmons and Jamall 1988). GSHPx catalyses the reduction of H 20 2 and various organic hydroperoxides with the simultaneous oxidation of glutathione (GSH) to glutathione disulphide (GSSG) (Ursini and Bindoli 1987). GSHPx activity has been identified in salmonid fish tissues (Bell et al. 1985, 1986). GSSG is reduced to GSH by the NADPH dependent en2
zyme glutathione reductase (ECI.6.4.2) thus maintaining a high GSH:GSSG ratio. The damaging effects of lipid peroxidation to cellular components are related to the degree of membrane lipid unsaturation (Tien and Aust 1982) and increasing unsaturation renders membrane fatty acids more susceptible to peroxidative damage (May and McCay 1968). Phosphatides from fish tissues are particularly rich in fatty acids of the (n-3) series especially the highly unsaturated species, docosahexaenoic acid (Henderson and Tocher 1987) which shows the highest rate of peroxidation in mammalian systems in vitro (Tien and Aust 1982). The presence of a highly efficient system for the removal of lipid hydroperoxides is therefore particularly important in fish. GSHPx activity in salmonid tissue homogenates has been demonstrated with various hydroperoxide
Present address: Department of Nutritional Sciences, University of Guelph, Ontario NIG 2W1, Canada
12 substrates (Bell et al. 1985). In this study the role of GSHPx as a component of a complex antioxidant system was investigated in an intact cell system so that the consequences of its action could be related to the overall metabolism of the cell. Isolated rainbow trout hepatocytes were challenged with t-butyl hydroperoxide (BuOOH) to observe the capacity of the GSHPx/GSSG reductase system and to assess the effect of oxidative challenge on cellular glutathione status and membrane integrity.
Materials and methods Fish Rainbow trout (200-800 g) were obtained from Almondbank Trout Farm, Perth, Scotland, and maintained in outdoor fibreglass tanks supplied with fresh water from the Oban town supply and given a commercial pelleted feed. Reagents D-[1- 14 C] and D-[6-14C] glucose and L-[U-14 C] lactate were obtained from Amersham International, U.K. Glutathione reductase type III, oxidised and reduced glutathione, t-butyl hydroperoxide and collagenase type IV were obtained from Sigma Chemical Company Ltd., U.K. 2-vinyl pyridine was obtained from Aldrich, U.K. All other reagents were of the highest grade available. Preparationof hepatocytes The procedure described by Moon et al. (1985) was used to isolate hepatocytes. The perfusion medium was a Modified Hanks medium containing 6 mM NaHCO 3. After final washing the hepatocytes were resuspended in Modified Hanks medium containing 1 mM CaC1 2 and 0.2 mM alanine, cysteine and glutamine (incubation medium). Hepatocyte viability was checked periodically by measuring gluconeogenesis from L-[U-1 4C] lactate as described by Walton and Cowey (1979). During incubations leakage of intracellular lactate dehydrogenase into the medium was measured as described
by Lush et al. (1969) to assess membrane integrity (Moon et al. 1985). Hepatocyte incubation Washed hepatocytes were resuspended in incubation medium at a concentration of approximately 100 mg fresh weight/ml of medium and placed in 25 ml 'Reacti-Flasks' which had been pre-treated with 'Sigmacote' to prevent cell adhesion. The cells were maintained in suspension by rotating the flasks using a 'Roto-torque' rotator (Cole-Parker Instruments, Illinois, USA). Incubations were carried out in air at 20°C. All experiments were started between 11.00 and 12.00 a.m. to avoid changes in cellular glutathione levels due to circadian rhythm. Glutathione measurement Total glutathione and GSSG were measured by the method of Griffith (1980) with the following adaptations. At various time intervals 1.0 ml samples of hepatocytes were removed from the flasks and centrifuged in a microcentrifuge (12000 x g for 1 min). 0.6 ml of the supernatant (medium) was removed and deproteinized with 0.3 ml 5°% (w/v) sulphosalicylic acid. The cell pellet was homogenized in 9 volumes of 2% (w/v) sulphosalicylic acid. Both were then recentrifuged (12000 x g for 1 min) to remove precipitated protein. The two supernatant fractions were treated as follows: (a) Intracellular glutathione: 150 1 of the homogenized cell supernatant was placed in each of two vials containing 10 /1l 3% (w/v) triethanolamine. One vial was stored on ice until assayed for total intracellular GSH equivalents. 3 1 of 2-vinyl pyridine was added to the second vial, the contents mixed and incubated at room temperature for 45 minutes prior to assay of GSSG. (b) Extracellular glutathione: 300 l of deproteinized medium supernatant was placed in each of 2 vials containing 20 Itl 3% triethanolamine. One vial was stored on ice until assayed for total extracellular GSH equivalents. 3 tl of 2-vinyl pyridine was added to the second vial, the contents mixed and incubated at room temperature for 45 minutes prior to the assay of GSSG. Total GSH equivalents and
13 H 2SO 4. Sixty minutes after acidification the filter paper wicks were removed and along with two cup washings (distilled H 20) were placed in scintillation vials containing 10 ml 'Instagel' scintillant (Packard, U.K.). The samples were counted in an LKB 'Rackbeta' No. 1219 scintillation counter. Counting efficiency was determined using an external standard.
E 0 0
Fatty acid analysis
-
time(min)
Fig. 1. Time course of GSSG content (nmol/g cells) of control C) and 10 mM BuOOH · , 1 mM BuOOH, ( (· A ) treated hepatocytes. Results are means + SEM from ( 3-5 different experiments.
GSSG were assayed by an enzymatic recycling procedure in which GSH is sequentially oxidised by 5,5' dithiobis-(2-nitrobenzoic acid) (DTNB) and reduced by NADPH in the presence of GSSG reductase as described by Tietze (1979) and adapted by Griffith (1980). A standard curve was produced with known amounts of GSSG. Each standard cuvette contained triethanolamine at a concentration equivalent to adding 100 tl of sample. Glucose metabolism One ml samples of hepatocyte suspension were placed in 25 ml 'Reacti-Flasks' containing 0.9 ml incubation medium. The flasks were incubated at 20°C for 15 minutes on a 'Roto-torque' rotator after which 0.1 ml (0.1 ItCi) of either [1- 14C] or [6-14 C] glucose was added to the flasks. The isotope had been diluted in cold glucose making the final flask concentration 3 mM. Zero time flasks had 1.0 ml of 5 M H 2SO4 added before isotope addition. The flasks were then stoppered but aeration was provided via a 19G syringe needle which also supported a plastic cup containing 5 cm 2 of filter paper soaked in 0.3 ml of 20% KOH to act as a CO 2 trap. The incubations were terminated at 30, 60, 90 and 180 minutes by addition of 1.0 ml of 5M
Approximately 100 mg of hepatocytes were homogenised in 4 ml chloroform:methanol (2:1, v:v) containing 0.00125% butlated hydroxytoluene. Water was added (0.2 volumes) and the contents extracted on a vortex mixer. The lower chloroform layer was removed, blown to dryness under N 2 and methyl esters of fatty acids prepared by acid catalysed transmethylation (Christie 1982), then purified by TLC. Fatty acid methyl esters were resolved and measured in a Carlo Erba HRGC 41 gas chromatograph equipped with an FFAP capillary column (50 m x 0.22 mm i.d.) and using hydrogen as carrier gas (Cowey et al. 1984). Identification was by comparison with known standards and by reference to published data (Ackman and Eaton, 1978). Results Control hepatocytes incubated up to 3 h have a relatively constant intracellular GSSG level of around 10 nmol/g representing between 1 and 2% of total cellular glutathione (see Figs. 1 and 2a). When BuOOH is added to the incubation medium intracellular GSSG increases indicating activity of glutathione peroxidase. BuOOH added at 1 mM concentration causes an initial rise in GSSG which is reduced to normal levels after about 1 hour. BuOOH added at 10 mM causes a large initial rise of intracellular GSSG which is gradually reduced to control levels after 90 minutes (Fig. 1). GSSG as a 70 of total glutathione increases to around 60% and remains at this level throughout the incubation period indicating that GSSG reductase is unable to restore the normal GSH:GSSG ratio when hydroperoxide is present at this concentration (Fig. 2a). Total intracellular GSH remains fairly constant
14 aN
0 0 0 9
A
E DI
a
c .0 c~
0
time(min)
Fig. 2a. Time course of percentage total GSH equivalents as GSSG in control (· ), 1 mM BuOOH ( [ ) and 10 mM BuOOH ( * ) treated hepatocytes. Results are means + SEM from 3-5 different experiments.
tlm®(min)
Fig. 3. Time course of GSSG release into the extracellular medium from control ( *), 1 mM BuOOH ( ) and 10 mM BuOOH ( ) treated hepatocytes. Results are means + SEM from 3-5 different experiments.
X aR o E
e e oI
aI 0
tim(min)
Fig. 4. Time course of extracellular lactate dehydrogenase (LDH) activity in control (· *), 1 mM BuOOH ( B) tlme(min)
Fig. 2b. Time course of total intracellular GSH equivalents (~Imol/g cells) of control ( *), 1 mM BuOOH, (D[ B), and 10 mM BuOOH ( A) treated hepatocytes. Results are means ± SEM from 3-5 different experiments.
(around 1.2 /Amol/g) in control cells during incubation and addition of 1 mM BuOOH causes no significant alterations. Addition of 10 mM BuOOH causes a rapid and irreversible loss of GSH (Fig. 2b). BuOOH at both concentrations causes an increased efflux of GSSG into the medium (Fig. 3). However, only 307o of GSSG formed intracellular-
and 10 mM BuOOH (A
A
) treated hepatocytes. Results are
means ± SEM from 3-5 different experiments.
ly on addition of 10 mM BuOOH appears in the medium, the majority is probably reacting with protein free thiols to produce glutathione-protein mixed disulphides. Leakage of lactate dehydrogenase into the medium during the period of incubation showed little change in control and 1 mM BuOOH treated cells but those incubated with 10 mM BuOOH showed a steady accumulation of this enzyme in the medium after I h incubation (Fig. 4). Analysis of cellular total lipid fatty acid composition showed no change during incubation in control
15 40
m2 o
o
B.uOOH
30
20
10
O
300
0
10
time(mln)
Fig. 5 Time course of docosahexaenoic acid (DHA) content of contr, ol ( *), 1 mM BuOOH ( 0) and 10 mM BuOC)H (A A) treated hepatocytes. Results are means SEM from 3-5 different experiments.
Fig. 5. The graph gives values for DHA as % by weight of total cell lipid. In molar terms initial DHA concentrations in control, 1 mM and 10 mM BuOOH treated cells were 48.2 + 2.42, 43.9 ± 4.11 and 45.7 4.47 tmol DHA/g wet weight of hepatocytes respectively. In control and 1 mM BuOOH treated cells there was no significant loss of DHA over the 3h incubation period, but in hepatocytes treated with 10 mM BuOOH DHA concentration fell to 32.2 3.04 tmol/g after 3h. Fig. 6 indicates that utilisation of [1-14C] glucose to 14CO 2 by hepatocytes was increased as a result of increased intracellular GSSG concentration. Cells treated with 10 mM BuOOH showed increased [1-14 C] glucose utilisation between 30 and 60 minutes but thereafter glucose utilisation tended to plateau suggesting that the metabolic pathways had been disrupted by oxidative damage (result not shown). Metabolism of [6-14C] glucose was not sigHIlcanILIy
altered In
UuJr
treated
cells.
Discussion
time(min)
Fig. 6. Time course of 14 CO2 release from either [1- 1 4C] or [6-14C] glucose by control and 1 mM BuOOH stimulated hepatocytes. [1-14 C] glucose; control (o o), [1-14 C] glucose; BuOOH ( *), [6-14C] glucose; control ( ), [6- 14 C] glucose; BuOOH ( -). Results are means + SEM from 3-5 different experiments.
and I mM BuOOH treated hepatocytes. However, those cells treated with 10 mM BuOOH showed decreased levels of highly unsaturated fatty acids (HUFA) particularly docosahexaenoic acid (DHA) with increased levels of monoenes and saturates. The effect on DHA levels versus time is shown in
In this study we have investigated the metabolism of BuOOH, a recognised substrate for rainbow trout GSHPx (Bell et al. 1984) in isolated hepatocytes. BuOOH at a concentration of 1 mM caused a reversible increase in cellular GSSG which did not enhance leakage of lactate dehydrogenase or damage the HUFA component of cellular lipids. Ten mM BuOOH, however, caused an irreversible oxidation of GSH which resulted in extensive leakage of lactate dehydrogenase and loss of DHA. Accumulation of intracellular GSSG was followed by efflux into the extracellular medium, a phenomenon also observed in rat hepatocytes (Sies and Summer 1975; Eklow et al. 1984) and human erythrocytes (Srivastava et al. 1974). In trout cells challenged with 10 mM BuOOH only 30% of intracellular GSSG appeared extracellularly, the remainder presumably forming mixed disulphides by reacting with free thiol groups on cellular proteins. It has been proposed that formation of proteinglutathione mixed disulphides may be an important regulatory mechanism in rat hepatocytes (Isaacs and Binkley 1977a, b) although excessive oxidation
16 of protein thiols could obviously result in indiscriminate enzyme deactivation causing a breakdown in metabolic pathways and a collapse of cellular integrity. A relatively small increase in intracellular GSSG resulting from addition of 1 mM BuOOH, caused an increased release of 14 CO 2 from the metabolism of [1- 14 C] glucose. This demonstrates the dependence of hydroperoxide removal on increased flux through the hexose monophosphate pathway. This dependence is linked to the ability of the hexose monophosphate pathway to supply NADPH, the limiting substrate in the reduction of GSSG to GSH via GSSG reductase (Sies et al. 1972). Stimulation of the hexose monophosphate pathway in mammals, and presumably also in fish, occurs by activation of glucose-6-phosphate dehydrogenase via formation of enzyme-glutathione mixed disulphides (Keeling et al. 1982). Accumulation of lipid peroxides within cell and organelle membranes results in reduced membrane fluidity, uncontrolled ion permeability and inactivation of membrane bound enzymes (Halliwell 1987). Incubation of trout hepatocytes with 10 mM BuOOH clearly overloads the cellular capacity to metabolise the hydroperoxide by the GSHPx/GSSG reductase couple. The reversal of the normal GSH:GSSG ratio will result in indiscriminate formation of mixed disulphides in cellular proteins resulting in their deactivation and ultimately in cell death. Furthermore, unmetabolised BuOOH will decompose to yield unstable free radical products capable of attacking membrane HUFA and thereby reducing cell integrity. These experiments demonstrate the activity of GSHPx in an intact cell system from rainbow trout and further elucidate its role in integrating with other enzymes and metabolic pathways to reduce intracellular hydroperoxides. Obviously, concentrations of endogenous hydroperoxides are not likely to reach a level of 10 mM normally. However instances which can compromise cellular hydroperoxide metabolism are not unknown and these could allow an accumulation of potentially damaging hydroperoxides, e.g. deficiencies in selenium, riboflavin or essential amino acids or loss of superoxide dismutase activity could reduce the ability to metabolise hydroperoxides thus predisposing the likeli-
hood of oxidative damage. In normal cells, however, the presence of hydroperoxide levels up to an equimolar concentration with respect to GSH, while causing a transient increase in GSSG, would appear to cause no significant damage to the fatty acid membrane components of rainbow trout hepatocytes. References cited Ackman, R.G. and Eaton, C.A. 1978. Some contemporary applications of open-tubular gas-liquid chromatography in analyses of methyl esters of longer-chain fatty acids. Fette Seifen Anstr. Mittel. 80: 21-37. Bell, J.G., Cowey, C.B. and Youngson, A. 1984. Rainbow trout liver microsomal lipid peroxidation; the effect of purified glutathione peroxidase, glutathione S-transferase and other factors. Biochim. Biophys. Acta. 795: 91-99. Bell, J.G., Cowey, C.B., Adron, J.W. and Shanks, A.M. 1985. Some effect of vitamin E and selenium deprivation on tissue enzyme levels and indices of tisue peroxidation in rainbow trout (Salmo gairdneri). Br. J. Nutr. 53: 149-157. Bell, J.G., Pirie, B.J.S., Adron, J.W. and Cowey, C.B. 1986. Some effects of selenium deficiency on glutathione peroxidase (EC1.11.19) activity and tissue pathology in rainbow trout (Salmo gairdneri). Br. J. Nutr. 55: 305-311. Christie, W.W. 1982. Lipid Analyses, 2nd edition. Pergamon Press, Oxford. Cowey, C.B., Degener, E., Tacon, A.G.J., Youngson, A. and Bell, J.G. 1984. The effect of vitamin E and oxidised fish oil on the nutrition of rainbow trout (Salmo gairdneri)grown at natural, varying water temperatures. Br. J. Nutr. 51: 443451. Eklow, L., Moldens, P. and Orrenius, S. 1984. Oxidation of glutathione during hydroperoxide metabolism. A study using hepatocytes and the glutathione reductase inhibitor 1,3-bis (2-chloroethyl)-nitrosourea. Eur. J. Biochem. 138: 459-463. Griffith, O.W. 1980. Determination of glutathione and glutathione disulphide using glutathione reductase and 2-vinyl pyridine. Anal. Biochem. 106: 207-212. Halliwell, B. and Gutteridge, J.M.C. 1985. Free radicals in biology and medicine. Oxford University Press, U.K. Halliwell, B. 1987. Free radicals and metal ions in health and disease. Proc. Nutr. Soc. 46: 13-26. Henderson, R.J. and Tocher, D.R. 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26: 281-347. Isaacs, J.T. and Binkley, F. 1977a. Glutathione dependent control of protein disulphide-sulphydryl content by subcellular fractions of hepatic tissue. Biochim. Biophys. Acta. 497: 192-204. Isaacs, J.T. and Binkley, F. 1977b. Cyclic AMP-dependent control of the rat hepatic glutathione disulphide-sulphydryl ratio. Biochim. Biophys. Acta. 298: 29-38.
17 Keeling, P.L., Smith, L.L. and Aldridge, W.N. 1982. The formation of mixed disulphides in rat lung following paraquat administration; correlation with changes in intermediary metabolism. Biochim. Biophys. Acta. 716: 249-257. Lush, I.E., Cowey, C.B. and Knox, D. 1969. The lactate dehydrogenase isozymes of 12 species of flatfish (Heterosomata). J. Exp. Zool. 171: 105-118. May, H.E. and McCay, P.B. 1968. Reduced triphosphopyridine nucleotide oxidase-catalysed alterations of membrane phospholipids. 11. Enzyme properties and stoichiometry. J. Biol. Chem. 243: 2296-2305. Moon, T.W., Walsh, P.J. and Mommsen, T.P. 1985. Fish hepatocytes: A model metabolic system. Can. J. Fish. Aquat. Sci. 42: 1772-1782. Sies, H., Gerstenecker, C., Menzel, H. and Flohe, L. 1972. Oxidation in the NADP system and release of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides. FEBS Lett. 27: 171-175. Sies, H. and Summer, K.-H. 1975. Hydroperoxide metabolizing
systems in rat liver. Eur. J. Biochem. 57: 503-512. Simmons, T.W. and Jamall, I.S. 1988. Significance of alterations in hepatic antioxidant enzymes; primacy of glutathione peroxidase. Biochem. J. 251: 913-917. Srivastava, S.K., Yogesh, C.A. and Beutler, E. 1974. Useful agents for the study of glutathione metabolism in erythrocytes. Biochem. J. 139: 289-295. Tien, M. and Aust, S.D. 1982. Rabbit liver microsomal lipid peroxidation; the effect of lipid on the rate of peroxidation. Biochim. Biophys. Acta. 712: 1-9. Tietze, F. 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidised glutathione; applications to mammalian blood and other tissues. Anal. Biochem. 27: 507-522. Ursini, F. and Bindoli, A. 1987. The role of selenium peroxidases in the protection against oxidative damage of membranes. Chem. Phys. Lipids 44: 255-276. Walton, M.J. and Cowey, C.B. 1979. Gluconeogenesis by isolated hepatocytes from rainbow trout Salmo gairdneri. Comp. Biochem. Physiol. 62B: 75-79.
