SHORT COMMUNICATION Research in Veterinary Science 1991, 51, 120-122
Effect of chemicals on glutathione peroxidase of chick liver S. MIYAZAKI, Feed Safety Research Division, National Institute o f Animal Health, 3-1-1, Kannondai, Tsukuba, Ibaraki 305, Japan
Chick liver glutathione peroxidase activity was separate~ into selenium-dependent and selenium-independent enzyme fractions and the effects of chemicals on the activities of each fraction were examined. Clofibrate induced the seleniumdependent enzyme, while phenobarbital, butylhydroxyanisole and trans.stilbene oxide elevated seleniumindependent peroxidase activity. A third enzyme, which showed glutathione peroxidase activity toward both hydrogen peroxide and cumene hydroperoxide, was found. GLUTATHIONE peroxidase catalyses the reduction of hydrogen peroxide or lipid hydroperoxides using reduced glutathione as a hydrogen source. This system is important for protecting cells from oxidative damage. As selenium (Se) is an essential component of glutathione peroxidase (Forstrom et al 1978), the enzyme content is decreased when the animals are fed a selenium-deficient diet. However, another glutathione peroxidase activity in selenium-deficient rat liver has been detected (Lawrence and Burk 1976). This selenium-independentglutathione peroxidase was identified as glutathione S-transferase by Prohaska and Ganther (1977). Glutathione S-transferase catalyses the reduction of lipid hydroperoxides but does not reduce hydrogen peroxide. In this study, the quantitative relationship was determined in glutathione peroxidase activity for broiler chick liver selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase, glutathione S-transferase (non-Se-GSH-Px). The effects of the treatment of chemicals on these two enzymes were also examined. A third glutathione peroxidase activity in chick liver cytosol was found in the course of the present examination. Male one-day-old broiler chicks, from the local hatchery, received vaccines for Marek's disease and fowl pox. Threeto four-week-old chicks were used for all experiments. To determine the effects of chemicals on glutathione peroxidase activity, sodium phenobarbital (PB), butylhydroxyanisole (BHA), 3-methylcholanthrene (MC), transstilbene oxide (sao) and clofibrate (CLF) were administered to chicks. PB was added in the drinking water (0-05 per cent w/v) for 10 days. BHAand CLF were added to the diet at concentrations of 6 g kg- ] for five days and 1-7 g kg- l for 12 days, respectively. SBO (400 mg kg-1 bodyweight) and MC (20 mg kg-1 bodyweight) were dissolved in corn oil and given by daily intraperitoneal injection for four consecutive days. One group of chicks was untreated. Each group consisted of seven chicks and they were allowed free access to the diet and water throughout the experiments. The chicks
were killed by bleeding and livers were perfused with ice-cold saline. The removed livers were frozen in liquid nitrogen and kept at - 7 0 ° C until analysis. Thawed livers were homogenised in ice-cold 0.25 M sucrose by means of a teflon homogeniser to obtain 20 per cent (w/v) homogenates: these were centrifuged at 100,000 g for 60 minutes at 4°C. The supernatants were used as cytosol fractions in the studies. Glutathione peroxidase activity was assayed by the method of Lawrence and Burk (1976). Hydrogen peroxide or cumene hydroperoxide was used as a substrate and final concentrations were 0"25 mM and 1-5 raM, respectively. Enzyme activity was expressed as nmoles nicotinamide adenine dinucleotide phosphate (NADPH) oxidised per minute. Protein concentrations were estimated according to Lowry et al (1951). Glutathione peroxidase activities in control- and drugtreated chicks are shown in Table 1. Glutathione peroxidase activity toward hydrogen peroxide represents the activity of Se-GSH-Px, while the activity toward cumene hydroperoxide shows the total activity of Se-GSH-Px and non-SeGSH-Px. PB, MC and SBO depressed Se-GSH-Px as reported in rats and mice (Carlberg et a11981, Di Simplicio et a11989). Increased production of hydrogen peroxide is expected in animals which receive inducers of drug metabolising enzymes as a result of induced oxidase activity in the microsome. It is interesting that Se-GSH-Px activity, one of the hydrogen peroxide r~ducing systems, was depressed in the animals receiving such chemicals. On the other hand, CLF significantly elevated Se-GSH-Px activity. This result does not agree with the decreased Se-GSH-Px activity in peroxisome proliferator-treated rats (Tamura et al 1990). CLF, a
TABLE 1: Liver glutathione peroxidase (GSH-Px) activity of normal and chicks treated with drugs
Treatment Control Phenobarbital Butylhydroxyanisole Clofibrate 3-methylcholanthrene
Trans-stilbeneoxide
GSH-Px activity (nmol rain - 1 m g - 1) Substrate CHPOCHPO H202 H202 174 304* * 265"* 222* 155 394**
104 86.6* 97.7 166" 74.2* 78-8*
70.0 217* * 167" 56.4 80.5 315"*
Significant difference * P < O. 05, * * P < O- 01 from control CHPO Cumene hydroperoxide H 2 0 2 Hydrogen peroxide
120
Glutathione peroxidase in chick liver
121
Px was reduced or unaffected in chicks treated with PB, BHA or SBO, elevated glutathione peroxidase activity toward cumene hydroperoxide in chicks which received these chemicals suggests the induction of glutathione S-transr ferase by these chemicals. In contrast with the present result, 100 Di Simplicio et al (1989) observed a reduction of non-SeGSH-Px in mice which received PB, BHA,MC or SBO. F~ To estimate the exact activity of non-Se-GSH-Px, non-SeGSH-Px was separated from Se-GSH-Px by gel permeation ,g 50 chromatography. For chromatographic analyses, cytosol fractions of each group were pooled and passed through Sepharose 12 HR, 10/30 column (Pharmacia). Proteins were eluted with 50 mM potassium phosphate, 150 mM potassium chloride, 0-5 mM EDTA, 5 mM 2-mercaptoethanol, pH 7" 6. 0 The flow rate was 0" 5 ml rain- 1and eluate was collected for 45 50 55 60 65 70 30 second fractions. Fractionnumber As shown in Fig la, normal chick liver glutathione peroxidase activity was separated into three different molecular weight peaks. The first peak and the third peak showed activities toward both hydrogen peroxide and cumene hydroperoxide, while the second peak showed activity only toward cumene hydroperoxide. Molecular weights of each peak were estimated as 80, 45 and 20 kDa, respectively, by using standard molecular weight proteins. E 200 From the estimated molecular weights and the activity toward hydrogen peroxide, the first peak and the second peak were considered to be Se-GSH-Px and non-Se-GSHPx, respectively. As for the third peak, a rnonomeric enzyme of 18"5 kDa was partially purified from liver cytosol (S. Miyazaki, unpublished data) and is now under examination. By the administration of eLF, glutathione peroxidase 0 ---T .... 1" activity in the first peak was significantly elevated (Fig lb) 55 60 65 70 45 50 Fraction number but the areas of the second peak and the third peak were not changed. In spite of the activity toward hydrogen peroxide BOO- c common to the first peak and the third peak in the controls, the third peak did not respond to CLF treatment. This indicates that different mechanisms are involved in the expression of these two enzymes which reduce hydrogen E 400 peroxide. i Following sso treatment, the second peak was increased, .=_ E while the areas of the first and third peaks were little changed (Fig lc). Similar results were obtained in chicks which received PB or BHA. In all groups fractions in the first and third peaks showed equal activity values toward both hydrogen peroxide and cumene hydroperoxide. This means that the differences between the activities toward cumene hydroperoxide and those toward hydrogen peroxide shown 0 in Table 1 are equal to the areas of the second peaks, namely 45 50 55 60 65 70 the activities of non-Se-GSH-Px. These observations conFraction number firmed the induction of non-Se-GSH-Px in PB, BHA or SBOFIG 1: Gel permeation chromatographic profiles of glutathione treated chicks. peroxidases of control (a), clofibrate (b) and trans-stilbene oxide The present results show that Se-GSH-Px activity and (c) treated chick liver. Enzyme activity was determined by using non-Se-GSH-Px activity are changed by the administration hydrogen peroxide (©) or cumene hydroperoxide ( 0 ) as a of chemicals in a different manner from that observed in substrate mammals. Ongoing work is being directed to examine the enzymatic nature of monomeric glutathione peroxidase peroxisome proliferator, is known to induce peroxisomal found in the third peak. fatty acyl-CoA oxidase activity followed by the increased generation of hydrogen peroxide (Tamura et al 1990). An increase in Se-GSH-Px activity of CLF administered chicks References might be a protective response against the oxidative stress in CARLBERG, I., DEPIERRE, J. W. & MANNERVIK, B. (1981) CLF-treated chicks. Effect of inducers of drug-metabolizingenzymeson glutathione Glutathione peroxidase activity toward cumene hydroreductase and glutathione peroxidase in rat liver. Bioehimica et Biophysica Acta 677, 140-145 peroxide was induced by PB, BHA,SBO and eLF. As Se-GSH150-
a
4001 b
°t
~"20(3
122
S. M i y a z a k i
DI SIMPLICIO, P., JENSSON, H. & MANNERVIK, B. (1989) Effects of inducers of drug metabolism on basic hepatic forms of mouse glutathione transferase. Biochemical Journal 263,679-685 FORSTROM, J. W., ZAKOWSKI, J. J. & TAPPEL, A. L. (1978) Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine. Biochemistry 17, 2639-2644 LAWRENCE, R. A. & BURK, R. F. (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochemical and Biophysical Research Communications 71,952-958 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275
PROHASKA, J. R. & GANTHER, H. E. (1977) Glutathione peroxidase activity of glutathione S-transferases purified from rat liver. Biochemical and Biophysical Research Communications 76, 437 -445 TAMURA, H., IIDA, T., WATANABE, T.& SUGA, T. (1990) Long-term effects of peroxisome proliferators on the balance between hydrogen peroxide-generating and scavenging capacities in the liver of Fischer-344 rats. Toxicology 63, 199-213
Received January 29, 1991 Accepted February 25, 1991
Effect of chemicals on glutathione peroxidase of chick liver S. MIYAZAKI, Feed Safety Research Division, National Institute o f Animal Health, 3-1-1, Kannondai, Tsukuba, Ibaraki 305, Japan
Chick liver glutathione peroxidase activity was separate~ into selenium-dependent and selenium-independent enzyme fractions and the effects of chemicals on the activities of each fraction were examined. Clofibrate induced the seleniumdependent enzyme, while phenobarbital, butylhydroxyanisole and trans.stilbene oxide elevated seleniumindependent peroxidase activity. A third enzyme, which showed glutathione peroxidase activity toward both hydrogen peroxide and cumene hydroperoxide, was found. GLUTATHIONE peroxidase catalyses the reduction of hydrogen peroxide or lipid hydroperoxides using reduced glutathione as a hydrogen source. This system is important for protecting cells from oxidative damage. As selenium (Se) is an essential component of glutathione peroxidase (Forstrom et al 1978), the enzyme content is decreased when the animals are fed a selenium-deficient diet. However, another glutathione peroxidase activity in selenium-deficient rat liver has been detected (Lawrence and Burk 1976). This selenium-independentglutathione peroxidase was identified as glutathione S-transferase by Prohaska and Ganther (1977). Glutathione S-transferase catalyses the reduction of lipid hydroperoxides but does not reduce hydrogen peroxide. In this study, the quantitative relationship was determined in glutathione peroxidase activity for broiler chick liver selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase, glutathione S-transferase (non-Se-GSH-Px). The effects of the treatment of chemicals on these two enzymes were also examined. A third glutathione peroxidase activity in chick liver cytosol was found in the course of the present examination. Male one-day-old broiler chicks, from the local hatchery, received vaccines for Marek's disease and fowl pox. Threeto four-week-old chicks were used for all experiments. To determine the effects of chemicals on glutathione peroxidase activity, sodium phenobarbital (PB), butylhydroxyanisole (BHA), 3-methylcholanthrene (MC), transstilbene oxide (sao) and clofibrate (CLF) were administered to chicks. PB was added in the drinking water (0-05 per cent w/v) for 10 days. BHAand CLF were added to the diet at concentrations of 6 g kg- ] for five days and 1-7 g kg- l for 12 days, respectively. SBO (400 mg kg-1 bodyweight) and MC (20 mg kg-1 bodyweight) were dissolved in corn oil and given by daily intraperitoneal injection for four consecutive days. One group of chicks was untreated. Each group consisted of seven chicks and they were allowed free access to the diet and water throughout the experiments. The chicks
were killed by bleeding and livers were perfused with ice-cold saline. The removed livers were frozen in liquid nitrogen and kept at - 7 0 ° C until analysis. Thawed livers were homogenised in ice-cold 0.25 M sucrose by means of a teflon homogeniser to obtain 20 per cent (w/v) homogenates: these were centrifuged at 100,000 g for 60 minutes at 4°C. The supernatants were used as cytosol fractions in the studies. Glutathione peroxidase activity was assayed by the method of Lawrence and Burk (1976). Hydrogen peroxide or cumene hydroperoxide was used as a substrate and final concentrations were 0"25 mM and 1-5 raM, respectively. Enzyme activity was expressed as nmoles nicotinamide adenine dinucleotide phosphate (NADPH) oxidised per minute. Protein concentrations were estimated according to Lowry et al (1951). Glutathione peroxidase activities in control- and drugtreated chicks are shown in Table 1. Glutathione peroxidase activity toward hydrogen peroxide represents the activity of Se-GSH-Px, while the activity toward cumene hydroperoxide shows the total activity of Se-GSH-Px and non-SeGSH-Px. PB, MC and SBO depressed Se-GSH-Px as reported in rats and mice (Carlberg et a11981, Di Simplicio et a11989). Increased production of hydrogen peroxide is expected in animals which receive inducers of drug metabolising enzymes as a result of induced oxidase activity in the microsome. It is interesting that Se-GSH-Px activity, one of the hydrogen peroxide r~ducing systems, was depressed in the animals receiving such chemicals. On the other hand, CLF significantly elevated Se-GSH-Px activity. This result does not agree with the decreased Se-GSH-Px activity in peroxisome proliferator-treated rats (Tamura et al 1990). CLF, a
TABLE 1: Liver glutathione peroxidase (GSH-Px) activity of normal and chicks treated with drugs
Treatment Control Phenobarbital Butylhydroxyanisole Clofibrate 3-methylcholanthrene
Trans-stilbeneoxide
GSH-Px activity (nmol rain - 1 m g - 1) Substrate CHPOCHPO H202 H202 174 304* * 265"* 222* 155 394**
104 86.6* 97.7 166" 74.2* 78-8*
70.0 217* * 167" 56.4 80.5 315"*
Significant difference * P < O. 05, * * P < O- 01 from control CHPO Cumene hydroperoxide H 2 0 2 Hydrogen peroxide
120
Glutathione peroxidase in chick liver
121
Px was reduced or unaffected in chicks treated with PB, BHA or SBO, elevated glutathione peroxidase activity toward cumene hydroperoxide in chicks which received these chemicals suggests the induction of glutathione S-transr ferase by these chemicals. In contrast with the present result, 100 Di Simplicio et al (1989) observed a reduction of non-SeGSH-Px in mice which received PB, BHA,MC or SBO. F~ To estimate the exact activity of non-Se-GSH-Px, non-SeGSH-Px was separated from Se-GSH-Px by gel permeation ,g 50 chromatography. For chromatographic analyses, cytosol fractions of each group were pooled and passed through Sepharose 12 HR, 10/30 column (Pharmacia). Proteins were eluted with 50 mM potassium phosphate, 150 mM potassium chloride, 0-5 mM EDTA, 5 mM 2-mercaptoethanol, pH 7" 6. 0 The flow rate was 0" 5 ml rain- 1and eluate was collected for 45 50 55 60 65 70 30 second fractions. Fractionnumber As shown in Fig la, normal chick liver glutathione peroxidase activity was separated into three different molecular weight peaks. The first peak and the third peak showed activities toward both hydrogen peroxide and cumene hydroperoxide, while the second peak showed activity only toward cumene hydroperoxide. Molecular weights of each peak were estimated as 80, 45 and 20 kDa, respectively, by using standard molecular weight proteins. E 200 From the estimated molecular weights and the activity toward hydrogen peroxide, the first peak and the second peak were considered to be Se-GSH-Px and non-Se-GSHPx, respectively. As for the third peak, a rnonomeric enzyme of 18"5 kDa was partially purified from liver cytosol (S. Miyazaki, unpublished data) and is now under examination. By the administration of eLF, glutathione peroxidase 0 ---T .... 1" activity in the first peak was significantly elevated (Fig lb) 55 60 65 70 45 50 Fraction number but the areas of the second peak and the third peak were not changed. In spite of the activity toward hydrogen peroxide BOO- c common to the first peak and the third peak in the controls, the third peak did not respond to CLF treatment. This indicates that different mechanisms are involved in the expression of these two enzymes which reduce hydrogen E 400 peroxide. i Following sso treatment, the second peak was increased, .=_ E while the areas of the first and third peaks were little changed (Fig lc). Similar results were obtained in chicks which received PB or BHA. In all groups fractions in the first and third peaks showed equal activity values toward both hydrogen peroxide and cumene hydroperoxide. This means that the differences between the activities toward cumene hydroperoxide and those toward hydrogen peroxide shown 0 in Table 1 are equal to the areas of the second peaks, namely 45 50 55 60 65 70 the activities of non-Se-GSH-Px. These observations conFraction number firmed the induction of non-Se-GSH-Px in PB, BHA or SBOFIG 1: Gel permeation chromatographic profiles of glutathione treated chicks. peroxidases of control (a), clofibrate (b) and trans-stilbene oxide The present results show that Se-GSH-Px activity and (c) treated chick liver. Enzyme activity was determined by using non-Se-GSH-Px activity are changed by the administration hydrogen peroxide (©) or cumene hydroperoxide ( 0 ) as a of chemicals in a different manner from that observed in substrate mammals. Ongoing work is being directed to examine the enzymatic nature of monomeric glutathione peroxidase peroxisome proliferator, is known to induce peroxisomal found in the third peak. fatty acyl-CoA oxidase activity followed by the increased generation of hydrogen peroxide (Tamura et al 1990). An increase in Se-GSH-Px activity of CLF administered chicks References might be a protective response against the oxidative stress in CARLBERG, I., DEPIERRE, J. W. & MANNERVIK, B. (1981) CLF-treated chicks. Effect of inducers of drug-metabolizingenzymeson glutathione Glutathione peroxidase activity toward cumene hydroreductase and glutathione peroxidase in rat liver. Bioehimica et Biophysica Acta 677, 140-145 peroxide was induced by PB, BHA,SBO and eLF. As Se-GSH150-
a
4001 b
°t
~"20(3
122
S. M i y a z a k i
DI SIMPLICIO, P., JENSSON, H. & MANNERVIK, B. (1989) Effects of inducers of drug metabolism on basic hepatic forms of mouse glutathione transferase. Biochemical Journal 263,679-685 FORSTROM, J. W., ZAKOWSKI, J. J. & TAPPEL, A. L. (1978) Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine. Biochemistry 17, 2639-2644 LAWRENCE, R. A. & BURK, R. F. (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochemical and Biophysical Research Communications 71,952-958 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275
PROHASKA, J. R. & GANTHER, H. E. (1977) Glutathione peroxidase activity of glutathione S-transferases purified from rat liver. Biochemical and Biophysical Research Communications 76, 437 -445 TAMURA, H., IIDA, T., WATANABE, T.& SUGA, T. (1990) Long-term effects of peroxisome proliferators on the balance between hydrogen peroxide-generating and scavenging capacities in the liver of Fischer-344 rats. Toxicology 63, 199-213
Received January 29, 1991 Accepted February 25, 1991
