Fish Physiology and Biochemistry vol. 14 no. 4 pp 259-266 (1995) Kugler Publications, Amsterdam/New York
Influence of growth hormone on the hepatic mixed function oxidase and transferase systems of rainbow trout J.P. Cravedil, A. Parisl, E. Perdu-Durandl and P. Prunet 2 1INRA, Laboratoiredes Xinobiotiques, B.P., 3 180, chemin de Tournefeuille, 31931 Toulouse C'dex, France;2 INRA, Laboratoirede Physiologie des Poissons, Campus de Beaulieu, 35042 Rennes, France Accepted: January 12, 1995 Keywords: rainbow trout, cytochrome P450, glutathione-transferase, glucuronosyl-transferase, growth hormone
Abstract The effect of GH treatment on hepatic cytochrome P450 content, aryl hydrocarbon hydroxylase (AHH), aminopyrine-N-demethylase (AND), testosterone hydroxylase, testosterone 5a- and 51-reductase, UDP-glucuronyl transferase (UDPGT) and glutathione S-transferase (GST) activities in immature rainbow trout were investigated. Hepatic cytochrome P450 content, AHH and GST activities were measured in both GH implanted and GH injected animals whereas other activities were assayed in GH implanted trout only. GH implants significantly decreased cytochrome P450 content at 15 days compared to the control but no significant effect was observed at 15 or 30 d when GH was injected biweekly. In both cases, AHH activity was significantly decreased by GH treatment compared to the control whereas GST remained unchanged. Compared to the control, GH implanted fish exhibited a pronounced inhibition of AND, a decreased 61 and 1613-testosterone hydroxylation, an inhibition of UDPGT with testosterone as substrate and an enhanced 17[Itestosterone oxidation.
Introduction In recent years, there have been several studies examining the effect of growth hormone (GH) on mammalian hepatic drug and steroid metabolism, especially through its action on cytochrome P450 (Lund et al. 1991). These studies indicate that GH modulate the expression of several members of the P450 family including P450 1A (Kato et al. 1986), P450 2A (Waxman et al. 1988) P450 2A and 2B (Yamazoe et al. 1987), P450 2C (Yamazoe et al. 1986a; Mode et al. 1989), P450 2E (Williams and Simonet 1987) and P450 3A (Yamazoe et al. 1986a; Waxman et al. 1988). In contrast, the role of this hormone on the regulation of transferases in liver is little known. Staffas et al. (1992) and Srivastava
and Waxman (1993) recently demonstrated in the rat that hepatic glutathione transferases are regulated by GH, whereas Yamazoe et al. (1989) showed that cortisol sulfotransferase in rat liver is under the control of GH. Except for the role of endogenous steroids, little is known in fish about the regulation of the mixed function oxidase and transferase systems by hormones. The present study was designed to determine quantitative differences, if any, in the activity of phase I and phase II steroid and xenobiotic metabolizing enzymes in rainbow trout (Oncorhynchus mykiss) treated with ovine GH. This mammalian GH is known to promote growth (Foster et al. 1991) and to bind to hepatic GH receptors (Yao et al. 1991) in trout.
260 The hormonal regulation of hepatic cytochrome P450 - dependent monooxygenases, UDP-glucuronyltransferases and glutathione transferases was investigated by measuring the catalytic activities of these enzymes.
Materials and methods Chemicals 4-Nitro[U-14C]phenol was obtained from 3 Amersham, Les Ulis, France. [ H]benzo(a)pyrene and [4-14C]testosterone were purchased from C.E.A., Saclay, France. [4-14 C] oestrone was from NEN, Boston, MA, USA. Unlabelled 4-nitrophenol and benzo(a)pyrene (BP) were from Fluka, Bucks, Switzerland. Aminopyrine was from Carlo Erba, Milano, Italy. Unlabelled steroids were from Sigma-Chimie, excepted 6-OH-testosterone and 1613-OH-testosterone from Steraloids Inc, Wilton, N.H., U.S.A. Uridine 5' -diphosphoglucuronic acid, reduced glutathione, cytochrome c and NADPH were obtained from Boehringer-Mannheim, Meylan, France. l-chloro2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) were from Sigma. trans-4-Phenyl-3-buten-2one (tPBO) was from Aldrich-Chimie, Steinheim, Germany. Ovine GH (oGH) was a generous gift from Dr. S. Raiti, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (National Hormone and Pituitary Program, NIH, USA).
Animal experiments Two separate experiments were conducted, using 35-37 g (experiment I) and 140-150 g (experiment II) rainbow trout. Once a day and throughout the experiments animals were fed ad libitum a pelleted commercial feed. The water temperature was 14 ±+ 1°C and the fish were maintained under natural photo-periodic conditions. In Experiment I, 80 trout were treated biweekly by one of the following i.p. injection: saline (NaCI 0.15 M, NaHCO 3 0.03 M, pH 9.5) or oGH (5 pg
g-' body weight dissolved in NaHCO 3 0.03 M, NaCl 0.15 M, pH 9.5). Fish were sampled on days 15 and 30 of the trial and the liver was excised, rinsed in ice-cold 100 mM potassium phosphate (pH 7.4) containing 1.15% KCI and immediately frozen in liquid nitrogen until further processed. In Experiment II, oGH was administered to 50 trout via an intraperitoneal cholesterol implant (Foster et al. 1991) containing 1 mg of hormone. The animals of the control group (n= 50) each received a single cholesterol pellet containing no oGH. Fish were maintained under these conditions for 15 days, after which they were killed. The liver was removed from each animal and stored as indicated in Experiment I.
Enzyme assays Microsomes and cytosol were prepared from perfused livers as previously described (Perdu-Durand and Cravedi 1989). Cytochrome P450 was measured by the method of Johannesen and Depierre (1978). The microsomal aryl hydrocarbon hydroxylase (AHH) was assayed with [3 H]-benzo(a)pyrene as substrate (DePierre et al. 1975). Aminopyrine-N-demethylase (AND) was assayed spectrophotometrically by determination of formaldehyde (La Du et al. 1955). Testosterone hydroxylation was measured by incubation of microsomes (2 mg of protein, 1 ml 0.1 M HEPES buffer pH 7.4) with 100 pIM labeled testosterone (in 10 pl methanol), NADPH and NADPH-generating system containing glucose 6-phosphate and glucose 6-phosphate dehydrogenase, for 2h at 25°C. The reaction was stopped by addition of dichloromethane. The metabolites and parent compound were immediately extracted with 2 x 3 ml of dichloromethane. The extraction was completed with 3 ml ethyl acetate. Testosterone metabolites were separated by HPLC as described by Wood et al. (1983), and by TLC as described by Waxman et al. (1983), identified by co-chromatography with authentic standards or GC/MS analysis and quantitated using 14C activity monitoring. p-Nitrophenol-UDP-glucuronyl-transferase (UDPGT) was measured by a radiochemical
261 lation counter (Packard Instrument Co., Downers Grove, IL, USA) after use of Ultima Gold (Packard Instrument Co.) for the cocktail. HPLC was performed on a Pye Unicam 4110 system equipped with reverse phase ODS 2 column (180 x 4.6 mm, 3 gim). A flow one/jB-radioactivity flow detector (Packard Instrument Co.) was used for 14C monitoring. GC/MS analyses were performed on a Delsi DN 200 gas chromatograph interfaced with a Nermag R10- 10 mass spectrometer working in the electron
0 0 c
o U
0 z
Z O-
P450
AHH
GST
P450
AHH
GST
Fig. 1. Effect of animal treatment with oGH (5 mg/kg, i.p., biweekly) on hepatic drug-metabolizing enzymes. Values are means + SD expressed as percent of concomitant controls and are calculated from 3 pools of 6 samples each. Open bars represent control samples and striped bars are for 0.025 treated samples. The mean control values were 0.157 and 0.257 + 0.050 nmol/mg protein for d15 and d30 cytochrome P450 levels respectively, 103.3 20.8 and 120.0 + 26.5 pmol/min/mg protein for d15 and d30 AHH activities respectively and 784.0 95.8 and 883.0 + 80.3 nmol/min/mg protein for d15 and d30 GST activities respectively. Asterisks (*) indicate significant (p
Influence of growth hormone on the hepatic mixed function oxidase and transferase systems of rainbow trout J.P. Cravedil, A. Parisl, E. Perdu-Durandl and P. Prunet 2 1INRA, Laboratoiredes Xinobiotiques, B.P., 3 180, chemin de Tournefeuille, 31931 Toulouse C'dex, France;2 INRA, Laboratoirede Physiologie des Poissons, Campus de Beaulieu, 35042 Rennes, France Accepted: January 12, 1995 Keywords: rainbow trout, cytochrome P450, glutathione-transferase, glucuronosyl-transferase, growth hormone
Abstract The effect of GH treatment on hepatic cytochrome P450 content, aryl hydrocarbon hydroxylase (AHH), aminopyrine-N-demethylase (AND), testosterone hydroxylase, testosterone 5a- and 51-reductase, UDP-glucuronyl transferase (UDPGT) and glutathione S-transferase (GST) activities in immature rainbow trout were investigated. Hepatic cytochrome P450 content, AHH and GST activities were measured in both GH implanted and GH injected animals whereas other activities were assayed in GH implanted trout only. GH implants significantly decreased cytochrome P450 content at 15 days compared to the control but no significant effect was observed at 15 or 30 d when GH was injected biweekly. In both cases, AHH activity was significantly decreased by GH treatment compared to the control whereas GST remained unchanged. Compared to the control, GH implanted fish exhibited a pronounced inhibition of AND, a decreased 61 and 1613-testosterone hydroxylation, an inhibition of UDPGT with testosterone as substrate and an enhanced 17[Itestosterone oxidation.
Introduction In recent years, there have been several studies examining the effect of growth hormone (GH) on mammalian hepatic drug and steroid metabolism, especially through its action on cytochrome P450 (Lund et al. 1991). These studies indicate that GH modulate the expression of several members of the P450 family including P450 1A (Kato et al. 1986), P450 2A (Waxman et al. 1988) P450 2A and 2B (Yamazoe et al. 1987), P450 2C (Yamazoe et al. 1986a; Mode et al. 1989), P450 2E (Williams and Simonet 1987) and P450 3A (Yamazoe et al. 1986a; Waxman et al. 1988). In contrast, the role of this hormone on the regulation of transferases in liver is little known. Staffas et al. (1992) and Srivastava
and Waxman (1993) recently demonstrated in the rat that hepatic glutathione transferases are regulated by GH, whereas Yamazoe et al. (1989) showed that cortisol sulfotransferase in rat liver is under the control of GH. Except for the role of endogenous steroids, little is known in fish about the regulation of the mixed function oxidase and transferase systems by hormones. The present study was designed to determine quantitative differences, if any, in the activity of phase I and phase II steroid and xenobiotic metabolizing enzymes in rainbow trout (Oncorhynchus mykiss) treated with ovine GH. This mammalian GH is known to promote growth (Foster et al. 1991) and to bind to hepatic GH receptors (Yao et al. 1991) in trout.
260 The hormonal regulation of hepatic cytochrome P450 - dependent monooxygenases, UDP-glucuronyltransferases and glutathione transferases was investigated by measuring the catalytic activities of these enzymes.
Materials and methods Chemicals 4-Nitro[U-14C]phenol was obtained from 3 Amersham, Les Ulis, France. [ H]benzo(a)pyrene and [4-14C]testosterone were purchased from C.E.A., Saclay, France. [4-14 C] oestrone was from NEN, Boston, MA, USA. Unlabelled 4-nitrophenol and benzo(a)pyrene (BP) were from Fluka, Bucks, Switzerland. Aminopyrine was from Carlo Erba, Milano, Italy. Unlabelled steroids were from Sigma-Chimie, excepted 6-OH-testosterone and 1613-OH-testosterone from Steraloids Inc, Wilton, N.H., U.S.A. Uridine 5' -diphosphoglucuronic acid, reduced glutathione, cytochrome c and NADPH were obtained from Boehringer-Mannheim, Meylan, France. l-chloro2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) were from Sigma. trans-4-Phenyl-3-buten-2one (tPBO) was from Aldrich-Chimie, Steinheim, Germany. Ovine GH (oGH) was a generous gift from Dr. S. Raiti, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (National Hormone and Pituitary Program, NIH, USA).
Animal experiments Two separate experiments were conducted, using 35-37 g (experiment I) and 140-150 g (experiment II) rainbow trout. Once a day and throughout the experiments animals were fed ad libitum a pelleted commercial feed. The water temperature was 14 ±+ 1°C and the fish were maintained under natural photo-periodic conditions. In Experiment I, 80 trout were treated biweekly by one of the following i.p. injection: saline (NaCI 0.15 M, NaHCO 3 0.03 M, pH 9.5) or oGH (5 pg
g-' body weight dissolved in NaHCO 3 0.03 M, NaCl 0.15 M, pH 9.5). Fish were sampled on days 15 and 30 of the trial and the liver was excised, rinsed in ice-cold 100 mM potassium phosphate (pH 7.4) containing 1.15% KCI and immediately frozen in liquid nitrogen until further processed. In Experiment II, oGH was administered to 50 trout via an intraperitoneal cholesterol implant (Foster et al. 1991) containing 1 mg of hormone. The animals of the control group (n= 50) each received a single cholesterol pellet containing no oGH. Fish were maintained under these conditions for 15 days, after which they were killed. The liver was removed from each animal and stored as indicated in Experiment I.
Enzyme assays Microsomes and cytosol were prepared from perfused livers as previously described (Perdu-Durand and Cravedi 1989). Cytochrome P450 was measured by the method of Johannesen and Depierre (1978). The microsomal aryl hydrocarbon hydroxylase (AHH) was assayed with [3 H]-benzo(a)pyrene as substrate (DePierre et al. 1975). Aminopyrine-N-demethylase (AND) was assayed spectrophotometrically by determination of formaldehyde (La Du et al. 1955). Testosterone hydroxylation was measured by incubation of microsomes (2 mg of protein, 1 ml 0.1 M HEPES buffer pH 7.4) with 100 pIM labeled testosterone (in 10 pl methanol), NADPH and NADPH-generating system containing glucose 6-phosphate and glucose 6-phosphate dehydrogenase, for 2h at 25°C. The reaction was stopped by addition of dichloromethane. The metabolites and parent compound were immediately extracted with 2 x 3 ml of dichloromethane. The extraction was completed with 3 ml ethyl acetate. Testosterone metabolites were separated by HPLC as described by Wood et al. (1983), and by TLC as described by Waxman et al. (1983), identified by co-chromatography with authentic standards or GC/MS analysis and quantitated using 14C activity monitoring. p-Nitrophenol-UDP-glucuronyl-transferase (UDPGT) was measured by a radiochemical
261 lation counter (Packard Instrument Co., Downers Grove, IL, USA) after use of Ultima Gold (Packard Instrument Co.) for the cocktail. HPLC was performed on a Pye Unicam 4110 system equipped with reverse phase ODS 2 column (180 x 4.6 mm, 3 gim). A flow one/jB-radioactivity flow detector (Packard Instrument Co.) was used for 14C monitoring. GC/MS analyses were performed on a Delsi DN 200 gas chromatograph interfaced with a Nermag R10- 10 mass spectrometer working in the electron
0 0 c
o U
0 z
Z O-
P450
AHH
GST
P450
AHH
GST
Fig. 1. Effect of animal treatment with oGH (5 mg/kg, i.p., biweekly) on hepatic drug-metabolizing enzymes. Values are means + SD expressed as percent of concomitant controls and are calculated from 3 pools of 6 samples each. Open bars represent control samples and striped bars are for 0.025 treated samples. The mean control values were 0.157 and 0.257 + 0.050 nmol/mg protein for d15 and d30 cytochrome P450 levels respectively, 103.3 20.8 and 120.0 + 26.5 pmol/min/mg protein for d15 and d30 AHH activities respectively and 784.0 95.8 and 883.0 + 80.3 nmol/min/mg protein for d15 and d30 GST activities respectively. Asterisks (*) indicate significant (p
