- Email: [email protected]
A comparative study of some oxidative and conjugative drug metabolizing enzymes in liver, lung and kidney of sheep
Camp. Biochem. Physiol.Vol. 89C, No. 2, pp. 225-228, 1988
0306-4492/88 $3.00 + 0.00
0 1988Pergamon Press plc
Printed in Great Britain
A COMPARATIVE STUDY OF SOME OXIDATIVE AND CONJUGATIVE DRUG METABOLIZING ENZYMES IN LIVER, LUNG AND KIDNEY OF SHEEP G. LARRIEU and P. GALTIER Laboratoire de Pharmacologic-Toxicologic,
INRA, 31931 Toulouse, France (Tel.: 1661-49-1686)
(Received 4 March 1987)
The comparative distribution of cytochrome P-450 monooxygenase system, glucuronyltransferase, glutathione S-transferase and N-acetyltransferase was studied in the liver, lung and kidney of young male sheep. 2. The sheep liver was characterized by a lack in glutathione S-transferase activity with isoniazid as substrate. 3. The oxidative drug metabolizing enzymes of lung were generally close to those of liver; benzphetamine N-demethylase and ethoxycoumarin O-deethylase were even found to be higher in lung (213 and 148%, respectively). 4. Pulmonary conjugative and both renal oxidative and conjugative systems accounted only for 9-38% of hepatic corresponding enzymes. 5. The enzyme determination in various sampling sites of the three organs, demonstrated the homogeneous distribution of all investigated monooxygenases and transferases in liver, lung and kidney of sheep.
Abstract-l.
INTRODUCTION
the liver has long been acknowledged as the major site of drug metabolism, however, many recent studies have emphasized the importance of extrahepatic tissues in the biotransformation of xenobiotics and endogenous substances. These studies resulted generally in comparisons of the enzymatic equipment of liver with that of organs which have been recognized as the portals of entry or excretion of the body, namely the lung, kidney, skin or gastrointestinal mucosa. Thus, foreign compounds are converted by the extrahepatic metabolism to either active or inactive forms preceding their entry into the circulating system and access to the target tissue. In the case of ruminant breeding species, there is a need for information concerning such comparative data because of the increasing rise of veterinary drugs and the involvement of these enzymatic systems in the occurrence of drug residues in edible tissues. In sheep, most of the studies described the liver oxidative and conjugative drug metabolizing enzymes in control animals (Smith et al., 1984) and in animals under particular physiological states (Dvorchik et al., 1986). Recently, detailed kinetic properties of sheep liver and lung microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase were described (Arinc and Iscan, 1983; Arinc, 1985). In investigating the species susceptibility to the lung toxicity of perilla ketone, Garst et al. (1985) provided relevant information concerning ovine pulmonary monooxygenases. Therefore, in attempting to obtain comparative information on the activities of drug metabolizing enzymes in ovine tissues, we have studied the ability of microsomal and cytosolic fractions of liver, lung and kidney from young male sheep to metabolize a
variety of drug substrates. Findings have been discussed in relation to data obtained in other animal species.
In mammals,
MATERIALS
AND METHODS
Chemicals
Ethoxycoumarin and acetylcoenzyme A were obtained from Boehringer Mannheim, Meylan, France. Aminopyrine, aniline, 1-chloro-2,4-dinitrobenzene, isoniazid, pnitrophenol and cytochrome C were purchased from Sigma Chemical Co., St Louis, MO, USA. Benzphetamine was kindly provided by Upjohn Laboratories, Le Vaudreuil, France. Glycerol, sodium dithionite (Na,S,O,) and glutathione (reduced) were the products of E. Merck, Darmstadt, Germany. Dichloroacetyl (1, 14C) chloramphenicol (52 mCi/nmol) was purchased from DuPont NEN France, Paris, France. Complete scintillation cocktail Lumagel was obtained from Kontron, Velizy, France. All other chemicals were the highest quality available. Desionized distilled water was used in all studies. Preparation of microsomal and cytosolic fractions The livers, lungs and kidneys from well bled Lacaune young male sheep (about 3-4 months old) were obtained immediately after killing in the laboratory. The organs were weighed, washed with ice-cold saline solution and blotted free of excess moisture. All subsequent operations were carried out at 04°C. The livers were then divided into left, right and caudate lobes in order to check the enzyme distribution within the hepatic parenchyma. Tissue blocks were obtained from 10 different sites of the lamb liver: 3 from the left lobe, 6 from the right lobe and 1 from the caudate lobe. In the case of lungs, 3 tissue blocks were samples from the right lobe and 2 from the left whereas for the kidneys 3 blocks were selected from each left or right kidney.
225
226
G. LARRIEUand P. GALTIER
Randomized 8 g samples from each tissue block were homogenized with 22ml of ice-cold solution of EDTA (I mM) and hydroxytoluene (20pM) in 0.1 M potassium phosphate buffer (PH 7.4) in a glass Potter homogenizer with a Teflon pestle. The homogenate was centrifuged at 10,000 g for 30 min in a Jouan model K 101 centrifuge. The supematant fraction was centrifuged for 1 hr at 105,OOOgin a Beckman model LS-50 ultracentrifuge, washed with 7.5 ml of 0.1 M sodium pyrophosphate-HCl buffer (pH 7.5) and recentrifuged for 30 min in order to obtain a cleaner microsomal fraction. The microsomal pellets were suspended in an ice-cold solution consisting of EDTA (0.1 mM), glycerol (20%) and potassium phosphate buffer (0.1 M, pH 7.4). The microsomal and cytosolic (first 105,OOOgsupernatant) proteins were determined by the procedure of Lowry et al. (1951), crystalline bovine serum albumin was used as a standard. When enzyme assays were not performed on the day the animals were killed, cytosolic fractions and microsomal pellets were frozen at -80°C. Enzyme assays
The contents of cytochromes P-450 and b5 were measured from the difference spectra between a microsomal sample reduced with sodium dithionite and one gassed with CO (Omura and Sato, 1964). NADPH cytochrome C reductase, aniline hydroxylase and aminopyrine N-demethylase microsomal activities were determined as previously described (Mazel, 1971). Benzphetamine N-demethylase was measured using 2SmM benzphetamine, 5mg of microsomal proteins and an NADPH-generating system (0.5 mM NADP). The formation of formaldehyde was estimated by the Nash reaction modified by Cochin and Axelrod (1959). Ethoxycoumarin 0-deethylase was determined by direct fluorimetry (Aitio, 1978). Microsomal UDP-glucuronyltransferase was assayed employing either 0.7mM pnitrophenol (Frei ef al., 1970) or 10 mM labelled chloramphenicol (Young et al., 1978). I-Chloro-2,4_dinitrobenzene was used as the substrate in the determination of glutathione transferase of liver cytosol (Habig ef al., 1974), and cytosolic acetyltransferase was measured with isoniazid as substrate (Weber, 1971). All enzymes reactions were performed under conditions of maximal velocity with appropriate blanks and were linear with time and protein concentration. Radioactivity was measured by liquid scintillation spectrometry (Intertechnique, model SL 33). Quenching was estimated by automatic external standardization. Statislics
Data were analysed using a two-way analysis of variance with interaction. The two ways corresponded respectively to the distribution of activities between animals and within the various organs or lobes. A complementary range test was Table I.
Microsomal
protein
used to compare the means. A probability of P -C0.05 was considered significant. RESULTS
Under our experimental conditions, microsomal protein concentrations were found to be similar (16-19 mg/g of tissues) in liver, lung and kidney (Table 1) whereas cytochrome P-450 levels were around tenfold lower in extrahepatic organs as compared to its hepatic concentration (0.24 nmol mg-’ of microsomal protein). Although, in general, NADPH cytochrome c reductase, aminopyrine N-demethylase and aniline hydroxylase activities of liver microsomes (26.9,0.202 and 0.156 nmol mg-’ min-‘, respectively) were higher than those of lung microsomes, in some preparations enzymatic activity of lung were comparable to those of liver. Likewise, pulmonary benzphetamine Ndemethylase and ethoxycoumarin 0 -deethylase were 1.5 and 2.1-fold greater than hepatic corresponding monooxygenases (0.760 and 0.645 nmol mg-’ min-‘, respectively). Concerning renal mixed function oxidases, NADPH cytochrome c reductase, aminopyrine N-demethylase and benzphetamine N-demethylase activities corresponded respectively to 27, 19 and 18% of the hepatic enzymes (Fig. 1) whereas ethoxycoumarin 0-deethylase exhibited remarkably low activities (around 0.01 nmol mg-’ min-‘) and aniline hydroxylase was not detected. Microsomal and cytosolic phase II enzymatic systems from sheep liver, lung and kidney are presented in Table 2. As compared to hepatic corresponding activities, pulmonary and renal UDP-glucuronyltransferase accounted for 1S-38% whatever the substrate was used, i.e. p-nitrophenol or chloramphenicol. Liver cytosolic proteins (43.7 mg/g) were 1.2-fold higher than in lung or kidney. Cytosolic glutathione S-transferase activity using 1-chloro-2-4 dinitrobenzene as substrate was about 4-fold and IO-fold more active in liver than in lung and kidney, respectively (Fig. 1). On the other hand, cytosolic N-acetyltransferase activity employing isoniazid as substrate was found to be at a level below the detection limits of the method (0.05nmol mg-’ min-I) regardless of which tissue was investigated. The two-way analysis of variance failed to exhibit any measurable difference between the animals used or
and cytochrome concentrations, and mixed function in liver. luna and kidnev of she&
Parameters Microsomal proteins (mgg-‘) Cytochrome P-450 (nmolmg* microsomal proteins-‘) NADPH cytochrome c reductase (nmolmg-‘min-‘) Aminopytine N-demethylase (nmolmg-‘min-‘) Benzophetamine N-demethylase (nmolmg-‘min-‘) Aniline hydroxylase (nmolmg-‘min-‘) Ethoxycoumarin O-deethylase (nmolmg-‘min-‘) *Values are means f SD from 4 animals liver, lung and kidney, respectively. ND, not detected value.
oxidase systems
Liver
Lung
Kidney
19.5 + 0.6 0.240 f 0.020
18.1 +0.8 0.024 * 0.003
16.3 k 0.5 0.020 + 0.001
26.9 rt 1.5
18.9 f I.0
7.2 f 0.1
0.202 f 0.043
0.108 iO.022
0.038 f 0.004
0.760 5 0.053
1.620 k 0.023
0. I36 It 0.020
0.156 f 0.007
0.086 f 0.005
ND
0.645 + 0.030
0.956 f 0.033
0.014 + 0.002
corresponding
to 40, 20 and 24 tissue samples in case of
Drug metabolism in ovine tissues P-450 GST
NCR
__
EdOD
Fig. 1. Relative enzymatic activity in sheep liver (-), lung(--) and kidney (. . .). Abbreviations: P-450, microsomal cytochrome P-450; NCR, NADPH cytochrome c reductase; AND, aminopyrine N-demethylase; BND, benzphetamine N-demethylase; ECOD, ethoxycoumarin O-deethylase; AH, aniline hydroxylase; GCC, glucuronyl-transferase (substrate:chloramphenical); GCP, glucuronyltransferase (substrate:p-nitrophenol); GST, glutathione S-transferase.
between the sampling tissue sites or lobes; this result led to the homogeneous distribution of all determined activity within each investigated tissue. DISCUSSION
Comparative species studies of tissue oxidative and conjugative biotransformations resulted in numerous data regarding almost all the hepatic activities of rodents, whereas only sporadic data were reported for ruminants. The present study provides information involving 10 enzyme systems and should become a baseline for comparisons of ovine hepatic, pulmonary and renal systems to other animals species. Hepatic enzyme parameters of sheep determined in the present paper are in good agreement with data from control animals used in an experiment describing the incidence of ovine fascioliasis on such activities (Galtier et al., 1986). NADPH cytochrome c reductase activity was similar to the value reported by Dvorchik et al. (1986) in freshly isolated microsomes from the livers of maternal sheep; these authors likewise detected significant N-dealkylating and hydroxylating activities towards methadone, mepiridine, hexobarbital and benzopyrene. Concern-
221
ing hepatic conjugative enzymes in sheep, there is a large variability of response in relation to the nature of substrate, as previously observed by Smith et al. (1984). In this paper, glutathione S-transferase level was close to our values while acetyltransferase activity with 2naphtylamine or 2-aminofluorene as substrates was not found in liver tissues from the bovine or ovine species. This could correspond with our lack of sensitivity for measuring this cytosolic activity. The determination of enzymes in 10 sampling sites resulted in any significant difference between the liver lobes and led to the homogeneous distribution of both oxidative and conjugative drug metabolizing activities within the sheep liver. This conclusion contrasts with the demonstration of a heterogenous distribution of the cytochrome P-450 monooxygenase system in rat liver lobes (Matsubara et al., 1984). Generally, there is a lack of literature noting comparisons with other data concerning the extrahepatic drug metabolism in ovine tissues. Effectively, most information concerns the characterization and kinetic properties of sheep lung microsomal NADPH cytochrome c reductase (Iscan and Arinc, 1986), aniline hydroxylase (Arinc and Iscan, 1983) or ethylmorphine N-demethylase (Arinc, 1985). This interest for ovine pulmonary monooxygenases is in accordance with our observation of high oxidizing-enzyme levels in sheep lung. However, lung microsomal cytochrome P-450 appeared IO-fold lower than in liver. Such lung/liver ratios have been reported in goats (Burley and Bray, 1983), lactating cattle (Shull et al., 1986) and in rodents (Litterst et al., 1975). The role of cytochrome P-450 and related enzymes in the pulmonary metabolism of xenobiotics has been recently reviewed by Philpot and Smith (1984). The best understood pulmonary P-450 system is that of the rabbit. In this species, one of the major forms, cytochrome P-450, isozyme, has been determined to be the same as form 2 from the liver. Since this form has been recognized to be involved in both benzphetamine N-demethylation and ethoxycoumarin O-deethylation, this could be related to the high efficiency of these dealkylation activities determined in ovine lung. In sheep conjugative enzymes such as glutathione S-transferase or glucuronyltransferases are characterized by a low lung/liver ratio; similar results were obtained in other animal species, i.e. rat, rabbit and guinea pig (Litterst et al., 1975). Concerning the sheep renal drug metabolizing activities, our data are in agreement with cytochrome P-450 and NADPH-cytochrome c reductase level
Table 2. Some microsomal and cytosolic phase II systems in liver, lunp. and kidnev in sheeD* Parameters Microsomal Glucuronyltransferase (PNP) (nmolmg-‘min-‘) Glucuronyltransferase (CAP) (nmolmg-‘min-‘) Cytosolic Cytosolic proteins (mgg-’ ) Glutathione S-transferase (nmolmg-‘min-‘) Acetyltransferase
Liver
Lunn
Kidnev
2.88 k 0.10
0.95 + 0.20
0.72 + 0.08
0.180~0.007
0.027 f 0.007
0.068 * 0.004
43.7 + 0.5
34.8 + 2. I
33.2 + 1.7
0.893 It 0.063
0.218+0.048
0.090 f 0.006
(nmolmg-‘min-‘) *For legend, see Table I. PNP, p-nitrophenol; CAP, chloramphenicol.
ND*
ND
ND
G. LARRIEU and P. GALT~ER
228
determined by Garst et al. (1985) whereas the low enzyme activities in this organ confirmed the observation previously reported in rodents (Litterst et al., 1975). The results of this study permit us to classify sheep tissues by decreasing order of their enzymatic systems for metabolizing xenobiotics: liver > lung > kidney. Such a classification agrees generally with findings obtained in other animals species. However, lung monooxygenases appeared sometimes as high or higher than the corresponding liver enzymes. This conclusion is of importance because of the increasing use of veterinary drugs in livestock species and the pulmonary localization of many parasitic diseases which can interfere with lung disposition of endogenous substances or xenobiotics. Ac~no~Zedgerne~~s-The authors are indebted to A. E. Tufenkji and C, Eeckhoutte for skillful assistance in statistical and biochemical analysis.
REFERENCES Aitio A. (1978) A simple and sensitive assay of 7-ethoxycoumarin deethylation. Anal. B&hem. 85, 48&491. Arinc E. (1985) Chara~e~~tion of sheep liver and lung microsomal ethyhnorphine N-demethylase. Camp. Biothem. Physiol.
&oB,389-399.
Arinc E. and Iscan M. Y. (1983) Comparative studies of sheep liver and lung microsomal aniline 4-hydroxylase. Comp. Biochem. Physiol. 74C, 151-158. Burley F. E. and Bray T. M. (1983) Effect of dietary vitamin A on the mixed function oxidases and the pneumotoxi~ty of 3-methylindole of goats. Can. J. Physiol. Pharmac. 61, 81&821.
Cochin J. and Axelrod J. (1959) Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmac. exp. Ther. 125, 105-115. Dvorchik B. H., Woodward G., Sitar D. S. and Tweed W. A. (1986) Hydroxylation and glucuronidation of various xenobiotics by hepatic microsomes from the fetal lamb, pregnant ewe and human fetus. Devel. Pharmac. Ther. 9, 282-289.
Frei J. (1970) Multiplicity and specificity of UDPglucuronyl transferase I. Effect of divalent cations and EDTA on the activity of UDP-glucuronyl transferase assayed with bilirubin, 4-methyl-um~lliferone and pnitrophenol. Enzymol. Biol. Cl&. 11, 385401. Galtier P., Larrieu G., Tufenkji A. E. and Franc M. (1986)
Incidence of experimental fascioliasis on the activity of drug metabolizing enzymes in lamb liver. Drug Merab. Dis. 14, 137-141. Garst J. E., Wilson W. C., Kristensen N. C., Harrison P. C., Corbin J. E., Simon J., Philpot R. M. and Szabo R. R. (1985) Species susceptibility to the pulmonary toxicity of 3-furylisoamylketone (Perilla ketone): in viuo support for involvement of the lung monooxygenase system. J. Animal Sci. 60, 248-257.
Habig W. H., Pabst M. J. and Jacoby W. B. (1974) Glutathione transferase. The first enzymatic step in marcapturic acid formation. J. biol. Chem. 249, 7130-7r39. Iscan M. and Arinc E. (1986) Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase. Int. J. Biochem. 18, 731-741.
Litterst C. L., Mimnaugh E. G., Reagan R. L. and Gram T. E. (1975) Comparison of in uitro drug metabolism by lung, liver, and kidney of several common laboratory species. Drug Meiab. D&p. 3, 259-265. Lo&y 0. H.,RosebroughON. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin nhenol . reagent. J. ‘biol. Chem. 193, 265-275. Matsubara T., Touchi A. and Ogawa A. (1982) Heterogeneous distribution of the cytochrome P-450 monooxygenase system in rat liver lobes. Jap. J. Pharmac. 32, 99-1011.
Maze1 P. (1971) Experiments illustrating drug metabolism in vitro. In Fundamentals of Drug Metabolism and Drug Disposition (Edited by La Du B. N., Mandel H. G. and Way E. L.), pp. 546-577. Williams & Wilkins, Baltimore. Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes. J. biol. Chem. 239, 2379-2384.
Philpot R. M. and Smith B. R. (1984) Role of cytochrome P-450 and related enzymes in the pulmonary metabolism of xenobiotics. Envir. Hlth Pers. 55, 359-367. Schull L. R., Olson B. A., Hughes B. J., McKenzie R. M. and Kinzell J. H. (1986) Effect of pentachlorophenol on microsomal mixed-function oxidases in cattle. Pestic. Biochem. Physioi. 25, 31-39.
Smith G. S., Watkins J. B., Thompson T. N., Rozman K. and Klaassen C. D. (1984) Oxidative and conjugative metabolism of xenobiotics by liver of cattle, sheep, swine and rats. J. Animal. Sci. 58, 386-395. Weber W. W. (1971) N-Acetyltransferase (mammalian liver). Methods Enzvmol. 17B. 805-811. Young-W. S. and Lieimann P. s. (1978) Chloramphenicol glucuronyl transferase: assay, ontogeny and inductibility. J. Pharmac. exp. Ther. 204, 203-211.
0306-4492/88 $3.00 + 0.00
0 1988Pergamon Press plc
Printed in Great Britain
A COMPARATIVE STUDY OF SOME OXIDATIVE AND CONJUGATIVE DRUG METABOLIZING ENZYMES IN LIVER, LUNG AND KIDNEY OF SHEEP G. LARRIEU and P. GALTIER Laboratoire de Pharmacologic-Toxicologic,
INRA, 31931 Toulouse, France (Tel.: 1661-49-1686)
(Received 4 March 1987)
The comparative distribution of cytochrome P-450 monooxygenase system, glucuronyltransferase, glutathione S-transferase and N-acetyltransferase was studied in the liver, lung and kidney of young male sheep. 2. The sheep liver was characterized by a lack in glutathione S-transferase activity with isoniazid as substrate. 3. The oxidative drug metabolizing enzymes of lung were generally close to those of liver; benzphetamine N-demethylase and ethoxycoumarin O-deethylase were even found to be higher in lung (213 and 148%, respectively). 4. Pulmonary conjugative and both renal oxidative and conjugative systems accounted only for 9-38% of hepatic corresponding enzymes. 5. The enzyme determination in various sampling sites of the three organs, demonstrated the homogeneous distribution of all investigated monooxygenases and transferases in liver, lung and kidney of sheep.
Abstract-l.
INTRODUCTION
the liver has long been acknowledged as the major site of drug metabolism, however, many recent studies have emphasized the importance of extrahepatic tissues in the biotransformation of xenobiotics and endogenous substances. These studies resulted generally in comparisons of the enzymatic equipment of liver with that of organs which have been recognized as the portals of entry or excretion of the body, namely the lung, kidney, skin or gastrointestinal mucosa. Thus, foreign compounds are converted by the extrahepatic metabolism to either active or inactive forms preceding their entry into the circulating system and access to the target tissue. In the case of ruminant breeding species, there is a need for information concerning such comparative data because of the increasing rise of veterinary drugs and the involvement of these enzymatic systems in the occurrence of drug residues in edible tissues. In sheep, most of the studies described the liver oxidative and conjugative drug metabolizing enzymes in control animals (Smith et al., 1984) and in animals under particular physiological states (Dvorchik et al., 1986). Recently, detailed kinetic properties of sheep liver and lung microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase were described (Arinc and Iscan, 1983; Arinc, 1985). In investigating the species susceptibility to the lung toxicity of perilla ketone, Garst et al. (1985) provided relevant information concerning ovine pulmonary monooxygenases. Therefore, in attempting to obtain comparative information on the activities of drug metabolizing enzymes in ovine tissues, we have studied the ability of microsomal and cytosolic fractions of liver, lung and kidney from young male sheep to metabolize a
variety of drug substrates. Findings have been discussed in relation to data obtained in other animal species.
In mammals,
MATERIALS
AND METHODS
Chemicals
Ethoxycoumarin and acetylcoenzyme A were obtained from Boehringer Mannheim, Meylan, France. Aminopyrine, aniline, 1-chloro-2,4-dinitrobenzene, isoniazid, pnitrophenol and cytochrome C were purchased from Sigma Chemical Co., St Louis, MO, USA. Benzphetamine was kindly provided by Upjohn Laboratories, Le Vaudreuil, France. Glycerol, sodium dithionite (Na,S,O,) and glutathione (reduced) were the products of E. Merck, Darmstadt, Germany. Dichloroacetyl (1, 14C) chloramphenicol (52 mCi/nmol) was purchased from DuPont NEN France, Paris, France. Complete scintillation cocktail Lumagel was obtained from Kontron, Velizy, France. All other chemicals were the highest quality available. Desionized distilled water was used in all studies. Preparation of microsomal and cytosolic fractions The livers, lungs and kidneys from well bled Lacaune young male sheep (about 3-4 months old) were obtained immediately after killing in the laboratory. The organs were weighed, washed with ice-cold saline solution and blotted free of excess moisture. All subsequent operations were carried out at 04°C. The livers were then divided into left, right and caudate lobes in order to check the enzyme distribution within the hepatic parenchyma. Tissue blocks were obtained from 10 different sites of the lamb liver: 3 from the left lobe, 6 from the right lobe and 1 from the caudate lobe. In the case of lungs, 3 tissue blocks were samples from the right lobe and 2 from the left whereas for the kidneys 3 blocks were selected from each left or right kidney.
225
226
G. LARRIEUand P. GALTIER
Randomized 8 g samples from each tissue block were homogenized with 22ml of ice-cold solution of EDTA (I mM) and hydroxytoluene (20pM) in 0.1 M potassium phosphate buffer (PH 7.4) in a glass Potter homogenizer with a Teflon pestle. The homogenate was centrifuged at 10,000 g for 30 min in a Jouan model K 101 centrifuge. The supematant fraction was centrifuged for 1 hr at 105,OOOgin a Beckman model LS-50 ultracentrifuge, washed with 7.5 ml of 0.1 M sodium pyrophosphate-HCl buffer (pH 7.5) and recentrifuged for 30 min in order to obtain a cleaner microsomal fraction. The microsomal pellets were suspended in an ice-cold solution consisting of EDTA (0.1 mM), glycerol (20%) and potassium phosphate buffer (0.1 M, pH 7.4). The microsomal and cytosolic (first 105,OOOgsupernatant) proteins were determined by the procedure of Lowry et al. (1951), crystalline bovine serum albumin was used as a standard. When enzyme assays were not performed on the day the animals were killed, cytosolic fractions and microsomal pellets were frozen at -80°C. Enzyme assays
The contents of cytochromes P-450 and b5 were measured from the difference spectra between a microsomal sample reduced with sodium dithionite and one gassed with CO (Omura and Sato, 1964). NADPH cytochrome C reductase, aniline hydroxylase and aminopyrine N-demethylase microsomal activities were determined as previously described (Mazel, 1971). Benzphetamine N-demethylase was measured using 2SmM benzphetamine, 5mg of microsomal proteins and an NADPH-generating system (0.5 mM NADP). The formation of formaldehyde was estimated by the Nash reaction modified by Cochin and Axelrod (1959). Ethoxycoumarin 0-deethylase was determined by direct fluorimetry (Aitio, 1978). Microsomal UDP-glucuronyltransferase was assayed employing either 0.7mM pnitrophenol (Frei ef al., 1970) or 10 mM labelled chloramphenicol (Young et al., 1978). I-Chloro-2,4_dinitrobenzene was used as the substrate in the determination of glutathione transferase of liver cytosol (Habig ef al., 1974), and cytosolic acetyltransferase was measured with isoniazid as substrate (Weber, 1971). All enzymes reactions were performed under conditions of maximal velocity with appropriate blanks and were linear with time and protein concentration. Radioactivity was measured by liquid scintillation spectrometry (Intertechnique, model SL 33). Quenching was estimated by automatic external standardization. Statislics
Data were analysed using a two-way analysis of variance with interaction. The two ways corresponded respectively to the distribution of activities between animals and within the various organs or lobes. A complementary range test was Table I.
Microsomal
protein
used to compare the means. A probability of P -C0.05 was considered significant. RESULTS
Under our experimental conditions, microsomal protein concentrations were found to be similar (16-19 mg/g of tissues) in liver, lung and kidney (Table 1) whereas cytochrome P-450 levels were around tenfold lower in extrahepatic organs as compared to its hepatic concentration (0.24 nmol mg-’ of microsomal protein). Although, in general, NADPH cytochrome c reductase, aminopyrine N-demethylase and aniline hydroxylase activities of liver microsomes (26.9,0.202 and 0.156 nmol mg-’ min-‘, respectively) were higher than those of lung microsomes, in some preparations enzymatic activity of lung were comparable to those of liver. Likewise, pulmonary benzphetamine Ndemethylase and ethoxycoumarin 0 -deethylase were 1.5 and 2.1-fold greater than hepatic corresponding monooxygenases (0.760 and 0.645 nmol mg-’ min-‘, respectively). Concerning renal mixed function oxidases, NADPH cytochrome c reductase, aminopyrine N-demethylase and benzphetamine N-demethylase activities corresponded respectively to 27, 19 and 18% of the hepatic enzymes (Fig. 1) whereas ethoxycoumarin 0-deethylase exhibited remarkably low activities (around 0.01 nmol mg-’ min-‘) and aniline hydroxylase was not detected. Microsomal and cytosolic phase II enzymatic systems from sheep liver, lung and kidney are presented in Table 2. As compared to hepatic corresponding activities, pulmonary and renal UDP-glucuronyltransferase accounted for 1S-38% whatever the substrate was used, i.e. p-nitrophenol or chloramphenicol. Liver cytosolic proteins (43.7 mg/g) were 1.2-fold higher than in lung or kidney. Cytosolic glutathione S-transferase activity using 1-chloro-2-4 dinitrobenzene as substrate was about 4-fold and IO-fold more active in liver than in lung and kidney, respectively (Fig. 1). On the other hand, cytosolic N-acetyltransferase activity employing isoniazid as substrate was found to be at a level below the detection limits of the method (0.05nmol mg-’ min-I) regardless of which tissue was investigated. The two-way analysis of variance failed to exhibit any measurable difference between the animals used or
and cytochrome concentrations, and mixed function in liver. luna and kidnev of she&
Parameters Microsomal proteins (mgg-‘) Cytochrome P-450 (nmolmg* microsomal proteins-‘) NADPH cytochrome c reductase (nmolmg-‘min-‘) Aminopytine N-demethylase (nmolmg-‘min-‘) Benzophetamine N-demethylase (nmolmg-‘min-‘) Aniline hydroxylase (nmolmg-‘min-‘) Ethoxycoumarin O-deethylase (nmolmg-‘min-‘) *Values are means f SD from 4 animals liver, lung and kidney, respectively. ND, not detected value.
oxidase systems
Liver
Lung
Kidney
19.5 + 0.6 0.240 f 0.020
18.1 +0.8 0.024 * 0.003
16.3 k 0.5 0.020 + 0.001
26.9 rt 1.5
18.9 f I.0
7.2 f 0.1
0.202 f 0.043
0.108 iO.022
0.038 f 0.004
0.760 5 0.053
1.620 k 0.023
0. I36 It 0.020
0.156 f 0.007
0.086 f 0.005
ND
0.645 + 0.030
0.956 f 0.033
0.014 + 0.002
corresponding
to 40, 20 and 24 tissue samples in case of
Drug metabolism in ovine tissues P-450 GST
NCR
__
EdOD
Fig. 1. Relative enzymatic activity in sheep liver (-), lung(--) and kidney (. . .). Abbreviations: P-450, microsomal cytochrome P-450; NCR, NADPH cytochrome c reductase; AND, aminopyrine N-demethylase; BND, benzphetamine N-demethylase; ECOD, ethoxycoumarin O-deethylase; AH, aniline hydroxylase; GCC, glucuronyl-transferase (substrate:chloramphenical); GCP, glucuronyltransferase (substrate:p-nitrophenol); GST, glutathione S-transferase.
between the sampling tissue sites or lobes; this result led to the homogeneous distribution of all determined activity within each investigated tissue. DISCUSSION
Comparative species studies of tissue oxidative and conjugative biotransformations resulted in numerous data regarding almost all the hepatic activities of rodents, whereas only sporadic data were reported for ruminants. The present study provides information involving 10 enzyme systems and should become a baseline for comparisons of ovine hepatic, pulmonary and renal systems to other animals species. Hepatic enzyme parameters of sheep determined in the present paper are in good agreement with data from control animals used in an experiment describing the incidence of ovine fascioliasis on such activities (Galtier et al., 1986). NADPH cytochrome c reductase activity was similar to the value reported by Dvorchik et al. (1986) in freshly isolated microsomes from the livers of maternal sheep; these authors likewise detected significant N-dealkylating and hydroxylating activities towards methadone, mepiridine, hexobarbital and benzopyrene. Concern-
221
ing hepatic conjugative enzymes in sheep, there is a large variability of response in relation to the nature of substrate, as previously observed by Smith et al. (1984). In this paper, glutathione S-transferase level was close to our values while acetyltransferase activity with 2naphtylamine or 2-aminofluorene as substrates was not found in liver tissues from the bovine or ovine species. This could correspond with our lack of sensitivity for measuring this cytosolic activity. The determination of enzymes in 10 sampling sites resulted in any significant difference between the liver lobes and led to the homogeneous distribution of both oxidative and conjugative drug metabolizing activities within the sheep liver. This conclusion contrasts with the demonstration of a heterogenous distribution of the cytochrome P-450 monooxygenase system in rat liver lobes (Matsubara et al., 1984). Generally, there is a lack of literature noting comparisons with other data concerning the extrahepatic drug metabolism in ovine tissues. Effectively, most information concerns the characterization and kinetic properties of sheep lung microsomal NADPH cytochrome c reductase (Iscan and Arinc, 1986), aniline hydroxylase (Arinc and Iscan, 1983) or ethylmorphine N-demethylase (Arinc, 1985). This interest for ovine pulmonary monooxygenases is in accordance with our observation of high oxidizing-enzyme levels in sheep lung. However, lung microsomal cytochrome P-450 appeared IO-fold lower than in liver. Such lung/liver ratios have been reported in goats (Burley and Bray, 1983), lactating cattle (Shull et al., 1986) and in rodents (Litterst et al., 1975). The role of cytochrome P-450 and related enzymes in the pulmonary metabolism of xenobiotics has been recently reviewed by Philpot and Smith (1984). The best understood pulmonary P-450 system is that of the rabbit. In this species, one of the major forms, cytochrome P-450, isozyme, has been determined to be the same as form 2 from the liver. Since this form has been recognized to be involved in both benzphetamine N-demethylation and ethoxycoumarin O-deethylation, this could be related to the high efficiency of these dealkylation activities determined in ovine lung. In sheep conjugative enzymes such as glutathione S-transferase or glucuronyltransferases are characterized by a low lung/liver ratio; similar results were obtained in other animal species, i.e. rat, rabbit and guinea pig (Litterst et al., 1975). Concerning the sheep renal drug metabolizing activities, our data are in agreement with cytochrome P-450 and NADPH-cytochrome c reductase level
Table 2. Some microsomal and cytosolic phase II systems in liver, lunp. and kidnev in sheeD* Parameters Microsomal Glucuronyltransferase (PNP) (nmolmg-‘min-‘) Glucuronyltransferase (CAP) (nmolmg-‘min-‘) Cytosolic Cytosolic proteins (mgg-’ ) Glutathione S-transferase (nmolmg-‘min-‘) Acetyltransferase
Liver
Lunn
Kidnev
2.88 k 0.10
0.95 + 0.20
0.72 + 0.08
0.180~0.007
0.027 f 0.007
0.068 * 0.004
43.7 + 0.5
34.8 + 2. I
33.2 + 1.7
0.893 It 0.063
0.218+0.048
0.090 f 0.006
(nmolmg-‘min-‘) *For legend, see Table I. PNP, p-nitrophenol; CAP, chloramphenicol.
ND*
ND
ND
G. LARRIEU and P. GALT~ER
228
determined by Garst et al. (1985) whereas the low enzyme activities in this organ confirmed the observation previously reported in rodents (Litterst et al., 1975). The results of this study permit us to classify sheep tissues by decreasing order of their enzymatic systems for metabolizing xenobiotics: liver > lung > kidney. Such a classification agrees generally with findings obtained in other animals species. However, lung monooxygenases appeared sometimes as high or higher than the corresponding liver enzymes. This conclusion is of importance because of the increasing use of veterinary drugs in livestock species and the pulmonary localization of many parasitic diseases which can interfere with lung disposition of endogenous substances or xenobiotics. Ac~no~Zedgerne~~s-The authors are indebted to A. E. Tufenkji and C, Eeckhoutte for skillful assistance in statistical and biochemical analysis.
REFERENCES Aitio A. (1978) A simple and sensitive assay of 7-ethoxycoumarin deethylation. Anal. B&hem. 85, 48&491. Arinc E. (1985) Chara~e~~tion of sheep liver and lung microsomal ethyhnorphine N-demethylase. Camp. Biothem. Physiol.
&oB,389-399.
Arinc E. and Iscan M. Y. (1983) Comparative studies of sheep liver and lung microsomal aniline 4-hydroxylase. Comp. Biochem. Physiol. 74C, 151-158. Burley F. E. and Bray T. M. (1983) Effect of dietary vitamin A on the mixed function oxidases and the pneumotoxi~ty of 3-methylindole of goats. Can. J. Physiol. Pharmac. 61, 81&821.
Cochin J. and Axelrod J. (1959) Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmac. exp. Ther. 125, 105-115. Dvorchik B. H., Woodward G., Sitar D. S. and Tweed W. A. (1986) Hydroxylation and glucuronidation of various xenobiotics by hepatic microsomes from the fetal lamb, pregnant ewe and human fetus. Devel. Pharmac. Ther. 9, 282-289.
Frei J. (1970) Multiplicity and specificity of UDPglucuronyl transferase I. Effect of divalent cations and EDTA on the activity of UDP-glucuronyl transferase assayed with bilirubin, 4-methyl-um~lliferone and pnitrophenol. Enzymol. Biol. Cl&. 11, 385401. Galtier P., Larrieu G., Tufenkji A. E. and Franc M. (1986)
Incidence of experimental fascioliasis on the activity of drug metabolizing enzymes in lamb liver. Drug Merab. Dis. 14, 137-141. Garst J. E., Wilson W. C., Kristensen N. C., Harrison P. C., Corbin J. E., Simon J., Philpot R. M. and Szabo R. R. (1985) Species susceptibility to the pulmonary toxicity of 3-furylisoamylketone (Perilla ketone): in viuo support for involvement of the lung monooxygenase system. J. Animal Sci. 60, 248-257.
Habig W. H., Pabst M. J. and Jacoby W. B. (1974) Glutathione transferase. The first enzymatic step in marcapturic acid formation. J. biol. Chem. 249, 7130-7r39. Iscan M. and Arinc E. (1986) Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase. Int. J. Biochem. 18, 731-741.
Litterst C. L., Mimnaugh E. G., Reagan R. L. and Gram T. E. (1975) Comparison of in uitro drug metabolism by lung, liver, and kidney of several common laboratory species. Drug Meiab. D&p. 3, 259-265. Lo&y 0. H.,RosebroughON. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin nhenol . reagent. J. ‘biol. Chem. 193, 265-275. Matsubara T., Touchi A. and Ogawa A. (1982) Heterogeneous distribution of the cytochrome P-450 monooxygenase system in rat liver lobes. Jap. J. Pharmac. 32, 99-1011.
Maze1 P. (1971) Experiments illustrating drug metabolism in vitro. In Fundamentals of Drug Metabolism and Drug Disposition (Edited by La Du B. N., Mandel H. G. and Way E. L.), pp. 546-577. Williams & Wilkins, Baltimore. Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes. J. biol. Chem. 239, 2379-2384.
Philpot R. M. and Smith B. R. (1984) Role of cytochrome P-450 and related enzymes in the pulmonary metabolism of xenobiotics. Envir. Hlth Pers. 55, 359-367. Schull L. R., Olson B. A., Hughes B. J., McKenzie R. M. and Kinzell J. H. (1986) Effect of pentachlorophenol on microsomal mixed-function oxidases in cattle. Pestic. Biochem. Physioi. 25, 31-39.
Smith G. S., Watkins J. B., Thompson T. N., Rozman K. and Klaassen C. D. (1984) Oxidative and conjugative metabolism of xenobiotics by liver of cattle, sheep, swine and rats. J. Animal. Sci. 58, 386-395. Weber W. W. (1971) N-Acetyltransferase (mammalian liver). Methods Enzvmol. 17B. 805-811. Young-W. S. and Lieimann P. s. (1978) Chloramphenicol glucuronyl transferase: assay, ontogeny and inductibility. J. Pharmac. exp. Ther. 204, 203-211.