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Drug Metabolizing Enzymes in the Ostrich (Struthio camelus): Comparison with the Chicken and the Rat
Comp. Biochem Physiol. Vol. 116C, No. 1, pp. 47–50, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0742–8413/97/$17.00 PII S0742–8413(96)00136-3
Drug Metabolizing Enzymes in the Ostrich (Struthio camelus): Comparison with the Chicken and the Rat E. Amsallem-Holtzman and Z. Ben-Zvi Department of Clinical Pharmacology, The Corob Center for Health Sciences, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beersheva 84105, Israel ABSTRACT. The activities of selected hepatic and renal drug-metabolizing enzymes of the ostrich, chicken and rats were compared. The concentration of glutathione in the liver and kidneys of the avian species was significantly lower than that in the rat. The activity of ostrich hepatic glutathione S-transferase was 2-fold higher than that of the chicken and the rat and the renal glutathione S-transferase of the ostrich was 10 times higher than that of the rat. The activity of ostrich hepatic UDP-glucuronyl transferase was significantly lower than that of the rat. The activities of hepatic cytochrome P450 1A and 2B1/2 as measured by the dealkylation of ethoxy- and methoxyresorufin, respectively, were higher in the avian species than the rat; no difference was noticed in the activity of aniline hydroxylase. The results show that the activity of ostrich drug-metabolizing enzyme system is quantitatively different from the rat and in many cases also from the chicken. Copyright 1997 Elsevier Science Inc. comp biochem physiol 116C;1:47–50, 1997. KEY WORDS. Ostrich, chicken, rat, glutathione S-transferase, UDP-glucuronyl transferase, EROD, MROD, PROD
INTRODUCTION
MATERIALS AND METHODS
The intensive production of ostriches (Struthio camelus) in big flocks in various parts of the world is becoming an important economic enterprise, mainly because of the high value of the ostrich leather. Ostrich meat is also gaining popularity because of its low cholesterol contents compared with beef (9,10,14). Because of the high cost of each individual bird, intensive production of ostriches involves the use of various medicines, both for treatment and prevention of diseases (28). However, very sparse data are available on drug use in these birds (23,29). There is a lack of knowledge concerning the pharmacokinetic profiles of drugs in ostriches or any data pertaining to the ability of the ostrich liver to detoxify drugs. Thus, the dosage of drugs in ostriches is extrapolated from doses used in small domesticated birds like chicken, ducks, geese and turkeys. Such a practice is very inaccurate and may be even dangerous because of the significant difference in the activities of drug-metabolizing enzymes among avian species (2,22,25,26). In the present study, the activity of selected hepatic and renal drug-metabolizing enzymes in the ostrich was compared with the activity of these enzymes in chickens and rats.
Liver and kidneys from 10-month-old male and female ostriches were obtained from the France Croco Ostrich Farm, Ltd. (Kezioth, Israel). Male Sprague-Dawley rats, 12 weeks old, were received from Harlan Industries (Jerusalem, Israel). Livers and kidneys from adult male and female white Leghorn chicken were received from a local slaughter house (Of-Hanegev, Netivot, Israel). For the determination of hepatic and renal glutathione concentrations and glutathione S-transferase activity, liver and kidney aliquots of the three species were homogenized with five volumes of 1.15% KCl solution. For the determination of cytochrome P450-mediated mixed-function oxidase activity and UDP-glucuronyl transferase activity, liver aliquots were homogenized in 7.5 volumes of 10 mM HEPES buffer containing 250 mM sucrose, 1 mM dithiothreitol, 25 mM KCl, 0.5 mM EDTA and 10% glycerol at pH 7.4. The homogenates were centrifuged for 20 min at 9000 g. The 9000 g supernatants in the HEPES buffer were stored at 280°C pending analysis. Microsomes were prepared by the calcium aggregation method according to Cinti et al. (6). Glutathione concentrations in liver and kidney cytosolic fractions were determined according to Mitchell et al. (21). Glutathione S-transferase activity in hepatic and renal cytosolic fractions were determined with 1-chloro-2,4dinitrobenzene according to Baars et al. (1). Aniline hydroxylase was determined according to Mazel (19). Methoxy-, ethoxy- and pentoxyresorufin-O-dealkylase activi-
Correspondence to: Z. Ben-Zvi, Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beersheva 84105, Israel. Tel. 972-7-400845; Fax 972-7-277342; email [email protected]. Received 27 April 1996; accepted 14 July 1996.
E. Amsallem-Holtzman and Z. Ben-Zvi
48
TABLE 1. Concentration of Protein in Liver Fractions of the Ostrich, Chicken and Rat
TABLE 3. Activity of Glutathione S-Transferase and UDPGlucuronyl Transferase in the Ostrich, Chicken and Rat
Liver protein concentration (mg/g liver)
Glutathione S-transferase*
UDP-glucuronyl transferase
Species
Cytosolic
Microsomal
Species
Liver
Kidney
Liver
Ostrich Chicken Rat
71.51 6 1.82* 70.89 6 4.43* 86.01 6 1.87
9.24 6 0.41*† 13.97 6 0.33* 18.08 6 0.41
Ostrich Chicken Rat
2.50 6 0.14†‡ 1.23 6 0.09 1.02 6 0.07
1.90 6 0.14†‡ 1.00 6 0.06† 0.09 6 0.01
17.60 6 2.59†‡ 27.94 6 2.68† 35.36 6 2.64
*Significantly different from the rat, P , 0.05. †Significantly different from the chicken, P , 0.05.
ties (MROD, EROD and PROD, respectively) were determined according to Burke et al. (4). UDP-glucuronyl transferase activity with p-nitrophenol as a substrate was determined according to Bock et al. (3). Protein concentration in the microsomal and cytosolic fractions was determined according to Lowry et al. (17). The results, presented as mean 6 SEM (n 5 10 in each group), were compared for statistical analysis by ANOVA followed by Tukey-Kramer test. Differences were considered significant when P , 0.05. RESULTS The concentration of hepatic protein in the microsomal and cytosolic fractions of the ostrich, chicken and rat are presented in Table 1. The concentration of renal cytosolic protein of the ostrich (10.30 6 2.78 mg/g kidney) was significantly lower than those of the chicken and rat (39.01 6 0.75 and 54.02 6 1.32 mg/g kidney, respectively). Hepatic and renal glutathione concentrations in the ostrich were significantly lower than the concentrations in the rat (Table 2). The activity of ostrich renal and hepatic glutathione S-transferase was significantly higher than that of the chicken and the rat; also, the activity of chicken renal glutathione S-transferase was significantly higher than that of the rat (Table 3). The activity of UDP glucuronyl-transferase was significantly lower in the ostrich than in the rat (Table 3). MROD, EROD, PROD and aniline hydroxylase activities are presented in Table 4. The O-deethylation of ethoxyresorufin in the ostrich was significantly higher than that of the rat and the chicken. The activity of EROD and MROD in chicken microsomes, however, was significantly TABLE 2. Glutathione Concentrations in the Kidney and
Liver of the Ostrich, Chicken and Rat Glutathione concentration (mmol/g tissue) Species
Liver
Kidney
Ostrich Chicken Rat
2.70 6 0.10* 2.70 6 0.10* 6.08 6 0.18
1.49 6 0.13* 2.08 6 0.10* 2.80 6 0.04
*Significantly different form GSH concentration in the rat, P , 0.05.
*The activity of glutathione S-transferase is expressed in µmol of conjugate formed/min/mg prot. The activity of UDP-glucuronyl transferase is expressed in nmol of conjugate formed/min/mg prot. †Significantly different from the rat, P , 0.05. ‡Significantly different from the chicken, P , 0.05
lower than that of the rat. There was no difference in the activity of aniline hydroxylase between the avian species and the rat. DISCUSSION In the present investigation, we studied the activity of the ostrich drug-metabolizing enzymes in comparison with those of the chicken and the rat. Although several studies show that the concentration of cytochrome P450 in rat hepatic microsomes is higher than in birds (11–13), the activity of many cytochrome P450-mediated mixed function oxidases was lower in the rat than in many avian species. There is some discrepancy as to the activity of aniline hydroxylase in the chicken as compared with the rat in various studies. Our results, showing that the activity of aniline hydroxylase is similar in the avian species and in rats, are in agreement with those of Dalvi et al. (7,8); however, other studies have shown that the activity of hepatic aniline hydroxylase was higher in the chicken than in the rat (11,12). The activities of cytochrome P450 1A1 and 1A2, as measured by the dealkylation of ethoxy- and methoxyresorufin, respectively, were higher in ostrich hepatic microsomes than in chicken and rat hepatic microsomes. On the other hand, the activity of hepatic cytochrome P450 2B1/2, measured by pentoxyresorufin-O-depentylation, was lower in the ostrich than in the chicken and the rat. Although such a comparison between ostrich, chicken and rat was not reported previously, Ronis et al. (24) showed that the activity of cytochrome P450 1A1, 2B1, 4A and testosterone hydroxylase were higher in the rat than in the bobwhite quail; however, the activities of cytochrome P450 2E1 and 3A were comparable in the rat and quail. The activity of the hepatic glutathione S-transferase was higher in the ostrich than in the rat, whereas the activity of ostrich hepatic UDP-glucuronyl transferase was lower than that of the chicken and the rat. These results are in line with observations previously published on several wild birds, chicken and quail as compared with the rat (5,15,16,25). The activity of renal glutathione S-transferase in the avian species was significantly and impressively
Drug Metabolism in the Ostrich
49
TABLE 4. Activity of Hepatic Mixed Function Oxidase in the Ostrich, Chicken and Rat
Species
Aniline hydroxylase*
MROD
EROD
PROD
Ostrich Chicken Rat
62.19 6 7.14 50.84 6 3.98 54.70 6 5.10
188.6 6 22.4†‡ 9.6 6 0.7† 32.4 6 4.8
130.3 6 2.8†‡ 12.1 6 1.0† 65.4 6 5.4
3.0 6 0.3†‡ 4.2 6 0.4† 5.0 6 0.4
*The activity of aniline hydroxylase is expressed ass nmol of products formed/min/mg/prot. The activity of MROD, EROD and PROD is expressed as pmol/min/mg prot. †Significantly different from the rat, P,0.05 ‡Significantly different from the chicken, P,0.05
higher than that of the rat renal enzyme activity. Maurice et al. (18) previously showed that in a few breeds of chicken and in the quail, the activity of renal glutathione S-transferase was one order of magnitude higher than in the rat. The ostrich belongs to the family of ratites, which includes several other flightless birds like the rhea, cassowary and the kiwi. Phylogenetically, this family is the oldest offshoot of the avian stem, as evidenced by biochemical and anatomical studies (20,27). Thus, several developmental changes could have occurred in the ostrich enzymatic machinery, as compared with other avian families like the galliformes (chicken and turkey) and the anseriformes (duck), due to environmental, nutritional or other developmental factors. The help of Prof. R. Yagil in obtaining the ostrich tissues is highly acknowledged.
References 1. Baars, A.J.; Jansen, M.; Breimer, D.D. The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin on glutathione S-transferase activity of rat liver cytosol. Biochem. Pharmacol. 7:2487–2494;1978. 2. Bartlet, A.L.; Kirinya, L.M. Activities of mixed function oxidases, UDP-glucuronyl transferase and sulfate conjugating enzymes in galliformes and anseriformes. Q.J.Exp. Physiol. 61; 105–119;1976. 3. Bock, K.W.; Burchell, B.; Dutton, G.J.; Haninnen, O.; Mulder, G.J.; Owens, I.S.; Siest, G.; Tephley, T.R. UDP-glucuronosyl transferase activities: guidelines for consistent interim terminology and assay conditions. Biochem. Pharmacol. 32:953–955;1983. 4. Burke, M.D.; Thomson, S.; Elcombe, C.R.; Halpert, J.; Haaparanta, J.; Mayer, R.T. Ethoxy, pentoxy and benzyloxyphenazones and homologues: a series of substrates to distinguish between different induced cytochromes P450. Biochem. Pharmacol. 34:3337 –3345;1985. 5. Chang, L.H.; Fan, J.Y.; Liu, L.F.; Tsai, S.P.; Tan M.F. Cloning and expression of chick liver glutathione S-transferase CL3 subunit with the use of baculovirus expression system. Biochem. J. 281:545–551;1992. 6. Cinti, D.L.; Moldeus, P.; Schenkman, J.B. Kinetic parameters of drug metabolizing enzymes in Ca12 sedimented microsomes from rat liver. Biochem. Pharmacol. 21:3249 –3256;1972. 7. Dalvi, R.R.; Nunn, V.A.; Juskevich, J. Studies on comparative drug metabolism by hepatic cytochrome P450 containing microsomal enzymes in quail, duck, geese, chicken, turkeys and rats. Comp. Biochem. Physiol. 87C:421–424;1987.
8. Dalvi, R.R.; Gawai, K.R.; Dalvi, P.S. Lack of in vivo and in vitro effects of fenbendazole on phase I and phase II biotransformation enzymes in rat, mice and chicken. Vet. Hum. Toxicol. 33:548–550;1991. 9. Deeming, D.C.; Ayres, L.; Ayres, F.J. Observation on the commercial production of ostrich (Struthio camelus) in the United Kingdom: incubation. Vet. Rec. 132:602–607;1993. 10. Deeming, D.C.; Ayres, L.; Ayres, F.J. Observation on the commercial production of ostrich (Struthio camelus) in the United Kingdom: rearing of chicks. Vet. Rec. 132:627–631; 1993. 11. Ehrich, M.; Larsen, C. Drug metabolism in adult white Leghorn hens—response to enzyme inducers. Comp. Biochem. Physiol. 74C:383–386;1983. 12. Gawai, K.K.; Vodek, J.K.; Dalvi, P.S.; Dalvi, R.R. Comparative assessment of the effect of aflotoxin B1 on hepatic disfunction in some mammalian and avian species. Comp. Biochem. Physiol. 10C;414–418;1992. 13. Gay, L.; Ehrich, M. A comparative study of drug metabolizing enzymes in adrenal glands and livers of rats and chicken. Int. J. Biochem. 22:15–18;1990. 14. Gobbel, T. Ostriches—an agricultural domestic animal? Dtsch. Tierarztl. Wochenschr. 101:88–91;1994. 15. Gregus, Z.; Watkins, J.B.; Thompson, T.N.; Harvey, M.J.; Rozman, K.; Klaassen, C.D. Hepatic phase I and phase II biotransformation in quail and trout: comparison to other species commonly used in toxicity testen. Toxicol. Appl. Pharmacol. 67:430–441;1983. 16. Husain, M.M.; Kumar, A.; Mukhtar, H.; Krishna Murti, C.R. Xenobiotic biotransformation in wild birds: activity, induction and comparison with rat and mouse microsomal enzymes. Xenobiotica 11:785–793;1981. 17. Lowry, O.M.; Rosebrough, N.J.; Farr, A.L.; Randal, R.J. Protein measurement with folin phenol reagent. J. Biol. Chem. 193;265–275;1951. 18. Maurice, D.V.; Lightsey, S.F.; Tung, H.K.; Rhoades, J.F. Comparison of glutathione S-transferase activity in rat and birds: tissue distribution and rhythmicity in chicken liver. Comp. Biochem. Physiol. 100B:471–474;1991. 19. Mazel, P. Experiments illustrating drug metabolism in vitro. In: LaDu, B.N.; Mandel, G.H.; Way, E.L. (eds). Fundamentals of Drug Metabolism and Drug Disposition. Baltimore: Williams and Wilkins; 1971: pp. 569–572. 20. McGowan, C. Evolutionary relationship of ratites and carinates: evidence from ontogeny of the tarsus. Nature 307:733– 735;1984. 21. Mitchell, J.R.; Jollow, D.J.; Potter, W.Z.; Gillette, J.R.; Brodie, B.B. Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187:211–217; 1973. 22. Pan, H.P.; Fouts, J.R. Drug metabolism in birds. Drug Metab. Rev. 7:1–253;1978.
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23. Raath, J.P.; Quandt, S.K.; Malan, J.H. Ostrich immobilization using carfentanil and xylazine and reversal with yohimbine and naltrexone. J. S. Afr. Vet. Assoc. 63:138–140;1992. 24. Ronis, M.J.J.; Ingelman-Sundberg, M.; Badger, T.M. Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail by ergosterol biosynthesis inhibiting fungicides (EBIFs). Biochem. Pharmacol. 48:1953–1965;1994. 25. Short, C.R.; Flory, W.; Hsieh, L.C.; Aranas, T.; Ou, S.P.; Weissinger, J. Comparative hepatic drug metabolizing enzyme activities in several agricultural species. Comp. Biochem. Physiol. 91C:419–424;1988. 26. Sifri, M.; Sell, J.L.; and Davison, K.L. Comparative effects of p,p′-DDT and pentobarbital on hepatic microsomal enzyme
E. Amsallem-Holtzman and Z. Ben-Zvi
in the young quail chicks and ducklings. Comp. Biochem. Physiol. 51B:213–219;1971. 27. Stapel, S.O.; Leunissen, J.A.M.; Versteeg, M.; Wattel, J.; de Jong, W.W. Ratites as oldest offshoot of avian stem-evidence from α-crystallin A sequences. Nature 311:257–259;1984. 28. Terzich, M.; Vanhooser, S. Postmortem findings of ostriches submitted to the Oklahoma Animal Disease Diagnostic Laboratory. Avian Dis. 37:1136–1141;1993. 29. van Heerden, J.; Keffen, R.H. A preliminary investigation into the immobilizing potential of tiletamine/zolazepam mixture, metomidate, a metomidate and azaperone combination and medetomidine in ostriches. J.S. Afr. Vet. Assoc. 62;114– 117;1991.
ISSN 0742–8413/97/$17.00 PII S0742–8413(96)00136-3
Drug Metabolizing Enzymes in the Ostrich (Struthio camelus): Comparison with the Chicken and the Rat E. Amsallem-Holtzman and Z. Ben-Zvi Department of Clinical Pharmacology, The Corob Center for Health Sciences, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beersheva 84105, Israel ABSTRACT. The activities of selected hepatic and renal drug-metabolizing enzymes of the ostrich, chicken and rats were compared. The concentration of glutathione in the liver and kidneys of the avian species was significantly lower than that in the rat. The activity of ostrich hepatic glutathione S-transferase was 2-fold higher than that of the chicken and the rat and the renal glutathione S-transferase of the ostrich was 10 times higher than that of the rat. The activity of ostrich hepatic UDP-glucuronyl transferase was significantly lower than that of the rat. The activities of hepatic cytochrome P450 1A and 2B1/2 as measured by the dealkylation of ethoxy- and methoxyresorufin, respectively, were higher in the avian species than the rat; no difference was noticed in the activity of aniline hydroxylase. The results show that the activity of ostrich drug-metabolizing enzyme system is quantitatively different from the rat and in many cases also from the chicken. Copyright 1997 Elsevier Science Inc. comp biochem physiol 116C;1:47–50, 1997. KEY WORDS. Ostrich, chicken, rat, glutathione S-transferase, UDP-glucuronyl transferase, EROD, MROD, PROD
INTRODUCTION
MATERIALS AND METHODS
The intensive production of ostriches (Struthio camelus) in big flocks in various parts of the world is becoming an important economic enterprise, mainly because of the high value of the ostrich leather. Ostrich meat is also gaining popularity because of its low cholesterol contents compared with beef (9,10,14). Because of the high cost of each individual bird, intensive production of ostriches involves the use of various medicines, both for treatment and prevention of diseases (28). However, very sparse data are available on drug use in these birds (23,29). There is a lack of knowledge concerning the pharmacokinetic profiles of drugs in ostriches or any data pertaining to the ability of the ostrich liver to detoxify drugs. Thus, the dosage of drugs in ostriches is extrapolated from doses used in small domesticated birds like chicken, ducks, geese and turkeys. Such a practice is very inaccurate and may be even dangerous because of the significant difference in the activities of drug-metabolizing enzymes among avian species (2,22,25,26). In the present study, the activity of selected hepatic and renal drug-metabolizing enzymes in the ostrich was compared with the activity of these enzymes in chickens and rats.
Liver and kidneys from 10-month-old male and female ostriches were obtained from the France Croco Ostrich Farm, Ltd. (Kezioth, Israel). Male Sprague-Dawley rats, 12 weeks old, were received from Harlan Industries (Jerusalem, Israel). Livers and kidneys from adult male and female white Leghorn chicken were received from a local slaughter house (Of-Hanegev, Netivot, Israel). For the determination of hepatic and renal glutathione concentrations and glutathione S-transferase activity, liver and kidney aliquots of the three species were homogenized with five volumes of 1.15% KCl solution. For the determination of cytochrome P450-mediated mixed-function oxidase activity and UDP-glucuronyl transferase activity, liver aliquots were homogenized in 7.5 volumes of 10 mM HEPES buffer containing 250 mM sucrose, 1 mM dithiothreitol, 25 mM KCl, 0.5 mM EDTA and 10% glycerol at pH 7.4. The homogenates were centrifuged for 20 min at 9000 g. The 9000 g supernatants in the HEPES buffer were stored at 280°C pending analysis. Microsomes were prepared by the calcium aggregation method according to Cinti et al. (6). Glutathione concentrations in liver and kidney cytosolic fractions were determined according to Mitchell et al. (21). Glutathione S-transferase activity in hepatic and renal cytosolic fractions were determined with 1-chloro-2,4dinitrobenzene according to Baars et al. (1). Aniline hydroxylase was determined according to Mazel (19). Methoxy-, ethoxy- and pentoxyresorufin-O-dealkylase activi-
Correspondence to: Z. Ben-Zvi, Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beersheva 84105, Israel. Tel. 972-7-400845; Fax 972-7-277342; email [email protected]. Received 27 April 1996; accepted 14 July 1996.
E. Amsallem-Holtzman and Z. Ben-Zvi
48
TABLE 1. Concentration of Protein in Liver Fractions of the Ostrich, Chicken and Rat
TABLE 3. Activity of Glutathione S-Transferase and UDPGlucuronyl Transferase in the Ostrich, Chicken and Rat
Liver protein concentration (mg/g liver)
Glutathione S-transferase*
UDP-glucuronyl transferase
Species
Cytosolic
Microsomal
Species
Liver
Kidney
Liver
Ostrich Chicken Rat
71.51 6 1.82* 70.89 6 4.43* 86.01 6 1.87
9.24 6 0.41*† 13.97 6 0.33* 18.08 6 0.41
Ostrich Chicken Rat
2.50 6 0.14†‡ 1.23 6 0.09 1.02 6 0.07
1.90 6 0.14†‡ 1.00 6 0.06† 0.09 6 0.01
17.60 6 2.59†‡ 27.94 6 2.68† 35.36 6 2.64
*Significantly different from the rat, P , 0.05. †Significantly different from the chicken, P , 0.05.
ties (MROD, EROD and PROD, respectively) were determined according to Burke et al. (4). UDP-glucuronyl transferase activity with p-nitrophenol as a substrate was determined according to Bock et al. (3). Protein concentration in the microsomal and cytosolic fractions was determined according to Lowry et al. (17). The results, presented as mean 6 SEM (n 5 10 in each group), were compared for statistical analysis by ANOVA followed by Tukey-Kramer test. Differences were considered significant when P , 0.05. RESULTS The concentration of hepatic protein in the microsomal and cytosolic fractions of the ostrich, chicken and rat are presented in Table 1. The concentration of renal cytosolic protein of the ostrich (10.30 6 2.78 mg/g kidney) was significantly lower than those of the chicken and rat (39.01 6 0.75 and 54.02 6 1.32 mg/g kidney, respectively). Hepatic and renal glutathione concentrations in the ostrich were significantly lower than the concentrations in the rat (Table 2). The activity of ostrich renal and hepatic glutathione S-transferase was significantly higher than that of the chicken and the rat; also, the activity of chicken renal glutathione S-transferase was significantly higher than that of the rat (Table 3). The activity of UDP glucuronyl-transferase was significantly lower in the ostrich than in the rat (Table 3). MROD, EROD, PROD and aniline hydroxylase activities are presented in Table 4. The O-deethylation of ethoxyresorufin in the ostrich was significantly higher than that of the rat and the chicken. The activity of EROD and MROD in chicken microsomes, however, was significantly TABLE 2. Glutathione Concentrations in the Kidney and
Liver of the Ostrich, Chicken and Rat Glutathione concentration (mmol/g tissue) Species
Liver
Kidney
Ostrich Chicken Rat
2.70 6 0.10* 2.70 6 0.10* 6.08 6 0.18
1.49 6 0.13* 2.08 6 0.10* 2.80 6 0.04
*Significantly different form GSH concentration in the rat, P , 0.05.
*The activity of glutathione S-transferase is expressed in µmol of conjugate formed/min/mg prot. The activity of UDP-glucuronyl transferase is expressed in nmol of conjugate formed/min/mg prot. †Significantly different from the rat, P , 0.05. ‡Significantly different from the chicken, P , 0.05
lower than that of the rat. There was no difference in the activity of aniline hydroxylase between the avian species and the rat. DISCUSSION In the present investigation, we studied the activity of the ostrich drug-metabolizing enzymes in comparison with those of the chicken and the rat. Although several studies show that the concentration of cytochrome P450 in rat hepatic microsomes is higher than in birds (11–13), the activity of many cytochrome P450-mediated mixed function oxidases was lower in the rat than in many avian species. There is some discrepancy as to the activity of aniline hydroxylase in the chicken as compared with the rat in various studies. Our results, showing that the activity of aniline hydroxylase is similar in the avian species and in rats, are in agreement with those of Dalvi et al. (7,8); however, other studies have shown that the activity of hepatic aniline hydroxylase was higher in the chicken than in the rat (11,12). The activities of cytochrome P450 1A1 and 1A2, as measured by the dealkylation of ethoxy- and methoxyresorufin, respectively, were higher in ostrich hepatic microsomes than in chicken and rat hepatic microsomes. On the other hand, the activity of hepatic cytochrome P450 2B1/2, measured by pentoxyresorufin-O-depentylation, was lower in the ostrich than in the chicken and the rat. Although such a comparison between ostrich, chicken and rat was not reported previously, Ronis et al. (24) showed that the activity of cytochrome P450 1A1, 2B1, 4A and testosterone hydroxylase were higher in the rat than in the bobwhite quail; however, the activities of cytochrome P450 2E1 and 3A were comparable in the rat and quail. The activity of the hepatic glutathione S-transferase was higher in the ostrich than in the rat, whereas the activity of ostrich hepatic UDP-glucuronyl transferase was lower than that of the chicken and the rat. These results are in line with observations previously published on several wild birds, chicken and quail as compared with the rat (5,15,16,25). The activity of renal glutathione S-transferase in the avian species was significantly and impressively
Drug Metabolism in the Ostrich
49
TABLE 4. Activity of Hepatic Mixed Function Oxidase in the Ostrich, Chicken and Rat
Species
Aniline hydroxylase*
MROD
EROD
PROD
Ostrich Chicken Rat
62.19 6 7.14 50.84 6 3.98 54.70 6 5.10
188.6 6 22.4†‡ 9.6 6 0.7† 32.4 6 4.8
130.3 6 2.8†‡ 12.1 6 1.0† 65.4 6 5.4
3.0 6 0.3†‡ 4.2 6 0.4† 5.0 6 0.4
*The activity of aniline hydroxylase is expressed ass nmol of products formed/min/mg/prot. The activity of MROD, EROD and PROD is expressed as pmol/min/mg prot. †Significantly different from the rat, P,0.05 ‡Significantly different from the chicken, P,0.05
higher than that of the rat renal enzyme activity. Maurice et al. (18) previously showed that in a few breeds of chicken and in the quail, the activity of renal glutathione S-transferase was one order of magnitude higher than in the rat. The ostrich belongs to the family of ratites, which includes several other flightless birds like the rhea, cassowary and the kiwi. Phylogenetically, this family is the oldest offshoot of the avian stem, as evidenced by biochemical and anatomical studies (20,27). Thus, several developmental changes could have occurred in the ostrich enzymatic machinery, as compared with other avian families like the galliformes (chicken and turkey) and the anseriformes (duck), due to environmental, nutritional or other developmental factors. The help of Prof. R. Yagil in obtaining the ostrich tissues is highly acknowledged.
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