- Email: [email protected]
Xenobiotic conjugation systems in deer compared with cattle and rat
Comparative Biochemistry and Physiology Part C 134 (2003) 169–173
Xenobiotic conjugation systems in deer compared with cattle and rat Susila Sivapathasundaramb, Maurice J. Sauera, Costas Ioannidesb,* a
Department of Risk Research, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK Molecular Toxicology Group, School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
b
Received 6 June 2002; received in revised form 8 August 2002; accepted 20 October 2002
Abstract The ability of cattle and deer liver to catalyse xenobiotic conjugation reactions was investigated and compared with that of the rat. Marked differences in the activity of these enzymes were noted between the domestic animals and rats. Hepatic microsomal epoxide hydrolase activity in cattle and deer, determined using benzowaxpyrene 4,5-oxide as substrate, was nearly twice that of the rat. In contrast, glutathione S-transferase activity in hepatic cytosol, determined with 1chloro-2,4-dinitrobenzene as substrate, was significantly lower in the cattle and deer. When 1,2-dichloro-4-nitrobenzene served as the accepting substrate, no activity was detectable in the cattle and deer. Similarly, glutathione reductase activity and total glutathione levels were markedly lower in the cattle and deer compared with the rat. Cytosolic sulfotransferase activity, monitored using 2-naphthol as substrate, was higher in cattle compared with the rat. Finally, microsomal UDP-glucuronosyl transferase activity, determined using 1-napththol as substrate, did not differ significantly among the three species. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Cattle; Deer; Bovine; Conjugation; Epoxide hydrolase; Glutathione S-transferase; Phase II metabolism
1. Introduction In order to eliminate xenobiotics, living organisms have developed enzyme systems that metabolise these to hydrophilic, readily excretable metabolites. Such metabolism usually proceeds through two distinct stages, phase I metabolism which introduces a functional group into the molecule, and phase II metabolism which involves conjugation of the phase I metabolites, through the functional group, with endogenous substrates such as sulfate, glutathione, glucuronic acid and amino acids (Ioannides, 2002). The principal enzyme system involved in the phase I metabolism of xenobiotics are the microsomal cytochromes P450, *Corresponding author. Tel.: q44-1483-689709; q441483-576978. E-mail address: [email protected] (C. Ioannides).
whereas the phase II metabolism is catalysed by a number of microsomal and cytosolic enzyme systems such as the UDP-glucuronosyl transferases and sulfotransferases. These enzyme systems have been extensively studied for decades in humans and laboratory animals. Very few studies, however, have been devoted to assessing the ability of domestic animals to handle xenobiotics. Such information will make possible the appreciation of potential species differences in bioactivation, and would facilitate the extrapolation of metabolic and toxicological data from one species to another, and thus allow the rationale extension of veterinary medicines originally licensed in a major species, for use in minor or exotic food-producing species. Furthermore, this information would facilitate the risk assessment of drug and other chemical residues in edible tissues
1532-0456/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 2 2 4 - 7
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S. Sivapathasundaram et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 169–173
Table 1 Microsomal conjugation enzymes in bovine and cervine liver in comparison with the rat Enzyme activity
Rat
Cattle
Deer
Epoxide hydrolase (nmolymin per mg protein) Glucuronosyl transferase (nmolymin per mg protein) Microsomal protein (mgyg liver)
3.46"0.34
6.82"0.25*
6.90"0.63*
6.0"0.9
6.2"0.5
6.1"1.0
25.8"2.1
35.0"3.2
29.6"1.9
Results are presented as mean"S.E.M. for five male animals. Epoxide hydrolase and UDP-glucuronosyl transferase activities were determined using benzowaxpyrene 4,5-oxide and 1-naphthol as substrates, respectively. *P-0.05 (t-test).
and milk that reach the consumer. We have recently defined the cytochrome P450 system in the liver of cattle and deer, and observed major differences in the cytochrome P450 profile of these animals and rat (Sivapathasundaram et al., 2001, 2002). Here we report the activities of microsomal and cytosolic conjugation enzyme systems in the liver of cattle and deer in comparison with the rat. 2. Materials and methods Benzowaxpyrene 4,5-epoxide and benzowaxpyrene 4,5-diol (Mid-West Research Institute, Kansas, USA), adenosine 39-phosphate 59-phosphosulfate (PAPS), UDP-glucuronic acid, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro4-nitrobenzene (DCNB), 1- and 2-naphthol, glutathione reductase and glutathione (Sigma Co. Ltd., Poole, Dorset, UK) were all purchased. Male Wistar albino rats (Rattus norvegicus, 200 g) were purchased from the Experimental Breeding Unit, University of Surrey. Bovine liver, from Friesian–Hol stein steers (Bos taurus, 11–12 months old, 416–474 kg), and cervine liver from red deer stags (Cervus elaphus, approx. 18 months old), were obtained from Chitty’s abattoir, Guildford, Surrey and Wadhurst Park, Tunbridge Wells, Kent, respectively. The livers of the domestic animals were removed within 30 min of the animals being killed, perfused with ice-cold EBSS and kept on ice until processing, which was 10 min in the case of the bovine liver and 90 min in the case of the cervine liver. Liver microsomal and cytosolic preparations were prepared as previously described (Ioannides and Parke, 1975). The following assays were performed on the microsomal fraction; epoxide hydrolase using benzowaxpyrene 4,5-epoxide as substrate (Dansette et al., 1979) and UDP-glucuronosyl transferase using 1-naphthol as substrate (Bock and White, 1974). On the
cytosolic fraction: glutathione S-transferase using CDNB and DCNB as substrates (Habig et al., 1974), glutathione reductase (Carlberg and Mannervik, 1975), total glutathione levels (Akerboom and Sies, 1981) and sulfotransferase using 2naphthol as substrate (Sekura et al., 1981). Protein was determined on both fractions (Lowry et al., 1951). Statistical analysis was carried out using Student’s t-test. 3. Results Table 1 shows the activities of microsomal conjugating enzyme systems in the three species. Epoxide hydrolase activity in the cattle and deer was nearly twice that of the rat, whereas there was no difference in UDP-glucuronosyl transferase activity. Sulfotransferase activity in the liver cytosol was significantly higher in the cattle compared with the rat, but there was no significant difference between the two ruminant species (Table 2). Glutathione S-transferase activity, determined using DCNB as the accepting substrate, was not detectable in the cattle and deer but was present in the rat. When the activity was monitored using CDNB, glutathione S-transferase activity was present in all three animal species, being highest in the rat and lowest in the cattle (Table 2). Glutathione reductase activity was low in the cattle and deer compared to the rat and; similarly, total glutathione levels were markedly lower in the domestic species, especially deer, compared with the rat (Table 2). 4. Discussion The fate of a xenobiotic in the living organism depends largely on the profile of the metabolising enzyme systems at the time of exposure. In turn,
S. Sivapathasundaram et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 169–173
171
Table 2 Cytosolic conjugation enzymes in bovine and cervine liver in comparison with the rat Enzyme activity
Rat
Cattle
Deer
Sulfotransferase (pmolymin per mg protein)
46"4
67"6*
57"6
51.03"3.52
ND
ND
1.28"0.14
0.50"0.09**
0.97"0.07*
Glutathione reductase (nmolymin per mg protein)
224"5
121"3**
103"4**
Total glutathione (mM)
5.53"0.30
2.8610.31**
1.32"O.I2**
Cytosolic protein (mgyg liver)
76 "1
76"5
100 "3
Glutathione S-transferase DCNB (nmolymin per mg protein) CDNB (mmolymin per mg protein)
Results are presented as mean"S.E.M. for five male animals. Sulfotransferase was determined using 2-naphthol as substrate. ND, not detectable; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene. *P-0.05, **P-0.001 (t-test).
the expression of individual enzymes is genetically determined (Wormhoudt et al., 1999), but can also be modulated by prior exposure to chemicals, both naturally-occurring and anthropogenic (Lin and Lu, 1998; Ioannides, 1999), and by pathophysiological conditions such as the presence of disease (Ioannides et al., 1996). If an enzyme on which a chemical relies on for its metabolism is not expressed, then the half-life of this compound will be prolonged. For example, in persons that do not express a functional CYP2D6, the half-life and pharmacological activity of drugs such as debrisoquine are increased (Ingelman-Sundberg et al., 1999). Patients with Gilbert’s syndrome, where the activity of UGT1A1 is low resulting in mild hyperbilirubinaemia, exhibit higher incidence of adverse effects after treatment with the topoisomerase inhibitor irinotecan, presumably as a consequence of their inability to eliminate its active metabolite by glucuronidation (Iyer et al., 1998). In order to facilitate extrapolation of metabolic and toxicological data from one species to another, it is vital that the enzyme systems that metabolise xenobiotics in the target species are defined. The two most active phase II conjugation enzyme systems are the UDP-glucuronosyl transferases (Bock, 2002) and sulfotransferases (Glatt, 2002). These enzyme systems largely conjugate nucleophilic metabolites produced as a result of phase I metabolism. The mammalian UDP-glucuronosyl transferases exist as two distinct families. 1-Naphthol, the probe employed in the present study, is a substrate of UGT1A isoforms (Bock, 2002). Both the cattle and deer appear to express this enzyme
to the same extent as the rat. In previous studies, activity was reported to be lower in the cattle compared with the rat (Smith et al., 1984; Watkins and Klaassen, 1986). Cytosolic sulfotransferase activity, however, was significantly higher in the cattle compared with the rat, in concordance with previous studies (Smith et al., 1984; Watkins and Klaassen, 1986; Short et al., 1988). All cytosolic sulfotransferases belong to the same superfamily (Glatt, 2000). Epoxides are toxicologically very important structures since, being electrophiles, they can interact with vital cellular macromolecules giving rise to genotoxicity and cytotoxicity. For example, the carcinogenicity of chemical carcinogens such as bromobenzene, vinyl chloride, aflatoxin B1 and polycyclic aromatic hydrocarbons is mediated by epoxides, which are generated following oxidation of the parent compounds by cytochromes P450. However, two enzyme systems are present in the living organism that can detoxicate these reactive metabolites, the epoxide hydrolases and glutathione S-transferases. The former adds water to the molecule to generate the corresponding dihydrodiol whereas the latter catalyses the conjugation of the epoxide with glutathione, and the resulting conjugate is further metabolically processed and often excreted as the N-acetylcysteine derivative (mercapturate). Microsomal epoxide hydrolase activity, the major xenobiotic-metabolising form of the enzyme, in cattle and deer was nearly double that seen in the rat, indicating that the two ruminants favour this pathway for the detoxication of epoxides. High bovine epoxide hydrolase activity,
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when compared with the rat, has also been reported in studies where styrene oxide was employed as the substrate (Smith et al., 1984; Watkins and Klaassen, 1986). In contrast, glutathione S-transferase activity is significantly lower in the cattle and deer. In fact, when the enzyme was monitored using DCNB no activity was detectable in the bovine and cervine livers. Very low or no activity in cattle liver has also been reported previously by other workers using the same substrate (Smith et al., 1984; Aceto et al., 1986; Hayes et al., 1989; Asaoka, 1984). Clearly both cattle and deer lack the glutathione S-transferase isoform(s) that conjugate this substrate. When CDNB was used, a general substrate of a number of glutathione Stransferase isoenzymes (Sherratt and Hayes, 2002), activity was detectable in all three species but was significantly lower in cattle and deer. Low glutathione S-transferase activity in bovine liver towards CDNB has also been reported previously (Smith et al., 1984; Watkins and Klaassen, 1986). Moreover, the levels of glutathione were markedly lower in the domestic animals compared with the rat. Similarly, glutathione reductase, the enzyme that maintains glutathione in the reduced state, was markedly lower in the bovine and cervine livers compared with the rat. These observations would suggest that cattle and deer, similar to the human, but contrary to the rat (Parke and Ioannides, 1990), favour detoxication of epoxides through hydrolysis rather than through glutathione conjugation. In this way, their limited amounts of glutathione are protected. However, this hypothesis needs to be confirmed by studying the metabolism of epoxides via hydrolysis and glutathione conjugation. In conclusion, marked differences in hepatic phase II conjugation reactions were noted between cattle and deer on one hand, and rat on the other. However, no major changes were observed between the cattle and deer. Similar observations have been made in the case of the cytochrome P450 enzymes, in that the rat differs markedly in its hepatic cytochrome P450 composition from deer and cattle whereas the two domestic species were more closely related (Sivapathasundaram et al., 2001, 2002). These observations would imply that metabolic and toxicological data may be extrapolated from cattle to deer and vice versa, but not from the rat to the two ruminants. However, some caution must be exercised since not all isoforms of the conjugation enzymes have been
investigated in the present study, and additional work would be required, before such conclusions can be confidently reached. To our knowledge, this is the first study concerned with a study of the conjugation enzymes in deer. Acknowledgments This work was supported by the Quality Initiative Scheme from the Veterinary Laboratory Agency, Weybridge, UK. References Aceto, A., Di cola, D., Casalone, E., Sacchetta, P., Federici, G., 1986. Glutathione S-transferase from bovine tissues: relationship between multiple forms, distribution and catalytic activity. Free Rad. Commun. 1, 379–386. Akerboom, T.P.H., Sies, H., 1981. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 7, 373–382. Asaoka, K., 1984. Affinity purification and characterization of glutathione S-transferases from bovine liver. J. Biochem. 95, 685–696. Bock, K.W., 2002. UDP-Glucuronosyl transferases. In: Ioannides, C. (Ed.), Enzyme Systems that Metabolise Drugs and Other Xenobiotics. Wiley, Chichester, pp. 281–318. Bock, K.W., White, I.N.H., 1974. UDP-glucuronosyltransferase in perfused rat liver and in microsomes: influence of phenobarbital and 3-methylcholanthrene. Eur. J. Biochem. 46, 451–459. Carlberg, I., Mannervik, B., 1975. Purification and characterisation of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 250, 5475–5480. Dansette, P.M., DuBois, G.C., Jerina, D.M., 1979. Continuous fluorometric assay of epoxide hydratase activity. Anal. Biochem. 97, 340–345. Glatt, H., 2000. Sulfotransferases in the bioactivation of xenobiotics. Chem-Biol. Int. 129, 141–170. Glatt, H., 2002. Sulphotransferases. In: Ioannides, C. (Ed.), Enzyme Systems that Metabolise Drugs and Other Xenobiotics. Wiley, Chichester, pp. 353–439. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione Stransferase, the first enzymic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Hayes, J.D., Milner, S.W., Walker, S.W., 1989. Expression of glyoxalase, glutathione peroxidase and glutathione S-transferase isoenzymes in different bovine tissues. Biochim. Biophys. Acta 994, 21–29. Ingelman-Sundberg, I., Oscarson, M., McLellan, R.A., 1999. Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends Pharm. Sci. 20, 342–349. Ioannides, C., 1999. Effect of diet and nutrition on the expression of cytochromes P450. Xenobiotica 29, 109–154. Ioannides, C., 2002. Enzyme Systems that Metabolise Drugs and Other Xenobiotics. Wiley, Chichester. Ioannides, C., Parke, D.V., 1975. Mechanism of induction of hepatic drug metabolising enzymes by a series of barbiturates. J. Pharm. Pharmacol. 27, 739–749.
S. Sivapathasundaram et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 169–173 Ioannides, C., Barnett, C.R., Irizar, A., Flatt, P.R., 1996. Expression of cytochrome P450 proteins in disease. In: Ioannides, C. (Ed.), Cytochromes P450: Metabolic and Toxicological Aspects. CRC Press, Boca Raton, FL, pp. 301–327. Iyer, L., King, C.D., Whitington, P.F., et al., 1998. Genetic predisposition to the metabolism of irinotecan (CPT-11): role of uridine diphosphate glucuronosyl transferase 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes. J. Clin. Invest. 101, 847–854. Lin, J.H., Lu, A.Y.H., 1998. Inhibition and induction of cytochrome P450 and the clinical implications. Clin. Pharmacokinet. 35, 361–390. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 263–275. Parke, D.V., Ioannides, C., 1990. The role of cytochromes P450 in mouse liver tumour production. In: Stevenson, D.E., McClain, R.M., Popp, J.A., Slaga, T.J., Ward, J.M., Pitot, H.C. (Eds.), Mouse Liver Carcinogenesis. Alan L. Liss, New York, pp. 215–230. Sekura, R.D., Duffel, M.W., Jakoby, W.B., 1981. Aryl sulfotransferases. Methods Enzymol. 77, 197–199. Sherratt, P.J., Hayes, J.D., 2002. Glutathione S-transferases. In: Ioannides, C. (Ed.), Enzyme Systems that Metabolise
173
Drugs and Other Xenobiotics. Wiley, Chichester, pp. 319–352. Short, C.R., Barker, S.A., Flory, W., 1988. Comparative drug metabolism and disposition in minor species. Vet. Hum. Toxicol. 30, 2–8. Sivapathasundaram, S., Magnisali, P., Coldham, N.G., Howells, L.C., Sauer, M.J., Ioannides, C., 2001. A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver. Biochem. Pharmacol. 62, 635–645. Sivapathasundaram, S., Magnisali, P., Coldham, N.G., Howells, L.C., Sauer, M.J., Ioannides, C., 2002. Cytochrome P450 expression and testosterone metabolism in the liver of deer. Submitted. Smith, G.S., Watkins, J.B., Thompson, T.N., Rozman, K., Klassen, C.D., 1984. Oxidative and conjugative metabolism of Xenobiotics by liver of cattle, sheep, swine and rats. J. Anim. Sci. 58, 386–395. Watkins, J.B., Klaassen, C.D., 1986. Xenobiotic biotransformation in livestock-comparison to other species commonly used in toxicity testing. J. Anim. Sci. 63, 933–942. Wormhoudt, L.W., Commandeur, J.N.M., Vermeulen, N.P.E., 1999. Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione S-transferase, and epoxide hydrolase enzymes; relevance to xenobiotic metabolism and toxicity. Crit. Rev. Toxicol. 29, 59–124.
Xenobiotic conjugation systems in deer compared with cattle and rat Susila Sivapathasundaramb, Maurice J. Sauera, Costas Ioannidesb,* a
Department of Risk Research, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK Molecular Toxicology Group, School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
b
Received 6 June 2002; received in revised form 8 August 2002; accepted 20 October 2002
Abstract The ability of cattle and deer liver to catalyse xenobiotic conjugation reactions was investigated and compared with that of the rat. Marked differences in the activity of these enzymes were noted between the domestic animals and rats. Hepatic microsomal epoxide hydrolase activity in cattle and deer, determined using benzowaxpyrene 4,5-oxide as substrate, was nearly twice that of the rat. In contrast, glutathione S-transferase activity in hepatic cytosol, determined with 1chloro-2,4-dinitrobenzene as substrate, was significantly lower in the cattle and deer. When 1,2-dichloro-4-nitrobenzene served as the accepting substrate, no activity was detectable in the cattle and deer. Similarly, glutathione reductase activity and total glutathione levels were markedly lower in the cattle and deer compared with the rat. Cytosolic sulfotransferase activity, monitored using 2-naphthol as substrate, was higher in cattle compared with the rat. Finally, microsomal UDP-glucuronosyl transferase activity, determined using 1-napththol as substrate, did not differ significantly among the three species. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Cattle; Deer; Bovine; Conjugation; Epoxide hydrolase; Glutathione S-transferase; Phase II metabolism
1. Introduction In order to eliminate xenobiotics, living organisms have developed enzyme systems that metabolise these to hydrophilic, readily excretable metabolites. Such metabolism usually proceeds through two distinct stages, phase I metabolism which introduces a functional group into the molecule, and phase II metabolism which involves conjugation of the phase I metabolites, through the functional group, with endogenous substrates such as sulfate, glutathione, glucuronic acid and amino acids (Ioannides, 2002). The principal enzyme system involved in the phase I metabolism of xenobiotics are the microsomal cytochromes P450, *Corresponding author. Tel.: q44-1483-689709; q441483-576978. E-mail address: [email protected] (C. Ioannides).
whereas the phase II metabolism is catalysed by a number of microsomal and cytosolic enzyme systems such as the UDP-glucuronosyl transferases and sulfotransferases. These enzyme systems have been extensively studied for decades in humans and laboratory animals. Very few studies, however, have been devoted to assessing the ability of domestic animals to handle xenobiotics. Such information will make possible the appreciation of potential species differences in bioactivation, and would facilitate the extrapolation of metabolic and toxicological data from one species to another, and thus allow the rationale extension of veterinary medicines originally licensed in a major species, for use in minor or exotic food-producing species. Furthermore, this information would facilitate the risk assessment of drug and other chemical residues in edible tissues
1532-0456/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 2 2 4 - 7
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Table 1 Microsomal conjugation enzymes in bovine and cervine liver in comparison with the rat Enzyme activity
Rat
Cattle
Deer
Epoxide hydrolase (nmolymin per mg protein) Glucuronosyl transferase (nmolymin per mg protein) Microsomal protein (mgyg liver)
3.46"0.34
6.82"0.25*
6.90"0.63*
6.0"0.9
6.2"0.5
6.1"1.0
25.8"2.1
35.0"3.2
29.6"1.9
Results are presented as mean"S.E.M. for five male animals. Epoxide hydrolase and UDP-glucuronosyl transferase activities were determined using benzowaxpyrene 4,5-oxide and 1-naphthol as substrates, respectively. *P-0.05 (t-test).
and milk that reach the consumer. We have recently defined the cytochrome P450 system in the liver of cattle and deer, and observed major differences in the cytochrome P450 profile of these animals and rat (Sivapathasundaram et al., 2001, 2002). Here we report the activities of microsomal and cytosolic conjugation enzyme systems in the liver of cattle and deer in comparison with the rat. 2. Materials and methods Benzowaxpyrene 4,5-epoxide and benzowaxpyrene 4,5-diol (Mid-West Research Institute, Kansas, USA), adenosine 39-phosphate 59-phosphosulfate (PAPS), UDP-glucuronic acid, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro4-nitrobenzene (DCNB), 1- and 2-naphthol, glutathione reductase and glutathione (Sigma Co. Ltd., Poole, Dorset, UK) were all purchased. Male Wistar albino rats (Rattus norvegicus, 200 g) were purchased from the Experimental Breeding Unit, University of Surrey. Bovine liver, from Friesian–Hol stein steers (Bos taurus, 11–12 months old, 416–474 kg), and cervine liver from red deer stags (Cervus elaphus, approx. 18 months old), were obtained from Chitty’s abattoir, Guildford, Surrey and Wadhurst Park, Tunbridge Wells, Kent, respectively. The livers of the domestic animals were removed within 30 min of the animals being killed, perfused with ice-cold EBSS and kept on ice until processing, which was 10 min in the case of the bovine liver and 90 min in the case of the cervine liver. Liver microsomal and cytosolic preparations were prepared as previously described (Ioannides and Parke, 1975). The following assays were performed on the microsomal fraction; epoxide hydrolase using benzowaxpyrene 4,5-epoxide as substrate (Dansette et al., 1979) and UDP-glucuronosyl transferase using 1-naphthol as substrate (Bock and White, 1974). On the
cytosolic fraction: glutathione S-transferase using CDNB and DCNB as substrates (Habig et al., 1974), glutathione reductase (Carlberg and Mannervik, 1975), total glutathione levels (Akerboom and Sies, 1981) and sulfotransferase using 2naphthol as substrate (Sekura et al., 1981). Protein was determined on both fractions (Lowry et al., 1951). Statistical analysis was carried out using Student’s t-test. 3. Results Table 1 shows the activities of microsomal conjugating enzyme systems in the three species. Epoxide hydrolase activity in the cattle and deer was nearly twice that of the rat, whereas there was no difference in UDP-glucuronosyl transferase activity. Sulfotransferase activity in the liver cytosol was significantly higher in the cattle compared with the rat, but there was no significant difference between the two ruminant species (Table 2). Glutathione S-transferase activity, determined using DCNB as the accepting substrate, was not detectable in the cattle and deer but was present in the rat. When the activity was monitored using CDNB, glutathione S-transferase activity was present in all three animal species, being highest in the rat and lowest in the cattle (Table 2). Glutathione reductase activity was low in the cattle and deer compared to the rat and; similarly, total glutathione levels were markedly lower in the domestic species, especially deer, compared with the rat (Table 2). 4. Discussion The fate of a xenobiotic in the living organism depends largely on the profile of the metabolising enzyme systems at the time of exposure. In turn,
S. Sivapathasundaram et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 169–173
171
Table 2 Cytosolic conjugation enzymes in bovine and cervine liver in comparison with the rat Enzyme activity
Rat
Cattle
Deer
Sulfotransferase (pmolymin per mg protein)
46"4
67"6*
57"6
51.03"3.52
ND
ND
1.28"0.14
0.50"0.09**
0.97"0.07*
Glutathione reductase (nmolymin per mg protein)
224"5
121"3**
103"4**
Total glutathione (mM)
5.53"0.30
2.8610.31**
1.32"O.I2**
Cytosolic protein (mgyg liver)
76 "1
76"5
100 "3
Glutathione S-transferase DCNB (nmolymin per mg protein) CDNB (mmolymin per mg protein)
Results are presented as mean"S.E.M. for five male animals. Sulfotransferase was determined using 2-naphthol as substrate. ND, not detectable; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene. *P-0.05, **P-0.001 (t-test).
the expression of individual enzymes is genetically determined (Wormhoudt et al., 1999), but can also be modulated by prior exposure to chemicals, both naturally-occurring and anthropogenic (Lin and Lu, 1998; Ioannides, 1999), and by pathophysiological conditions such as the presence of disease (Ioannides et al., 1996). If an enzyme on which a chemical relies on for its metabolism is not expressed, then the half-life of this compound will be prolonged. For example, in persons that do not express a functional CYP2D6, the half-life and pharmacological activity of drugs such as debrisoquine are increased (Ingelman-Sundberg et al., 1999). Patients with Gilbert’s syndrome, where the activity of UGT1A1 is low resulting in mild hyperbilirubinaemia, exhibit higher incidence of adverse effects after treatment with the topoisomerase inhibitor irinotecan, presumably as a consequence of their inability to eliminate its active metabolite by glucuronidation (Iyer et al., 1998). In order to facilitate extrapolation of metabolic and toxicological data from one species to another, it is vital that the enzyme systems that metabolise xenobiotics in the target species are defined. The two most active phase II conjugation enzyme systems are the UDP-glucuronosyl transferases (Bock, 2002) and sulfotransferases (Glatt, 2002). These enzyme systems largely conjugate nucleophilic metabolites produced as a result of phase I metabolism. The mammalian UDP-glucuronosyl transferases exist as two distinct families. 1-Naphthol, the probe employed in the present study, is a substrate of UGT1A isoforms (Bock, 2002). Both the cattle and deer appear to express this enzyme
to the same extent as the rat. In previous studies, activity was reported to be lower in the cattle compared with the rat (Smith et al., 1984; Watkins and Klaassen, 1986). Cytosolic sulfotransferase activity, however, was significantly higher in the cattle compared with the rat, in concordance with previous studies (Smith et al., 1984; Watkins and Klaassen, 1986; Short et al., 1988). All cytosolic sulfotransferases belong to the same superfamily (Glatt, 2000). Epoxides are toxicologically very important structures since, being electrophiles, they can interact with vital cellular macromolecules giving rise to genotoxicity and cytotoxicity. For example, the carcinogenicity of chemical carcinogens such as bromobenzene, vinyl chloride, aflatoxin B1 and polycyclic aromatic hydrocarbons is mediated by epoxides, which are generated following oxidation of the parent compounds by cytochromes P450. However, two enzyme systems are present in the living organism that can detoxicate these reactive metabolites, the epoxide hydrolases and glutathione S-transferases. The former adds water to the molecule to generate the corresponding dihydrodiol whereas the latter catalyses the conjugation of the epoxide with glutathione, and the resulting conjugate is further metabolically processed and often excreted as the N-acetylcysteine derivative (mercapturate). Microsomal epoxide hydrolase activity, the major xenobiotic-metabolising form of the enzyme, in cattle and deer was nearly double that seen in the rat, indicating that the two ruminants favour this pathway for the detoxication of epoxides. High bovine epoxide hydrolase activity,
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when compared with the rat, has also been reported in studies where styrene oxide was employed as the substrate (Smith et al., 1984; Watkins and Klaassen, 1986). In contrast, glutathione S-transferase activity is significantly lower in the cattle and deer. In fact, when the enzyme was monitored using DCNB no activity was detectable in the bovine and cervine livers. Very low or no activity in cattle liver has also been reported previously by other workers using the same substrate (Smith et al., 1984; Aceto et al., 1986; Hayes et al., 1989; Asaoka, 1984). Clearly both cattle and deer lack the glutathione S-transferase isoform(s) that conjugate this substrate. When CDNB was used, a general substrate of a number of glutathione Stransferase isoenzymes (Sherratt and Hayes, 2002), activity was detectable in all three species but was significantly lower in cattle and deer. Low glutathione S-transferase activity in bovine liver towards CDNB has also been reported previously (Smith et al., 1984; Watkins and Klaassen, 1986). Moreover, the levels of glutathione were markedly lower in the domestic animals compared with the rat. Similarly, glutathione reductase, the enzyme that maintains glutathione in the reduced state, was markedly lower in the bovine and cervine livers compared with the rat. These observations would suggest that cattle and deer, similar to the human, but contrary to the rat (Parke and Ioannides, 1990), favour detoxication of epoxides through hydrolysis rather than through glutathione conjugation. In this way, their limited amounts of glutathione are protected. However, this hypothesis needs to be confirmed by studying the metabolism of epoxides via hydrolysis and glutathione conjugation. In conclusion, marked differences in hepatic phase II conjugation reactions were noted between cattle and deer on one hand, and rat on the other. However, no major changes were observed between the cattle and deer. Similar observations have been made in the case of the cytochrome P450 enzymes, in that the rat differs markedly in its hepatic cytochrome P450 composition from deer and cattle whereas the two domestic species were more closely related (Sivapathasundaram et al., 2001, 2002). These observations would imply that metabolic and toxicological data may be extrapolated from cattle to deer and vice versa, but not from the rat to the two ruminants. However, some caution must be exercised since not all isoforms of the conjugation enzymes have been
investigated in the present study, and additional work would be required, before such conclusions can be confidently reached. To our knowledge, this is the first study concerned with a study of the conjugation enzymes in deer. Acknowledgments This work was supported by the Quality Initiative Scheme from the Veterinary Laboratory Agency, Weybridge, UK. References Aceto, A., Di cola, D., Casalone, E., Sacchetta, P., Federici, G., 1986. Glutathione S-transferase from bovine tissues: relationship between multiple forms, distribution and catalytic activity. Free Rad. Commun. 1, 379–386. Akerboom, T.P.H., Sies, H., 1981. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 7, 373–382. Asaoka, K., 1984. Affinity purification and characterization of glutathione S-transferases from bovine liver. J. Biochem. 95, 685–696. Bock, K.W., 2002. UDP-Glucuronosyl transferases. In: Ioannides, C. (Ed.), Enzyme Systems that Metabolise Drugs and Other Xenobiotics. Wiley, Chichester, pp. 281–318. Bock, K.W., White, I.N.H., 1974. UDP-glucuronosyltransferase in perfused rat liver and in microsomes: influence of phenobarbital and 3-methylcholanthrene. Eur. J. Biochem. 46, 451–459. Carlberg, I., Mannervik, B., 1975. Purification and characterisation of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 250, 5475–5480. Dansette, P.M., DuBois, G.C., Jerina, D.M., 1979. Continuous fluorometric assay of epoxide hydratase activity. Anal. Biochem. 97, 340–345. Glatt, H., 2000. Sulfotransferases in the bioactivation of xenobiotics. Chem-Biol. Int. 129, 141–170. Glatt, H., 2002. Sulphotransferases. In: Ioannides, C. (Ed.), Enzyme Systems that Metabolise Drugs and Other Xenobiotics. Wiley, Chichester, pp. 353–439. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione Stransferase, the first enzymic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Hayes, J.D., Milner, S.W., Walker, S.W., 1989. Expression of glyoxalase, glutathione peroxidase and glutathione S-transferase isoenzymes in different bovine tissues. Biochim. Biophys. Acta 994, 21–29. Ingelman-Sundberg, I., Oscarson, M., McLellan, R.A., 1999. Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends Pharm. Sci. 20, 342–349. Ioannides, C., 1999. Effect of diet and nutrition on the expression of cytochromes P450. Xenobiotica 29, 109–154. Ioannides, C., 2002. Enzyme Systems that Metabolise Drugs and Other Xenobiotics. Wiley, Chichester. Ioannides, C., Parke, D.V., 1975. Mechanism of induction of hepatic drug metabolising enzymes by a series of barbiturates. J. Pharm. Pharmacol. 27, 739–749.
S. Sivapathasundaram et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 169–173 Ioannides, C., Barnett, C.R., Irizar, A., Flatt, P.R., 1996. Expression of cytochrome P450 proteins in disease. In: Ioannides, C. (Ed.), Cytochromes P450: Metabolic and Toxicological Aspects. CRC Press, Boca Raton, FL, pp. 301–327. Iyer, L., King, C.D., Whitington, P.F., et al., 1998. Genetic predisposition to the metabolism of irinotecan (CPT-11): role of uridine diphosphate glucuronosyl transferase 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes. J. Clin. Invest. 101, 847–854. Lin, J.H., Lu, A.Y.H., 1998. Inhibition and induction of cytochrome P450 and the clinical implications. Clin. Pharmacokinet. 35, 361–390. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 263–275. Parke, D.V., Ioannides, C., 1990. The role of cytochromes P450 in mouse liver tumour production. In: Stevenson, D.E., McClain, R.M., Popp, J.A., Slaga, T.J., Ward, J.M., Pitot, H.C. (Eds.), Mouse Liver Carcinogenesis. Alan L. Liss, New York, pp. 215–230. Sekura, R.D., Duffel, M.W., Jakoby, W.B., 1981. Aryl sulfotransferases. Methods Enzymol. 77, 197–199. Sherratt, P.J., Hayes, J.D., 2002. Glutathione S-transferases. In: Ioannides, C. (Ed.), Enzyme Systems that Metabolise
173
Drugs and Other Xenobiotics. Wiley, Chichester, pp. 319–352. Short, C.R., Barker, S.A., Flory, W., 1988. Comparative drug metabolism and disposition in minor species. Vet. Hum. Toxicol. 30, 2–8. Sivapathasundaram, S., Magnisali, P., Coldham, N.G., Howells, L.C., Sauer, M.J., Ioannides, C., 2001. A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver. Biochem. Pharmacol. 62, 635–645. Sivapathasundaram, S., Magnisali, P., Coldham, N.G., Howells, L.C., Sauer, M.J., Ioannides, C., 2002. Cytochrome P450 expression and testosterone metabolism in the liver of deer. Submitted. Smith, G.S., Watkins, J.B., Thompson, T.N., Rozman, K., Klassen, C.D., 1984. Oxidative and conjugative metabolism of Xenobiotics by liver of cattle, sheep, swine and rats. J. Anim. Sci. 58, 386–395. Watkins, J.B., Klaassen, C.D., 1986. Xenobiotic biotransformation in livestock-comparison to other species commonly used in toxicity testing. J. Anim. Sci. 63, 933–942. Wormhoudt, L.W., Commandeur, J.N.M., Vermeulen, N.P.E., 1999. Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione S-transferase, and epoxide hydrolase enzymes; relevance to xenobiotic metabolism and toxicity. Crit. Rev. Toxicol. 29, 59–124.