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Influence of urethane and ketamine on rat hepatic cytochrome P450 in vivo
Exp Toxic Patho11999; 51: 392-396 URBAN & FISCHER http://www.urbanfischer.de/journals/exptoxpath
Departement of Biochemical Pharmacology, Laboratory of Pharmacology, Istituto Superiore di Sanita, Roma, Italia
Influence of urethane and ketamine on rat hepatic cytochrome P4S0
. .
In VlVO ANNARITA MENEGUZ, STEFANO FORTUNA, PAOLA LORENZINI, and MARIA TERESA VOLPE With 1 figure and 1 table Address for correspondence: Dr. Annarita Meneguz, Department of Biochemical Pharmacology, Laboratory of Pharmacology, Istituto Superiore di Sanita, Viale Regina Elena 299, 00163 Roma, Italia; fax +39 6 49387104. Key words: Hepatic, cytochrome P-4S0; Liver, cytochrome P-4S0; Cytochrome P-4S0, liver; Urethane; Ketamine; Ethylmorphine; Aniline; 7-Pentoxyresorufin; 7 -ethoxyresorufin.
Summary The aim of this study was to identify the effects of widely used laboratory anaesthetics on cytochrome (CYP) activity in male Sprague Dawley rats in vivo. The anaesthetics used were urethane and ketamine. 7-Ethoxyresorufin (EROD), 7-pentoxyresorufin (PROD), aniline and ethylmorphine were used as substrates for CYP lA, CYP 2B, CYP 2El and CYP 3A, respectively. Urethane increased EROD (CYP lA) activity by 40 % (p < 0.01), and hydroxylation of aniline (CYP 2El) by 14 % in the early phase of anaesthesia and by 60 % (p < 0.01) in the later one. Urethane also reduced the demethylation of ethylmorphine by 37 % (p < 0.01), but did not affect CYP 2B activity significantly. Ketamine did not significantly affect CYP lA, 2B or 2EI. However, it reduced the demethylation of ethylmorphine (i.e. CYP 3A) by 32 % (p < 0.01). From these data, we concluded that a single dose of urethane inhibits CYP 3A but increases CYP 2EI and CYP lA, and that a single dose of ketamine inhibits the activity of CYP 3A.
brane-bound heme containing proteins localised in the smooth endoplasmic reticulum of hepatocytes, and to a lesser extent in other tissues, that in most cases generate more polar hydrophilic metabolites that are readily excreted from the body (13). Some commonly used laboratory anaesthetics undergo biotransformation by the hepatic metabolism (S, 6, 11), and can inhibit liver CYP P4S0 monooxygenase (19). Injectable anaesthetics such as urethane and ketamine are reported to be substrates and interact with CYP P4S0 (12, 18,23, 34). It can be hypothesised that these anaesthetics alter the biodisposition of drugs administered during and/or after anaesthesia by their interactions with CYP P4S0 enzymes. The aim of the present study was to characterise the in vivo effects of urethane and ketamine on the rat liver monooxygenase system using 7-ethoxyresorufin (EROD), 7pentoxyresorufin (PROD), aniline and ethylmorphine as substrates for CYP lA, CYP 2B, CYP 2El and CYP 3A, respectively.
Introduction
Material and methods
Ethyl carbamate (EC, urethane) and ketamine are widely used laboratory anaesthetics that provide a sufficiently long period of anaesthesia for surgical procedures in rat stereotaxic surgery and in the rat experimental model of ischemia (3, 8, 31). The potential influence of anaesthetics is a concern when pharmacokinetic parameters and the effect of drug co-administration must be estimated. The administration of two or more drugs is often a common practice to obtain a desired pharmacological effect in an experimental model. The co-administration is associated with changes in the clearance of the administered agents, largely dependent on phase I metabolism through the cytochrome P4S0 monooxygenase system. The cytochrome (CYP) P4S0 enzyme family comprises a system of mem-
Animals: Male-Sprague Dawley rats (200-2S0 g body weight from Charles River, Calco-Co Italy), were housed in polypropylene cages at 22°C and SO % humidity, with lighting from 07.00 a.m. to 10.00 p.m. and were given standard chow and drinking water ad libitum. Rats were divided randomly into four groups of six rats each:
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Group 1: received a single dose of 1.2 g/kg via intraperitoneal (i.p.) injection of urethane (40 % w/v solution in normal saline 0.9 % Nacl) Group 2: received normal saline 0.9 % NaCl and served as control Group 3: received a single dose of 100 mg/kg via i.p. injection of ketamine (SO % w/v solution in distilled water) Group 4: received distilled water and served as control.
The doses of anaesthetics were consistent with those actually used in the laboratory practice (8, 31), and we followed the principles set forth in the Directive of the Council of the European Communities (86/609/EEC) on animal care and use. Preparation of microsomes: Immediately following the loss of the righting reflex (about 15') and 60, 90 and 120 minutes after urethane and/or ketamine administration, rats were killed by cervical dislocation, liver was removed as quickly as possible and immersed in a cool 0.25 M sucrose solution. All subsequent steps were performed at 4°C. Liver was homogenised with a motor drive teflon/glass Potter - Elvehjem homogenizer and the homogenate was centrifuged at 9000 g for 20 min. The supernatants were than centrifuged at 100.000 g for 60 min. The pellets obtained were suspended in 1 mM EDTA (0,15 KCI, pH 704) and centrifuged again at 100.000 g for 60 min. The final microsomal pellets were resuspended in a freezing phosphate buffer solution (0,1 M, pH 704) and EDTA (1 mM), and stored at-80 °C. Drugs: Resorufin, 7-ethoxyresorufin, 7-pentoxyresorufin, aniline, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase were obtained from Boehringer-Mannheim GmbH and Sigma Chemical, Ethylmorphine was from RhonePoulenc, and dimethylsulfoxide (DMSO) from Merck (Darmstadt, Germany). Assays: Total cytochrome P450 was determined spectrophotometrically as the carbon monoxide complex by the method of OMURA and SATO (28). The enzymatic activity of cytochrome P450 IA (CYP IA) was determined by measuring resorufin production by the specific 7-ethoxyresorufin O-deethylase (EROD) method by BURKE and MAYER (7), and cytochrome P450 2B (CYP 2B) was similarly determined using the 7-pentoxyresorufin 0deethylase (PROD) method by LUBERT et al. (22). CYP 2EI was determined by measuring aniline hydroxylation by p-arninophenol (PAP) formation according to MAZEL' s method (24) as described elsewhere (25); the enzymatic activity of ethylmorphine-N-demethylase associated with CYP 3A was also determined by MAZEL'S method, by measure of formaldehyde (HCOH) production.
The kinetic parameters of ethylmorphine inhibition (Km and V ma,) were evaluated using different concentrations of the substrate in the absence and presence of a constant concentration of urethane and ketamine according to the method by MAZEL, in a pool of hepatic microsomes from untreated animals. Statistical analysis: Depending on the experiment, statistical analysis was carried out either by Student's t paired test or by ANOV A. Student's test was used for comparison of two groups, whereas ANOV A in case of multiple comparisons.
Results As shown in table 1 when urethane- and ketamine-treated animals started to lose the righting reflex they showed no change in the basal total content of cytochrome P450. In contrast, changes in CYP lA (+ 63 % EROD activity, p < 0.01), CYP 2EI (+ 15 % aniline hydroxylase activity, not reaching statistical significance), and CYP 3A (- 40 %, p < 0.01) were observed after urethane. In ketamine-treated rats only a decrease (- 30 %, P < 0.01) in CYP 3A was evident. Neither urethane nor ketamine influenced PROD activity. In urethane-treated rats the changes in CYP lA and CYP 3A occurred within 15 min, to return to the basal value 60-90 min after injection. At this point the increase in CYP 2E 1 attained statistical significance and appeared as a dominant microsomal enzyme activity from this time points onwards (fig. 1). Preliminary in vitro studies on ethylmorphine inhibition indicated that in the absence of preincubation both urethane and ketamine display similar V max values and dissimilar Km values (mean ± SE of 3 rats per group: V max 22 ± 4 without anaesthetic and 18 ± 3.8 and 20 ± 3.6 nmol HCOHlmg prot/min in the presence of urethane and ketamine, respectively; ~ 0.29 ± 0.06 without anaesthetic and 0.31 ± 0.06 and 0.75 ± 0.09 mM in the presence of urethane and ketamine, respectively). These data, though
Table 1. Changes in cytochrome P450 activities of rat liver following acute urethane and ketamine anaesthesia. Treatment
Total CYP
EROD (CYP IA)
PROD (CYP 2B)
Aniline Hydroxylase (CYP 2EI)
Urethane Control Ketamine Control
0.68 0.71 0.70 0.67
*15.6 ± 1.8 9.5 ± 2.1 9.8 ± 1.9 lOA ± 2.5
1.85 1.7 2.0 1.9
0.75 0.65 0.68 0.71
± 0.05
± 0.04 ± 0.05 ± 0.06
± 0.29
±0.3 ± 0.35 ±0.3
± 0.05
± 0.07 ± 0.005 ± 0.03
Ethylmorphine-Ndemethylase (CYP 3A) *8.2 12.85 *9.0 13.2
±0.3 ± 0.2 ±0.3 ± 0.18
Results are mean value ± SEM of six rats in each of the groups killed 15' minuts after treatment; CYP IA was determined as EROD activity, CYP 2B as PROD activity, CYP 2EI as aniline hydroxylase activity and CYP 3A as ethylmorphine-Ndemethylase activity. Units for total CYP are nmol/mg protein, for CYP lA and CYP 2B are pmol resorufin/min/mg protein for CYP 2EI are nmol PAP/min/mg protein and for CYP 3A nmol HCHO/mg prot/min. * p < 0.01 compared with control rats. Exp Toxic Pathol51 (1999) 4-5
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mJnuI •
Fig. 1. Time course effects of urethane on rat liver cytochrome P450s activities in rats. Data points represent means ± SE of 6 rats per group and are expressed as percentage of control values, that are for EROD (CYP lA) 10.6 ± 1.7 pmol resorufin/min/mg protein, for aniline hydroxylase (CYP 2E1) 0.63 ± 0.08 nmol PAP/min/mg protein and for ethylmorphineN-demethylase (CYP 3A) 14 ± 0.4 nmol HCHO/min/mg protein. * p < 0.01 versus control value.
obtained in few animals, seem to indicate that both anaesthetics induce a competitive inhibition.
Discussion Several anaesthetics can affect the liver monooxygenase system (20, 21, 23). In the present study, the effects of two commonly used laboratory anaesthetics, urethane and ketamine on the CYP monooxygenase have been described. Urethane was found i) to increase the O-deethylation of ethoxyresorufin, extensively used as a diagnostic substrate for CYP 1A proteins (29), and the hydroxylation of aniline, a substrate for CYP 2E1; ii) to decrease the N-demethylation of ethylmorphine, an activity associated with the CYP 3A family (2). Ketamine was found to reduce the CYP 3A activity, inducing about a 30 % reduction ofthe N-demethylation of ethylmorphine. The choice selection of substrates of CYP activities was based on their relatively selective oxidation by those CYP families (CYP I-CYP3), among the numerous existing, that are mostly involved in the metabolism of drugs and xenobiotics. The CYP 1A family (EROD) is responsible for the metabolism of polycyclin aromatic hydrocarbons and for O-deethylation of phenactin, and it has long been referred to as the "phenobarbital-inducible cytochrome P450" (26, 33). Under our experimental conditions the CYP 1A family appeared to be affected only by urethane administration. In fact, this anaesthetic induces about 60 % increase in EROD activity, while ketamine does not cause any modifications with respect to controls. These data are consistent with those by LOCK et al. (21), that reported an increased activity of CYP 1A as measured by theophylline 394
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clearance, which is a good probe for CYP 1A activity (1) by over 90 %. The induction of cytochrome P450 IA is considered an indicator of potential chemical carcinogenesis (16). Interstingly the metabolic activation of ethylcarbamate is necessary to effect the alkylation of cellular constituents, which is responsible for urethane carcinogenic activity when administered in rodents for long time at high doses. Cytochrome 2El (aniline hydroxylase), an ethanolinducible form, is active in the metabolism of a variety of small organic molecules and is capable of affecting oxidative events because it has a high rate of oxidase activity even in the absence of substrate (4, 15). Thus, the level of P450 2El expression is likely correlated with the production of reacti ve metabolic intermediates generated by a wide variety of small organic compounds, and the induction ofP450 2El produces a high propensity to generate reactive oxygen species (ROS) and promote lipid peroxidation, with the destruction of microsomal membranes (17). In the present study urethane, but not ketamine, modified CYP 2E1 activity. In fact, aniline hydroxylation after urethane administration is stimulated later than EROD and ethylmorphine-N-demethylation are, and the induction is still evident after two hours. This is probably the start of an effect that persists for a longer period. In fact, GLAZER et al. (12) reported that aniline hydroxylation increased significantly (140 %-174 %) from 6 to 48 hours, with a maximum (209 %) increase 12 hours after urethane administration. Ethylmorphine N-demethylase is a microsomal activity associated with the CYP 3A family, the most important family in the human hepatic drug metabolism (16). Cytochrome P450 enzymes of the 3A gene have been isolated from rat, dog and man (9, 14), and they all share similar substrates and selective inhibitors with the consequent similarity in the clearance mechanism of all these species. Antibiotics, Ca++ antagonist, steroids, midazolam, diazepam (for references see Smith, 30) are some of the substrates of CYP 3A. The two anaesthetics used in this study seem competitively and reversibly inhibit the CYP 3A enzyme in the early stage of administration, and consequently modify the basal metabolic pattern of the experimental model. The action of urethane on CYP 450 activity in rats appears to indicate that: I) aniline hydroxylation after administration of urethane is stimulated later than EROD activity is, with no significant alteration in the total level of cytochrome P450; this result is in agreement with those by GLAZER et al. (12) that demonstrated an induction effect from 6 to 48 hours; 2) the early depression of ethylmorphine N-demethylase seems to be competitive and does not require a metabolic activation; in fact, the apparent Km increased in in vitro experiments without preincubation, and the calculated Vmax values were relatively close to each other. These results indicate that in rats in the early phase of anaesthesia the activity ofCyp lA is enhanced and that of CYP 3A is depressed. It can be therefore supposed that the biodisposition of systemically co-administered drugs can be somehow modified or affected in urethane - an-
aesthetised rats. For example, Ca++ antagonists drugs are often administered in experimental models of ischemia in rats anaesthetised with urethane (31, 32). Since the metabolism of all dihydropyridine calcium channel blockers is mediated by CYP 3A to form the pyridine metabolite as the initial clearance step (30) an inhibition of CYP 3A activity could result in higher plasmatic levels of the parent drug and prolonged pharmacological effects, that have to be taken into account when conducting the experiment and when evaluating results. The temporal response of CYP 450 to urethane anaesthesia indicated that the effect on CYP 1A and CYP 3A were reversible; the enzymatic activity returned to control values in about 1 hour. From then onwards CYP 2E1 became the dominant microsomal enzyme activity, which seems to persist for at least 48 hours (12). This effect is important in those cases in which anaesthesia is to be maintained for long time and the ensuing generation of ROS, associated with anaesthetic toxicity (10), can be hypothesised. The marked ability of urethane to interact with the monooxygenase system is probably due to the parent compound and not to a metabolite. In fact, in rats low doses « 5 mg/kg) of urethane are almost exclusively metabolised to CO 2 that is then eliminated through exhaled air. At higher doses the percentage of exhaled CO 2 decreases progressively (27). The dose used in the present experiment falls within the range 400-1800 mg/kg, at which urethane elimination from blood is saturated and it is excreted as such (27). As regard ketamine, the only effect we observed was a reversible and competitive decrease of ethylmorphine N-demethylation (p < 0.01), indicating an influence on the CYP 3A family. MARIETIA et al. (23) demonstrated that pre-treatment of rats with ketamine increases both in vitro hepatic microsomal metabolism ofketamine and the in vivo rate of its plasma decay. Moreover, the metabolism of ketamine to nor-ketamine can be induced by chronic pre-treatment with the anaesthetic itself or with phenobarbital, a well-known P450 inducer, and can be inhibited by SKF 525A a well-known P450 inhibitor, suggesting that the anaesthetic is a substrate for CYP enzymes. It is possible that the effect on CYP 3A may also be due to the metabolite. All these findings are in general agreement with those of LOCH et al. (21) who examined the effect of a series of anaesthetics on CYP 450 activities in vivo, and reported for urethane an increased theophylline clearance (CYP 1A activity), and a decreased CYP 3A activity, which was inferred from the decline (40 % ) in ethosuximide clearance. Ketamine did not significantly alter CYP IA or CYP 2B/ 2C activities, but reduced the clearance of flecamide (CYP 2D1) and ethosuximide (CYP 3A). In conclusion, single large doses of urethane and ketamine similar to those used to anaesthetize experimental animals can affect CYP 450 activity. Our results militate against the use of these anaesthetics in experimental rat models in which systemic administration of drugs is foreseen, because drug pharmacokinetics and metabolic parameters
could be significantly altered. This effect has to be taken into account because it could have as a consequence some modifications of the expected pharmacological activity. Acknowledgements: We thank STEFANO FIDANZA and ADRIANO URCIOLI for the excellent work in animal care. We also thank Mrs. CONCETIINA CORALLO for her prompt assistance in typing this manuscript.
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15. INGELMAN-SUNDBERG M, 10HANSSON I, YIN H, et al.: Ethanol-inducible cytochrome P450 2El: genetic polymorphism, regulation, and possible role in the ethiology of alcohol-induced liver disease. Alcohol 1993; 10: 447-452. 16. IOANNIDES C, PARKE DV: Induction of cytochrome P450 1 as an indicator of potential chemical carcinogenesis. Drug Metab Rev 1993; 25: 485-501. 17. KIM ND, KWAK MK, KIM SG: Inhibition of cytochrome P450 2El expression by 2-(allylthio) pyrazine, a potential chemoprotective agent: hepatoprotective effects. Biochern Pharmacol1997; 53: 261-269. 18. KURATA N, HURST HE, BENZ FW, et al.: Studies on inhibition and induction of methabolism of ethyl carbamate by acetone and related compounds. Drug Metab Disp 1991; 19: 388-393. 19. LABELLA FS, QUEEN G: General anesthetics inhibit cytochrome P450 monooxygenases and arachidonic acid metabolism. Can 1 Physiol PharmacolI992; 71: 48-53. 20. Lm PT, IOANNIDES C, SHAVILA 1, et al.: Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney. Biochem Pharmacol 1993; 45: 871-877. 21. LOCH 1M, POTTER 1, BACHMANN KA: The influence of anesthetic agents on rat hepatic cytochromes P450 in vivo. Pharmacology 1995; 50: 146-153. 22. LUBERT RA, MAYER RT, CAMERON JW, et al.: Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P450 by phenobarbital and other xenobiotics in the rat. Arch Bioche Biophys 1985; 238: 43-48. 23. MARIETTA MP, WHITE PF, PuDWILL CR, et al.: Biodisposition of ketamine in the rat: self-induction of metabolism. 1 Pharmacol Exp Ther 1975; 196: 536-544. 24. MAZEL P: Experiments illustrating drug metabolism in vitro. In: LA Du BN, MANDEL HG, WAY EL (eds): Fundamentals of drug Metabolism and Disposition. Williams and Wilkins, Baltimore. 1972; p. 546.
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25. MENEGUZ A, MICHALEK H: Effect of Diisopropyl Fluorophosphate on hepatic microsomal systems in two strain of rats. Bull Environ Contam Toxicol1990; 44: 924-931. 26. NELSON DR, KAMATAKI T, WAXMAN Dl, et al.: The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes and nomenclature. DNA Cell BioI 1993; 12: 1-51. 27. NOMEIR AA, IOANNOU YM, SANDERS 1M, MATTHEWS HB: Comparative metabolism and disposition of ethyl carbamate (urethane) in male fischer 344 rats and male B6C3Fl mice. Toxicol Appl Pharmacol1989; 97: 203-215. 28. OMURA T, SATO R: The carbon monoxide pigment of liver micro somes I Evidence for its haemoprotein nature. 1 BioI Chern 1964; 239: 2370-2378. 29. RYAN DE, LEWIN W: Purification and characterization of hepatic cytochrome P450. Pharmacol Ther 1990; 45: 153-239. 30. SMITH DA: Species differences in metabolism and pharmacokinetics: are we close to an understanding? Drug Metab Rev 1991; 23: 355-373. 31. SORRENTI V, DI GIACOMO C, RENIS M, et al.: Lipid peroxidation and survival in rats following cerebral postischaernic reperfusion: effect of drugs with different molecular mechanisms. Drugs Exp Clin Res 1994; XX (5): 185-189. 32. VANELLA A, SORRENTI V, CASTORINA C, CAMPISI A, et al.: Lipid peroxidation in rat cerebral cortex during postischaernic reperfusion: effect of exogenous antioxidants and Ca2++ antagonist drugs. Int 1 Devl Neurosci 1992; 10: 75-80. 33. WAXMAN Dl, AZAROFF L: Phenobarbital induction of cytochrome P450 gene expression Biochem 11992; 281: 577-592. 34. YAMAMOTO T, PIERCE lr WM, HURST HE, et al.: Ethyl carbamate metabolism: in vivo and in vitro enzymatic systems. Drug Metab Disp 1990; 18: 276-280.
Departement of Biochemical Pharmacology, Laboratory of Pharmacology, Istituto Superiore di Sanita, Roma, Italia
Influence of urethane and ketamine on rat hepatic cytochrome P4S0
. .
In VlVO ANNARITA MENEGUZ, STEFANO FORTUNA, PAOLA LORENZINI, and MARIA TERESA VOLPE With 1 figure and 1 table Address for correspondence: Dr. Annarita Meneguz, Department of Biochemical Pharmacology, Laboratory of Pharmacology, Istituto Superiore di Sanita, Viale Regina Elena 299, 00163 Roma, Italia; fax +39 6 49387104. Key words: Hepatic, cytochrome P-4S0; Liver, cytochrome P-4S0; Cytochrome P-4S0, liver; Urethane; Ketamine; Ethylmorphine; Aniline; 7-Pentoxyresorufin; 7 -ethoxyresorufin.
Summary The aim of this study was to identify the effects of widely used laboratory anaesthetics on cytochrome (CYP) activity in male Sprague Dawley rats in vivo. The anaesthetics used were urethane and ketamine. 7-Ethoxyresorufin (EROD), 7-pentoxyresorufin (PROD), aniline and ethylmorphine were used as substrates for CYP lA, CYP 2B, CYP 2El and CYP 3A, respectively. Urethane increased EROD (CYP lA) activity by 40 % (p < 0.01), and hydroxylation of aniline (CYP 2El) by 14 % in the early phase of anaesthesia and by 60 % (p < 0.01) in the later one. Urethane also reduced the demethylation of ethylmorphine by 37 % (p < 0.01), but did not affect CYP 2B activity significantly. Ketamine did not significantly affect CYP lA, 2B or 2EI. However, it reduced the demethylation of ethylmorphine (i.e. CYP 3A) by 32 % (p < 0.01). From these data, we concluded that a single dose of urethane inhibits CYP 3A but increases CYP 2EI and CYP lA, and that a single dose of ketamine inhibits the activity of CYP 3A.
brane-bound heme containing proteins localised in the smooth endoplasmic reticulum of hepatocytes, and to a lesser extent in other tissues, that in most cases generate more polar hydrophilic metabolites that are readily excreted from the body (13). Some commonly used laboratory anaesthetics undergo biotransformation by the hepatic metabolism (S, 6, 11), and can inhibit liver CYP P4S0 monooxygenase (19). Injectable anaesthetics such as urethane and ketamine are reported to be substrates and interact with CYP P4S0 (12, 18,23, 34). It can be hypothesised that these anaesthetics alter the biodisposition of drugs administered during and/or after anaesthesia by their interactions with CYP P4S0 enzymes. The aim of the present study was to characterise the in vivo effects of urethane and ketamine on the rat liver monooxygenase system using 7-ethoxyresorufin (EROD), 7pentoxyresorufin (PROD), aniline and ethylmorphine as substrates for CYP lA, CYP 2B, CYP 2El and CYP 3A, respectively.
Introduction
Material and methods
Ethyl carbamate (EC, urethane) and ketamine are widely used laboratory anaesthetics that provide a sufficiently long period of anaesthesia for surgical procedures in rat stereotaxic surgery and in the rat experimental model of ischemia (3, 8, 31). The potential influence of anaesthetics is a concern when pharmacokinetic parameters and the effect of drug co-administration must be estimated. The administration of two or more drugs is often a common practice to obtain a desired pharmacological effect in an experimental model. The co-administration is associated with changes in the clearance of the administered agents, largely dependent on phase I metabolism through the cytochrome P4S0 monooxygenase system. The cytochrome (CYP) P4S0 enzyme family comprises a system of mem-
Animals: Male-Sprague Dawley rats (200-2S0 g body weight from Charles River, Calco-Co Italy), were housed in polypropylene cages at 22°C and SO % humidity, with lighting from 07.00 a.m. to 10.00 p.m. and were given standard chow and drinking water ad libitum. Rats were divided randomly into four groups of six rats each:
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Group 1: received a single dose of 1.2 g/kg via intraperitoneal (i.p.) injection of urethane (40 % w/v solution in normal saline 0.9 % Nacl) Group 2: received normal saline 0.9 % NaCl and served as control Group 3: received a single dose of 100 mg/kg via i.p. injection of ketamine (SO % w/v solution in distilled water) Group 4: received distilled water and served as control.
The doses of anaesthetics were consistent with those actually used in the laboratory practice (8, 31), and we followed the principles set forth in the Directive of the Council of the European Communities (86/609/EEC) on animal care and use. Preparation of microsomes: Immediately following the loss of the righting reflex (about 15') and 60, 90 and 120 minutes after urethane and/or ketamine administration, rats were killed by cervical dislocation, liver was removed as quickly as possible and immersed in a cool 0.25 M sucrose solution. All subsequent steps were performed at 4°C. Liver was homogenised with a motor drive teflon/glass Potter - Elvehjem homogenizer and the homogenate was centrifuged at 9000 g for 20 min. The supernatants were than centrifuged at 100.000 g for 60 min. The pellets obtained were suspended in 1 mM EDTA (0,15 KCI, pH 704) and centrifuged again at 100.000 g for 60 min. The final microsomal pellets were resuspended in a freezing phosphate buffer solution (0,1 M, pH 704) and EDTA (1 mM), and stored at-80 °C. Drugs: Resorufin, 7-ethoxyresorufin, 7-pentoxyresorufin, aniline, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase were obtained from Boehringer-Mannheim GmbH and Sigma Chemical, Ethylmorphine was from RhonePoulenc, and dimethylsulfoxide (DMSO) from Merck (Darmstadt, Germany). Assays: Total cytochrome P450 was determined spectrophotometrically as the carbon monoxide complex by the method of OMURA and SATO (28). The enzymatic activity of cytochrome P450 IA (CYP IA) was determined by measuring resorufin production by the specific 7-ethoxyresorufin O-deethylase (EROD) method by BURKE and MAYER (7), and cytochrome P450 2B (CYP 2B) was similarly determined using the 7-pentoxyresorufin 0deethylase (PROD) method by LUBERT et al. (22). CYP 2EI was determined by measuring aniline hydroxylation by p-arninophenol (PAP) formation according to MAZEL' s method (24) as described elsewhere (25); the enzymatic activity of ethylmorphine-N-demethylase associated with CYP 3A was also determined by MAZEL'S method, by measure of formaldehyde (HCOH) production.
The kinetic parameters of ethylmorphine inhibition (Km and V ma,) were evaluated using different concentrations of the substrate in the absence and presence of a constant concentration of urethane and ketamine according to the method by MAZEL, in a pool of hepatic microsomes from untreated animals. Statistical analysis: Depending on the experiment, statistical analysis was carried out either by Student's t paired test or by ANOV A. Student's test was used for comparison of two groups, whereas ANOV A in case of multiple comparisons.
Results As shown in table 1 when urethane- and ketamine-treated animals started to lose the righting reflex they showed no change in the basal total content of cytochrome P450. In contrast, changes in CYP lA (+ 63 % EROD activity, p < 0.01), CYP 2EI (+ 15 % aniline hydroxylase activity, not reaching statistical significance), and CYP 3A (- 40 %, p < 0.01) were observed after urethane. In ketamine-treated rats only a decrease (- 30 %, P < 0.01) in CYP 3A was evident. Neither urethane nor ketamine influenced PROD activity. In urethane-treated rats the changes in CYP lA and CYP 3A occurred within 15 min, to return to the basal value 60-90 min after injection. At this point the increase in CYP 2E 1 attained statistical significance and appeared as a dominant microsomal enzyme activity from this time points onwards (fig. 1). Preliminary in vitro studies on ethylmorphine inhibition indicated that in the absence of preincubation both urethane and ketamine display similar V max values and dissimilar Km values (mean ± SE of 3 rats per group: V max 22 ± 4 without anaesthetic and 18 ± 3.8 and 20 ± 3.6 nmol HCOHlmg prot/min in the presence of urethane and ketamine, respectively; ~ 0.29 ± 0.06 without anaesthetic and 0.31 ± 0.06 and 0.75 ± 0.09 mM in the presence of urethane and ketamine, respectively). These data, though
Table 1. Changes in cytochrome P450 activities of rat liver following acute urethane and ketamine anaesthesia. Treatment
Total CYP
EROD (CYP IA)
PROD (CYP 2B)
Aniline Hydroxylase (CYP 2EI)
Urethane Control Ketamine Control
0.68 0.71 0.70 0.67
*15.6 ± 1.8 9.5 ± 2.1 9.8 ± 1.9 lOA ± 2.5
1.85 1.7 2.0 1.9
0.75 0.65 0.68 0.71
± 0.05
± 0.04 ± 0.05 ± 0.06
± 0.29
±0.3 ± 0.35 ±0.3
± 0.05
± 0.07 ± 0.005 ± 0.03
Ethylmorphine-Ndemethylase (CYP 3A) *8.2 12.85 *9.0 13.2
±0.3 ± 0.2 ±0.3 ± 0.18
Results are mean value ± SEM of six rats in each of the groups killed 15' minuts after treatment; CYP IA was determined as EROD activity, CYP 2B as PROD activity, CYP 2EI as aniline hydroxylase activity and CYP 3A as ethylmorphine-Ndemethylase activity. Units for total CYP are nmol/mg protein, for CYP lA and CYP 2B are pmol resorufin/min/mg protein for CYP 2EI are nmol PAP/min/mg protein and for CYP 3A nmol HCHO/mg prot/min. * p < 0.01 compared with control rats. Exp Toxic Pathol51 (1999) 4-5
393
mJnuI •
Fig. 1. Time course effects of urethane on rat liver cytochrome P450s activities in rats. Data points represent means ± SE of 6 rats per group and are expressed as percentage of control values, that are for EROD (CYP lA) 10.6 ± 1.7 pmol resorufin/min/mg protein, for aniline hydroxylase (CYP 2E1) 0.63 ± 0.08 nmol PAP/min/mg protein and for ethylmorphineN-demethylase (CYP 3A) 14 ± 0.4 nmol HCHO/min/mg protein. * p < 0.01 versus control value.
obtained in few animals, seem to indicate that both anaesthetics induce a competitive inhibition.
Discussion Several anaesthetics can affect the liver monooxygenase system (20, 21, 23). In the present study, the effects of two commonly used laboratory anaesthetics, urethane and ketamine on the CYP monooxygenase have been described. Urethane was found i) to increase the O-deethylation of ethoxyresorufin, extensively used as a diagnostic substrate for CYP 1A proteins (29), and the hydroxylation of aniline, a substrate for CYP 2E1; ii) to decrease the N-demethylation of ethylmorphine, an activity associated with the CYP 3A family (2). Ketamine was found to reduce the CYP 3A activity, inducing about a 30 % reduction ofthe N-demethylation of ethylmorphine. The choice selection of substrates of CYP activities was based on their relatively selective oxidation by those CYP families (CYP I-CYP3), among the numerous existing, that are mostly involved in the metabolism of drugs and xenobiotics. The CYP 1A family (EROD) is responsible for the metabolism of polycyclin aromatic hydrocarbons and for O-deethylation of phenactin, and it has long been referred to as the "phenobarbital-inducible cytochrome P450" (26, 33). Under our experimental conditions the CYP 1A family appeared to be affected only by urethane administration. In fact, this anaesthetic induces about 60 % increase in EROD activity, while ketamine does not cause any modifications with respect to controls. These data are consistent with those by LOCK et al. (21), that reported an increased activity of CYP 1A as measured by theophylline 394
Exp Toxic Pathol 51 (1999) 4-5
clearance, which is a good probe for CYP 1A activity (1) by over 90 %. The induction of cytochrome P450 IA is considered an indicator of potential chemical carcinogenesis (16). Interstingly the metabolic activation of ethylcarbamate is necessary to effect the alkylation of cellular constituents, which is responsible for urethane carcinogenic activity when administered in rodents for long time at high doses. Cytochrome 2El (aniline hydroxylase), an ethanolinducible form, is active in the metabolism of a variety of small organic molecules and is capable of affecting oxidative events because it has a high rate of oxidase activity even in the absence of substrate (4, 15). Thus, the level of P450 2El expression is likely correlated with the production of reacti ve metabolic intermediates generated by a wide variety of small organic compounds, and the induction ofP450 2El produces a high propensity to generate reactive oxygen species (ROS) and promote lipid peroxidation, with the destruction of microsomal membranes (17). In the present study urethane, but not ketamine, modified CYP 2E1 activity. In fact, aniline hydroxylation after urethane administration is stimulated later than EROD and ethylmorphine-N-demethylation are, and the induction is still evident after two hours. This is probably the start of an effect that persists for a longer period. In fact, GLAZER et al. (12) reported that aniline hydroxylation increased significantly (140 %-174 %) from 6 to 48 hours, with a maximum (209 %) increase 12 hours after urethane administration. Ethylmorphine N-demethylase is a microsomal activity associated with the CYP 3A family, the most important family in the human hepatic drug metabolism (16). Cytochrome P450 enzymes of the 3A gene have been isolated from rat, dog and man (9, 14), and they all share similar substrates and selective inhibitors with the consequent similarity in the clearance mechanism of all these species. Antibiotics, Ca++ antagonist, steroids, midazolam, diazepam (for references see Smith, 30) are some of the substrates of CYP 3A. The two anaesthetics used in this study seem competitively and reversibly inhibit the CYP 3A enzyme in the early stage of administration, and consequently modify the basal metabolic pattern of the experimental model. The action of urethane on CYP 450 activity in rats appears to indicate that: I) aniline hydroxylation after administration of urethane is stimulated later than EROD activity is, with no significant alteration in the total level of cytochrome P450; this result is in agreement with those by GLAZER et al. (12) that demonstrated an induction effect from 6 to 48 hours; 2) the early depression of ethylmorphine N-demethylase seems to be competitive and does not require a metabolic activation; in fact, the apparent Km increased in in vitro experiments without preincubation, and the calculated Vmax values were relatively close to each other. These results indicate that in rats in the early phase of anaesthesia the activity ofCyp lA is enhanced and that of CYP 3A is depressed. It can be therefore supposed that the biodisposition of systemically co-administered drugs can be somehow modified or affected in urethane - an-
aesthetised rats. For example, Ca++ antagonists drugs are often administered in experimental models of ischemia in rats anaesthetised with urethane (31, 32). Since the metabolism of all dihydropyridine calcium channel blockers is mediated by CYP 3A to form the pyridine metabolite as the initial clearance step (30) an inhibition of CYP 3A activity could result in higher plasmatic levels of the parent drug and prolonged pharmacological effects, that have to be taken into account when conducting the experiment and when evaluating results. The temporal response of CYP 450 to urethane anaesthesia indicated that the effect on CYP 1A and CYP 3A were reversible; the enzymatic activity returned to control values in about 1 hour. From then onwards CYP 2E1 became the dominant microsomal enzyme activity, which seems to persist for at least 48 hours (12). This effect is important in those cases in which anaesthesia is to be maintained for long time and the ensuing generation of ROS, associated with anaesthetic toxicity (10), can be hypothesised. The marked ability of urethane to interact with the monooxygenase system is probably due to the parent compound and not to a metabolite. In fact, in rats low doses « 5 mg/kg) of urethane are almost exclusively metabolised to CO 2 that is then eliminated through exhaled air. At higher doses the percentage of exhaled CO 2 decreases progressively (27). The dose used in the present experiment falls within the range 400-1800 mg/kg, at which urethane elimination from blood is saturated and it is excreted as such (27). As regard ketamine, the only effect we observed was a reversible and competitive decrease of ethylmorphine N-demethylation (p < 0.01), indicating an influence on the CYP 3A family. MARIETIA et al. (23) demonstrated that pre-treatment of rats with ketamine increases both in vitro hepatic microsomal metabolism ofketamine and the in vivo rate of its plasma decay. Moreover, the metabolism of ketamine to nor-ketamine can be induced by chronic pre-treatment with the anaesthetic itself or with phenobarbital, a well-known P450 inducer, and can be inhibited by SKF 525A a well-known P450 inhibitor, suggesting that the anaesthetic is a substrate for CYP enzymes. It is possible that the effect on CYP 3A may also be due to the metabolite. All these findings are in general agreement with those of LOCH et al. (21) who examined the effect of a series of anaesthetics on CYP 450 activities in vivo, and reported for urethane an increased theophylline clearance (CYP 1A activity), and a decreased CYP 3A activity, which was inferred from the decline (40 % ) in ethosuximide clearance. Ketamine did not significantly alter CYP IA or CYP 2B/ 2C activities, but reduced the clearance of flecamide (CYP 2D1) and ethosuximide (CYP 3A). In conclusion, single large doses of urethane and ketamine similar to those used to anaesthetize experimental animals can affect CYP 450 activity. Our results militate against the use of these anaesthetics in experimental rat models in which systemic administration of drugs is foreseen, because drug pharmacokinetics and metabolic parameters
could be significantly altered. This effect has to be taken into account because it could have as a consequence some modifications of the expected pharmacological activity. Acknowledgements: We thank STEFANO FIDANZA and ADRIANO URCIOLI for the excellent work in animal care. We also thank Mrs. CONCETIINA CORALLO for her prompt assistance in typing this manuscript.
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