Halothane metabolism. Impairment of hepatic omega-oxidation of leukotrienes in vivo and in vitro

Eur. J. Biochem. 206,869-879 (1992) 0 FEBS 1992

Halothane metabolism Impairment of hepatic o-oxidation of leukotrienes in vivo and in vitro

Jorg HUWYLER', Gabriele JEDLITSCHKY ', Dietrich KEPPLER2 and Josef GUT'

* Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany

(Received January 30, 1992) - EJB 92 0126

Department of Pharmacology, Biocenter of the University, Basel, Switzerland

o-Oxidation of leukotrienes is the initial step of hepatic degradation and thus inactivation of these proinflammatory mediators. w-Oxidation is followed by @-oxidation of leukotrienes from the w-end. After exposure of rats to a single dose of the anesthetic agent halothane, a transient decrease in leukotriene o-oxidation was induced both in vivo and in vitro. In untreated rats, 44.1 f 6.0% of N-[3H]acetylleukotriene E4 injected intravenously was recovered unchanged in bile collected for 60 min in vivo; 46.5 f 3.0% was recovered as w-/&oxidation products, of which 24.7 f 4.5% were associated with @-oxidation products only (mean f SEM; n = 5). In rats receiving a single dose of halothane 18 h before the experiment, recovery of unchanged N-[3H]acetylleukotriene E4 was significantly increased to 79.8 f 4.8%, while the fraction of w-/@-oxidation products decreased to 9.0 f 1.7% (n = 5 ) ; 90 h after exposure to halothane, N-[3H]acetylleukotriene E4 recovery decreased to 30.0 f 3.0% and o-/@-oxidation products amounted to 49.1 f 3.8%; the fraction of @-oxidation products was significantly increased to 43.1 f 3.4% (n = 5). Ten days after exposure of rats to halothane, the recoveries of N-[3H]acetylleukotriene E4, of o-/@-oxidation products, and of @-oxidation products alone, returned to almost normal values. Microsomal fractions obtained from rat hepatocytes catalyzed the NADPH- and 02-dependent leukotriene w-oxidation in vitro. The formation of w-hydroxy-metabolites of leukotriene B4, leukotriene E4, and N-acetylleukotriene E4 was decreased by 50% in microsomal fractions obtained from rats 18 h and 90 h after halothane treatment, and returned back to control levels in microsomal fractions obtained 10 days after halothane treatment. The K,,, value of leukotriene B4 w-oxidation revealed no significant change in enzyme affinity towards leukotriene B4; in contrast, as reflected by the reduction of the V,,, value by 65%, a decrease in the amount of the active enzyme in microsomes obtained from rats 18 h after halothane treatment was observed. Halothane-metabolism-dependent trifluoroacetylation of hepatic proteins may mediate this process. Thus, the time course of the density on immunoblots of trifluoroacetylated protein adducts paralleled that of the transient decrease in leukotriene o-oxidation. In contrast to its o-oxidation, leukotriene B4 synthesis from 5-hydroperoxyeicosatetraenoate was not inhibited in hepatocyte homogenates obtained from rats pretreated with halothane. The data suggest that metabolism of halothane causes a transient derangement of hepatic leukotriene homeostasis in vivo.

Correspondence to J. Gut, Department of Pharmacology, Bio- center of the University, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Abbreviations. RP, reverse-phase; SP, straight-phase; NaCl/P,, phosphate-buffered saline; PGB1, prostaglandin B1; i.p., intraperitoneally; CF3C0, trifluoroacetyl; LTB,, (6Z,8E,lOE,142)- (5S, 1 2R)-5,12-dihydroxyeicosa-6,8,10,14-tetraenoate ; LTE4, (7 E,9E, 11ZJ 4Z)-(5S,6R)-6-(cystein-~-yl)-5-hydroxyeicosa-7,9,11,14-tetraenoate; LTE4NAc, (7E,9E,llZ,14Z)-(5S,6R)-6-[(N-acetyl)cystein-S- yl]-5-hydroxyeicosa-7,9,11,I4-tetraenoate; LTE4[3H]NAc, (7E,9E, 1 1 Z, 14Z)-(5S,6R)- 6- { (N-[3H]acetyl)cystein-S-yl} - 5 - hydroxyeicosa- 7,9,11,14-tetraenoate; 20-OH-LTB4, (6Z,8E,lOE,14Z)-(5S,12R)- 5,12,20-trihydroxyeicosa-6,8,10,14-tetraenoate; 20-COOH-LTB4, (62,8 E, 1 OE, 14Z)-(5S. I2R) -5. It-dihydroxyeicosa-6.8,10,14- tetraen- I .20-dioate; 20-OH-LTE4, (7E,9E,11 Z,14Z)-(5S,6R)-6-(cystein-S-y1)- 5,20-dihydroxyeicosa-7,9,11,14-tetraenoate; 20-COOH-LTE,, (7E,

9E,11 Z,14Z)-(5S,6R)-6-(cystein-S-yl)- 5 - hydroxyeicosa-7,9,11,14- tetraen-I ,20-dioate; 20-OH-LTE4NAc, (7E,9E,1 IZ,14Z)-(5S,6R)-6- [(N-acetyl)cystein-S-yl]-5,20-dihydroxyeicosa-7,9,11,14-tetraenoate; 20-COOH-LTE,NAc, (7E,9E,llZ,14Z)-(5S,6R)-6-[(N-acetyl)cystein-S-yl]-5-hydroxyeicosa-7,9,11,14-tetraen-1,20-dioate; 18-COOH- dinor-LTEaNAc, (7E,9E,1 lZ,l4Z)-(5S,6R)-6-[(N-acetyl)cystein-S- yl]-5-hydroxyoctadeca-7,9,11,14-tetraen-l,l8-dioate; 16-COOH-tetranor-LTE,NAc, (7E,9E,1 lZ)-(SS,6R)-6-[(N-acetyl)cystein-S-yl]-5- hydroxyhexadeca-7,9,11 -trien-1,16-dioate.

Enzyme. Leukotriene-B4 20-monooxygenase, (6Z,8E,1 OE,l42)- (5S,12R)-5,12-dihydroxyeicosa-6,8,10,14-tetraenoate, O2 oxidoreductase (20-hydroxylating), LTB, w-hydroxylase (EC 1.14.13.30); leukotriene-E4 20-monooxygenase, (7,5941 1 E,142)-(5S,6R)-6-(cystein-S-yl)-5-hydroxyeicosa-7,9,11 JCtetraenoate, O2 oxidoreductase (20-hydroxylating), LTE4 a-hydroxylase (EC 1.14.1 3.34); NADPH -cytochrome P450 reductase (EC 1.6.2.4).

870

Intrahepatic o-oxidation is an initial event in the stepwise inactivation of LTB4 and the degradation of LTE4 and LTE4NAc [I - 61. Microsomal cytochrome-P450-dependent o-oxidation results in formation of leukotrienes hydroxylated at the position 20 of the carbon skeleton and precedes the formation of o-carboxylated metabolites. Further degradation from the w-end through fl-oxidation leads to a shortening of the fatty acid chain giving rise to the formation of dinor, tetranor, and hexanor metabolites of leukotrienes [3, 41.

The liver is considered as the main organ for leukotriene uptake and inactivation [6]. Increased local leukotriene concentrations due to an impairment of w-oxidation of leukotrienes may contribute to the development of liver injury. Levels of cysteinyl-leukotrienes are elevated in endotoxin- induced acute liver injury [7] and inhibition of leukotriene biosynthesis or receptor antagonism result in hepatoprotec- tion in experimental liver disease [7, 81. An LTB4-like com- pound was discussed as a mediator in acute alcoholic hepatitis since both rat and human hepatocytes produced LTB4-like activity when challenged with ethanol [9, 101. Recent in vivo experiments showed that ethanol impaired the inactivation of cysteinyl leukotrienes by o-oxidation; identical results were obtained for oxidative LTB4 inactivation in hepatocyte suspensions in vitro. The major site of inhibition was identified as the oxidation of 20-OH-LTE4NAc to 20-COOH-LTE4NAc and 20-OH-LTB4 to 20-COOH-LTB4, respectively, leading to an accumulation of the corresponding 20-OH-leukotrienes [Ill. In fact, separate experiments in vitro had revealed that hepatic alcohol dehydrogenase and acetaldehyde dehydrogenase catalyzed the NAD +-dependent oxidation of 20-OH-LTB4 to 20-COOH-LTB4, which was inhibitable by ethanol [I I].

Metabolism of the anesthetic agent halothane (CF,CHCIBr) causes a mild form of hepatotoxicity in about 20% of patients 1121. In rare cases, patients develop a fulminant hepatitis which is believed to have an immunological basis; most convincingly, sera of patients, but not sera of the corresponding control individuals, contain antibodies against prominent liver microsomal heterologous proteins, tnflUOrdWty1 adducts (CF,CO-adducts ', which may comprise the offending immunogen(s) in susceptible individuals [13 - 171. CF,CO-adducts arise upon cytochrome-P450-dependent oxidative metabolism of halothane, which yields, among other metabolites, trifluoroacetyl chloride (CF,COCI) as a reactive intermediate that covalently modifies hepatic proteins through trifluoroacetylation [13 - 171. Critical cellular proteins might be among the targets of CF,CO-adduct formation and, consequently, an ensuing disturbance of their enzymic functions might occur. In this study, using a rat model system, we tested the hypothesis that hepatic leukotriene w-hydroxylase might be one of these functionally affected cellular targets.

EXPERIMENTAL PROCEDURES

Materials

Prostaglandin B (PGB trypsin-chymotrypsin inhibitor, phenylmethanesulfonyl fluoride, phenobarbital, cytochrome

The term CF,CO-adduct denotes any protein carrying trifluoroacetylated amino acid residues with no reference made to function and identity of that particular protein. The term anti-CF,CO antibody refers to the affinity-purified monoform antibody specifi- cally recognizing CF,CO-protein adducts, and cross-reactive proteins of 52 kDa and 64 kDa.

c, collagenase and cumene hydroperoxide were purchased rom Sigma (St. Louis, MO, USA). Collagenase/dispase was from Boehringer (Mannheim, FRG). NADPH was from Fluka (Switzcrkand). Percoll was from Pharmacia (Sweden). Halothane was purchased from Halocarbon Laboratories (New Jersey, USA). Synthetic LTB4 and LTE4 were generous gifts fiom the Center for Therapeutic Research of Merck Frosst (Canada). Synthetic 20-OH-LTB4, 20-COOH-LTB4, 20-OH -LTE4, 20-COOH-LTE4, LTE4NAc and 20-OH- LTE4hAc were obtained from Cascade Chemicals, UK. LTE4['HH]NAc (kindly provided by Dr. B. Glanzer, Heidel- berg) was synthesized from LTE, by chemical N-acetylation with ["Hlacetic anhydride, purified by RP-HPLC and was more than 98% pure [18]. (6E,SZ,llZ,14Z)-(SS)-hydroperoxyeicosa-6,8,11,14-tetraenoate was synthesized enzy- matically from arachidonic acid (Calbiochem, San Diego, CA, USA) using potato tuber 5-lipoxygenase and purified by pre- parati1 e SP-HPLC using a LiChrosorb Si-60 column (Merck, Darmstadt, FRG) as described [19]. Debrisoquine and 4-hydr8.,xy-debrisoquine were obtained from U. A. Meyer as gifts from Hoffmann-LaRoche (Basel, Switzerland).

Animals

Male Sprague-Dawley rats (250 g) were obtained from the animal breeding facility of the Biocenter (Basel, Switzerland). The animals were treated intraperitoneally (i.p.) with phenobarbital (80 mg/kg of body mass) on four successive days. On the fifth day, the animals received halothane (i.p., 10 mmol/ kg boc!y mass) in Sesam oil ( l : l , by vol.). Control animals were nt)t treated with halothane. After the time intervals indicated, .inimals were subjected to measurement of leukotriene o-oxidation in vivo or to preparation of hepatocytes, which were cised both to investigate in vitro w-oxidation of leukotrienes and to evaluate the halothane-metabolism dependent C F,CO-adduct formation.

In vivo animal experiments

In bjivo experiments with rats that had been starved for 12 h p ~ i o r to the experiment were performed under anesthesia with kztamine (80 mg/kg body mass) and xylazine (12 mg/ kg body mass) injected intraperitoneally. The bile duct was cannulded with polyethylene tubing (outer diameter 0.6 mni, inner diameter 0.3 mm). LTE4[3H]NAc (150 kBq/ kg; 2.5 nmol/kg) was injected into the femoral vein. Bile was sampled continuously into ice-cold 90% (by vol.) aqueous methanol (containing 1 mM 4-hydroxy-2,2,6,6-tetramethyl- piperidine-1-oxyl and 0.5 mM EDTA at pH 7.4) and stored at -20°C under argon. After 3 h at -2O"C, bile fractions were centrifuged for 15 min at 10000 x g; the supernatants were then evaporated to dryness, resuspended in 30% (by vol.) aqueous methanol, and separated by RP-HPLC with continuous on-line determination of tritium radioactivity in the eluate (LB 500 C, Berthold). The total amount of radioactivity injected for rach chromatogram was about 3.7 kBq. LTE4NAc and its metabolites were separated on a linear gradient of 40- 80% aqueous methanol over 35 min followed by a hold at 80% aqueous methanol for 20 min. All solvents contained 0.1 % (by vol.) acetic acid and 1 mM EDTA and were adjusted to pH 5.0 with ammonium hydroxide. The identification of metabc Ilites was by co-chromatography with synthetic standards in several standard systems as described [20]. Ab- sorbance of standards was followed at 280 nm.

871

Preparation of microsomal fractions from rat hepatocytes

Animals received an unrestricted diet and were starved for 12 h prior to the experiment. Single-cell suspensions of hepatocytes were prepared by in situ perfusion of collagenase/ dispase. Separation of hepatocytes from non-parenchymal liver cells was performed according to Smedsrerd and Pertoft [21] with slight modifications. Briefly, cell suspensions were filtered through nylon mesh and then centrifuged for 4 min at 50 x g , yielding a pellet enriched in hepatocytes and a supernatant fraction enriched in non-parenchymal cells. The pellet, containing mostly hepatocytes, was washed in NaCl/Pi (10 mM Na2HP04, 3 mM KH2P04, 137 mM NaCl, pH 7.4) and diluted aliquots of resuspended hepatocytes (25 ml) were immediately layered on top of a Percoll cushion (25 ml) with a density of 1.070 g/ml and centrifuged at 130 x g for 10 min. Viable hepatocytes, which only penetrated the Percoll layer, were collected and washed once more with NaC1/Pi. Finally, the hepatocytes obtained were resuspended in 50 mM Tris HCl, 150 mM NaC1, pH 7.4, containing 500 pM phenyl- methylsulfonyl fluoride and 60 pg/ml trypsin-chymotrypsin inhibitor. Hepatocytes were disrupted by ten strokes in a Pot- ter Elvehjem homogenizer at 4°C followed by sonication for 15 rnin in a bath-type sonifier (Branson) in an ice water bath. The homogenate obtained was centrifuged at 3000 x g for 20 min to remove nuclei and cell debris. The supernatant was then centrifuged at 105 000 x g for 60 min. The resulting pellet was washed, centrifuged as above and resuspended in 50 mM Tris HCl, 150 mM NaCl, pH 7.4 containing 500 pM phenyl- methylsulfonyl fluoride and 60 pg/ml trypsin-chymotrypsin inhibitor. The hepatocyte microsomal fractions obtained were stored in aliquots at - 80°C and typically thawed only once just prior to the experiment.

w-Oxidation of LTB.,, LTE4, and LTE4NAc by hepatocyte microsomal fractions

Hepatocyte microsomal fractions (0.19 mg protein in 200 pl 10 mM potassium phosphate pH 7.4, 150 mM NaC1, 50 pM EDTA) were incubated for 30 rnin at 37°C with LTB4, LTE4, or LTE4NAc at the concentrations indicated in presence of 1mM NADPH. When LTB4 (kept in methanol) was used as a substrate, it was taken to dryness under a stream of nitrogen in the reaction vessel directly followed by the addition of the incubation mixture. In contrast, LTE4 and LTE4NAc were added directly to the incubation mixture from an aqueous stock solution. The reaction was started by the addition of NADPH. In studies where kinetic or inhibition parameters were determined, substrate and inhibitor concentrations were varied as described in the figure legends. Termination of the reaction and extraction of metabolites was done as described previously [19]. Metabolites were analyzed by RP-HPLC using a modified method of Shak and Goldstein [22]. Briefly, separation of products was achieved on a Nucleosil 100 C18 column (4.6 x 250 mm, 5 pm; operated with a guard column, at a flow rate of 0.9 ml/min) using a gradient system comprising a linear gradient of methanol/acetonitrile/water/acetic acid from 310:221:469:0.2 (by vol.) to 481 :226:293:0.2 (by vol.) over 6 rnin followed by a hold of 12 min at this condition. A linear gradient over 2 min to methanol/acetonitrile (750:250, by vol.) followed and, after 16 min, the column was reequilib- rated to the initial conditions. Retention times determined with synthetic standards were 10.3 min, 11.3 min, 12.2 min, 12.6 min, 13.9 min, 21.0 min, 25.2min, 26.9 rnin and 27.7 min for 20-COOH-LTB4, 20-OH-LTB4, 20-COOH-LTE4, 20-OH-

LTE4, 20-OH-LTE4NAc, PGBl, LTB4, LTE4NAc and LTE4, respectively. Absolute recovery was determined by comparing (in percentages) peak heights of metabolites and the internal standard PGBl with those of the directly injected synthetic standards. An area height percentage method using baseline projection from the last baseline reference point after the previous peak to the next point satisfying baseline criteria (Perkin-Elmer LCI 100 computing integrator software, Code 2) was used to calculate peak heights.

In double inhibition experiments, metabolites were isocratically separated at methanol/water/acetic acid (620 : 380: 1.5, by vol.) followed after 10 min by a linear gradient to 755:245:1.0 (by vol.) over 6 min. The flow was kept at 0.8 ml/min and all eluents used were adjusted to pH 4.7 with triethylamine and included 50 pM EDTA throughout. In this chromatographic system, retention times were 14.0 min, 17.0 min, 20.0 min, and 28.0 rnin for 20-OH-LTB4, 20-OH- LTE4, 20-OH-LTE4NAc, and PGB1, respectively.

Biosynthesis of LTB4 by rat hepatocytes

Incubations were carried out in sealed Reacti-vials under an argon atmosphere at room temperature for 5 min. Hepatocyte homogenates were incubated (typically 0.1 9 mg protein in 200 p1 of 10 mM potassium phosphate pH 7.4, 150 mM NaC1, 50 pM EDTA) with 5-hydroperoxyeicosatetraenoate (100 pM). Reaction products were extracted and analyzed by RP-HPLC as described [19].

Gel electrophoresis and immunoblotting

Hepatocyte homogenates (5 - 10 mg protein/ml) were mixed 1 : 1 (by vol.) with dissociation buffer [0.5 M Tris HCl pH 6.8, 8% (masslvol.) SDS, 20% glycerol, 4 mM EDTA, 40 mM dithiothreitol] and heated to 95°C for 10 min. SDS PAGE was performed according to Laemmli [23] using a 4% stacking and a 10% separating gel. Protein loading was 250 pg/cm slot width. Electrophoresis was typically for 4 h at 30 mA/gel. Resolved polypeptides were transferred elec- trophoretically to nitrocellulose according to Towbin [24] at 360 V x h using a transfer buffer comprising 15.6 mM Tris, 120 mM glycine and 20% (by vol.) methanol at pH 8.3. After transfer, the nitrocellulose was stained with amido black for visualizing proteins, destained, and blocked for 2 h at room temperature with NaC1/Pi containing 2% (mass/vol.) dried milk powder and 0.02% (masslvol.) Thimerosal. The nitrocellulose was then cut into strips (3 mm width) and used for antibody overlay. Incubation with the first antibody (appro- priately diluted into blocking solution) was for 18 h at room temperature under constant shaking. After five 5-min washes with blocking solution, incubation with horseradish-peroxidase-conjugated second antibody (diluted into blocking solution) was for 2 h at room temperature. After one wash with blocking solution and four washes with NaCl/Pi for 5 rnin each, CF,CO-adducts were visualized by enhanced chemiluminescence detection. The anti-CF3C0 antibody used in these experiments was obtained according to Christen et al. [14, 251 by affinity chromatography of an antiserum raised against trifluoroacetylated rabbit serum albumin on an Affi-Gel102 amino-terminal agarose column (1.0 x 10 cm), to which Nc-trifluoroacetyl-L-lysine had been coupled by carbodiimide-facilitated chemistry according to the manufac- turer’s guidelines (Bio-Rad).

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Microsomal debrisoquine hydroxylation

Microsomal cytochrome-P-450 dependent hydroxylation of debrisoquine was determined according to Kronbach et al. [26] with slight modifications. Briefly, hepatocyte microsomal fractions (0.3 mg in 100 pl of 100 mM sodium phosphate pH 7.4) were incubated for 15 min at 2 5 T in the presence of 2 mM debrisoquine and 125 mM cumene hydroperoxide. In a separate set of experiments, cumene hydroperoxide was replaced by 1 mM NADPH and the incubation time was prolonged to 30 min. Reactions were terminated by the addition of 10 pl 60% perchloric acid. After centrifugation (3 min, 10 000 x g), the supernatant was analyzed by RP- HPLC. Separation of metabolites was achieved isocratically on a Nucleosil 5 C18 column (125 x 4.6 mm, 5 pm, operated at a flow rate of 1.2 ml/min) with an eluent comprising acetonitrile/20 mM perchlorate, pH 2.5 (15:85, by mass). De- tection of metabolites was by fluorescence (Perkin Elmer 650- 10s fluorescence spectrophotometer, excitation wavelength 219 nm, emission wavelength 286 nm).

Other methods

The concentrations of the leukotrienes, of the o-hydroxylated metabolites thereof and of S-hydroperoxyeicosatetraenoate were estimated by ultraviolet spectroscopy using an Uvikon 820 spectrophotometer. Typically, absorption spectra were recorded over 220-330 nm. Absorption coefficients were E~~~ = 40000 M-' cm-' for LTB4 and its metabolites, &280 = 39900 M- ' cm-' for LTE4, LTE4NAc, and their metabolites and ~ 2 3 5 = 28 000 M-' cm-' for 5- hydroperoxyeicosatetraenoate. The measurements were carried out in ethanol. During RP-HPLC, on-line spectra of leukotrienes were taken and compared to those in a library obtained from synthetic standards for spectral identification. NADPH - cytochrome P-450 reductase was assayed according to Strobel et al. [27]. Cytochrome c reduction rates were calculated using an absorption coefficient of 21 mM-' cm-' at 550 nm. Measurements were carried out at 31°C in an Aminco DW-2 spectrophotometer. Protein concentrations were estimated by the Bio-Rad assay procedure using bovine serum albumin as a standard.

RESULTS AND DISCUSSION

The liver is the main organ for uptake and degradation of circulating leukotrienes [6] . An initial event in the pathway of the stepwise inactivation of leukotrienes B4, E4, and E4NAc is intrahepatic w-oxidation [l - 51. The very first step in this process is a cytochrome-P450-dependent hydroxylation at position 20 of the carbon backbone of leukotrienes, leading to the formation of the corresponding o-hydroxy-metabolites. This transformation is followed by further oxidation of the w- hydroxy-metabolites to the o-carboxy-m'etabolites, a reaction involving cytosolic alcohol dehydrogenase and aldehyde dehydrogenase [l ll. The cytochrome-P450-dependent transformation of w-hydroxy- to w-carboxy-metabolites, as known for neutrophils [28], seemed of little importance in the hepatocyte suspensions [ll]. The fatty acid chain of leukotrienes is then further degraded through /?-oxidation from the o-carboxy-end to dinor, tetranor and hexanor metabolites [3 - 51. In the study presented here, we have inves- tigated the influence of in vivo metabolism of the anesthetic

LTE,NAc

20-COOH- LTE,NAc

18-COOH-dinor-

tetranor- LTE,NAc 7 -

v m " 0

t

40

1 , 2ou 0

0 20 40 60

RETENTION TIME (rnin)

Fig. 1. HPLC separation of biliary LTE4I3HJNAc metabolites. Rats were pretreated with four daily injections (i.p.) of phenobarbital (80 mg 'kg body mass) followed by a single dose (i.p.) of halothane (10 mnbol/kg body mass) 18 h or 90 h prior to the experiment. Control animals did not receive halothane. Anesthesized bile duct cdnnulated rats reccived LTE4[3H]NAc (150 kBq/kg; 2.5 nmol/kg) intravenously and bile was collected over 0 - 30 min and analyzed by RP-HPLC. Retention times of synthetic standards are indicated by arrows.

agent halothane on leukotriene w-oxidation both in vivo and in vitro.

I n vivo inhibition of LTE4NAc degradation by halothane

The major circulating metabolite of LTC4 in vivo is LTE4 which is predominantly taken up by the liver (i.e. hepatocytes), partially degraded and excreted into bile [6]. Besides LTE4, LTC4 m d LTE4NAc are also taken up efficiently by the liver [29]. In the liver, rapid N-acetylation transforms LTE4 into LTEJJAc which represents the major metabolite in rat bile collected over the first 30 min after intravenous injection of LTE4. Additional reports indicate that LTB4 is also eliminated from the circulation by hepatic uptake and biliary excretion both i:i the isolated perfused rat liver and in vivo in the rat [30] and in the monkey [31]. Furthermore, extensive chain shortening through /?-oxidation from the wend of the cysteiIty1 leukotrienes and of LTB4 was localized to the peroxisomal compartment isolated from livers of normal and clofibi ate-treated rats [32].

In order to assess the effect of the anesthetic agent halothane on leukotriene w-oxidation in vivo, LTE4[3H]NAc was administered intravenously to untreated rats or rats that had been pretreated with a single dose of halothane (10 mmol/

873

* I T

LTE4 r3 HINAc 0 - I p -oxidation products p -oxIdation products

Control 18 h 90 h 10 d

Time after halothane exposure

Fig. 2. Metabolism in vivo of LTE,NAc in rats treated with halothane. Rats wcrc pretreated with four daily injections of phenobarbital (80 mg/kg body mass, i.p.) followed by a single dose of halothane (10 mmol/kg body mass, i.p.). Control animals did not receive halothane. Rats were anesthesized and bile duct cannulation was performed as described in Experimental Procedures. LTE4[3H]NAc (1 50 kBq/kg; 2.5 nmol/kg) was injected intravenously and bile samples were collected continuously over 0 - 30 min and over 30 - 60min and analyzed separately on HPLC as described [20]. The data represent the percentage of the injected dose recovered in the combined 0 - 60 min bile sample. w-/P-Oxidation products comprise 20-OH-LTE4[3H]NAc. 20-COOH-LTE4[3H]NAc, 18-COOH-dinor- LTE,[’H]NAc, 16-COOH-tetranor-LTEJ[fH]NAc, and more polar products of /?-oxidation (see Fig. 1); /?-oxidation products comprise ~ ~ - C O O H - ~ ~ ~ O ~ - L T E , [ ~ H ] N A C , 16-C00H-tetran0r-LTE,[~H]NAc, and more polar products of /?-oxidation only. Data represent means 5 SEM of values obtained from five animals. Asterisk (*) indicates a statistically significant difference from control value by Student’s t-test (P < 0.01).

kg body mass) and its metabolites recovered in bile were analyzed by RP-HPLC (Fig. 1). Halothane treatment did not interfere with hepatobiliary elimination of LTE4[3H]NAc: 90.6 f 3.8%0, 88.8 & 3.3%, 79.1 f 5.6%, and 79.6 f 5.0% (mean values f SEM, n = 5 ) of administered radioactivity were excreted in bile within 60 min in rats not treated, or rats treated with halothane 18 h, 90 h, and 10 days before the experiment, respectively (Fig. 2). In the bile of rats not treated with halothane, LTE4[3H]NAc was transformed to w-/fl-oxidation products’ (Fig. 1, control) that amounted to 46.5 f 3.0% of recovered radioactivity. The fraction of p-oxidation products was 24.7 + 4.5%, while 44.1 6.0% of LTE4[3H]NAc was recovered unchanged (Fig. 2, control). Very low amounts of the initial o-oxidation product, 20-OH- LTE4[3H]NAc, were detected in these in vivo experiments. In striking contrast, in the bile of rats treated with halothane 18 h before the experiment, 79.8 f 4.8% of LTE4[3H]NAc was recovered unchanged and 9.0 ? 1.7% was recovered as w-/S-oxidation products, of which 3.5 1.2% was associated with P-oxidation products (Figs. 1 and 2). These data indicate a strong block in w-oxidation of LTE,[’H]NAc in halothane

The term w-//?-oxidation products used in this report refers to the products of hepatic LTE,NAc w-oxidation in vivo, comprising 20-OH-LTE4NAc, 20-COOH-LTE4NAc, 18-COOH-dinor- LTE,NAc, 16-COOH-tetranor-LTE,NAc, and more polar, not yet structurally elucidated products of b-oxidation (see Fig. 1, control). Thc term P-oxidation products refers to 18-COOH-dinor-LTE4NAc, 16-COOH-tetranor-LTE,NAc, and the more polar products of p- oxidation combined.

treated rats (Fig. 2), leading to an increased amount of intact LTE4[3H]NAc recovered in bile.

Recovery from impairment of LTEJ’HlNAc o-oxidation in vivo

We next tested the persistence in vivo of halothane-associated impaired LTE,[’H]NAc o-oxidation. In rats treated with halothane 90 h before the experiment, a pattern of leukotriene metabolites very different from that in rats exposed to halothane 18 h before the experiment was observed. Only 30.0 k 3.0% of LTE4[3H]NAc was recovered unchanged after 90 h (Fig. 2). Strikingly, 49.1 f 3.8% of recovered radioactivity was associated with w-/p-oxidation products, of which the majority (i.e. 43.1 f 3.4%) was associated with P-oxidation products (Figs. 1 and 2). When compared to rats treated with halothane 18 h before the experiment, these data indicated, based on the significant decrease in recovery of intact LTE4[3H]NAc, a slightly increased capacity for w-oxidation of LTE4[3H]NAc, and, in parallel, a far higher capacity for the P-oxidation of 0-oxidation products (primarily 20-COOH- LTE,[3H]NAc) in rats treated 90 h before the experiment. When compared to control rats not exposed to halothane (Fig. 2), both w- and the ensuing P-oxidation of LTE4[3H]NAc were slightly, but significantly, higher in rats treated with halothane 90 h before the experiment. Both activities returned to almost normal values in rats treated with halothane 10 days before the experiment (Fig. 2).

o-Oxidation of leukotrienes in vitro

The previous in vivo experiments suggested that in rats treated with halothane 18 h prior to the experiment, the first step in w-oxidation of LTE,NAc, namely its transformation into 20-OH-LTE4NAc, was severely affected. In order to assess this impairment in vitro, we prepared hepatocyte microsomal fractions from untreated rats, and rats pretreated with a single dose of halothane 6 h, 12 h, 18 h 90 h, and 10 days before the experiment. These preparations were used to measure in vitro the NADPH-dependent transformation of LTB4, LTE4 , and LTE4NAc into their corresponding w-hydroxy-metabolites. The formation rates of 20-OH-LTE4 and 20-OH-LTE4NAc from 20pM LTE, and 10pM LTE4NAc, respectively (Table 1) and those of 20-OH-LTB4 formation from 5 pM LTB4 (Fig. 3B) decreased by about 50% in microsomal fractions of rats treated with halothane 18 h or 90 h before the experiment. The impairment of w-oxidation of leukotrienes (as shown for LTB4 in Fig. 3B) was present as early as 6 h and 12 h after exposure of rats to halothane; at the same time points, extensive trifluoroacetylation of proteins was also evident (Fig. 3A). In contrast, the formation rates of o-hydroxy-metabolites of all three leukotrienes in microsomal fractions obtained from rats 10 days after halothane exposure were almost identical to those in microsomal preparations from rats not treated with halothane. Virtually no w-carboxy- metabolites of leukotrienes were detected in these incubations, probably for two reasons. First, NADPH was the only source of reducing equivalents employed; NAD’ as a cofactor for alcohol dehydrogenase, which is participating in the transformation of 20-OH-LTB4 to 20-COOH-LTB4 was omitted. Second, very low activity of transformation of 20-OH-LTB4 to 20-COOH-LTB, has been found by other investigators [ll] in rat liver microsomes. In our incubations, the transient concentrations of w-hydroxy-metabolites formed from the parent LTB, and LTE4 remained rather low; therefore, forma-

874

Table 1. w-Hydroxylation of LTE4 and LTE4NAc by rat hepatocyte microsomal fractions. Rats were pretreated with four daily injections (i.p.) of phenobarbital (80 mg/kg body mass). 12 h after the last phenobarbital injection, a single dose of halothane (10 mmol/kg body mass) was administered (i.p.) a t the time intervals indicated before the isolation of hepatocytes. Control animals did not receive halothane. Hepatocyte microsomal fractions were incubated (0.19 mg protein/ 200 pl) in presence of 1 mM NADPH with LTE4 (20 pM) and LTE4NAc (10 pM) for 30 min at 37°C. Reaction products were extracted and analyzed by RP-HPLC. Microsomal fractions from livers of the same animals as used in the corresponding in vivo experiments on LTE4NAc w-oxidation were used here. Figures are means f SEM of three experiments. A statistically significant difference from the control values by Student's t-test (P < 0.01) is indicated by (*); P < 0.05 is indicated by (**); a statistically not significant difference (P > 0.9) is indicated by (***).

Halothane Metabolites formed exposure

20-OH-LTE4 ~O-OH-LTE~NAC

pmol x (mg protein)-' x min-'

Control 17.5 f 1.5 9.2 0.4 1 8 h 8.6 0.4* 7.4 0.3** 90 h 8.6 f 1.3" 4.4 f 0.2* 10 days 20.3 f 0.9*** 10.9 f 1.0***

tion of w-carboxy-metabolites was barely detected above the limit ( M 2 ng 20-COOH-LTB4) of our RP-HPLC-based system coupled with diode-array-based ultraviolet-spectroscopy .

One should note that the data of in vitro o-oxidation of parent leukotrienes do not exactly reflect the changes occur- ring in vivo after halothane treatment, as assessed via the o-oxidation of LTE4NAc. From the in vitro data, an accumulation of parent LTE4NAc due to decreased w-oxidation last- ing for > 90 h after halothane treatment would be anticipated. However, in vivo, the transient period of reduced w-oxidation of LTE4NAc is clearly less than 90 h. Thus, 90 h after halothane, LTE4NAc is transformed to a higher extent into w-/j-oxidation products, including more polar but not yet structurally identified products of B-oxidation (Figs. 1, and 2) than in rats not treated with halothane (Fig. 2, control). We do not have an explanation for this discrepancy at present. It remains to be established whether the increase in /%oxidation products recovered from the bile of these rats is due to a) increased (i. e. induced) B-oxidation only, b) increased o-oxidation in vivo only, c) compensatory pathway(s) for leukotriene inactivation in vivo only, or d) a combination of the three possibilities. In addition, hepatic microsomal LTB4 w-hydroxylase activity was shown to be rather labile in vitro (i.e. t1/2 x 1 h at 37°C [5 ] ) ; an increased lability in vitro of the enzyme after interaction with halothane in vivo is feasible and could be reflected by our in vitro data. Regardless, the extent of o-oxidation of LTE4, LTE4NAc, and LTB4 in hepatocyte microsomal fractions in vitro obtained from rats treated with halothane 10 days before the experiment (Table 1 and Fig. 3 B) faithfully reflected the reversal to a normal pattern of leukotriene metabolism (Fig. 2) as reflected by the analysis of LTE4[3H]NAc and its w-/fl-oxidation products in the bile of these rats.

Romano and coworkers have indicated [5] that phenobarbital pretreatment of rats results in a slight decrease rather than induction of LTB4 o- and w-1-hydroxylase activity in rat liver microsomal membranes. Those data are corroborated

I T T

O h 6 h 1 2 h 1 8 h 90h 1 0 d

Time after halothane exposure

Fig. 3. CF3CO-adduct formation (A) and w-oxidation of LTB4 (B) in hepatorytes of rats treated with halothane. Rats were pretreated with four d d y injections (i.p.) of phenobarbital (80 mg/kg body mass) followed by a single dose (i.p.) of halothane (10 mmol/kg body mass). Control animals did not receive halothane. (A) Hepatocyte homogenates obtained from untreated rats (0 h) or rats treated with a single dose of halothane at the indicated time intervals before the experiment were subjected to SDS PAGE followed by immunoblotting. Visualization of CF,CO-adducts was by using, sequentially, anti-CI :&O antibody, horseradish-peroxidase-conjugated goat anti- rabbit second antibody, and enhanced chemiluminescence detection. The apparent molecular mass of CF,CO-adducts were estimated by compaiing their relative migration distances on SDS PAGE with those of marker proteins of known molecular mass: myosin, rabbit muscle subunit (205 kDa); Escherichia coli a-galactosidase, (116 k1)a); phosphorylase b, rabbit muscle subunit (97.4 kDa); albumin, bovine plasma (66 kDa); albumin, egg ovalbumin (45 kDa); glyceraldehyde-3-phosphate dehydrogenase, rabbit muscle (36 kDa); carbonic anhydrase, bovine erythrocytes (29 kDa). (B) Hepatocyte microsomal fractions were incubated (0.19 mg protein/200 pl) in the presence of 1 mM NADPH and 5 pM LTB4 for 30 min at 37°C. Reaction products were extracted and analyzed by RP-HPLC. Figures are means f SEM of three experiments. Asterisk (*) indicates significmt difference from control value (0 h) by Student's t-test (P < 0.01).

875

in the present study in that the extent of LTE4[,H]NAc w- oxidation in vivo (Fig. 2) and of LTE4, LTE4NAc, as well as of LTB4 o-oxidation in vitro (Table 1 and Fig. 3A), 10 days after exposure of rats to halothane (where any inductive effects due to phenobarbital should be negligible), is identical to that at time 0 (i.e. 12 h after the last exposure to phenobarbital, but no exposure to halothane).

Trifluoroacetylation of liver proteins and leukotriene o-oxidation

Earlier studies [13 - 171 have indicated that oxidative metabolism of halothane in vivo leads to extensive covalent modification of cellular macromolecules. Antisera raised against trifluoroacetylated rabbit serum albumin recognized prominent liver microsomal proteins (so-called CF3CO-adducts) of about 100 kDa, 76 kDa, 59 kDa, 57 kDa and 54 kDa on immunoblots. These adducts are preferentially trifluoroacetylated at the primary 8-amine moieties of lysines by the reactive halide CF,COCl, a major metabolite of oxidative halothane metabolism. Some of these liver microsomal adducts have been isolated [33] and the gene of at least one of the corresponding native proteins has been cloned [34]. Recently, our laboratory has obtained a monoform anti- CF3C0 antibody through purification on an Nc-trifluoroacetyl-l-lysine affinity matrix [14, 251. This anti-CF,CO antibody was used to detect CF3CO-adducts on immunoblots of hepatocyte homogenates obtained from untreated rats, or rats treated with halothane 6 h, 12 h, 18 h, 90 h or 10 days before the experiment. A striking parallelism was observed for extent of recognition of CF,CO-adducts by anti-CF3C0 antibody (Fig. 3A) and impaired formation of 20-OH-LTB4 (Fig. 3B). The persistence of these adducts (they were present as early as 6 h and lasted > 90 h but < 10 d after halothane exposure) correlated exactly with the transient impairment of leukotriene o-oxidation (Fig. 3B). These data provide circumstantial evidence for a role of CF3CO-adduct formation in the transient decrease of leukotriene o-oxidation capacity of the liver of halothane-treated rats.

Note here that, in rats not exposed to halothane (Fig. 3A, 0 h) or rats treated with halothane 10 days before the experiment (Fig. 3A, 10 d), no CF,CO-adducts were detected; however, cross-reactive proteins of 52 kDa and 64 kDa are recognized by anti-CF3C0 antibody on these immunoblots. The expression of these constitutive, cross-reactive proteins is totally unrelated to halothane metabolism; they confer molecular mimicry to epitopes present on CF3CO-adducts [25] and a role for these proteins in immunological tolerance of organ- isms towards these adducts has been suggested [25].

Cytochrome P450 isozymes involved in o-oxidation of LTB4, LTE4, and LTE4NAc could be among the many target enzymes which are chemically modified in vivo by halothane- induced trifluoroacetylation. However, the transformation of LTB4 to 20-OH-LTB4 by hepatocyte microsomal fractions of rats treated with halothane 18 h before the experiment was not inhibited by increasing amounts of anti-CF,CO antibody nor was that of hepatocyte microsomal fractions obtained from control rats (data not shown), although a high density of CF3CO-adducts was confirmed in liver homogenates obtained from rats 18 h after halothane exposure (Fig. 3A, 18 h). This observation is compatible with two likely possibilities. First, only a fraction of the LTB4 o-hydroxylase is affected by CF3CO-adduct formation and is completely inactivated. Se- cond, although covalently modified, the function of the enzyme is not affected by the presence of the anti-CF,CO anti-

body; however, covalent modification might lead to decreased affinity of the enzyme(s) for LTB4.

Kinetic analysis of LTB., o-oxidation by hepatocyte microsomal fractions in vitro

The incubations of increasing amounts of LTB4 with microsomal fractions obtained from rats' not treated with halothane revealed a Michealis-Menten type of saturation (Fig. 4A). Transformation of the data and analysis in Lineweaver-Burk plots (Fig. 4A, inset) revealed an apparent K, of 25 f 2.6 pM and an apparent V,,, of 332 f 19.9 pmol 20-OH-LTB4 x (mg protein)-' x min-' (n = 3 experiments; each was performed with microsomes obtained from a distinct animal). Treatment of rats with a single dose of halothane 18 h before the experiment reduced the amount of the active enzyme, as reflected by a decrease in VmaX to 119.6 k 37.6 pmol 20-OH-LTB4 x (mg protein)-' x min-' (n = 3; significantly different from control value by Student's t-test: P < O.OOS), but not the affinity of the active enzyme towards the substrate LTB4 (K, 24.6 f 8.0 pM; n = 3, P > 0.9). In separate experiments, we confirmed that microsomal fractions obtained from rats 10 days after exposure to halothane had almost recovered from the impairment of the LTB4 o-oxidation activity; an only slightly lowered V,,, (x 15%) was found in those preparations (data not shown). The K, values obtained in these experiments are in good agreement with values determined by Romano et al. [S] ( K , x 40 pM) and Sumimoto et al. [35] (K, x 42 pM) for rat liver microsomal LTB4 o-oxidation, while the apparent V,,,-values differed considerably, possibly due to inter-strain variations in the pertinent enzyme levels as extensively documented for cytochrome P450dbl [36].

The data presented here lend further support to the idea that a transient loss of functional enzyme, possibly due to trifluoroacetylation, rather than a decreased affinity towards the substrate, is the basis of impaired LTB4 w-oxidation in the liver of halothane-treated rats. In fact, in earlier experiments, at least one cytochrome P450 isozyme was identified as a target in halothane-dependent trifluoroacetylation of liver microsomal proteins [15]. Note that instead of protein trifluoroacetylation, halothane-dependent rapid ( < 6 h), yet sustained (> 90 h), down-regulation of LTB4 w-hydroxylase could also be responsible for the observed decrease in V,,,. Furthermore, the transient formation of inhibitory carbene and/or carbanion complexes [37, 381, or of suicidal interactions [39] of halothane with either bulk cytochrome P450 or cytochrome P450 isozymes specific for leukotriene o-oxidation might affect leukotriene o-oxidation. Obviously, such interactions would have to be persistent for > 90 h when assayed in vitro but < 90 h in vivo. In control experiments, a reduction of spectrally determined [40] bulk cytochrome P450 from about 1.3 nmol P450 x (mg protein)-' to about 1 .O nmol P450 x (mg protein)- in microsomal membranes obtained from rats not treated with halothane or rats treated with halothane 18 h before the experiment was observed (data not shown).

It is noteworthy that the K, values obtained here for liver microsomal LTB4 o-hydroxylase are significantly higher than those reported for LTB4 o-hydroxylase in human polymorphonuclear leukocytes. The latter is also a cytochrome P450 isozyme [41], however, it exhibits a much higher affinity for LTB4 (i.e. K, m 1 pM [41, 421) than its counterpart of hepatocyte-derived microsomal membranes. Further experimentation will reveal if such differences mirror

876

B o.2 1

4 c 2 0 0 1 / - 3 ._ 3 Lo] e fqF1 3 0.02

0.00 a 8 100 0.00 0.10 0.20

I - I / I 11s

A Control 1% halothane

" I I

0 20 40 6 0 80

I I I I I

-1110.0 -80.0 -60.0 -40.0 -20.0 0.0 20.0 40.0

C

0 20 pM LTB4

A 10 pM LTB4

rn 5 p M LTB4

r I I

-60.0 -40.0 -20.0 0 . 0 20.0 40.0

LTE4 @M)

Fig. 4. Kinetic parameters of LTB4 o-oxidation (A) and inhibition of LTB4 w-oxidation by LTE4 (B, C). (A) Rats were pretreated with a single dose of halothane (10 mmol/kg body mass, i.p.). Control animals did not receive halothane. Liver microsomes were incubated (0.095 mg protein/100 pl) for 30 min at 25°C in presence of 1 mM NADPH and the indic.ited concentrations of LTB,. Extraction of reaction products and HPLC-based analysis was as described in Experimental Procedures. Insel : transformation of the data according to Lineweaver-Burk. Values are means of two experiments which differed less than 5%. The figure is representative for an experiment performed with microsomes obtained from a rat halothane-treated at 18 h (B) and a control (A) rat. (B) Microsomes of untreated rats were incubated (0.19 mg protein/ 200 pl) for 30 min at 37°C in presence of 1 mM NADPH and, concomitantly, LTB4 and LTE4 at the concentrations indicated. Analysis of formation of 20-OH LTB4 and of 20-OH-LTE4 within the same chromatogram was performed as described in Experimental Procedures. Dixon plot analysis of the data with LTB4 as the substrate and LTE, as the inhibitor is shown. V represents reaction velocity. (C) Analysis of the data by means of Cornish-Bowden plotting. S represents substrate concentration, V the reaction velocity. Figures are means of two experiments.

variations in the tasks of the two cellular system; while hepatic LTB4 w-hydroxylase may act to clear hepatic tissue and pe- ripheral blood of LTB4, polymorphonuclear leukocyte LTB4 w-hydroxylase may play a major role in fine-tuning of LTB4 concentrations at inflammatory sites.

Inhibition of LTB4 o-oxidation by LTE, and LTE4NAc in vitro

The similarity in the extent of decrease, and in the time course of recovery, of w-oxidation of LTB4, LTE4, and LTE4NAc suggested that a single enzyme might be involved in transformation of all three substrates. In double-inhibition experiments, we have analyzed the influence of varying concentrations of LTE4 on the formation rates of 20-OH-LTB4 at varying concentrations of LTB4. Within the same experiment,

LTE4 was transformed to 20-OH-LTE4; therefore, the RP- HPLC separation of products was designed to allow the con- comitant quantitation of both 20-OH-LTB4 and 20-OH- LTE4. Analysis of data in Dixon (Fig. 4B) and Cornish- Bowdcn plots (Fig. 4C) revealed a competitive type of inhibition of LTB4 w-oxidation by LTE4, and vice versa. An apparent inhibitory constant Ki of 80 pM was determined for inhibition by LTE4 of 20-OH-LTB4 formation (Fig. 4B); the same value was obtained for inhibition by LTB4 of 20-OH- LTE4 formation (not shown). Unfortunately, an instability over time, i.e. % 50% decrease in intact LTE4NAc over 30 min, which affected its concentration in double-inhibition experiments with LTB4, did not allow a determination of the type of inhibition in Dixon or Cornish-Bowden plots. Never1 heless, at apparent LTE4NAc concentrations of 7.5

877

Table 2. Influence of halothane metabolism on microsomal NADPH - cytochrome P450 reductase activity and on debrisoquine 4- hydroxylase activity. Rats were pretreated with four daily injections (i.p.) of phenobarbital (80mg/kg body mass); 12 h after the last phenobarbital injection, a single dose of halothane 10 mmol/kg body mass was administered (i. p.) at the time intervals indicated prior to isolation of hepatocytes. Control animals did not receive halothane. Hepatocyte microsomal fractions were tested for NADPH - cytochrome P450 reductase activity (measured as rate of reduction of cytochrome c) or debrisoquine 4-hydroxylase activity (measured as rate of 4-hydroxydebrisoquine production) as described in Exper- imental Procedures. Figures are means f SD of two experiments, each with duplicate incubations. Asterisk (*) indicates a standard deviation below detection limit of the assay in duplicate experiments.

Halothane Debrisoquine 4-hydroxylase NADPH -cytochrome treatment dependent on P450 reductase

cumene NADPH hydroperoxide

pmol x (mg protein)-' x min-' x min-'

pnol x (mg protein)-'

None 3.1 f 0.2 9.8 f 0.0 0.172* 18 h 9.9 f 0.7 3.3 0.1 0.052 * 90 h 22.1 1.4 10.6 2 0.1 0.141 * 10 d 22.3 & 0.3 3.1 -t 0.1 0.045 *

pM, 15 pM and 30 pM, an inhibition of 20-OH-LTB4 formation by about 10 - 20% as compared to that in the absence of LTE4NAc was observed, indicating some degree of interaction of metabolism of LTB4 and LTE4NAc by o-oxidation in vitro, suggesting that the observed impairment in vivo of LTE4NAc w-oxidation in rats 18 h after halothane exposure might also be true for both LTE4 and LTB4.

The data obtained here, indicating a single P450 isozyme acting on LTB4 and LTE4 as substrates, are in contrast to earlier reports [2] suggesting that LTB4 w-hydroxylase and LTE4 w-hydroxylase were distinct enzymes based on apparent absence of inhibition of metabolism of either substrate in the presence of the other. We do not yet have an explanation for this discrepancy. However, the preparation method of microsomal fractions from hepatocytes (our experiments) or microsomal membranes from whole liver tissue [2], as well as the substrate concentrations used, may affect enzymatic activity. Alternatively, leukotriene o-hydroxylases present in distinct cells of the liver may have characteristics largely different from those in hepatocytes separated from the reminder of hepatic cells. Only the purification, molecular cloning and heterologous functional expression of these activities might eventually clarify this discrepancy.

Influence of halothane metabolism on microsomal monooxygenation

Microsomal monooxygenases comprise NADPH - cytochrome P450 reductase and structurally distinct cytochrome P450 isozymes [43]. The experiments presented so far did not rule out that the function of NADPH - cytochrome P450 reductase could be affected and become the limiting component in leukotriene w-hydroxylation. Consequently, we have examined the activity of NADPH -cytochrome P450 reductase itself. When assayed in vitro with cytochrome c, the

Table 3. Conversion of 5-hydroperoxyeicosatetraenoate to LTB, by rat hepatocyte homogenates. Rats were pretreated with four daily injections (i.p.) of phenobarbital (80 mg/kg body mass) followed by a single dose (i.p.) of halothane (10 mmol/kg body mass). Control animals did not receive halothane. Heptocyte homogenates were incubated (0.19 mg protein/200 pl) with 100 pM 5-hydroperoxyeicosatetraenoate under an argon atmosphere at room temperature for 5 min. HPLC-based separation and quantitation of reaction products was as described [19]. All-trans LTBI reflects the combined amounts of A6-truns -LTB4 and A6-truns-l 2-epi-LTB4 formed. Figures are means f SEM of three experiments.

Halothane Products formed exposure

all-trans LTB4 LTB4

pmol x (mg protein)-' x min-'

Control 18 h 90 h

28.1 f 4 . 5 15.1 f 2.2 26.9 f 0.4 13.8 f 1.1 19.4 & 0.1 13.8 f 1.1

apparent activity of NADPH - cytochrome P450 reductase was affected by halothane pretreatment of rats (Table 2). However, the pattern of changes in activity of the reductase did not parallel that observed with NADPH-dependent w-oxidation of LTB4, LTE4, and LTE4NAc measured in vitro (Table 2 and Fig. 3B). In contrast, NADPH-dependent debrisoquine 4-hydroxylase activity [44] followed exactly the activity pattern of NADPH - cytochrome P450 reductase. Debrisoquine 4-hydroxylase can be assayed uncoupled from NADPH - cytochrome P450 reductase using cumene hydroperoxide as electron donor [45]. As compared to the control, a sevenfold increase in uncoupled debrisoquine 4-hydroxylase activity was detected (Table 2) in microsomal fractions obtained from rats 10 days after halothane treatment. The increase in cumene-hydroperoxide-dependent debrisoquine 4- hydroxylase activity may reflect an upregulated expression of cytochrome P450db1[43] in that P450dbl was shown to ex- hibit sex-related [46] and strain-related [36] differences in re- sponse to a variety of cytochrome-P450-inducing agents. One should note that attempts to measure LTB4 o-hydroxylation in an uncoupled fashion with cumene hydroperoxide as electron donor failed. These data indicate that reductase activity is the limiting component in debrisoquine 4-hydroxylation but not in o-hydroxylation of LTB4, LTE4, and LTE4NAc in vitro. Furthermore, although LTE4NAc w-oxidation in vivo seems to be linked to total reductase activity at 0 h, 18 h, and 90 h (Fig. 2) , this is not the case 10 days after exposure of rats to halothane in that the extent of w-/B-oxidation of LTE4NAc is not significantly different at the latter time points. Thus, together with the data of the kinetic analysis (Fig. 4A), the decrease in activity of leukotriene w-hydroxylation truly reflects a decrease in the amount of functional leukotriene o-hydroxylase.

Synthesis of LTB4 from 5-hydroperoxyeicosatetraenoate by hepatocyte homogenates

Previous work indicated that hepatocyte homogenates were capable of synthesis of biologically active LTB4 from exogenous 5-hydroperoxyeicosatetraenoate [47 - 491 ; when supplemented with glutathione, LTC4 was also among the transformation products [49]. In contrast to its w-oxidation,

878

the synthesis of LTB4 (and that of all-trans LTB4, i.e. d6-trans- LTB4 and A6-trans-l 2-epi-LTB4 combined) was not affected in hepatocyte homogenates obtained from rats 18 h or 90 h after halothane pretreatment (Table 3), suggesting that distinct enzymatic steps of LTB4 synthesis within hepatocytes (i.e. transformation of 5-hydroperoxyeicosatetraenoate to LTA4 and LTA4 hydrolysis [47 -491) are not sensitive to protein trifluoroacetylation or any of the mechanisms resulting in transiently decreased o-oxidation of leukotrienes under these conditions.

In conclusion, we present several lines of evidence that the anesthetic agent halothane transiently impairs hepatic homeostasis of the biologically active leukotrienes. First, treatment of rats with a single dose of halothane resulted in a transient decrease in liver o-oxidation of LTE4NAc in vivo. When compared to that of rats not treated with halothane, the formation of 20-COOH-LTE4NAc decreased by a factor of about 3.5 in rats treated with halothane 18 h before the experiment and returned back to normal in rats 10 days after halothane treatment. Second, decreases in the in vitro rates of w-oxidation of LTB4, LTE4, and LTE4NAc in hepatocyte microsomal fractions were induced by treatment of rats with a single dose of halothane. The major site of inhibition of o- oxidation of LTB4, LTE4, and LTE4NAc was their conversion to the corresponding w-hydroxy-metabolites. o-Oxidation in vitro was decreased by about 50% in hepatocyte microsomal fractions obtained from rats 18 h or 90 h after a single dose of halothane and also returned to normal levels 10 days after halothane treatment. Kinetic analysis revealed that the decrease in w-oxidation capacity was due to a decrease in the amount of active leukotriene w-hydroxylase; circumstantial evidence suggested that halothane metabolism-dependent triffuoroacetylation of hepatic target proteins might play a role in the inactivation of this enzyme. Third, in contrast to the o-oxidation of LTB4, its synthesis from exogenous 5- hydroperoxyeicosatetraenoate was not inhibited in hepatocyte homogenates obtained from halothane-treated rats. Increased concentrations of leukotrienes may be a contributing factor to the hepatotoxicity associated with halothane in about 20% of patients receiving this agent [12]. The exact molecular mechanisms by which leukotrienes elicit toxicity in hepatocytes [50], particularly under hypoxic conditions, remain to be elucidated.

We wish to thank U. Christen and M. Biirgin for help in hepatocyte preparation and in affinity purification of anti-CF,CO antibody, and J. Miiller for her excellent help in surgical procedures and RP-HPLC analysis of leukotriene metabolites obtained in vivo. We also thank Dr. B. Glanzer (Heidelberg) for kindly synthesizing and providing LTE4[3H]NAc. The help of M. Beer in assays of debrisoquine-4-hydroxylase is acknowledged. We are indebted to the Center of Therapeutic Research of Merck-Frosst (Canada) for gener- ously providing synthetic LTB4 and LTE4. The continuous support of Prof. U. A. Meyer is acknowledged. This work was supported by the Swiss National Science Foundation (grant 3-109.0.88 to J. G.), the Forschungsschwerpunkt Transplantation, Heidelberg (grant to D. K.) and the very generous help of the Roche Research Foundation. Josef Gut is the recipient of a START Research Career Development Award (no. 3-018.0.87) from the Swiss National Science Foundation.

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