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Effects of Cold Preservation and Reperfusion on Microsomal Cytochrome P-450-Linked Monooxygenase System of the Rat Liver
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
61, 361–366 (1996)
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Effects of Cold Preservation and Reperfusion on Microsomal Cytochrome P-450-Linked Monooxygenase System of the Rat Liver KUNIHIKO IZUISHI, M.D., YOSHIYUKI ICHIKAWA, M.D.,* MOHAMMAD AKRAM HOSSAIN, M.D., TAKASHI MAEBA, M.D., HAJIME MAETA, M.D., AND SATOSHI TANAKA, M.D. First Department of Surgery and *Department of Biochemistry, Kagawa Medical School, Miki-cho, Kita-gun, Kagawa, 761-07, Japan Submitted for publication April 4, 1994
The effects of cold preservation and reperfusion of the liver on the hepatic microsomal cytochrome P-450linked monooxygenase system (P-450 system) were investigated. Rat livers were preserved with cold University of Wisconsin solution for 0, 12, 24, 36, and 48 hr. Half of them in 0-, 24-, and 48-hr groups were reperfused for 1 hr at 377C with oxygenated Krebs–Henseleit buffer. After preservation or reperfusion, the liver microsomes were prepared and the concentration of each component of the P-450 system [NADPH-cytochrome b5 reductase (b5 reductase), cytochrome b5 (b5), NADPH-cytochrome P-450 reductase (P-450 reductase) and cytochrome P-450 (P-450)] and their drug metabolizing activities and concentration of apo-cytochrome P-450 2E1 (apo-P-450 2E1) were measured. After 48-hr preservation, b5 concentration did not decrease, whereas the concentration of P-450, P-450 reductase, and b5 reductase decreased from 0.865 to 0.676 nmole/ mg protein, from 0.262 to 0.233 mmole/mg protein/min and from 5.34 to 4.86 mmole/mg protein/min, respectively. During cold preservation, the activities of pnitroanisole O-demethylase and aniline p-hydroxylase did not change. Aminopyrine N-demethylase activity was inhibited from 4.45 to 3.34 nmole/mg protein/min after 48-hr cold preservation. Apo-P-450 2E1 was gradually decreased during cold preservation. Reperfusion caused a further decrease in the activities and concentration of the components of the P-450 system and concentration of apo-P-450 2E1 to 80–90% after 1-hr reperfusion. It was suggested that the prolonged preservation caused deterioration of the P-450 system and the loss of the abilities of metabolism and detoxication of xenobiotics. q 1996 Academic Press, Inc.
INTRODUCTION
The hepatic microsomal cytochrome P-450-linked monooxygenase system (P-450 system) plays important roles in synthesizing steroid hormones [1, 2] and prostaglandin [3, 4], activating vitamin D3 [5, 6], producing bile acid [7], and metabolizing and detoxicating xenobiotics [8]: chemical components that are foreign to the
metabolic network of an organism. The P-450 system is composed of four components: cytochrome b5 (b5), NADH-cytochrome b5 reductase (b5 reductase), cytochrome P-450 (P-450), and NADPH-cytochrome P-450 reductase (P-450 reductase), as shown in Fig. 1. Many reports have suggested that drug metabolizing abilities are a reliable indicator of liver function after liver transplantation in vivo [9–14]. They have also discussed that the causes of decreased drug metabolizing ability were hepatic blood flow and the P-450 related activities in liver microsomes. There is evidence that hepatic blood flow is one of the most important factors affecting the drug metabolizing ability [16]. Poor blood flow causes hypoxia, and hypoxia causes degeneration of the P-450 structure [17]. The P-450 system reflects the intrinsic ability of the liver to metabolize drugs. In addition, oxygen may act as a terminal electron acceptor in the generation of high-energy bonds or may be required as a direct substrate for oxygenase such as those in the P-450 system. However, no study has shown directly the effects on the P-450 system of liver microsomes after cold ischemia and reperfusion. In this study, we hypothesized that cold ischemia/reperfusion injury might affect the P-450 system in drug metabolism, and perfusion ‘‘ex vivo’’ experiments were conducted to exclude hemodynamic factors after transplantation in vivo. MATERIALS AND METHODS Surgical procedure. Male Sprague–Dawley rats weighing 200– 250 g (Clea Japan Inc., Tokyo, Japan) were housed under standard conditions with normal lighting cycle and allowed free access to food and water. Between 7:00 and 10:00 PM, livers were harvested under inhalation anesthesia with ethyl ether. After opening the abdomen, the common bile duct was cannulated with a polyethylene catheter (PE-10; Becton Dickinson & Co., Parsippany, NJ) and secured. The animals were then heparinized with 200 U of heparin from the penile vein, the abdominal aorta was cannulated with a 20-gauge intravenous catheter, and 10 ml of chilled University of Wisconsin (UW) solution was injected. When the injection was started, the thoracic cavity was opened, the descending aorta was clamped, and the right atrium was cut. After this procedure, the portal vein was immediately cannulated with an 18-gauge intravenous catheter, secured, and 5 ml of cold UW solution was slowly injected. All the connective 0022-4804/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Cytochrome P-450-linked monooxygenase systems. It is composed of four components: cytochrome b5 , cytochrome b5 reductase, cytochrome P-450 reductase, and cytochrome P-450. An electron is transferred from NADH or NADPH through cytochrome P-450 monooxygenase system to a substrate into which molecular oxygen is incorporated.
tissues and ligaments were dissected, the liver was removed as quickly as possible, immersed in UW solution in an ice water bath and stored for 12, 24, 36, and 48 hr. After storage, the liver was reperfused at 377C with Krebs–Henseleit buffer. Reperfusion of the liver. The preserved liver was reperfused by a recirculating fashion as described by Hamamoto et al. [18]. Warmed Krebs–Henseleit buffer at 377C was delivered from a reservoir to the liver through the portal vein at a constant perfusion rate (3.5 ml/min/g wet tissue) with a peristaltic pump (Model 312; Gilson Inc., Villier-le-Bel, France). The perfusate was oxygenated with a gas mixture of 95% O2 / 5% CO2 through a Hamilton lung [19], which was made of 10 m of silastic tube (1.47 mm internal diameter) and inserted into the circuit. The perfusate was then drained from the hepatic vein. During reperfusion, bile was collected in a graduated tube and volume was recorded at 1-hr reperfusion. Aliquots of perfusate were collected at 1-hr reperfusion for enzyme determinations, which were assayed using standard colorimetric techniques with a Sigma Diagnostic Kit. Preparation of the liver microsomes. All procedures were carried out at 47C. The liver was homogenized in 5 vol of ice-cold 0.25 M sucrose adjusted to pH 7.4 with 100 mM potassium phosphate buffer and 1 mM of ethylenediaminetetraacetic acid, disodium salt with a motor-driven Teflon pestle and a glass tube kept in an ice bath. The homogenate was centrifuged at 9000 g for 20 min at 47C with a centrifuge (Model 20RP-52, Hitachi Co., Ltd., Tokyo, Japan). The supernatant was collected and centrifuged at 10,500 g for 90 min at 47C with an ultracentrifuge (Model 55P-72, Hitachi Co., Ltd., Tokyo, Japan). The pellet was collected and stored as microsomal fraction at 0807C for further analysis. Experimental groups. Thirty rat livers were preserved with UW solution for 0, 12, 24, 36, and 48 hr (six livers each). Another 18 livers were preserved with UW solution for 0, 24, and 48 hr (six livers each) and then reperfused with Krebs–Henseleit solution at 377C for 1 hr. The microsomes of these livers were prepared for analysis.
tase activity of P-450 reductase were measured by the decrease in absorbance at 420 nm of ferricyanide [22]. Drug metabolizing activities. The activities of aminopyrine Ndemethylase, aniline p-hydroxylase and p-nitroanisole O-demethylase were assayed as follows: The reaction mixture in 1.5 ml of 50 mM Tris-HCl buffer, pH 7.4 containing 5 mM of MgCl2 , 5 mM of glucose 6-phosphate, 0.5 mM of NADP/, 1.5 U of glucose 6-phosphate dehydrogenase and 1 mg protein/ml of microsomal suspension was preincubated for 5 min at 377C. Enzyme reaction was started by adding 100 ml of 30 mM aminopyrine or 15 mM of aniline for aminopyrine N-demethylase or aniline phydroxylase activities, respectively. The reaction mixture was incubated in a shaking water bath at 377C for 15 min. Respective enzyme activities were calculated with the concentrations of released formaldehyde or p-aminophenol, respectively. Formaldehyde was measured by the method of Nash [23]. p-Aminophenol was assayed by the method of Imai et al. [24]. One and one-half milliliters of the reaction mixture of 70 mM of potassium phosphate buffer, pH 7.4 containing 1 mM p-nitroanisole and 1 mg of microsomal protein, was preincubated for 5 min at 377C. The enzyme reaction was started by the addition of 5 mM of MgCl2 , 5 mM of glucose 6-phosphate, 0.5 mM of NADP/, and 1.5 U of glucose 6-phosphate dehydrogenase. The reaction mixture was incubated for 10 min at 377C in a shaking water bath. The reaction was terminated by adding 1.5 ml of 0.5 M NaOH. The mixture was then centrifuged at 10,500 g for 30 min and p-nitrophenol concentration of the supernatant was measured to obtain the activity of p-nitroanisole O-demethylase according to the method of Netter et al. [25]. Concentration of apo-P-450 2E1. Concentration of apo-P-450 2E1 was estimated by Western blotting method with a cytochrome P450 2E1 commercial kit (Oxygene, Dallas, TX). Briefly, microsomal proteins separated by polyacrylamide gel electrophoresis were transferred onto a nitrocellulose paper. The separated P-450 2E1 was then stained using a rabbit anti-rat-P-450 2E1 and sheep-anti-rabbit IgG conjugated with horse radish peroxidase (Sigma chemical Co., St. Louis, MO) [26]. The absorption of apo-P-450 2E1 band was measured densitometrically with a dual-wavelength chromatoscanner (Model CS-910, Shimadzu Co., Kyoto, Japan) to estimate its concentration. Relative value compared with the mean value (cm2/mg protein) of 0 hr was indicated. Effect of perfusate on P-450 and P-450 reductase. Frozen rats liver microsomes (n Å 6) were homogenated with Krebs–Henseleit buffer adjusted to pH 7.4 with 100 mM potassium phosphate buffer and to make 10 mg protein/ml of microsomal suspension. This suspension was incubated in a water bath at 377C for 1 hr. After incubation, P-450 concentration and P-450 reductase activity were examined, and compared with concentration and activity before incubation to examine the possibility of toxicity associated with perfusate on P450 and P-450 reductase. Statistical analysis. In the groups of preservation, significance was tested by one-way analysis of variance. Differences between groups at specific time points were considered significant at P õ 0.05 with Dunnett’s test as a post hoc analysis. Between the groups of before and after reperfusion, and before and after incubation, statistical comparison was performed by Student’s t test. Differences with P õ 0.05 were listed as significant. All values were presented as mean { S.D.
Analytical Procedure Microsomal suspension. Frozen microsomes were homogenated with 0.1 M potassium phosphate buffer pH 7.4 to make 10 mg protein/ml of microsomal suspension. Protein concentration. Protein concentration of microsomal suspension was estimated by the method of Bradford with a protein assay kit (Bio-Rad Lab., Hercules, CA) [20]. Concentration of P-450, b5 , b5 reductase, and P-450 reductase. The concentration of P-450 and b5 were measured spectrophotometricaly with a molar extinction coefficient, 91,000 and 185,000, respectively [21]. The concentration of b5 reductase and P-450 reductase were estimated by their ferricyanide reductase activities. NADH-ferricyanide reductase activity of b5 reductase and NADPH-ferricyanide reduc-
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RESULTS
Bile Production and Enzyme Release into the Perfusate after 1-hr Reperfusion Following Preservation Bile production (Table 1). Freshly harvested livers produced 0.061 { 0.007 ml/g tissue/hr bile 1 hr after reperfusion, and after 24- and 48-hr preservation decreased significantly to 0.045 { 0.007 and 0.025 { 0.007 ml/g tissue/hr, respectively (P õ 0.05).
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TABLE 1 Bile Production, Release of LDH and GOT after 1-hr Reperfusion Following Cold Ischemia Cold ischemia time (hr)
Bile production (ml/g tissue/hr)
LDH release (IU/g tissue/hr)
GOT release (IU/g tissue/hr)
0 24 48
0.061 { 0.007 0.045 { 0.007* 0.025 { 0.007*
0.48 { 0.17 1.08 { 0.19 3.65 { 1.06*
0.058 { 0.027 0.122 { 0.029 0.380 { 0.103*
Note. ∗ vs corresponding value of 0-hr group.
LDH and GOT release (Table 1). Release of LDH and GOT 1 hr after reperfusion following 48-hr preservation increased significantly more than the control group (P õ 0.05), to 3.65 { 1.06 and 0.380 { 0.103 IU/ g tissue/hr, respectively. Concentration of the Components of the P-450 System NADH-cytochrome b5 reductase (Table 2). Concentration of b5 reductase assessed by its NADH-ferricyanide reductase activity was reduced from 5.34 { 0.36 to 4.86 { 0.35 mmole/mg protein/min after 48-hr preservation. Only reperfusion subsequent to 48-hr preservation suppressed the concentration from 4.86 { 0.35 to 4.28 { 0.36 mmole/mg protein/min (P õ 0.05). NADPH-cytochromeP-450reductase (Table 2). Concentration of P-450 reductase assessed with its activity of NADPH-ferricyanide reductase was decreased by 48hr cold preservation from 0.262 { 0.020 to 0.233 { 0.023 mmole/mg protein/min. Reperfusion after 24- and 48-hr cold preservation reduced it significantly (P õ 0.05). Cytochrome b5 (Fig. 2). Cytochrome b5 concentration determined spectrophotometrically showed no change during cold preservation. After reperfusion following 24- and 48-hr preservation, the concentration was reduced from 0.408 { 0.016 to 0.357 { 0.047 and 0.419 { 0.034 to 0.317 { 0.043 nmole/mg protein, respectively (P õ 0.05). Cytochrome P-450 (Fig. 3). In contrast to b5 concentration, 48-hr cold preservation significantly reduced PTABLE 2
FIG. 2. Changes in the concentration of cytochrome b5 after cold preservation and reperfusion. Broken lines indicate reperfusion. ∗ vs corresponding value of 0-hr preserved liver (P õ 0.05); † vs before reperfusion (P õ 0.05).
450 concentration from 0.865 { 0.074 to 0.676 { 0.043 nmole/mg protein. In 0-, 24-, and 48-hr preservation groups, reperfusion decreased the concentration from 0.865 { 0.073 to 0.776 { 0.053, from 0.801 { 0.041 to 0.674 { 0.074 and from 0.676 { 0.043 to 0.542 { 0.049 nmole/mg protein, respectively (P õ 0.05). Drug Metabolizing Activities Aminopyrine N-demethylase (Table 3). The activity decreased from 4.45 { 0.25 to 3.34 { 0.41 nmole/mg protein/min 48 hr after preservation. After reperfusion, activity following 48-hr preservation was significantly inhibited compared with that before reperfusion, to 2.66 { 0.32 nmole/mg protein/min. p-nitroanisole O-demethylase (Table 3). During cold preservation, the activity was kept constant up to 48 hr. Reperfusion did not change the activity in each group. Aniline p-hydroxylase (Table 3). The activity was gradually decreased during 48 hr cold preservation, although no significant difference was detected among these groups. After reperfusion following 48-hr cold preservation, activity was reduced from 1.81 { 0.24 to 1.51 { 0.21 nmole/mg protein/min (P õ 0.05). Concentration of Apo-P-450 2E1 (Fig. 4). After cold preservation and reperfusion, concentration of apo-P-450 2E1 was decreased, but this failed to reach statistical significance.
Changes in NADH0 and NADPH-Ferricyanide Reductase Activities Group
A
0 hr 0-hr reperfusion 12 hr 24 hr 24-hr reperfusion 36 hr 48 hr 48-hr reperfusion
5.34 5.10 5.41 5.20 5.12 4.84 4.86 4.28
{ { { { { { { {
B 0.36 0.50 0.26 0.41 0.52 0.34* 0.35* 0.36†
0.262 0.241 0.252 0.249 0.271 0.254 0.233 0.189
{ { { { { { { {
0.020 0.021 0.013 0.013 0.027† 0.018 0.023* 0.028†
Note. A, NADH-ferricyanide reductase activity (mmole/mg protein/ min); B, NADPH-ferricyanide reductase activity (mmole/mg protein/ min); ∗ vs corresponding value of 0-hr group; † vs before reperfusion.
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FIG. 3. Changes in the concentration of cytochrome P-450 after cold preservation and reperfusion. Broken lines indicate reperfusion. ∗ vs corresponding value of 0-hr preserved liver (P õ 0.05); † vs before reperfusion (P õ 0.05).
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TABLE 3
TABLE 4
Changes in Activities of Aminopyrine N-Demethylase, p-Nitroanisole O-Demethylase, and Aniline p-Hydroxylase
Effect of Perfusate on P-450 and P-450 Reductase
Group 0h 0 hr reperfusion 12 hr 24 hr 24 hr reperfusion 36 hr 48 hr 48 hr reperfusion
A 4.45 4.53 3.92 4.19 4.06 3.35 3.34 2.66
{ { { { { { { {
B 0.25 0.32 0.25* 0.37 0.58 0.35* 0.41* 0.32†
1.78 1.79 1.82 1.81 1.78 1.65 1.61 1.51
{ { { { { { { {
C 0.05 0.25 0.05 0.09 0.21 0.08 0.07 0.22
1.91 1.87 1.94 1.85 1.92 1.98 1.81 1.51
{ { { { { { { {
0.39 0.27 0.21 0.20 0.31 0.19 0.24 0.21†
Note. A, aminopyrine N-demethylase activity (nmole/mg protein/ min); B, p-nitroanisole O-demethylase activity (nmole/mg protein/ min); C, aniline p-hydroxylase (nmole/mg protein/min). ∗ vs corresponding value of 0-hr group; † vs before reperfusion.
Effect of Perfusate on P-450 and P-450 Reductase (Table 4) Statistical significance was not found between before incubation and after incubation. P-450, which has maximum absorbency at 450 nm, is a heme protein and is easily converted reversibly or nonreversibly to its inactive form, cytochrome P-420, which has maximum absorbency at 420 nm [27]. After incubation, cytochrome P-420 was not found. The possibility of the toxicity of the perfusate to P-450 and P-450 reductase was excluded. DISCUSSION
In order to estimate the liver viability and predict the prognosis of the patient after liver transplantation, drug metabolizing activities have been used in some institutes [9–14]. Oellerich et al. reported that lidocaine metabolizing ability of the donor patients was the most reliable indicator for early graft survival after transplantation [11]. However, nothing more than speculation is known about the causes of decreased drug metabolizing abilities during cold preservation and after reperfusion of the liver.
FIG. 4. Changes in concentration of apo-P-450 2E1 after cold preservation and reperfusion. Broken lines indicate reperfusion. Relative values compared with the mean value (cm2/mg protein) at 0 hr was indicated. These values failed to reach statistical significance.
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Before incubation After incubation
Concentration of P-450 (nmole/mg protein)
Activity of P-450 reductase (mmole/mg protein/min)
0.922 { 0.062 0.908 { 0.116
0.264 { 0.030 0.247 { 0.013
Note. Statistical significance was not found between before incubation and after incubation.
Molecular oxygen is incorporated into substrates by P-450, which receives electrons from NADPH or NADH through P-450 reductase, or b5 and b5 reductase, respectively, as shown in Fig. 1 [28]. In this experiment, we examined these four components of the P-450 system, because damage to one of these components causes severe loss of activity of the P-450 system. In addition, drug metabolizing activities by the P-450 system were examined. In the present study, concentration of P-450, b5 reductase, and P-450 reductase decreased during 48-hr cold preservation, whereas b5 concentration was not affected. A similar tendency to our study was observed only in the lung in which concentration of P-450 and b5 decreased during cold preservation [29]. Reduced P450 concentration was paralleled to the inhibition of aminopyrine N-demethylase activity. In contrast, it is of special interest that aniline p-hydroxylase and pnitroanisole O-demethylase activities were not affected during cold preservation. In previous reports, a specific alteration of activities of P-450 isozymes was demonstrated in a model of warm ischemia/reperfusion injury [30]. Studies using the isolated liver preparation show that the elimination of theophylline [31], propranolol [32], and omeprazole [33] may be impaired by hypoxia but that elimination of misonidasole [34] is enhanced. The mechanism of this remains unclear. However, it is well established that multiple isozymes of P-450 exist and that each isozyme is associated with a different spectrum of substrate specificity of P-450 system. The regulation of each isozyme appears to be subject to independent control mechanisms [35, 36] and different affinity of isozyme for oxygen [37]. In addition, several investigators have shown that the different subpopulations of P-450 can be localized to different areas within the liver lobule [38, 39]. The enzymes that are distributed in the pericentral zone are most susceptible to hypoxic tissue damage [40]. These reports strongly suggest that the most likely explanation for nonuniform effect in activities of the P-450 system is that individual P-450 isozymes are differentially affected by cold ischemia/reperfusion injury. This study provides the data that after cold preservation and reperfusion concentration of apoprotein of P450 2E1, which is one of the isozymes of P-450 and also one of the isozymes metabolizing aminopyrine, was decreased, but this failed to reach statistical significance. However, reduction of P-450 concentration and inhibition of aminopyrine N-demethylase activity were paral-
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leled to the degeneration of apo-P-450 2E1. One of the reasons for deterioration of the P-450 system might be the degeneration of apoprotein of P-450 after cold preservation and reperfusion. On the other hand, previous data suggested another reason for the deterioration of the P-450 system. Phosphatidylcholine is an important stabilizer of the P-450 system and thereby sustains the activity of the system [41]. It has been clearly demonstrated that warm ischemia accelerates membrane phospholipid degradation caused by Ca2/ influx and subsequent activation of intrinsic phospholipase A2 [42–46]. In cold ischemia for up to 48 hr with UW solution, accumulation and release of free fatty acids in the liver tissue were observed [18]. These findings strongly suggest that the microsomal membrane is considerably degraded during cold preservation even at low temperatures. The change of phospholipid composition may cause activity loss of the P450 system [47] and acceleration of degradation of the components of the P-450 system. Takei et al. reported that the liver function after transplantation was improved using protease inhibitors: leupeptin, pepstatin A, phenylmethylsulfonyl fluoride [48]. Membranelinked proteases also have to be considered as a possible cause of the activity loss of the P-450 system. Reperfusion of the preserved liver caused significant decrease in the concentration of components of the P450 system, and its drug metabolizing activities. Many hypotheses to elucidate the mechanism of so-called ischemia/reperfusion injury have been proposed [49– 51]. Reactive oxygen substances produced by ischemia/ reperfusion induce lipid peroxidation [52]. This cascade reaction then induces platelet activation [53], neutrophil infiltration [54], and finally increased coagulation [55]. The sequence of the tissue damage ultimately brings tissue hypoperfusion. Once tissue ischemia is introduced, Ca2/ influx and phospholipase activation occur as indicated above and the instability of the P450 system is accelerated. The lipid peroxidation may also cause disturbance of the microsomal membrane and instability and deactivation of the P-450 system. In this study, deteriorative effects of cold preservation and reperfusion of the liver on microsomal cytochrome P-450-linked monooxygenase system were clearly demonstrated. Until now, little attention has been paid to the P-450 system of the preserved liver for transplantation. We should consider the loss of the abilities of metabolism and detoxication of xenobiotics, especially in long-preserved livers in clinical transplantation. REFERENCES 1. Tsubaki, M., Tomita, S., Tsuneoka, Y., and Ichikawa, Y. Characterization of two cysteine residues in cytochrome P-450scc : Chemical identification of the heme-binding cysteine residue. Biochim. Biophys. Acta 870: 564, 1986. 2. Hiwatashi, A., and Ichikawa, Y. Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes. Biochim. Biophys. Acta 664: 33, 1981.
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22. Hiwatashi, A., Ichikawa, Y., Maruya, N., Yamano, T., and Aki, K. Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. I. Physiochemical properties of horo- and apo-NADH-adrenodoxin reductase and interaction between non-heme iron proteins and the reductase. Biochemistry 15: 3082, 1976. 23. Nash, T. Colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55: 416, 1953. 24. Imai, Y., Ito, A., and Sato, R. Evidence for biologically different types of vesicles in the hepatic microsomal fraction. J. Biochem. 60: 417, 1966. 25. Netter, K. J., and Seidel, G. An adaptively stimulated o-demethylating system in rat liver microsomes and its system in rat liver microsomes and its kinetic properties. J. Pharmacol. Exp. Ther. 146: 61, 1964. 26. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350, 1979. 27. Ichikawa, Y., and Yamano, T. The role of the hydrophobic bonding in P-450 and the effect of organic compounds on the conversion of P-450 to P-420. Biochim. Biophys. Acta 147: 518, 1967. 28. Estabrook, R. W., Hildebrandt, A. G., Baron, J., Netter, K. J., and Leibman, K. A new spectral intermediate associated with cytochrome P-450 function in liver microsomes. Biochem. Biophys. Res. Commun. 42: 132, 1971. 29. Kawaguchi, H., Maeda, M., Nakamoto, K., Hamakubo, S., Hayashi, E., Taniguchi, K., Tubota, N., Okada, T., Katura, H., Kaseki, M., and Miyatake, A. Injury to microsomal electron transport system compornents caused by preservation of donor lung. Construction of a monogram relating marginal preservation temperature and marginal preservation time. J.J.A.C.S. 6: 130, 1992. 30. Lee, S-M., and Clemens, M. G. Effect of a-tocopherol on hepatic mixed function oxidase in hepatic ischemia/reperfusion. Hepatology 15: 276, 1992. 31. Nakatsu, K. Limitation of theophylline elimination by reduced oxygen availability in mouse hepatocytes and rat isolated livers. Can. J. Physiol. Pharmacol. 63: 903, 1984. 32. Jones, D. B., Mihaly, G. W., Smallwood, R. A., Webster, L. K., Morgan, D. J., and Madsen, N. P. Differential effect of hypoxia on the disposition of propranolol and sodium taurocholate by the isolated perfused rat liver. Hepatology 4: 205, 1984. 33. Webster, L., Jones, D. B., Mihaly, G. W., Morgan, D. J., and Smallwood, R. A. Effect of hypoxia on oxidative and reductive pathways of omeprasole metabolizm by isolated perfused rat liver. Biochem. Pharmacol. 34: 1239, 1985. 34. Smith, R. B., Born, J. L., and Garcia, D. J. Influence of hypoxia on the metabolizm and excretion of misonidazole by the isolated perfused rat liver-model system. Biochem. Pharmacol. 32: 1609, 1983. 35. Guengerich, F. P. CytochromeP-450 enzymes and drug metabolizm. Prog. Drug Metab. 10: 3, 1987. 36. Guengerich, F. P., Umbenhauer, D. R., and Churchill, P. F. Polymorphism of human cytochrome P-450. Xenobiotica 17: 311, 1987. 37. Jones, D. P. Hypoxia and drug metabolizm. Biochem. Pharmacol. 30: 1019, 1981. 38. Baron, J., Redick, J. A., and Guengerich, F. P. Immunohistochemical localization of cytochrome P-450 in the rat liver. Life Sci. 23: 2627, 1978.
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39. Guengerich, F. P., Annan, G. A., Wright, S. T., Martin, M. V., and Kaminsky, L. S. Purification and characterization of liver microsomal cytochrome P-450s. Xenobiotica 12: 701, 1982. 40. Lemasters, J. J., and Thurman, R. G. Centrilobular injury following hypoxia in isolated, perfused rat liver. Science 213: 661, 1981. 41. Strobel, H. W., Lu, A. Y., Heidema, J., and Coon, M. J. Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation. J. Biol. Chem. 245: 4851, 1970. 42. Chien, K. R., Abrams, J., Serroni, A., Martin, J. T., and Farber, J. L. Accerated phospholipid degradation and associated membrane dysfunction in irreversible, ischemic liver cell injury. J. Biol. Chem. 253: 4809, 1978. 43. Scherphof, G. L., Scarpa, A., and Toorenenbergn, A. The effect of local anesthetics on the hydrolysis of free and membranebound phospholioids catalyzed by various phospholipase. Biochim. Biophys. Acta 270: 226, 1972. 44. Kunze, H., Nahas, N., Traynor, L. R., and Wurl, M. Effect of local anaesthetics on phospholipase. Biochim. Biophys. Acta 441: 93, 1976. 45. Kim, S-K., Belzer, F. O., and Southard, J. H. Loss of mitochondrial respiratory function and its suppression during cold ischemic preservation of rat livers with University of Wisconsin solution. Hepatology 16: 742, 1992. 46. Harrison, D. C., Lemasters, J. J., and Herman, B. A pH-dependent phospholipase A2 contributes to loss of plasma membrane integrity during chemical hypoxia in rat hepatocytes. Biochem. Biophys. Res. Commun. 174: 654, 1991. 47. Hamamoto, I., Tanaka, S., Maeba, T., Cikaishi, K., and Ichikawa, Y. Microsomal cytochrome P-450-linked monooxygenase systems and lipid composition of human hepatocellular carcinoma. Br. J. Cancer 59: 6, 1989. 48. Takei, Y., Marzi, I., Kauffman, F. C., Currin, R. T., Lemasters, J. J., and Thurman, R. G. Increase in survival time of liver transplants by protease inhibitors and a calcium channel blocker, Nisoldipine. Transplantation 50: 14, 1990. 49. Chazouilleres, O., Vaubourdolle, M., Legendre, C., Balladur, P., Laribi, A., Giboudeau, J., Parc, R., Poupon, R., and Hannoun, L. Biochemical and morphological assessment of sinusoidal damage in human liver graft. Gastroenterology 52: 225, 1991. 50. Caldwell-Kenkel, J. C., Currin, R. T., Tanaka, Y., Thurman, R. G., and Lemasters, J. J. Kupffer cell activation and endothelial cell damage after storage of rat livers: Effects of reperfusion. Hepatology 13: 83, 1991. 51. Marubayashi, S., Dohi, K., Ezaki, H., Hayashi, K., and Kawasaki, T. Preservation of ischemic rat liver mitochondrial functions and liver viability with CoQ10. Surgery 91: 631, 1982. 52. Younes, M., Mohr, A., Schoenberg, M. H., and Schildberg, F. W. Inhibition of lipid peroxidation by superoxide dismutase following regional intestinal ischemia and reperfusion. Res. Exp. Med. 187: 9, 1987. 53. McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. Leukotrienes C4 and D4 stimulate human endothelial cells to synthetize platelet-activating factor and bind neutrophils. Proc. Natl. Acad. Sci. U.S.A. 51: 2204, 1986. 54. Jaeschke, H., Farhood, A., and Smith, C. W. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB. J. 4: 3355, 1990. 55. Ogawa, S., Gerlach, H., Esposito, C., Pasagian-Macaulay, A., Brett, J., and Stern, D. Hypoxia modulates the barrier and coagulant function of cultured bovine endothelium. J. Clin. Invest. 85: 1090, 1990.
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Effects of Cold Preservation and Reperfusion on Microsomal Cytochrome P-450-Linked Monooxygenase System of the Rat Liver KUNIHIKO IZUISHI, M.D., YOSHIYUKI ICHIKAWA, M.D.,* MOHAMMAD AKRAM HOSSAIN, M.D., TAKASHI MAEBA, M.D., HAJIME MAETA, M.D., AND SATOSHI TANAKA, M.D. First Department of Surgery and *Department of Biochemistry, Kagawa Medical School, Miki-cho, Kita-gun, Kagawa, 761-07, Japan Submitted for publication April 4, 1994
The effects of cold preservation and reperfusion of the liver on the hepatic microsomal cytochrome P-450linked monooxygenase system (P-450 system) were investigated. Rat livers were preserved with cold University of Wisconsin solution for 0, 12, 24, 36, and 48 hr. Half of them in 0-, 24-, and 48-hr groups were reperfused for 1 hr at 377C with oxygenated Krebs–Henseleit buffer. After preservation or reperfusion, the liver microsomes were prepared and the concentration of each component of the P-450 system [NADPH-cytochrome b5 reductase (b5 reductase), cytochrome b5 (b5), NADPH-cytochrome P-450 reductase (P-450 reductase) and cytochrome P-450 (P-450)] and their drug metabolizing activities and concentration of apo-cytochrome P-450 2E1 (apo-P-450 2E1) were measured. After 48-hr preservation, b5 concentration did not decrease, whereas the concentration of P-450, P-450 reductase, and b5 reductase decreased from 0.865 to 0.676 nmole/ mg protein, from 0.262 to 0.233 mmole/mg protein/min and from 5.34 to 4.86 mmole/mg protein/min, respectively. During cold preservation, the activities of pnitroanisole O-demethylase and aniline p-hydroxylase did not change. Aminopyrine N-demethylase activity was inhibited from 4.45 to 3.34 nmole/mg protein/min after 48-hr cold preservation. Apo-P-450 2E1 was gradually decreased during cold preservation. Reperfusion caused a further decrease in the activities and concentration of the components of the P-450 system and concentration of apo-P-450 2E1 to 80–90% after 1-hr reperfusion. It was suggested that the prolonged preservation caused deterioration of the P-450 system and the loss of the abilities of metabolism and detoxication of xenobiotics. q 1996 Academic Press, Inc.
INTRODUCTION
The hepatic microsomal cytochrome P-450-linked monooxygenase system (P-450 system) plays important roles in synthesizing steroid hormones [1, 2] and prostaglandin [3, 4], activating vitamin D3 [5, 6], producing bile acid [7], and metabolizing and detoxicating xenobiotics [8]: chemical components that are foreign to the
metabolic network of an organism. The P-450 system is composed of four components: cytochrome b5 (b5), NADH-cytochrome b5 reductase (b5 reductase), cytochrome P-450 (P-450), and NADPH-cytochrome P-450 reductase (P-450 reductase), as shown in Fig. 1. Many reports have suggested that drug metabolizing abilities are a reliable indicator of liver function after liver transplantation in vivo [9–14]. They have also discussed that the causes of decreased drug metabolizing ability were hepatic blood flow and the P-450 related activities in liver microsomes. There is evidence that hepatic blood flow is one of the most important factors affecting the drug metabolizing ability [16]. Poor blood flow causes hypoxia, and hypoxia causes degeneration of the P-450 structure [17]. The P-450 system reflects the intrinsic ability of the liver to metabolize drugs. In addition, oxygen may act as a terminal electron acceptor in the generation of high-energy bonds or may be required as a direct substrate for oxygenase such as those in the P-450 system. However, no study has shown directly the effects on the P-450 system of liver microsomes after cold ischemia and reperfusion. In this study, we hypothesized that cold ischemia/reperfusion injury might affect the P-450 system in drug metabolism, and perfusion ‘‘ex vivo’’ experiments were conducted to exclude hemodynamic factors after transplantation in vivo. MATERIALS AND METHODS Surgical procedure. Male Sprague–Dawley rats weighing 200– 250 g (Clea Japan Inc., Tokyo, Japan) were housed under standard conditions with normal lighting cycle and allowed free access to food and water. Between 7:00 and 10:00 PM, livers were harvested under inhalation anesthesia with ethyl ether. After opening the abdomen, the common bile duct was cannulated with a polyethylene catheter (PE-10; Becton Dickinson & Co., Parsippany, NJ) and secured. The animals were then heparinized with 200 U of heparin from the penile vein, the abdominal aorta was cannulated with a 20-gauge intravenous catheter, and 10 ml of chilled University of Wisconsin (UW) solution was injected. When the injection was started, the thoracic cavity was opened, the descending aorta was clamped, and the right atrium was cut. After this procedure, the portal vein was immediately cannulated with an 18-gauge intravenous catheter, secured, and 5 ml of cold UW solution was slowly injected. All the connective 0022-4804/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Cytochrome P-450-linked monooxygenase systems. It is composed of four components: cytochrome b5 , cytochrome b5 reductase, cytochrome P-450 reductase, and cytochrome P-450. An electron is transferred from NADH or NADPH through cytochrome P-450 monooxygenase system to a substrate into which molecular oxygen is incorporated.
tissues and ligaments were dissected, the liver was removed as quickly as possible, immersed in UW solution in an ice water bath and stored for 12, 24, 36, and 48 hr. After storage, the liver was reperfused at 377C with Krebs–Henseleit buffer. Reperfusion of the liver. The preserved liver was reperfused by a recirculating fashion as described by Hamamoto et al. [18]. Warmed Krebs–Henseleit buffer at 377C was delivered from a reservoir to the liver through the portal vein at a constant perfusion rate (3.5 ml/min/g wet tissue) with a peristaltic pump (Model 312; Gilson Inc., Villier-le-Bel, France). The perfusate was oxygenated with a gas mixture of 95% O2 / 5% CO2 through a Hamilton lung [19], which was made of 10 m of silastic tube (1.47 mm internal diameter) and inserted into the circuit. The perfusate was then drained from the hepatic vein. During reperfusion, bile was collected in a graduated tube and volume was recorded at 1-hr reperfusion. Aliquots of perfusate were collected at 1-hr reperfusion for enzyme determinations, which were assayed using standard colorimetric techniques with a Sigma Diagnostic Kit. Preparation of the liver microsomes. All procedures were carried out at 47C. The liver was homogenized in 5 vol of ice-cold 0.25 M sucrose adjusted to pH 7.4 with 100 mM potassium phosphate buffer and 1 mM of ethylenediaminetetraacetic acid, disodium salt with a motor-driven Teflon pestle and a glass tube kept in an ice bath. The homogenate was centrifuged at 9000 g for 20 min at 47C with a centrifuge (Model 20RP-52, Hitachi Co., Ltd., Tokyo, Japan). The supernatant was collected and centrifuged at 10,500 g for 90 min at 47C with an ultracentrifuge (Model 55P-72, Hitachi Co., Ltd., Tokyo, Japan). The pellet was collected and stored as microsomal fraction at 0807C for further analysis. Experimental groups. Thirty rat livers were preserved with UW solution for 0, 12, 24, 36, and 48 hr (six livers each). Another 18 livers were preserved with UW solution for 0, 24, and 48 hr (six livers each) and then reperfused with Krebs–Henseleit solution at 377C for 1 hr. The microsomes of these livers were prepared for analysis.
tase activity of P-450 reductase were measured by the decrease in absorbance at 420 nm of ferricyanide [22]. Drug metabolizing activities. The activities of aminopyrine Ndemethylase, aniline p-hydroxylase and p-nitroanisole O-demethylase were assayed as follows: The reaction mixture in 1.5 ml of 50 mM Tris-HCl buffer, pH 7.4 containing 5 mM of MgCl2 , 5 mM of glucose 6-phosphate, 0.5 mM of NADP/, 1.5 U of glucose 6-phosphate dehydrogenase and 1 mg protein/ml of microsomal suspension was preincubated for 5 min at 377C. Enzyme reaction was started by adding 100 ml of 30 mM aminopyrine or 15 mM of aniline for aminopyrine N-demethylase or aniline phydroxylase activities, respectively. The reaction mixture was incubated in a shaking water bath at 377C for 15 min. Respective enzyme activities were calculated with the concentrations of released formaldehyde or p-aminophenol, respectively. Formaldehyde was measured by the method of Nash [23]. p-Aminophenol was assayed by the method of Imai et al. [24]. One and one-half milliliters of the reaction mixture of 70 mM of potassium phosphate buffer, pH 7.4 containing 1 mM p-nitroanisole and 1 mg of microsomal protein, was preincubated for 5 min at 377C. The enzyme reaction was started by the addition of 5 mM of MgCl2 , 5 mM of glucose 6-phosphate, 0.5 mM of NADP/, and 1.5 U of glucose 6-phosphate dehydrogenase. The reaction mixture was incubated for 10 min at 377C in a shaking water bath. The reaction was terminated by adding 1.5 ml of 0.5 M NaOH. The mixture was then centrifuged at 10,500 g for 30 min and p-nitrophenol concentration of the supernatant was measured to obtain the activity of p-nitroanisole O-demethylase according to the method of Netter et al. [25]. Concentration of apo-P-450 2E1. Concentration of apo-P-450 2E1 was estimated by Western blotting method with a cytochrome P450 2E1 commercial kit (Oxygene, Dallas, TX). Briefly, microsomal proteins separated by polyacrylamide gel electrophoresis were transferred onto a nitrocellulose paper. The separated P-450 2E1 was then stained using a rabbit anti-rat-P-450 2E1 and sheep-anti-rabbit IgG conjugated with horse radish peroxidase (Sigma chemical Co., St. Louis, MO) [26]. The absorption of apo-P-450 2E1 band was measured densitometrically with a dual-wavelength chromatoscanner (Model CS-910, Shimadzu Co., Kyoto, Japan) to estimate its concentration. Relative value compared with the mean value (cm2/mg protein) of 0 hr was indicated. Effect of perfusate on P-450 and P-450 reductase. Frozen rats liver microsomes (n Å 6) were homogenated with Krebs–Henseleit buffer adjusted to pH 7.4 with 100 mM potassium phosphate buffer and to make 10 mg protein/ml of microsomal suspension. This suspension was incubated in a water bath at 377C for 1 hr. After incubation, P-450 concentration and P-450 reductase activity were examined, and compared with concentration and activity before incubation to examine the possibility of toxicity associated with perfusate on P450 and P-450 reductase. Statistical analysis. In the groups of preservation, significance was tested by one-way analysis of variance. Differences between groups at specific time points were considered significant at P õ 0.05 with Dunnett’s test as a post hoc analysis. Between the groups of before and after reperfusion, and before and after incubation, statistical comparison was performed by Student’s t test. Differences with P õ 0.05 were listed as significant. All values were presented as mean { S.D.
Analytical Procedure Microsomal suspension. Frozen microsomes were homogenated with 0.1 M potassium phosphate buffer pH 7.4 to make 10 mg protein/ml of microsomal suspension. Protein concentration. Protein concentration of microsomal suspension was estimated by the method of Bradford with a protein assay kit (Bio-Rad Lab., Hercules, CA) [20]. Concentration of P-450, b5 , b5 reductase, and P-450 reductase. The concentration of P-450 and b5 were measured spectrophotometricaly with a molar extinction coefficient, 91,000 and 185,000, respectively [21]. The concentration of b5 reductase and P-450 reductase were estimated by their ferricyanide reductase activities. NADH-ferricyanide reductase activity of b5 reductase and NADPH-ferricyanide reduc-
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RESULTS
Bile Production and Enzyme Release into the Perfusate after 1-hr Reperfusion Following Preservation Bile production (Table 1). Freshly harvested livers produced 0.061 { 0.007 ml/g tissue/hr bile 1 hr after reperfusion, and after 24- and 48-hr preservation decreased significantly to 0.045 { 0.007 and 0.025 { 0.007 ml/g tissue/hr, respectively (P õ 0.05).
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TABLE 1 Bile Production, Release of LDH and GOT after 1-hr Reperfusion Following Cold Ischemia Cold ischemia time (hr)
Bile production (ml/g tissue/hr)
LDH release (IU/g tissue/hr)
GOT release (IU/g tissue/hr)
0 24 48
0.061 { 0.007 0.045 { 0.007* 0.025 { 0.007*
0.48 { 0.17 1.08 { 0.19 3.65 { 1.06*
0.058 { 0.027 0.122 { 0.029 0.380 { 0.103*
Note. ∗ vs corresponding value of 0-hr group.
LDH and GOT release (Table 1). Release of LDH and GOT 1 hr after reperfusion following 48-hr preservation increased significantly more than the control group (P õ 0.05), to 3.65 { 1.06 and 0.380 { 0.103 IU/ g tissue/hr, respectively. Concentration of the Components of the P-450 System NADH-cytochrome b5 reductase (Table 2). Concentration of b5 reductase assessed by its NADH-ferricyanide reductase activity was reduced from 5.34 { 0.36 to 4.86 { 0.35 mmole/mg protein/min after 48-hr preservation. Only reperfusion subsequent to 48-hr preservation suppressed the concentration from 4.86 { 0.35 to 4.28 { 0.36 mmole/mg protein/min (P õ 0.05). NADPH-cytochromeP-450reductase (Table 2). Concentration of P-450 reductase assessed with its activity of NADPH-ferricyanide reductase was decreased by 48hr cold preservation from 0.262 { 0.020 to 0.233 { 0.023 mmole/mg protein/min. Reperfusion after 24- and 48-hr cold preservation reduced it significantly (P õ 0.05). Cytochrome b5 (Fig. 2). Cytochrome b5 concentration determined spectrophotometrically showed no change during cold preservation. After reperfusion following 24- and 48-hr preservation, the concentration was reduced from 0.408 { 0.016 to 0.357 { 0.047 and 0.419 { 0.034 to 0.317 { 0.043 nmole/mg protein, respectively (P õ 0.05). Cytochrome P-450 (Fig. 3). In contrast to b5 concentration, 48-hr cold preservation significantly reduced PTABLE 2
FIG. 2. Changes in the concentration of cytochrome b5 after cold preservation and reperfusion. Broken lines indicate reperfusion. ∗ vs corresponding value of 0-hr preserved liver (P õ 0.05); † vs before reperfusion (P õ 0.05).
450 concentration from 0.865 { 0.074 to 0.676 { 0.043 nmole/mg protein. In 0-, 24-, and 48-hr preservation groups, reperfusion decreased the concentration from 0.865 { 0.073 to 0.776 { 0.053, from 0.801 { 0.041 to 0.674 { 0.074 and from 0.676 { 0.043 to 0.542 { 0.049 nmole/mg protein, respectively (P õ 0.05). Drug Metabolizing Activities Aminopyrine N-demethylase (Table 3). The activity decreased from 4.45 { 0.25 to 3.34 { 0.41 nmole/mg protein/min 48 hr after preservation. After reperfusion, activity following 48-hr preservation was significantly inhibited compared with that before reperfusion, to 2.66 { 0.32 nmole/mg protein/min. p-nitroanisole O-demethylase (Table 3). During cold preservation, the activity was kept constant up to 48 hr. Reperfusion did not change the activity in each group. Aniline p-hydroxylase (Table 3). The activity was gradually decreased during 48 hr cold preservation, although no significant difference was detected among these groups. After reperfusion following 48-hr cold preservation, activity was reduced from 1.81 { 0.24 to 1.51 { 0.21 nmole/mg protein/min (P õ 0.05). Concentration of Apo-P-450 2E1 (Fig. 4). After cold preservation and reperfusion, concentration of apo-P-450 2E1 was decreased, but this failed to reach statistical significance.
Changes in NADH0 and NADPH-Ferricyanide Reductase Activities Group
A
0 hr 0-hr reperfusion 12 hr 24 hr 24-hr reperfusion 36 hr 48 hr 48-hr reperfusion
5.34 5.10 5.41 5.20 5.12 4.84 4.86 4.28
{ { { { { { { {
B 0.36 0.50 0.26 0.41 0.52 0.34* 0.35* 0.36†
0.262 0.241 0.252 0.249 0.271 0.254 0.233 0.189
{ { { { { { { {
0.020 0.021 0.013 0.013 0.027† 0.018 0.023* 0.028†
Note. A, NADH-ferricyanide reductase activity (mmole/mg protein/ min); B, NADPH-ferricyanide reductase activity (mmole/mg protein/ min); ∗ vs corresponding value of 0-hr group; † vs before reperfusion.
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FIG. 3. Changes in the concentration of cytochrome P-450 after cold preservation and reperfusion. Broken lines indicate reperfusion. ∗ vs corresponding value of 0-hr preserved liver (P õ 0.05); † vs before reperfusion (P õ 0.05).
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TABLE 3
TABLE 4
Changes in Activities of Aminopyrine N-Demethylase, p-Nitroanisole O-Demethylase, and Aniline p-Hydroxylase
Effect of Perfusate on P-450 and P-450 Reductase
Group 0h 0 hr reperfusion 12 hr 24 hr 24 hr reperfusion 36 hr 48 hr 48 hr reperfusion
A 4.45 4.53 3.92 4.19 4.06 3.35 3.34 2.66
{ { { { { { { {
B 0.25 0.32 0.25* 0.37 0.58 0.35* 0.41* 0.32†
1.78 1.79 1.82 1.81 1.78 1.65 1.61 1.51
{ { { { { { { {
C 0.05 0.25 0.05 0.09 0.21 0.08 0.07 0.22
1.91 1.87 1.94 1.85 1.92 1.98 1.81 1.51
{ { { { { { { {
0.39 0.27 0.21 0.20 0.31 0.19 0.24 0.21†
Note. A, aminopyrine N-demethylase activity (nmole/mg protein/ min); B, p-nitroanisole O-demethylase activity (nmole/mg protein/ min); C, aniline p-hydroxylase (nmole/mg protein/min). ∗ vs corresponding value of 0-hr group; † vs before reperfusion.
Effect of Perfusate on P-450 and P-450 Reductase (Table 4) Statistical significance was not found between before incubation and after incubation. P-450, which has maximum absorbency at 450 nm, is a heme protein and is easily converted reversibly or nonreversibly to its inactive form, cytochrome P-420, which has maximum absorbency at 420 nm [27]. After incubation, cytochrome P-420 was not found. The possibility of the toxicity of the perfusate to P-450 and P-450 reductase was excluded. DISCUSSION
In order to estimate the liver viability and predict the prognosis of the patient after liver transplantation, drug metabolizing activities have been used in some institutes [9–14]. Oellerich et al. reported that lidocaine metabolizing ability of the donor patients was the most reliable indicator for early graft survival after transplantation [11]. However, nothing more than speculation is known about the causes of decreased drug metabolizing abilities during cold preservation and after reperfusion of the liver.
FIG. 4. Changes in concentration of apo-P-450 2E1 after cold preservation and reperfusion. Broken lines indicate reperfusion. Relative values compared with the mean value (cm2/mg protein) at 0 hr was indicated. These values failed to reach statistical significance.
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Before incubation After incubation
Concentration of P-450 (nmole/mg protein)
Activity of P-450 reductase (mmole/mg protein/min)
0.922 { 0.062 0.908 { 0.116
0.264 { 0.030 0.247 { 0.013
Note. Statistical significance was not found between before incubation and after incubation.
Molecular oxygen is incorporated into substrates by P-450, which receives electrons from NADPH or NADH through P-450 reductase, or b5 and b5 reductase, respectively, as shown in Fig. 1 [28]. In this experiment, we examined these four components of the P-450 system, because damage to one of these components causes severe loss of activity of the P-450 system. In addition, drug metabolizing activities by the P-450 system were examined. In the present study, concentration of P-450, b5 reductase, and P-450 reductase decreased during 48-hr cold preservation, whereas b5 concentration was not affected. A similar tendency to our study was observed only in the lung in which concentration of P-450 and b5 decreased during cold preservation [29]. Reduced P450 concentration was paralleled to the inhibition of aminopyrine N-demethylase activity. In contrast, it is of special interest that aniline p-hydroxylase and pnitroanisole O-demethylase activities were not affected during cold preservation. In previous reports, a specific alteration of activities of P-450 isozymes was demonstrated in a model of warm ischemia/reperfusion injury [30]. Studies using the isolated liver preparation show that the elimination of theophylline [31], propranolol [32], and omeprazole [33] may be impaired by hypoxia but that elimination of misonidasole [34] is enhanced. The mechanism of this remains unclear. However, it is well established that multiple isozymes of P-450 exist and that each isozyme is associated with a different spectrum of substrate specificity of P-450 system. The regulation of each isozyme appears to be subject to independent control mechanisms [35, 36] and different affinity of isozyme for oxygen [37]. In addition, several investigators have shown that the different subpopulations of P-450 can be localized to different areas within the liver lobule [38, 39]. The enzymes that are distributed in the pericentral zone are most susceptible to hypoxic tissue damage [40]. These reports strongly suggest that the most likely explanation for nonuniform effect in activities of the P-450 system is that individual P-450 isozymes are differentially affected by cold ischemia/reperfusion injury. This study provides the data that after cold preservation and reperfusion concentration of apoprotein of P450 2E1, which is one of the isozymes of P-450 and also one of the isozymes metabolizing aminopyrine, was decreased, but this failed to reach statistical significance. However, reduction of P-450 concentration and inhibition of aminopyrine N-demethylase activity were paral-
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leled to the degeneration of apo-P-450 2E1. One of the reasons for deterioration of the P-450 system might be the degeneration of apoprotein of P-450 after cold preservation and reperfusion. On the other hand, previous data suggested another reason for the deterioration of the P-450 system. Phosphatidylcholine is an important stabilizer of the P-450 system and thereby sustains the activity of the system [41]. It has been clearly demonstrated that warm ischemia accelerates membrane phospholipid degradation caused by Ca2/ influx and subsequent activation of intrinsic phospholipase A2 [42–46]. In cold ischemia for up to 48 hr with UW solution, accumulation and release of free fatty acids in the liver tissue were observed [18]. These findings strongly suggest that the microsomal membrane is considerably degraded during cold preservation even at low temperatures. The change of phospholipid composition may cause activity loss of the P450 system [47] and acceleration of degradation of the components of the P-450 system. Takei et al. reported that the liver function after transplantation was improved using protease inhibitors: leupeptin, pepstatin A, phenylmethylsulfonyl fluoride [48]. Membranelinked proteases also have to be considered as a possible cause of the activity loss of the P-450 system. Reperfusion of the preserved liver caused significant decrease in the concentration of components of the P450 system, and its drug metabolizing activities. Many hypotheses to elucidate the mechanism of so-called ischemia/reperfusion injury have been proposed [49– 51]. Reactive oxygen substances produced by ischemia/ reperfusion induce lipid peroxidation [52]. This cascade reaction then induces platelet activation [53], neutrophil infiltration [54], and finally increased coagulation [55]. The sequence of the tissue damage ultimately brings tissue hypoperfusion. Once tissue ischemia is introduced, Ca2/ influx and phospholipase activation occur as indicated above and the instability of the P450 system is accelerated. The lipid peroxidation may also cause disturbance of the microsomal membrane and instability and deactivation of the P-450 system. In this study, deteriorative effects of cold preservation and reperfusion of the liver on microsomal cytochrome P-450-linked monooxygenase system were clearly demonstrated. Until now, little attention has been paid to the P-450 system of the preserved liver for transplantation. We should consider the loss of the abilities of metabolism and detoxication of xenobiotics, especially in long-preserved livers in clinical transplantation. REFERENCES 1. Tsubaki, M., Tomita, S., Tsuneoka, Y., and Ichikawa, Y. Characterization of two cysteine residues in cytochrome P-450scc : Chemical identification of the heme-binding cysteine residue. Biochim. Biophys. Acta 870: 564, 1986. 2. Hiwatashi, A., and Ichikawa, Y. Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes. Biochim. Biophys. Acta 664: 33, 1981.
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