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Naproxen-induced oxidative stress in the isolated perfused rat liver
Chemico-Biological Interactions 160 (2006) 150–158
Naproxen-induced oxidative stress in the isolated perfused rat liver Hiroyuki Yokoyama a , Toshiharu Horie b,∗ , Shoji Awazu a a
Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0355, Japan b Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Received 26 July 2005; received in revised form 28 December 2005; accepted 11 January 2006 Available online 10 February 2006
Abstract We previously showed that naproxen induced the oxidative stress in the liver microsomes and the isolated hepatocytes of rats. In this study, the in situ effect of naproxen on the rat liver tissue was investigated, using the isolated perfused liver from the viewpoint of the naproxen-induced hepatotoxicity. The leakage of glutamic-oxaloacetic transaminase (GOT) from the perfused liver and appearance of thiobarbituric acid reactive substances (TBARS) in the perfusate increased with the progress of perfusion after a lag time of about 1 h. The naproxen-perfusion of the liver decreased the biliary excretion of glutathione (GSH) and oxidized glutathione, glutathione disulfide (GSSG) prior to TBARS production and GOT leakage. GSSG content in the naproxen-perfused liver was significantly higher than in the control. TBARS appeared in the perfusate of the naproxen-perfused liver for 30 min, but not in the control. The biliary excretion clearance (CLbile ) of indocyanine green (ICG), a reagent for testing the liver function, in the liver perfused with naproxen decreased to a half of that in the liver perfused without naproxen. Thus, the naproxen-induced oxidative stress in the liver was shown to affect the physiological function of liver through the impairment of biliary excretion, which is recognized as a detoxification system. © 2006 Published by Elsevier Ireland Ltd. Keywords: Naproxen; Oxidative stress; Liver perfusion; Liver injury; Glutathione
1. Introduction Naproxen, (S)-6-methoxy-␣-methyl-2-naphthalene acetic acid, is a widely used non-steroidal anti-inflamAbbreviations: TBARS, thiobarbituric acid reactive substances; GSH, glutathione; GSSG, oxidized glutathione; KHBB, Krebs–Henseleit bicarbonate buffer; MDA, malondialdehyde; TBA, thiobarbituric acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; ICG, indocyanine green; t-BuOOH, tert-butylhydroperoxide; MRP2, multidrug resistance-associated protein; HNE, 4-hydroxynonenal ∗ Corresponding author. Tel.: +81 43 226 2886; fax: +81 43 226 2886. E-mail address: [email protected] (T. Horie). 0009-2797/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2006.01.003
matory drug in the treatment of rheumatoid arthritis [1–3]. This drug is recognized to be highly effective and clinically safe, but some side-effects such as gastrointestinal toxicity [4], nephrotoxicity [5,6], jaundice [7–9] and hepatotoxicity [10] have been reported. The hypersensitive response is suggested to contribute to the hepatic injury associated with naproxen therapy [6,9], but little has been clarified on the mechanism of the side-effects other than the highpersensitive response. We reported that naproxen induced lipid peroxidation in the liver microsomes and the isolated hepatocytes of rats, which was caused by the reactive oxygens produced during naproxen oxidative metabolism but not by naproxen or its oxidative metabolite [11–13]. Interestingly, ferrous
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ion was released from the liver microsomes underwent lipid peroxidation caused by naproxen [14]. The intracellular glutathione (GSH) decreased and the oxidized glutathione, glutathione disulfide (GSSG) increased during naproxen metabolism in the isolated rat hepatocytes [15]. The depletion of GSH in the hepatocytes makes them susceptible to the oxidative stress [16]. The production of GSSG indicates an early stage of the naproxeninduced oxidative stress, which leads to the occurrence of lipid peroxidation and lethal cell injury. These results obtained from the in vitro studies clearly showed that naproxen provoked the oxidative stress in liver cells. The next subject to be studied is how naproxen affects the in vivo liver functions. Thus, in this paper, we investigated the naproxen-induced oxidative stress in the isolated rat perfused liver, since the isolated perfused liver maintains the spatial and anatomical architechture and is a useful experimental system to study the physiological function of liver.
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naproxen were assayed according to Buege and Aust [18] and expressed as nmol of malondialdehyde (MDA) equivalents per mL of the perfusate. Briefly, 2 mL of thiobarbituric acid (TBA) stock reagent containing 15% TCA, 0.375% TBA, and 0.25 N HCl were added to 1 mL of the perfusate mixture, followed by boiling for 15 min. The mixture was then cooled and centrifuged at 1000 × g for 15 min. The absorbance of the supernatant at 535 nm was determined, using 1,1,3,3-tetraethoxypropane as the standard. 2.4. Assay of GOT GOT leaked from the naproxen perfused liver was assayed using GOT-UV-Test Wako, a kit for the measurement of GOT activity [19] and expressed as Karmen unit. 2.5. Assay of high molecular weight protein aggregates
2. Materials and methods 2.1. Materials Naproxen sodium salt was purchased from Sigma Chemical Co. (St. Louis, MO, USA). GOT-UV-Test Wako was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were of the highest purity available. 2.2. Isolated rat liver perfusion Isolated rat liver perfusion was conducted by the recirculation method [17]. Wistar male rats (170–250 g, 8–12 weeks of age) were anesthetized by sodium pentobarbital (50 mg/kg) and heparin solution (200 units/rat) was injected via femoral vein. In the case of bile collection, the bile duct was cannulated with a polyethylene tube (PE-10). The liver was perfused with Krebs–Henseleit bicarbonate buffer (KHBB) (pH 7.4) saturated by 95%O2 /5%CO2 through a polyethylene tube (PE-50) cannulated into the portal vein. The perfusate was collected from a polyethylene tube (PE-50) cannulated into superior vena cava via the right atrium. The flow rate of the perfusate was 20 mL/min, otherwise stated, and the volume of the perfusate was 200 mL. 2.3. Assay of TBARS Thiobarbituric acid reactive substances (TBARS) appeared in the perfusate during the liver perfusion with
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to Weber and Osborn [20], as reported elsewhere [21]. After perfusion, the isolated liver was homogenized with KHBB. An aliquot of the homogenate was mixed with 0.2 M sodium phosphate buffer (pH 7.4) containing 5% SDS, 20% glycerol, 2 mM 2-mercaptoethanol and 0.06% phenol red and was applied to the gel. The gel bands stained with 0.01% Coomassie Brilliant Blue were analysed with a chromatoscanner (CS-9000, Shimadzu, Japan) by dual wavelengths (550 nm as a sample and 490 nm as a reference). 2.6. Assay of biliary glutathione (GSH and GSSG) The bile collected in 150 L of 5% metaphospholic acid was centrifuged at 10,000 rpm for 1 min in Beckman microfuge® . The supernatant was mixed with ␥-glutamylaminomethylsulfonic acid solution as an internal standard and injected into HPLC. HPLC was performed under the following conditions: column, TSK-gel DEAE-2SW (4.6 nm × 250 nm); mobile phase, 5 mM citric acid solution containing 1 mM EDTA; flow rate, 1.0 mL/min; detection, o-phthalaldehyde fluorescence (excitation wavelength 355 nm, Emission wavelength 425 nm) by postlabeling method; solution for postlabeling, 18.6 mM o-phthalaldehyde and 17.1 mM 2-mercaptoethanol in 10% methanol and 90% 0.1 M NaHCO3 –NaOH buffer (pH 10.5); reaction temperature, 70 ◦ C; stainless steel coil length, 2 m; flow rate 0.5 mL/min.
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2.7. Assay of GSSG in the perfused liver Immediately after the liver perfusion, the liver was isolated and frozen in liquid nitrogen. The liver was freeze-dried and powderized. Five milliliters of 1 M HClO4 containing 2 mM EDTA and 50 mM N-ethylmaleimide (NEM) were added to 0.5 g of the powderized liver, and the mixture was homogenized. A part of the homogenate (0.5 mL) was mixed with 0.45 mL of 2 M KOH containing 0.3 M 3[N-morpholino]propanesulfonic acid, and the mixture was centrifuged at 10,000 rpm for 1 min in Beckman microfuge® . Five hundred microliters of the supernatant were passed through C18 -cartridge (TOYOPAK® , TOSOH Corporation, Tokyo, Japan) to remove NEM that interferes glutathione reductase activity from the sample. The cartridge was washed with 500 L of 0.1 M sodium phosphate buffer containing 1 mM EDTA (pH 7.0). The content of GSSG in the eluate was determined according to Tietze [22]. Briefly, an aliquot of the eluate was added to the mixture consisting of 0.1 M sodium phosphate buffer, 1 mM EDTA, 0.1 mM 5,5 -dithiobis(2-nitrobenzoic acid), 0.15 mM NADPH, and 0.5 U/mL glutathione reductase. After mixing, the production rate of 5-thio-2-nitrobenzoate was measured at 25 ◦ C as an increase of absorbance at 412 nm. The calibration curve was produced by authentic GSSG dissolved in 1 M HClO4 containing 2 mM EDTA and 50 mM NEM as described above. The GSSG concentration was calculated from the calibration curve.
bile by the area under the curve (AUC) of ICG concentration in the perfusate. 2.10. Statistical analysis Statistical analysis was performed by Student’s t-test. Differences were considered to be statistically significant when P < 0.05. 3. Results 3.1. GOT leakage in the naproxen perfused liver The isolated rat liver was perfused with buffer containing naproxen (0, 1, 5 and 10 mM) (Fig. 1). Time course of GOT leakage from the liver was examined by perfusing the liver without and with 10 mM naproxen (Fig. 1, inset). The GOT activity in the perfusate was the same level as that of control perfusion (without naproxen), up to about 30 min after the start of liver perfusion with 10 mM naproxen solution. After that, the GOT level in the perfusate increased with the progress of perfusion and reached about 1000 Karmen units at 2 h after the start of perfusion. The concentration depencence of naproxen on the GOT leakage from the perfused liver was examined at 2 h after the start of perfusion. The GOT leakage in the liver perfused with 1 mM naproxen was the same level as that of the control and increased markedly with 5 mM and 10 mM naproxen perfusion.
2.8. Assay of ICG in bile and perfusate The contents of indocyanine green (ICG) in bile and perfusate during ICG liver perfusion were determined according to Iga et al. [23]. Briefly, bile was mixed with an equal volume of rat plasma, and diluted with purified water. The absorbance of the mixture at 795 nm was determined. The perfusate (300 L) was mixed with 30 L of rat plasma. The absorbance of the mixture at 800 nm was determined. 2.9. Determination of clearance of ICG The elimination clearance of ICG (CLorg ) was calculated as the product of the elimination rate constant (kel ) and the volume of perfusate (200 mL). The elimination rate constant was calculated from the following equation using a least square regression program: C = C0−kel t . The biliary excretion clearance of ICG (CLbile ) was calculated by dividing the total amount of ICG excreted into
Fig. 1. The leakage of GOT from the perfused liver of rat. The rat liver was perfused for 2 h without and with 1, 5, and 10 mM naproxen. (inset) Time course of the leakage of GOT from the rat liver perfused without () and with (䊉) 10 mM naproxen. Data represent the mean ± S.E. of three to eight experiments, except for the data with naproxen at 100 and 110 min which are mean of two experiments in the inset. ** P < 0.01, *** P < 0.001, significantly different from the data without naproxen.
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Fig. 2. Appearance of TBARS in the perfusate of rat liver perfusion. The rat liver was perfused for 2 h without and with 1, 5 and 10 mM naproxen. (inset) Time course of the appearance of TBARS in the perfusate of rat liver perfused without () and with (䊉) 10 mM naproxen. Data represent the mean ± S.E. of three to eight experiments, except for the data with naproxen at 100 and 110 min which are mean of two experiments in the inset. *** P < 0.001, significantly different from the data without naproxen.
Fig. 3. Change in bile flow in the perfused liver of rat. The rat liver was perfused without () and with (䊉) 10 mM naproxen. Data were expressed as the percentage of the value without naproxen at 15 min. Data represent the mean ± S.E. of three to four experiments. * P < 0.05, significantly different from the data without naproxen.
3.2. TBARS production in the naproxen perfused liver
ous work [11]. The ratio of the high molecular weight protein aggregates to total proteins in the liver perfused with 10 mM naproxen (2.47%) was higher than that of the control (without naproxen) (1.37%).
The isolated rat liver was perfused with buffer containing naproxen (0, 1, 5 and 10 mM) (Fig. 2). Time course of TBARS production was examined by perfusing the liver without and with 10 mM naproxen (Fig. 2, inset). The TBARS production was the same level as that of control perfusion (without naproxen), up to about 30 min after the start of liver perfusion with 10 mM naproxen solution. After that, TBARS in the perfusate increased with the progress of perfusion and reached about 6 nmol MDA equivalents/mL at 2 h after the start of the perfusion. TBARS in the perfusate at 2 h after the start of 1 mM and 5 mM naproxen perfusion was slightly higher than that of the control, although not significant. TBARS were produced markedly by the 10 mM naproxen perfusion of the liver. 3.3. High molecular weight protein aggregates in the naproxen perfusued liver The isolated rat liver was perfused for 2 h with and without 10 mM naproxen. After the perfusion, the liver was homogenized. The homogenate was analyzed by SDS–PAGE according to the same way as we showed in the rat liver microsomes incubated with naproxen and the high molecular weight protein aggregates appeared at the top of the gel (data not shown), which was the similar pattern of SDS–PAGE as shown in the previ-
3.4. Bile flow in the naproxen perfused liver The isolated rat liver was perfused with and without 10 mM naproxen. The bile was collected at 15 min intervals, and the bile flow was estimated from a weight of collected bile on the assumption that its gravity is one. As shown in Fig. 3, up to about 60 min after the liver perfusion was started, the bile flow in the naproxenperfused liver was almost the same level as that of the control (without naproxen). After that, the bile flow decreased with the progress of naproxen perfusion, while the bile flow in the control was maintained at a constant level. 3.5. Biliary excretion of glutathione in the naproxen perfused liver Fig. 4A shows the biliary excretion of GSSG in the liver during the naproxen perfusion. When the liver was perfused with 0.01 mM naproxen, GSSG excreted into bile was comparable to that of the control. When perfused with 0.1 mM naproxen, the GSSG excretion was less than that of the control. When perfused with 1 and 10 mM naproxen, the GSSG excretion was much less than that of the control. After the perfusion for 30 min with 10 mM naproxen, GSSG scarcely appeared in the
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3.6. GSSG content in the naproxen perfused liver The GSSG content in the liver perfused with 10 mM naproxen for 30 min (0.135 ± 0.013 mol/g liver, mean ± S.E., n =3) was significantly higher than that of the control (0.058 ± 0.012 mol/g liver, mean ± S.E., n = 3) (p < 0.05). 3.7. Biliary excretion of ICG in the liver perfused with and without naproxen
Fig. 4. Biliary excretion of GSSG and GSH in the perfused liver of rat: (A) biliary excretion of GSSG in the perfused liver of rat and (B) biliary excretion of GSH in the perfused liver of rat. The rat liver was perfused without (; n = 3) and with 0.01 mM (; n = 1), 0.1 mM (; n = 1), 1 mM (; n = 1), and 10 mM (; n = 2 for GSSG, n = 3 for GSH) naproxen. n is the number of experiments.
bile, while the bile flow between 30 min and 60 min after the start of perfusion was almost the same level as that of the control. Fig. 4B shows the biliary excretion of GSH in the liver during the naproxen perfusion. When the liver was perfused with 0.01 and 0.1 mM naproxen, the GSH excreted into bile was comparable to that of the control. When perfused with 1 mM naproxen, the GSH excretion was much less than that of the control. When perfused with 10 mM naproxen, the GSH excretion was further lowered compared with that of 1 mM naproxen perfusion. After the perfusion for 30 min with 10 mM naproxen, GSH scarcely appeared in the bile, while the bile flow between 30 and 60 min after the start of perfusion was almost the same level as that of the control.
The liver was perfused with ICG (0.5 mg in 200 mL perfusate) and either without naproxen (control) or with 10 mM naproxen at the perfusion rate of 30 mL/min. Since the bile flow decreased by perfusing the liver with ICG (data not shown), sodium taurocholate (3.75 mol) was added to the perfusate every 15 min of the perfusion. The addition of sodium taurocholate maintained the bile flow in the liver perfused with ICG alone almost at a constant level during the perfusion (Fig. 5A). In contrast, the bile flow in the liver perfused with ICG and 10 mM naproxen increased temporarily immediately after the start of the perfusion and thereafter decreased rapidly. The ICG biliary excretion of the liver perfused with ICG and 10 mM naproxen was much less than that of the control during the perfusion (Fig. 5B). Although the bile flow was maintained almost at the control level or at higher level during the perfusion, the ICG biliary excretion of the naproxen perfused liver already decreased at the early stage of perfusion (within 20 min), suggesting that naproxen itself may possibly affect the biliary excretion of ICG directly. 3.8. Effect of oxidative stress on biliary excretion of ICG in the naproxen perfused liver The liver was perfused with 10 mM naproxen or without naproxen (control) for 60 min. Then, the liver was perfused with KHBB for 10 min to minimize the effect of naproxen itself on the biliary excretion of ICG. Subsequently, the liver was perfused with ICG alone (0.5 mg in 200 mL perfusate) (without naproxen) for 60 min at the perfusion rate of 30 mL/min. After the perfusion of 10 mM naproxen for 60 min, TBARS in the perfusate were 2.84 nmole MDA equivalents/mL and not detectable in case of the control perfusion (without naproxen). GOT in the perfusate in case of 10 mM naproxen perfusion (502.60 ± 152.03 karmen unit, mean ± S.E., n = 3) was significantly higher than that in case of the control perfusion (55.64 ± 7.54 karmen unit, mean ± S.E., n = 3) (p < 0.05).
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0.383 mg (76.6% of dose). CLbile value in the liver perfused with 10 mM naproxen was 1.27 ± 0.03 mL/min/g liver (mean ± S.E., n = 3), which was less than that of the control (without naproxen) (2.62 ± 0.08 mL/min/g liver; mean ± S.E., n = 3). 4. Discussion
Fig. 5. Effects of naproxen on bile flow and biliary excretion of ICG in the perfused liver of rat. The rat liver was perfused with ICG (), and with ICG and 10 mM naproxen (䊉). (A) bile flow and (B) biliary excretion of ICG.
These indicated that lipid peroxidation and liver damage occurred in the liver perfused with naproxen. The bile flow in the liver perfused without naproxen was maintained at a constant level during the perfusion (Fig. 6A). In contrast, the bile flow in the liver perfused with 10 mM naproxen increased temporarily immediately after the liver perfusion and thereafter gradually decreased below the control level. Fig. 6B shows the disappearance of ICG from the perfusate. CLorg value of ICG in the liver perfused with 10 mM naproxen was 3.43 ± 0.10 mL/min/g liver (mean ± S.E., n = 3) and that without naproxen was 3.42 ± 0.27 mL/min/g liver (mean ± S.E., n = 3), indicating no difference between them. As shown in Fig. 6C, the biliary excretion of ICG decreased in the liver perfused with 10 mM naproxen, compared with that without naproxen, and total amount of ICG excreted into bile after the perfusion for 60 min with 10 mM naproxen was 0.188 mg (37.6% of dose) and that without naproxen was
TBARS are water-soluble and released into medium and blood from their production site in the in vitro and in vivo systems [21,24,25]. TBARS are used to evaluate the t-butylhydroperoxide (t-BuOOH)- and allyl alcohol-induced lipid peroxidation in the perfused liver [26,27]. TBARS appeared in the perfusate of the liver perfused with 10 mM naproxen. High molecular weight protein aggregates were also produced in the membranes underwent lipid peroxidation induced by naproxen. The high molecular weight protein aggregates can be a useful index, which substantiates the occurrence of lipid peroxidation in tissues, since they are retained in the membranes after produced [21,25]. Further the formation of high molecular weight protein aggregates suggests the depression of the physiological function of tissues, since the physiologically important proteins such as cytochrome P-450 are included in the high molecular weight protein aggregates [21]. The ratio of high molecular weight protein aggregates to total proteins in the liver perfused with 10 mM naproxen was higher than that of the control (without naproxen), indicating the occurrence of lipid peroxidation in the membranes. These results suggested that lipid peroxidation occurred in the liver perfused with naproxen. The GOT activity in the perfusate increased in parallel with appearance of TBARS, and bile flow from the liver decreased with the proceeding of perfusion, suggesting that the liver was injured. The biliary GSSG efflux in the perfused liver increased under the oxidative stress induced by tBuOOH [28,29], diquat [29,30], and menadione [31]. However, contrary to the above results, the biliary GSSG efflux decreased under the oxidative stress induced by the naproxen perfusion. In addition, the biliary GSH efflux also decreased by the naproxen perfusion. It should be noted that both decreases of biliary GSSG and GSH excretion, of which excretion is reported to be mediated by multidrug resistance-associated protein 2 (MRP2) [32,33], were observed prior to the appearance of TBARS and GOT in the perfusate, suggesting that these are the preceding events leading to lipid peroxidation and liver injury. On the other hand, the GSSG content in the liver perfused with naproxen for 30 min was almost two-fold higher than that without naproxen
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Fig. 6. Bile flow, disappearance of ICG from the perfusate, and biliary excretion of ICG in the perfused liver of rat. After the rat liver was perfused without () and with (䊉) 10 mM naproxen for 60 min by the re-circulation mode (0–60 min), the liver was perfused with KHBB for 10 min by the single pass mode (60–70 min). Then, the liver was perfused with ICG solution that did not contain naproxen for 60 min by the re-circulation mode (70–130 min). (A) bile flow; (B) disappearance of ICG from the perfusate; and (C) Biliary excretion of ICG. Data represent the mean ± S.E. of three experiments.
(control). We previously reported that GSSG increased at early incubation time before TBARS production and LDH release were observed in the isolated rat hepatocytes with naproxen, indicating the occurrence of the naproxen-induced oxidative stress before the resulting lipid peroxidation and lethal cell injury [15]. TBARS value in the perfusate after the naproxen-perfusion of liver for 30 min was 0.045 ± 0.070 nmol MDA equivalents/mL (mean ± S.E., n = 3) and that for the control was little (not detectable). GOT in the perfusate after the perfusion was 10.28 ± 2.94 karmen unit for naproxen and 5.31 ± 3.80 karmen unit for the control (mean ± S.E., n = 3), and these variations were small. The increase of GSSG in the liver perfused with naproxen at an early incubation period may possibly be due to not only the inhibition of biliary GSSG efflux mediated by MRP2 but also the increase of GSSG production.
The effect of oxidative stress induced by the naproxen-liver perfusion on the biliary excretion of ICG, a reagent for testing liver function, was examined. In the liver perfused together with ICG and 10 mM naproxen, the biliary excretion of ICG decreased at the early stage of perfusion (within 20 min), while the bile flow was maintained almost at the control level during the same perfusion period. This suggests that naproxen itself may possibly affect the biliary excretion of ICG, although the mechanism is unknown at present. Therefore, to minimize the effect of naproxen itself on the biliary excretion of ICG, naproxen was washed out with KHBB from the liver underwent lipid peroxidation induced by the 10 mM naproxen perfusion for 60 min. After that, the liver was perfused with ICG, and the bile flow, disappearance of ICG from the perfusate and biliary excretion of ICG were examined. Disappearance of ICG from
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the perfusate after the 10 mM naproxen perfusion was comparable to that after the control perfusion. CLorg value of ICG in the perfusate after perfusion of 10 mM naproxen was almost the same as that after the control perfusion. Since CLorg value of ICG was similar to the rate of perfusion (4.12 mL/min/g liver), uptake of ICG into the liver may be dependent on the rate of perfusion. Therefore, the change in uptake of ICG into the liver would not be observed. On the other hand, the biliary excretion of ICG decreased in the liver perfused with 10 mM naproxen, compared with that without naproxen. The total amounts of ICG excreted into bile after the 10 mM naproxen perfusion for 60 min decreased to 47.2% of that without naproxen. CLbile value in the liver perfused with 10 mM naproxen decreased to a half of that without naproxen. These results suggest that the biliary excretion of ICG decreased in the liver underwent the naproxen-induced lipid peroxidation. However, the reason why the ICG biliary excretion decreased by the naproxen-induced oxidative stress is unknown. The change in redox state of thiol in the liver was reported to induce cholestasis [34]. Yoshida et al. [35] reported that the diquat administration to rats did not change the ICG uptake but decreased the ICG biliary excretion, and that those might be caused by the change in redox state of thiol in the liver. Therefore, the decrease of biliary excretion of ICG in the 10 mM naproxen perfused liver may possibly be due to the change in redox state of thiol in the liver caused by the naproxen-induced oxidative stress. The oxidative stress is suggested to be a trigger for internalization of MRP2 in rat liver perfused with tBuOOH and chlorodinitrobenzene which deplete GSH [36]. The reduction of MRP2 expression and the change in its localization would reduce the bile flow, because MRP2 is a biliary transporter involved in bile saltindependent bile flow. We have recently, reported the internalization of MRP2 under the ethacrynic acidinduced acute oxidative stress [37–39]. On the other hand, naproxen causes the oxidative stress in the liver as was shown here and elsewhere [11–15]. Therefore, the internalization of MRP2 would be one possible mechanism for the reduction of bile flow observed in the naproxen perfused liver, although the internalization of MRP2 has not been examined here. In addition to the changes in localization and/or molecular structure of transporter, which may be caused by the naproxeninduced oxidative stress, the resulting lipid peroxidation would damage the bile canalicular membranes and affect the biliary excretion. The protective role of MRPs against toxins is wellknown. For example, 4-hydroxynonenal (HNE) is a
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toxic byproduct of lipid peroxidation and is suggested to be involved in enzyme inactivation, inhibition of DNA, RNA and protein synthesis [40–42]. HNE is also involved in several diseases [43–45]. HNE forms a glutathione conjugate and MRP2 plays an important role in its biliary excretion [46,47]. Therefore, the naproxeninduced oxidative stress in liver would affect the biological system through the impairment of biliary excretion, which is recognized as a detoxification system. Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] D.W. Hallesy, L.D. Shott, R. Hill, Comparative toxicity of naproxen, Scand. J. Rheumatol. Suppl. 2 (1973) 20–28. [2] E. Kucharz, B. Stawiarska, M. Droztz, Influence of anti-rheumatic drugs on the activity of collagenolytic in hepatic culture, Acta. Biol. Med. Germ. 41 (1982) 39–46. [3] B. Stawiarska, E. Kucharz, M. Droztz, in: E. Kaiser, F. Gabl, M.M. Mueller (Eds.), Proceeding of the eleventh international congress on clinical chemistry, Walter de Gruyer, Berlin, 1982, pp. 817–827. [4] L. Aabakken, J.H. Dybdahl, W. Eidsaunet, A. Haaland, S. Larsen, M. Dsnes, Optimal assessment of gastrointestinal side effects induced by non-steroidal anti-inflammatory drugs, Scand. J. Gastroenterol. 24 (1989) 1007–1013. [5] P.G.F. Cox, W.M. Moons, F.G.M. Russel, C.A.M. Van Ginneken, Renal disposition and effects of naproxen and its l-enantiomer in the isolated perfused rat kidney, J. Pharmacol. Exp. Ther. 255 (1990) 491–496. [6] M.J. Dunn, L. Scharschmidt, E. Zambraski, Mechanism of the nephrotoxicity of non-steroidal anti-inflammatory drugs, Arch. Toxicol. Suppl. 7 (1984) 328–337. [7] B.H. Bass, Jaundice associated with naproxen, Lancet 1 (1974) 998. [8] I.P. Law, H. Knight, Jaundice associated with naproxen, N. Eng. J. Med. 295 (1976) 1201. [9] R.M.M. Victorino, J.C.B. Silveira, A. Baptista, M.C. de Moura, Jaundice associated with naproxen, Postgrad. Med. J. 56 (1980) 368–370. [10] L. Giarelli, G. Falconieri, M. Delendi, Fluminant hepatitis following naproxen administration, Hum. Pathol. 17 (1986) 1079. [11] H. Yokoyama, T. Horie, S. Awazu, Lipid peroxidation in rat liver microsomes during naproxen metabolism, Biochem. Pharmacol. 45 (1993) 1721–1724. [12] H. Yokoyama, T. Horie, S. Awazu, Lipid peroxidation and chemiluminescence during naproxen metabolism in rat liver microsomes, Hum. Exp. Toxicol. 13 (1994) 831–838. [13] H. Yokoyama, T. Horie, S. Awazu, Oxidative stress in isolated rat hepatocytes during naproxen metabolism, Biochem. Pharmacol. 49 (1995) 991–996. [14] B. Ji, Y. Masubuchi, T. Horie, A possible mechanism of naproxeninduced lipid peroxidation in rat liver microsomes, Pharmacol. Toxicol. 89 (2001) 43–48.
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Naproxen-induced oxidative stress in the isolated perfused rat liver Hiroyuki Yokoyama a , Toshiharu Horie b,∗ , Shoji Awazu a a
Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0355, Japan b Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Received 26 July 2005; received in revised form 28 December 2005; accepted 11 January 2006 Available online 10 February 2006
Abstract We previously showed that naproxen induced the oxidative stress in the liver microsomes and the isolated hepatocytes of rats. In this study, the in situ effect of naproxen on the rat liver tissue was investigated, using the isolated perfused liver from the viewpoint of the naproxen-induced hepatotoxicity. The leakage of glutamic-oxaloacetic transaminase (GOT) from the perfused liver and appearance of thiobarbituric acid reactive substances (TBARS) in the perfusate increased with the progress of perfusion after a lag time of about 1 h. The naproxen-perfusion of the liver decreased the biliary excretion of glutathione (GSH) and oxidized glutathione, glutathione disulfide (GSSG) prior to TBARS production and GOT leakage. GSSG content in the naproxen-perfused liver was significantly higher than in the control. TBARS appeared in the perfusate of the naproxen-perfused liver for 30 min, but not in the control. The biliary excretion clearance (CLbile ) of indocyanine green (ICG), a reagent for testing the liver function, in the liver perfused with naproxen decreased to a half of that in the liver perfused without naproxen. Thus, the naproxen-induced oxidative stress in the liver was shown to affect the physiological function of liver through the impairment of biliary excretion, which is recognized as a detoxification system. © 2006 Published by Elsevier Ireland Ltd. Keywords: Naproxen; Oxidative stress; Liver perfusion; Liver injury; Glutathione
1. Introduction Naproxen, (S)-6-methoxy-␣-methyl-2-naphthalene acetic acid, is a widely used non-steroidal anti-inflamAbbreviations: TBARS, thiobarbituric acid reactive substances; GSH, glutathione; GSSG, oxidized glutathione; KHBB, Krebs–Henseleit bicarbonate buffer; MDA, malondialdehyde; TBA, thiobarbituric acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; ICG, indocyanine green; t-BuOOH, tert-butylhydroperoxide; MRP2, multidrug resistance-associated protein; HNE, 4-hydroxynonenal ∗ Corresponding author. Tel.: +81 43 226 2886; fax: +81 43 226 2886. E-mail address: [email protected] (T. Horie). 0009-2797/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2006.01.003
matory drug in the treatment of rheumatoid arthritis [1–3]. This drug is recognized to be highly effective and clinically safe, but some side-effects such as gastrointestinal toxicity [4], nephrotoxicity [5,6], jaundice [7–9] and hepatotoxicity [10] have been reported. The hypersensitive response is suggested to contribute to the hepatic injury associated with naproxen therapy [6,9], but little has been clarified on the mechanism of the side-effects other than the highpersensitive response. We reported that naproxen induced lipid peroxidation in the liver microsomes and the isolated hepatocytes of rats, which was caused by the reactive oxygens produced during naproxen oxidative metabolism but not by naproxen or its oxidative metabolite [11–13]. Interestingly, ferrous
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ion was released from the liver microsomes underwent lipid peroxidation caused by naproxen [14]. The intracellular glutathione (GSH) decreased and the oxidized glutathione, glutathione disulfide (GSSG) increased during naproxen metabolism in the isolated rat hepatocytes [15]. The depletion of GSH in the hepatocytes makes them susceptible to the oxidative stress [16]. The production of GSSG indicates an early stage of the naproxeninduced oxidative stress, which leads to the occurrence of lipid peroxidation and lethal cell injury. These results obtained from the in vitro studies clearly showed that naproxen provoked the oxidative stress in liver cells. The next subject to be studied is how naproxen affects the in vivo liver functions. Thus, in this paper, we investigated the naproxen-induced oxidative stress in the isolated rat perfused liver, since the isolated perfused liver maintains the spatial and anatomical architechture and is a useful experimental system to study the physiological function of liver.
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naproxen were assayed according to Buege and Aust [18] and expressed as nmol of malondialdehyde (MDA) equivalents per mL of the perfusate. Briefly, 2 mL of thiobarbituric acid (TBA) stock reagent containing 15% TCA, 0.375% TBA, and 0.25 N HCl were added to 1 mL of the perfusate mixture, followed by boiling for 15 min. The mixture was then cooled and centrifuged at 1000 × g for 15 min. The absorbance of the supernatant at 535 nm was determined, using 1,1,3,3-tetraethoxypropane as the standard. 2.4. Assay of GOT GOT leaked from the naproxen perfused liver was assayed using GOT-UV-Test Wako, a kit for the measurement of GOT activity [19] and expressed as Karmen unit. 2.5. Assay of high molecular weight protein aggregates
2. Materials and methods 2.1. Materials Naproxen sodium salt was purchased from Sigma Chemical Co. (St. Louis, MO, USA). GOT-UV-Test Wako was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were of the highest purity available. 2.2. Isolated rat liver perfusion Isolated rat liver perfusion was conducted by the recirculation method [17]. Wistar male rats (170–250 g, 8–12 weeks of age) were anesthetized by sodium pentobarbital (50 mg/kg) and heparin solution (200 units/rat) was injected via femoral vein. In the case of bile collection, the bile duct was cannulated with a polyethylene tube (PE-10). The liver was perfused with Krebs–Henseleit bicarbonate buffer (KHBB) (pH 7.4) saturated by 95%O2 /5%CO2 through a polyethylene tube (PE-50) cannulated into the portal vein. The perfusate was collected from a polyethylene tube (PE-50) cannulated into superior vena cava via the right atrium. The flow rate of the perfusate was 20 mL/min, otherwise stated, and the volume of the perfusate was 200 mL. 2.3. Assay of TBARS Thiobarbituric acid reactive substances (TBARS) appeared in the perfusate during the liver perfusion with
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to Weber and Osborn [20], as reported elsewhere [21]. After perfusion, the isolated liver was homogenized with KHBB. An aliquot of the homogenate was mixed with 0.2 M sodium phosphate buffer (pH 7.4) containing 5% SDS, 20% glycerol, 2 mM 2-mercaptoethanol and 0.06% phenol red and was applied to the gel. The gel bands stained with 0.01% Coomassie Brilliant Blue were analysed with a chromatoscanner (CS-9000, Shimadzu, Japan) by dual wavelengths (550 nm as a sample and 490 nm as a reference). 2.6. Assay of biliary glutathione (GSH and GSSG) The bile collected in 150 L of 5% metaphospholic acid was centrifuged at 10,000 rpm for 1 min in Beckman microfuge® . The supernatant was mixed with ␥-glutamylaminomethylsulfonic acid solution as an internal standard and injected into HPLC. HPLC was performed under the following conditions: column, TSK-gel DEAE-2SW (4.6 nm × 250 nm); mobile phase, 5 mM citric acid solution containing 1 mM EDTA; flow rate, 1.0 mL/min; detection, o-phthalaldehyde fluorescence (excitation wavelength 355 nm, Emission wavelength 425 nm) by postlabeling method; solution for postlabeling, 18.6 mM o-phthalaldehyde and 17.1 mM 2-mercaptoethanol in 10% methanol and 90% 0.1 M NaHCO3 –NaOH buffer (pH 10.5); reaction temperature, 70 ◦ C; stainless steel coil length, 2 m; flow rate 0.5 mL/min.
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2.7. Assay of GSSG in the perfused liver Immediately after the liver perfusion, the liver was isolated and frozen in liquid nitrogen. The liver was freeze-dried and powderized. Five milliliters of 1 M HClO4 containing 2 mM EDTA and 50 mM N-ethylmaleimide (NEM) were added to 0.5 g of the powderized liver, and the mixture was homogenized. A part of the homogenate (0.5 mL) was mixed with 0.45 mL of 2 M KOH containing 0.3 M 3[N-morpholino]propanesulfonic acid, and the mixture was centrifuged at 10,000 rpm for 1 min in Beckman microfuge® . Five hundred microliters of the supernatant were passed through C18 -cartridge (TOYOPAK® , TOSOH Corporation, Tokyo, Japan) to remove NEM that interferes glutathione reductase activity from the sample. The cartridge was washed with 500 L of 0.1 M sodium phosphate buffer containing 1 mM EDTA (pH 7.0). The content of GSSG in the eluate was determined according to Tietze [22]. Briefly, an aliquot of the eluate was added to the mixture consisting of 0.1 M sodium phosphate buffer, 1 mM EDTA, 0.1 mM 5,5 -dithiobis(2-nitrobenzoic acid), 0.15 mM NADPH, and 0.5 U/mL glutathione reductase. After mixing, the production rate of 5-thio-2-nitrobenzoate was measured at 25 ◦ C as an increase of absorbance at 412 nm. The calibration curve was produced by authentic GSSG dissolved in 1 M HClO4 containing 2 mM EDTA and 50 mM NEM as described above. The GSSG concentration was calculated from the calibration curve.
bile by the area under the curve (AUC) of ICG concentration in the perfusate. 2.10. Statistical analysis Statistical analysis was performed by Student’s t-test. Differences were considered to be statistically significant when P < 0.05. 3. Results 3.1. GOT leakage in the naproxen perfused liver The isolated rat liver was perfused with buffer containing naproxen (0, 1, 5 and 10 mM) (Fig. 1). Time course of GOT leakage from the liver was examined by perfusing the liver without and with 10 mM naproxen (Fig. 1, inset). The GOT activity in the perfusate was the same level as that of control perfusion (without naproxen), up to about 30 min after the start of liver perfusion with 10 mM naproxen solution. After that, the GOT level in the perfusate increased with the progress of perfusion and reached about 1000 Karmen units at 2 h after the start of perfusion. The concentration depencence of naproxen on the GOT leakage from the perfused liver was examined at 2 h after the start of perfusion. The GOT leakage in the liver perfused with 1 mM naproxen was the same level as that of the control and increased markedly with 5 mM and 10 mM naproxen perfusion.
2.8. Assay of ICG in bile and perfusate The contents of indocyanine green (ICG) in bile and perfusate during ICG liver perfusion were determined according to Iga et al. [23]. Briefly, bile was mixed with an equal volume of rat plasma, and diluted with purified water. The absorbance of the mixture at 795 nm was determined. The perfusate (300 L) was mixed with 30 L of rat plasma. The absorbance of the mixture at 800 nm was determined. 2.9. Determination of clearance of ICG The elimination clearance of ICG (CLorg ) was calculated as the product of the elimination rate constant (kel ) and the volume of perfusate (200 mL). The elimination rate constant was calculated from the following equation using a least square regression program: C = C0−kel t . The biliary excretion clearance of ICG (CLbile ) was calculated by dividing the total amount of ICG excreted into
Fig. 1. The leakage of GOT from the perfused liver of rat. The rat liver was perfused for 2 h without and with 1, 5, and 10 mM naproxen. (inset) Time course of the leakage of GOT from the rat liver perfused without () and with (䊉) 10 mM naproxen. Data represent the mean ± S.E. of three to eight experiments, except for the data with naproxen at 100 and 110 min which are mean of two experiments in the inset. ** P < 0.01, *** P < 0.001, significantly different from the data without naproxen.
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Fig. 2. Appearance of TBARS in the perfusate of rat liver perfusion. The rat liver was perfused for 2 h without and with 1, 5 and 10 mM naproxen. (inset) Time course of the appearance of TBARS in the perfusate of rat liver perfused without () and with (䊉) 10 mM naproxen. Data represent the mean ± S.E. of three to eight experiments, except for the data with naproxen at 100 and 110 min which are mean of two experiments in the inset. *** P < 0.001, significantly different from the data without naproxen.
Fig. 3. Change in bile flow in the perfused liver of rat. The rat liver was perfused without () and with (䊉) 10 mM naproxen. Data were expressed as the percentage of the value without naproxen at 15 min. Data represent the mean ± S.E. of three to four experiments. * P < 0.05, significantly different from the data without naproxen.
3.2. TBARS production in the naproxen perfused liver
ous work [11]. The ratio of the high molecular weight protein aggregates to total proteins in the liver perfused with 10 mM naproxen (2.47%) was higher than that of the control (without naproxen) (1.37%).
The isolated rat liver was perfused with buffer containing naproxen (0, 1, 5 and 10 mM) (Fig. 2). Time course of TBARS production was examined by perfusing the liver without and with 10 mM naproxen (Fig. 2, inset). The TBARS production was the same level as that of control perfusion (without naproxen), up to about 30 min after the start of liver perfusion with 10 mM naproxen solution. After that, TBARS in the perfusate increased with the progress of perfusion and reached about 6 nmol MDA equivalents/mL at 2 h after the start of the perfusion. TBARS in the perfusate at 2 h after the start of 1 mM and 5 mM naproxen perfusion was slightly higher than that of the control, although not significant. TBARS were produced markedly by the 10 mM naproxen perfusion of the liver. 3.3. High molecular weight protein aggregates in the naproxen perfusued liver The isolated rat liver was perfused for 2 h with and without 10 mM naproxen. After the perfusion, the liver was homogenized. The homogenate was analyzed by SDS–PAGE according to the same way as we showed in the rat liver microsomes incubated with naproxen and the high molecular weight protein aggregates appeared at the top of the gel (data not shown), which was the similar pattern of SDS–PAGE as shown in the previ-
3.4. Bile flow in the naproxen perfused liver The isolated rat liver was perfused with and without 10 mM naproxen. The bile was collected at 15 min intervals, and the bile flow was estimated from a weight of collected bile on the assumption that its gravity is one. As shown in Fig. 3, up to about 60 min after the liver perfusion was started, the bile flow in the naproxenperfused liver was almost the same level as that of the control (without naproxen). After that, the bile flow decreased with the progress of naproxen perfusion, while the bile flow in the control was maintained at a constant level. 3.5. Biliary excretion of glutathione in the naproxen perfused liver Fig. 4A shows the biliary excretion of GSSG in the liver during the naproxen perfusion. When the liver was perfused with 0.01 mM naproxen, GSSG excreted into bile was comparable to that of the control. When perfused with 0.1 mM naproxen, the GSSG excretion was less than that of the control. When perfused with 1 and 10 mM naproxen, the GSSG excretion was much less than that of the control. After the perfusion for 30 min with 10 mM naproxen, GSSG scarcely appeared in the
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3.6. GSSG content in the naproxen perfused liver The GSSG content in the liver perfused with 10 mM naproxen for 30 min (0.135 ± 0.013 mol/g liver, mean ± S.E., n =3) was significantly higher than that of the control (0.058 ± 0.012 mol/g liver, mean ± S.E., n = 3) (p < 0.05). 3.7. Biliary excretion of ICG in the liver perfused with and without naproxen
Fig. 4. Biliary excretion of GSSG and GSH in the perfused liver of rat: (A) biliary excretion of GSSG in the perfused liver of rat and (B) biliary excretion of GSH in the perfused liver of rat. The rat liver was perfused without (; n = 3) and with 0.01 mM (; n = 1), 0.1 mM (; n = 1), 1 mM (; n = 1), and 10 mM (; n = 2 for GSSG, n = 3 for GSH) naproxen. n is the number of experiments.
bile, while the bile flow between 30 min and 60 min after the start of perfusion was almost the same level as that of the control. Fig. 4B shows the biliary excretion of GSH in the liver during the naproxen perfusion. When the liver was perfused with 0.01 and 0.1 mM naproxen, the GSH excreted into bile was comparable to that of the control. When perfused with 1 mM naproxen, the GSH excretion was much less than that of the control. When perfused with 10 mM naproxen, the GSH excretion was further lowered compared with that of 1 mM naproxen perfusion. After the perfusion for 30 min with 10 mM naproxen, GSH scarcely appeared in the bile, while the bile flow between 30 and 60 min after the start of perfusion was almost the same level as that of the control.
The liver was perfused with ICG (0.5 mg in 200 mL perfusate) and either without naproxen (control) or with 10 mM naproxen at the perfusion rate of 30 mL/min. Since the bile flow decreased by perfusing the liver with ICG (data not shown), sodium taurocholate (3.75 mol) was added to the perfusate every 15 min of the perfusion. The addition of sodium taurocholate maintained the bile flow in the liver perfused with ICG alone almost at a constant level during the perfusion (Fig. 5A). In contrast, the bile flow in the liver perfused with ICG and 10 mM naproxen increased temporarily immediately after the start of the perfusion and thereafter decreased rapidly. The ICG biliary excretion of the liver perfused with ICG and 10 mM naproxen was much less than that of the control during the perfusion (Fig. 5B). Although the bile flow was maintained almost at the control level or at higher level during the perfusion, the ICG biliary excretion of the naproxen perfused liver already decreased at the early stage of perfusion (within 20 min), suggesting that naproxen itself may possibly affect the biliary excretion of ICG directly. 3.8. Effect of oxidative stress on biliary excretion of ICG in the naproxen perfused liver The liver was perfused with 10 mM naproxen or without naproxen (control) for 60 min. Then, the liver was perfused with KHBB for 10 min to minimize the effect of naproxen itself on the biliary excretion of ICG. Subsequently, the liver was perfused with ICG alone (0.5 mg in 200 mL perfusate) (without naproxen) for 60 min at the perfusion rate of 30 mL/min. After the perfusion of 10 mM naproxen for 60 min, TBARS in the perfusate were 2.84 nmole MDA equivalents/mL and not detectable in case of the control perfusion (without naproxen). GOT in the perfusate in case of 10 mM naproxen perfusion (502.60 ± 152.03 karmen unit, mean ± S.E., n = 3) was significantly higher than that in case of the control perfusion (55.64 ± 7.54 karmen unit, mean ± S.E., n = 3) (p < 0.05).
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0.383 mg (76.6% of dose). CLbile value in the liver perfused with 10 mM naproxen was 1.27 ± 0.03 mL/min/g liver (mean ± S.E., n = 3), which was less than that of the control (without naproxen) (2.62 ± 0.08 mL/min/g liver; mean ± S.E., n = 3). 4. Discussion
Fig. 5. Effects of naproxen on bile flow and biliary excretion of ICG in the perfused liver of rat. The rat liver was perfused with ICG (), and with ICG and 10 mM naproxen (䊉). (A) bile flow and (B) biliary excretion of ICG.
These indicated that lipid peroxidation and liver damage occurred in the liver perfused with naproxen. The bile flow in the liver perfused without naproxen was maintained at a constant level during the perfusion (Fig. 6A). In contrast, the bile flow in the liver perfused with 10 mM naproxen increased temporarily immediately after the liver perfusion and thereafter gradually decreased below the control level. Fig. 6B shows the disappearance of ICG from the perfusate. CLorg value of ICG in the liver perfused with 10 mM naproxen was 3.43 ± 0.10 mL/min/g liver (mean ± S.E., n = 3) and that without naproxen was 3.42 ± 0.27 mL/min/g liver (mean ± S.E., n = 3), indicating no difference between them. As shown in Fig. 6C, the biliary excretion of ICG decreased in the liver perfused with 10 mM naproxen, compared with that without naproxen, and total amount of ICG excreted into bile after the perfusion for 60 min with 10 mM naproxen was 0.188 mg (37.6% of dose) and that without naproxen was
TBARS are water-soluble and released into medium and blood from their production site in the in vitro and in vivo systems [21,24,25]. TBARS are used to evaluate the t-butylhydroperoxide (t-BuOOH)- and allyl alcohol-induced lipid peroxidation in the perfused liver [26,27]. TBARS appeared in the perfusate of the liver perfused with 10 mM naproxen. High molecular weight protein aggregates were also produced in the membranes underwent lipid peroxidation induced by naproxen. The high molecular weight protein aggregates can be a useful index, which substantiates the occurrence of lipid peroxidation in tissues, since they are retained in the membranes after produced [21,25]. Further the formation of high molecular weight protein aggregates suggests the depression of the physiological function of tissues, since the physiologically important proteins such as cytochrome P-450 are included in the high molecular weight protein aggregates [21]. The ratio of high molecular weight protein aggregates to total proteins in the liver perfused with 10 mM naproxen was higher than that of the control (without naproxen), indicating the occurrence of lipid peroxidation in the membranes. These results suggested that lipid peroxidation occurred in the liver perfused with naproxen. The GOT activity in the perfusate increased in parallel with appearance of TBARS, and bile flow from the liver decreased with the proceeding of perfusion, suggesting that the liver was injured. The biliary GSSG efflux in the perfused liver increased under the oxidative stress induced by tBuOOH [28,29], diquat [29,30], and menadione [31]. However, contrary to the above results, the biliary GSSG efflux decreased under the oxidative stress induced by the naproxen perfusion. In addition, the biliary GSH efflux also decreased by the naproxen perfusion. It should be noted that both decreases of biliary GSSG and GSH excretion, of which excretion is reported to be mediated by multidrug resistance-associated protein 2 (MRP2) [32,33], were observed prior to the appearance of TBARS and GOT in the perfusate, suggesting that these are the preceding events leading to lipid peroxidation and liver injury. On the other hand, the GSSG content in the liver perfused with naproxen for 30 min was almost two-fold higher than that without naproxen
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Fig. 6. Bile flow, disappearance of ICG from the perfusate, and biliary excretion of ICG in the perfused liver of rat. After the rat liver was perfused without () and with (䊉) 10 mM naproxen for 60 min by the re-circulation mode (0–60 min), the liver was perfused with KHBB for 10 min by the single pass mode (60–70 min). Then, the liver was perfused with ICG solution that did not contain naproxen for 60 min by the re-circulation mode (70–130 min). (A) bile flow; (B) disappearance of ICG from the perfusate; and (C) Biliary excretion of ICG. Data represent the mean ± S.E. of three experiments.
(control). We previously reported that GSSG increased at early incubation time before TBARS production and LDH release were observed in the isolated rat hepatocytes with naproxen, indicating the occurrence of the naproxen-induced oxidative stress before the resulting lipid peroxidation and lethal cell injury [15]. TBARS value in the perfusate after the naproxen-perfusion of liver for 30 min was 0.045 ± 0.070 nmol MDA equivalents/mL (mean ± S.E., n = 3) and that for the control was little (not detectable). GOT in the perfusate after the perfusion was 10.28 ± 2.94 karmen unit for naproxen and 5.31 ± 3.80 karmen unit for the control (mean ± S.E., n = 3), and these variations were small. The increase of GSSG in the liver perfused with naproxen at an early incubation period may possibly be due to not only the inhibition of biliary GSSG efflux mediated by MRP2 but also the increase of GSSG production.
The effect of oxidative stress induced by the naproxen-liver perfusion on the biliary excretion of ICG, a reagent for testing liver function, was examined. In the liver perfused together with ICG and 10 mM naproxen, the biliary excretion of ICG decreased at the early stage of perfusion (within 20 min), while the bile flow was maintained almost at the control level during the same perfusion period. This suggests that naproxen itself may possibly affect the biliary excretion of ICG, although the mechanism is unknown at present. Therefore, to minimize the effect of naproxen itself on the biliary excretion of ICG, naproxen was washed out with KHBB from the liver underwent lipid peroxidation induced by the 10 mM naproxen perfusion for 60 min. After that, the liver was perfused with ICG, and the bile flow, disappearance of ICG from the perfusate and biliary excretion of ICG were examined. Disappearance of ICG from
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the perfusate after the 10 mM naproxen perfusion was comparable to that after the control perfusion. CLorg value of ICG in the perfusate after perfusion of 10 mM naproxen was almost the same as that after the control perfusion. Since CLorg value of ICG was similar to the rate of perfusion (4.12 mL/min/g liver), uptake of ICG into the liver may be dependent on the rate of perfusion. Therefore, the change in uptake of ICG into the liver would not be observed. On the other hand, the biliary excretion of ICG decreased in the liver perfused with 10 mM naproxen, compared with that without naproxen. The total amounts of ICG excreted into bile after the 10 mM naproxen perfusion for 60 min decreased to 47.2% of that without naproxen. CLbile value in the liver perfused with 10 mM naproxen decreased to a half of that without naproxen. These results suggest that the biliary excretion of ICG decreased in the liver underwent the naproxen-induced lipid peroxidation. However, the reason why the ICG biliary excretion decreased by the naproxen-induced oxidative stress is unknown. The change in redox state of thiol in the liver was reported to induce cholestasis [34]. Yoshida et al. [35] reported that the diquat administration to rats did not change the ICG uptake but decreased the ICG biliary excretion, and that those might be caused by the change in redox state of thiol in the liver. Therefore, the decrease of biliary excretion of ICG in the 10 mM naproxen perfused liver may possibly be due to the change in redox state of thiol in the liver caused by the naproxen-induced oxidative stress. The oxidative stress is suggested to be a trigger for internalization of MRP2 in rat liver perfused with tBuOOH and chlorodinitrobenzene which deplete GSH [36]. The reduction of MRP2 expression and the change in its localization would reduce the bile flow, because MRP2 is a biliary transporter involved in bile saltindependent bile flow. We have recently, reported the internalization of MRP2 under the ethacrynic acidinduced acute oxidative stress [37–39]. On the other hand, naproxen causes the oxidative stress in the liver as was shown here and elsewhere [11–15]. Therefore, the internalization of MRP2 would be one possible mechanism for the reduction of bile flow observed in the naproxen perfused liver, although the internalization of MRP2 has not been examined here. In addition to the changes in localization and/or molecular structure of transporter, which may be caused by the naproxeninduced oxidative stress, the resulting lipid peroxidation would damage the bile canalicular membranes and affect the biliary excretion. The protective role of MRPs against toxins is wellknown. For example, 4-hydroxynonenal (HNE) is a
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