Biochemical and Biophysical Research Communications 263, 537–542 (1999) Article ID bbrc.1999.1403, available online at http://www.idealibrary.com on
Ursodeoxycholic Acid Protects Hepatocytes against Oxidative Injury via Induction of Antioxidants Hironori Mitsuyoshi, 1 Toshiaki Nakashima, Yoshio Sumida, Takaharu Yoh, Yoshiki Nakajima, Hiroki Ishikawa, Koji Inaba, Yoshikuni Sakamoto, Takeshi Okanoue, and Kei Kashima Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
Received August 23, 1999
The therapeutic efficacy of ursodeoxycholic acid (UDCA) has been widely demonstrated in various liver diseases, suggesting that UDCA might protect hepatocytes against common mechanisms of liver damage. A candidate for such protection is oxidative injury induced by reactive oxygen species. This study was designed to assess the effects of UDCA on oxidative injury and antioxidative systems in cultured rat hepatocytes. The viability of the hepatocytes dosedependently decreased after hydrogen peroxide or cadmium administration. Pretreatment with UDCA significantly prevented this decrease in viability. The amounts of glutathione (GSH) and protein thiol increased significantly, but the activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and catalase were unchanged in UDCA-treated hepatocytes. The mRNA levels of g-glutamylcysteine synthetase and metallothionein (MT) were significantly higher in UDCA-treated hepatocytes than in controls. In conclusion, UDCA increased hepatocyte levels of GSH and thiol-containing proteins such as MT, thereby protecting hepatocytes against oxidative injury. Our results provide a new perspective on the hepatoprotective effect of UDCA. © 1999 Academic Press
Hepatocytes are well recognized as being continuously exposed to reactive oxygen species (ROS) in various liver diseases including cholestasis (1) and viral hepatitis (2). ROS are derived from many sources in the liver; e.g. the mitochondrial electron transport system (3), microsomal cytochrome P450 system (4), and cytosolic xanthine-xanthine oxidase system (5) of hepatocytes, and non-parenchymal cells including inflammatory and Kupffer cells (2). Excessively produced 1
To whom correspondence should be addressed at Department of Digestive Disease, Osaka Dai-Ichi Hospital, Mitejima 6-2-2, Nishiyodogawa-ku, Osaka 555-0012, Japan. Fax: 181 06-6472-1717.
ROS oxidize membrane lipids, critical cellular proteins, and DNA, result in lethal hepatocytic injuries (3). Antioxidant molecules such as glutathione (GSH), and antioxidative enzymes such as superoxide dismutase (SOD), GSH peroxidase (GPx), and catalase, ordinarily provide hepatocytes with resistance to oxidative stresses. A thiol-containing protein called metallothionein (MT), a cadmium detoxifying protein (6), has also been identified as an antioxidant (7). The therapeutic efficacy of ursodeoxycholic acid (UDCA) has been well discussed not only for chronic cholestatic liver diseases such as primary biliary cirrhosis (8) and primary sclerosing cholangitis (9), but also for viral hepatitis (10). A number of mechanisms have been suggested as being responsible for the effects of UDCA: hypercholeresis (11), protection of cell membranes from injuries by hydrophobic bile acids (12), and immune modulation (13). Although ROS are assumed to play a role in the progression of liver diseases developing under aerobic conditions (3), the relationship between the hepatic metabolism of ROS and the effects of UDCA has rarely been investigated. In the present study, we assessed whether UDCA can protect hepatocytes against oxidative injury, and if so, how UDCA decreases hepatocytic damage caused by ROS. MATERIALS AND METHODS Chemicals. UDCA was provided by Tokyo-Tanabe Pharmaceutical Co. (Tokyo, Japan). The chemicals used were obtained from the following commercial sources: collagenase, GSH, tert-butylhydroperoxide, and catalase from Sigma Chemical Co. (St. Louis, MO); Eagle’s MEM from Nissui Co. (Tokyo, Japan); GSH reductase, pyruvate kinase, lactate dehydrogenase (LDH), Cell Proliferation Kit II (XTT assay), and Cytotoxicity Detection Kit (LDH assay) from Boehringer Mannheim Co. (Indianapolis, IN); nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), and 5,59-dithiobis (2-nitrobenzoic acid) (DTNB) from Nacalai Tesque Co. (Kyoto, Japan); fetal calf serum and Superscript Preamplification System from GIBCO (Grand Island, NY). Other chemicals used were of analytical grade and obtained locally.
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Vulnerability of hepatocytes to hydrogen peroxide (H 2O 2) determined by (A) XTT assay and (B) LDH assay. Each value is represented as a percentage of the initial value (untreated with H 2O 2) and mean 6 S.D. (n 5 4). E, value for control hepatocytes; and F, value for UDCA-treated hepatocytes. *p , 0.05, **p , 0.01, ***p , 0.001, compared to each value for control hepatocytes.
Hepatocyte isolation and culture. Male Wistar strain rats (200 g b.w.) obtained from Shimizu Experimental Materials Co. (Kyoto, Japan) were maintained in cages on a 12 h light-dark cycle and received humane care under a study protocol approved by our institution’s animal research committee. Hepatocytes were isolated by the collagenase perfusion method as previously described (14) and then suspended on 60 mm culture dishes or 96 well microplates (Iwaki glass, Chiba, Japan) in Eagle’s MEM containing 10% fetal calf serum. After a 4-h incubation in a CO 2 incubator, the medium was exchanged for Eagle’s MEM containing 5% fetal calf serum with or without 100 mM UDCA. Assay for cell viability. After a 24-h incubation with or without UDCA in 96 well microplates, the medium was replaced with Eagle’s MEM containing hydrogen peroxide (H 2O 2) or cadmium chloride (CdCl 2) followed by a 1-h incubation. Then, the medium was replaced with fresh MEM containing 5% fetal calf serum for 12 h. The viability of hepatocytes was determined with a microplate reader (Benchmark, Bio-Rad Laboratories, CA) by reduction of sodium 39-[1(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate to formazan (XTT assay) (15), or by the measurement of LDH activity released from the cytosol of hepatocytes into the culture medium (LDH assay) (16). The protein concentration was determined by the method of Bradford (17). Assay for antioxidative capacity and antioxidant content of hepatocytes. After a 24-h incubation, hepatocytes were harvested with a rubber policeman in ice cold physiological saline, destroyed by repeated freezing and thawing, and then the supernatants were obtained after centrifugation at 24,000 3 g for 10 min at 4°C. The oxidation of ascorbic acid by the superoxides (O 22) generated by the xanthine-xanthine oxidase system in the supernatant was monitored by absorbance change for one min at 249.6 nm using a spectrophotometer (UV-2100S, Shimadzu, Kyoto) (18). In brief, 40 mM xanthine, 100 mM EDTA, 22 mg/ml catalase, and 46 mM ascorbic acid were added to the obtained supernatant, followed by addition of 10 mg/ml xanthine oxidase at 25°C. Thiol content in the supernatant was determined by the method of Ellman (19) before and after removing the protein fraction by the addition of 10% trichloroacetic acid. The thiol-containing protein content was determined from the difference between the total thiol
and the non-protein thiol contents. The concentration of GSH in the supernatant was measured by oxidation of DTNB in a reaction mixture containing 0.25 mM NADPH, 0.1 U/ml glutathione reductase and supernatant, using a spectrophotometer (20). Assay for enzymes in hepatocytes. Hepatocytes were harvested with a rubber policeman in ice cold physiological saline, homogenized with a Physcotron (NS-310E, Sogo Rikagaku, Kyoto, Japan), and centrifuged at 24,000 3 g for 10 min at 4°C. SOD activity was quantified according to the nitro blue tetrazolium method (21). GPx activity was quantified by following the oxidation of NADPH by tert-butylhydroperoxide in the presence of GSH and GSH reductase (22). Catalase activity was determined by a decrease in the H 2O 2 concentration of the supernatant using the absorbance at 240 nm (23). Purified catalase from bovine liver with well defined activity was used as the standard. The activity of g-glutamylcysteine synthetase (g-GCS) was quantified by following the oxidation of NADH in the presence of pyruvate kinase and LDH according to the method of Seelig and Meister (24). Quantification of mRNA levels ofg-GCS and MT by RT-PCR. Non-radioactive RT-PCR and a charge-coupled device (CCD) imaging system (25) were employed to quantify mRNA levels of g-GCS and MT-II. In brief, total RNA from cultured hepatocytes was isolated by a single step method (26). First strand cDNA was made from 5 mg of RNA using a Superscript preamplification system and following the manufacturer’s instructions. The PCR mixture contained first strand cDNA and primers for either g-GCS [sense: (59)-TGACATTCCAAGCCTGCAGT-(39), antisense: (59)-CTGGTCAGCAGTACCACAAA-(39)] or MT- II [sense: (59)-AACTGCTCCTGTGCCACAGA-(39), antisense: (59)-AGCAGCTGCACTTGTCCGAA(39)]. The complete reaction mixture was divided into 10 ml aliquots in a 0.2 ml microcentrifuge tube. PCR was performed in a PerkinElmer thermal cycler (PCR system 9600, Perkin-Elmer Japan Co., Ltd., Chiba, Japan) using a step-cycle program of 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 1 min of extension at 72°C. One sample was picked up at every cycle, and the products were then electrophoresed on a 1% agarose gel. The intensity of the ethidium bromide luminescence of each band was measured using a CCD image sensor (Densitograph, Atto Corp., Tokyo, Japan). Reaction cycle-PCR product yield curves of each reaction mixture were
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FIG. 3. Oxidation of ascorbic acid in supernatants of hepatocytes. Each value represents the mean 6 S.D. (n 5 4). *p , 0.05, compared to the value for control hepatocytes.
FIG. 2. Vulnerability of hepatocytes to cadmium determined by XTT assay. Each value is represented as a percent of the initial value (untreated with cadmium) and mean 6 S.D. (n 5 4). E, value for control hepatocytes; and F, value for UDCA-treated hepatocytes. *p , 0.05, **p , 0.01, compared to each value for control hepatocytes.
plotted on semilogarithmic graphs. For estimation of the initial amounts of cDNA, which reflect the mRNA levels of g-GCS or MT-II, regression equations of the form: y 5 I 3 E n (y, intensity of fluorescence; I, initial amounts of cDNA; E, efficiency of amplification; n, cycle number) were fitted to the data in the linear portion of the curves. The mRNA level of b-actin was also measured in the same manner to normalize the efficiencies of RNA extraction and cDNA synthesis. Statistics. ANOVA was employed for statistical analysis. Differences were considered statistically significant when the p-value was less than 0.05.
RESULTS The vulnerability of control and UDCA-treated hepatocytes to H 2O 2 was determined by XTT assay (Fig. 1A). The concentration of formazan produced by control hepatocytes decreased dose-dependently with the addition of H 2O 2. In contrast, the concentration of formazan produced by UDCA-treated hepatocytes was increased by the addition of H 2O 2 at a lower concentration, and maintained at a level above the initial value even at 700 mM of H 2O 2. The vulnerability of hepatocytes was also determined by LDH activity in the culture medium released from the hepatocytes (Fig. 1B). LDH release from hepatocytes increased dose-dependently with the addition of H 2O 2 in control and UDCA-treated hepatocytes, and was significantly lower in UDCA-treated hepatocytes than in control hepatocytes with the addition of 600 and 700 mM of H 2O 2. The vulnerability of control and UDCA-treated hepatocytes to cadmium was next determined by XTT assay (Fig. 2). The formazan formation decreased dose-
dependently with the addition of cadmium in the control and UDCA-treated hepatocytes, and was significantly higher in UDCA-treated hepatocytes than in control hepatocytes with the addition of 6, 12.5, and 25 mM of cadmium. The antioxidative capacity of hepatocytes was evaluated from the rate of oxidation of ascorbic acid coupled to the xanthine-xanthine oxidase system in supernatants of the hepatocytes (Fig. 3). The difference of absorbance at 249.6 nm before and after the addition of xanthine oxidase was significantly lower in UDCAtreated hepatocytes than in the controls. No absorbance change was observed in the sample without xanthine oxidase. Subsequent experiments were conducted to determine whether UDCA influences the levels of antioxidants and antioxidative enzymes in hepatocytes. The contents of GSH and thiol-containing proteins in UDCA-treated hepatocytes were significantly higher than those of control hepatocytes (Fig. 4). On the other hand, there were no significant differences in the activities of SOD, GPx, and catalase between control and UDCA-treated hepatocytes (Table 1).
FIG. 4. The contents of (A) glutathione (GSH) and (B) proteinthiol in hepatocytes. Each value represents the mean 6 S.D. (n 5 4). *p , 0.05, **p , 0.01, compared to the value for control hepatocytes.
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Effects of UDCA on Antioxidative Enzymes in Hepatocytes Enzyme (unit/mg protein)
SOD GPx Catalase
26.3 6 1.5 4.95 6 0.49 254.2 6 44.3
20.9 6 2.3 5.16 6 0.11 261.3 6 79.6
N.S. N.S. N.S.
Note. After 24 h incubation with or without 100 mM UDCA, activities of SOD, GPx and catalase were determined in cultured rat hepatocytes. Data represents the mean 6 S.D. (N 5 4). N.S. (not significant), compared to values for control hepatocytes.
The activity of g-GCS, a rate limiting enzyme in GSH synthesis (27), was measured in control and UDCAtreated hepatocytes. The g-GCS activities (n 5 4, mean 6 S.D., mU/mg protein) were 209 6 10 in control hepatocytes and 339 6 35 in UDCA-treated hepatocytes (p , 0.05). Quantitative RT-PCR analysis was performed to determine mRNA levels of g-GCS in control and UDCAtreated hepatocytes (Fig. 5). The amount of PCR product increased exponentially in the early cycles of the reaction, but subsequently reached a plateau. The linear portion of each curve was parallel. The mRNA level of g-GCS, which was calculated from the linear portion of the curve and normalized by the mRNA level of b-actin, was significantly higher in UDCA-treated hepatocytes than in the controls. Similarly, the mRNA level of MT-II, an antioxidant protein having an abundance of cysteine (thiol) (7), was measured in control and UDCA-treated hepatocytes by RT-PCR analysis (Fig. 6). The mRNA level of MT-II was significantly higher in UDCA-treated hepatocytes than in the controls.
FIG. 5. (A) RT-PCR analysis for mRNA of g-GCS and b-actin in hepatocytes. E, value for control hepatocytes; and F, value for UDCA-treated hepatocytes. (B) mRNA level of g-GCS in control hepatocytes and UDCA-treated hepatocytes (n 5 4, mean 6 S.D.). *p , 0.01, compared to the value for control hepatocytes.
FIG. 6. (A) RT-PCR analysis for mRNA of MT-II and b-actin in hepatocytes. E, value for control hepatocytes; and F, value for UDCA-treated hepatocytes. (B) mRNA level of MT-II in control hepatocytes and UDCA-treated hepatocytes (n 5 4, mean 6 S.D.). *p , 0.001, compared to the value for control hepatocytes.
DISCUSSION H 2O 2, one of the ROS, affects the macromolecules in hepatocytes directly or through the formation of hydroxyl radicals (3) and causes cell death. In the present study hepatocytes lost the capacity to reduce XTT to formazan after H 2O 2 treatment, but the reduction rate was maintained at a higher level in UDCA-pretreated hepatocytes than in controls. The viability of the hepatocytes was also estimated by the measurement of LDH activity in the culture medium released from the hepatocytes. As a result, UDCA treatment inhibited LDH release from hepatocytes exposed to H 2O 2. Since metabolism of XTT is related to the activity of mitochondrial enzymes (15) and UDCA is known to be capable of protecting mitochondrial membrane against various stimuli (28), the protection of mitochondria by UDCA might have contributed to the finding that the cytoprotective effect of UDCA was more remarkable in XTT assay than in LDH assay. The vulnerability of hepatocytes to cadmium was investigated next by using XTT assay. LDH assay was not employed in this experiment, because cadmium directly affects the activity of LDH. Cadmium does not generate free radicals, but depletes cellular GSH and protein-bound sulfhydryls, resulting in ROS production (29). UDCA-treated hepatocytes were found to be resistant to cadmium as well as H 2O 2. In order to exclude the direct effects of UDCA on organelles such as mitochondria, plasma membrane, and so on, the supernatant was used for the evaluation of the antioxidative capacity of UDCA-treated hepatocytes. The amount of ascorbic acid which was oxidized by O 22 in the supernatant was smaller in UDCAtreated hepatocytes than in control hepatocytes, indicating that the capacity of scavenging O 22 has increased in the supernatant of UDCA-treated hepatocytes.
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The present study clearly revealed that UDCA treatment significantly increased the concentrations of GSH and thiol-containing proteins in the supernatant, but did not induce the activities of antioxidative enzymes such as SOD, GPx and catalase in hepatocytes. GSH, a thiol-containing tripeptide, is synthesized predominantly in the liver, and can reduce hepatic ROS and lipid peroxides in concert with NADPH and catalytic enzymes such as GPx (3). Hepatic GSH synthesis is regulated mainly by the enzymatic activity of g-GCS in the cytosol (27). The present study also demonstrated that UDCA treatment significantly increased the activity of g-GCS at the transcriptional level. In addition to GSH, hepatocytes have an abundance of thiol-containing proteins such as albumin and MT. MT is characterized by a high cysteine (thiol) content (30 mole%) (7). Although the original role of MT had been considered to be the detoxification of cadmium (6), MT can also efficiently scavenge the most highly reactive ROS, hydroxyl radicals (30, 31). Because the direct quantification of MT requires environmentally hazardous 109Cd (32), the mRNA level of MT instead of MT content was measured in the present study. MT-II mRNA was found to be increased in UDCA-treated hepatocytes. In summary, UDCA promotes the synthesis of GSH and thiol-containing proteins such as MT at the transcriptional level in hepatocytes, thereby enhancing the resistance of hepatocytes to oxidative stress. One of the interesting findings obtained in the present study is that formazan formation in the XTT assay was higher at a lower H 2O 2 concentration in UDCA-treated hepatocytes (Fig. 1A); this phenomenon was not observed in control hepatocytes or cadmiumtreated hepatocytes. H 2O 2 generates ROS such as hydroxyl radicals (3), which are known to enhance the activity of mitogen-activated protein kinase (MAPK) (33), a member of the protein kinase family associated with cell proliferation (34). On the other hand, UDCA has recently been recognized as influencing the signal transduction pathways in hepatocytes through the modification of calcium ions (35), protein kinase C (36), or the glucocorticoid receptors (14, 37). It has also been established that cellular redox (reduction/oxidation) status regulates the interactions among various transcriptional regulatory factors and DNA; e.g. the DNAbinding property of NF-kB increases when disulfide bonds of the binding domain are reduced to thiol bonds by GSH or thioredoxin (38). Thus, it is possible that the thiol-containing antioxidants increased by UDCA enhance the proliferation signals from H 2O 2-induced ROS. Considering that so-called redox regulation of this kind plays important roles not only in cytoprotection from oxidative stress but also in signal transduction (39), the induction of thiol-containing antioxidants by UDCA may explain why this compound has a wide
spectrum of efficacy in liver diseases caused by various mechanisms. ACKNOWLEDGMENT Part of this work was presented at the 49th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), held in Chicago, Illinois, on November 8, 1998.
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