Vitamin E reduces oxidant injury to mitochondria and the hepatotoxicity of taurochenodeoxycholic acid in the rat

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Vitamin E Reduces Oxidant Injury to Mitochondria and the Hepatotoxicity of Taurochenodeoxycholic Acid in the Rat RONALD J. SOKOL,*,‡ JAMES M. MCKIM, Jr.,*,‡ M. COLBY GOFF,* STEPHANIE Z. RUYLE,§ MICHAEL W. DEVEREAUX,*,‡ DERICK HAN,\ LESTER PACKER,\ and GREGORY EVERSON‡,¶ *Pediatric Liver Center and Section of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, ¶Liver Section, Department of Medicine, §Department of Pathology, and ‡Hepatobiliary Research Center, The Children’s Hospital and University of Colorado School of Medicine, Denver, Colorado; and \The Membrane Bioenergetics Group, Lawrence Berkeley Laboratory, University of California at Berkeley, California

Background & Aims: Hydrophobic bile acids have been implicated in the pathogenesis of cholestatic liver injury. The hypothesis that hydrophobic bile acid toxicity is mediated by oxidant stress in an in vivo rat model was tested in this study. Methods: A dose-response study of bolus intravenous (IV) taurochenodeoxycholic acid (TCDC) in rats was conducted. Rats were then pretreated with parenteral a-tocopherol, and its effect on IV TCDC toxicity was evaluated by liver blood tests and by assessing mitochondrial lipid peroxidation. Results: Four hours after an IV bolus of TCDC (10 mmol/100 g weight), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels peaked, hepatic mitochondria showed evidence of increased lipid peroxidation, and serum bile acid analysis was consistent with a cholestatic injury. Liver histology at 4 hours showed hepatocellular necrosis and swelling and mild portal tract inflammation. Treatment with parenteral a-tocopherol was associated with a 60%–70% reduction in AST and ALT levels, improved histology, and a 60% reduction in mitochondrial lipid peroxidation in rats receiving TCDC. Conclusions: These data show that hepatocyte injury and oxidant damage to mitochondria caused by IV TCDC can be significantly reduced by pretreatment with the antioxidant vitamin E. These in vivo findings support the role for oxidant stress in the pathogenesis of bile acid hepatic toxicity.

holestatic liver disorders are a significant clinical problem in infants, children, and adults.1–3 Current medical therapies for cholestasis frequently fail to prevent progression to cirrhosis and other complications that occur in most patients. In 1993, approximately 270 adults and children underwent orthotopic liver transplantation in the United States because of complications of cholestatic disorders, at an estimated cost of more than $40,000,000.4 Thus, there is a considerable need for the development of new approaches to medical treatment of chronic cholestasis. The underlying cellular and molecular mechanisms by which cholestasis leads to liver injury and fibrosis are therefore of interest.

C

Although the various causes of chronic cholestasis include structural, genetic, immunologic, and inflammatory processes, one of the putative final common pathways leading to cholestatic liver injury is the intracellular accumulation of hydrophobic (toxic) bile acids, particularly chenodeoxycholic acid (CDC) and its conjugates.5–7 CDC has been shown to be hepatotoxic in rats,8 rabbits,9 nonhuman primates,10 and humans ingesting CDC for dissolution of gallstones11,12 and hepatotoxic to isolated human hepatocytes in cell culture.13 Moreover, a more hydrophilic bile acid, ursodeoxycholic acid, seems to provide some protection against cholestatic liver injury14–16 by displacing CDC from the bile acid pool17 through reduction of intestinal absorption of other bile acids18 or by a direct cytoprotective effect.13 Recent studies investigating the mechanisms of CDC toxicity have focused on its effects on hepatocyte membranes19,20 and hepatic mitochondria.21–25 We have postulated that oxidant stress may play a role in cholestatic liver injury.24,26,27 Our initial studies have shown that exogenously administered taurochenodeoxycholic acid (TCDC) causes freshly isolated rat hepatocytes in suspension and isolated rat hepatic mitochondria to generate increased amounts of hydroperoxides24 and to undergo lipid peroxidation in conjunction with cell injury27; these effects were reversed in vitro by preincubation of various free radical inhibitors and scavengers.24,27 Because of the difficulties inherent in extrapolating observations from isolated hepatocytes to the intact organism, investigations were needed to determine the role of oxidant injury in CDC hepatic toxicity in the intact rat. The objectives of the current study were (1) to develop an in vivo model Abbreviations used in this paper: CDC, chenodeoxycholic acid; GC-MS, gas chromatography–mass spectrometry; HPLC, highperformance liquid chromatography; MMPT, mitochondrial membrane permeability transition; TCDC, taurochenodeoxycholic acid. r 1998 by the American Gastroenterological Association 0016-5085/98/$3.00

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of acute TCDC hepatotoxicity in the rat; (2) to determine if intravenously (IV) administered TCDC causes oxidant injury of the liver and particularly to hepatic mitochondria; and (3) to determine if the antioxidant vitamin E administered parenterally could prevent the oxidant injury in this model.

Materials and Methods Materials All chemicals were obtained in analytical purity from the suppliers. Bovine serum albumin (fraction V) was obtained from Calbiochem (La Jolla, CA), bile acids from Sigma Chemical Co. (St. Louis, MO), digitonin from Sigma, and parenteral vitamin E (all-rac-a-tocopherol, Ephynal) from Hoffmann–LaRoche (Nutley, NJ).

Development of Experimental Model of IV TCDC Toxicity The first phase of this study was the development of an IV model of TCDC toxicity in the rat that produced a significant but reversible hepatic injury. For all experiments, young adult male Sprague–Dawley rats (150–180 g) purchased from Sasco Inc. (Omaha, NE) were fed Purina Lab Chow (Ralston Purina Co., Chicago, IL) ad libitum for 2–3 weeks and housed in polyethylene cages with stainless steel tops on a 12-hour light-dark cycle. All rats received humane care in compliance with the guidelines of the Animal Use in Research Committee of the University of Colorado Health Sciences Center. Rats were then randomly assigned (4–5/group) to receive one of the following four IV doses of TCDC solubilized in 5% dextrose, 10% bovine serum albumin, and 0.45% normal saline: 0 (vehicle only), 5, 10, and 20 mmol of TCDC/100 g body wt. Between 7 and 9 AM, the rats were given an intravenous bolus of the desired dose of TCDC in 1–2 minutes through the tail vein while they were under light methoxyflurane anesthesia. Rats recovered from the injection uneventfully, and blood was obtained at 0, 4, 8, 12, and 24 hours after the injection for determination of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, and alkaline phosphatase levels by an automated chemical analyzer. In addition, for the group receiving 10 mmol/100 g body wt of TCDC, total concentrations (conjugated plus free bile acids) of individual bile acids were measured in serum obtained at 4 hours by gas chromatography– mass spectrometry (GC-MS), as described previously.28 Briefly, after serum samples were extracted with the addition of the internal standard 7-a, 12-a, dihydroxy-5b cholanic acid, trimethylsilyl esterification was performed. Individual bile acids were then quantitated by GC or GC-MS using a Hewlett-Packard 5790 gas chromatograph (Hewlett-Packard Co., Wilmington, DE) with flame ionization detector equipped with a 30-m DB-1 capillary column with internal diameter of 0.25 mm and a film thickness of 0.25 mm (J & W Scientific, Folsom, CA). For GC-MS measurements, a Hewlett-Packard 5970-A mass-selective detector operating in selective ion

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monitoring mode was used. Results were expressed as micromoles per liter for each individual bile acid.

Mitochondrial Oxidant Injury in TCDC-Injected Rats Once it was determined that the 10-mmol/100 g body wt dose of TCDC given IV produced the desired reversible hepatic injury with peak injury occurring 4 hours after injection (see Results), the next goal was to determine if this hepatic injury was associated with oxidant damage to hepatic mitochondria. Hepatic mitochondria were chosen as the indicator of intracellular oxidant injury because of the prior demonstration of peroxidation of mitochondrial lipids in the bile duct–ligated rat model26,29 and the inverse relationship in this model of lipid peroxidation with activity of mitochondrial respiratory enzymes.29 Rats maintained as described above were randomly assigned to receive an IV bolus of vehicle or of 10 mmol/100 g weight of TCDC at time 0. Rats were killed 4 hours later. Blood was removed at 0 and 4 hours for determination of serum AST, ALT, alkaline phosphatase, and total bilirubin levels. The rats were killed, and the liver was rapidly removed and processed for light microscopy (buffered formalin fixation) and for isolation of mitochondria. Sections of liver tissue were stained with H&E and with trichrome and examined microscopically without knowledge of the treatment of the rats. Histology was graded from 0 (absent) to 41 (most severe) for the severity of hepatocellular necrosis, hepatocyte swelling, cholestasis, portal tract inflammation, lobular inflammation, and Kupffer cell reactivity. Fibrosis was not observed. Isolation of hepatic mitochondria. Mitochondria were isolated from 10 g of fresh liver from each rat by a modification of our previously described method.30 This method routinely yields highly pure hepatic mitochondria with minimal contamination from both normal rats and rats with various pathological states.26,30 Briefly, the liver was gently homogenized in a loose-fitting Potter–Elvehjem homogenizer in buffer containing 225 mmol/L mannitol, 70 mmol/L sucrose, 3 mmol/L KH2PO4, 2 mmol/L ethylene glycol-bis (b-amino ethyl ether)N,N,N8,N 8-tetraacetic acid, 0.1% bovine serum albumin, and 5 mmol/L MgCl2 (pH 7.0), filtered through cheesecloth, and centrifuged at 400g for 10 minutes. The supernatant was centrifuged at 10,000g for 10 minutes. The supernatant was aspirated, and the pellet was resuspended and centrifuged at 10,000g for 10 minutes. The resulting mitochondrial pellet was resuspended in 15 mL of buffer, divided into three tubes, and added to digitonin (0.29 mg for each gram of original liver) on wet ice for 20 minutes to minimize any contamination by lysosomes.31 The resulting mitochondrial preparation was washed two times in the original buffer and centrifuged at 10,000g for 10 minutes, and samples were taken for marker enzyme analysis. Two more washes were then performed using a buffer containing 50 mmol/L 3-(N-morpholino)propanesulfonic acid and 100 mmol/L KCl, yielding the final mitochondrial pellet.

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Enrichment and recovery of mitochondrial preparations. To determine if the mitochondrial fractions isolated

from the various experimental groups were of similar purity and lack of contamination, organelle-specific marker enzyme activity was measured in each fraction isolated and the percentage recovery and enrichment of mitochondria were calculated. After storage at 2707C, liver homogenate and mitochondrial fractions were analyzed for marker enzymes specific to mitochondria (citrate synthase30) and lysosomes (N-acetyl glucoseaminidase30) and for protein.32 Enrichment was calculated by dividing the specific activity of the organelle fraction by that of the original hepatic homogenate, and the percentage recovery was calculated by dividing the total activity of the organelle fraction by that of the homogenate. Lipid peroxidation analysis of mitochondria. Fresh mitochondria were analyzed for lipid peroxidation as an index of oxidant damage by the lipid-conjugated diene method of Recknagel and Ghoshal33 with the modification of Bacon et al.34 Briefly, lipids were extracted from mitochondria pellets with chloroform and methanol (2:1, vol/vol), and the lipid in chloroform was dried under a stream of oxygen-free nitrogen30 and redissolved in 1.5 mL of spectrophotometric-grade cyclohexane. Absorbance from 220 to 275 nm was recorded against a cyclohexane blank on a Lambda 2 UV/Vis Spectrometer (Perkin-Elmer Corp., Norwalk, CT). Lipid concentration was approximately 0.5 mg/mL of cyclohexane. After the UV measurements, a 250-mL aliquot from each sample was analyzed for total lipid content by the method of Chiang et al.,35 and absorbance measurements were normalized to a denominator of 1.0 mg lipid per milliliter of cyclohexane. Lipid peroxidation was estimated by calculating the difference in absorption at 233 nm (the peak absorbance for lipidconjugated dienes) between each rat and the mean of the values from the vehicle-treated control rats.

Effect of Vitamin E on Oxidant Injury of IV TCDC To determine if treatment with an antioxidant would reduce the oxidant injury caused by IV TCDC, in separate experiments, rats underwent pretreatment with or without vitamin E and then received either vehicle or TCDC (10 mmol/100 g body wt) by tail vein injection as described above. After 2 weeks of receiving laboratory chow ad libitum, rats were randomly assigned to treatment with either (1) parenteral vitamin E (all rac-a-tocopherol) by intraperitoneal injection (at a dose of 3 mg/100 g body wt, repeated every other day for a total of five doses) plus an identical IV dose immediately before the TCDC IV injection, or (2) to treatment with an equivalent volume of normal saline (control) given by the same route and schedule as the vitamin E. These intraperitoneal injections were administered under light methoxyflurane anesthesia. After the vitamin E or saline injections, rats were randomly assigned to receive a tail vein bolus injection of either TCDC or vehicle as described above. Rats were killed 4 hours later, and serum was analyzed for AST, ALT, total bilirubin, and alkaline phosphatase levels, for serum a-tocopherol levels by high-

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performance liquid chromatography with absorbance detection,36 and for total lipids by a colorimetric assay.37 The serum a-tocopherol/total lipids ratio (milligrams per gram) was calculated as an additional index of vitamin E status.38 Liver was removed at death and processed for light microscopy. Histological injury was assessed semiquantitatively by a scoring system devised for the histological changes observed during the development of the experimental model. At least 10 areas of each biopsy were examined, without knowledge of the treatment group for individual specimens. They were scored from 1 (absent or minimal) to 4 (severely abnormal) for the severity of hepatocellular necrosis, hepatocellular swelling, portal tract inflammation, and lobular inflammation, and a total score was then calculated. Specific criteria were used to establish the score for each of these histological variables. Other histological findings (such as cholestasis, fibrosis, bile duct changes, etc.) were absent in this model. In an additional group of rats that received either TCDC plus saline or TCDC plus vitamin E, serum was obtained 24 hours after the IV TCDC injection and analyzed for AST, ALT, and bilirubin concentrations. The liver was then removed and processed for histology. In rats killed after 4 hours, liver was rapidly removed and hepatic mitochondria were isolated by differential centrifugation and analyzed for lipid-conjugated dienes, as described above. A specimen of liver was also immediately frozen, protected from light, and stored at 2707C and then analyzed for a-tocopherol and reduced ubiquinol-9 content by highperformance liquid chromatography with electrochemical detection, as described previously.39 The results were expressed as nanomoles of a-tocopherol or nanomoles of reduced ubiquinol-9 per gram of wet weight of liver.

Statistical Analysis Comparisons between experimental groups were performed by the analysis of variance (ANOVA) with the Scheffe F test or the unpaired t test, and relationships between variables were sought by linear and nonlinear regression analyses. A P value of ,0.05 was considered statistically significant. All values are expressed as the mean 6 SEM.

Results In Vivo TCDC Toxicity Model and Mitochondrial Lipid Peroxidation IV bolus injections of TCDC in rats caused a significant increase in serum AST and ALT concentrations in a dose-dependent manner (Table 1), with peak values attained 4 hours after injection. There were no significant changes in serum alkaline phosphatase or total serum bilirubin concentrations in response to TCDC (data not shown). Because serum AST and ALT concentrations peaked at 4 hours, further evaluation of liver injury was performed at that time point. Analysis of serum for concentrations of individual bile acids obtained 4 hours after the IV injections (Table 2) showed significant

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Table 1. Dose-Response of IV TCDC and Hepatic Toxicity Dose of IV TCDC (mmol/100 g body wt) Time after IV TCDC dose 0h AST ALT 4h AST ALT 8h AST ALT 12 h AST ALT 24 h AST ALT

0 (n 5 4)

5 (n 5 4)

10 (n 5 5)

20 (n 5 4)

62 6 1 54 6 5

73 6 9 52 6 6

92 6 8 53 6 3

70 6 6 64 6 9

66 6 6 51 6 8

121 6 24 92 6 23

3679 6 1105 1855 6 701

1456 6 748 1712 6 554

59 6 14 56 6 10

110 6 12 88 6 12

2233 6 1906 1963 6 701

2057 6 939 1238 6 257

78 6 10 66 6 11

105 6 12 70 6 7

605 6 249 492 6 291

1282 6 375 851 6 167

85 6 3 81 6 6

89 6 3.5 72 6 7

328 6 82 198 6 82

399 6 41 362 6 75

NOTE. AST and AL values are expressed as IU/L. All values are means 6 SEM.

increases in most bile acids in the TCDC (10-mmol/100 g dose) group compared with vehicle-injected rats. Importantly, the CDC concentrations were elevated to a similar extent as the other bile acids, indicating that the dose of TCDC had been cleared by the liver and that a cholestatic hepatic insult had occurred. In addition, the lack of substantial elevation of lithocholate conjugates indicated that conversion of CDC to lithocholic acid in the intestinal tract had not been prominent and, hence, that lithocholic acid toxicity was not playing a major role in these experiments. Histology of the liver at 4 hours after the 10-mmol/100 g weight dose of TCDC showed mild hepatocyte swelling, variable necrosis of individual hepatocytes, mild portal tract and lobular inflammation with lymphocytes without bile duct injury, and accumulation of smooth eosinophilic intracytoplasmic globules (Figure Table 2. Serum Bile Acid Concentrations 4 Hours After IV TCDC Dose Experimental group

Lithocholic acid Deoxycholic acid a-Muricholic acid b-Muricholic acid CDC Cholic acid Ursodeoxycholic acid Total bile acids

TCDC (n 5 5)

Vehicle (n 5 4)

0.6 6 0.2 4.8 6 1.2 77.4 6 15.9 a 12.4 6 3.3 a 23.2 6 2.6 a 134.8 6 30.2 a 4.4 6 0.4 277.8 6 49.0 a

0 4.0 6 1.2 7.2 6 5.2 1.0 6 0.7 7.2 6 0.6 24.2 6 4.1 2.5 6 0.3 53.8 6 5.1

NOTE. All values are expressed as means 6 SEM of micromoles per liter of total concentration (conjugates plus unconjugates) of individual serum bile acids. aP , 0.05 vs. vehicle group by t test.

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1). Based on these biochemical and histological findings, the 4-hour time point was chosen for subsequent experiments because it represented a time of significant but reversible injury to hepatocytes. Hepatic mitochondria isolated at 4 hours after rats received either 10 mmol/100 g TCDC or vehicle were analyzed for evidence of oxidant damage. Lipid-conjugated dienes of mitochondria were significantly elevated in the TCDC group compared with the vehicle-treated group (Table 3), indicating that oxidant injury to the mitochondria accompanied the toxicity of TCDC. Effect of Vitamin E on TCDC Toxicity Parenteral vitamin E treatment led to significant increases in serum vitamin E concentrations, serum vitamin E/total lipid ratios, and hepatic a-tocopherol concentrations at 4 hours in rats receiving either TCDC or vehicle (Table 3). Hepatic reduced ubiquinol-9 was similar in all four treatment groups (Table 3). Pretreatment with parenteral vitamin E resulted in a significant decrease (approximately 60%–70%) in serum AST and ALT concentrations at 4 hours in rats receiving TCDC infusion (Figure 2 and Table 3). There was no effect on these values in rats receiving IV vehicle (Table 3), nor on alkaline phosphatase (data not shown) or serum bilirubin concentrations in any group. Hepatic mitochondrial fractions isolated from rats of the four experimental groups after 4 hours showed similar excellent enrichment and recovery of the mitochondrial marker enzyme with lack of contamination by lysosomes (Table 4). Pretreatment with vitamin E resulted in a significant reduction at 4 hours in mitochondrial lipidconjugated dienes in the IV TCDC group (Figure 2 and Table 3), accompanying the reduction in AST and ALT levels. Significant correlations (Figure 3) were found between mitochondrial lipid-conjugated diene values at 4 hours and concentrations of serum AST (r 5 0.625; P , 0.001) and ALT (r 5 0.651; P , 0.001). No changes in hepatic ubiquinol-9 concentrations were observed in relationship to TCDC or vitamin E treatment (Table 3). To ascertain if the effect of vitamin E treatment on the TCDC-induced increase in serum AST and ALT levels at 4 hours was one of merely delaying rather than ameliorating injury, we measured serum AST, ALT, and bilirubin concentrations in rats killed 24 hours after IV TCDC injection (Table 3). Values for these indices of hepatocellular injury decreased significantly and to a similar extent at 24 hours in rats receiving either vitamin E or saline; thus, there was no delayed increase in these values in rats treated with vitamin E. These data confirm that vitamin E treatment attenuated, rather than delayed, the hepatocellular injury caused by TCDC.

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Figure 1. Histology of liver in rats 4 hours after IV bolus infusion of 10 mmol/100 g TCDC. (A) Portal tract inflammation and individual hepatocyte necrosis (arrow) are evident. (B) Intracellular, smooth globules (arrows) were present in predominantly periportal hepatocytes. Councilman bodies (arrowheads) are scattered throughout the hepatic lobule (H&E; original magnification 403).

Effect of Vitamin E on Hepatic Histology Liver histology was assessed semiquantitatively 4 and 24 hours after IV TCDC or saline infusions (Table 5 and Figure 4). Cellular necrosis, lobular and portal tract inflammation, and total histological scores were increased in the TCDC-treated rats. Vitamin E treatment led to a significant reduction of scores for the histological injury in TCDC-treated rats at 4 hours and had no effect on the control saline-treated rats (Table 5 and Figure 4). Liver

histology was normal at 24 hours in rats receiving TCDC with and without vitamin E (total scores of 4.20 6 0.12 and 4.17 6 0.17, respectively), consistent with the marked improvement in serum AST and ALT values at 24 hours in both of these experimental groups.

Discussion Recent investigations into the pathogenesis of cholestatic liver injury have focused on the role of

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Table 3. Effect of Vitamin E Treatment on TCDC Hepatic Toxicity Treatment group Control/vehicle (n 5 8) Serum a-tocopherol (mg/mL) Serum a-tocopherol/total lipids (mg/g) Hepatic a-tocopherol (nmol/g liver) Hepatic ubiquinol-9 (reduced) (nmol/g liver) AST (IU/L) 4h 24 h ALT (IU/L) 4h 24 h Bilirubin (mg/dL) 4h 24 h Lipid-conjugated dienes (difference of absorbance at 233 nm per mg lipid )

Control/TCDC (n 5 13)

Vitamin E/vehicle (n 5 7)

6.0 6 0.2 a 2.04 6 0.06 a 34.0 6 7.4 a

5.5 6 0.3 a 1.47 6 0.13 a 35.5 6 7.4 a

9.9 6 0.7 b 3.71 6 0.20 b 84.4 6 11.2 b

72.8 6 5.3 a

72.3 6 6.0 a

67.7 6 4.7 a

Vitamin E/TCDC (n 5 12) 13.2 6 0.7 b 4.41 6 0.5 b 167.4 6 28.8 b 71.9 6 5.2 a

85 6 5 a

2472 6 505 b 235 6 21.3 (n 5 4)

81 6 2 a

870 6 278 c 211 6 5.4 (n 5 5)

74 6 3 a

1111 6 213 b 158.0 6 23.0 (n 5 4)

61 6 5 a

325 6 78 c 154 6 6.6 (n 5 5)

0.2 6 0.02 0.00 6 0.02 a

0.4 6 0.1 0.25 6 0.02 (n 5 4) 0.24 6 0.02 b

0.2 6 0.02 20.03 6 0.02 a

0.2 6 0.02 0.35 6 0.03 (n 5 5) 0.13 6 0.01 c

NOTE. Values in each row with different superscript letters are significantly different from each other (P , 0.05) by ANOVA. All values are expressed as mean 6 SEM after 4 hours of treatment unless otherwise indicated.

hydrophobic bile acids.22–27 In this study, we developed a novel in vivo model of bile acid toxicity that uses a single-bolus IV injection of TCDC, a conjugate of the bile acid most implicated in cholestatic liver injury.5–7 An IV dose of 10 mmol/100 g body wt in young adult male rats produced a significant but reversible injury to hepatocytes, which was maximum at 4 hours and much reduced by 24 hours after injection. Hepatocyte swelling, individual cellular necrosis, and portal and lobular inflammation were the predominant histological findings in this model. These histological findings are similar to those reported in a recently described rat model of continuous jugular vein infusion of TCDC at a rate of

0.4–0.6 mmol · min21 · 100 g weight21 over 60 minutes.40 In both models, a significant degree of hepatocyte injury was shown either by liver histology, elevated biliary excretion of lactate dehydrogenase, or elevated serum AST, ALT, and bile acid concentrations. In addition, the continuous TCDC infusion model produced a marked reduction in bile flow and of hepatic bile acid secretion,40 confirming a cholestatic process. In our study, evidence of oxidant injury to the liver was present 4 hours after the TCDC infusion, similar to our previous observations in isolated rat hepatocytes exposed to TCDC for 4 hours.24,27 Moreover, pretreatment with vitamin E attenuated both the hepatic injury (assessed both biochemically and histologically) and the severity of mitochondrial lipid peroxidation at 4 hours, consistent with our hypothesis that the bile acids caused hepatic injury, at least in part, by a free radical–mediated process. At 24 hours, significant biochemical and histological Table 4. Enrichment and Percentage Recovery of Organelle Marker Enzymes in Hepatic Mitochondria Citrate synthetase a Treatment group

Figure 2. Comparison of (A) serum ALT and (B) hepatic mitochondrial lipid-conjugated dienes in four treatment groups. Significant reductions in ALT and lipid-conjugated dienes were observed in vitamin E–treated rats that received IV TCDC compared with those receiving TCDC alone. Lipid-conjugated diene values are expressed as the difference in absorbance at 233 nm/mg lipid between experimental rats and the mean of the control (Contr) rats. Numbers in parentheses refer to number of rats tested in each group. *P , 0.05 vs. TCDC group.

Enrichment

% Recovery

Control/vehicle 12.9 6 0.9 100 6 0 Control/TCDC 8.2 6 0.6 85.3 6 5.8 Vitamin E/ vehicle 12.0 6 1.3 97.1 6 2.9 Vitamin E/TCDC 7.8 6 0.9 77.0 6 5.4

N-Acetyl glucoseaminidase b

Enrichment

% Recovery

1.1 6 0.2 1.2 6 0.1

11.2 6 1.4 13.8 6 1.6

1.1 6 0.1 1.3 6 0.2

12.4 6 1.3 14.8 6 1.7

NOTE. All values are expressed as 6 SEM for 6 rats in each group. marker enzyme. bLysosomal marker enzyme. aMitochondrial

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Figure 3. Relationship between serum ALT and lipid-conjugated dienes (expressed as difference in absorbance at 233 nm/mg lipid between experimental rats and the mean of the control rats) calculated by polynomial linear regression analysis. Curve defined by y 5 0.0292 1 0.00026x 2 7.27417E2832. r 5 0.651; P , 0.0001.

recovery from this process was present in rats regardless of treatment with vitamin E, indicating that the vitamin E attenuated, rather than delayed, the bile acid–induced oxidant injury at 4 hours. Vitamin E is a lipid-soluble antioxidant that accumulates in cellular membranes and scavenges superoxide, lipid-peroxy radicals, and the hydroxyl radical.41 Therefore, the protective effect of vitamin E treatment in this model was most likely mediated through scavenging of free radicals that were stimulated by the administration of TCDC and subsequent reduction of hepatocellular membrane damage. The protective effect of vitamin E in our in vivo model of bile acid hepatotoxicity is similar to one we have observed in isolated rat hepatocytes exposed to TCDC.24,27 Because the time point chosen for study was only 4 hours after the TCDC injections, we did not observe any decrease in hepatic ubiquinol-9 concentration as has been described in a more long-term bile duct ligation rat model of cholestasis.29 Parenteral vitamin E injections were used in our model to elevate hepatic levels of a-tocopherol

severalfold. Because of limited intestinal absorption of large doses of orally administered vitamin E,42 it is not known if a similar protective effect could have been achieved if the vitamin E had been administered enterally. The cellular and intracellular sources of the free radicals generated during bile acid toxicity are of considerable interest. Oxygen free radical generation has been shown to precede cellular necrosis in purified, isolated rat hepatocytes exposed to hydrophobic bile acids,24 indicating that the hepatocytes generated the free radicals. Further studies have shown that hydrophobic bile acids stimulate hydroperoxide generation in isolated rat hepatic mitochondria.24 In the present in vivo TCDC toxicity model and a prior bile duct–ligated model of cholestasis,26 the presence of increased peroxidation of mitochondrial lipids suggests that free radicals were either generated in the mitochondria causing autooxidation of membrane lipids or, alternatively, that the mitochondria were a susceptible target of free radicals generated elsewhere within the cell. Krahenbuhl et al.29 recently showed that endogenous mitochondrial antioxidant defenses (glutathione and ubiquinone) become depleted in the chronic bile duct–ligated rat, increasing the susceptibility of this organelle to oxidant damage. In our model of acute TCDC toxicity, ubiquinol-9 and a-tocopherol content of liver did not decrease. Therefore, the observed increase in lipid peroxidation was not caused by antioxidant deficiency but most likely represented an increase in the generation of oxygen free radicals in or near the hepatocyte mitochondria. Taken together, these observations suggest that the hepatocyte mitochondrion is both an important target and a potential source of oxygen free radicals generated in our model. Other nonparenchymal cells capable of generating free radicals (e.g., Kupffer cells, endothelial cells, and neutrophils43,44) have not been excluded as potential sources of free radicals in experimental models of cholestatic injury. However, nonparenchymal cells were absent in the

Table 5. Effect of Vitamin E Treatment on Hepatic Histology Experimental group a

Cellular necrosis Cellular swelling Portal inflammation Lobular inflammation Total score

Control/vehicle (n 5 10)

Control/TCDC (n 5 8)

Vitamin E/vehicle (n 5 8)

Vitamin E/TCDC (n 5 8)

1.05 6 0.05 a 1.30 6 0.15 1.0 6 0 a 1.05 6 0.05 a 4.40 6 0.16 a

3.38 6 0.26 b 1.75 6 0.31 2.13 6 0.13 b 2.0 6 0.19 b 9.25 6 0.49 b

1.0 6 0 a 1.19 6 0.13 1.0 6 0 a 1.0 6 0 a 4.19 6 0.13 a

1.94 6 0.36 c 1.31 6 0.16 1.50 6 0.16 c 1.50 6 0.19 a 6.25 6 0.66 c

NOTE. Values in each row with different superscript letters are significantly different from each other (P , 0.05) by ANOVA. Histology was scored from 1 (normal) to 4 (markedly abnormal) on liver specimens (see text) obtained 4 hours after IV infusion of TCDC or vehicle. All values are means 6 SEM.

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Figure 4. Representative histology from liver specimens obtained 4 hours after IV infusion of TCDC or saline, with or without treatment with vitamin E. (A) Liver from rat after TCDC treatment showing significant hepatocellular necrosis (arrowheads) and portal and lobular inflammation. (B) Liver from a rat receiving both vitamin E and TCDC, showing marked reduction of abnormal findings present in A. Arrowhead indicates hepatocellular necrosis. (C) Liver from rat receiving IV normal saline, showing no histological abnormalities. (D) Liver from rat receiving IV normal saline and vitamin E, showing no abnormalities (H&E; original magnification 403).

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isolated hepatocyte studies in which exposure to toxic bile acids led to generation of free radicals.24,26 In contrast, inhibition of xanthine oxidase activity has been shown in a preliminary report to reduce cholestatic injury in the bile duct–ligated rat without affecting lipid peroxidation of hepatocytes,45 suggesting that nonparenchymal cells might also participate in free radical generation during cholestasis. Although the precise mechanism(s) by which bile acids may lead to increased mitochondrial generation of free radicals is not understood, we propose the following sequence of events. Hydrophobic bile acids that accumulate intracellularly during cholestasis interfere with normal mitochondrial electron transport and reduce ATP synthesis by inhibiting activity of respiratory complexes I and III.22 This perturbation of normal electron flow may lead to increased electron leakage from the respiratory chain as substrates continue to donate reducing equivalents to sites I and II, as has been shown when electron flow is chemically blocked downstream at complex III by antimycin A46 or at complex IV by azide or KCN.47 Inasmuch as both complexes I and ubisemiquinone are capable of generating substantial amounts of superoxide when electron flow is interrupted,46,48,49 we propose that bile acids accelerate superoxide generation at these sites, overwhelming endogenous mitochondrial antioxidant defenses. In an analogous model, Dawson et al.47 showed that ubisemiquinone is capable of generating increased quantities of superoxide when electron flow at complex IV is interrupted chemically. The superoxide thus formed is reduced to H2O2 by manganese-superoxide dismutase in the mitochondria, which can then either (1) react with iron or copper and generate the hydroxyl radical that auto-oxidizes the mitochondrial lipids or proteins, (2) react with intramitochondrial glutathione and be reduced to H2O, or (3) diffuse into cytosol, in which conversion to the hydroxyl radical may lead to damage of other key cellular components. The low mitochondrial glutathione levels observed in cholestasis29 may limit this detoxification pathway, thus increasing the generation of damaging oxygen radicals. This proposed oxidative damage to mitochondria may cause cell necrosis by reduction of ATP synthesis,25 release of calcium into cytosol and activation of intracellular nonlysosomal proteases,25 or stimulation of the mitochondrial membrane permeability transition (MMPT),23 all of which have been observed in models of bile acid cytotoxicity. The MMPT is an abrupt increase of inner mitochondrial membrane permeability to electrolytes and low-molecular-weight compounds that precedes the onset of cell necrosis in many models of hepatocellular injury.50 During the MMPT, there is a collapse of the

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electrochemical gradient across the mitochondrial membrane leading to a reduction in oxidative phosphorylation. The MMPT seems to be caused by the opening of a specific megachannel in the inner membrane that leads to increased permeability to small-molecular-weight compounds.50 Recent studies by Botla et al.23 suggest that the MMPT may be the key event linking bile acids and cytotoxicity. Others have shown that chemically induced oxidative stress stimulates the MMPT in isolated hepatic mitochondria51 and in primary cultured rat hepatocytes.52 Recently, Kowaltowski et al.53 showed that the MMPT could be stimulated by uncoupling agents that increased generation of H2O2 from the mitochondrial respiratory chain. Based on these studies, we propose that accumulation of hydrophobic bile acids in hepatic mitochondria during cholestasis stimulates the MMPT through the generation of mitochondrially derived oxygen free radicals, resulting in cell necrosis. The beneficial effects of vitamin E in our in vivo and in vitro models of bile acid toxicity indicate that scavenging of free radicals may interrupt these processes and be a potential therapeutic approach for treatment of cholestasis that deserves further investigation. Preliminary data from our laboratory confirm that hydrophobic bile acid stimulation of the MMPT in isolated rat hepatic mitochondria can be attenuated by treatment with a-tocopherol.54 Patel et al.55 recently showed that low concentrations (50 mmol/L) of hydrophobic bile acids can cause hepatocyte injury by stimulating apoptosis, or programmed cell death. The presence of Councilman’s bodies (individual ‘‘necrotic’’ cells) in liver histology of TCDC-treated rats in our study suggests the likelihood that apoptosis is occurring in hepatocytes. However, the marked elevations of AST and ALT in the TCDC-treated rats indicate that cell injury is also being mediated by necrosis, inasmuch as apoptosis does not result in release of cell enzymes extracellularly.50 The reduced number of Councilman’s bodies after vitamin E treatment in our model may indicate a reduction in apoptosis as well as in cellular necrosis. Although the proposed pathogenesis of bile acid–induced apoptosis involves activation of endonucleases by an influx of Mg21 into the cell,55 recent studies suggest that oxidant stress and lipid peroxidation may be involved as well and that antioxidants might reduce bile acid–stimulated apoptosis.56 In addition, a preliminary report showed that oxidant stress reduced cellular Bcl-2 expression,57 which could increase the release of cytochrome c from mitochondria and activate intracellular apoptotic pathways.58,59 Thus, the beneficial effect of the antioxidant, vitamin E, in our model may involve an effect on both bile acid–stimulated cell necrosis and apoptosis.

January 1998

In summary, we have developed an in vivo model of parenterally administered TCDC hepatic toxicity that is associated with mitochondrial oxidant injury and that is significantly attenuated by pretreatment with vitamin E. These findings support the notion that free radical generation may be an important factor involved in the pathogenesis of bile acid toxicity and in cholestatic liver injury. Further investigations of the specific free radical pathways involved in this process and of the effect of fortification of antioxidant defenses in cholestatic liver injury are warranted.

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Received August 15, 1996. Accepted September 18, 1997. Address requests for reprints to: Ronald J. Sokol, M.D., Department of Pediatrics, The Children’s Hospital, Box B290, 1056 East 19th Avenue, Denver, Colorado 80218. Fax: (303) 764-8025. Supported in part by grants RO1DK38446 and IP30DK34914 from the National Institutes of Health and the Abbey Bennett Liver Research Fund. Presented in part at the American Gastroenterological Association Annual Meeting, May 1994, and at the American Association for the Study of Liver Disease Annual Meeting, November 1994, and published in abstract form (Gastroenterology 1994;106:A1029 and Hepatology 1994;20:178A).