Parallel intestinal and liver injury during early cholestasis in the rat: Modulation by bile salts and antioxidants

Free Radical Biology & Medicine 42 (2007) 1381 – 1391 www.elsevier.com/locate/freeradbiomed

Original Contribution

Parallel intestinal and liver injury during early cholestasis in the rat: Modulation by bile salts and antioxidants Piero Portincasa a,⁎, Ignazio Grattagliano a , Mario Testini b , Maria Lucia Caruso c , David Q.-H. Wang d , Antonio Moschetta a , Giuseppe Calamita e , Michele Vacca a , Anna Maria Valentini c , Giuseppe Renna f , Germana Lissidini b , Giuseppe Palasciano a a

Clinica Medica “A. Murri,” Department of Internal Medicine and Public Medicine (DIMIMP), University of Bari Medical School, Policlinico, 70124 Bari, Italy b Department of Surgery, University of Bari Medical School, Bari, Italy c Department of Pathology, Research Institute “S. De Bellis,” Castellana Grotte, Italy d Gastroenterology Division, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA, USA e Department of General and Environmental Physiology, University of Bari, Bari, Italy f Section of Pharmacology, Department of Physiology and Pharmacology, University of Bari, Bari, Italy Received 26 July 2006; revised 15 December 2006; accepted 23 January 2007 Available online 30 January 2007

Abstract Whereas long-term cholestasis results in intestinal alterations and increased permeability to hepatotoxins, the effect of short-term cholestasis is less known and was investigated in bile duct ligated (BDL) rats. In the intestinal mucosa, at Day 7 BDL, total glutathione and protein sulfhydryl contents had decreased, oxidized glutathione levels increased (P < 0.05 vs baseline), and a reduced epithelium thickness with dissolving crypt phenomena was observed in 40% of rats. At Day 10, total protein content, glutathione-related enzyme activities, and the transmural electrophysiological activity had decreased (−50%); by contrast, oxidized proteins doubled (P < 0.05), and histological changes were extended to 70% of rats. In vitro exposure to taurodeoxycholate at micellar concentrations determined dysepithelization in normal gut but dissolving crypt phenomena and necrosis in cholestatic bowels. In the liver, ongoing cholestasis was associated with early oxidative changes especially in mitochondria, where protein sulfhydryls were decreased and negatively correlated with glutathione-protein mixed disulfides (r = −0.807, P < 0.001). Daily oral administration of tauroursodeoxycholate, a hydrophilic bile salt, and glutathione to BDL rats improved intestinal histology, function, and redox state. In conclusion, short-term cholestasis results in distinctive functional, oxidative, and morphological changes of intestinal mucosa, determined increased vulnerability to toxic injury, and parallel hepatic oxidative damage. © 2007 Elsevier Inc. All rights reserved. Keywords: Bile duct ligation; Bile salts; Extrahepatic cholestasis; Glutathione; Intestinal mucosa; Mitochondria; Protein sulfhydryls; Protein-glutathione mixed disulfides; Ussing chamber

Introduction Cholestasis is characterized by accumulation of toxic bile salts in the liver and is associated with intracellular metabolic Abbreviations: BDL, bile duct ligation; GSH, glutathione; GSH-Px, glutathione peroxidase; GSSG-Rx, glutathione reductase; GSSG, oxidized glutathione; PD, potential difference; PSH, protein sulfhydryls; PSSG, proteinglutathione mixed disulfides; Isc, short-circuit current; SSA, sulfosalycilic acid; TDC, taurodesossicholate; TUDCA, tauroursodeoxycholate; Rt, transepithelial resistance. ⁎ Corresponding author. Fax: +39 80 5478 232. E-mail address: [email protected] (P. Portincasa). 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.01.039

disorders and reduced detoxification capacity [1–3]. Imbalance of mitochondrial energy metabolism [2], hepatic retention of hydrophobic bile salts [1], and inflammation [4] have been associated with enhanced generation of reactive oxygen species and oxidative stress in hepatocytes [5]. Such events are responsible for lipid and protein oxidation [6,7]. Both retention of bile salts and oxidative stress, however, cannot explain alone the fast progression of cholestatic livers toward organ failure. Mounting evidence suggests that tissues other than the liver may be altered during prolonged cholestasis [8] and that extrahepatic factors may participate in the determination of cholestatic liver injury. In this respect, prolonged

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interruption of entero-hepatic bile salt circulation impairs the intestinal permeability and favors portal endotoxemia [9] with promotion of hepatic damage [10]. These factors worsen hepatic oxidative stress, decrease glutathione stores, and impair the detoxification defenses [11–13]. Glutathione is the main intracellular detoxifying molecule and plays an important role in bile formation [14,15] and biliary excretion of toxic compounds [16]. Intracellular glutathione redox status is strategic for several cell functions, including maintenance of protein sulfhydryls (PSH) in the reduced form and mitochondrial function [17–19]. Low levels of mitochondrial glutathione have been associated with increased susceptibility to oxidative damage [20] and activation of death pathways [21,22]. Glutathione and its related enzymes exert important functions also in the intestinal mucosa by contributing to intestinal villi viability [23], and represent the first detoxification system against toxins ingested with the food or generated in the lumen [24]. Flattening of intestinal villi has been observed in rats depleted of glutathione and in those exposed to toxins [23,25]. Mucosal glutathione level is maintained by both intracellular ex novo synthesis and recycling of the biliary-derived glutathione. The latter aliquot may be dramatically affected by interruption of bile flow and may impair the intestinal glutathione-dependent antioxidant defense. As very little is known on these major points, despite both biological and clinical relevance, the present integrated study aimed to investigate the effects of bile deprivation on intestinal function, resistance to toxic injury and morphology, and on intestinal and liver redox status in bile duct ligated (BDL) rats. Moreover, the potential protective effects of the tauro-conjugated hydrophilic bile salt tauroursodeoxycholate (TUDCA) and of the antioxidant glutathione on intestinal changes were evaluated. Materials and methods Protocol Adult male Wistar rats (weighting 250–350 g; Harlan, S. Pietro al Natisone, Italy) were maintained on a standard diet and water ad libitum and kept in individual cages under controlled conditions of temperature and humidity and a constant 12-h light/dark cycle, according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Animals had access to standard rat chow and tap water for the whole study period. In the first part of the study, the short-term effects of BDL were investigated on both intestine and liver. For this, at Day 0, rats underwent general anesthesia (5.45 mg xylazine with 36.4 mg ketamine/kg im) and the common bile duct was double ligated and cut between ligatures. Control animals underwent a sham operation with exposure but not ligation of the common bile duct and were successively pair-fed with BDL rats. After 1, 3, 7, and 10 days of ligation, animals were killed by decapitation and blood samples taken for analyses. After the abdomen was opened, the liver was removed and the intestines were ex-

teriorized. A middle tract of the small intestine (∼ 15 cm) and the whole colon were immediately opened along the mesocolic ligament and rinsed with ice-cold saline. Mucosae were obtained separately by gentle scraping with a spatula. Livers were homogenized in ice-cold MSM buffer (440 M mannitol, 70 M sucrose, 5 mM 3-morpholinopropanesulfonic acid, pH 7.4) and mitochondria were isolated [26]. Cytosol was obtained from the postmitochondrial fraction. Liver homogenate and mitochondrial fractions were assayed for succinate dehydrogenase: the recovery in the mitochondrial pellet averaged 85–92%. Serum alkaline phosphatase, total bilirubin, and alanine aminotransferase levels were measured using commercial kits at baseline and Day 10. In the second part of the study, the potential protective effect of the hydrophilic bile salt TUDCA (30 mg/kg/day in saline 1 ml) and glutathione (5 mmol/kg/day in saline 1 ml), as compared to control saline (1 ml), was tested. For this, treatments were given at 9.00 AM by gastric gavage in three additional groups of animals and started simultaneously 2 days before BDL. Animals were sacrificed after 10 days of BDL with treatments stopped the day before. The 10-day BDL was chosen because maximum damage occurred at this time point, as shown in the first part of the study. Oxidative stress measurements The following antioxidant and oxidative stress parameters were measured both in the intestinal mucosa and in the liver homogenate and mitochondrial fraction. Enzymatic determination of total (GSH) and oxidized (GSSG) glutathione concentrations was performed by precipitating tissue homogenates with 15% sulfosalycilic acid (SSA). The supernatant was processed for GSH determination by the GSSG recycling procedure [27] and incubated with 2-vinylpiridine and triethanolamine for GSSG assay [28]. PSH were measured with a modification of Elmann's procedure in which the SSA-precipitated proteins were resuspended in 700 μl of 6 M guanidine, pH 6.0 [29]. Optical density was read spectrophotometrically at 412 and 530 nm before and after 30 min of incubation with 50 μl of 10 mM 5,5-dithiobis-2nitrobenzoic acid. Protein-glutathione mixed disulfides (PSSG) were measured as previously described [30]: proteins were precipitated with 15% SSA containing 0.02 M EDTA and then dissolved in 300 μl of 0.2 M ammonium bicarbonate containing 8 M urea and mixed with 5 mg Na2BH4. Pentanol (50 μl) was added to avoid frothing. After 20 min, proteins were precipitated with 100 μl of 15% SSA. The amount of GSH in the supernatant obtained after centrifugation at 45,000 g for 15 min was enzymatically measured [27]. Results are expressed as nanomole GSH per milligram protein. Glutathione peroxidase (GSH-Px) activity was assessed using the method described by Flohè [31]: one unit gave the amount of enzyme consuming 1.15 μmol of NADPH per minute at 37°C (pH 7.0). Glutathione reductase (GSSG-Rx) activity was determined by the method of Carlberg [32]: one unit defined the amount of

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enzyme catalyzing the oxidation of 1 μmol of NADPH per minute at 30°C (pH 7.0). Protein concentrations were measured by the method of Lowry [33]. Protein concentration in guanidine-solved samples was determined by using a Bio-Rad kit for protein measurement (Bio-Rad GmbH, Munich, Germany). Intestinal experiments Protein carbonyls were measured as previously described by Levine [34] immediately after mucosa separation as well as after 30 min incubation with 2, 4, or 8 mM of the hydrophobic bile salt tauroursodeoxycholate (TDC). Briefly, equal aliquots of proteins were incubated with 2 N HCl or 0.2% dinitrophenylhydrazine in 2 N HCl. Next, proteins were precipitated by adding 50% TCA and subsequently washed with 1:1 ethanol:ethylacetate. The final precipitate was dissolved in 6 M guanidine and the spectrum of absorbance of the hydrazone derivatives was followed spectrophotometrically. The extinction coefficient for aliphatic hydrazones (21.5 nM−1 cm−1) was used to calculate carbonyl group concentrations. Intestinal transport capacity was evaluated on colonic mucosa after careful dissection and peeling of the mucosacontaining lamina propria. Tissues were mounted in a Ussing chamber apparatus with a 0.8 cm2 opening surface area (Dipl.Ing. K. Mussler Scientific Instruments, Aachen, Germany). Each mucosal side was bathed in a modified Krebs solution containing (in mM): 107 NaCl, 25 NaHCO3, 1.25 CaCl2, 0.2 NaH2PO4, 1.8 Na2HPO4, 4.5 KCl, 1 MgCl2, 12 glucose, at pH 7.2 [35]. The solution was continuously oxygenated with O2/ CO2 (95%/5%) and, within each chamber, two pairs of Ag/ AgCl electrodes were used to monitor the transmural potential difference (PD, in mV) under open-circuit conditions or the short-circuit current (Isc, μA/cm2) with transmural PD clamped to zero. Changes in Isc depend on net active ion transport across the epithelium [36]. In this system, the transepithelial resistance (Rt, in Ω*cm2) is obtained from measurements of the transepithelial voltage (Vte) and of the short-circuit current (Isc), according to Ohm's law: (Isc = Vte/Rt, i.e., Rt = Vte /Isc). After an equilibration time of 40–60 min, PD, Isc, and Rt were measured in the basal state every 6 s under voltage-clamped conditions [37].

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bile ducts, biliary hyperplasia and neoductular genesis, and cell necrotic and apoptotic features). A semiquantitative analysis was then performed. Statistical analysis Results are presented as the mean ± SD. Data obtained from experiments carried out in BDL and sham-operated rats (n = 6–8 per group at each time point) were analyzed using the one-way ANOVA repeated measures followed by multiple comparison procedures (Tukey test), and Student's t test for unpaired data, as appropriate. The level of significance was set at the P < 0.05 value. All statistical calculations were performed with the NCSS 2004 software (Kaysville, UT). Chemicals All reagents were purchased from Sigma-Aldrich Chemical Co. (Milan, Italy) or otherwise indicated, and were of the highest purity grade commercially available. Results BDL resulted in extrahepatic cholestasis, and this was confirmed by simultaneous increase of total serum bilirubin (from 0.6 ± 0.1 to 7.4 ± 0.5 mg/dl, at Day 0 and Day 10, respectively), alkaline phosphatase, and ALT. At each time point, BDL and control animals had comparable body weight. At Day 10 liver weight was significantly increased in BDL rats (Table 1). Effect of BDL on intestinal mucosa No major changes were observed in the sham group and 1- to 3-day BDL rats. BDL produced significant changes on intestinal redox status starting from Day 7, in that concentration of GSH decreased in both ileal and colonic mucosa. By contrast, mucosal levels of GSSG showed an early increase (Day 1) in the ileum with subsequent increase in both intestinal segments from Day 3 to a maximum on Day 10 (Fig. 1). The protein contents of small and large intestine decreased in BDL rats (from 167 ± 12 to 130 ± 16 mg protein/g tissue at Day 10, P < 0.05), and also mucosal PSH decreased in the whole intestine from 29.6 ± 3.6

Histology Multiple segments of small and large intestine and liver specimens were fixed in 10% neutral buffered formalin and paraffin-embedded. Colonic sections were examined also after incubation with 2, 4, or 8 mM TDC in the continuously oxygenated (O2:CO2, 95%:5%) Krebs-solution, modified as reported above. Five sections of 4 μm thickness from each sample were cut and stained with hematoxylin-eosin. The following histologic features, on five low-power fields per specimen, were examined by two unbiased pathologists (A.M.V. and M.L.C.) blinded to the experimental design: for the intestine (presence of edema, grade and type of inflammation, mucosal thickness, and necrosis extension); for the liver (distorsion of

Table 1 Characteristics of sham-operated (SO) and bile duct ligated (BDL) rats before (T0) and 10 days (BDL) after surgery without or with tauroursodeoxycholate (TUDCA) or glutathione (GSH) treatment T0

SO

BDL

TUDCA

GSH

Body 301 ± 19 308 ± 22 330 ± 21 320 ± 18 310 ± 11 weight (g) Liver 13.8 ± 0.9 14.1 ± 0.8 16.2 ± 1.2* 15.2 ± 1.1^ 15.6 ± 1.2 weight (g) ALT (UI/L) 88 ± 34 93 ± 36 281 ± 29* 181 ± 24^^ 211 ± 28^^ AP (UI/L) 420 ± 34 464 ± 40 1190 ± 280** 690 ± 80^^ 890 ± 130^ Significantly different *P < 0.01 and **P < 0.001 compared to baseline and control rats at the same time point; ^P < 0.01 and ^^P < 0.001 compared to BDL rats at the same time point.

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Fig. 1. Time-related changes of total glutathione (GSH), oxidized glutathione (GSSG, expressed as GSSG/GSH ratio), and protein sulfhydryls (P-SH) in ileal and colonic mucosa of sham-operated (○) and bile duct ligated (●) rats. Data are means ± SD (n = 6–9). Significantly different (0.01 < P < 0.05) compared to *baseline and ^sham-operated rats at each time point.

(Day 0) to 20.8 ± 3.1 nmol/mg protein (Day 10, P < 0.001). A progressive decrease of both GSH-Px and GSSG-Rx activities was observed with cholestasis (Table 2), while the amount of protein carbonyls had doubled at Day 10 (Table 3). Concerning intestinal transport, BDL was associated with profound changes of intestinal ionic transport in the colon with a significant overall decrease of all electrophysiological parameters (Fig. 2): PD decreased by 25 and 55% at Day 1 and Day 10, respectively; Isc showed a prompt decrease immediately after surgery (−50%) and remained constantly low for 10 days; Rt, an index of intestinal integrity, decreased progressively up to 57% of control values on Day 10. The same parameters of intestinal transport function did not show significant changes in sham-operated animals, apart from a transient and early (Day 1) decrease in both PD and Isc, most likely consequent to operationinduced mucosal stress. Whereas no histologic changes were observed in shamoperated control rats, both small and large intestinal mucosa of BDL rats were affected. In particular, at Day 3 an increased number of lymphocytes had infiltrated the mucosal epithelium and an initial fibrosis of the lamina propria was simultaneously observed in most of the examined tissues. At Day 7, reduced thickness of the epithelium was observed in up to 40% of rats. At Day 10, changes occurred in 70% of the animals and were associated with edema and spot necrosis of the mucosal epithelium in more than 30% of rats (Fig. 3).

Effect of BDL on liver BDL decreased cytosolic concentrations of both GSH (from Day 7) and PSH (from Day 3). GSSG, by contrast, increased significantly from Day 1 and remained high until Day 7, when it started to decrease to prior values (Fig. 4). Whereas total protein concentrations remained unchanged in both groups (all days: 179 ± 21 vs 188 ± 17 mg protein/g wet weight tissue in BDL and control rats, respectively, P = NS), both GSH-Px and GSSG-Rx activities increased progressively with ongoing cholestasis (Table 2). A progressive decrease of mitochondrial GSH concentrations accompanied cholestasis (−30% at Day 7 and −45% at Day 10). The mitochondrial content of GSSG was doubled at Day 3 and threefold increased at Day 10 (Fig. 4). The amount of mitochondrial membrane PSH decreased with cholestasis from 37.1 ± 2.3 (Day 0) to 23.8 ± 2.9 nmol/mg protein (Day 10, P < 0.001) and was negatively correlated with PSSG (all time points of BDL, n = 32, r = -0.807, P < 0.001) (Fig. 5). Total mitochondrial protein content was significantly decreased in BDL rats (Day 10: 32.1 ± 4.1 vs 44.3 ± 5.4 mg/g liver, P < 0.001). In normal livers, the activity of both glutathionerelated enzymes was three times higher in the mitochondrial than in the cytosolic compartment, while in BDL rats, both enzyme activities were significantly increased (Table 2). Concerning histology, no major change of liver tissue was observed in sham-operated rats. By contrast, the appearance of

P. Portincasa et al. / Free Radical Biology & Medicine 42 (2007) 1381–1391 Table 2 Time-related changes of glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-Rx) activities in liver cytosolic and mitochondrial fraction, ileal and colonic mucosa of sham-operated (SO) and bile duct ligated (BDL) rats before (T0) and 3 (T3) and 10 (T10) days after surgery Liver GSH-Px cytosol GSH-Px mitochondria GSSG-Rx cytosol GSSG-Rx mitochondr.

Ileum GSH-Px GSSG-Rx

Colon GSH-Px GSSG-Rx

T0

T3

T10

SO BDL SO BDL SO BDL SO BDL

351 ± 32 352 ± 31 977 ± 27 976 ± 27 0.31 ± 0.11 0.30 ± 0.10 1.12 ± 0.17 1.11 ± 0.17

336 ± 35 504 ± 54* 913 ± 44 1061 ± 95 0.27 ± 0.09 0.37 ± 0.08* 0.93 ± 0.09 1.33 ± 0.12*

335 ± 20 566 ± 32*^ 890 ± 42 1568 ± 105*^ 0.28 ± 0.05 0.60 ± 0.10*^ 0.89 ± 0.07 1.98 ± 0.12*^

SO BDL SO BDL

121 ± 9 122 ± 8 0.24 ± 0.06 0.23 ± 0.05

125 ± 33 104 ± 18 0.20 ± 0.04 0.19 ± 0.04

136 ± 19 82 ± 5*^ 0.24 ± 0.03 0.14 ± 0.03*^

SO BDL SO BDL

107 ± 22 110 ± 20 0.26 ± 0.04 0.24 ± 0.05

105 ± 16 98 ± 17 0.27 ± 0.05 0.24 ± 0.07

114 ± 9 82 ± 11*^ 0.28 ± 0.03 0.17 ± 0.03*^

Enzymes activities are expressed as mU/min/mg protein. Significantly different (0.01 < P < 0.05) compared to *baseline and ^sham-operated rats at each time point.

biliary ductular hyperplasia and neoductulogenesis, periductular inflammation, focal hepatocyte necrosis, porto-portal septa, and ground glass/feathery degeneration of centrilobular area were noted with ongoing cholestasis (Fig. 6). Effect of TDC incubation on colonic mucosa Because the hydrophobic, lipophilic detergent secondary bile salt TDC is physiologically present in the colon lumen, this bile salt was challenged against colonic mucosa. In sham-operated rats, TDC determined dysepithelization of the mucosa. In BDL rats, a more striking and time-dependent (duration of BDL) effect was noted: addition of TDC 8 mM to the incubation buffer produced submucosal edema and lowered the amount of mucin at Day 3, and dissolving crypt phenomena and enlarged areas of necrosis at Day 7. Similar changes were observed at Day 10 with a lower concentration of TDC 4 mM. Mucosal contents of protein carbonyls were significantly increased (3- to 7-fold) after incubation with TDC (Table 3). Effect of TUDCA and glutathione on the intestinal mucosa of BDL rats Twelve days of oral TUDCA and glutathione induced significant changes in the intestinal mucosa redox status of BDL rats. In particular, GSH concentrations increased from 0.9 ± 0.1 μmol/ g tissue in untreated BDL rats to 1.5 ± 0.1 μmol/g tissue in rats receiving TUDCA and 1.9 ± 0.2 μmol/g tissue in rats receiving glutathione. With both treatments, the intestinal PSH content was doubled and those of GSSG and protein carbonyls decreased to

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33 and 66%, respectively, compared to the values of untreated BDL rats. The activity of glutathione-related enzymes was also increased by 30 and 60% with TUDCA and glutathione, respectively. The electrophysiological parameters measured at the colonic level documented a trend toward control values of PD, Isc, and Rt. In fact, at Day 10 of BDL, PD was increased by 30%, Isc by 40%, and Rt by 25% in BDL rats receiving TUDCA and glutathione compared to untreated BDL rats. Administration of TUDCA and glutathione to BDL rats resulted in an improvement of histological grade of inflammation of intestinal mucosa. In particular, at Day 10, the amount of goblet cells in intestinal mucosa was significantly higher than in untreated BDL rats, the epithelium thickness was unaltered, and spot necrosis was completely absent. Incubation of colonic mucosa from TUDCA and glutathione-treated BDL rats with 4 mM TDC resulted in a significantly lower increased protein carbonyl content and in milder histological changes (less pronounced dissolving crypt phenomena and minor necrosis) compared with the mucosa of untreated BDL rats. Discussion The complex changes of both liver metabolism and functions with cholestasis are partly dependent on retention of hydrophobic bile salts and reduced hepatic detoxification capacity [1,3,38]. Although some extrahepatic factors have been proposed to promote cholestatic liver injury, it still remains pending whether alterations of tissues different from the liver effectively participate in such events. Indeed, the present integrated study shows that the impairment of the intestinal GSH-dependent antioxidant system, mucosal permeability, transport capacity, and susceptibility to toxic injury occur early in cholestatic rats. Since these events can promote intestinal bacterial translocation and absorption of hepatotoxic molecules [39], our results point to the importance of parallel alterations at both hepatic and intestinal levels during cholestatic conditions. The observation that in BDL rats the intestinal GSH content declines as early as in the liver points to the existence of a close interrelation between liver and intestine. As shown for the liver, the reduced synthesis and the increased metabolic utilization represent plausible explanations for GSH decline. However, it is known that BDL rats progressively reduce the daily intake of food; as a consequence, a deficient availability of dietary preTable 3 Protein carbonyl content in the colonic mucosa of sham-operated (SO) and bile duct ligated (BDL) rats at Days 1 (T1), 3 (T3), and 10 (T10) after surgery before and after incubation with 2, 4, and 8 mM taurodeoxycholate (TDC)

Basal TDC 2 mM TDC 4 mM TDC 8 mM

SO

BDL - T1

BDL - T3

BDL - T7

BDL - T10

0.6 ± 0.2 1.4 ± 0.3* 2.7 ± 0.4*^ 4.2 ± 0.4*^

0.6 ± 0.2 1.3 ± 0.2* 2.4 ± 0.3*^ 3.9 ± 0.5*^

0.6 ± 0.2 1.5 ± 0.3* 2.7 ± 0.5*^ 5.3 ± 0.4*^

0.9 ± 0.3 2.2 ± 0.4* 3.8 ± 0.5*^ 6.8 ± 0.4*^

1.3 ± 0.3* 3.1 ± 0.2* 5.9 ± 0.5*^ 7.1 ± 0.5*^

Data are reported as nmol/mg protein. Significantly different (0.001 < P < 0.05) compared to *baseline and ^lower TDC concentrations in the same time group.

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Fig. 2. Time-related changes of transmural potential difference (PD, expressed in mV), short-circuit current (Isc expressed in μA/cm2) and transepithelial resistance (Rt, in Ω*cm2) in the colonic mucosa of sham-operated (○) and bile duct ligated (●) rats. Data are means ± SD (n = 6–9). Significantly different (0.001 < P < 0.05) compared to *baseline and ^sham-operated rats at each time point.

cursors is likely to occur earlier in the intestine and in the liver than in other organs. Accordingly, our experiments were not extended longer to avoid the negative impact of liver cirrhosis and severe malnutrition which inevitably develop with prolonged cholestasis. A decreased availability of biliary GSH, which currently represents a considerable source of luminal GSH, may also take part in the depletion process of intestinal GSH. The higher level of GSSG in the intestinal mucosa of BDL rats can be explained by decreased GSSG-Rx activity, most likely due to reduced synthesis of proteins; this condition, in turn, can also explain the decreased content of PSH. It must be noted that bile salts are known to promote gene expression and to regulate the synthesis of proteins, especially at the intestinal level [40,41]. Their absence, conversely, promotes mucosal injury [42] and a down-regulation of MRP2 expression at the duodenal level [43]. Therefore, bile salt deficiency is associated, or directly causes, a reduction in protein synthesis and expression in the intestinal mucosa of BDL rats. In line with these considerations, the present study points to a progressive impairment of the intestinal transmucosal ionic transport and increased intestinal permeability in BDL rats. Such condition appears to parallel the failure in the antioxidant defense and anticipate morphological changes appearing after 7–10 days of cholestasis. The lower Isc observed in the colonic mucosa of BDL rats points to a decrease of net active ion transport across the epithelium dependent on bile salt deficiency [36]. A bile salt-dependent increase of ion transport has been proposed, in fact, by previous observations in other models [35,44]. Hydrophobic bile salts are known to induce also electrolyte and water secretion [45,46]. A previous study of our group showed that TDC induced a concentration-dependent increase of Isc on colonic mucosa [35], and this effect was due to an increase of Cl- secretion (at doses below the critical micellar concentration) and mucosal damage (at higher doses). Moreover, the association of progressive time dependent Isc impairment with a decreased PD in BDL rats could be explained by a decreased stimulation of the mucosa likely due to a reduced presence of bile salts. These events may be explained, at least in

part, by an altered balance of nuclear receptor activity which could result in an altered transcription of target genes coding for carrier, enzymatic, and defensive proteins. Deprivation of bile salts is indeed associated with morphological alterations of the colonic epithelium after 7–10 days of cholestasis: this is in agreement with our results showing significant differences in Rt values, an index of epithelium integrity. Intestinal permeability is increased also in patients with PBC to a higher extent than in noncholestatic conditions [47]. Some authors [48] describe morphological changes only after 3 weeks from BDL, while others [10] reported a reduction of mucosal thickness and submicroscopical alterations (disruption of desmosomes, mitochondrial swelling, and cytoplasm vacuolation) already after 1 week of biliary obstruction. Also, a marked loss of occludin expression at ileal mucosa (tightjunction associated proteins) was found in rats with obstructive jaundice [49], while exposure of Caco-2 cells to toxic bile salts increased transepithelial permeability by promoting oxidative stress [50]. These antioxidant, morphological, and functional changes of the intestinal mucosa in BDL rats provide additional data to the hypothesis of a less active barrier against potential hepatotoxins [43]. Portal endotoxiemia was reported 7 days after BDL [51], while identical doses of bacteria-derived LPS caused significantly more severe liver damage in BDL compared to shamoperated rats [52]. The increased susceptibility of the intestinal mucosa to the direct damaging effect of TDC at a micellar concentration, as evidenced both by histology and by increased accumulation of protein carbonyls, strengthens the importance of the detoxifying systems at the intestinal level and points to supplementation with antioxidants and less toxic bile salts for the protection of intestinal mucosa and its barrier function in long-standing bile-deprived conditions. It has been noted, in fact, that bile replacement restores impaired intestinal barrier function [53]. Bile administration to BDL rats maintains integrity and permeability of the intestinal mucosa and prevents bacterial translocation [54,55], reverses the increased intestinal permeability [56], and has an important part in maintaining indigenous

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Fig. 3. Light micrographs of rat colon stained with H&E. (a) Normal colon from sham-operated rat (400×); (b) normal colon from sham-operated rat exposed to 8 mM taurodeoxycholate (TDC) showing deep mucosa disepithelization (200×); (c) colon from bile duct ligated (BDL) rat (Day 10) showing edema and reduced mucosa thickness (200x); (d) colon from BDL rat (Day 3) exposed to 8 mM TDC showing edema and reduced amount of mucin (400×); (e) colon from BDL rat (Day 10) exposed to 2 mM TDC showing dissolving crypt phenomena and necrosis (400×); (f) colon from BDL rat (Day 10) receiving 30 mg/kg/day of TUDCA showing only mild reduction of goblet cells and epithelium thickness, and absence of spot necrosis (200×); (g) colon from BDL rat (Day 10) receiving 5 mmol/kg/day of glutathione showing maintenance of goblet cells and epithelium thickness, and absence of spot necrosis (200×).

microecological homeostasis. Conversely, lack of bile promotes gram-negative overgrowth [48], endotoxin production, activation of Kupffer cells, and increased susceptibility to toxic liver injury [57]. In this regard, our results show that daily administration of 30 mg/kg of TUDCA, a hydrophilic bile salt, was able to maintain protein level and to avoid, at least in part,

the fall in antioxidants, the electrophysiological alterations, and the histological damage induced by cholestasis at the intestinal level. Of interest, from our study it emerges that also high dose glutathione was able to counteract the deleterious effect of cholestasis on the intestinal mucosa. Indeed, glutathione is taken up intact by enterocytes [58] and at such

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Fig. 4. Time-related changes of total glutathione (GSH), oxidized glutathione (GSSG, expressed as GSSG/GSH ratio) and protein sulfhydryls (PSH) in liver cytosol and mitochondria of sham-operated (○) and bile duct ligated (●) rats. Data are means ± SD (n = 6–9). Significantly different (0.01 < P < 0.05) compared to *baseline and ^sham-operated rats at each time point.

doses was able to improve also BDL-induced liver histological alterations. In the liver of BDL rats, in fact, selective damage impairs the capability of GSSG regeneration especially at the mitochondrial

Fig. 5. Correlations between protein sulfhydryls (PSH) and protein-glutathione mixed disulfides (PSSG) in liver mitochondria of sham-operated (○) and bile duct ligated rats (●) at different time points (n = 32; r = −0.807, r2 = −0.652, P < 0.001).

Fig. 6. Light micrographs of rat liver stained with H&E. (a) Normal liver from sham-operated rat (200×); (b) liver from bile duct ligated rat (Day 10) showing neoductular genesis, focal necrosis, and feathery degeneration (200×).

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level. We observed, even at an early stage of BDL, a progressive impairment of mitochondrial redox status and increased formation of oxidative proteins. Retention and accumulation of hydrophobic bile salts (i.e., tauro- and glicochenodeoxycholate, major bile salts in rat bile) may activate injuring pathways which could cause the decrease of GSH levels, stimulate GSH efflux from hepatocytes [59], and induce necrosis by activating the mitochondrial membrane permeability transition [60]. Indeed, the maintenance of intracellular GSH levels is important also for the regulation of bile formation [15,61]. In the present study, cholestasis was associated with a decreased intracellular content of PSH, which includes proteins participating in energy production and membrane function, i.e., proteins regulating the water transport, and increased formation of PSSG; this fact may suggest an oxidative damage of mitochondrial proteins. These changes in thiols status also account, at least in part, for the changes occurring in enzyme activities. The increase of both the GSHrelated enzyme activities a few days after surgery reflects an increased generation of free radicals and an increased oxidation of GSH. Our results show a basal enzyme activity which is three times higher in mitochondria than in the cytosol, indicating that oxygen free radicals are physiologically released to a higher extent in mitochondria and that there is a need for a more pronounced antioxidant activity at this level. Indeed, mitochondria, which are remarkably plastic organelles constantly changing their shape to fulfill various functional activities, by exhibiting an increase of oxidized proteins in cholestatic rats, may be less prone to change rapidly their shape and, therefore, to adapt their function in response to external stimuli [62]. In conclusion, the present study shows that early cholestasis markedly affects both liver and intestine. Intestinal functional alterations and oxidative changes anticipate the appearance of morphological alterations and are associated with increased vulnerability of the mucosa to toxic injury and oxidative damage. The protective effects afforded by TUDCA and glutathione against BDL-induced intestinal damage demonstrate the importance of maintaining the intestinal integrity in such conditions. Although our experience was only confined to short-term cholestasis, it supports the hypothesis that intestinal mucosa alteration represents an important extrahepatic factor contributing to the determination of cholestatic liver injury and that oral supplementation with TUDCA and glutathione may be taken into account for future studies on the maintenance of intestinal and liver function in long-lasting cholestasis. Acknowledgments This research was supported in part by the FIRB National Grant (Fondo per gli Investimenti della Ricerca di Base) from Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Roma and Fondi Progetto Ateneo 2004/2005, University of Bari Medical School. P.P. was a recipient of the Short Term Mobility Grant 2005 (Harvard Medical School, Boston, MA) from the Centro Nazionale delle Ricerche (CNR,

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