Introduction

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Cholestasis, an impairment in bile formation, is a common feature in many human liver diseases (Popper, 1968). Bile acids are compounds synthesized from cholesterol in the liver that are normally secreted into bile (Hofmann, 1976). Failure of biliary bile acid excretion in cholestasis leads to retention and accumulation of hydrophobic bile acids within the liver (Greim et al., 1972). Elevated hepatic tissue bile acid concentrations induce hepatocellular injury by causing hepatocyte apoptosis ( Kwo et al., 1995; Jones et al., 1997, 1998; Roberts et al., 1997; Rodrigues et al., 1998; Miyoshi et al., 1999; Sodeman et al., 2000; Yerushalmi et al., 2001). The pathophysiologic importance of bile acid-induced hepatocytes apoptosis is highlighted in children with subtype 2 of the progressive familial intrahepatic cholestasis syndromes (Strautnieks et al., 1998). These children have mutations in the canalicular transport protein for bile acid secretion into bile and develop progressive liver disease because of the inability to excrete bile acids from the hepatocyte (Strautnieks et al., 1998). Thus, the cellular and molecular mechanisms of bile acid-induced hepatocyte apoptosis are of clinical and scientific importance.

Apoptosis can be initiated either by death receptor or mitochondrial pathways (Hengartner, 2000). Both pathways involve the activation of caspases, a family of cysteine proteases that cleave on the carboxyl side of aspartic acid (Thornberry and Lazebnik, 1998). Death receptors initiate apoptosis by directly activating the initiator caspase 8 (Scaffidi et al., 1998). The mitochondrial pathway of apoptosis is associated with cytochrome c release from mitochondria, which then binds to apoptosis activating factor-1 (Apaf-1), resulting in activation of another initiator caspase, caspase 9. Caspase 8 and 9 can activate effector caspases such as caspases 3, 6, and 7 executing a cell death program (Green and Reed, 1998; Thornberry and Lazebnik, 1998). In hepatocytes, these two pathways are not mutually exclusive in that death receptor initiated cascades require mitochondrial dysfunction to fully induce apoptosis (Scaffidi et al., 1999). In hepatocytes, active caspase 8 cleaves cytosolic Bid, a proapoptotic Bcl-2 family member (Li et al., 1998; Luo et al., 1998). The carboxyl fragment of cleaved Bid translocates to mitochondria where it induces cytochrome c release (Li et al., 1998; Eskes et al., 2000; Wei et al., 2000). Indeed, hepatocytes from the Bid knockout mouse are resistant to apoptosis by death receptor ligands (Yin et al., 1999).

Glycochenodeoxycholate (GCDC) induces hepatocyte apoptosis at low concentrations by a mechanism associated with activation of the Fas death receptor (Faubion et al., 1999). Increased bile acid concentrations, however, will induce hepatocyte apoptosis in the absence of Fas, indicating that inhibition of Fas expression would not be sufficient to ameliorate cholestatic liver injury (Miyoshi et al., 1999). Recent observations have demonstrated that high concentrations of bile acids induce cell death in the absence of Fas by activating another death receptor, most probably tumor necrosis factor-related apoptosis inducing ligand receptor-2 (TRAIL-R2, also called Killer and DR5) (Higuchi et al., 2001). The dependence of both Fas and Fas-independent bile acid cytotoxicity on death receptors suggested to us that Bid would be an appropriate pharmacologic target for the prevention of bile acid-mediated liver injury. Indeed, we have recently shown Bid activation in Fas-associated bile acid cytotoxicity and inhibition of cell death by signaling events preventing Bid translocation to mitochondria (Takikawa et al., 2001). However, if Bid is to become an important target for treatment of cholestatic liver injury, its importance in bile acid cytotoxicity in the absence of Fas must also be demonstrated.

The overall objective of this study was to ascertain if Bid induced mitochondrial dysfunction was a key mechanism in bile acid cytotoxicity despite the absence of Fas. Using hepatocytes from the Fas-deficient lymphoproliferative (lpr) mouse, we sought to answer the following questions: 1) Do Bid cleavage and mitochondrial dysfunction occur following bile acid treatment? 2) Does mitochondrial cytochrome c occur in bile acid cells? and 3) Does inhibition of Bid expression by antisense treatment prevent bile acid-mediated apoptosis and liver injury in the bile duct-ligated lpr mouse? We choose GCDC as the toxic bile salt for these studies because it is a primary bile salt that accumulates intrahepatically during cholestasis and is a potent inducer of hepatocyte apoptosis (Greim et al., 1972; Kwo et al., 1995). Our results suggest Bid cleavage is an important mechanism in bile acid cytotoxicity. The data suggest that inhibition of Bid expression using antisense technology appears to be a promising approach for the amelioration of cholestatic liver injury.

	Experimental Procedures

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Isolation of Mouse Hepatocytes and Culture. Mouse Hepatocytes were isolated from lpr and wild-type (C57/BL/6J) male mice (Jackson Laboratories, Bar Harbor, ME), purified by Percoll gradient centrifugation, and cultured as described previously (Faubion et al., 1999). Viability of isolated hepatocytes was always > 95% as assessed by trypan blue exclusion.

Quantitation of Apoptosis. Apoptosis was quantitated by assessing the characteristic nuclear changes of apoptosis (i.e., chromatin condensation and nuclear fragmentation) using the nuclear binding dye 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and fluorescence microscopy (Kwo et al., 1995).

Plasmids and Transfection. Plasmids for cytochrome c-green fluorescent protein (pE-GFP-Cyt-c) (Higuchi et al., 2000) and Bid-GFP (pE-GFP-Bid) (Takikawa et al., 2001) have been previously described. Mouse hepatocytes were transiently transfected using LipofectAMINE as previously described by us (Roberts et al., 1997). In brief, cells grown in 3.5-cm dishes were transfected by adding 1 ml of OptiMEM I containing 6 µg of LipofectAMINE (Invitrogen, Carlsbad, CA), 6 µl of LipofectAMINE plus reagent (Invitrogen), and each plasmid; pE-GFP-Cyt-c (2 µg), pE-GFP-Bid (0.5 µg), and pE-GFP-Bax (2 µg). The cells were incubated in the above mixture for 3 h at 37°C in a 5% CO₂/95% air incubator. After this incubation, 1 ml of Eagle's minimal essential medium containing 20% fetal bovine serum was added to the transfection medium in each culture dish. The medium was aspirated 24 h later and replaced with 2 ml of Eagle's minimal essential medium containing 10% fetal bovine serum. The transfection efficiency was approximately 40 to 60% for all plasmids as estimated by the percentage of cells expressing GFP was visualized by fluorescence microscopy.

Confocal Microscopy. Confocal microscopy of expressed GFP-fusion proteins was performed using a laser scanning confocal microscope (Axiovert 100 M-LSM 510, Carl Zeiss Inc., Thornwood, NY). GFP fluorescence was imaged using excitation and emission wavelength of 488 and 505 nm, respectively. To identify mitochondria, cells were incubated in the presence of 200 nM tetramethylrhodamine methylester (TMRM) for 10 min at 37°C prior to observation. TMRM is a fluorescent cation that electrophoretically distributes into polarized mitochondria (Scaduto and Grotyohann, 1999).

Subcellular Fractionation. Cytosolic extracts for the immunoblot assay of cytochrome c were obtained as described by Leist et al. (1998). Briefly, at the desired time points, the culture medium was exchanged with permeabilization buffer (210 mM D-mannitol, 70 mM sucrose, 10 mM HEPES, 5 mM succinate, 0.2 mM EGTA, 0.15% bovine serum albumin, 80 µg/ml digitonin, pH 7.2). The permeabilization buffer was removed and centrifuged for 10 min at 13,000g. Supernatants representing the cytosolic extract were employed for the immunoblot analysis. Mitochondria were isolated from the cells using sucrose-Percoll gradient centrifugation as described by us previously (Lieser et al., 1998).

Immunoblot Analysis. Samples were prepared by boiling for 4 min in Laemli sample buffer, resolved by 14% SDS-page, transferred to nitrocellulose membrane, and blotted with appropriate primary antibody at dilution of 1:1000. Peroxidase-conjugated secondary antibodies (Biosource International, Camarillo, CA) were incubated at a dilution of 1:2000. Bound antibody was visualized using chemiluminescent substrate (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL) and exposed to Kodak X-OMAT film. Primary antibodies were mouse anti-cytochrome c (Pharmingen, San Diego, CA), goat-anti-Bid (R&D Systems, Minneapolis, MN), goat anti-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and mouse anticytochrome oxidase (Molecular Probes, Inc., Eugene, OR).

Quantitation of Mitochondrial Membrane Potential. The mitochondrial membrane potential () was measured using a fluorescence unquenching assay with multiparameter digitized fluorescence microscopy as previously described (Ribeiro et al., 1999). Cells cultured on collagen coated glass coverslips were loaded with 200 nM mitogreen for 30 min at 37°C. Mitogreen, a fluorescent cation, selectively loads into mitochondria based on the negative membrane potential of mitochondria (180 mV), and covalently reacts with mitochondrial proteins. Mitogreen, which fluorescences at a fluorescein wavelength, serves as the fluorescent donor in this resonance energy transfer assay. After the first incubation, the media was exchanged and the cells were loaded with 50 nM TMRM for 15 min at 37°C. TMRM is a fluorescent cation that also loads into mitochondria based on the . This fluorophore, however, does not react with mitochondrial constituents and rapidly diffuses out of mitochondria upon mitochondrial depolarization. TMRM, which fluorescences at rhodamine wavelength, serves as the fluorescent acceptor in this energy transfer based assay effectively quenching the fluorescence of mitogreen in fully polarized mitochondria. However, upon loss of , TMRM diffuses out of the cell resulting in the unquenching of mitogreen fluorescence. Mitogreen fluorescence was imaged using excitation and emission filters of 470 and 520 nm, respectively. After the initial fluorescence measurements, the nonfluorescent uncoupler 1799 (50 nM) was added to completely depolarize the mitochondria and obtain maximal mitogreen fluorescence. Change in fluorescence (fluorescence in the presence of uncoupler-initiated fluorescence without uncoupler) for GCDC-treated cells was expressed as a percentage of the change in fluorescence for controls. The advantage of this assay for the measurement of is that it is insensitive to the plasma membrane potential and, therefore, only measures .

Bid Antisense Oligodeoxynucleotides. 2'-MOE capped antisense oligodeoxynucleotides (ODNs) targeting mouse Bid (AS-Bid; 5'-GAC CAT GTC CTG GCC AGA AA-3') and scramble ODNs (5'-GAG CCG TCT GAA ACC ACT AG-3') were prepared as described previously (Yu et al., 2001). The AS-Bid and scramble ODNs were dissolved in saline, and 50 mg/kg of each ODN were administrated into the mouse by intraperitoneal injection. Injection was repeated every second day. After 6 times injections, hepatocytes were isolated and subjected to the following in vitro treatment with ODNs. Isolated hepatocytes were cultured in the presence of each ODN (10 µg/5 × 10⁵ cells) for 3 days.

Bile Duct Ligation and the Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) Assay. The use and care of the animals were reviewed and approved by the Institutional Animal Care and Use Committee at the Mayo Clinic. Common bile duct ligation (BDL) was performed as previously described (Miyoshi et al., 1999). In brief, mice were anesthetized and the peritoneal cavity was opened. The common bile duct was double ligated and cut between the ligatures. After 3 days of BDL, blood samples, and liver tissue were collected as described (Miyoshi et al., 1999). The blood samples were used for the measurements of serum alanine aminotransferase (ALT) levels using a commercially available assay kit following the manufacturer's instructions (Sigma Diagnostics Kit no.505; Sigma Chemical Co., St. Louis, MO). The liver tissues were fixed in 4% paraformaldehyde and embedded in Tissue Path (Curtin Matheson Scientific Inc., Houston, TX). Tissue sections (4 µm) were prepared, and TUNEL assay was performed following the instructions of a commercial kit (In Situ Cell Death Detection Kit; Roche Molecular Biochemicals, Summerville, NJ). Hepatocyte apoptosis in liver sections were quantitated by counting the number of TUNEL-positive cells in 30 random microscopic fields (630×) as described (Miyoshi et al., 1999).

Materials and Reagents. DAPI, mitogreen, and TMRM were obtained from Molecular Probes Inc. (Eugene, OR). GCDC was obtained from Sigma Chemicals Co. (St. Louis, MO). ZVAD-fmk was obtained from Enzyme Systems (Livermore, CA).

Statistical Analysis. All data represent at least three independent experiments and are expressed as the mean ± S.D. unless otherwise indicated. Differences between groups were compared using analysis of variance for repeated measures and a post hoc Bonferroni test to correct for multiple comparisons.

	Results

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GCDC Induces Apoptosis via Fas-Dependent and -Independent Mechanisms in a Concentration-Dependent Manner. To develop our models for Fas-dependent and -independent apoptosis, we first examined the concentration- and time-dependence of GCDC-induced apoptosis in wild-type and lpr mice hepatocytes (Fig. 1). GCDC at a concentration of <100 µM rapidly induced apoptosis in wild-type mouse hepatocytes, whereas these concentrations of GCDC did not induce apoptosis in Fas-deficient, lpr mouse hepatocytes (Fig. 1A). In contrast, concentrations of GCDC  150 µM induced apoptosis in both wild-type and lpr hepatocytes (Fig. 1A). The magnitude of GCDC-induced apoptosis in wild-type hepatocytes was similar between GCDC concentrations of 50 to 100 µM, but increased further at concentrations of 150 to 300 µM (Fig. 1A). The time-dependence of apoptosis during treatment with GCDC was also examined (Fig. 1B). At a concentration of 200 µM, GCDC induced apoptosis in lpr hepatocytes beginning at 2 h but increasing over 12 h to a maximum of 38.7 ± 5.4%. For comparison purposes, the time course for GCDC-induced apoptosis in wild-type cells is shown in Fig. 1C. Thus, concentrations of bile acids <50 induce apoptosis via Fas, whereas concentrations >150 cause apoptosis in the absence of Fas.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   GCDC induces apoptosis in wild-type and lpr mouse hepatocytes. Mouse hepatocytes were incubated in the presence of GCDC (50-300 µM). Apoptosis was then assessed using DAPI (1 µM) and fluorescent microscopy. A, dose-response of GCDC-induced apoptosis of wild-type (WT, ) and lpr mouse hepatocytes () treated by GCDC for 12 h. B and C, time course alterations of apoptosis of lpr and wild-type mouse hepatocytes, respectively. In lpr mice, 200 µM GCDC () induced apoptosis while 50 µM GCDC () did not. In contrast, GCDC induced apoptosis in wild-type mice at both concentrations although 200 µM GCDC was more toxic than 50 µM GCDC. Data were expressed as mean ± S.D. from three individual experiments.

GCDC Induces Bid Transmigration to Mitochondria in lpr Mouse Hepatocytes. Because we have previously reported Bid translocation to mitochondria and mitochondrial cytochrome c release in Fas associated bile acid cytotoxicity, we limited our examination of these events in this study to bile acid cytotoxicity in lpr mouse hepatocytes. In control lpr hepatocytes transfected with the plasmid pE-GFP-Bid, Bid-GFP fluorescence did not colocalize with mitochondria that were identified by TMRM fluorescence (Fig. 2A). One hour after GCDC treatment, however, Bid-GFP became punctate and completely colocalized with mitochondria, consistent with Bid transmigration to mitochondria. Moreover, immunoblot analysis demonstrated that the 15-kDa active fragment of Bid was observed in the mitochondrial fraction of lpr mouse hepatocytes treated with GCDC (Fig. 2B). These data demonstrate that Bid is cleaved and translocates to mitochondria even in the absence of Fas.

View larger version (80K): [in this window] [in a new window]   Fig. 2.   Bid is cleaved into 15-kDa active fragment and translocates to mitochondria in lpr mouse hepatocytes treated with GCDC. A, lpr mouse hepatocytes were transfected with Bid-GFP and observed by confocal microscopy after treatment with media (control) and GCDC (200 µM, 2 h). Mitochondria were stained with TMRM (200 nM, 10 min) prior to the observation. B, mitochondrial extracts from lpr mouse hepatocytes were analyzed by immunoblot using anti-Bid antibody. Cleaved form of Bid migrates at a molecular mass of 15 kDa within 1 h after GCDC treatments.

View larger version (69K): [in this window] [in a new window]   Fig. 3.   GCDC induces mitochondrial cytochrome c release and depolarization in lpr mouse hepatocytes. A, lpr mouse hepatocytes were transfected with cytochrome c-GFP and observed by confocal microscopy after treatment with media (control) and GCDC (200 µM, 2 h). Cells were loaded with TMRM (200 nM, 10 min) prior to the observation to identify mitochondria. B, immunoblot analysis of cytosolic extracts from lpr mouse hepatocytes was performed using anti-cytochrome c mouse monoclonal antibody after treatment with media (control) and GCDC (200 µM). Cyt-c, cytochrome c. C, mitochondrial membrane potential of GCDC (200 µM)-treated lpr mouse hepatocytes in the presence () and absence of 50 µM z-VAD-fmk () and control hepatocytes () was monitored using mitogreen and TMRM fluorescence unquenching assay as described under Experimental Procedures. Data were expressed as mean ± S.D. from three individual experiments. Note the initial cytochrome c release (1 h) is prior to the mitochondrial depolarization (8 h).

Bid Is a Pharmacological Target to Prevent Bile Acid-Induced Apoptosis. Having documented that Bid is activated and induces cytochrome c release in the Fas-deficient lpr mouse hepatocytes, we next determined if inhibition of Bid expression attenuates bile acid cytotoxicity. The 23-kDa proform of Bid was readily detected by immunoblot analysis using whole cell lysates from lpr mouse hepatocytes. Treatment with Bid antisense ODNs blocked Bid expression, while scrambled ODNs did not alter Bid expression level (Fig. 4A). These lpr mouse hepatocytes lacking 23-kDa Bid were resistant to GCDC (200 µM) induced apoptosis (Fig. 4B). Inhibition of Bid expression and attenuation of GCDC-mediated apoptosis by Bid antisense ODNs was also observed in wild-type mouse hepatocytes (Fig. 4C). Thus, both Fas-associated and Fas-independent bile acid cytotoxicity requires Bid.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   AS-Bid effectively depletes 23-kDa Bid and the cells without Bid protein are resistant to both Fas-dependent and -independent apoptosis induced by GCDC. A, whole cell extract from lpr mouse hepatocytes was analyzed by immunoblot using anti-Bid antibody. The lpr (B) and wild-type (C) mouse hepatocytes treated with AS-Bid or scramble ODNs were exposed to GCDC (200 µM) for 12 h. Apoptosis was assessed by DAPI staining and fluorescent microscopy. Data were expressed as mean ± S.D. from three individual experiments. AS-Bid, antisense oligodeoxynucleotides against Bid; scramble, scramble oligodeoxynucleotides.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   AS-Bid attenuates hepatocyte apoptosis induced by common bile duct ligation in vivo. Wild-type mice were injected with AS-Bid or scramble ODNs (25 mg/kg, i.p. at day 0, 2, 4, and 6). At day 7, mice were subjected to common bile duct ligation, and then sacrificed at day 10. Hepatocyte apoptosis (A) and serum ALT levels (B) were quantitated. Data were expressed as mean ± S.D. from six mice for each group. AS-Bid, antisense oligodeoxynucleotides against Bid; scramble, scramble oligodeoxynucleotides.

	Discussion

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The principal findings of this study relate to the cellular mechanisms of Fas-independent apoptosis by toxic bile salts. The results demonstrate that GCDC-induced hepatocyte apoptosis in lpr mouse hepatocytes requires greater bile acid concentrations than in wild-type Fas-expressing hepatocytes, is associated with Bid cleavage and translocation to mitochondria and cytochrome c release, and is attenuated by inhibiting Bid protein expression using an antisense ODN approach. Taken together, these observations suggest that Bid is an appropriate pharmacological target for preventing bile acid-induced liver injury.

These current findings suggesting that bile acid-mediated hepatocyte toxicity is Bid-dependent extend our knowledge of how these toxic agents signal cell death. Bile acids at concentrations <100 µM cause apoptosis by promoting ligand-independent Fas aggregation (Faubion et al., 1999). Although bile acids at concentrations >150 µM have been shown to cause mitochondrial dysfunction with enhanced cellular generation of reactive oxygen species, inhibition of caspase 8 attenuated this oxidative stress and cytotoxicity (Yerushalmi et al., 2001). The dependence of bile acid cytotoxicity on caspase 8 even at high concentrations suggested a death receptor-mediated process was occurring. Indeed, we have recently observed induction of the TRAIL-R2 death receptor and its oligomerization by toxic bile acids in the absence of Fas (Higuchi et al., 2001). Coupled with our current data, it would appear that like Fas, TRAIL-R2 also induces hepatocyte apoptosis by a Bid-dependent pathway. In this respect, hepatocytes would appear to signal as type II cells following TRAIL-R2 activation. In type II cells, caspase 8 induces apoptosis by a mitochondrial step requiring Bid rather than direct interaction of caspase 8 with caspase 3. Because the Bid knockout animal has no identifiable phenotype and is indispensable for bile acid cytotoxicity, targeting of Bid would be rational in cholestatic liver diseases. Such an approach would be expected to not only inhibit cell injury but would likely be well tolerated.

Our studies showing translocation of Bid to mitochondria in GCDC-associated Fas-independent apoptosis are consistent with the studies of others in cell systems and our observations in the bile duct-ligated mouse that Bax is associated with mitochondria in this process. Although Bid had been shown to directly cause release of cytochrome c in cell free systems (Luo et al., 1998), Martinou and coworkers have recently demonstrated that Bid and Bax cooperate to induce mitochondrial cytochrome c release (Eskes et al., 1998, 2000). Moreover, Fas-mediated apoptosis is inhibited in the Bax/Bak knockout animal despite Bid cleavage and mitochondrial translocation (Wei et al., 2001). Bid either helps chaperone Bax to the mitochondria or, by directly allosterically modifying Bax, promotes its insertion into the outer mitochondrial membrane, a requisite step for Bax-induced cytochrome c release. Taken together with the present results, cytochrome c release during GCDC-induced apoptosis may be triggered by Bid cooperating with Bax or perhaps Bak to disrupt the integrity of the outer mitochondrial membrane (Wei et al., 2001).

The mitochondrial membrane permeability transition (a phenomenon characterized by permeability of the inner membrane to solutes, collapse of the membrane potential, and uncoupling of mitochondrial oxidative phosphorylation) has been implicated as a mechanism in bile acid cytotoxicity. We and others, for example, have shown that in cell free systems, toxic bile acids will induce the permeability transition in isolated mitochondria (Botla et al., 1995; Rodrigues et al., 1999). However, in the current study, the mitochondria maintained the membrane potential despite cytochrome c release in hepatocytes and only depolarized late in the apoptotic process as a secondary event. These data suggest that previous studies using high concentrations of toxic bile salts with isolated mitochondria cannot be extrapolated to in vivo cytotoxicity. The observation that mitochondria can maintain the membrane potential (a property of the inner mitochondrial membrane) despite cytochrome c release (due to loss of integrity of the outer mitochondrial membrane) has been reported by others (Eskes et al., 1998; Jurgensmeier et al., 1998). The release of cytochrome c in bile acid cytotoxicity may explain the observations by us and others that bile acid cytotoxicity is associated with generation of reactive oxygen species (Sokol et al., 1995; Patel and Gores, 1997). Loss of cytochrome c from the mitochondria results in impairment of electron flow in the respiratory chain enhancing reduction of oxygen by electron carriers (Fernandez-Checa et al., 1997). The resultant formation of superoxide anion and generation of other reactive oxygen species would then ensue, causing oxidative stress and further contributing to the toxic effects of the bile acids.

2'-MOE modified antisense ODN were employed in these studies. These ODN are relatively resistant to degradation by intracellular endonucleases, rendering them more potent than phosphorothioate ODN. Moreover, 2'-MOE containing ODN also accumulate in the liver, making them useful for affecting hepatic protein expression. For example, Fas antisense ODN strongly inhibits liver Fas expression and blocks liver injury by acetaminophen and Fas agonists (Zhang et al., 2000). This latter proof of principle study demonstrates that 2'-MOE modified ODN are useful compounds for inhibiting acute liver injury and, therefore, potentially therapeutically useful for ameliorating chronic human liver diseases (Yu et al., 2001; Zhang et al., 2000). The current observations extend these studies by showing Bid can also be effectively targeted using this technology. The therapeutic use of Bid antisense merits further study in chronic models of cholestasis associated with hepatocellular apoptosis (e.g., Mdr 2 / mice). If this approach proved beneficial in animal models of chronic cholestasis, human trials could be contemplated and justified. However, it remains to be determined whether inhibition of apoptosis by this approach may be tumorigenic in type II cells using death receptor-mediated processes to control cell numbers. We are optimistic that this is not the case given the normal phenotype of the Bid knockout mouse.