Fig. 1. (A) Cirrhotic hepatocytes were pretreated with exogenous antioxidants including deferoxamine (DEF; 1.5 mM), glutathione (GLUT; 5 mM), DPPD (5 μM), or N-acetylcysteine (NAC; 2.5 mM) for 1 h prior to administration of TGFβ (5 ng/ml) and ROS activity was measured 90 min after TGFβ administration. The ROS activity, expressed as fold difference, returned to baseline at time zero (data not shown), but increased in response to TGFβ treatment for all antioxidants tested. These findings suggest involvement of multiple ROS-generating pathways in increased cirrhotic hepatocyte basal ROS activity. (B) The percentage of condensed nuclei, indicative of morphologic apoptosis, was determined after cirrhotic hepatocytes were pretreated with exogenous antioxidants (agents and dose identical to Fig. 1A) for 1 h prior to treatment with or without TGFβ (5 ng/ml) and apoptosis was determined at 48 h. Pretreatment with the antioxidants alone did not induced apoptosis, but antioxidant treatment followed by TGFβ administration increased markedly cirrhotic hepatocyte apoptosis for each antioxidant tested.

Fig. 2. (A) Cirrhotic hepatocytes were transfected with adenoviruses (MOI 100) expressing luciferase (AdLuc; control), catalase (AdCat), or MnSOD (AdMnSOD) for 24 h and ROS activity was fluorometrically determined using DCF. Some hepatocytes were untreated with adenovirus (control). Transfection with AdCat and AdMnSOD decreased significantly ROS formation (P < 0.05 vs control). (B) Cirrhotic hepatocytes were transfected with adenoviruses (MOI 100) expressing luciferase (AdLuc), catalase (AdCat), or MnSOD (AdMnSOD) 24 h prior to TGFβ (5 ng/ml) treatment and incubated with DCF. Generation of ROS, expressed as fold difference, was fluorometrically determined at 90 min following TGFβ administration. The adenoviruses expressing the antioxidants, catalase and MnSOD, permitted a ROS burst at 90 min, indicating ROS responsiveness to TGFβ administration in cirrhotic hepatocytes. (C) The percentage of condensed nuclei, indicative of morphologic apoptosis, was determined at 48 h in cirrhotic hepatocytes in response to treatment with or without TGFβ (5 ng/ml) after pretreatment for 24 h with adenoviruses (100 MOI) expressing luciferase (AdLuc), catalase (AdCat), or MnSOD (AdMnSOD). Transfection of cirrhotic hepatocytes with adenoviruses expressing the antioxidants, catalase and MnSOD, followed by treatment with TGFβ increased significantly the percentage of apoptotic hepatocytes compared to control (P < 0.05 vs control).

Fig. 3. Cleavage of caspase-3 by immunoblot was assessed at 48 h to confirm apoptotic cell death in cirrhotic hepatocytes transfected with adenoviruses (100 MOI) expressing luciferase (AdLuc), catalase (AdCat), or MnSOD (AdMnSOD) for 24 h prior to treatment with TGFβ (5 ng/ml). (A) Cirrhotic hepatocytes were transfected with AdLuc alone (lane 1), AdLuc followed by TGFβ (lane 2), AdMnSOD alone (lane 3), or AdMnSOD followed by TGFβ (lane 4). As expected, only infection with AdMnSOD followed by TGFβ treatment resulted in the cleaved product indicating caspase-3 activity. (B) Cirrhotic hepatocytes were transfected with AdLuc alone (lane 1), AdLuc followed by TGFβ (lane 2), AdCat alone (lane 3), or AdCat followed by TGFβ (lane 4). The cleaved caspase-3 fragment was present only in AdCat-infected hepatocytes treated with TGFβ. (C) Caspase-3 activity, expressed as fold difference, was measured at 48 h to confirm apoptotic cell death in cirrhotic hepatocytes transfected with adenviruses (100 MOI) expressing luciferase, catalase, or MnSOD for 24 h prior to treatment with or without TGFβ (5 ng/ml). Infection with adenoviruses alone did not alter caspase-3 activity whereas transduction of cirrhotic hepatocytes with antioxidant expressing adenoviruses (AdCat and AdMnSOD) followed by TGFβ increased significantly caspase-3 activity. (D) The percentage of condensed nuclei was determined 48 h following TGFβ treatment in cirrhotic hepatocytes infected with adenoviruses (MOI 100) expressing luciferase (AdLuc), catalase (AdCat), or MnSOD (AdMnSOD) for 24 h prior to TGFβ treatment (5 ng/ml). These hepatocytes were also pretreated with the pan-caspase inhibitor, zVAD (5 μM), for 1 h prior to treatment with or without TGFβ. Pretreatment with zVAD inhibited apoptosis in cirrhotic hepatocytes infected with AdCat and AdMnSOD confirming a caspase-mediated form of cell death.

Fig. 4. Normal and cirrhotic hepatocytes were loaded with the fluorophores H₂-DCFDA (2 μM; green) to assess ROS formation and MitoTracker Red (MTR 500 nM; red) to localize respiring mitochondria. Confocal microscopy was performed at 90 min following TGFβ (5 ng/ml) administration. (A) Normal (left) and cirrhotic (right) hepatocytes were imaged without treatment. Note a low mitochondrial ROS formation in normal hepatocytes while diffused ROS formation in cirrhotic hepatocytes. (B) Normal hepatocytes were treated with TGFβ for 90 min. Some hepatocytes were transfected with AdMnSOD for 24 h prior to TGFβ treatment. Confocal imaging revealed a ROS burst in the mitochondria (yellow fluorescence, left panel), which was suppressed by AdMnSOD transfection (middle and right panel). (C) Confocal imaging of cirrhotic hepatocytes. TGFβ alone did not caused a mitochondrial ROS burst (left panel). Although AdMnSOD alone (middle panel) decreased ROS generation, subsequent TGFβ administration induced a mitochondrial ROS burst (yellow, right panel). (D) Quantification of yellow fluorescence, an indication of mitochondrial generation of ROS for control and cirrhotic hepatocytes treated with TGFβ, AdMnSOD, or TGFβ and AdMnSOD.

Fig. 5. (A) Whole tissue lysates were prepared from normal and cirrhotic livers and levels of Bcl-xL and MCL-1 were determined by Western blot analysis. Actin levels were also immunoblotted to ensure an equal protein loading. (B) Hepatocytes isolated from normal and cirrhotic livers were incubated in HDM in the presence or absence of 5 ng/ml TGFβ, as described under Materials and methods. After 48 h, cell lysates were prepared and total protein tyrosine phosphatase activity was determined spectrometrically.

Fig. 6. (A) ROS fluorescent units (FU) were determined over time in normal, cirrhotic, and normal-H₂O₂-converted hepatocytes. Normal hepatocytes (open circles) have a low basal level of ROS activity whereas cirrhotic hepatocytes (black diamonds) have a high steady-state level of ROS activity. Normal hepatocytes exposed to 10 μM H₂O₂ for 10 min (black triangles) failed to maintain a sustained ROS response. However, normal hepatocytes exposed to 10 μM H₂O₂ for 20 min (black squares) demonstrate sustained increased ROS activity similar to basal cirrhotic hepatocytes over the study period. These hepatocytes had 97% viability (data not shown). (B) The percentage of condensed nuclei was determined in normal and H₂O₂-converted hepatocytes at 48 h after pretreatment (or not) with trolox (2 μM) followed by treatment with or without TGFβ (5 ng/ml). Treatment with H₂O₂ alone did not increase apoptosis, and, similar to cirrhotic hepatocytes, H₂O₂-converted cells were resistant to TGFβ-induced apoptosis at 48 h. However, H₂O₂-converted hepatocytes that were pretreated with trolox prior to TGFβ exposure underwent apoptosis similar to normal hepatocyte controls and antioxidant-treated cirrhotic hepatocytes.

Fig. 7. Hepatocytes isolated from normal livers were incubated with 5 ng/ml TGFβ for 24 h. Some hepatocytes were treated with 30 μM 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) for 20 min prior to TGFβ administration. After washing once, hepatocytes were further incubated with TGFβ. Apoptosis was evaluated by chromatin condensation and nuclear fragmentation of PI-stained nuclei (arrows), as described under Materials and methods. TGFβ alone induced a substantial apoptosis (A), which was reversed by a brief treatment with DMNQ (B).