Cristina E. Carnovale

Nitric oxide release and enhancement of lipid peroxidation in regenerating rat liver.

BACKGROUND/AIMS Clarification of the role of lipid peroxidation in the onset of liver proliferation has been hampered by the fact that both higher and lower lipid peroxidation have been reported after two-thirds partial hepatectomy. Recently, it has been shown that nitric oxide might be involved in the control of early responses after partial hepatectomy. We analysed the possible involvement of nitric oxide production in lipid peroxidation levels during liver regeneration. METHODS Sham-operated, hepatectomised and sham and hepatectomised rats pretreated with two inhibitors of oxide nitric synthesis (aminoguanidine or N(G)-monomethyl-L-arginine) were used throughout. Animals were killed at 1, 3, 5 and 15 h after surgery. Cytosolic superoxide dismutase and microsomal-lysosomal catalase activities were measured. Lipid peroxidation levels were measured as thiobarbituric acid-reactive substances and conjugated dienes. Cytosolic nitrate (a stable metabolic product of nitric oxide) was enzymatically determined. Inducible-type nitric oxide synthase (iNOS) was analysed in hepatic cytosol by immunoblotting. DNA synthesis 24 and 48 h after surgery was assessed by [3H]thymidine incorporation. RESULTS Increased lipid peroxidation was found in total homogenate, cytosol and microsomes. The hepatic cytosolic content of nitrates increased, reaching the highest values at 5 h posthepatectomy. Aminoguanidine or N(G)-monomethyl-L-arginine pretreatment blocked the rise of nitric oxide production and lipid peroxidation levels and decreased the DNA synthesis. The increase in hepatic iNOS protein expression at 5 h after partial hepatectomy disappeared with aminoguanidine pretreatment. CONCLUSIONS Our experiments suggest that nitric oxide plays a role in the proliferation mechanism, although it is responsible, at least in part, for the enhanced lipid peroxidation.

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability.

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H(2)O(2) release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H(2)O(2) release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (-80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H(2)O(2) release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.

Hyperglycemia induces apoptosis in rat liver through the increase of hydroxyl radical: new insights into the insulin effect

In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated by hyperglycemia through the Bax-caspase pathway and the effects of insulin protection against the apoptosis induced by hyperglycemia. Male adult Wistar rats were randomized in three groups: control (C) (sodium citrate buffer, i.p.), streptozotocin (STZ)-induced diabetic (SID) (STZ 60 mg/kg body weight, i.p.), and insulin-treated SID (SID+I; 15 days post STZ injection, SID received insulin s.c., twice a day, 15 days). Rats were autopsied on day 30. In liver tissue, diabetes promoted a significant increase in hydroxyl radical production which correlated with lipid peroxidation (LPO) levels. Besides, hyperglycemia significantly increased mitochondrial BAX protein expression, cytosolic cytochrome c levels, and caspase-3 activity leading to an increase in apoptotic index. Interestingly, the treatment of diabetic rats with desferoxamine or tempol (antioxidants/hydroxyl radical scavengers) significantly attenuated the increase in both hydroxyl radical production and in LPO produced by hyperglycemia, preventing apoptosis by reduction of mitochondrial BAX and cytosolic cytochrome c levels. Insulin treatment showed similar results. The finding that co-administration of antioxidants/hydroxyl radical scavengers together with insulin did not provide any additional benefit compared with those obtained using either inhibitors or insulin alone shows that it is likely that insulin prevents oxidative stress by reducing the effects of hydroxyl radicals. Importantly, insulin significantly increased apoptosis inhibitor protein expression by induction of its mRNA. Taken together, our studies support that, at least in part, the hydroxyl radical acts as a reactive intermediate, which leads to liver apoptosis in a model of STZ-mediated hyperglycemia. A new anti-apoptosis signal for insulin is shown, given by an increase of apoptosis inhibitor protein.

Tumor necrosis factor alpha pathways develops liver apoptosis in type 1 diabetes mellitus.

We analyzed the contribution of TNF-α intracellular pathway in the development of apoptosis in the liver of streptozotocin-induced diabetic rats. In liver tissue, diabetes promoted a significant increase of TNF-α/TNF-R1, and led to the activation of caspase-8, of nuclear factor kappa B (NFκB), and JNK signaling pathways. The activation of NFκB led to an induction of iNOS and consequent increase in NO production. As a consequence of such changes a significant increase of caspase-3 activity and of apoptotic index were observed in the liver of diabetic animals. Importantly, the treatment in vivo of diabetic rats with etanercept (TNF-α blocking antibody) or aminoguanidine (selective iNOS inhibitor) significantly attenuated the induction of apoptosis by reduction of caspase-3 activity. Overall, we demonstrated that in the diabetes enhances TNF-α in the liver, which may be a fundamental key leading to apoptotic cell death, through activation of caspase-8, NFκB and JNK pathways.

Reversible impairment of hepatobiliary function induced by streptozotocin in the rat

The effect of streptozotocin (SZ) on hepatobiliary function was studied in rats on the 1st, 7th and 15th days of treatment. Serum glucose increased significantly on the 1st day, and then remained high. Bile flow, bile acids output and BSP biliary excretion were significantly decreased on the 1st day of treatment, whereas serum sorbitol dehydrogenase was increased. All the parameters tested apart from serum glucose tended to normalize with time. The results suggested a transient toxic effect of SZ on the hepatocyte.

Putrescine decreases cytochrome P450 3A4 levels during liver regeneration in the rat

BACKGROUND/AIMS The mechanism by which many cytochrome P450 (CYP) isozymes decrease during liver regeneration is unclear. Peptides and growth factors are thought to be involved. Putrescine, the first polyamine synthesised by ornithine decarboxylase, peaks early following partial hepatectomy and is known to play an essential role in hepatic regeneration. Gamma amino butyric acid was reported as a physiologic inhibitor of ornithine decarboxylase. In this work we studied the possible involvement of putrescine in the CYP reduction during liver regeneration. METHODS Hepatectomised, putrescine-treated sham, and GABA-treated hepatectomised rats were used throughout. Total hepatic cytochrome P450, o-dealkylase activities (CYP1A1 and CYP2B1/2), nifedipine oxidase activity (CYP3A4), and Western blot assays of their respective apoproteins were analysed in liver microsomes. Putrescine levels in hepatic tissue were also measured. RESULTS Partial hepatectomy and putrescine treatment induced a significant diminution in total CYP (50% and 30% of sham-operated rats, respectively). Gamma amino butyric acid treatment prevented this decrease in partially hepatectomised rats. Nifedipine oxidase activity of partially hepatectomised and putrescine-treated rats significantly decreased to 43% and 60% of that in sham-operated rats, respectively. Again, gamma amino butyric acid prevented the diminution in partially hepatectomised rats. No significant changes were observed in o-dealkylase activities. CONCLUSIONS These results show that inducible CYP1A1 and CYP2B1/2, which are important in carcinogen metabolisation, are preserved after partial hepatectomy. However, constitutive CYP3A4, which represents 50% of total CYP and metabolises drugs like nifedipine, warfarin, acetaminophen, cyclosporin and FK-506, is reduced during liver regeneration. Our experiments suggest that endogenous putrescine is, at least, partly responsible for this decrease.

Interferon α-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor β1

In previous work we showed that interferon alfa‐2b (IFN‐α2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor β1 (TGF‐β1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2‐phase model (diethylnitrosamine plus 2‐acetylaminofluorene) of preneoplasia development (group 1); treated with IFN‐α2b during the 2 phases (group 2); treated with IFN‐α2b during initiation with diethylnitrosamine (group 3); treated with IFN‐α2b during 2‐acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN‐α2b during the initiation period (group 6). Serum TGF‐β1 levels were increased in IFN‐α2b–treated rats. Immunohistochemical studies showed that IFN‐α2b significantly increased the quantity of TGF‐β1–positive hepatocytes in groups 2 to 4. Phosphorylated‐Smads‐2/3 (p‐Smads‐2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF‐β1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN‐α2b. IFN‐α2b stimulus induced several‐fold increases of TGF‐β1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF‐β1 levels when they were treated with IFN‐α2b. IFN‐α2b–stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase‐3 activity. They presented higher nuclear accumulation of p‐Smads‐2/3, indicating increased TGF‐β1 signaling. When anti–TGF‐β1 was added to the culture media, TGF‐β1 activation and apoptosis induced by IFN‐α2b were blocked. In conclusion, IFN‐α2b–induced production of TGF‐β1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci. (HEPATOLOGY 2004;40:394–402.)

Quercetin prevents liver carcinogenesis by inducing cell cycle arrest, decreasing cell proliferation and enhancing apoptosis

SCOPE Quercetin is the most abundant flavonoid in human diet. It has special interest as it holds anticancerous properties. This study aims to clarify the mechanisms involved in quercetin effects during the occurrence of preneoplastic lesions in rat liver. METHODS AND RESULTS Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted group). Initiated-promoted animals also received quercetin 10 and 20 mg/kg body weight (IPQ10 and IPQ20 groups, respectively). Antioxidant defenses were modified by quercetin administration at both doses. However, only IPQ20 group showed a reduction in number and volume of preneoplastic lesions. This group showed increased apoptosis and a reduction in the proliferative index. In addition, IPQ20 group displayed a reduction of cell percentages in G₁ and S phases, accumulation in G₂, and decrease in M phase, with reduced expression of cyclin D1, cyclin A, cyclin B, and cyclin-dependent kinase 1. Interestingly, peroxisome proliferator activated receptor-α levels were reduced in IPQ20 group. CONCLUSION The outcomes of this study represent a significant contribution to the current understanding on the preventive mechanisms of quercetin during the early stages of liver cancer development, demonstrating that in addition to its known proapoptotic characteristics, the flavonoid modulates the expression of critical cell cycle regulators and peroxisome proliferator activated receptor-α activity.

Hepatic Cyclooxygenase-2 Expression Protects Against Diet-Induced Steatosis, Obesity, and Insulin Resistance

Accumulation evidence links obesity-induced inflammation as an important contributor to the development of insulin resistance, which plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and nonalcoholic fatty liver disease. Cyclooxygenase (COX)-1 and -2 catalyze the first step in prostanoid biosynthesis. Because adult hepatocytes fail to induce COX-2 expression regardless of the proinflammatory stimuli used, we have evaluated whether this lack of expression under mild proinflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX-2 (hCOX-2) in hepatocytes protects against adiposity, inflammation, and, hence, insulin resistance induced by a high-fat diet, as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin-to-leptin ratio, and decreased levels of proinflammatory cytokines, together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX-2 transgenic mice exhibited increased whole-body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptor–mediated Akt phosphorylation in hCOX-2 transgenic mice. In conclusion, our results point to COX-2 as a potential therapeutic target against obesity-associated metabolic dysfunction.

Interferon-α2b and transforming growth factor-β1 treatments on HCC cell lines: Are Wnt/β-catenin pathway and Smads signaling connected in hepatocellular carcinoma?

Wnt/β-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated β-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-β1 (TGF-β1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/β-catenin and TGF-β1/Smads pathways is the association of Smads with β-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-β1 treatments on Wnt/β-catenin pathway, Smads proteins levels, β-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-β1 attenuated Wnt/β-catenin signal by decreasing β-catenin and Frizzled7 receptor proteins contents and the interaction of β-catenin with TCF4. Truncated β-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates β-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-β1 proved to be effective as modulators of Wnt/β-catenin pathway in HCC cell lines holding both wild-type and truncated β-catenin. Since the inhibition of β-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-β1 could be useful as potential treatments in patients with HCC.