Effects of proanthocyanidin enriched extract from Ligaria cuneifolia on plasma cholesterol and hemorheological parameters. In vivo and In vitro studies.

UNLABELLED: It was demonstrated that Ligaria cuneifolia (Lc) crude extract increased blood viscosity and decreased plasma cholesterol in rats. In the present study, we analyzed the Lc proanthocyanidin enriched fraction (PLc) to determine if it is capable of altering the hemorheological parameters while diminishing the plasma cholesterol. In vivo studies in adult male Wistar rats, randomized in three groups (n = 6 each one) were performed: 1. CONTROL: saline intraperitoneal (i.p.); 2. PLc 0.6 mg/100 g body weight (b.w.) i.p. and 3. PLc 3 mg/100 g b.w. i.p., every 24 hours during 3 days. IN VITRO STUDIES: with blood obtained by cardiac puncture, separated in aliquots and incubated with: 1. Saline solution (Control); 2. PLc 0.1 mg/mL, and 3. PLc 1.0 mg/mL, equivalent to doses in vivo experiments. The results demonstrated that in vivo PLc 0.6 and PLc 3 reduced plasma cholesterol (Cho) and LDL-Cho. Neither blood nor plasma viscosity was altered. Decrease of plasma cholesterol could be due to an increase of cholesterol and bile salts excretion leading to an increase of bile flow. In vitro experiments showed a direct interaction of PLc, at high concentration, with the erythrocyte membrane, inducing a switch from discocyte to stomatocyte. Only, PLc without hepatic metabolism produces hemorheological changes. Thus, PLc in vivo might be a pharmacological agent capable of decreasing plasma cholesterol.

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.

Evidence for necrosis, but not apoptosis, in human hepatoma cells with knockdown of mitochondrial aquaporin-8.

We previously found that mitochondrial aquaporin-8 (mtAQP8) channels facilitate mitochondrial H2O2 release in human hepatoma HepG2 cells and that their knockdown causes oxidant-induced mitochondrial dysfunction and loss of viability. Here, we studied whether apoptosis or necrosis is involved as the mode of cell death. We confirmed that siRNA-induced mtAQP8 knockdown significantly decreased HepG2 viability by MTT assay, LDH leakage, and trypan blue exclusion test. Analysis of mitochondrial proapoptotic Bax-to-antiapoptotic BclXL ratio, mitochondrial cytochrome c release and caspase-3 activation showed no alterations in mtAQP8-knockdown cells. This indicates a primary mechanism of cell death other than the intrinsic mitochondrial apoptotic pathway. Thus, nuclear staining with DAPI did not reveal any increase of apoptotic features, i.e. chromatin condensation or nuclear fragmentation. Flow cytometry studies after double cell staining with annexin V and propidium iodide confirmed lack of apoptosis and suggested necrosis as the primary mechanism of death in mtAQP8-knockdown HepG2 cells. Necrosis was further supported by the increased nuclear delocalization and extracellular release of the High Mobility Group Box 1 protein. The knockdown of mtAQP8 in another human hepatoma-derived cell line, i.e. HuH-7 cells, also induced necrotic but not apoptotic death. Our data suggest that mtAQP8 knockdown induces necrotic cell death in human neoplastic hepatic cells, a finding that might be relevant to therapeutic strategies against hepatoma cells.

FoxO3a modulation and promotion of apoptosis by interferon-α2b in rat preneoplastic liver.

BACKGROUND: FoxO3a, a member of the FOXO family of transcription factors, is expressed in adult liver and modulates the expression of genes involved in apoptosis. FoxO3a is post-translationally regulated, negatively by PI3K/Akt and MAPK/Erk and positively by oxidative stress/JNK pathways. In previous works, we have demonstrated that interferon-α2b (IFN-α2b) induces apoptosis of hepatic preneoplastic foci through the production of reactive oxygen species (ROS). AIMS: To investigate the post-translational signal events triggered by the oxidative stress induced by IFN-α2b and the modulation of FoxO3a transcriptional activity during these events in rat preneoplastic liver. METHODS: Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis. A group of animals received IFN-α2b and another group received IFN-α2b and ascorbic acid (ASC), by intraperitoneal injection. Lipid peroxidation, immunohistochemistry, immunoblotting, co-immunoprecipitation and sqRT-PCR assays were performed to explore the role of ROS, JNK, Akt, Erk, FoxO3a, ß-catenin and PUMA in the IFN-α2b-mediated apoptotic mechanism. RESULTS: In vivo IFN-α2b treatment induced endogenous production of ROS which activated JNK. IFN-α2b blocked the activation of Akt and Erk, avoiding FoxO3a activity repression. Activated JNK was responsible for the nuclear translocation and transcriptional activity of FoxO3a which positively modulated the expression of PUMA, a proapoptotic player. In addition, nuclear FoxO3a competed for the nuclear ß-catenin associated to TCF, inhibiting the canonical Wnt signalling pathway. CONCLUSIONS: The data presented here propose a model in which in vivo IFN-α2b treatment induces nuclear translocation and transcriptional activity of FoxO3a, triggering the mitochondrial apoptotic pathway in hepatic preneoplastic foci.

Cyclooxygenase-2 over-expression inhibits liver apoptosis induced by hyperglycemia.

Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.

Role of nitric oxide in liver regeneration.

The liver has a remarkable ability to regenerate in response to surgical removal or chemical insult. The mechanisms regulating regenerative processes are complex, and incompletely understood. A large number genes, which are not normally expressed in the quiescent liver, are activated. Immediately after partial hepatectomy (PH) (1-6 h), nitric oxide (NO) is synthesized by liver parenchymal and nonparenchymal cells from L-arginine, via induction of the inducible form of nitric oxide synthase (iNOS). NO is a highly reactive molecule, known to be involved in diverse biological processes in nearly all aspects of life. Liver regeneration is a major area within the field of NO research. Our review describes several processes that have been suggested to be modulated by the NO released following PH, including proliferation, apoptosis and angiogenesis in the remnant tissue. Because iNOS up regulation has such profound physiologic effects, its regulation is strictly controlled. The up regulation of iNOS after PH and the subsequent production of NO induce positive effects on the regulation of early stages of the regenerative process. However, overproduction (> 100%) can have detrimental effects, including apoptosis. Thus, the iNOS induction after PH is necessary, and enough to allow for the normal regenerative process.

Role of reactive oxygen species in the early stages of liver regeneration in streptozotocin-induced diabetic rats.

Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.

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.

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.

Attenuation of the Wnt/beta-catenin/TCF pathway by in vivo interferon-alpha2b (IFN-alpha2b) treatment in preneoplastic rat livers.

Wnt/beta-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, promoting cell growth and proliferation. Forkhead box containing protein class O (FOXO) transcription factors compete with TCF for beta-catenin binding, particularly under cellular oxidative stress conditions. Contrary to beta-catenin/TCF, beta-catenin/FOXO promotes the transcription of genes involved in cell cycle arrest and apoptosis. We have previously demonstrated that in vivo interferon-alpha2b (IFN-alpha2b) administration induces apoptosis in preneoplastic livers, a mechanism mediated by reactive oxygen species (ROS) and transforming growth factor-beta(1) (TGF-beta(1)). This study was aimed to assess the status of the Wnt/beta-catenin/TCF pathway in a very early stage of rat hepatocarcinogenesis and to further evaluate the effects of in vivo IFN-alpha2b treatment on it. We demonstrated that the Wnt/beta-catenin/TCF pathway is activated in preneoplastic rat livers. More important, in vivo IFN-alpha2b treatment inhibits Wnt/beta-catenin/TCF pathway and promotes programed cell death possibly providing a link with FOXO pathway.

Tumor necrosis factor alpha induced by Trypanosoma cruzi infection mediates inflammation and cell death in the liver of infected mice.

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFalpha). TNFalpha has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFalpha. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 microg of antihuman TNFalpha blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFalpha treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFalpha production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.

Interferon-alpha2b (IFN-alpha2b)-induced apoptosis is mediated by p38 MAPK in hepatocytes from rat preneoplastic liver via activation of NADPH oxidase.

It is still unclear how Interferon-alfa (IFN-alpha) acts on preventing the appearance of hepatocarcinogenesis. We have demonstrated that IFN-alpha2b induces hepatocytic transforming growth factor-beta1 (TGF-beta(1)) production and secretion by inducing reactive oxygen species (ROS) formation through the activation of NADPH oxidase. This TGF-beta(1), alters antioxidant defences and induces programmed cell death. Since it was demonstrated that IFN-alpha induces apoptosis through the activation of p38 mitogen-activated protein kinase (p38 MAPK), this study was aimed to assess the role of this kinase in the IFN-alpha2b-induced apoptosis in rat liver preneoplasia; and to further evaluate the participation of NADPH oxidase. p38 MAPK pathway was activated during the IFN-alpha2b-induced apoptosis in rat liver preneoplasia. This activation was accompanied with phosphorylation of different transcription factors, depending on the time of IFN-alpha2b stimulus. Our data suggest that NADPH oxidase is activated by IFN-alpha2b through p38 MAPK. p38 MAPK-induced activation of NADPH oxidase is accomplished by a two-step pathway: first, ROS-independent and second ROS- and TGF-beta(1)-dependent.

Cross-talk between IFN-alpha and TGF-beta1 signaling pathways in preneoplastic rat liver.

Interferon-gamma/transforming growth factor-beta (IFN-gamma/TGF-beta) pathways have opposite effects on diverse cellular functions. However, little is known about interactions between IFN-alpha/TGF-beta. In previous studies, we showed that IFN-alpha2b increases TGF-beta(1) production and secretion in hepatocytes from preneoplastic rat livers. Here, the interaction between IFN-alpha/TGF-beta(1) pathways was explored. We observed a positive cross-talk between IFN-alpha and TGF-beta(1) signaling, with activation of both pathways. p300 protein levels in hepatocytes from preneoplastic livers were enough to interact with both activated Stat1 and Smad2/3. Besides, Smad7 was not directly related with TGF-beta(1) and IFN-alpha signals. Interestingly, we reported the novel finding that the autocrine TGF-beta(1) up-regulates TGF-betaRII at protein and mRNA levels. In conclusion, the intracellular signals triggered by IFN-alpha2b and by autocrine TGF-beta(1) are integrated at the nuclear level, where activated Stat1 and Smad2/3 are capable of interact with p300, present in no restrictive cellular amounts.

Oxidative stress in primary culture hepatocytes isolated from partially hepatectomized rats.

The aim of this study was to evaluate the influence of partial hepatectomy prior to cell isolation on hepatocytes in vitro. We characterized the possible changes of various stress oxidative parameters within the first 24 h after seeding. Male Wistar rats served as donors. Hepatocytes were isolated by collagenase digestion from either liver of simulated surgery (SH) or from liver 1 h after 70% hepatectomy (PH), and the changes in stress parameters were analyzed after 1, 3, 18, and 24 h in culture. At 24 h, only hepatocytes from PH maintained significantly increased reactive oxygen species production, oxidized glutathione percentage, and Cu/Zn superoxide dismutase and catalase activities. Our results show that hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells from SH metabolically recover from this stress after 18 h. After this time, primary culture hepatocytes primed by PH maintain their in vivo-like metabolic activities (increase in both oxidative stress and antioxidant status).

Study of iron homeostasis following partial hepatectomy in rats with chronic aluminum intoxication.

Effects of both chronic aluminum (Al) exposure and partial hepatectomy on iron (Fe) homeostasis were studied. Male Wistar rats were intraperitoneally administered either 27 mg Al/kg body weight (as aluminum hydroxide) or the vehicle saline, three times a week for 3 mo. After this time, half of the rats of each group was sham operated (SH) and the other half was partially hepatectomized (PH). Animals of the four experimental groups (vehicle+SH [SH]; Al+SH; vehicle+PH [PH], and Al+PH) were killed 48 h after the surgical procedure. Serum, hepatic, and intestinal Al levels were found to be increased both for Al+SH and Al+PH. The serum Fe concentration and transferrin saturation percentage were significantly diminished in the rats of the Al+PH group, thus showing interaction between Al administration and PH. The 59Fe mucosal-to-serosal transport, studied in the intestinal loop in situ, was not affected by Al or PH. The malregulation of intestinal Fe absorption in Al exposure and/or PH when the serum Fe concentration was diminished could be the result of the increased lipid peroxidation (thiobarbituric acid-reactive substances [TBARS]) observed in this tissue. Mucosal TBARS were increased by Al exposure (+26%) and PH (+37%) and interaction between Al and PH was observed (+44%). These results show that when liver surgery is performed after prolonged Al exposure, it leads to impairment of Fe homeostasis. We underline the importance of the exposure to Al, a potentially toxic element, in the study of risk assessment in patients who must be submitted to major liver resection.

Involvement of reactive oxygen species on the apoptotic mechanism induced by IFN-alpha2b in rat preneoplastic liver.

Interferon-alpha2b (IFN-alpha2b) is an important component in the preventive treatment of patients who have severe hepatic illness such as hepatitis B or C and hepatocarcinomas. In a previous work, using a rat liver preneoplastic model, we have demonstrated that IFN-alpha2b reduces the number and volume of altered hepatic foci (AHF) inducing apoptosis through a mechanism mediated by TGF-beta(1). In this study, the implication of hepatocytes redox status of IFN-alpha2b-treated preneoplastic liver in the TGF-beta(1)-induced apoptotic death was analyzed. Results indicate that IFN-alpha2b induces hepatocytic TGF-beta(1) production and secretion by induction of reactive oxygen species (ROS) formation through the activation of a membrane bound NADPH oxidase complex. TGF-beta(1), in turn, reduces hepatocytes antioxidant defenses and induces programmed cell death. On the other hand, it was also demonstrated that treatment of rats with IFN-alpha2b plus a ROS scavenger such as ascorbic acid, abolishes the apoptotic effect of IFN-alpha2b in rat preneoplastic livers, leading to an increase of the foci volume. In conclusion, these findings strongly suggest that ROS have a fundamental role as signaling and/or regulator molecules in the IFN-alpha2b-induced apoptosis in hepatic preneoplastic cells.

Study of hemorheological parameters following partial hepatectomy in rats with chronic aluminium intoxication.

The aim of our work was to analyze the hemorheological parameters following partial hepatectomy in rats with chronic Al-intoxication (Al). Male Wistar rats were randomly assigned into four experimental groups (n=6 each one): Sham (rats subjected to simulated surgery); Al+Sham; Partial Hepatectomy (animals subjected to 65% liver resection) and Al+Partial Hepatectomy. Our results show that both Partial Hepatectomy and Al treatment produce a decrease of plasma cholesterol level, which showed a negative association with Rigidity Index increase (r(s)=-0.6475, p<0.05). The increase of Rigidity Index observed in Partial Hepatectomy, Al+Sham and Al+Partial Hepatectomy could be related to the increase of the proportion of non-discocytic erythrocytes, particularly stomatocytes, which determines a diminution of the Morphological Index. In the Altreated groups, greater changes in Rigidity Index and Morphological Index were observed. The relative viscosity of blood at a standard haematocrit of 40% was increased in Partial Hepatectomy, Al+Sham and Al+Partial Hepatectomy as compared to Sham, due to erythrocyte rigidity. On the other hand, we observed that the increase of plasma fibrinogen concentration correlates with augmentation of plasma viscosity (r(s)=0.689, p=0.004) for all the experimental groups studied. The results indicate that both administration of Al and Partial Hepatectomy induce microcytic hypocromic anaemia in the rats reflected by a significant decrease of haematocrit, mean corpuscular volume and mean corpuscular haemoglobin concentration. From these results, we conclude that in partially hepatectomized, Al-overloaded rats the decrease in erythrocyte deformability may be an important factor leading to the installation of anaemia.

Time-dependent onset of Interferon-alpha2b-induced apoptosis in isolated hepatocytes from preneoplastic rat livers.

We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.

Interferon alpha-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor beta(1).

In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) 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-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-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-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-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-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.

Modulation of balance between apoptosis and proliferation by lipid peroxidation (LPO) during rat liver regeneration.

BACKGROUND: This work aims to investigate the role of lipid peroxidation (LPO) at early stages of liver regeneration and to evaluate the balance between apoptosis and cell proliferation during this process. METHODS: Sham and partial hepatectomized (PH) male Wistar rats were randomized in seven groups: Control (untreated), E-Control (injected with vitamin E-vehicle), C-Control (injected with vitamin C-vehicle), E1 (vitamin E 100 mg/kg body weight), E2 (vitamin E 600 mg/kg body weight), C1 (vitamin C 30 mg/kg body weight), C2 (vitamin C 100 mg/kg body weight). RESULTS: Vitamin treatments attenuated the increase of LPO level observed in total homogenate and microsomes at 3 and 5 hr after PH. Both antioxidant vitamins attenuated the increase in Bax pro-apoptotic protein and augmented Bcl-xL antiapoptotic protein levels (35%) at 3 and 5 hr post-PH; Bcl-xL/Bax ratio was, therefore, increased. A direct linear relationship between LPO levels and Bax mitochondrial protein levels was seen. Vitamin-treatments diminished the apoptosis index with respect to PH-Control values, so that this parameter showed a linear relationship with LPO levels. At 24 hr after PH, the vitamin treatments increased the peak of [(3) H]-thymidine incorporation into DNA and the proliferative index (PI), measured as PCNA expression; an inverse relationship between PI and LPO levels could be demonstrated. CONCLUSION: Our data show that the diminution of LPO levels by vitamin-treatment post-PH produces both an attenuation of cellular apoptosis and a marked increase in the proliferation process, suggesting that the modulation of LPO has a role in liver regeneration process.