Cristina E Carnovale - Academia.edu (original) (raw)
Papers by Cristina E Carnovale
PubMed, 1984
Hepatic blood flow was measured in 10 cirrhotic patients by a constant infusion of Indocyanine Gr... more Hepatic blood flow was measured in 10 cirrhotic patients by a constant infusion of Indocyanine Green (ICG) and details of the technique are analysed. A decrease in total hepatic blood flow (0.777 +/- 0.38 l/min.) was found in most of the patients. Different variations in hepatic blood flow were observed in three patients after the administration of Cimetidine (300 mg IV). The response in hepatic blood flow in another patient in whom a peritoneo-jugular valve (Le Veen shunt) was inserted in analysed.
Cellular and Molecular Life Sciences, Mar 1, 1984
Toxicology and Applied Pharmacology, Oct 1, 2012
Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membran... more 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.
Journal of Endocrinology, Feb 17, 2010
In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated b... more 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 insulintreated SID (SIDCI; 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.
Toxicology Letters, 1990
The effect of aflatoxin B, (AFB,) on the glutathione S-transferase activity (GST) and on non-prot... more The effect of aflatoxin B, (AFB,) on the glutathione S-transferase activity (GST) and on non-protein thiol levels of different tissues was studied in adult male Wistar rats. Animals received a single dose of the toxin (100 or 500 ,ug/kg body wt., p.o.), and were studied 6 or 24 h after administration. GST was determined in liver, renal cortex, duodenum, jejunum-ileum and distal ileum, using 3 substrates: l-chloro-2.4-dinitrobenzene (CDNB), trans-4-phenyl-3-buten-2-one (PBO) and 1,2-epoxyethylbenzene (STOX). The non-protein thiol content of all tissues tested increased with the lowest dose at 6 h, returning to normal values at 24 h, while the higher dose produced a significant decrease in reduced thiol levels at 6 h, returning to normal values at 24 h. AFB, administration induced, independently of dose and tissue, total CST (CDNB) and epoxide-transferase activity (STOX) while A-C-type transferases (PBO) were inhibited. Almost all activities returned to normal values at 24 h. In cases of enzyme induction there was in general an increase in V,,, and a decrease in apparent K,. The opposite was seen in cases of inhibition. In conclusion, the results provide evidence that extrahepatic GST could be important in the overall process of detoxification of AFB,. The behavior seen in hepatic and extrahepatic tissues revealed the functions of catalysis (B-type transferases) and covalent bond formation, as well as inactivation by probable AFB, metabolites (A-C-type transferases).
Annals of Hepatology, Sep 1, 2012
Molecular Nutrition & Food Research, Oct 1, 2013
Scope: Quercetin is the most abundant flavonoid in human diet. It has special interest as it hold... more 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 G 1 and S phases, accumulation in G 2 , 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-␣ 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-␣ activity.
Growth Factors Journal, 2009
Interferon-gamma/transforming growth factor-beta (IFN-gamma/TGF-beta) pathways have opposite effe... more 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.
Journal of Hepatology, Apr 1, 2012
Background and Aims: Treatment of hepatocellular carcinoma (HCC) in diethylnitrosamine (DEN)-indu... more Background and Aims: Treatment of hepatocellular carcinoma (HCC) in diethylnitrosamine (DEN)-induced mice with antibodies directed against the Placental Growth Factor (PlGF) causes a substantial improvement in mortality, tumor burden and vascular normalization. Increased expression of multi-antennary N-glycans is correlated with oncogenic development and we observed a significant decrease of this glycomic fraction following anti-PlGF treatment. To elucidate the molecular mechanism behind this down-regulation, we focused on E26 transformation specific sequence 1 (Ets-1), a transcription factor essential for the glycomic and angiogenic changes in malignant transformation. Ets-1 is a downstream target of two signaling pathways: the MAP kinase (MAPK) pathway and a Ca 2+-dependent pathway. Activation of the latter pathway inhibits Ets-1 DNA-binding. Methods: 129S2/SvPasCrl mice were injected with DEN for 25w followed by 5w injections with IgG or anti-PlGF. Ets-1 mRNA expression levels were assessed by qPCR analysis. Phosphorylated and non-phosphorylated forms of Ets-1, MAPK pathway (JNK, p38 and ERK1/2) and Ca 2+-dependent pathway (PKCa and CAMKII) were investigated by Western Blot analysis. Results: Number of Ets-1 transcripts doubled surrounding the tumor and tripled in tumor liver tissue. However, a non-significant decrease in Ets-1 transcription was observed surrounding or in the tumor of anti-PlGF treated mice. On the protein level, anti-PlGF treatment resulted in a substantial increase of the phosphorylated forms of all three MAPK subfamilies in tumor tissue. Similar results were obtained surrounding the tumor with exception of JNK activation. Importantly, the most distinct activation was observed for p38. In the Ca 2+-dependent pathway, activated forms of PKCa and CAMKII displayed a substantial increase in signal intensity in tumor tissue after anti-PlGF treatment. Ets-1 was significantly upregulated surrounding the tumor in anti-PlGF treated mice. Signal intensity of phosphorylated form pSer251 increased with 60% in tumor tissue (downstream Ca 2+-dependent pathway) and tripled for pThr38 surrounding the tumor (downstream MAPK pathway). Conclusions: Ca 2+-dependent Ets-1 phosphorylation in tumor tissue of anti-PlGF treated mice might be the molecular basis for the down-regulation of multi-antennary N-glycans. Our data indicate no role for MAPK Ets-1 phosphorylation in this process. However, p38 activation could play an important part in the molecular mechanism of anti-PlGF treatment independent of Ets-1.
Pharmacology & Toxicology, Nov 1, 1995
In a previous study we demonstrated that the administration of 20 pg/kg b.wt. of glucagon to rats... more In a previous study we demonstrated that the administration of 20 pg/kg b.wt. of glucagon to rats caused a significant diminution of hepatic cytosolic glutathione S-transferase (GST) activity. This inhibition was non-competitive and reversible. We suggested that the effect would be mediated by cytosolic effectors. The present work was performed to characterize the mechanism involved in this inhibition. Liver tissue slices (1 70 to 200 mg) were incubated during different periods of time (0, 5, 10, 15, 20 and 30 min.) with several concentrations of glucagon (IO-'M, IOWM and 10-I0M), dibutiryl cyclic AMP (10-4M, 10-6M and 10-9M), divalent cation ionophore A23187 (10-4M, 10-6M and 10-9M) or vasopressin (lO-'M, 5X IO-'M and 10-*M). The incubation was done with or without calcium in the medium. In all cases the cytosolic GST activity were determined in liver slices. The percentage of inhibition of GST activity was directly related to the increase of concentration of the test substances. An inhibition between 40% to 45% after 10 min. of incubation with the highest concentrations was observed (except vasopressin which caused 10% of inhibition). 10-'OM glucagon did not produce a decrease of GST activity. The inhibition disappeared in calcium-free incubated slices, but direct relationship between plasma-membrane calcium influx and inhibition of GST activity (r=0.950, P<O.OOI, n=24) could be obtained. By using calmodulin antagonists, we conclude that the inhibition process of the enzyme was mediated by calmodulin. in summary, we propose that plasma-membrane calcium influx induced by high concentrations of glucagon activates calmodulin, which promotes a modification (actually a methylation, according to other authors) on GST, thereby causing a decrease in its activity.
Toxicology Letters, Feb 1, 1988
The effect of streptozotocin (SZ) administration on sodium [14C]taurocholate (TC) transmural tran... more The effect of streptozotocin (SZ) administration on sodium [14C]taurocholate (TC) transmural transfer was studied in the everted rat ileum. The excretion of fecal bile acids was also studied in living rats injected with that compound. The viability of the preparation used for the in vitro experiments was evaluated by light microscopy and by the rate of glucose uptake by tissue from the mucosal fluid. The results obtained showed that TC transfer to the serosal fluid was impaired after 24 h of SZ injection, as well as the active transport observed in control preparations. The amount of TC accumulated in the intestinal tissue was also diminished. In addition, total ATPase activity of tissue was decreased, and intracellular electrolyte concentration was altered. Therefore, a slower saturation of binding sites could be responsible for the effects of SZ on TC tissue accumulation, and a decreased ATPase activity for the impairment of the TC concentrative transport system. The results observed in vitro were supported by data in vivo because fecal bile acid excretion was significantly diminished in SZ-treated rats.
Experimental and Toxicologic Pathology, Oct 1, 1992
The mechanisms involved in bile salt-induced choleresis are poorly known. To give an insight in t... more The mechanisms involved in bile salt-induced choleresis are poorly known. To give an insight in this physiological process, bile salt-associated electrolyte secretion was studied following relief of a short-term (2 h) biliary .obstruction in the rat, an experimental model that shows an important diminution of bile salt choleretic efficiency. For this purpose, biliary excretion of total bile salts and electrolytes (sodium, chloride and bicarbonate) were studied in such a model during taurocholate infusion at increasing rates. The results showed that bile flow, bile salt output and electrolyte secretion stimulated by taurocholate,administration were decreased in the rats that were subjected to biliary obstruction. Besides, the choleretic efficiency of the excreted bile salts, as estimated by the slope of the regression line of bile flow vs. bile salt output, was diminished by 46 % (p < 0.005). Multiple regression analysis of bile flow vs. bile salt and electrolyte outputs allowed to detect a selective diminution of the fraction of bile flow related to bile salt-associated electrolyte secretion ("secretory fraction" of the choleretic efficiency of bile salts) (3.2 ± 0.3 vs. 2.5 ± 0.2L1mol, p < 0.05) whereas the "osmotic fraction" of the choleretic efficiency of bile salts was not modified by the treatment (5. a ± 0.4 vs. 5.1 ± 0.3 Llmol, p> 0.05). Since both chloride and bicarbonate biliary concentrations in the volume of bile stimulated by taurocholate were reduced by 53 % and 52 % respectively, a role of these anions in the generation of bile salt-induced choleresis was suggested. Possible mechanisms involved in such a process and in its early impairment during cholestasis are discussed.
Toxicology and Applied Pharmacology, 2017
Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen sp... more Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity. Previous studies demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, has immunomodulatory effects, increasing survival in C57BL/6 mice in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The mechanism by which BZL inhibits inflammatory response in sepsis is poorly understood. Also, our group recently reported that BZL is able to activate the nuclear factor erytroide-derived 2-Like 2 (NRF2) in vitro. The aim of the present work was to delineate the beneficial role of BZL during sepsis, analyzing its effects on the cellular redox status and the possible link to the innate immunity receptor TLR4. Specifically, we analyzed the effect of BZL on Nrf2 regulation and TLR4 expression in liver of mice 24 hours post-CLP. BZL was able to induce NRF2 nuclear protein localization in CLP mice. Also, we found that protein kinase C (PKC) is involved in the NRF2 nuclear accumulation and induction of its target genes. In addition, BZL prompted a reduction in hepatic CLP-induced TLR4 protein membrane localization, evidencing its immunomodulatory effects. Together, our results demonstrate that BZL induces hepatic NRF2 activation with the concomitant increase in the antioxidant defenses, and the attenuation of inflammatory response, in part, by inhibiting TLR4 expression in a murine model of sepsis.
Journal of Cellular Biochemistry, Jan 22, 2013
Increased expression of COX-2 has been linked to inflammation and carcinogenesis. The constitutiv... more Increased expression of COX-2 has been linked to inflammation and carcinogenesis. The constitutive expression of hCOX-2 protects hepatocytes from several pro-apoptotic stimuli. It is established that an increase in hepatic apoptosis exists in experimental models of diabetes. The aim of this work was to analyze the role of COX-2 as a regulator of apoptosis in liver of diabetic mice. Mice of C57BL/6 strain Wild Type (Wt) and transgenic in hCOX-2 (COX-2 Tg) were separated into Control (vehicle) and SID (Streptozotocin Induced Diabetes, 200mg/kg). 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 proteins as Bad and Bax, release of cytochrome c and activities of caspases-3 and-9, leading to an increase in apoptotic index. This situation was improved in COX-2 Tg animals. In addition, SID COX-2 Tg showed increased expression of Mcl-1 and XIAP anti-apoptotic proteins. In summary, there is a pro-apoptotic state in the liver of diabetic animals, situation improved by over-expression of COX-2. Also, we analized the contribution of increased glucose on apoptosis and the role of hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured 72 hours at glucose 5 mM and 25 mM. At 25 mM there was an increase in apoptosis in non-transfected cells vs those exposed to 5 mM. This increase was prevented in part in transfected cells at 25 mM. The above suggest a protective role of hCOX-2 to apoptosis induced by hyperglycemia in vitro and in vivo.
Background/Aims: Clarification of the role of lipid peroxidation in the onset of liver proliferat... more 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 munoblotting. DNA synthesis 24 and 48 h after surgery was assessed by (3mthymidine incorporation. Results: Increased lipid peroxidation was found in total homogenate, cytosol and microsomes. The he- patic cytosolic content of nitrates increased, reaching the highest values at 5 h posthepatectomy. Amino- guanidine or NC-monomethyl-L-arginine pretreat- ment blocked the rise of nitric oxide production and lipid peroxidation levels and decreased the DNA syn- thesis. The increase in hepatic iNOS protein express- ion at 5 h after partial hepatectomy disappeared with aminoguanidiue preatreatment. Conchsions: Our experiments suggest that nitric ox- ide plays a role in the proliferation mechanism, al- though it is responsible, at least in part, for the en- hanced lipid peroxidation.
Apoptosis, Jan 12, 2014
Your article is protected by copyright and all rights are held exclusively by Springer Science +B... more Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com".
Canadian Journal of Physiology and Pharmacology, Oct 1, 2007
The aim of this study was to evaluate the influence of partial hepatectomy prior to cell isolatio... more 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).
Journal of Hepatology, 2017
However, the relationship between β-catenin and CXCL10 in FPC cholangiocytes as well as the relev... more However, the relationship between β-catenin and CXCL10 in FPC cholangiocytes as well as the relevance of CXCL10 in the progression of the disease remains unclear. Methods: WT and FPC cholangiocytes were treated with inhibitors or siRNA of intracellular signaling. Secretion and gene expression of CXCL10 were evaluated by ELISA and RT-PCR, respectively. Pkhd1 del4/ del4 mice were treated with the CXCR3 antagonist AMG-487 (5 mg/ Kg/day) for three months. Results: FPC-cholangiocytes, both in vitro and in vivo (LCM microscope), showed increased pro-IL-1β expression and IL-1β secretion, and an activated inflammasome (increased caspase 1 and Nrlp3). Administration of IL1-ß to FPC-cholangiocytes strongly upregulated the production of CXCL10, through a JAK/STAT-dependent pathway (JAK inhibitor I; 10 μM). Inhibition of β-Catenin (by ICG-001; 10 μM and by siRNA) and of cAMP signaling (by PKA inhibitor 14-22 amide; 1 μM), significantly reduced CXCL10 gene expression and secretion following IL1-ß. Furthermore, as assesses by Western blot, immunofluorescence and FACS analysis, β-catenin inhibition prevented the nuclear translocation of pSTAT3(Tyr 705) indicating that β-catenin signaling is necessary for STAT3-dependent CXCL10 gene transcription. Finally, in Pkhd1 del4/del4 mice, inhibition of CXCR3, the cognate receptor of CXCL10, significantly reduced macrophage infiltration (CD45 + cells), cyst growth (K19 + area), spleen size and liver fibrosis (Sirius red). Conclusions: In FPC-defective cholangiocytes, an increased production and secretion of IL1ß is responsible for β-catenin and STAT3dependent secretion of CXCL10. In vivo experiments show that this mechanism is of pathophysiological relevance, as targeting the CXCL10/CXCR3 axis prevents the recruitment of macrophages and halts the progression of the disease.
PubMed, 1984
Hepatic blood flow was measured in 10 cirrhotic patients by a constant infusion of Indocyanine Gr... more Hepatic blood flow was measured in 10 cirrhotic patients by a constant infusion of Indocyanine Green (ICG) and details of the technique are analysed. A decrease in total hepatic blood flow (0.777 +/- 0.38 l/min.) was found in most of the patients. Different variations in hepatic blood flow were observed in three patients after the administration of Cimetidine (300 mg IV). The response in hepatic blood flow in another patient in whom a peritoneo-jugular valve (Le Veen shunt) was inserted in analysed.
Cellular and Molecular Life Sciences, Mar 1, 1984
Toxicology and Applied Pharmacology, Oct 1, 2012
Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membran... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05) and loss of mitochondrial membrane potential (-80%, p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;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.
Journal of Endocrinology, Feb 17, 2010
In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated b... more 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 insulintreated SID (SIDCI; 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.
Toxicology Letters, 1990
The effect of aflatoxin B, (AFB,) on the glutathione S-transferase activity (GST) and on non-prot... more The effect of aflatoxin B, (AFB,) on the glutathione S-transferase activity (GST) and on non-protein thiol levels of different tissues was studied in adult male Wistar rats. Animals received a single dose of the toxin (100 or 500 ,ug/kg body wt., p.o.), and were studied 6 or 24 h after administration. GST was determined in liver, renal cortex, duodenum, jejunum-ileum and distal ileum, using 3 substrates: l-chloro-2.4-dinitrobenzene (CDNB), trans-4-phenyl-3-buten-2-one (PBO) and 1,2-epoxyethylbenzene (STOX). The non-protein thiol content of all tissues tested increased with the lowest dose at 6 h, returning to normal values at 24 h, while the higher dose produced a significant decrease in reduced thiol levels at 6 h, returning to normal values at 24 h. AFB, administration induced, independently of dose and tissue, total CST (CDNB) and epoxide-transferase activity (STOX) while A-C-type transferases (PBO) were inhibited. Almost all activities returned to normal values at 24 h. In cases of enzyme induction there was in general an increase in V,,, and a decrease in apparent K,. The opposite was seen in cases of inhibition. In conclusion, the results provide evidence that extrahepatic GST could be important in the overall process of detoxification of AFB,. The behavior seen in hepatic and extrahepatic tissues revealed the functions of catalysis (B-type transferases) and covalent bond formation, as well as inactivation by probable AFB, metabolites (A-C-type transferases).
Annals of Hepatology, Sep 1, 2012
Molecular Nutrition & Food Research, Oct 1, 2013
Scope: Quercetin is the most abundant flavonoid in human diet. It has special interest as it hold... more 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 G 1 and S phases, accumulation in G 2 , 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-␣ 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-␣ activity.
Growth Factors Journal, 2009
Interferon-gamma/transforming growth factor-beta (IFN-gamma/TGF-beta) pathways have opposite effe... more 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.
Journal of Hepatology, Apr 1, 2012
Background and Aims: Treatment of hepatocellular carcinoma (HCC) in diethylnitrosamine (DEN)-indu... more Background and Aims: Treatment of hepatocellular carcinoma (HCC) in diethylnitrosamine (DEN)-induced mice with antibodies directed against the Placental Growth Factor (PlGF) causes a substantial improvement in mortality, tumor burden and vascular normalization. Increased expression of multi-antennary N-glycans is correlated with oncogenic development and we observed a significant decrease of this glycomic fraction following anti-PlGF treatment. To elucidate the molecular mechanism behind this down-regulation, we focused on E26 transformation specific sequence 1 (Ets-1), a transcription factor essential for the glycomic and angiogenic changes in malignant transformation. Ets-1 is a downstream target of two signaling pathways: the MAP kinase (MAPK) pathway and a Ca 2+-dependent pathway. Activation of the latter pathway inhibits Ets-1 DNA-binding. Methods: 129S2/SvPasCrl mice were injected with DEN for 25w followed by 5w injections with IgG or anti-PlGF. Ets-1 mRNA expression levels were assessed by qPCR analysis. Phosphorylated and non-phosphorylated forms of Ets-1, MAPK pathway (JNK, p38 and ERK1/2) and Ca 2+-dependent pathway (PKCa and CAMKII) were investigated by Western Blot analysis. Results: Number of Ets-1 transcripts doubled surrounding the tumor and tripled in tumor liver tissue. However, a non-significant decrease in Ets-1 transcription was observed surrounding or in the tumor of anti-PlGF treated mice. On the protein level, anti-PlGF treatment resulted in a substantial increase of the phosphorylated forms of all three MAPK subfamilies in tumor tissue. Similar results were obtained surrounding the tumor with exception of JNK activation. Importantly, the most distinct activation was observed for p38. In the Ca 2+-dependent pathway, activated forms of PKCa and CAMKII displayed a substantial increase in signal intensity in tumor tissue after anti-PlGF treatment. Ets-1 was significantly upregulated surrounding the tumor in anti-PlGF treated mice. Signal intensity of phosphorylated form pSer251 increased with 60% in tumor tissue (downstream Ca 2+-dependent pathway) and tripled for pThr38 surrounding the tumor (downstream MAPK pathway). Conclusions: Ca 2+-dependent Ets-1 phosphorylation in tumor tissue of anti-PlGF treated mice might be the molecular basis for the down-regulation of multi-antennary N-glycans. Our data indicate no role for MAPK Ets-1 phosphorylation in this process. However, p38 activation could play an important part in the molecular mechanism of anti-PlGF treatment independent of Ets-1.
Pharmacology & Toxicology, Nov 1, 1995
In a previous study we demonstrated that the administration of 20 pg/kg b.wt. of glucagon to rats... more In a previous study we demonstrated that the administration of 20 pg/kg b.wt. of glucagon to rats caused a significant diminution of hepatic cytosolic glutathione S-transferase (GST) activity. This inhibition was non-competitive and reversible. We suggested that the effect would be mediated by cytosolic effectors. The present work was performed to characterize the mechanism involved in this inhibition. Liver tissue slices (1 70 to 200 mg) were incubated during different periods of time (0, 5, 10, 15, 20 and 30 min.) with several concentrations of glucagon (IO-'M, IOWM and 10-I0M), dibutiryl cyclic AMP (10-4M, 10-6M and 10-9M), divalent cation ionophore A23187 (10-4M, 10-6M and 10-9M) or vasopressin (lO-'M, 5X IO-'M and 10-*M). The incubation was done with or without calcium in the medium. In all cases the cytosolic GST activity were determined in liver slices. The percentage of inhibition of GST activity was directly related to the increase of concentration of the test substances. An inhibition between 40% to 45% after 10 min. of incubation with the highest concentrations was observed (except vasopressin which caused 10% of inhibition). 10-'OM glucagon did not produce a decrease of GST activity. The inhibition disappeared in calcium-free incubated slices, but direct relationship between plasma-membrane calcium influx and inhibition of GST activity (r=0.950, P<O.OOI, n=24) could be obtained. By using calmodulin antagonists, we conclude that the inhibition process of the enzyme was mediated by calmodulin. in summary, we propose that plasma-membrane calcium influx induced by high concentrations of glucagon activates calmodulin, which promotes a modification (actually a methylation, according to other authors) on GST, thereby causing a decrease in its activity.
Toxicology Letters, Feb 1, 1988
The effect of streptozotocin (SZ) administration on sodium [14C]taurocholate (TC) transmural tran... more The effect of streptozotocin (SZ) administration on sodium [14C]taurocholate (TC) transmural transfer was studied in the everted rat ileum. The excretion of fecal bile acids was also studied in living rats injected with that compound. The viability of the preparation used for the in vitro experiments was evaluated by light microscopy and by the rate of glucose uptake by tissue from the mucosal fluid. The results obtained showed that TC transfer to the serosal fluid was impaired after 24 h of SZ injection, as well as the active transport observed in control preparations. The amount of TC accumulated in the intestinal tissue was also diminished. In addition, total ATPase activity of tissue was decreased, and intracellular electrolyte concentration was altered. Therefore, a slower saturation of binding sites could be responsible for the effects of SZ on TC tissue accumulation, and a decreased ATPase activity for the impairment of the TC concentrative transport system. The results observed in vitro were supported by data in vivo because fecal bile acid excretion was significantly diminished in SZ-treated rats.
Experimental and Toxicologic Pathology, Oct 1, 1992
The mechanisms involved in bile salt-induced choleresis are poorly known. To give an insight in t... more The mechanisms involved in bile salt-induced choleresis are poorly known. To give an insight in this physiological process, bile salt-associated electrolyte secretion was studied following relief of a short-term (2 h) biliary .obstruction in the rat, an experimental model that shows an important diminution of bile salt choleretic efficiency. For this purpose, biliary excretion of total bile salts and electrolytes (sodium, chloride and bicarbonate) were studied in such a model during taurocholate infusion at increasing rates. The results showed that bile flow, bile salt output and electrolyte secretion stimulated by taurocholate,administration were decreased in the rats that were subjected to biliary obstruction. Besides, the choleretic efficiency of the excreted bile salts, as estimated by the slope of the regression line of bile flow vs. bile salt output, was diminished by 46 % (p < 0.005). Multiple regression analysis of bile flow vs. bile salt and electrolyte outputs allowed to detect a selective diminution of the fraction of bile flow related to bile salt-associated electrolyte secretion ("secretory fraction" of the choleretic efficiency of bile salts) (3.2 ± 0.3 vs. 2.5 ± 0.2L1mol, p < 0.05) whereas the "osmotic fraction" of the choleretic efficiency of bile salts was not modified by the treatment (5. a ± 0.4 vs. 5.1 ± 0.3 Llmol, p> 0.05). Since both chloride and bicarbonate biliary concentrations in the volume of bile stimulated by taurocholate were reduced by 53 % and 52 % respectively, a role of these anions in the generation of bile salt-induced choleresis was suggested. Possible mechanisms involved in such a process and in its early impairment during cholestasis are discussed.
Toxicology and Applied Pharmacology, 2017
Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen sp... more Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity. Previous studies demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, has immunomodulatory effects, increasing survival in C57BL/6 mice in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The mechanism by which BZL inhibits inflammatory response in sepsis is poorly understood. Also, our group recently reported that BZL is able to activate the nuclear factor erytroide-derived 2-Like 2 (NRF2) in vitro. The aim of the present work was to delineate the beneficial role of BZL during sepsis, analyzing its effects on the cellular redox status and the possible link to the innate immunity receptor TLR4. Specifically, we analyzed the effect of BZL on Nrf2 regulation and TLR4 expression in liver of mice 24 hours post-CLP. BZL was able to induce NRF2 nuclear protein localization in CLP mice. Also, we found that protein kinase C (PKC) is involved in the NRF2 nuclear accumulation and induction of its target genes. In addition, BZL prompted a reduction in hepatic CLP-induced TLR4 protein membrane localization, evidencing its immunomodulatory effects. Together, our results demonstrate that BZL induces hepatic NRF2 activation with the concomitant increase in the antioxidant defenses, and the attenuation of inflammatory response, in part, by inhibiting TLR4 expression in a murine model of sepsis.
Journal of Cellular Biochemistry, Jan 22, 2013
Increased expression of COX-2 has been linked to inflammation and carcinogenesis. The constitutiv... more Increased expression of COX-2 has been linked to inflammation and carcinogenesis. The constitutive expression of hCOX-2 protects hepatocytes from several pro-apoptotic stimuli. It is established that an increase in hepatic apoptosis exists in experimental models of diabetes. The aim of this work was to analyze the role of COX-2 as a regulator of apoptosis in liver of diabetic mice. Mice of C57BL/6 strain Wild Type (Wt) and transgenic in hCOX-2 (COX-2 Tg) were separated into Control (vehicle) and SID (Streptozotocin Induced Diabetes, 200mg/kg). 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 proteins as Bad and Bax, release of cytochrome c and activities of caspases-3 and-9, leading to an increase in apoptotic index. This situation was improved in COX-2 Tg animals. In addition, SID COX-2 Tg showed increased expression of Mcl-1 and XIAP anti-apoptotic proteins. In summary, there is a pro-apoptotic state in the liver of diabetic animals, situation improved by over-expression of COX-2. Also, we analized the contribution of increased glucose on apoptosis and the role of hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured 72 hours at glucose 5 mM and 25 mM. At 25 mM there was an increase in apoptosis in non-transfected cells vs those exposed to 5 mM. This increase was prevented in part in transfected cells at 25 mM. The above suggest a protective role of hCOX-2 to apoptosis induced by hyperglycemia in vitro and in vivo.
Background/Aims: Clarification of the role of lipid peroxidation in the onset of liver proliferat... more 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 munoblotting. DNA synthesis 24 and 48 h after surgery was assessed by (3mthymidine incorporation. Results: Increased lipid peroxidation was found in total homogenate, cytosol and microsomes. The he- patic cytosolic content of nitrates increased, reaching the highest values at 5 h posthepatectomy. Amino- guanidine or NC-monomethyl-L-arginine pretreat- ment blocked the rise of nitric oxide production and lipid peroxidation levels and decreased the DNA syn- thesis. The increase in hepatic iNOS protein express- ion at 5 h after partial hepatectomy disappeared with aminoguanidiue preatreatment. Conchsions: Our experiments suggest that nitric ox- ide plays a role in the proliferation mechanism, al- though it is responsible, at least in part, for the en- hanced lipid peroxidation.
Apoptosis, Jan 12, 2014
Your article is protected by copyright and all rights are held exclusively by Springer Science +B... more Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com".
Canadian Journal of Physiology and Pharmacology, Oct 1, 2007
The aim of this study was to evaluate the influence of partial hepatectomy prior to cell isolatio... more 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).
Journal of Hepatology, 2017
However, the relationship between β-catenin and CXCL10 in FPC cholangiocytes as well as the relev... more However, the relationship between β-catenin and CXCL10 in FPC cholangiocytes as well as the relevance of CXCL10 in the progression of the disease remains unclear. Methods: WT and FPC cholangiocytes were treated with inhibitors or siRNA of intracellular signaling. Secretion and gene expression of CXCL10 were evaluated by ELISA and RT-PCR, respectively. Pkhd1 del4/ del4 mice were treated with the CXCR3 antagonist AMG-487 (5 mg/ Kg/day) for three months. Results: FPC-cholangiocytes, both in vitro and in vivo (LCM microscope), showed increased pro-IL-1β expression and IL-1β secretion, and an activated inflammasome (increased caspase 1 and Nrlp3). Administration of IL1-ß to FPC-cholangiocytes strongly upregulated the production of CXCL10, through a JAK/STAT-dependent pathway (JAK inhibitor I; 10 μM). Inhibition of β-Catenin (by ICG-001; 10 μM and by siRNA) and of cAMP signaling (by PKA inhibitor 14-22 amide; 1 μM), significantly reduced CXCL10 gene expression and secretion following IL1-ß. Furthermore, as assesses by Western blot, immunofluorescence and FACS analysis, β-catenin inhibition prevented the nuclear translocation of pSTAT3(Tyr 705) indicating that β-catenin signaling is necessary for STAT3-dependent CXCL10 gene transcription. Finally, in Pkhd1 del4/del4 mice, inhibition of CXCR3, the cognate receptor of CXCL10, significantly reduced macrophage infiltration (CD45 + cells), cyst growth (K19 + area), spleen size and liver fibrosis (Sirius red). Conclusions: In FPC-defective cholangiocytes, an increased production and secretion of IL1ß is responsible for β-catenin and STAT3dependent secretion of CXCL10. In vivo experiments show that this mechanism is of pathophysiological relevance, as targeting the CXCL10/CXCR3 axis prevents the recruitment of macrophages and halts the progression of the disease.