Jindřich Martínek

Resveratrol attenuates lipopolysaccharide-induced hepatitis in D-galactosamine sensitized rats: role of nitric oxide synthase 2 and heme oxygenase-1.

The goal of study was directed to investigate the effects of resveratrol (RES) pretreatment on the enhancing action of D-galactosamine (D-GalN; 800 mg/kg) on lipopolysaccharide (LPS; 0.5 microg/kg) inducing liver failure in rats. Liver function was assessed by determination of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alpha-glutathione S-transferase (alpha GST) and bilirubin (BILI). Plasma NO(2)(-) was assessed by NO(2)(-)/NO(3)(-) colorimetric kit. The estimation of nonenzymatic and enzymatic antioxidants (glutathione and catalase) was performed in plasma and liver homogenate. Lipid peroxidation was evaluated by the thiobarbituric acid reacting substances (TBARS) and the conjugated dienes (CD). Morphological examinations using light and electron microscopy were performed. Observations related to pharmacological increases of inducible nitric oxide synthase (NOS-2)/nitric oxide (NO) and inducible heme oxygenase (HO-1) in fulminant hepatic failure and modulation by resveratrol were followed up by real-time reverse transcription PCR (RT-PCR) in liver tissue. In the present study we found that among the mechanisms responsible for the hepatoprotective effect of resveratrol in the LPS/D-GalN liver toxicity model are reduction in NO, downregulation of NOS-2, modification of oxidative stress parameters and modulation of HO-1 which led to overall improvement in hepatotoxic markers and morphology after the hepatic insult.

EFFECTS OF RESVERATROL PRETREATMENT ON TERT-BUTYLHYDROPEROXIDE INDUCED HEPATOCYTE TOXICITY IN IMMOBILIZED PERIFUSED HEPATOCYTES: INVOLVEMENT OF INDUCIBLE NITRIC OXIDE SYNTHASE AND HEMOXYGENASE-1

The aim of this work was to study the effects of resveratrol (RES) as compared to silymarin (SM) pretreatments on tert-butylhydroperoxide (tBH) induced apoptotic/necrotic markers in hepatocytes. Hepatocyte in cultures (48 h) and in perifused immobilized agarose threads (5h) were used as cellular systems. Hepatocyte apoptosis was estimated morphologically using Annexin-V combined with propidium iodide, or toluidine blue staining. Hepatocyte viability and functionality were evaluated by ALT and urea synthesis. Nitric oxide (NO) and carbon monoxide involvements were also examined. Resveratrol and silymarin reduced tBH-induced hepatocyte toxic effects in short term experiments (5h) as measured by a significant reduction in ALT and NO increase produced by tBH. Both inducible nitric oxide synthase (NOS-2) and hemoxygenase-1 (HO-1) gene expression were increased by tBH and reduced by both RES and SM pretreatments. Morphologically, there were ameliorations in both apoptotic and necrotic markers under RES treatment and were similar to biochemical findings. In addition, RES improved hepatocyte stability in both cellular systems. It may be concluded that resveratrol and sylimarin ameliorative effects on tBH hepatocyte toxicity are comparable; involve NOS-2 and HO-1 expression and should be re-evaluated in various in vitro and in vivo experimental conditions.

Nitric oxide synthase inhibitors modulate lipopolysaccharide-induced hepatocyte injury: dissociation between in vivo and in vitro effects

Effects of endotoxemia-induced NO production on rat liver and hepatocytes in culture were investigated. Rats were treated intraperitoneally with saline, lipopolysaccharide (LPS, 10 mg/kg), L-nitroarginine methyl ester (L-NAME)+LPS, aminoguanidine (AG)+LPS, FK 506+LPS, S-nitroso-N-acetyl penicillamine (SNAP)+L-NAME+LPS and SNAP+FK 506+LPS. Mortality, hepatocyte viability and liver function test were estimated. Liver morphology was observed by light and electron microscopy. Hepatocyte cultures were treated with LPS, cytokine mixture (CM) with or without FK 506, L-NAME or AG. Hepatocyte function and inducible form of NOS (iNOS) expression were evaluated. Twenty-four hours after treatments with saline, LPS, L-NAME+LPS, AG+LPS, FK 506+LPS, SNAP+L-NAME+LPS and SNAP+FK 506+LPS, rat mortalities were 0%, 10%, 48%, 8%, 20%, 38% and 0%, and hepatocyte viabilities were 93+/-3%, 80+/-3%, 52+/-8%, 88+/-1%, 70+/-3%, 80+/-4% and 82+/-3%, respectively. AG+LPS or L-NAME+LPS administration was followed by excessive vacuolization of hepatocytes with lesions in the intermediary lobule zone characterized by features of secondary necrosis as a continuation of apoptotic processes. SNAP+L-NAME+LPS resulted in a well-preserved structure of central vein lobules with sparse signs of apoptosis. Treatment with LPS or CM increased iNOS expression in hepatocyte culture, which was inhibited by L-NAME, FK 506 or AG. AG reduced LPS-induced rise in alanine aminotransferase leakage. LPS-induced NO exerts cytoprotective effects in vivo, while LPS-induced NO in vitro appears to be toxic. Based on the data of this report, one cannot use in vitro results to predict in vivo responses to LPS-induced NO production. The pharmacological modulation of iNOS expression or NO production in vivo or in vitro, therefore, by the development of specific NO donors or inhibitors is promising for improvement of hepatocyte functions under the two experimental conditions, respectively.

Thapsigargin, a selective inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases, modulates nitric oxide production and cell death of primary rat hepatocytes in culture

Increased cytosolic calcium ([Ca2+]i) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca2+]i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca2+-ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 μmol/L) for 0–24 or 24–48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca2+]i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0–24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 μmol/L produced secondary necrosis. During 24–48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 μmol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24–48 h. Our data suggest that the extent of the [Ca2+]i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.

Circulating ovarian autoantibodies and FSH and LH levels in adolescent girls with primary menstrual cycle disorders.

Seventy three adolescent patients with primary menstrual disturbances were studied by immunofluorescent methods for prevalence of ovarian autoantibodies (O-Ab), the enzyme immunoassay (EIA) method for examination of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) hormonal levels was used. Clinically healthy girls (40) served as controls. Patients were divided into a group of 13 girls with primary amenorrhea (PA) and a group of 60 girls with oligo and/or secondary amenorrhea (OSA). In the PA group 38.5% positivity linked to ooplasm (OO), zona pellucida (ZP), and membrana granulosa cells (MG), as well as 46.2% to theca folliculi interna (TI) and 53.8% to lutein cells (LC), was detected. Statistically significant differences (p < .05) of LH levels between OO immunopositive and negative girls (19.0 and 9.4 mU/ml) were found, while FSH values were not different. In the OSA group a 16.7% positivity linked to OO, 23.3% to ZP and MG, 38.3% to TI, and 58.3% to LC were detected. Significant linkage between MG immunopositive and negative girls and FSH:LH ratio values were estimated. FSH values were significantly different (p < .05) for PA and OSA groups (23.7 and 6.7 IU/l) which corresponded particularly with higher findings of Ab in germ line-cells (OO-, ZP-, and MG-Ab). A striking correlation between evidence of O-Ab and menstrual cycle irregularities was found. It could support a possible coincidence of autoimmune mechanism in these dysfunctions. Localization of O-Ab-binding was verified at the electron microscopic level.

Modulation of spontaneous and lipopolysaccharide-induced nitric oxide production and apoptosis by d-galactosamine in rat hepatocyte culture: the significance of combinations of different methods.

ABSTRACT Apoptotic markers and signals produced by xenobiotics as hepatotoxic D-galactosamine (D-GalN) and lipopolysaccharide (LPS) are extensively investigated in vivo. The contribution of various cells and factors as nitric oxide (NO) in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia was reported. Therefore, the aim of the present work was to study the in vitro effect of D-GalN on nonstimulated or LPS-treated rat hepatocytes in culture and the potential involvement of NO in this process. Our results showed that the spontaneous and LPS-induced NO production was completely blocked by D-GalN during 0 to 24 hours. However, D-GalN slightly enhanced NO production during 24 to 48 hours. D-GalN was more potent to induce hepatocyte apoptosis and necrosis during 24 to 48 than 0 to 24 hours as evidenced morphologically (Annexin V/propidium iodide staining) and biochemically (caspase-3-like activity, alanine-aminotransferase leakage, MTT test). Interestingly, D-GalN treatment suppressed mitochondrial cytochrome C release throughout the study. LPS addition to D-GalN considerably aggravated apoptotic/necrotic markers only during 0 to 24 hours. Surprisingly, a share of apoptotic cells was distinctly lower after LPS + GalN treatment than after LPS alone during 0 to 24 hours, while 24- to 48-hour incubation produced massive apoptotic/necrotic hepatocytes. It may be concluded that there is a significant modulation of NO production by D-GalN. Because the role of NO is only partly decisive in the apoptotic/necrotic events, and considering the fraction of the cells completing apoptosis while others that turn toward necrosis (aponecrosis), caution should be exercised in apoptosis data interpretation and combinations of different test methods should be applied.

Use of Mild Periodic Acid-Thiosemicarbazide-Silver Proteinate Staining for Ultrahistochemical Demonstration of Glycoconjugates in the Human Uterine Epithelium*)

The human uterine epithelium has been studied by means of mild (0.1, 0.01% respectively) periodic acid (PA) oxidation followed by thiosemicarbazide-silver proteinate staining. Distribution of silver deposits provided evidence for glycoconjugates and was in the case of oxidation with 0.01% PA selective for sialic acid (N-acetylneuraminic acid) complexes. The electron dense silver particles were detectable at the outer side of apical plasma membranes and on the inner surface of lysosomal membranes. Also the marginal zone of lipid droplets reacted regularly. Since the lower concentration of PA proves sialogycoconjugates selectively, the positive staining depicted not only those in the membrane compartments but also as a layer between the hydrophobic content and the hydrophilic cytoplasm. In the granular endoplasmic reticulum of the individual microvillous cells a special glycoprotein of paracrystalline organisation was detected. The functional significance of this substance remains unclear. Nuclear channel systems (NCS) are frequently observed in the epithelial cells in the uterine glands. Similar staining properties of the NCS and nuclear envelope support the hypothesis of its nuclear origin.