Félix V. Vega

Resveratrol protects primary rat hepatocytes against oxidative stress damage : Activation of the Nrf2 transcription factor and augmented activities of antioxidant enzymes

Oxidative stress is recognized as an important factor in the development of liver pathologies. The reactive oxygen species endogenously generated or as a consequence of xenobiotic metabolism are eliminated by enzymatic and nonenzymatic cellular systems. Besides endogen defences, the antioxidant consumption in the diet has an important role in the protection against the development of diseases product of oxidative damage. Resveratrol is a naturally occurring compound which is part of the human diet. This molecule has been shown to have many biological properties, including antioxidant activity. We decided to test if resveratrol could protect primary hepatocytes in culture from oxidative stress damage and if so, to determine if this compound affects the cellular detoxifying systems and their regulation through the Nrf2 transcription factor that regulates the expression of antioxidant and phase II detoxifying enzymes. Cell death by necrosis was detected by measuring the activity of lactate dehydrogenase liberated to the medium. The activities of antioxidant and phase II enzymes were measured using previously described methods. Activation of the Nrf2 transcription factor was studied by confocal microscopy and the Nrf2 and its coding mRNA levels were determined by western blot and quantitative PCR respectively. Resveratrol pre-treatment effectively protected hepatocytes in culture exposed to oxidative stress, increasing the activities of catalase, superoxide dismutase, glutathione peroxidase, NADPH quinone oxidoreductase and glutathione-S-transferase. Resveratrol increases the level of Nrf2 and induces its translocation to the nucleus. Also, it increases the concentration of the coding mRNA for Nrf2. In this work we show that resveratrol could be a useful drug for the protection of liver cells from oxidative stress induced damage.

Leptin, reproduction and sex steroids.

Leptin is a hormone secreted mainly by the adipose cells with a primary role in the regulation of body weight by establishing a feedback loop between the energy reserves and the hypothalamic centers that control food intake. Recent data suggest that, in addition, leptin interacts with other endocrine systems to provide critical information about the size of the fat stores, acting as a permissive factor that allows the triggering of energy-demanding situations, as the onset of puberty and the reproduction, only when the size of the fuel reserve is large enough to guarantee its success. In addition, leptin appears to play a role during pregnancy and lactation, as it is produced by the placenta and is present in maternal milk. The fact that leptin levels are always higher in females, even after correcting for body fat content, suggests that the interaction between the adipose tissue and the reproductive system is modulated in a different way in males and females by androgenic and estrogenic hormones. In fact, adipose tissue samples taken from male donors are completely refractory in vitro to the action of both estrogens and androgens. On the contrary, dihydrotestosterone, androstenedione and dehydroepiandrosterone-S are potent inhibitors of leptin secretion, while estradiol induces a strong stimulation in adipose tissue taken from women. Testosterone is devoid of activity in either gender.

Characterization of proton-transporting membranes from resting pig gastric mucosa.

Membrane vesicles were purified from resting corpus mucosa of pig stomachs by velocity-sedimentation on a sucrose-Ficoll step gradient. Two vesicular fractions containing the (H+ + K+)-ATPase were obtained. One fraction was tight towards KCl, the other was leaky. At 21 degrees C maximal (H+ + K+)-ATPase activities of 0.8 and 0.4 mumol X mg-1 X min-1, respectively, were observed in lyophilized vesicles. The vesicles contained a membrane-associated carbonic anhydrase, the activity of which was in 100-fold excess of the maximal ATPase activity. Both vesicular fractions were rich in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol. The characteristics of ion permeability and transport in the tight vesicles were in agreement with corresponding data for vesicles of a tubulovesicular origin in the parietal cell. Measurement of the rate of K+ uptake into the vesicles was based on the ability of K+ to promote H+ transport. The uptake was slow and dependent on the type of anion present. The effectiveness in promoting uptake of K+ by anions was SCN- greater than NO3- greater than Cl- much greater than HCO3- greater than SO4(2-). Uptake of K+ was much more rapid at alkaline pH than at neutral or at acidic pH. Addition of CO2 at alkaline pH strongly stimulated the rate of H+ accumulation in the vesicles. The initial part of this stimulation was sensitive to acetazolamide, an inhibitor of carbonic anhydrase. A model how the (H+ + K+)-ATPase and the carbonic anhydrase may co-operate is presented. It is concluded that membrane vesicles of a tubulovesicular origin can produce acid.

Glyceraldehyde-3-phosphate dehydrogenase exerts different biologic activities in apoptotic and proliferating hepatocytes according to its subcellular localization

Recent evidences indicate new roles for the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in essential mammalian cell processes, such as apoptosis and proliferation. To clarify the involvement of this protein in growth and programmed cell death in the liver, cell models of hepatocytes in culture were used to study GAPDH expression, localization and enzymatic activity in hepatocyte proliferation and apoptosis. GAPDH expression in cell compartments was studied by Western blot. Nuclear expression of GAPDH increased in apoptosis, and cytoplasmic expression was elevated in apoptosis and proliferation. Subcellular localization was determined by GAPDH immunostaining and confocal microscopic analysis. Quiescent and proliferating hepatocytes showed cytoplasmic GAPDH, while apoptotic cells showed cytoplasmic but also some nuclear staining. The glycolytic activity of GAPDH was studied in nuclear and cytoplasmic cell compartments. GAPDH enzymatic activity increased in the nucleus of apoptotic cells and in cytoplasms of apoptotic and proliferating hepatocytes. Our observations indicate that during hepatocyte apoptosis GAPDH translocates to the nucleus, maintaining in part its dehydrogenase activity, and suggest that this translocation may play a role in programmed hepatocyte death. GAPDH over-expression and the increased enzymatic activity in proliferating cells, with preservation of its cytoplasmic localization, would occur in response to the elevated energy requirements of dividing hepatocytes. In conclusion, GAPDH plays different roles or biological activities in proliferating and apoptotic hepatocytes, according to its subcellular localization.

“Fluorescent glycogen” formation with sensibility for in vivo and in vitro detection

There are presently many methods of detecting complex carbohydrates, and particularly glycogen. However most of them require radioisotopes or destruction of the tissue and hydrolysis of glycogen to glucose. Here we present a new method based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose), a d-glucose fluorescent derivative, into glycogen. Two kinds of approaches were carried out by using Clone 9 rat hepatocytes as a cellular model; (1) Incubation of cell lysates with 2-NBDG, carbohydrate precipitation in filters and measurement of fluorescence in a microplate reader (2) Incubation of living hepatocytes with 2-NBDG and recording of fluorescence images by confocal microscopy. 2-NBDG labeled glycogen in both approaches. We confirmed this fact by comparison to the labeling obtained with a specific monoclonal anti-glycogen antibody. Also drugs that trigger glycogen synthesis or degradation induced an increase or decrease of fluorescence, respectively. This is a simple but efficient method of detecting glycogen with 2-NBDG. It could be used to record changes in glycogen stores in living cells and cell-free systems and opens the prospect of understanding the role of this important energy reserve under various physiological and pathophysiological conditions.

Yessotoxin induces ER-stress followed by autophagic cell death in glioma cells mediated by mTOR and BNIP3.

Yessotoxin at nanomolar concentrations can induce programmed cell death in different model systems. Paraptosis-like cell death induced by YTX in BC3H1 cells, which are insensitive to several caspase inhibitors, has also been reported. This makes yessotoxin of interest in the search of molecules that target cancer cells vulnerabilities when resistance to apoptosis is observed. To better understand the effect of this molecule at the molecular level on tumor cells, we conducted a transcriptomic analysis using 3 human glioma cell lines with different sensitivities to yessotoxin. We show that the toxin induces a deregulation of the lipid metabolism in glioma cells as a consequence of induction of endoplasmic reticulum stress. The endoplasmic reticulum stress in turn arrests the cell cycle and inhibits the protein synthesis. In the three cell lines used we show that YTX induces autophagy, which is involved in cell death. The sensibility of the cell lines used towards autophagic cell death was related to their doubling time, being more resistant the cell line with the lowest proliferation rate. The involvement of mTOR and BNIP3 in the autophagy induction was also determined.

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane.

Protein synthesis inhibition and oxidative stress induced by cylindrospermopsin elicit apoptosis in primary rat hepatocytes.

The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce protein synthesis inhibition as well as inhibition of glutathione synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between protein synthesis inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, protein synthesis inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant response was observed at the transcriptional and translational levels.

Cytoskeletal toxicity of pectenotoxins in hepatic cells

Pectenotoxins are macrocyclic lactones found in dinoflagellates of the genus Dinophysis, which induce severe liver damage in mice after i.p. injection. Here, we have looked for the mechanism(s) underlying this hepatotoxicity.

Crambescidin-816 Acts as a Fungicidal with More Potency than Crambescidin-800 and -830, Inducing Cell Cycle Arrest, Increased Cell Size and Apoptosis in Saccharomyces cerevisiae

In this paper, we show the effect of crambescidin-816, -800, and -830 on Saccharomyces cerevisiae viability. We determined that, of the three molecules tested, crambescidin-816 was the most potent. Based on this result, we continued by determining the effect of crambescidin-816 on the cell cycle of this yeast. The compound induced cell cycle arrest in G2/M followed by an increase in cell DNA content and size. When the type of cell death was analyzed, we observed that crambescidin-816 induced apoptosis. The antifungal effect indicates that crambescidins, and mostly crambescidin-816, could serve as a lead compound to fight fungal infections.