Carlos Eduardo Alves de Souza

The FXR agonist 6ECDCA reduces hepatic steatosis and oxidative stress induced by ethanol and low-protein diet in mice

BACKGROUND AND AIM Excessive ethanol consumption can lead to development of hepatic steatosis. Since the FXR receptor regulates adipose cell function and liver lipid metabolism, the aim of this work was to examine the effects of the FXR agonist 6ECDCA on alcoholic liver steatosis development and on oxidative stress induced by ethanol consumption. METHODS Swiss mice (n=24) received a low-protein diet (6%) and a liquid diet containing 10% ethanol or water for 6weeks. In the last 15days mice received oral treatment with 6ECDCA (3mgkg(-1)) or 1% tween (vehicle). The experimental groups (n=6) were: water+tween, water+6ECDCA, ethanol+tween and ethanol+6ECDCA. Moreover, as a diet control, we used a basal group (n=6), fed by a normal-proteic diet (23%) and water. After the treatment period, the animals were anesthetized for sample collection to perform plasma biochemistry assays, hepatic oxidative stress assays, hepatic cholesterol and triglycerides measurements, liver histology and hepatic gene expression. RESULTS Ethanol associated with low-protein diet induced hepatic oxidative stress, increased plasma transaminases and induced hepatic lipid accumulation. Many of these parameters were reversed by the administration of 6ECDCA, including amelioration of lipid accumulation and lipoperoxidation, and reduction of reactive oxygen species. These effects were possibly mediated by regulation of Srebpf1 and FAS gene expression, both reduced by the FXR agonist. CONCLUSIONS Our data demonstrated that 6ECDCA reverses the accumulation of lipids in the liver and decreases the oxidative stress induced by ethanol and low-protein diet. This FXR agonist is promising as a potential therapy for alcoholic liver steatosis.

Uncaria tomentosa Exerts Extensive Anti-Neoplastic Effects against the Walker-256 Tumour by Modulating Oxidative Stress and Not by Alkaloid Activity

This study aimed to compare the anti-neoplastic effects of an Uncaria tomentosa (UT) brute hydroethanolic (BHE) extract with those of two fractions derived from it. These fractions are choroformic (CHCl3) and n-butanolic (BuOH), rich in pentacyclic oxindole alkaloids (POA) and antioxidant substances, respectively. The cancer model was the subcutaneous inoculation of Walker-256 tumour cells in the pelvic limb of male Wistar rat. Subsequently to the inoculation, gavage with BHE extract (50 mg.kg−1) or its fractions (as per the yield of the fractioning process) or vehicle (Control) was performed during 14 days. Baseline values, corresponding to individuals without tumour or treatment with UT, were also included. After treatment, tumour volume and mass, plasma biochemistry, oxidative stress in liver and tumour, TNF-α level in liver and tumour homogenates, and survival rates were analysed. Both the BHE extract and its BuOH fraction successfully reduced tumour weight and volume, and modulated anti-oxidant systems. The hepatic TNF-α level indicated a greater effect from the BHE extract as compared to its BuOH fraction. Importantly, both the BHE extract and its BuOH fraction increased the survival time of the tumour-bearing animals. Inversely, the CHCl3 fraction was ineffective. These data represent an in vivo demonstration of the importance of the modulation of oxidative stress as part of the anti-neoplastic activity of UT, as well as constitute evidence of the lack of activity of isolated POAs in the primary tumour of this tumour lineage. These effects are possibly resulting from a synergic combination of substances, most of them with antioxidant properties.

Sydnone SYD-1 affects the metabolic functions of isolated rat hepatocytes.

Previously, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) impairs the mitochondrial functions linked to energy provision and suggested that this effect could be associated with its antitumor activity. Herein, we evaluated the effects of SYD-1 (25 and 50 μM) on rat hepatocytes to determine its cytotoxicity on non-tumor cells. SYD-1 (25 and 50 μM) did not affect the viability of hepatocytes in suspension after 1-40 min of incubation. However, the viability of the cultured hepatocytes was decreased by ∼66% as a consequence of treatment with SYD-1 (50 μM) for 18 h. Under the same conditions, SYD-1 promoted an increase in the release of LDH by ∼19%. The morphological changes in the cultured cells treated with SYD-1 (50 μM) were suggestive of cell distress, which was demonstrated by the presence of rounded hepatocytes, cell fragments and monolayer impairment. Furthermore, fluorescence microscopy showed an increase in the annexin label after treatment with SYD-1 (50 μM), suggesting that apoptosis had been induced in these cells. SYD-1 did not affect the states of respiration in the suspended hepatocytes, but the pyruvate levels were decreased by ∼36%, whereas the lactate levels were increased by ∼22% (for the 50 μM treatment). The basal and uncoupled states of respiration of the cultured hepatocytes were inhibited by ∼79% and ∼51%, respectively, by SYD-1 (50 μM). In these cells, SYD-1 (50 μM) increased the pyruvate and lactate levels by ∼84% and ∼16%, respectively. These results show that SYD-1 affects important metabolic functions related to energy provision in hepatocytes and that this effect was more pronounced on cells in culture than those in suspension.

Effects of statins on liver cell function and inflammation in septic rats.

BACKGROUND Several studies suggest that the presence of statins may be beneficial during sepsis, but this idea is controversial. The aim of this study was to investigate the effects of long-term statin treatment in the livers of septic animals, focusing on its antioxidant, antiinflammatory, and metabolic properties. MATERIALS AND METHODS Male Wistar rats were treated orally with simvastatin, atorvastatin, or vehicle once a d. After 30 d, sepsis was induced by cecal ligation and puncture (CLP) in Control, Simvastatin-treated, and Atorvastatin-treated groups, while the Sham group underwent only laparotomy. The Basal Simvastatin and Basal Atorvastatin groups received only their respective drugs without surgery. Twenty-four h after CLP or laparotomy, samples were collected from anesthetized rats for evaluation of hepatic oxidative stress, liver histology, hepatic mitochondria enzyme activity, leukocyte counts in blood and peritoneal cavity, gene expression of hepatic superoxide dismutase and TNF-2, and plasma biochemistry. RESULTS Most parameters that we tested exhibited expected changes upon sepsis induction. However, statin treatment only improved liver mitochondrial enzymatic activity. In other parameters, simvastatin and atorvastatin failed to protect the liver against injuries incurred upon the CLP-induced polymicrobial sepsis model. CONCLUSIONS Pretreatment with simvastatin or atorvastatin alone before sepsis induction improved mitochondrial activity in the liver; however, this result was not reproduced in other biomarkers of liver function and leukocyte migration during sepsis. Future studies should be performed to evaluate whether statins can be combined with other drugs to increase the efficacy of sepsis therapy.

Ruthenium complex exerts antineoplastic effects that are mediated by oxidative stress without inducing toxicity in Walker-256 tumor-bearing rats

Abstract The present study evaluated the in vivo antitumor effects and toxicity of a new Ru(II) compound, cis‐(Ru[phen]2[ImH]2)2+ (also called RuphenImH [RuC]), against Walker‐256 carcinosarcoma in rats. After subcutaneous inoculation of Walker‐256 cells in the right pelvic limb, male Wistar rats received 5 or 10 mg kg−1 RuC orally or intraperitoneally (i.p.) every 3 days for 13 days. A positive control group (2 mg kg−1 cisplatin) and negative control group (vehicle) were also used. Tumor progression was checked daily. After treatment, tumor weight, plasma biochemistry, hematology, oxidative stress, histology, and tumor cell respiration were evaluated. RuC was effective against tumors when administered i.p. but not orally. The highest i.p. dose of RuC (10 mg kg−1) significantly reduced tumor volume and weight, induced oxidative stress in tumor tissue, reduced the respiration of tumor cells, and induced necrosis but did not induce apoptosis in the tumor. No clinical signs of toxicity or death were observed in tumor‐bearing or healthy rats that were treated with RuC. These results suggest that RuC has antitumor activity through the modulation of oxidative stress and impairment of oxidative phosphorylation, thus promoting Walker‐256 cell death without causing systemic toxicity. These effects make RuC a promising anticancer drug for clinical evaluation. Graphical abstract Figure. No caption available. HighlightsThe compound RuphenImH (RuC) presented antitumor effect against the Walker‐256 tumor in rats.RuC induced oxidative stress and reduced the respiration of tumor cells.RuC presented antitumor effect by i.p. but not by p.o. administration.RuC has antitumor effect without causing systemic toxicity.

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.

Remote Monitoring for Forest Management in the Brazilian Amazon

Timber harvesting is an important economic activity in the Brazilian Amazon. In 2009, the timber industry produced 5.8 million cubic meters of logwood and generated US

Characterization of an alcoholic hepatic steatosis model induced by ethanol and high-fat diet in rats

2.5 billion in gross income along with 203,705 direct and indirect jobs (Pereira et al., 2010). Logging in the region is predominantly predatory, and is commonly known as Conventional. Only a small proportion occurs in a managed fashion (planned), known as Reduced Impact Logging (RIL) (Asner et al., 2002; Gerwing, 2002; Pereira Jr. et al., 2002; Verissimo et al., 1992). In the conventional method activities are not planned (opening of roads and log decks1, tree felling and log skidding), while with RIL planned management techniques are applied at all stages of harvesting (Amaral et al., 1998). The two methods cause impacts ranging from low to severe on the structure and composition of the remaining forest (Gerwing, 2002; John et al., 1996; Pereira Jr. et al., 2002). However, the impacts of predatory logging are two times greater than those of managed logging (John et al., 1996). Among the main impacts are: greater reduction in living aboveground biomass (Gerwing, 2002; Monteiro et al., 2004), risk of extinction for highvalue timber species (Martini et al., 1994), greater susceptibility to forest fires (Holdsworth & Uhl, 1997), increase of vines and pioneer vegetation (Gerwing, 2002; Monteiro et al., 2004) and substantial reduction in carbon stocks (Gerwing, 2002; Putz et al., 2008). The impact of timber harvesting can be described by means of forest inventories carried out in the field, with which it is possible to evaluate the structure and composition of the remaining forest (Gerwing, 2002; John et al., 1996; Monteiro et al., 2004). Another method employed is remote sensing, which has advanced over the last decade. In the Amazon, there have been successful tests with satellite images to detect and quantify forest degradation brought about by logging activities in the region (Asner et al., 2005; Matricardi et al., 2007; Souza Jr. et al., 2005). Images with moderate spatial resolution, such as Landsat (30 m) and Spot (20 m), have been used to detect types of logging, damages to the canopy and roads and log decks for harvesting (Asner et al., 2002; Matricardi et al., 2007; Souza Jr. & Roberts, 2005). As for images with high spatial resolution, such as Ikonos (1 to 4 m), they are capable of detecting smaller features of logging, such as small clearings (Read et al., 2003), as well as making it possible to determine the size of log decks and width of roads (Monteiro et al.,

CLASSIFICAÇÃO ORIENTADA A OBJETO PARA DETECÇÃO DA EXPLORAÇÃO SELETIVA DE MADEIRA NA AMAZÔNIA

Alcoholic liver disease is characterized by a wide spectrum of liver damage, which increases when ethanol is associated with high-fat diets (HFD). This work aimed to establish a model of alcoholic hepatic steatosis (AHS) by using a combination of 10% ethanol and sunflower seeds as the source of HFD. Male rats received water or 10% ethanol and regular chow diet and/or HFD, which consisted of sunflower seeds. The food consumption, liquid intake and body weight of the rats were monitored for 30 days. After this period, blood was collected for biochemical evaluation, and liver samples were collected for histological, mitochondrial enzyme activity and oxidative stress analyses. Our results indicated that the combination of 10% ethanol and HFD induced micro- and macrosteatosis and hepatocyte tumefaction, decreased the levels of reduced glutathione and glutathione S-transferase activity and increased the level of lipoperoxidation and superoxide dismutase activity. The mitochondrial oxidation of NADH and succinate were partially inhibited. Complexes I and II were the main inhibition sites. Hepatic steatosis was successfully induced after 4 weeks of the diet, and the liver function was modified. The combination of 10% ethanol and sunflower seeds as an HFD produced an inexpensive model to study AHS in rats.