Study the effect of 3,4-Methylenedioxy methamphetamine on cytochrome P450 2E1 activity

Abstract Evaluating the effects of ecstasy on CYP2E1 activity is of great concern, mainly due to growing trends in abuse and co-administration of MDMA with ethanol and the dominant role of this isoenzyme on ethanol metabolism. This study aimed to evaluate the effects of MDMA on CYP2E1 activity. A total of 24 male rats were selected and divided into three groups. The first and second groups consisted of 12 rats and were employed to optimize the perfusion method, and the third group was employed for studying the alteration of CYP2E1 activity after liver exposure to MDMA (300 and 600 ng/ml). The amount of chlorzoxazone and 6-hydroxy chlorzoxazone in a sample obtained from liver perfusion before and after exposure to a buffer containing MDMA was determined by HPLC-FL. The enzymatic activity of rat CYP2E1 decreased after liver perfusion with a buffer containing 600 ng/ml of MDMA. However, no significant changes were observed in chlorzoxazone and 6-hydroxy chlorzoxazone concentration in perfusate before and after liver perfusion with a buffer containing 300 ng/ml of MDMA. Our findings suggest that the activity of CYP2E1 in rats might decrease only after administration of MDMA at a lethal dose. However, further animal and human studies are needed to confirm our assumption.

The photodynamic and intrinsic effects of Azure B on mitochondrial bioenergetics and the consequences of its intrinsic effects on hepatic energy metabolism.

BACKGROUND: The present study aimed to characterize the intrinsic and photodynamic effects of azure B (AB) on mitochondrial bioenergetics, as well as the consequences of its intrinsic effects on hepatic energy metabolism. METHODS: Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. RESULTS: AB interacted with mitochondria regardless of photostimulation, but its binding degree was reduced by mitochondrial energization. Under photostimulation, AB caused lipid peroxidation and protein carbonylation and decreased the content of reduced glutathione (GSH) in mitochondria. AB impaired mitochondrial bioenergetics in at least three distinct ways: (1) uncoupling of oxidative phosphorylation; (2) photoinactivation of complexes I and II; and (3) photoinactivation of the FoF1-ATP synthase complex. Without photostimulation, AB also demonstrated mitochondrial toxicity, which was characterized by the induction of lipid peroxidation, loss of inner mitochondrial membrane integrity, and uncoupling of oxidative phosphorylation. The perfused rat liver experiments showed that mitochondria were one of the major targets of AB, even in intact cells. AB inhibited gluconeogenesis and ureagenesis, two biosynthetic pathways strictly dependent on intramitochondrially generated ATP. Contrariwise, AB stimulated glycogenolysis and glycolysis, which are required compensatory pathways for the inhibited oxidative phosphorylation. Similarly, AB reduced the cellular ATP content and the ATP/ADP and ATP/AMP ratios. CONCLUSIONS: Although the properties and severe photodynamic effects of AB on rat liver mitochondria might suggest its usefulness in PDT treatment of liver tumors, this possibility should be considered with precaution given the toxic intrinsic effects of AB on mitochondrial bioenergetics and energy-linked hepatic metabolism.

Metabolic activation enhances the cytotoxicity, genotoxicity and mutagenicity of two synthetic alkaloids with selective effects against human tumour cell lines.

The pharmacological potential of drugs must be evaluated to establish their potential therapeutic benefits and side effects. This evaluation includes assessment of the effects of hepatic enzymes that catalyse their metabolic activation. Previously, our research group synthesized and characterized a set of synthetic 3-alkyl pyridine alkaloid (3-APA) analogues that cause in vitro cytotoxic, genotoxic, and mutagenic effects in various human cancer cell lines. The present study aimed to evaluate these activities with the two most promising synthetic 3-APAs (3-APA 1 and 3-APA 2) against cell lines derived from breast cancer (MDA-MB-231), ovarian cancer (TOV-21 G) and lung fibroblasts (WI-26-VA4) with and without metabolic activation (S9 fraction). The cytotoxicity of the compounds was evaluated employing MTT and clonogenic assays. In addition, comet assays, γH2AX immunocytochemistry labelling assays and cytokinesis-block micronucleus tests were carried out to evaluate the potential of these compounds to induce chromosomal damage. The results obtained in the MTT assay showed that compound 3-APA 2 exhibited high selectivity index (SI) values (ranging between 21.0 and 92.6). In addition, the cytotoxicity of the compounds was clearly enhanced by metabolic activation. Moreover, both compounds were genotoxic and induced double-strand breaks in DNA and chromosomal lesions with and without S9. The cancer cell lines tested showed higher genotoxic sensitivity to the compounds than did the non-tumour cell line used as a reference. The genotoxic and mutagenic effects of the compounds were potentiated in experiments with metabolic activation. The data obtained in this study indicate that compound 3-APA 2 is more active against the human cancer cell lines tested, both with and without metabolic activation, and can therefore be considered a candidate drug to treat human ovarian and breast cancer.

Effects of Moringa oleifera Leaf Extract on Diabetes-Induced Alterations in Paraoxonase 1 and Catalase in Rats Analyzed through Progress Kinetic and Blind Docking.

In our study, we aimed to evaluate the effects of Moringa oleifera leaves extract on rat paraoxonase 1 (rPON1) and catalase (rCAT) activities in alloxan-induced diabetic rats. Our study included three groups; group C (control, n = 5); group D (diabetic, n = 5); and group DM (M. oleifera extract-supplemented diabetic rats, n = 5). Daily oral administration of M. oleifera extract at 200 mg/kg doses produced an increase in endogenous antioxidants. Serum rPON1 (lactonase) and liver cytosol catalase activities were determined by a spectrophotometric assay using progress curve analysis. We found a decrease in the Vm value of rPON1 in diabetic rats, but dihydrocoumarin (DHC) affinity (Km) was slightly increased. The value of Vm for the DM group was found to be reduced approximately by a factor of 3 compared with those obtained for group C, whereas Km was largely changed (96 times). Catalase activity was significantly higher in the DM group. These data suggest that the activation of rPON1 and rCAT activities by M. oleifera extracts may be mediated via the effect of the specific flavonoids on the enzyme structure. In addition, through molecular blind docking analysis, rPON1 was found to have two binding sites for flavonoids. In contrast, flavonoids bound at four sites in rCAT. In conclusion, the data suggest that compounds from M. oleifera leaves extract were able to influence the catalytic activities of both enzymes to compensate for the changes provoked by diabetes in rats.

Glycemic homeostasis and hepatic metabolism are modified in rats with global cerebral ischemia.

Cerebral ischemia-induced hyperglycemia has been reported to accentuate neurological damage following focal or global cerebral ischemia. Hyperglycemia found in rats following focal brain ischemia occurs in the first 24 h and has been claimed to be caused by increased liver gluconeogenesis and insulin resistance. However, liver gluconeogenesis and the mechanisms leading to hyperglycemia after global cerebral ischemia remain uncertain. This study investigated the glycemic homeostasis and hepatic metabolism in rats after transient four-vessel occlusion (4-VO)-induced global cerebral ischemia, an event that mimics to a certain degree the situation during cardiac arrest. Several metabolic fluxes were measured in perfused livers. Activities and mRNA expressions of hepatic glycolysis and glyconeogenesis rate-limiting enzymes were assessed as well as respiratory activity of hepatic isolated mitochondria. Global cerebral ischemia was associated with hyperglycemia and hyperinsulinemia 24 h after ischemia. Insulin resistance developed later and was prominent after the 5th day. Hepatic anabolism and catabolism were both modified in a complex and time-dependent way. Gluconeogenesis, ß-oxidation, ketogenesis and glycolysis were diminished at 24 h after ischemia. At 5 days after ischemia glycolysis had normalized, but gluconeogenesis, ketogenesis and ß-oxidation were accelerated. The overall metabolic modifications suggest that a condition of depressed metabolism was established in response to the new conditions generated by the cerebral global ischemia. Whether the modifications in the liver metabolism found in rats after the ischemic insult can be translated to individuals following global brain ischemia remains uncertain, but the results of this study are hoped to encourage further investigations.

Acidic and hepatic derivatives of bioactive clerodane diterpenes casearins J and O.

Clerodane diterpenes from Casearia sylvestris are antiulcerogenic and anti-inflammatory. The finding that they may undergo acid degradation or hepatic metabolization led to an investigation of their degradation products. Purified clerodane diterpenes (casearins J and O) were subjected to in vitro assays to simulate their oral administration. Resulting derivatives were identified using chromatographic and spectrometric techniques. Nitric oxide synthesis by LPS-stimulated macrophages was assayed to verify whether structural modifications alter the anti-inflammatory activity of diterpenes. Nine compounds (1-9) were identified after acid degradation remaining 5.05% of casearin J. Besides the remaining casearin O (13.1%), eight compounds (10-17) were identified. The dialdehydes from each casearin were the major constituents. S9 rat liver treatment of casearins J and O generated two compounds identical to some of those produced by acid degradation, which remained 36.8% and 36.5% intact, respectively. Both casearins and its derivatives were not cytotoxicity at concentrations lower than 0.312 µg/mL (0.555 µM for casearin J and 0.516 µM for casearin O) and did not inhibit the nitric oxide production in this concentration. Thus, the structural modifications conducted did not alter the activity of casearins and the anti-inflammatory pathway of diterpenes probably is not involved on nitric oxide modulation.

Inclusion of memory effects in a dynamic model of electroporation in biological tissues.

This article presents experimental and computational results of electroporation in rat liver. The experiments were performed using different forms of electrodes and waveforms of applied electric pulses. For the numerical simulation, the electroporation model proposed by Ramos and Weinert in a previous publication was used. Dynamic adjustments were used for obtaining a good modeling of the electric current. A single set of model parameters was obtained to fit the simulated current response for different waveforms and electrodes. These parameters were obtained with the use of a genetic algorithm that minimized the error between the simulated and experimental currents. The electroporation model with dynamic adjustment proved to be an appropriate simulation tool to predict the tissue conductivity during stimulation by intense electrical fields.

Fatty acids uptake and oxidation are increased in the liver of rats with adjuvant-induced arthritis.

Severe rheumatoid cachexia is associated with pronounced loss of muscle and fat mass in patients with advanced rheumatoid arthritis. This condition is associated with dyslipidemia and predisposition to cardiovascular diseases. Circulating levels of triglycerides (TG) and free fatty acids (FFA) have not yet been consistently defined in severe arthritis. Similarly, the metabolism of these lipids in the arthritic liver has not yet been clarified. Aiming at filling these gaps this study presents a characterization of the circulating lipid profile and of the fatty acids uptake and metabolism in perfused livers of rats with adjuvant-induced arthritis. The levels of TG and total cholesterol were reduced in both serum (10-20%) and liver (20-35%) of arthritic rats. The levels of circulating FFA were 40% higher in arthritic rats, possibly in consequence of cytokine-induced adipose tissue lipolysis. Hepatic uptake and oxidation of palmitic and oleic acids was higher in arthritic livers. The phenomenon results possibly from a more oxidized state of the arthritic liver. Indeed, NADPH/NADP+ and NADH/NAD+ ratios were 30% lower in arthritic livers, which additionally presented higher activities of the citric acid cycle driven by both endogenous and exogenous FFA. The lower levels of circulating and hepatic TG possibly are caused by an increased oxidation associated to a reduced synthesis of fatty acids in arthritic livers. These results reveal that the lipid hepatic metabolism in arthritic rats presents a strong catabolic tendency, a condition that should contribute to the marked cachexia described for arthritic rats and possibly for the severe rheumatoid arthritis.

Experimental bio-artificial liver: Importance of the architectural design on ammonia detoxification performance.

AIM: To determine the influence of the construction design over the biological component's performance in an experimental bio-artificial liver (BAL) device. METHODS: Two BAL models for liver microorgans (LMOs) were constructed. First, we constructed a cylindrical BAL and tested it without the biological component to establish its correct functioning. Samples of blood and biological compartment (BC) fluid were taken after 0, 60, and 120 min of perfusion. Osmolality, hematocrit, ammonia and glucose concentrations, lactate dehydrogenase (LDH) release (as a LMO viability parameter), and oxygen consumption and ammonia metabolizing capacity (as LMO functionality parameters) were determined. CPSI and OTC gene expression and function were measured. The second BAL, a "flat bottom" model, was constructed using a 25 cm2 culture flask while maintaining all other components between the models. The BC of both BALs had the same capacity (approximately 50 cm3) and both were manipulated with the same perfusion system. The performances of the two BALs were compared to show the influence of architecture. RESULTS: The cylindrical BAL showed a good exchange of fluids and metabolites between blood and the BC, reflected by the matching of osmolalities, and glucose and ammonia concentration ratios after 120 min of perfusion. No hemoconcentration was detected, the hematocrit levels remained stable during the whole study, and the minimal percentage of hemolysis (0.65% ± 0.10%) observed was due to the action of the peristaltic pump. When LMOs were used as biological component of this BAL they showed similar values to the ones obtained in a Normothermic Reoxygenation System (NRS) for almost all the parameters assayed. After 120 min, the results obtained were: LDH release (%): 14.7 ± 3.1 in the BAL and 15.5 ± 3.2 in the NRS (n = 6); oxygen consumption (µmol/min·g wet tissue): 1.16 ± 0.21 in the BAL and 0.84 ± 0.15 in the NRS (n = 6); relative expression of Cps1 and Otc: 0.63 ± 0.12 and 0.67 ± 0.20, respectively, in the BAL, and 0.86 ± 0.10 and 0.82 ± 0.07, respectively, in the NRS (n = 3); enzymatic activity of CPSI and OTC (U/g wet tissue): 3.03 ± 0.86 and 222.0 ± 23.5, respectively, in the BAL, and 3.12 ± 0.73 and 228.8 ± 32.8, respectively, in the NRS (n = 3). In spite of these similarities, LMOs as a biological component of the cylindrical BAL were not able to detoxify ammonia at a significant level (not detected vs 35.1% ± 7.0% of the initial 1 mM NH4 + dose in NRS, n = 6). Therefore, we built a second BAL with an entirely different design that offers a flat base BC. When LMOs were placed in this "flat bottom" device they were able to detoxify 49.3% ± 8.8% of the initial ammonia overload after 120 min of perfusion (n = 6), with a detoxification capacity of 13.2 ± 2.2 µmol/g wet tissue. CONCLUSION: In this work, we demonstrate the importance of adapting the BAL architecture to the biological component characteristics to obtain an adequate BAL performance.

Neonatal treatment with fluoxetine improves mitochondrial respiration and reduces oxidative stress in liver of adult rats.

Recent studies have shown that exposure to fluoxetine treatment induces excessive production of ROS, and alters the antioxidant defense system in various tissues and cell types, mainly the liver. When fluoxetine is administered intraperitoneally, the drug rapidly reaches high concentrations in the liver, has potentially multiple toxic effects on energy metabolism in rat liver mitochondria. The aim of this study was to evaluate the effect of pharmacological treatment with fluoxetine during critical period for development on the mitochondrial bioenergetics and oxidative stress in liver of rat adult. To perform this study, the rat pups received Fx, or vehicle (Ct) from postnatal day 1 to postnatal day 21 (ie, during lactation period). We evaluated mitochondrial oxygen consumption, respiratory control ratio, ROS production, mitochondrial swelling by pore opening, oxidative stress biomarkers, and antioxidant defense in liver of rats at 60 days of age. Our studies have shown, that treatment with Fx during the lactation period resulted in reduced body mass gain, improvement of the mitochondrial respiratory capacity, induced higher mitochondrial resistance to calcium ion preventing the mitochondrial permeability transition pore opening, as well as decreased oxidative stress biomarkers, and increased the SH levels and enzymes antioxidant activities (SOD, CAT, GST) in liver of treated rats at 60 days of age. These findings suggest that pharmacological treatment with fluoxetine during critical period of development result in positive changes in liver of rats, as improvement of the mitochondrial bioenergetics and hepatic oxidative metabolism that persist in adulthood.

Identification of biotransformation products of disperse dyes with rat liver microsomes by LC-MS/MS and theoretical studies with DNA: Structure-mutagenicity relationship using Salmonella/microsome assay.

Azo dyes are known as a group of substances with DNA damage potential that depend on the nature and number of azo groups connected to aromatic rings (benzene and naphthalene), chemical properties, e.g. solubility and reactive functional groups, which significantly affect their toxicological and ecological risks. In this paper, we used in vitro models to evaluate the metabolism of selected textile dyes: Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) and Disperse Red 167 (DR 167). To evaluate the mutagenic potential of the textile dyes, the Salmonella mutagenicity assay (Ames test) with strains TA 98 and TA 100 in the presence and absence of the exogenous metabolic system (S9) was used. DR73 was considered the most mutagenic compound, inducing both replacement base pairs (TA 100) and also changing frameshift (TA 98) mutations that are reduced in the presence of the S9 mixture. Furthermore, we used rat liver microsomes in the same experimental conditions of the S9 mixture to metabolize the dyes and the resultant solutions were analyzed using a liquid chromatography coupled to a quadrupole linear ion trap mass spectrometry (LC-MS/MS) to investigate the metabolites formed by the in vitro biotransformation. Based on this experiment, we detected and identified two biotransformation products for each textile dye substrate analyzed. Furthermore, to evaluate the interaction and reactivity of these compounds with DNA, theoretical calculations were also carried out. The results showed that the chemical reaction occurred preferentially at the azo group and the nitro group, indicating that there was a reduction in these groups by the CYP P450 enzymes presented in the rat microsomal medium. Our results clearly demonstrated that the reduction of these dyes by biological systems is a great environmental concern due to increased genotoxicity for the body of living beings, especially for humans.

Beneficios de la fibra dietética y poder antioxidante del salvado de arroz en ratas deficientes en Vitamina E / Benefits of fiber and antioxidant power of rice bran in Vitamin E deficient rats / Beneficios da fibra alimentar e poder antioxidante do farelo de arroz em ratos deficientes em Vitamina E

Las recomendaciones de consumo de fibra no se cumplen y hay una necesidad por alimentos con fibra. El salvado de arroz (SA) tiene fibra y propiedades antioxidantes. Aquí se evaluaron estas propiedades en ratas suficientes (+) y deficientes (-) en Vitamina E (VitE) con o sin SA. Las ratas fueron divididas en cuatro grupos. Dos consumieron dietas +VitE y uno tenía SA. Los restantes consumieron dietas -VitE y uno tenía SA. El consumo de alimento, su eficiencia y el crecimiento, fueron similares entre los 4 grupos pero la masa fecal húmeda o seca fue 3 veces superior en los SA+. La hemoglobina en sangre y el hierro hepático fueron similares entre grupos, pero en los grupos (SA-) la VitE hepática fue 10 veces menor en las ratas -VitE que en las +VitE. Sin embargo, en las ratas -VitE/SA+, la VitE hepática fue sólo 2,6 veces menor. Este efecto del SA también se detectó en los eritrocitos, ya que la catalasa y la glutatión reductasa aumentaron en el grupo -VitE/SA-pero no en el grupo -VitE/SA+. El estudio muestra que SA no interfirió con el crecimiento y el metabolismo del hierro, sino que tuvo un efecto laxante y previno parcialmente la deficiencia de VitE.

Dietary fiber requirements are met by only a small fraction of the population. There is need for supplemented foods to fill this gap. Rice bran (RB) is high in fiber and has antioxidant properties. The effects of rice bran fiber on several metabolic indicators and the antioxidant capacity of rice bran in rats was reported. Rats were divided into 4 dietary groups: Vitamin E-sufficient with (+VitE/RB+) or without (+VitE/RB-) rice bran; Vitamin E-deficient with (-VitE/RB+) or without (-VitE/RB-) rice bran. Food intake, growth and feed efficiency were similar in all groups but wet and dry fecal mass of the RB+ groups were 3 times higher than the RB- groups. Blood hemoglobin and liver iron were also similar among all groups. However, the liver VitE concentration of the rats of (-VitE/RB-) group was 10x lower than the (+VitE/RB-) group. In contrast, liver VitE of the rats (-VitE/RB+) was only 2.6x lower. This effect of RB was also seen in erythrocytes since, catalase and glutathione reductase increased in the VitE deficient rats but RB prevented this increase. This study shows that dietary RB did not interfere with growth, feed efficiency and iron metabolism, it provided dietary fiber and laxation and partially prevented VitE deficiency.

As recomendações de ingestão de fibras não são cumpridos e existe uma necessidade de alimentos ricos em fibras. Farelo de arroz (FA) tem fibra e propriedades antioxidantes. Aqui, estas propriedades foram avaliadas em ratos suficientes (+) e pobres (-) em Vitamina E (VitE) com ou sem FA. Os ratos foram divididos em 4 grupos. Dois consumiram dietas +VitE e um tinha FA. Os restantes consumiram dietas -ViteE e um tinha FA. O consumo de alimento, sua eficiência e crescimento foram semelhantes entre os 4 grupos, mas nos grupos (FA-) a VitE hepática foi 10 vezes menor nos ratos -VitE que nos +VitE. Entretanto, nos ratos -VitE/FA+, a VitE hepática foi apenas 2,6 vezes menor. Este efeito do FA também foi detectado nos eritrócitos, visto que catalase e glutationa redutase aumentaram no grupo -VitE/FA-, mas não no grupo -VitE/FA+. O estudo mostra que FA não interferiu no crescimento ou no metabolismo do ferro, porém teve um efeito laxante e impediu parcialmente a deficiência de VitE.

Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver.

AIM: To develop a simplified bioartificial liver (BAL) device prototype, suitable to use freshly and preserved liver Microorgans (LMOs) as biological component. METHODS: The system consists of 140 capillary fibers through which goat blood is pumped. The evolution of hematocrit, plasma and extra-fiber fluid osmolality was evaluated without any biological component, to characterize the prototype. LMOs were cut and cold stored 48 h in BG35 and ViaSpan® solutions. Fresh LMOs were used as controls. After preservation, LMOs were loaded into the BAL and an ammonia overload was added. To assess LMOs viability and functionality, samples were taken to determine lactate dehydrogenase (LDH) release and ammonia detoxification capacity. RESULTS: The concentrations of ammonia and glucose, and the fluids osmolalities were matched after the first hour of perfusion, showing a proper exchange between blood and the biological compartment in the minibioreactor. After 120 min of perfusion, LMOs cold preserved in BG35 and ViaSpan® were able to detoxify 52.9% ± 6.5% and 53.6% ± 6.0%, respectively, of the initial ammonia overload. No significant differences were found with Controls (49.3% ± 8.8%, P < 0.05). LDH release was 6.0% ± 2.3% for control LMOs, and 6.2% ± 1.7% and 14.3% ± 1.1% for BG35 and ViaSpan® cold preserved LMOs, respectively (n = 6, P < 0.05). CONCLUSION: This prototype relied on a simple design and excellent performance. It's a practical tool to evaluate the detoxification ability of LMOs subjected to different preservation protocols.

The effect of a hydrogen sulfide releasing molecule (Na2S) on the cold storage of livers from cardiac dead donor rats. A study in an ex vivo model.

Liver transplantation is currently the preferred treatment option for end-stage liver disease. Donation after cardiac death was a common practice in the early years of organ donation before brain death criteria were established. Those organs were subjected to variable periods of warm ischemia that might intensify cold ischemia/reperfusion injuries. In the present, shortage of brain dead donors has led to the reassessment of organ donation after cardiac death. Since many cytoprotective roles have been describe for H2S during ischemia/reperfusion on a variety of tissues, we hypothesized that graft exposure to this bioactive gas might improve preservation of non-heart beating donated organs. Therefore, to establish a rat model of donation post-cardiac arrest and using this approach to judge H2S delivery effects on graft hypothermic preservation, were the main objectives of this investigation. Cardiopulmonary arrest was induced in sedated rats by overload of potassium (K(+)). Livers were surgically removed and subsequently stored in HTK Solution (Histidine-tryptophan-ketoglutarate) at 0-4°C. After 24 h of hypothermic preservation, livers were rewarmed in an ex vivo model. Three experimental groups were established as follows: I--Livers procured before cardiac death and cold stored 24 h in HTK (BCD); II--Livers procured after cardiac death (45 min) and cold stored 24 h in HTK (ACD); III--Livers procured after cardiac death (45 min) and cold stored 24 h in HTK+10 µM Sodium Sulfide (Na2S) (ACD-SS). Data suggest that after 45 min of warm ischemia, viability parameters assessed during reperfusion in the ex vivo model were significantly impaired. Real time PCR revealed that after ex vivo reperfusion there is an increased expression of HO-1 and TNF-α and a modest drop in Bcl-2 mRNA, which could be interpreted as the cellular response to the hypoxic insult sustained during warm ischemia. On the other hand, warm ischemic livers exposed to H2S during cold storage, improved microcirculation, morphology and viability parameters during ex vivo reperfusion and showed significant modulation of HO-1 mRNA expression. In conclusion, HTK supplementation with Na2S arose as a potential treatment to recover non-heart beating harvested organs. Furthermore, an appropriate model of cardiac dead liver donors was successfully developed.

Soybean flour induces a greater increase of the antioxidant defenses in rats fed with a normocaloric diet compared with a hypercaloric diet.

BACKGROUND: Soybeans, due to their antioxidant properties, present beneficial health effects. The objective was to evaluate if replacing casein with soy flour, modifies antioxidant defenses in rat liver, compared to animals that continued being fed with casein based diets (normocaloric and hypercaloric). RESULTS: Four groups of rats were used: CC (control casein), CS (control soy), HC (hypercaloric casein) and HS (hypercaloric soy). Malondialdehyde, in serum and liver, did not present differences. In liver, when comparing CS vs. CC: increased superoxide dismutase 1 (P < 0.001), catalase (P < 0.01) and glutathione reductase (P < 0.05) activities, the total glutathione (P < 0.001) and reduced glutathione (P < 0.05) content and decreased oxidized glutathione content (P < 0.05). In HS vs. HC: increased carbonyl groups (P < 0.01) and superoxide dismutase 1 activity (P < 0.05), and decreased glutathione peroxidase activity (P < 0.01), total glutathione (P < 0.05) and oxidized glutathione content (P < 0.001). In HS vs. CS: decreased glutathione reductase activity (P < 0.01), total glutathione (P < 0.001) and reduced glutathione (P < 0.01) content, and increased oxidized glutathione content (P < 0.05). CONCLUSION: Replacing casein by soybean flour improves antioxidant defenses, mainly in normocaloric diets.

Biotransformation of isoimperatorin by rat liver microsomes and its quantification by LC-MS/MS method.

The aim of the present research was to establish a comprehensive strategy to identify the metabolites of isoimperatorin after biotransformation with rat liver microsomes in vitro, and further describe metabolic kinetic characteristics of isoimperatorin and its main metabolites. Utilizing liquid chromatography with time of flight mass spectrometry (LC-TOF-MS), 18 metabolites (M 1-18) were characterized according to the typical fragment ions and literature data. Among them, M-2, 3, 5, 9, 10, and 15 were new compounds. To further verify structures of the metabolites, five main metabolites were obtained from the magnifying biotransformation incubation system, and their chemical structures were elucidated as 8-hydroxyoxypeucedanin (M-3), hydroxypeucedanin hydrate (M-4), E-5-(4-hydroxy-3-methyl-2-alkenyloxy)-psoralen (M-11), Z-5-(4-hydroxy-3-methyl-2-alkenyloxy)-psoralen (M-12), and oxypeucedanin (M-16) by various spectroscopy methods including IR, MS and NMR. A simple new liquid chromatography with triple quadrupole tandem mass spectrometry (LC-QqQ-MS) method was developed for the simultaneous determination of isoimperatorin and its main metabolites. The analysis was performed on a Diamonsil™ ODS C18 column with acetonitrile-water containing 0.1% formic acid as mobile phase. Total run time was 20.0 min. The results suggested that the method we exhibited was successfully applied for analysis of isoimperatorin and its metabolites. The study provides essential data for proposing metabolite pathway and further pharmacological study of isoimperatorin.

Proteomic analysis of the regenerating liver following 2/3 partial hepatectomy in rats

BACKGROUND: Liver regeneration (LR) after 2/3 partial hepatectomy (PH) is one of the most studied models of cell, organ, and tissue regeneration. Although the transcriptional profile analysis of regenerating liver has been carried out by many reserachers, the dynamic protein expression profile during LR has been rarely reported up to date. Therefore, this study aims to detect the global proteomic profile of the regenerating rat liver following 2/3 hepatectomy, thereby gaining some insights into hepatic regeneration mechanism. RESULTS: Protein samples extracted from the sham-operated and the regenerating rat livers at 6, 12, 24, 72, 120 and 168 h after PH were separated by IEF/SDS-PAGE and then analyzed by MALDI-TOF/TOF mass spectrometry. Compared to sham-operated groups, there were totally 220 differentially expressed proteins (including 156 up-regulated, 62 down-regulated, and 2 up/down-regulated ones) identified in the regenerating rat livers, and most of them have not been previously related to liver regeneration. According to the expression pattern analysis combined with gene functional analysis, it showed that lipid and carbohydrate metabolism were enhanced at the early phase of LR and continue throughout the regeneration process. Ingenuity Pathway Analysis indicated that YWHAE protein (one of members of the 14-3-3 protein family) was located at the center of pathway networks at all the timepoints after 2/3 hepatectomy under our experimental conditions, maybe suggesting a central role of this protein in regulating liver regeneration. Additionally, we also revealed the role of Cdc42 (cell division cycle 42) in the termination of LR. CONCLUSIONS: For the first time, our proteomic analysis suggested an important role of YWHAE and pathway mediated by this protein in liver regeneration, which might be helpful in expanding our understanding of LR amd unraveling the mechanisms of LR.

Effect of different provenances and production conditions on antioxidant properties in Buddleja globosa leaves / Efecto de diferentes procedencias y condiciones de cultivo sobre propiedades antioxidantes en hojas de Buddleja globosa

Buddleja globosa Hope (matico) is a medicinal shrub native to Chile whose leaves have been traditionally used for wound and ulcer healing, pathologies associated to oxidative stress. Matico leaves display a high content of polyphenols, compounds with recognized antioxidant capacity, which may contribute to its therapeutic properties. Several factors, however, can modify the polyphenol content of matico leaf extracts, including plant material, production techniques, provenances, leaf age, harvest time, irrigation, and desiccation procedures. Thus, standardized leaf extracts prepared with plants from different provenances and harvest conditions were compared in terms of polyphenol content and their protecting antioxidant effects on rat liver microsomal lipids and thiol groups. All factors tested, but irrigation, changed both polyphenol content and antioxidant properties of matico extracts; water stress only affected their antioxidant properties without changing their polyphenol content. Correlation between polyphenol content and lipid peroxidation inhibition was only significant in the provenance study.

Buddleja globosa Hope (matico) es un arbusto medicinal nativo de Chile cuyas hojas han sido utilizadas en la medicina tradicional como cicatrizante en caso de patologías relacionadas con el estrés oxidativo. Las hojas tienen un alto contenido de polifenoles, compuestos con reconocidos efectos antioxidantes relacionados con la inhibición de la lipoperoxidación. Varios factores pueden afectar a su contenido, entre ellos el origen de la planta, edad de la hoja, momento de cosecha, riego y métodos de secado. En extractos estandarizados preparados de hojas de diferentes tratamientos se compararon el contenido de polifenoles y los efectos antioxidantes protectores de lipidos y grupos tioles microsomales. Todos los ensayos de cultivo y postcosecha mostraron diferencias significativas entre los tratamientos, excepto para la inhibición de lipoperoxidación en el tratamiento de riego. Plantas de diferente origen muestran que el contenido de polifenoles en las hojas es determinado genéticamente y sufre variaciones por efectos ambientales.

Positive effects of Green Tea on hepatic dysfunction, lipid peroxidation and antioxidant defence depletion induced by cadmium

Cadmium (Cd) is a highly toxic environmental and industrial cumulative pollutant that affects many organs,especially the liver. The present study was designed to evaluate the antioxidant effect of green tea oncadmium-induced hepatic dysfunction and oxidative stress in rats. Adult male Wistar rats were administeredcadmium by injection of 20 ìmoles /kg bw/ every 3 days for six months. This study revealed significant (p <0.05) liver dysfunction, lipid peroxidation and a decline in antioxidant enzyme activities in the liver of cadmium-treated rats compared to control animals. Compared to control rats, the activities of lactate dehydrogenase (LDH), gammaglutamyl transferase (GGT), acid phosphatase (PAC), phosphatase alkaline (PAL), as well as bilirubin and thiobarbituric acid-reactive substances (TBARs), were significantly (p < 0.05)increased in Cd-treated rats. Moreover, antioxidant enzyme activities, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase, were significantly (p < 0.05) decreased in the liver of cadmiumtreatedrats. The oral administration of 5% aqueous green tea extract, along with cadmium treatment for six months, caused a significant (p < 0.05) improvement in cadmium-induced toxicity by significantly decreasing(p < 0.05) the activities of enzymatic markers of liver dysfunction (LDH, GGT, PAC, PAL activities, as well as the bilirubin rate). Indeed, green tea extract significantly increased (p < 0.05) antioxidant enzymatic activities (SOD, Catalase, GPX) in rat liver, compared to those given cadmium alone. Thus, the oral administration of green tea, along with cadmium significantly (p < 0.05) improves cadmium-induced liverdysfunction and stress oxidant in rats’ liver.

Differentially expressed genes in the liver of thioacetamide treated rats

Thioacetamide is a hepatotoxic and hepatocarcinogenic compound that affects liver metabolism, inhibits mRNA transport and induces enlargement of the nucleolus. To investigate the effect of thioacetamide at the molecular level, differential display RT-PCR was conducted. Analysis of nineteen differentially expressed genes demonstrated that ten cDNAs have their expression inhibited while the other nine were positively affected by thioacetamide. Two of the cDNAs were homologous to known genes-TAP and ankyrin-binding glycoprotein-1, two corresponded to repetitive sequences and seven were homologous to expressed sequence tags. The differential expression of some of the isolated cDNAs was confirmed by northern hybridization. It is proposed that since the product of TAP is involved in mRNA transport, thioacetamide inhibition of TAP expression might, at least partially, explain the thioacetamide-induced swelling of the nucleolus.