Ana I. Galán

EFFECTS OF AGING ON THE SUSCEPTIBILITY TO THE TOXIC EFFECTS OF CYCLOSPORIN A IN RATS. CHANGES IN LIVER GLUTATHIONE AND ANTIOXIDANT ENZYMES

Free radicals are involved in aging and cyclosporin A-induced toxicity. The age-related changes in the liver oxidative status of glutathione, lipid peroxidation, and the activity of the enzymatic antioxidant defense system, as well as the influence of aging on the susceptibility to the hepatotoxic effects of cyclosporin (CyA) were investigated in rats of different ages (1, 2, 4, and 24 months). The hepatic content of reduced glutathione (GSH) increased with aging, peaked at 4 months, and decreased in senescent rats. By contrast, glutathione disulfide (GSSG) and thiobarbituric acid-reactive substances (TBARS) concentrations and superoxide dismutase, catalase, and glutathione peroxidase activities were higher in the oldest than in the youngest rats. CyA treatment, besides inducing the well-known cholestatic syndrome, increased liver GSSG and TBARS contents and the GSSG/GSH molar ratio, and altered the nonenzymatic and enzymatic antioxidant defense systems. The CyA-induced cholestasis and hepatic depletion of GSH, and the increases in the GSSG/GSH ratio, and in GSSG and TBARS concentrations were higher in the older than the mature rats. Moreover, superoxide dismutase and catalase activities were found to be significantly decreased only in treated senescent rats. The higher CyA-induced oxidative stress, lipoperoxidation, and decreases in the antioxidant defense systems in the aged animals render them more susceptible to the hepatotoxic effects of cyclosporin.

CYCLOSPORINE A HEPATOTOXICITY: EFFECT OF PROLONGED TREATMENT WITH CYCLOSPORINE ON BILIARY LIPID SECRETION IN THE RAT

1. The effects of cyclosporine A (CyA) treatment on liver morphology, bile flow and biliary secretion of bile acid, cholesterol and phospholipid and some plasma biochemical indicators of liver function were examined.

Inhibition of biliary glutathione secretion by cyclosporine A in the rat: possible mechanisms and role in the cholestasis induced by the drug.

BACKGROUND/AIMS Biliary glutathione appears to be a major osmotic factor in the generation of bile acid-independent bile flow. This study was designed to investigate its importance in cyclosporine A-induced cholestasis in both acute and short-term-treated rats. METHODS Adult male Wistar rats were treated as follows: (i) with a single i.v. dose of cyclosporine or its vehicle (acute assays); (ii) with cyclosporine, its vehicle or physiological saline, i.p., for 7 days once per day (short-term treatment assays). Bile flow and biliary glutathione levels were determined under anesthesia both before and after intrabiliary hydrolysis of the tripeptide had been inhibited. RESULTS Acute cyclosporine administration, at a dose of 20 mg/kg, brought about an abrupt and marked fall in bile flow and bile acid secretion simultaneously with a rapid decrease in the biliary concentration and secretion rates of total, reduced and oxidized glutathione. When the rats were treated with cyclosporine A for 1 week, at a dose of 10 mg/kg per day, similar cholestatic and inhibitory effects on the biliary secretion of glutathione were noted both before and after the intrabiliary catabolism of the tripeptide had been inhibited with acivicin; in addition, the hepatic content of glutathione was also reduced. The cholestatic effect of the drug was associated with reductions in the four bile flow fractions evaluated: bile acid- and glutathione-dependent bile flow and bile acid- and glutathione-independent bile flow. CONCLUSIONS These findings indicate that cyclosporine-induced cholestasis in the rat is due not only to alterations in the hepatobiliary transport of bile acids but also to an impairment of bile formation dependent on the biliary secretion of glutathione, possibly through inhibition of the canalicular transport of the tripeptide.

Inhibition of biliary lipid and protein secretion by cyclosporine A in the rat

We investigated the effect of cyclosporine A (CyA) administered as a single i.v. dose of 20 and 40 mg/kg body wt, on biliary secretion of cholesterol, phospholipid, bile acid, and lysosomal marker and canalicular plasma membrane marker enzymes in anaesthetized Wistar rats. CyA reduced the concentration and biliary secretion of cholesterol, phospholipid and bile acid to a considerable extent; the inhibitory effect of CyA on the biliary secretion of phospholipid and bile acid was greater than that on cholesterol. The biliary outputs of acid phosphatase (AcP) and gamma-glutamyltransferase (gamma-GT) were also diminished by the drug, all these effects being dose-dependent. Maximum decreases in bile acid secretion were observed 10 min after administration, whereas those of cholesterol and phospholipid were delayed. Bile acid concentrations and secretion returned to pretest values at 30-50 min after CyA injection whereas those of cholesterol and phospholipid remained significantly reduced at this time point. The greater inhibitory effect of CyA on the biliary outputs of phospholipid and bile acid relative to cholesterol secretion together with the asynchronous fall and recovery of bile acid, cholesterol and phospholipid concentrations and secretion alter the cholesterol/bile acid, phospholipid/bile acid and cholesterol/phospholipid molar ratios as well as the lithogenic index, thus suggesting that CyA would uncouple biliary lipid secretion from bile acid secretion. Since under physiological conditions biliary lipid and gamma-GT secretion is related to and dependent upon bile acid secretion, we propose that the CyA-induced inhibition on lipid and gamma-GT secretion is, at least partly, secondary to the fall in bile acid output caused by the drug. However, since CyA inhibits secretory processes independent of the hepatobiliary flux of bile acid, such as the exocytic discharge of AcP, and because it also uncouples biliary lipid from bile acid secretion, other mechanisms and factors involved in lipid and protein secretion (such as intracellular transport, canalicular membrane fluidity and/or intracanalicular events) might also be altered by this drug.

Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study

The efficacy of long-term intake of a novel functional food supplement Funciona™ containing vitamins and juiced fruits was evaluated in order to assess the net effect of physical activity and antioxidant potentials in healthy older adult population. The long-term (2 years) and large-scale (400 older adult subjects) interventional study was based on both moderate-intensity exercise practice and concurrent supplementation. Sustained exercise-induced oxidative stress as reflected in significantly increased blood thiobarbituric acid-reactive substances (TBARS) (+15%), protein carbonyl groups (PC) (+18%) and oxidized glutathione (GSSG) (+112%) concentrations, and leukocyte 8-OHdG contents (23%). Exercise decreased the reduced/oxidized glutathione (GSH/GSSG) molar ratio (-43%) and plasma vitamin C levels (-22%). Supplementation with Funciona™ was significant in preventing oxidative damage to lipid, protein and DNA, and normalizing blood GSSG, GSH/GSSG and vitamin C levels. Thus daily intake of the antioxidant functional beverage counteracts the exercise-induced oxidative stress in free-living older subjects, and might be necessary to restore impaired antioxidant balance due long-term regular exercise.

Glutathione Metabolism In Cyclosporine A‐Treated Rats: Dose‐ And Time‐Related Changes In Liver And Kidney

1. We investigated the simultaneous effects of cyclosporine A (CsA) treatment in rats on glutathione metabolism, oxidative status and their interorgan relationship in the liver and kidney.

Effects of ursodeoxycholate on maximal biliary secretion of bilirubin in the rat

The effect of sodium ursodeoxycholate (0.5 and 1.0 mumol/min/100 g) on the maximal biliary secretion (Tm) of bilirubin and on the concentration of bilirubin in liver and plasma at the end of a bilirubin load was studied in Wistar rats. Administration of ursodeoxycholate at 0.5 mumol/min/100 g caused a 0.8-fold increase in bile flow and a significant increase in the bilirubin Tm (+24%). This was associated with a significant reduction of liver and plasma bilirubin concentrations (-16% and -17%, respectively). Bilirubin UDP-glucuronosyltransferase activity was not significantly enhanced. There was a significant increase in the biliary excretion of bilirubin conjugates (+30%) and in the diconjugates/monoconjugates ratio in bile (+31%). When ursodeoxycholate was given at 1.0 mumol/min/100 g, it produced a 1.7-fold increase in bile flow, but the bilirubin Tm was significantly reduced (-21%). Liver bilirubin concentrations were decreased (-20%) and there was a significant enhancement in total pigment concentration in plasma (+19%). Both the excretion of unconjugated bilirubin and that of bilirubin conjugates were significantly reduced (-60% and -18%, respectively). There was a significant decrease in the bilirubin-UDP glucuronosyltransferase activity and the diconjugates/monoconjugates ratio in bile (-27% and -27%, respectively). These results indicate that ursodeoxycholate is able to increase maximal bilirubin secretion only when administered at low doses and that infusion at higher rates can significantly interfere with different steps in the hepatobiliary transport of the pigment.

Cyclosporin A-Induced Cholestasis in the Rat

SummaryCyclosporin A, a powerful immunosuppressor drug, induces nephrotoxicity and hepatotoxicity. The purpose of this study was to evaluate the ability of S-adenosyl-L-methionine (SAMe) to antagonise the cyclosporin A-induced hepatotoxicity in rats treated with cyclosporin A plus SAMe. Cyclosporin A treatment for 1 or 2 weeks increases plasma bilirubin, alters some plasma biochemical indicators of hepatic and renal function, causes cholestasis and reduces the biliary concentration and secretion of bile acid and other bile components. SAMe pretreatment and simultaneous treatment with SAMe plus cyclosporin A suppresses bilirubin increases in plasma, attenuates cholestasis and totally antagonises the adverse effects of cyclosporin A on bile acid secretion. Although cyclosporin A-induced hepatotoxicity in the rat is a multifactorial phenomenon, we suggest that the hepatoprotective effects of SAMe against cyclosporin A could be related to its regulatory function of membrane lipid composition and fluidity, either alone or combined with stimulation of the hepatic synthesis of thiol compounds.

Antioxidantes, atividade física e estresse oxidativo em mulheres idosas

OBJECTIVE: To verify the influence of dietary antioxidant supplementation in older women who regularly practice physical activities, on the occurrence of oxidative stress, physical health and risk of cardiovascular diseases (CVD). METHOD: Two groups (S and C) of women, with age ranging from 60 to 80 years old, were observed. Both groups took part in a physical activity program for 58 weeks, three times a week, for about 50 to 55 minutes each session. The diet of group S (n=36) was daily supplemented with 330 ml of a functional antioxidant beverage, FuncionaTM; Group C (n=32) ingested water and was used as Control. As oxidative stress indicators, the plasmatic concentrations of reduced (GSH) and oxidized (GSSG) glutathione were determined; the molar GSH/GSSG ratio was calculated, and the oxidative damage in lipids and proteins was evaluated. The physical and cardiovascular conditions were evaluated through routine anthropometric parameters (weight, stature and BMI) and blood pressure. RESULTS: Group C presented significant increases of oxidative stress, reduction in the blood pressure and in the indicators of cardiovascular risks. Group S presented significant reduction of the oxidative stress and increment of the cardiovascular gains. Significance concerning the ergogenic effects has not been identified. CONCLUSION: Our data suggest that regular exercise in older women can improve physical and cardiovascular conditions. Moreover, daily intake of functional antioxidant supplement can minimize harmful effects of the reactive oxygen species.

Role of S-adenosylmethionine on the hepatobiliary homeostasis of glutathione during cyclosporine A treatment.

The effects of cyclosporine A (CyA) treatment on the hepatic content and biliary output of reduced (GSH) and oxidized (GSSG) glutathione and lipid peroxidation in the liver, and the ability of S-adenosylmethionine (SAMe) to antagonize the CyA-induced alterations were studied in male Wistar rats. To evaluate the efficacy of SAMe, three CyA and SAMe protocols were used: cotreatment with SAMe plus CyA, pretreatment with SAMe before starting cotreatment, and post-treatment with SAMe after beginning treatment with CyA alone. CyA treatment for one and four weeks depleted liver GSH, decreased the GSH/GSSG ratio and significantly reduced GSH and GSSG biliary concentrations and secretion rates. Additionally, long-term treatment enhanced lipid peroxidation. By contrast, when the rats were treated with CyA plus SAMe using any of the administration protocols, SAMe was seen to be efficient in antagonizing the GSH hepatic depletion, the changes in hepatic GSH/GSSG ratio and the increase induced by CyA in lipid peroxidation. Furthermore, SAMe also abolished the effects of CyA on the biliary secretion rates of GSH and GSSG. The efficacy of SAMe was similar, regardless of the administration protocols used. In conclusion, our results clearly demonstrate that SAMe is good for preventing, antagonizing and reversing the CyA-induced alterations in the hepatobiliary homeostasis of glutathione.ResumenSe estudian los efectos del tratamiento con ciclosporina A (CyA) sobre el contenido hepático y la concentración y secreción biliar de glutatión reducido (GSH) y oxidado (GSSG), y los niveles hepáticos de TBARS, y la capacidad de la S-adenosilmetionina (SAMe) para antagonizar las alteraciones inducidas por la CyA en ratas Wistar. Con objeto de evaluar la eficacia de la SAMe, se han utilizado tres tipos de protocolos de administración: cotratamiento o tratamiento simultáneo con CyA y SAMe, pretratamiento con SAMe antes de iniciar el cotratamiento, y post-tratamiento con SAMe después de iniciar el tratamiento con CyA. El tratamiento durante un periodo corto (una semana) o largo (cuatro semanas) con CyA depleciona el GSH, disminuye la relación GSH/GSSG y reduce significativamente la concentración y secreción biliar de GSH y GSSG. Además, el tratamiento largo aumenta la peroxidación lipídica. Por el contrario, cuando los animales se tratan con CyA y SAMe siguiendo cualquiera de los protocolos de administración, la SAMe muestra ser eficaz para antagonizar la depleción hepática de GSH, normalizar la relación GSH/GSSG y suprimir el incremento inducido por la CyA sobre la peroxidación lipídica. Además, la SAMe normaliza los efectos de la CyA sobre la secreción biliar de GSH y GSSG. La eficacia de la SAMe es similar en todos los tipos de protocolos de tratamiento utilizados. En resumen, nuestros resultados demuestran claramente que la SAMe es eficaz para prevenir, antagonizar y revertir las alteraciones inducidas por la CyA en la homeostasis hepatobiliar del glutatión.