M.E. Muñoz

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 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.

Influence of acute ethanol administration on hepatic glutathione metabolism in the rat.

The effect of acute ethanol administration on the hepatic metabolism of glutathione was studied in male Wistar rats. Animals fasted for 18 hr received ethanol (5 g/kg body wt.) through a gastric tube as a 20% (w/v) solution in 0.154 NaCl. Four hours after administration of ethanol liver glutathione content was decreased by 21% when compared to saline-treated controls. A significant reduction (28%) was also found in gamma-glutamylcysteine synthetase activity and plasma glutathione levels were increased non significantly by 17% with respect to control rats. Glutathione S-transferase activity in the liver of ethanol-treated animals was decreased by 28% but no change was found in total glutathione peroxidase activity. The results indicate that the lowered glutathione synthesis could be an important factor contributing to the reduction of hepatic glutathione concentration following the acute ingestion of ethanol.

Effect of chronic ethanol feeding on glutathione and glutathione-related enzyme activities in rat liver

The effects of chronic ethanol consumption on liver glutathione concentrations and glutathione-related enzyme activities were studied in rats over a period of 1-9 weeks. The animals received a liquid diet containing 36% of calories as ethanol or isocaloric carbohydrate. Glutathione concentrations were significantly enhanced following ethanol intake with increases of 99% after 3 weeks and a progressive decrease thereafter. Glutathione S-transferase activity reached a maximum increase of 36% after 2 weeks of ethanol feeding. Glutathione peroxidase activity remained unchanged for the first 6 weeks of treatment, with a tendency to decrease in the last weeks of ethanol consumption. Our findings indicate that chronic ethanol administration profoundly modifies the hepatic metabolism of glutathione and may thus have important effects on the detoxification of xenobiotics by the liver.

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.

Bile pigment formation and excretion in the rabbit

Bile and plasma levels of biliverdin and bilirubin, together with the hepatic biliverdin reductase and bilirubin UDP-glucuronosyl transferase activities, were studied in the rabbit. No biliverdin could be detected in the blood plasma. The bilirubin concentration in blood was 7.81 +/- 0.79 mumol/l. Biliverdin was the predominant pigment in bile (63%). Hepatic biliverdin reductase activity was 0.086 +/- 0.016 nmol/mg protein/hr. The synthesis of bilirubin was apparently limited by the enzyme activity. Most of the bilirubin in bile was conjugated (90%) with monoconjugates predominating (75%). Hepatic UDP-glucuronosyl transferase activity was 2.65 +/- 0.18 and 1.14 +/- 0.16 mumol/mg protein/hr with and without activation, respectively.

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.

Role of glucose reabsorption from bile on hyperglycaemia-induced cholestasis in the rabbit

The effect of glucose administration on bile secretion of glucose and bile flow and composition was studied in the rabbit. After intravenous glucose infusion at 83 mumol/kg/min a mean bile concentration of 12.7 +/- 1.8 mg/dl was reached. Intraportal administration of phlorizin enhanced bile glucose concentration to 169.6 +/- 18.1 mg/dl, suggesting the presence of a system for transferring glucose from bile to liver in the biliary tree of the rabbit. A significant correlation between bile flow and plasma glucose levels could be demonstrated. A cholestatic effect appeared in glucose-infused rabbits with a decrease in bile flow by about 40% during the second hour of infusion. Both bile acid and inorganic electrolyte output were significantly lowered. Cholestasis was maintained after phlorizin administration. Possible explanations for this effect are discussed.