N-acetylcysteine protects from glutathione depletion in rats exposed to hyperoxia.

BACKGROUND: N-acetylcysteine (NAC) may protect against oxidative injury by providing cysteine for glutathione (GSH) biosynthesis or by direct reactions with electrophiles. We have recently shown that hyperoxic exposure of rats prior to liver perfusion is associated with significant decreases in hepatic GSH and significant changes in biliary amino acid concentrations. We hypothesized that NAC administration during hyperoxic exposure would prevent depletion of hepatic GSH by providing cysteine for GSH biosynthesis. METHODS: NAC was administered during two conditions known to induce GSH depletion: hyperoxic exposure and biochemical inhibition of GSH synthesis using buthionine sulfoximine (BSO). After 48 hours, GSH concentrations in bile, liver and perfusate and biliary amino acid concentrations were determined using isolated perfused liver preparations. RESULTS: Administration of NAC to rats maintained in normoxic or hyperoxic conditions, prior to liver perfusion, resulted in dose-dependent increases in GSH concentrations in bile, liver and perfusate, increases in bile flow rates and changes in biliary amino acid concentrations. When BSO was given concurrently with NAC in normal or hyperoxic conditions, these effects were not observed, and oxidant stress was evident. CONCLUSIONS: NAC prevents oxidant stress during hyperoxic exposure, most likely by supplying cysteine as a precursor for GSH synthesis.

Hyperoxia and glutathione depletion in the isolated perfused rat liver.

BACKGROUND: Hepatic stores of glutathione may be depleted by hyperoxic exposure or poor nutritional status. We studied the effects of hyperoxia or hepatic glutathione depletion on bile flow rates, and on biliary concentrations of glutathione and amino acids. METHODS: Glutathione depletion was induced in vivo by 1) hyperoxic exposure (O2) for 48 hours, 2) inhibition of glutathione synthesis by treatment with buthionine sulfoximine (BSO), 3) a combination of BSO + O2, or 4) inhibition of cysteine synthesis by propargyglycine (PPG). Livers were then isolated and perfused. RESULTS: Glutathione concentrations in bile, liver, and perfusate were significantly decreased by all treatments. Bile flow was significantly decreased in groups treated with BSO or O2 + BSO, and perfusate LDH was increased by O2 + BSO or PPG. Significant changes in biliary amino acid concentrations included decreased sulfur-containing amino acids and increased branched-chain amino acids in groups treated with BSO, PPG, or O2; and increased essential amino acids in groups treated with O2 or PPG. CONCLUSION: Oxygen exposure or inhibition of glutathione synthesis results in significant decreases in hepatic, perfusate and biliary glutathione concentrations, and increases in biliary amino acids. A decrease in bile flow rate was associated only with the most severe glutathione depletion.

The acute effects of TNF-alpha on the isolated perfused rat liver.

BACKGROUND: TNF-alpha mediates the hepatic response to sepsis by mechanisms which are not well understood. TNF-alpha is known to stimulate the hepatocellular uptake of specific amino acids in vivo; however, little is known about the direct effects of TNF-alpha on hepatic amino acid or glutathione homeostasis, which is a potential factor in the acute hepatic response to sepsis. METHODS: Using the isolated perfused rat liver, we characterized the effects of TNF-alpha on the secretion of amino acids and glutathione into bile and perfusate. Livers taken from adult male Sprague-Dawley rats were perfused with TNF-alpha at a dose of 1 or 2 micrograms. Bile and perfusate were collected for the quantitation of amino acid and glutathione concentrations. RESULTS: Administration of 2 micrograms TNF-alpha resulted in significant increases in biliary and perfusate concentrations of branched chain, gluconeogenic, and total amino acids. TNF-alpha was also associated with dose-related increases in oxygen uptake, and greater biliary concentrations of glutathione. CONCLUSION: TNF-alpha has direct effects upon hepatic amino acid metabolism, which represent some of the early events involved in the mechanism of response to sepsis.

Biliary amino acid and glutathione secretion in response to amino acid infusion in the isolated rat liver.

The intravenous infusion of amino acid solutions has been associated with cholestatic liver injury in hospitalized patients and in laboratory animals. In the isolated rat liver, we recently showed that the acute decrease in bile flow, previously reported by other investigators, is dose related, reversible, and associated with dose-related increases in total biliary amino acid concentrations. In the present study, we characterized the effects of graded infusions of amino acid solutions, with and without taurocholate, on biliary secretion of individual amino acids and glutathione, an important regulator of bile flow. Livers from young adult male rats were perfused with an amino acid solution for 1 hour and allowed to recover for 30 minutes. Infusion of the amino acid solution was associated with dose-related increases in biliary concentrations of most amino acids included in the amino acid solution. Infusion of amino acid solutions resulted in a decreased bile/perfusate ratio of most amino acids, which were secreted into bile in amounts approximating their calculated uptake from the infusate. The inclusion of taurocholate in the infusate was associated with lower biliary concentrations of each individual amino acid and significant decreases in biliary total, reduced, and oxidized glutathione. Further investigation of the relationship between these changes in biliary amino acids and glutathione concentrations and the cholestasis associated with infusion of amino acid solutions may provide insights into the mechanism by which amino acids induce such cholestasis.

Amino acid infusions induce reversible, dose-related decreases in bile flow in the isolated rat liver.

Parenteral infusion of amino acid solutions is known to produce cholestasis in experimental animal models and in the isolated perfused rat liver. To characterize the dose responsiveness and reversibility of amino acid-induced cholestasis, isolated rat livers were perfused with solutions containing 1.5, 3.0, or 6.0 g of amino acids for 1 hour and allowed to recover for 30 minutes. Perfusion of livers resulted in a rapid, dose-related decrease in bile flow (p < .0001 at doses of 3.0 and 6.0 g). When the amino acid solution was discontinued, bile flow recovered to near control rates. Infusion of taurocholate reduced the magnitude of the decrease in bile flow associated with amino acid infusion but did not prevent it. Infusion of amino acid solutions was associated with the following changes in bile: (1) dose-related increases in total free amino acid concentrations; (2) increased osmolarity; (3) increased glucose concentrations; (4) increased potassium concentrations; (5) decreased chloride concentrations; (6) increased oxygen uptake in livers not perfused with added taurocholate; and (7) increased total bile acid concentrations in livers perfused with added taurocholate. Additional investigations are needed to determine whether these associations are attributable to individual amino acids or the total metabolic load of the amino acids.