Kristina E. Hill

Influence of vitamin E and selenium on glutathione-dependent protection against microsomal lipid peroxidation

A GSH-dependent microsomal protein which inhibits lipid peroxidation has been described [R. F. Burk, Biochim. biophys. Acta 757, 21 (1983)]. Studies of its mechanism indicate that it scavenges free radicals. Vitamin E (alpha-tocopherol) and selenium are micronutrients which protect against lipid peroxidation. The effect of nutritional deficiencies of these substances on the GSH-dependent protection against rat liver microsomal lipid peroxidation was studied to determine whether GSH, selenium and alpha-tocopherol function through separate or shared mechanisms. In the ascorbate-iron microsomal lipid peroxidation system, there is a 1-3 min lag phase before lipid peroxidation begins. The length of the lag correlated well (r = 0.87) with the microsomal alpha-tocopherol content as measured by high pressure liquid chromatography. Thus, the selenium-deficient microsomes, which had a shorter lag than controls, had a somewhat lower alpha-tocopherol content. The vitamin E-deficient microsomes, which had no detectable alpha-tocopherol, had the shortest lag, but a distinct lag was present. Addition of 0.1 mM GSH to control microsomes prolonged the lag by 270%. In selenium-deficient and vitamin E-deficient microsomes, which had shorter initial lags, GSH addition caused 345 and 280% increases respectively. This suggests that the function of the GSH-dependent protective mechanism is unimpaired in these deficiencies. Trypsin digestion of microsomes, which abolished the lag completely and destroyed the GSH-dependent protection, had no effect on microsomal alpha-tocopherol content, however. These experiments illustrate the importance of two defenses against microsomal lipid peroxidation: the GSH-dependent protein which is responsible for the existence of the lag, and alpha-tocopherol which affects the length of the lag. They suggest that these defenses function separately to prevent peroxidation of membrane polyunsaturated fatty acids. Selenium appears to affect microsomal alpha-tocopherol content but to have no other effect on the microsomal lipid peroxidation system.

Effect of selenium deficiency on the disposition of plasma glutathione

Selenium deficiency causes increased hepatic synthesis and release of GSH into the blood. The purpose of this study was to examine the effect of selenium deficiency on the disposition of plasma glutathione. Plasma glutathione concentration was 40 +/- 3.4 nmol GSH equivalents/ml in selenium-deficient rats and 17 +/- 5.4 nmol GSH equivalents/ml in control rats. The half-life and systemic clearance of plasma glutathione were found to be the same in selenium-deficient and control rats (t1/2 = 3.4 +/- 0.7 min). Because selenium-deficient plasma glutathione concentration was twice that of control, the determination that selenium deficiency did not affect glutathione plasma systemic clearance indicated that the flux of glutathione through the plasma was doubled by selenium deficiency. It has been proposed that the kidney is responsible for the removal of a major fraction of plasma glutathione. In these studies, renal clearance accounted for 24% of plasma systemic glutathione clearance in controls and 44% in selenium-deficient rats. This indicates that a significant amount of glutathione is metabolized at extrarenal sites, especially in control animals. More than half of the increased plasma glutathione produced in selenium deficiency was removed by the kidney. Thus, selenium deficiency results in a doubling of cysteine transport in the form of glutathione from the liver to the periphery as well as a doubling of plasma glutathione concentration.

Toxicity studies in isolated hepatocytes from selenium-deficient rats and vitamin E-deficient rats.

Isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats were treated with cumene hydroperoxide (CuOOH), phorone (diisopropylene acetone), acetaminophen, and diquat. The effect of these chemicals on cell viability, glutathione synthesis and release, and lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances was determined during a 4-hr incubation in a complete medium under 95% O2:5% CO2 at 37 degrees C. CuOOH-treated (0.5 mM) selenium-deficient and vitamin E-deficient hepatocytes lost viability sooner than control hepatocytes. Thus, loss of selenium or vitamin E from the hepatocyte resulted in a cell more susceptible to damage by CuOOH. Phorone treatment (1.65 mM) resulted in depletion of intracellular glutathione in all three groups to approximately 20% of that in untreated hepatocytes. Cell viability and TBA-reactive substances were the same in treated and untreated hepatocytes. Thus, lowering of intracellular glutathione did not result in the spontaneous loss of cell viability or increased lipid peroxidation in selenium-deficient or in vitamin E-deficient hepatocytes. Acetaminophen appeared to be less toxic to selenium-deficient hepatocytes than to controls. This finding is in agreement with whole animal studies reported previously showing that selenium deficiency protects rats against acetaminophen hepatotoxicity. A potential explanation of this result is stimulation of glutathione synthesis by selenium deficiency. Severely vitamin E-deficient hepatocytes were protected from cell death by 12.5 and 25.0 mM acetaminophen, apparently by its antioxidant properties, while 50.0 mM acetaminophen was toxic to them. At all concentrations used, acetaminophen decreased the TBA-reactive substances present in the hepatocyte suspensions. Diquat (0.1 mM) caused more rapid cell death and higher levels of TBA-reactive substances in selenium-deficient hepatocytes than in control hepatocytes. Diquat toxicity in selenium-deficient isolated hepatocytes was not as severe as its toxicity in selenium-deficient whole animals, however.

Effect of inhibition of γ-glutamyltranspeptidase by AT-125 (Acivicin) on glutathione and cysteine levels in rat brain and plasma

SummaryAT-125 (Acivicin) is an inhibitor of γ-glutamyltranspeptidase (γ-GTP) which initiates glutathione catabolism to cysteine. We measured plasma and brain glutathione and cysteine in rats treated with AT-125. Six h after AT-125 treatment, plasma glutathione had increased 6-fold and plasma cysteine had fallen significantly. Brain cysteine fell after 24 h of AT-125 treatment, and brain glutathione had also decreased 18%. AT-125 pretreatment inhibited brain uptake of 35S when it was given as 35S-GSH but had no effect when it was given as 35S-cysteine. These results suggest that plasma glutathione is catabolized by γ-GTP, and cysteine derived from it is taken up by the brain. N-acetylcysteine was administered to AT-125 treated rats in an attempt to supply cysteine to the brain in the face of γ-GTP inhibition. N-acetylcysteine supported brain glutathione levels, suggesting that it can serve as a source of cysteine under these conditions.

Studies on the biliary efflux of GSH from rat liver due to the metabolism of aminopyrine

The biliary efflux of GSH and GSSG due to aminopyrine was studied using perfused rat livers. The infusion of 0.8 mM aminopyrine led to a rapid rise in the amount of GSH released into the bile with only a small increase in the amount of GSSG released; caval GSH + GSSG efflux was unaffected. N-Benzylimidazole, an inhibitor of cytochrome P-450, completely blocked the response while phenobarbital pretreatment of the rats doubled the rate of GSH efflux. H2O2 and selenium-containing glutathione peroxidase were not involved since livers from selenium-deficient rats perfused with aminopyrine released GSH at the same rate as control livers. Aminopyrine injected i.p. into conscious rats also stimulated biliary GSH efflux to the same extent as with perfused livers. Biliary release of GSH in the perfused livers could be duplicated by infusing formaldehyde. It is proposed that formaldehyde produced during the N-demethylation of aminopyrine by cytochrome P-450 combines reversibly with GSH to form S-hydroxymethylglutathione which is oxidized by formaldehyde dehydrogenase to S-formylglutathione. Formaldehyde formed in excess of its capacity to be metabolized enzymatically is released into the bile as S-hydroxymethylglutathione which then dissociates to its initial reactants.

Influence of selenium deficiency on glutathione disulfide metabolism in isolated perfused rat heart

Selenium deficiency causes a fall in rat cardiac glutathione peroxidase activity. As a consequence, isolated perfused selenium-deficient heart does not release increased amounts of GSSG when hydroperoxide is infused. However, the total amount of glutathione measured as intracellular GSH, intracellular GSSG and GSSG released from the heart when hydroperoxide is infused does not equal the total glutathione measured in these pools in untreated hearts (Xia, Y., Hill, K.E. and Burk, R.F. (1985) J. Nutr. 115, 733-742). GSSG can react with protein sulfhydryl groups to form glutathione-protein mixed disulfides (PrS-SG). PrS-SG were measured in perfused selenium-deficient and control hearts infused with t-butylhydroperoxide and were found to account for the previously unmeasured glutathione. The ability of the selenium-deficient heart to transport GSSG was also examined. GSSG was produced non-enzymatically by infusing diamide. The diamide-treated selenium-deficient heart formed GSSG and released it at the same rate as similarly-treated control heart. Thus although selenium deficiency decreases GSSG formation by glutathione peroxidase, it does not affect cardiac GSSG transport.