Abstract
Selenium deficiency and vitamin E deficiency both affect xenobiotic metabolism and toxicity. In addition, selenium deficiency causes changes in the activity of some glutathione-requiring enzymes. We have studied glutathione metabolism in isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats. Cell viability, as measured by trypan blue exclusion, was comparable for all groups during the 5-h incubation. Freshly isolated hepatocytes had the same glutathione concentration regardless of diet group. During the incubation, however, the glutathione concentration in selenium-deficient hepatocytes rose to 1.4 times that in control hepatocytes. The selenium-deficient cells also released twice as much glutathione into the incubation medium as did the control cells. Total glutathione (intracellular plus extracellular) in the incubation flask increased from 47.7 +/- 8.9 to 152 +/- 16.5 nmol/10(6) selenium-deficient cells over 5 h compared with an increase from 46.7 +/- 7.1 to 92.0 +/- 17.4 nmol/10(6) control cells and from 47.7 +/- 11.7 to 79.5 +/- 24.9 nmol/10(6) vitamin E-deficient cells. This overall increase in glutathione concentration suggested that glutathione synthesis was accelerated by selenium deficiency. The activity of gamma-glutamylcysteine synthetase was twice as great in selenium-deficient liver supernatant (105,000 X g) as in vitamin E-deficient or control liver supernatant (105,000 X g). Hemoglobin-free perfused livers were used to determine the form of glutathione released and its route. Selenium-deficient livers released 4 times as much GSH into the caval perfusate as did control livers. Plasma glutathione concentration in selenium-deficient rats was found to be 2-fold that in control rats, suggesting that increased GSH synthesis and release is an in vivo phenomenon associated with selenium deficiency.
Highlights
Selenium deficiency and vitamin E deficiency both affect xenobiotic metabolism and toxicity (1, 2)
Lipid peroxidation in the cells, as measured by thiobarbituric acid-reactive substances, was low and not significantly different in the three major groups, but pl aliquot of hepatocyte suspension was mixed with 2.0 ml of thio- it was markedly elevated in the hepatocytes isolated from the barbituric acid-trichloroacetic acid solution containing 0.01% butyl- severely vitamin E-deficient rats (Fig. 2)
With the exception of cells isolated from severely vitamin E-deficient rats, the viabilities of deficient hepatocytes were similar to the viability of control hepatocytes
Summary
Materials-Collagenasec, lass II (145 units/mg) was obtained from Worthington. All amino acids, GSH, GSSG, glutathione reductase, NADPH, and ATP were purchased from Sigma. Selenium deficiency was verified by measurement of glutathione peroxidase in the livers of some of the rats fed the selenium-deficient diet (7). Solution B: 119 mM NaCI, 4.7 mM KCI, 0.8 mM NH4C1, 1.2 mM KH2PO 4, 1.3 mM MgSO4, 24.8 mM NaHC03, 48.8 mM lactate, 5.3 mM fumarate, 48.8 mM L-glutamate, 11.4 mM a-D(+)-glucose and amino acid mixture, pH 7.4. Solution C: 118 mM NaCl, 4.7 mM KCI, 1.0 mM NH4CI, 1.2 mM KH2PO 4, 1.2 mM MgSO 4, 24.7 mM NaHCO 3, 2.5 mM CaC 2 , 48.7 mM lactate, 5.3 mM fumarate, 48.7 mM L-glutamate, 11.4 mM a-D(+)glucose, and amino acid mixture, pH 7.4. The minimum amount of GSSG opened; the portal vein was cannulated; and solution A was perfused detectable with this assay was 0.6 nmol of GSSG/g of liver/min.
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