Decreased cellular permeability to H2O2 protects Saccharomyces cerevisiae cells in stationary phase against oxidative stress.

The higher resistance of stationary-phase Saccharomyces cerevisiae to H2O2 when compared with exponential phase is well characterized, but the molecular mechanisms underlying it remain mostly unknown. By applying the steady-state H2O2-delivery model, we show that (a) cellular permeability to H2O2 is five times lower in stationary--than in exponential phase; (b) cell survival to H2O2 correlates with H2O2 cellular gradients for a variety of cells; and, (c) cells in stationary phase are predicted to be more susceptible to intracellular H2O2 than in exponential phase. In conclusion, limiting H2O2 diffusion into cells is a key protective mechanism against extracellular H2O2.

Glutathione conjugation of 4-hydroxy-trans-2,3-nonenal in the rat in vivo, the isolated perfused liver and erythrocytes.

The formation of glutathione (GSH) conjugates of racemic 4-hydroxy-trans-2,3-nonenal (4-HNE) in the rat in vivo in the perfused rat liver and rat erythrocytes has been studied. An HPLC system was developed for the assay of 4-HNE-glutathione conjugates (HNE-SG). The very sensitive electrochemical detection method (detection limit 5 pmol) can also be used to study endogenously formed HNE-SG. Three diastereomeric HNE-SG conjugates could be separated by this system. Rat liver cytosol catalyzed the formation of 2 of the 3 conjugates. When 17 micromol/kg [(3)H] 4-HNE was injected intravenously in the rat, 21% of the radioactivity was excreted within 90 min in bile and 37% in urine. Most of the 4-HNE in bile was present as 2 of the HNE-SG conjugates (molecular mass 463). In addition, 25% was excreted as a third GSH conjugate (molecular mass of 461), which was identified as the lactone of the 4-hydroxynonenoic acid glutathione conjugate. Erythrocytes in vitro eliminated 4-HNE very rapidly, in part by GSH conjugation, suggesting that they may also play an important role in vivo. To study the role of the liver selectively, we used the recirculating perfused rat liver without erythrocytes in the perfusion medium; the same conjugates were found, but the third conjugate was a minor component. These results present direct evidence for the in vivo formation of 4-HNE glutathione conjugates in which the liver may play an important role.

Glutathione metabolism in hepatomous liver of rats treated with diethylnitrosamine.

Glutathione metabolism was studied in rat liver during diethylnitrosamine (DEN) carcinogenesis. Some studies were also made in foetal rat liver. Endogenous GSH and non-protein thiols concentrations are increased in DEN-treated rats when compared to non-treated rats but no differences were found in cysteine, total thiols and protein thiols concentration. In foetal liver GSH concentration is only 35% of that in DEN-treated rat liver. The activities of several enzymes involved in glutathione metabolism are changed in DEN-treated rats. gamma-Glutamyl transferase activity and cysteine formation from GSH by liver homogenates is increased sevenfold. gamma-Glutamylcysteine synthetase activity, initial rate of [35S]cysteine incorporation in gamma-glutamylcysteine and initial rate of GSH formation from [35S]cysteine are increased two-fold. Cytosolic GSH S-transferase activity is increased twofold in DEN-treated rats and so GSH S-conjugates concentration is probably also increased. In foetal rat liver gamma-glutamyl transferase activity is about the same but gamma-glutamylcysteine synthetase activity is only 10% of that in DEN-treated rat liver. The increased GSH concentration in DEN-treated rat liver is probably due to the simultaneous increase in the activities of gamma-glutamyl transferase and gamma-glutamylcysteine synthetase. Blood plasma total glutathione is increased 1.4 times in DEN-treated rats, but no differences are found in GSH hepatic arteriovenous gradient. This associated with the increased gamma-glutamyl transferase activity suggests that sinusoidal GSH efflux is increased in DEN-treated rats.

Role of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase in the reduction of lysophospholipid hydroperoxides.

1-linoleoyl lysophosphatidylcholine hydroperoxide is a substrate of GSH peroxidase (GPx) both purified from bovine erythrocytes and nonpurified from rat liver. The initial reaction rate for bovine erythrocyte GPx with 1-linoleoyl lysophosphatidylcholine hydroperoxide is about 76 and 95% of the reaction rate for hydrogen peroxide and linoleic acid hydroperoxide respectively. For rat liver GPx these initial reaction rates are about 66 and 75%, respectively. The rate constants for the reaction of GPx with 1-linoleoyl lysophosphatidylcholine hydroperoxide were calculated to be approximately 3 x 10(7) M-1s-1 and approximately 2 x 10(6) M-1s-1 for the bovine erythrocyte and the rat liver enzymes, respectively. By using kinetic models of lipid peroxidation we found by simulation that: (1) the main source of lysophospholipid hydroperoxides in vivo is the peroxidation of lysophospholipids, both in mitochondrial inner membranes and in endoplasmic reticulum; (2) a specialized enzyme able to reduce directly lysophospholipid hydroperoxides is important for the reduction of these hydroperoxides, because the detoxification of these species mediated by the action of acyl ester bond cleaving enzymes is not efficient; (3) the reduction through GPx predominates over phospholipid hydroperoxide glutathione peroxidase (PHGPx) in mitochondrial inner membranes and in the cytosolic phase of the endoplasmic reticulum; (4) in the luminal phase of endoplasmic reticulum PHGPx is predominant.

Lipid peroxidation in mitochondrial inner membranes. I. An integrative kinetic model.

An integrative mathematical model was developed to obtain an overall picture of lipid hydroperoxide metabolism in the mitochondrial inner membrane and surrounding matrix environment. The model explicitly considers an aqueous and a membrane phase, integrates a wide set of experimental data, and unsupported assumptions were minimized. The following biochemical processes were considered: the classic reactional scheme of lipid peroxidation; antioxidant and pro-oxidant effects of vitamin E; pro-oxidant effects of iron; action of phospholipase A2, glutathione-dependent peroxidases, glutathione reductase and superoxide dismutase; production of superoxide radicals by the mitochondrial respiratory chain; oxidative damage to proteins and DNA. Steady-state fluxes and concentrations as well as half-lives and mean displacements for the main metabolites were calculated. A picture of lipid hydroperoxide physiological metabolism in mitochondria in vivo showing the main pathways is presented. The main results are: (a) perhydroxyl radical is the main initiation agent of lipid peroxidation (with a flux of 10(-7)MS-1); (b) vitamin E efficiently inhibits lipid peroxidation keeping the amplification (kinetic chain length) of lipid peroxidation at low values (approximately equal to 10); (c) only a very minor fraction of lipid hydroperoxides escapes reduction via glutathione-dependent peroxidases; (d) oxidized glutathione is produced mainly from the reduction of hydrogen peroxide and not from the reduction of lipid hydroperoxides.