Susan M. Deneke

Involvement of glutathione enzymes in O2 tolerance development by diethyldithiocarbamate.

Abstract Diethyldithiocarbamate (DDC) in high doses (⩾ 250 mg/kg) accelerated the onset of death in adult rats exposed to 95% O2. At lower doses (100 and 200 mg/kg) no increase in O2 toxicity was seen. We observed that, at a level of 250 mg/kg, DDC accelerated the death of 26 per cent of the animals but a large number (50 per cent) developed resistance to the lethal effects of 95% O2. These animals also demonstrated an acceleration of the rate of increase in activity of glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase activity in response to O2, compared to untreated controls. No acceleration in the rate of increase of superoxide dismutase activity was seen. These data support a role of the glutathione-related enzymes in promoting O2 tolerance in rats.

Metallothionein, glutathione, and cystine transport in pulmonary artery endothelial cells and NIH/3T3 cells

Both glutathione (γ-glutamylcysteinylglycine; GSH) and the metalloprotein metallothionein (MT) are composed of approximately one-third cysteine. Both have antioxidant activity and are induced by oxidant stresses and heavy metals. Intracellular cysteine levels may depend on uptake and reduction of extracellular cystine. GSH synthesis can be limited by the activity of the [Formula: see text] cystine transport system, which is induced by oxidants and other stresses. MT is induced by treatments that also increase GSH levels and may compete with GSH for intracellular cysteine. We investigated the induction of MT and GSH and cystine transport in NIH/3T3 cells and bovine pulmonary artery endothelial cells exposed to cadmium (Cd) or arsenite. Cd and arsenite increased MT and GSH in both cells. Increases in MT and GSH were accompanied by increases in cystine uptake. Inhibition of cystine transport by glutamate decreased GSH levels and blocked Cd-induced GSH increases in both cell types. MT levels were not significantly affected, suggesting that MT synthesis is less sensitive to intracellular cysteine levels than GSH synthesis.Both glutathione (gamma-glutamylcysteinylglycine; GSH) and the metalloprotein metallothionein (MT) are composed of approximately one-third cysteine. Both have antioxidant activity and are induced by oxidant stresses and heavy metals. Intracellular cysteine levels may depend on uptake and reduction of extracellular cystine. GSH synthesis can be limited by the activity of the xc- cystine transport system, which is induced by oxidants and other stresses. MT is induced by treatments that also increase GSH levels and may compete with GSH for intracellular cysteine. We investigated the induction of MT and GSH and cystine transport in NIH/3T3 cells and bovine pulmonary artery endothelial cells exposed to cadmium (Cd) or arsenite. Cd and arsenite increased MT and GSH in both cells. Increases in MT and GSH were accompanied by increases in cystine uptake. Inhibition of cystine transport by glutamate decreased GSH levels and blocked Cd-induced GSH increases in both cell types. MT levels were not significantly affected, suggesting that MT synthesis is less sensitive to intracellular cysteine levels than GSH synthesis.

Ultrastructural changes in bovine pulmonary artery endothelial cells exposed to 80% O2 in vitro.

SummaryBovine pulmonary artery endothelial cells in culture were exposed for up to 7 d to a gas mixture containing 80% O2, 5% CO2, and 15% N2 (hyperoxia) and were compared by phase contrast and electron microscopy to cells exposed to a gas mixture containing 20% O2, 5% CO2, and 75% N2. Cells exposed to hyperoxia became enlarged and showed vacuolization and increased lysosomes within 24 to 48 h. These changes were progressive over the 7 d period of exposure. Between 3 and 7 d of exposure to hyperoxia the cells showed reductions in polysomes and endoplasmic reticulum. Despite the other marked cytoplasmic changes, the appearance of mitochondria of oxygen-exposed cells remained unchanged from those of air-exposed cells throughout the 7 d period. Preconfluent and confluent cells responded qualitatively similarly to hyperoxia, but morphological evidence of injury occurred more rapidly for preconfluent cells. We conclude that the initial early structural injury of the endothelial cell exposed to hyperoxia occurs in lysosomes and that the mitochondrial structure is relatively resistant to injury.

Protein Deficiency Potentiates Oxygen Toxicity

Male rats (Charles River COBS-CD derived) fed protein-deficient diets showed enhanced toxicity with failure of elevation of lung glutathione levels with exposure to greater than 98% O2. Replenishment of S-containing amino acids in the protein-deficient diets allowed elevation of lung glutathione and prevention of enhanced toxicity. Studies with endothelial cell cultures exposed to hyperoxia showed elevation of cellular glutathione coupled with enhanced uptake of amino acid precursors of glutathione. We postulate that hyperoxia causes an enhancement of uptake of S-containing amino acids necessary for glutathione synthesis, overriding glutathione feedback of its own synthesis. Limitation of available S-containing amino acids prevents elevation of glutathione synthesis and is detrimental to the cell exposed to hyperoxia.