Abstract
Human keratinocytes in culture were harvested at different stages of differentiation. Both the level of antioxidants and the response of cells to oxidative stress were measured as a function of growth and differentiation. As the keratinocyte cultures became confluent and began to differentiate, the cellular levels of glutathione, glutathione peroxidase, glutathione S transferase, and glucose-6-phosphate dehydrogenase increased. This higher level of antioxidants was maintained until the cells began to lose viability. Further, as the keratinocyte cultures began to differentiate, they became more resistant to the toxic effect of cumene hydroperoxide in terms of both of the rate of loss of cell mass and total glutathione and of the rate of decline in the activity of oxidation-sensitive enzymes. To determine how tightly the observed effects are linked to the calcium-dependent aspects of differentiation and to rule out effects related to time in culture, the cells were switched from 1.2 mM Ca++ to 0.03 mM Ca++ to suppress Ca(++)-dependent differentiation. After 4 d, these cells were then treated with 0.5 mM cumene hydroperoxide. The switch to 0.03 mM Ca++ blocked the normal increases in both glutathione peroxidase and glucose-6-phosphate dehydrogenase activities. Further, cells in 0.03 mM Ca++ had reduced resistance to cumene hydroperoxide relative to cells cultured for the same length of time in 1.2 mM Ca++. This indicates that there is a differentiation-associated, Ca(++)-specific increase in both the level of antioxidants and in tolerance to organic hydroperoxides.
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