DISULFIDE CYTOTOXICITY UNDER HYPOXIA

The cytotoxicity of the disulfide n-butyl 2-imidazolyl disulfide (III- 2) was determined to be the result of a disruption in the cellular red ox state and inhibition of critical membrane enzymes. These events cau se perturbations in Ca2+ homeostasis, which may affect the cell signal ling machinery and cause the activation of catabolic enzymes. Exposure of EMT6 cells to III-2 resulted in depletion of nonprotein and protei n thiols. Under hypoxic conditions, the depletion of reduced glutathio ne was less than that measured when cells were treated in air, whereas following an exposure to 500 mu M III-2 for 2 h the enzymes glutathio ne S-transferase and glutathione reductase were inhibited to a greater extent under hypoxia. Ca2+ homeostasis was disrupted with an initial shift from the mitochondrial to the cytoplasmic pool. The inhibition o f plasma membrane Ca2+-ATPase resulted in accumulation of Ca2+ in the cytoplasm. At higher concentrations, further disruption was seen as a net loss of Ca2+ of the cytoplasmic excess with no change in the mitoc hondrial levels, resulting in lower total cellular Ca2+. Neither the i nhibition of Ca2+-ATPase nor the disruption of Ca2+ homeostasis were d ifferent under hypoxic vs. oxic conditions. Due to these observations, HL60 cells were used to measure whether III-2 stimulated apoptosis. M orphologic changes and DNA laddering were observed following exposure to the disulfide, with lower concentrations required to stimulate the cellular changes under hypoxia. These events may be the result of the disruption in Ca2+ homeostasis due to thiolation or alteration in redo x status of the cells. Based on the present findings along with previo us reports of enzyme inhibition and limited NADPH generation under hyp oxia, it has been concluded that hypoxia makes the cells less able to cope with the oxidative stress caused by III-2. As a consequence, cell s under these conditions are more susceptible to the cytotoxic effects of III-2.