Spindle disturbances in mammalian cells. IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism

The glutathione-specific agents diamide, diethyl maleate and 1-chloro-2,4-dinitrobenzene were found to induce a low frequency of c-mitosis (15%) at non-toxic concentrations concomitant with a 30-40% decrease of non-protein sulfhydryls. The frequency of c-mitosis did not increase further with increased concentrations until non-protein sulfhydryl levels were obtained suggesting depletion of reduced glutathione. The observed shape of the concentration-response curve for c-mitosis is particular to these 3 agents and caffeine among 22 different compounds being tested under comparable conditions. This suggests a similar mechanism of action and from what is known about caffeine this mechanism probably involves an impaired control of cytoplasmic free Ca2+. It is speculated that this impairment with the glutathione-specific agents is primarily due to depletion of a particular pool of reduced glutathione. Tertiary butylhydroperoxide which is a substrate for glutathione peroxidase(s) also causes c-mitosis when there is no significant decrease of non-protein sulfhydryls. The c-mitotic response was found to be biphasic with maintained control levels at an intermediate concentration. The humps in the concentration-response curve for c-mitosis appeared coincident with a mitogenic response (G1----S). Since the latter type of effect most probably is Ca2+ dependent and since the spindle is sensitive to Ca2+ it is tentatively suggested that the c-mitotic effect of tertiary butylhydroperoxide is due to an increase of cytoplasmic Ca2+. Measurements performed imply that an increase of glutathione disulfide (diamide) is more inhibitory to uptake and incorporation of thymidine than a decrease of reduced glutathione per se (diethyl maleate). This difference is probably due to secondary effects on pertinent protein sulfhydryls with diamide, one possible target being the ribonucleotide reductase. All compounds were found to cause an increase of ATP with some of the applied concentrations. The results with diethyl maleate suggest that an increase of ATP is favored by an attack on mitochondrial reduced glutathione. The possible analogy between this effect and an increase of ATP and Ap4A in bacteria during oxidative stress is considered.