Stimulation of Oxygen Consumption Promotes Mitochondrial Calcium Accumulation, a Process Associated with, and Causally Linked to, Enhanced Formation of tert-Butylhydroperoxide-Induced DNA Single-Strand Breaks

Abstract

The NADH-linked substrates pyruvate, l-glutamine, and β-hydroxybutyrate, while enhancing the rate of oxygen consumption, also increased the formation of DNA single-strand breaks induced by tert-butylhydroperoxide in intact U937 cells. A cause–effect relationship between these two parameters was established by showing that: (a) rotenone, an inhibitor of complex I, abolished respiration and prevented the enhancement of the DNA-damaging response under all the above circumstances; (b) the membrane-impermeant, complex I-activating substrate l-malate gave similar results in permeabilized cells; and (c) none of the NADH-linked substrates affected the DNA-damaging response to tert-butylhydroperoxide in respiration-deficient cells. Stimulation of electron transport potentiated the DNA-cleaving ability of tert-butylhydroperoxide via a process involving enforced mitochondrial calcium accumulation in the absence of a discernible elevation in the cytosolic concentration of free Ca 2+. Finally, mitochondrial calcium was found to promote the mitochondrial formation of DNA-damaging levels of hydrogen peroxide. In conclusion, the data herein presented define a previously unexpected role of respiratory substrates in the control of the deleterious effects of an organic hydroperoxide at the level of genomic DNA. The enhanced DNA cleavage mediated by NADH-linked substrates in response to tert-butylhydroperoxide would appear to depend on a sequence of events involving stimulation of electron transport, mitochondrial accumulation of Ca 2+, and mitochondrial formation of DNA-damaging levels of hydrogen peroxide via a Ca 2+-dependent process.