NAD(P)H, a primary target of 1O2 in mitochondria of intact cells

Abstract

Direct reaction of NAD(P)H with oxidants like singlet oxygen (1O2) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2′,w4′,5′,7′-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of 1O2, which readily oxidized NAD(P)H to NAD(P)+. In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating 1O2 within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 ± 8.2 and 30.2 ± 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow reincrease. Prolonged photoactivation (≥15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for 1O2 in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.