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 ( 1 O 2 ) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2,4,5,7-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 1 O 2 , 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 1 O 2 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 TMRMloaded 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 1 O 2 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.Pyridine nucleotides, i.e. NAD(H) and NADP(H), play a central role in metabolism; they are the most important coenzymes acting as hydride (hydrogen anion) donors of various cellular dehydrogenases (e.g. glutathione reductase), functioning as reducing/oxidizing equivalents in essential reactions such as energy supply (aerobic or anaerobic) and photosynthesis, and are required for DNA repair.The ability of an organism to counteract reactive oxygen species (ROS) 1 or reactive nitrogen species depends on its antioxidative capabilities, which involves destroying of both prooxidants (e.g. ROOH, H 2 O 2 , ONOOH) and oxidants (e.g. radicals and reactive intermediates like singlet oxygen, 1 O 2 ). Whereas pro-oxidants are typically degraded by enzymes (e.g. catalase, glutathione peroxidase, and superoxide dismutase), oxidants are scavenged by relatively small biomolecules (e.g. ascorbic acid, glutathione, and ␣-tocopherol); these are termed directly operating antioxidants. In this context, NAD(P)H is crucial to maintaining the cellular redox state and/or antioxidative capacity, because of its essential role as a coenzyme in the enzymatic re-reduction of directly operating antioxidants (1, 2). Consequently, NAD(P)H deficiencies are linked with an increased sensitivity to oxidative stress (2, 3).The capability of NAD(P)H to additionally act as a directly operating antioxidant, i.e. to donate only one electron, was sharply underestimated by various biochemical researchers, a fact that is probably because of the observation that a biochemical standard one-electron oxidant, [Fe(CN) The H 2 O 2 -consuming enzymes catalase and glutathione peroxidase (GPx) strongly limi...

POS5 Gene of Saccharomyces cerevisiae Encodes a Mitochondrial NADH Kinase Required for Stability of Mitochondrial DNA

“…More recently, photoactivation of rhodamine dyes was utilized to generate in situ singlet oxygen within the mitochondrial matrix of cultured hepatocytes. Depletion of NAD(P)H was dependent on the dose of this endogenous ROS, and higher doses induced the mitochondrial permeability transition and apoptotic cell death (55). Both studies strongly suggest that the reducing equivalents of NAD(P)H function as an endogenous antioxidant to protect mitochondria from the injurious effects of ROS.…”

The Radical Scavenger Edaravone (3-Methyl-1-phenyl-2-pyrazolin-5-one) Reacts with a Pterin Derivative and Produces a Cytotoxic Substance That Induces Intracellular Reactive Oxygen Species Generation and Cell Death

“…Singlet oxygen is known to induce neuronal and hepatic cell death (Cutrìn et al, 2000;Petrat et al, 2003;Valencia and Moran, 2004). Furthermore, 1 O 2 is tumoricidal and plays an important role in anticancer photodynamic therapy (Dougherty et al, 1998;Dolmans et al, 2003).…”

“…Cells (1 ϫ 10 5 cells/ml) were incubated overnight in a 35-mm glass base dish (IWAKI, Funabashi, Japan) in 2 ml of medium. The cells were then incubated with 100 nM TMRM, a mitochondrial membrane potential-sensitive dye (Petrat et al, 2003), for 20 min. The medium was then changed, and the cells were incubated with combination of DFP and edaravone and 2 M DCFH-DA, an ROS-sensitive dye (Royall and Ischiropoulos, 1993) at 37°C and 5% CO 2 in the timelapse imaging system, and the phase-contrast images and fluorescent images were acquired every 15 min for 4 h. Green fluorescence images for DCF and red fluorescence images for TMRM were acquired using fluorescein isothiocyanate and tetramethylrhodamine B isothiocyanate filters (Nikon), respectively.…”

Investigating photoexcitation-induced mitochondrial damage by chemotherapeutic corroles using multimode optical imaging

“…Photoactivation of TMRM itself induces singlet oxygen 21 and thus prolonged photoactivation of TMRM-loaded cells resulted in stronger NAD(P)H oxidation and the rapid onset of mitochondrial permeability transition. Thus, in the measurement of mitochondrial membrane potential using TMRM, careful determination of light dose for excitation of TMRM is needed.…”

“…1], and the apparent disruption observed at 535 nm is likely to be caused mainly by reactive oxygen species generated by TMRM although it is somewhat affected by HerGa photo-reactivity. 21 In addition, light at 424 nm can induce moderate mitochondrial damage in S2Ga-treated cells, but considerably less compared to HerGa-treated cells [ Fig. 3].…”