Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

Abstract

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications

“…Regulation of GDH activity by ThTP and AThTP is consistent with cellular synthesis of these non-coenzyme thiamine derivatives under metabolic stress, including amino acid and carbon starvation [118,119]. The biological significance of the thiamine regulation of GDH is supported in cellular experiments which demonstrated similar changes in GDH upon incubation of cells with thiamine and its antagonist, oxythiamine [120]. …”

Monitoring NAD(P)H by an ultrasensitive fluorescent probe to reveal reductive stress induced by natural antioxidants in HepG2 cells under hypoxia

“…Insufficient NAD(P)H induces oxidative stress due to increased ROS production, while excessive NAD(P)H leads to reductive stress because it cannot be properly oxidized. 20 Besides, NAD(P)H is a highly reducible dynamic molecule with very high reactivity and instability which cannot be easily detected in cell and animal models. Therefore, an ultrasensitive and rapid method to selectively determine NAD(P)H in vivo must be developed to investigate the reductive stress.…”

Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance

“…Figure 1 depicts the physiological reactions catalyzed by the three enzymatic components of these complexes (Eqs. [1][2][3][4][5]), along with the overall process of oxidative decarboxylation of 2-oxo acids (Eq. [6]).…”