title: 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.
authors: Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia. aleshin_vasily@mail.ru.
authors: Vasily A
authors: VA
authors: Aleshin
authors: Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia. whitelord32br@gmail.com.
authors: Artem V
authors: AV
authors: Artiukhov
authors: Neurosurgical clinic and polyclinic, Leipzig University Clinic, Leipzig 04103, Germany. henry.oppermann@medizin.uni-leipzig.de.
authors: Henry
authors: H
authors: Oppermann
authors: Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119234, Russia. mak@org.chem.msu.ru.
authors: Alexey V
authors: AV
authors: Kazantsev
authors: Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119234, Russia. nvlukashev@gmail.com.
authors: Nikolay V
authors: NV
authors: Lukashev
authors: Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia. bunik@belozersky.msu.ru.|Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia. bunik@belozersky.msu.ru.
authors: Victoria I
authors: VI
authors: Bunik
keywords: 2-oxo acid dehydrogenase; T98G; U87; cellular NAD(P)H-dependent oxidoreductase; glioblastoma viability; metabolon; oxythiamin; phosphonate analog of 2-oxo acid; resazurin; thiamin