Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays
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 incre...
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MDPI AG
2015-08-01
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| Series: | Cells |
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| Online Access: | http://www.mdpi.com/2073-4409/4/3/427 |
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| author | Vasily A. Aleshin Artem V. Artiukhov Henry Oppermann Alexey V. Kazantsev Nikolay V. Lukashev Victoria I. Bunik |
| author_facet | Vasily A. Aleshin Artem V. Artiukhov Henry Oppermann Alexey V. Kazantsev Nikolay V. Lukashev Victoria I. Bunik |
| author_sort | Vasily A. Aleshin |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T06:57:34Z |
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| id | doaj.art-e97d1b017e594019aa90c327b924a7d3 |
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| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-12T06:57:34Z |
| publishDate | 2015-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-e97d1b017e594019aa90c327b924a7d32023-09-02T23:59:32ZengMDPI AGCells2073-44092015-08-014342745110.3390/cells4030427cells4030427Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability AssaysVasily A. Aleshin0Artem V. Artiukhov1Henry Oppermann2Alexey V. Kazantsev3Nikolay V. Lukashev4Victoria I. Bunik5Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, RussiaFaculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, RussiaNeurosurgical clinic and polyclinic, Leipzig University Clinic, Leipzig 04103, GermanyFaculty of Chemistry, Lomonosov Moscow State University, Moscow 119234, RussiaFaculty of Chemistry, Lomonosov Moscow State University, Moscow 119234, RussiaFaculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, RussiaCellular 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.http://www.mdpi.com/2073-4409/4/3/427glioblastoma viabilitycellular NAD(P)H-dependent oxidoreductasemetabolonthiaminoxythiamin2-oxo acid dehydrogenasephosphonate analog of 2-oxo acidresazurinT98GU87 |
| spellingShingle | Vasily A. Aleshin Artem V. Artiukhov Henry Oppermann Alexey V. Kazantsev Nikolay V. Lukashev Victoria I. Bunik Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays Cells glioblastoma viability cellular NAD(P)H-dependent oxidoreductase metabolon thiamin oxythiamin 2-oxo acid dehydrogenase phosphonate analog of 2-oxo acid resazurin T98G U87 |
| title | Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays |
| title_full | Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays |
| title_fullStr | Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays |
| title_full_unstemmed | Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays |
| title_short | Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays |
| title_sort | mitochondrial impairment may increase cellular nad p h resazurin oxidoreductase activity perturbing the nad p h based viability assays |
| topic | glioblastoma viability cellular NAD(P)H-dependent oxidoreductase metabolon thiamin oxythiamin 2-oxo acid dehydrogenase phosphonate analog of 2-oxo acid resazurin T98G U87 |
| url | http://www.mdpi.com/2073-4409/4/3/427 |
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