Direct measurement of NAD(P)H:quinone reductase from cells cultured in microtiter wells: a screening assay for anticarcinogenic enzyme inducers.
We describe a rapid and direct assay of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) activity in cultured cells suitable for identifying and purifying inducers of this detoxication enzyme. Hepa 1c1c7 murine hepatoma cells are plated in 96-well microtiter plates, grown for 24 h, and exposed to inducing agents for another 24 h. The cells are then lysed and quinone reductase activity is assayed by the addition of a reaction mixture containing an NADPH-generating system, menadione (2-methyl-1,4-naphthoquinone), and MTT [3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide]. Quinone reductase catalyzes the reduction of menadione to menadiol by NADPH, and MTT is reduced nonenzymatically by menadiol resulting in the formation of a blue color which can be quantitated on a microtiter plate absorbance reader. The reaction is more than 90% dicoumarol inhibitable and menadione dependent. The results are comparable to those obtained by harvesting cells from larger plates, preparing cytosols, and carrying out spectrophotometric measurements.
Prochaska
H J
HJ
Department of Pharmacology and Molecular Sciences, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Santamaria
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AB
D000818:Animals; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004790:Enzyme Induction / Q000187:drug effects; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D010084:Oxidation-Reduction / Q000187:drug effects; D011808:Quinone Reductases / Q000032:analysis*; D014407:Tumor Cells, Cultured; D014812:Vitamin K / Q000494:pharmacology
D014812:Vitamin K; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases