Isolation of DT-diaphorase [NAD(P)H dehydrogenase (quinone)] from rat liver cytosol:: Identification of new enzyme substrates, carcinogenic aristolochic acids

Cytosolic fractions isolated from liver and kidney of rats treated with P-naphthoflavone, Sudan I, ellipticine, phenobarbital, ethanol, acetone and natural carcinogenic and nephrotoxic nitroaromatics, aristolochic acids, were tested for the activity of DT-diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2]. While the most efficient inducers of DT-diaphorase in liver were Sudan I, ellipticine and aristolochic acids, the highest increase in the DT-diaphorase activity in kidney was produced by aristolochic acids. No increase in the enzyme activity was determined after treatment of rats with acetone. DT-Diaphorase was isolated from liver cytosol of Sudan I-treated rats by the procedure consisting of fractionation with ammonium sulfate, gel permeation chromatography on a Sephadex G-150 column, affinity chromatography on an Affi-Gel Blue (Cibracron Blue Agarose) column and re-chromatography on Sephadex G-150. Pat DT-diaphorase catalyzed NAD(P)H-dependent reduction of menadione (vitamin K-3), vitamin K-1 and 4-nitrosophenol as substrates. Moreover, we newly identified two carcinogenic nitroaromatic compounds, aristolochic acids, as other substrates of DT-diaphorase. A selective inhibitor of the human DT-diaphorase, dicoumarol, inhibited the catalytic activity of the rat purified enzyme.