Collect. Czech. Chem. Commun. 2001, 66, 959-972
doi:10.1135/cccc20010959

Isolation of DT-Diaphorase [NAD(P)H Dehydrogenase (Quinone)] from Rat Liver Cytosol: Identification of New Enzyme Substrates, Carcinogenic Aristolochic Acids

Marie Stiborováa,*, Miroslav Hájeka, Hana Vošmikováa, Eva Freib and Heinz H. Schmeiserb

a Department of Biochemistry, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic
b Department of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

Abstract

Cytosolic fractions isolated from liver and kidney of rats treated with β-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. Rat DT-diaphorase catalyzed NAD(P)H-dependent reduction of menadione (vitamin K3), vitamin K1 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.

Keywords: NAD(P)H dehydrogenase (quinone); DT-Diaphorase; Purification procedure; Inducers; Drugs; Carcinogens; Aristolochic acids; Reductive activation; Chinese herbs nephropathy; Carcinogenesis; Enzymes; Oxidases.

References: 50 live references.