The oxidation of the highly tumorigenic benz[a]anthracene 3,4-dihydrodiol by rat liver dihydrodiol dehydrogenase.
Rat liver dihydrodiol dehydrogenase (DDH, EC 1.3.1.20) has been shown to reduce the mutagenicity of benz[a]anthracene (BA) in the bacterial Ames test. BA-3,4-dihydrodiol is a highly mutagenic and tumorigenic metabolite of BA. In order to test the hypothesis that this dihydrodiol may be a substrate of DDH, we established two novel assay systems for the NADP(+)-dependent oxidation of BA-3,4-dihydrodiol by rat liver DDH, an HPLC-based assay procedure and a radiometric assay with specifically labelled [3,4-3H]-BA-3,4-dihydrodiol as substrate. With the HPLC-based assay, the kinetic constants of the enzymatic catalysis were as follows: Km(app) = 21 microM for BA-3,4-dihydrodiol and Vmax = 20.0 nmol/min.mg enzyme. The reaction product was identified by cochromatography, fluorimetry and mass spectroscopy as BA-3,4-catechol, but interconversions between the catechol and the corresponding o-quinone during the analytical procedures were detected. With the radiolabelled substrate, a linear relationship between substrate concentration and reaction velocity was found. The V/K value for labelled substrate was 0.155 ml/min.mg enzyme and a (V/K)H/(V/K)T kinetic isotope effect of 6.7 was observed. The non-labelled substrate acted as a competitive inhibitor of the enzymatic oxidation of tritiated BA-3,4-dihydrodiol with a Ki value of 56.4 microM. The reaction rates determined in this study suggest an important role of DDH activity in the metabolism of BA.
Klein
J
J
Institute of Toxicology, University of Mainz, F.R.G.
Post
K
K
Thomas
H
H
Wörner
W
W
Setiabudi
F
F
Frank
H
H
Oesch
F
F
Platt
K L
KL
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001551:Benz(a)Anthracenes / Q000737:chemistry / Q000493:pharmacokinetics*; D002273:Carcinogens / Q000737:chemistry / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid; D008658:Inactivation, Metabolic; D008099:Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats; D012588:Scintillation Counting
D001551:Benz(a)Anthracenes; D002273:Carcinogens; C015149:benzanthracene-3,4-dihydrodiol; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C023125:dihydrodiol dehydrogenases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C116788:cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase