Formation of 4-hydroxyochratoxin A from ochratoxin A by rat liver microsomes

Hydroxyochratoxin A was isolated and identified from the urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate, one major (90%) and two minor metabolites, more polar than ochratoxin A, were formed. Thin-layer chromatography revealed that the major metabolite had Rf values identical to those of hydroxyochratoxin A in six different solvent systems. Formation of the metabolites in vitro was inhibited by carbon monoxide and by metyrapone, and the rate of formation increased after pretreatment of the rats with phenobarbital. A type I spectrum appeared upon binding of ochratoxin A to microsomes with a spectral dissociation constant (Ks) of 37.6 microM. These findings strongly suggest the involvement of a cytochrome P-450 in the hydroxylation of ochratoxin A by rat liver microsomes. Apparent Km and Vmax values for the formation of hydroxyochratoxin A were determined to 50 microM and 5.5 nmol/mg of protein per h, respectively.