Detection and identification of sulfhydryl conjugates of rho-benzoquinone in microsomal incubations of benzene and phenol.

The glutathione and cysteine conjugates of rho-benzoquinone are detected and conclusively identified in microsomal incubations of benzene and phenol using liquid chromatography/electrochemistry (LCEC). Identification of the compounds is based on retention time, electrochemical behavior and acid hydrolysis. The fact that both of these compounds can be detected easily in a benzene incubation provides further evidence that rho-benzoquinone or the corresponding semiquinone is a product of benzene metabolism in vivo. The conjugation of rho-benzoquinone with glutathione is predominantly a non-enzymatic process. This is illustrated by the fact that the addition of cytosolic glutathione-S-transferases do not significantly increase the amount of glutathione conjugate produced in a phenol incubation containing glutathione. The kinetic constants for phenol metabolism to hydroquinone by microsomal protein are calculated. As suspected, the rate of metabolism of phenol is significantly higher than the rate of benzene metabolism. The Vmax for phenol metabolism was calculated to be 7.1 nmol/min/mg protein and the KM was found to be 0.38 mM. The further oxidation of hydroquinone to rho-benzoquinone appears to be primarily an enzymatic process. Incubations of just hydroquinone with glutathione at 37 degrees C produced only a small amount of the glutathione conjugate. The addition of cytosolic protein increases the amount of rho-benzoquinone produced about 10-fold. This could be due to the peroxidases found in that medium. The addition of microsomal protein and NADPH increases the amount of glutathione conjugate produced to over 100-fold of that produced non-enzymatically. This indicates that a microsomal enzyme is responsible for the oxidation of hydroquinone to rho-benzoquinone in vitro and the subsequent covalent binding to macromolecules.