IMMUNOCHEMICAL DETECTION OF CYTOCHROME-P450 ISOZYMES INDUCED IN RAT-LIVER BY N-HEXANE, 2-HEXANONE AND ACETONYL ACETONE

Cytochrome P450 isozymes induced in rat liver by treatment with n-hexane, 2-hexanone and acetonyl acetone (given intraperitoneally 5 mmol/kg for 4 days) were investigated using enzyme assays (benzene, toluene, 7-ethoxyresorufin and 7-pentoxyresorufin metabolism) and monoclonal antibodies (anti-P450IA1/2, anti-P450IIB1/2, anti-P450IIC11/6, anti-P450IIE1(91) and anti-P450IIE1(98)). n-Hexane treatment enhanced the activities of low-K(m) benzene aromatic hydroxylase and toluene side-chain oxidase, but not 7-ethoxyresorufin O-deethylase or 7-pentoxyresorufin O-depentylase. 2-Hexanone or acetonyl acetone treatment enhanced the activities of low- and high-K(m) benzene aromatic hydroxylases, toluene side-chain oxidase and 7-pentoxyresorufin O-depentylase, but not of 7-ethoxyresorufin O-deethylase. Immunoblot analysis showed that anti-P450IA1/2 did not bind liver microsomal protein from either control and treated rats in the region of cytochrome P450s, whereas with anti-P450IIE1(98) a clear-cut band was seen in liver microsomes from control and treated rats, with intensities in the following order: 2-hexanone = acetonyl acetone greater-than-or-equal-to n-hexane > control > phenobarbital. With anti-P450IIB1/2, a band was detected in microsomes from phenobarbital-treated rats, and to a lesser extent, in microsomes from 2-hexanone- and acetonyl acetone-treated rats. Like the immunoblot analysis, anti-P450IIE1(91) inhibited toluene side-chain hydroxylase activity in all microsomes, except in preparations from phenobarbital-treated rats and anti-P450IIB1 in microsomes from phenobarbital-, 2-hexanone- and acetonyl acetone-treated rats. Anti-P450IIC11/6 also inhibited toluene side-chain hydroxylase activity: the inhibited activity in the five different microsome preparations was as follows: n-hexane = control > acetonyl acetone = 2-hexanone = phenobarbital. These results indicate that n-hexane induces only quantitative alterations in the constitutive cytochrome P450 isozyme (P450IIE1), whereas its metabolites 2-hexanone and acetonyl acetone induce not only quantitative changes in constitutive cytochrome P450 (P450IIE1 and P450IIC11/6) but also a different type of isozyme (P450IIB1/2).