RAT-LIVER MICROSOMAL CYTOCHROME P450-DEPENDENT OXIDATION OF 3,5-DISUBSTITUTED ANALOGS OF PARACETAMOL

1. The cytochrome P450-dependent binding of paracetamol and a series o f 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -CH3, -C2H5, -iC(3)H(7)) have been determined with beta-naphthoflavone (bet a NF)-induced rat liver microsomes and produced reverse type I spectra l changes. K-s,K-app varied from 0.14 mM for 3,5-diiC(3)H(7)-paracetam ol to 2.8 mM for paracetamol. 2. All seven analogues underwent rat liv er microsomal cytochrome P450-dependent oxidation, as reflected by the formation of GSSG in the presence of GSH. The GSSG-formation was incr eased in all cases upon pretreatment of rats by beta-naphthoflavone (b eta NF) and was generally decreased upon pretreatment by phenobarbital (PB). 3. Rat liver microsomal cytochrome P450 as well as horseradish peroxidase catalysed the formation of 3,5-disubstituted NAPQI analogue s from the corresponding parent compounds, as identified by UV-spectro photometry of the NAPQI analogues and by GC/MS detection of the follow ing GSH-conjugates: lutathione-S-yl-3,5-dimethyl-1,4-dihydroxybenzene, 2-glutathione-S-yl-3,5-dichloro-paracetamol, and 2-glutathione-S-yl-3 ,5-dibromo-paracetamol. 4. In liver microsomal (beta NF-induced) incub ations, apparent K,values, as determined for the cytochrome P450 catal ysis-dependent oxidation of GSH, for seven 3,5-disubstituted paracetam ol analogues (R = -F, -Cl, -Br, -I, -CH3, -C2H5, iC(3)H(7)) varied fro m 0.07 to 0.64 mM. Paracetamol exhibited an apparent K-m of 0.73 mM. A pparent V-max values for the cytochrome P450 catalysis dependent oxida tion of GSH varied from 0.66 nmol min(-1) mg(-1)protein for paracetamo l to 3.0 nmol min(-1) mg(-1) protein for 3,5-dimethyl-paracetamol.