Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol

Abstract

1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -CH₃, -C₂H₅, -iC₃H₇) have been determined with β-naphthoflavone (βNF)-induced rat liver microsomes and produced reverse type I spectral changes. K_s,app varied from 0·14 mM for 3,5-diiC₃H₇-paracetamol to 2·8 mM for paracetamol.

2. All seven analogues underwent rat liver microsomal cytochrome P450-dependent oxidation, as reflected by the formation of GSSG in the presence of GSH. The GSSG-formation was increased in all cases upon pretreatment of rats by β-naphthoflavone (βNF) 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 analogues from the corresponding parent compounds, as identified by UV-spectrophotometry of the NAPQI analogues and by GC/MS detection of the following GSH-conjugates: 2-glutathione-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 (βNF-induced) incubations, apparent K_m values, as determined for the cytochrome P450 catalysis-dependent oxidation of GSH, for seven 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -CH₃, -C₂H₅, iC₃H₇) varied from 0·07 to 0·64 mM. Paracetamol exhibited an apparent K_m of 0·73 mM. Apparent V_max values for the cytochrome P450 catalysis dependent oxidation of GSH varied from 0·66 nmol min^-1 mg^-1 protein for paracetamol to 3·0 nmol min^-1 mg^-1 protein for 3,5-dimethyl-paracetamol.