Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol.
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 beta-naphthoflavone (beta NF)-induced rat liver microsomes and produced reverse type I spectral changes. Ks,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 beta-naphthoflavone (beta 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 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 (beta NF-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 Vmax 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.
Bessems
J G
JG
Leiden/Amsterdam Center for Drug Research, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Te Koppele
J M
JM
Van Dijk
P A
PA
Van Stee
L L
LL
Commandeur
J N
JN
Vermeulen
N P
NP
D000082:Acetaminophen / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005978:Glutathione / Q000378:metabolism; D007097:Imines / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D013056:Spectrophotometry, Ultraviolet; D019324:beta-Naphthoflavone / Q000494:pharmacology
D016227:Benzoquinones; D007097:Imines; D000082:Acetaminophen; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; C028473:N-acetyl-4-benzoquinoneimine; D005978:Glutathione