Cytochrome P-450-mediated oxidation of substrates by electron-transfer; role of oxygen radicals and of 1- and 2-electron oxidation of paracetamol.
The mechanism by which the hepatic cytochrome P-450 (Cyt. P-450) containing mixed-function oxidase system oxidizes the analgesic drug paracetamol (PAR) to a hepatotoxic metabolite was studied. Since previous studies excluded the possibility of oxygenation of PAR, three other mechanisms, namely direct 1-electron oxidation by a Cyt. P-450-ferrous-dioxygen complex under concomitant formation of H2O2 to N-acetyl-p-semiquinone imine (NAPSQI), direct 2-electron oxidation by a Cyt. P-450-ferric-oxene complex to N-acetyl-p-benzoquinone imine (NAPQI) and indirect oxidation by active oxygen species released from Cyt. P-450, were considered. Indirect oxidation by active oxygen species was not involved, as active oxygen scavengers such as superoxide dismutase, catalase and DMSO did not affect the oxidation of PAR in hepatic microsomes. No reaction products characteristic for a direct 1-electron oxidation of PAR by Cyt. P-450 were observed: neither NAPSQI radical formation was detectable by ESR, nor PAR-dimer formation, nor stimulation of the microsomal H2O2 production was found to occur. In fact, PAR inhibited the spontaneous microsomal H2O2 formation. Studies on the reactions of NAPSQI with glutathione (GSH) revealed that NAPSQI hardly conjugated with GSH to a 3-glutathionyl-paracetamol conjugate (PAR-GSH) conjugate. The reactions of the elusive reactive metabolite formed during microsomal oxidation of PAR in the presence of GSH closely resembled those of synthetic NAPQI: both PAR-GSH and oxidized glutathione (GSSG) formation occurred. Furthermore, in agreement with a 2-electron oxidation hypothesis, iodosobenzene-dependent oxidation of PAR by cyt. P-450 in the presence of GSH resulted in the formation of the PAR-GSH conjugate. It is concluded that bioactivation of PAR by the Cyt. P-450 containing mixed-function oxidase system consists of a direct 2-electron oxidation to NAPQI.
van de Straat
R
R
Department of Pharmacochemistry, Molecular Toxicology, Free University, Amsterdam, The Netherlands.
Vromans
R M
RM
Bosman
P
P
de Vries
J
J
Vermeulen
N P
NP
D000082:Acetaminophen / Q000378:metabolism*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D005609:Free Radicals; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D005609:Free Radicals; D011725:Pyridines; C013109:metapyrone; D000082:Acetaminophen; D009249:NADP; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; D010100:Oxygen; D019803:Glutathione Disulfide