Binding of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to protein- and low molecular weight thiols and its role in ring hydroxylation.
The N-oxidized species of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have been shown to react with thiols. We have previously characterized a glutathione conjugate of PhIP linked via the C2 of PhIP with apparent loss of the amino group, in rat hepatocytes and PhIP exposed rats. This metabolite was possibly formed from 1-methyl-2-nitro-6-phenylimidazo[4,5-b]pyridine (nitro-PhIP). Upon reacting nitro-PhIP with rat albumin, both in the presence and absence of a reducing system, four products were observed after enzymic proteolysis. One of them was markedly increased after 2-mercaptoethanol treatment of the protein. This adduct was linked to a cysteine-S via C2 of PhIP. Using N2-acetoxy-PhIP as a starting material, an unstable protein adduct was observed which degraded to 50% of the original concentration (t 1/2) after 3 days. This is compatible with the finding that serum PhIP adducts decline rapidly in PhIP exposed rats. Unstable adducts were also formed following the reaction of N2-acetoxy-PhIP with glutathione or cysteine. Based on mass spectroscopy and UV spectra analysis, the suggested structures were RS(-S-)-(H)N2-PhIP. In all cases a degradation product identified as 5-hydroxy-PhIP was formed as characterized by mass spectrometry and NMR spectroscopy. 5-hydroxy-PhIP and its glucuronyl derivative were also observed in rat hepatocytes incubated in vitro with PhIP. In bile of PhIP-exposed rats, only the glucuronyl derivative was observed. Depletion of glutathione reduced the amount excreted in bile and experiments with microsomes indicate that hydroxylation directly at the 5 position is not mediated by cytochrome P-450 mono-oxygenase system. This indicates that 5-hydroxy-PhIP may be formed from N-acetoxy-PhIP via binding to thiols also in cells.
Alexander
J
J
Department of Environmental Medicine, National Institute of Public Health, Oslo, Norway.
Reistad
R
R
Frandsen
H
H
Grivas
S
S
D000818:Animals; D005978:Glutathione / Q000737:chemistry; D006900:Hydroxylation; D007093:Imidazoles / Q000737:chemistry*; D008099:Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D007093:Imidazoles; D011506:Proteins; D013438:Sulfhydryl Compounds; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D005978:Glutathione