BINDING OF 2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE (PHIP) TO PROTEIN-THIOLS 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]pyrid ine (PhIP) have been shown to react with thiols, We have previously ch aracterized a glutathione conjugate of PhIP linked via the C2 of PhIP with apparent loss of the amino group, in rat hepatocytes and PhIP exp osed rats. This metabolite was possibly formed from 1-methyl-2-nitro-6 -phenylimidazo[4,5-b]pyridine (nitro-PhIP). Upon reacting nitro-PhIP w ith rat albumin, both in the presence and absence of a reducing system , four products were observed after enzymic proteolysis. One of them w as markedly increased after 2-mercaptoethanol treatment of the protein . This adduct was linked to a cysteine-S via C2 of PhIP. Using N-2-ace toxy-PhIP as a starting material, an unstable protein adduct was obser ved which degraded to 50% of the original concentration (t(1/2)) after 3 days. This is compatible with the finding that serum PhIP adducts d ecline rapidly in PhIP exposed rats. Unstable adducts were also formed following the reaction of N-2-acetoxy-PhIP with glutathione or cystei ne. Based on mass spectroscopy and UV spectra analysis, the suggested structures were RS(-S-)-(H)N-2-PhIP. In all cases a degradation produc t identified as 5-hydroxy-PhIP was formed as characterized by mass spe ctrometry and NMR spectroscopy. 5-hydroxy-PhIP and its glucuronyl deri vative were also observed in rat hepatocytes incubated in vitro with P hIP. 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 s ystem. This indicates that 5-hydroxy-PhIP may be formed from N-acetoxy -PhIP via binding to thiols also in cells.