Formation of a glutathione conjugate and a semistable transportable glucuronide conjugate of N2-oxidized species of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat liver.

We have previously shown that 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine(2-h ydroxamino-PhIP) is the principal metabolite leading to mutations in Salmonella typhimurium TA98 and DNA damage in mammalian cells. In rat hepatocytes this metabolite can be further conjugated to 2-(N-beta-D-glucuronopyranosyl (hydroxamino)-1-methyl-6-phenylimidazo[4, 5-b]pyridine[N(OH)-gluc-PhIP]. Its rate of formation was increased in hepatocytes from polychlorinated biphenyl (PCB)-pretreated animals. This metabolite is the main metabolite of PhIP in bile and it is hydrolyzed both by human and rat intestinal bacteria. Smaller amounts are excreted into urine. The evidence for the proposed structure is based on 1H- and 13C-NMR, beta-glucuronidase-lability giving 2-hydroxamino-PhIP upon hydrolysis and on the results obtained by using biochemical enzyme inhibitors. N(OH)-gluc-PhIP may be important for genotoxic lesions and tumors of 2-amino-1methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in extrahepatic tissue. In hepatocytes and bile from PCB-pretreated rats a PhIP-glutathione conjugate, 2-glutathionyl-1-methyl-6-phenylimidazo[4,5-b]pyridine (GSH-PhIP) was also found. The evidence for the proposed structure is based on 1H-NMR and high-resolution mass spectrometry. The metabolite can also be produced by a direct nucleophilic substitution of the nitro group in 2-nitro-PhIP by glutathione (GSH) in vitro. The metabolite did not form from 2-hydroxamino-PhIP and GSH either directly or in the presence of glutathione S-transferase. The formation of GSH-PhIP in rat liver and isolated cells only at a high rate of 2-hydroxamino-PhIP formation (PCB-treated animals) indicates that 2-nitro-PhIP may be formed in the liver during such N-oxidation of PhIP.

Differential rates of metabolic activation and detoxication of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by different cytochrome P450 enzymes.

Rat liver microsomes metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to the genotoxic metabolite 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-hydroxamino-PhIP) and to the detoxified product 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP). A 25-fold higher rate of metabolism was measured in microsomes from polychlorinated-biphenyl-treated rats (94 nmol/mg proteins/30 min) in comparison with those from untreated rats. Other effective inducers of PhIP metabolism were beta-naphthoflavone and isosafrole (ISF), whereas phenobarbital was ineffective. About twice as much 2-hydroxamino-PhIP as 4'-hydroxy-PhIP was formed in microsomes irrespective of the inducer the rats had been treated with. The metabolism was dependent on NADPH and was abolished by the cytochrome P450 inhibitor alpha-naphthoflavone. In a reconstituted enzyme system purified rat cytochrome P450 IA2 (P450ISF-G) had the highest N-hydroxylation rate (30 nmol/nmol P450/30 min) closely followed by the rat cytochrome P450 IA1 (P450BNF-B). Less activity was seen with rat P450 IIC11 (P450UT-A) and rabbit P450 IA2 (P450 LM4). Rat P450 IIE1 (P450j), P450 IIB1 (P450PB-B) and rabbit P450 IIB4 (P450 LM-2) and P450 IIE1 (P450 LM3a) were essentially inactive. Rat P450 IA1 (P450BNF-B) produced five times more 4'-hydroxy-PhIP (32 +/- 2 nmol/nmol P450/30 min) than did P450 IA2 (P450ISF-G). Hence, the measured ratio of activation to detoxication for rat P450 IA2 (P450ISF-G) enzyme was 7-fold higher than that of the other active P450 enzymes.