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Metabolic activation of mutagenic tryptophan pyrolysis products by rat liver microsomes
Abstract:
In an attempt to determine whether cytochrome P-450 metabolizes tryptophan pyrolysis products, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) to active forms, studies were done using microsomes and the 9000 x g supernatant (S-9) from polychlorinated biphenyl mixture-treated rat livers together with Salmonella typhimurium TA 98 as a tester strain. The number of revertants increased with increments in the amount of S-9 fraction added. The highest mutation was seen with an amount of S-9 that is equivalent to 2.5 mg of liver tissue; however, further increase in the S-9 fraction resulted in a sharp decline in the number of revertants. There was a similar biphasic response when a relatively large amount of S-9 fraction was incubated for an increasing length of time. Microsomes showed parallel biphasic responses, and, in addition, the 105,000 x g supernatant fraction was ineffective in increasing the number of revertants. These results suggested that most if not all of the ability of the S-9 fraction to convert Trp-1 and Trp-P-2 to active forms resided in the microsomes. The involvement of microsomal cytochrome P-450 in this process was further confirmed by the following evidence. The treatment of rats with polychlorinated biphenyl mixture and 3-methylcholanthrene resulted in a marked increase in the ability of microsomes to activate Trp-P-1 and Trp-P-2, and the activation reaction required reduced nicotinamide adenine dinucleotide phosphate as a cofactor and was inhibited by carbon monoxide, 7,8-benzoflavone, n-octylamine, and 2-diethylaminoethyl-2,2-diphenylvalerate. The mutagenic metabolite formed as a result of microsomal metabolism of Trp-P-2 was fairly stable and survived heating at 90 degrees for 7 min. With a high-performance liquid chromatography, an active metabolite of Trp-P-2 was purified. A preliminary analysis showed this molecule to be an N-hydroxylated derivative of Trp-P-2.