Electrochemical Determination for Enzymatic Production of Ultimate Carcinogen from Tryptophan Pyrolysate by Rat Hepatic Microsomes

This study established a rapid and sensitive method of determining the level of the ultimate carcinogen from 3-amino-1-methyl-5H-prido[4,3- b]indole (Trp-P-2) produced by rat hepatic microsomes. An electrochemical detector (ECD) used with high-performance liquid chromatography (HPLC) gave a linear calibration curve for synthetic N-hydroxy-Trp-P-2 (the ultimate carcinogenic form) at concentrations ranging between 0.3 and 340 pmol. The enzymic production of N-hydroxy-Trp-P-2 from Trp-P-2 was also determined by the ECD with HPLC. Hepatic microsomes (0.2 mg as protein) from rats treated with methylcholanthrene (MC) and phenobarbital (PB) were incubated with Trp-P-2 for 5 min. The mixture was centrifuged with acetonitrile and the supernatant was then analyzed using HPLC. The ECD determined the level of N-hydroxy-Trp-P-2 to levels nearing 1 pmol, and the preparation before submission to the HPLC took such a short time (5 min) that N-hydroxy-Tr-P-2 did not have sufficient time to decompose. Then, the microsomal N-hydroxylation activity on Trp-P-2 was compared with five different sources of microsomes. The microsomes from rats treated with MC plus PB, MC, PB, or polychlorinated biphenyl showed an activity level (mol/min/mol P450 enzymes) of 2.41 ± 0.19, 1.92 ± 0.21, 0.048 ± 0.017, and 1.79 ± 0.15, respectively, and those from untreated rats showed no activity. This method was useful for evaluating the N-hydroxylation activity of P450 enzymes.