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 electrochemi cal detector (ECD) used with high-performance liquid chromatography (H PLC) 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 phenoba rbital (PB) were incubated with Trp-P-2 for 5 min. The mixture was cen trifuged with acetonitrile and the supernatant was then analyzed using HPLC. The ECD determined the level of N-hydroxy-Trp-P-2 to levels nea ring 1 pmol, and the preparation before submission to the HPLC took su ch 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 Tr p-P-2 was compared with five different sources of microsomes. The micr osomes from rats treated with MC plus PB, MC, PB, or polychlorinated b iphenyl showed an activity level (mol/min/mel P450 enzymes) of 2.41 +/ - 0.19, 1.92 +/- 0.21, 0.048 +/- 0.017, and 1.79 +/- 0.15, respectivel y, and those from untreated rats showed no activity. This method was u seful for evaluating the N-hydroxylation activity Of P450 enzymes. (C) 1995 Academic Press,Inc.