Electrochemical determination for enzymic 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.
Minamoto
S
S
Department of Industrial Education, Ashiya University, Japan.
Kanazawa
K
K
D000818:Animals; D001711:Biotransformation; D002243:Carbolines / Q000032:analysis* / Q000378:metabolism*; D002273:Carcinogens / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004563:Electrochemistry / Q000379:methods; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D002243:Carbolines; D002273:Carcinogens; C027210:3-hydroxyamino-1-methyl-5H-pyrido(4,3-b)indole; C019133:3-amino-1-methyl-5H-pyrido(4,3-b)indole; D003577:Cytochrome P-450 Enzyme System