Identification of cytochrome P-450s involved in the formation of APNH from norharman with aniline.

Abstract

Mutagenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), formed from norharman and aniline in the presence of S9 mix, is thought to be accountable for the co-mutagenic action of norharman. Our previous studies suggest that cytochrome P-450s (CYPs) are involved in the generation of APNH. In order to identify the responsible CYP species in the present study, norharman (8 mg) and aniline (4 mg) were incubated with individual recombinant human CYPs (2 nmol) at 37 degrees C for 20 min. Formation of APNH was observed with CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2D6, CYP2E1 and CYP3A4, but not with CYP2A6, CYP2C9 and CYP2C19. The amounts of APNH from norharman and aniline were 33 ng for CYP1A1, 15 ng for CYP3A4, 7 ng for CYP2D6, 6 ng for CYP1A2 and 5 ng for CYP2B6. APNH formation in the presence of CYP1B1 and CYP2E1 was very low at around one fiftieth of that with CYP3A4. When CYP selective chemical inhibitors, such as furafylline for CYP1A2 and ketoconazole for CYP3A4, were added to the reaction mixture of norharman, aniline and human microsomes, formation of APNH was decreased to 14 and 16% of the control level, respectively. Moreover, human lung microsomes also showed the activity of APNH formation from norharman and aniline, albeit at only one hundredth of that with liver microsomes. In general, content in human liver microsomes is rather high for CYP3A4 and CYP1A2 but relatively low for CYP2D6 and CYP2B6, at about 30, 10, 1.5% and less than 1% of the total CYP, respectively. Although CYP1A1 showed the highest APNH formation activity, its expression in human liver is reported to be below the level of detection. Based on these observations, it is suggested that the practical major contributors to the formation of APNH from norharman and aniline are CYP3A4 and CYP1A2, the responsible reactions mainly occurring in the liver.