N-hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver.
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [13C,15N2]MeIQx and [14C]MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M+H]+) from the parent compound, MeIQx and at 230/233 ([M+H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M+H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.
Rich
K J
KJ
Clinical Pharmacology, Royal Postgraduate Medical School, London, UK.
Murray
B P
BP
Lewis
I
I
Rendell
N B
NB
Davies
D S
DS
Gooderham
N J
NJ
Boobis
A R
AR
D002273:Carcinogens / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004032:Diet; D006801:Humans; D006900:Hydroxylation; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism*; D009153:Mutagens / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011810:Quinoxalines / Q000378:metabolism*; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology
D002273:Carcinogens; D065607:Cytochrome P-450 Enzyme Inhibitors; D009153:Mutagens; D011810:Quinoxalines; C036990:2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline; D003577:Cytochrome P-450 Enzyme System; D013806:Theophylline; C050131:furafylline; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2