The binding affinity of carcinogenic N-nitrosodimethylamine and N-nitrosomethylaniline to cytochromes P450 2B4, 2E1 and 3A6 does not dictate the rate of their enzymatic N-demethylation.
The interaction of carcinogenic N-nitrosodimethylamine (NDMA) and N-nitrosomethylaniline (NMA) with cytochromes P450 (CYP), CYP2B4, CYP2E1 and CYP3A6, and their metabolism by these enzymes reconstituted with NADPH-CYP reductase in liposomes were studied. Using the difference spectroscopy, the lowest values of spectral dissociation constants (KS) of the binary complex of NDMA and NMA with the enzyme were found for CYP2E1. Both N-nitrosamines bind to the heme iron atom as ligands. On the contrary, the binding of NDMA and NMA to CYP2B4 gives the type I spectra. NDMA is bound to CYP3A6 analogously as to CYP2B4, whereas no difference spectra were acquired with NMA and CYP3A6. All tested CYPs oxidize NDMA and NMA. CYP2E1 exhibits the lowest Km values, 7.5 and 5.0 micromol/l for NDMA and NMA, respectively, and for CYP3A6 we found 30.0 and 10.0 micromol/l for NDMA and NMA, respectively, while CYP2B4 exhibits the lowest affinity for both carcinogens. In spite of different binding affinities of NDMA and NMA, the values of Vmax for their oxidation were, however, similar for all tested CYPs. The results demonstrate that investigations utilizing several enzymatic approaches are necessary to properly evaluate the mechanism and efficiency of NDMA and NMA oxidation by CYPs in vitro.
Sulc
Miroslav
M
Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 43 Prague 2, Czech Republic.
Hodek
Petr
P
Stiborová
Marie
M
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism*; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004128:Dimethylnitrosamine / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008862:Microsomes, Liver / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits
D002273:Carcinogens; D008081:Liposomes; D009602:Nitrosamines; D003577:Cytochrome P-450 Enzyme System; C014102:N-methyl-N-nitrosoaniline; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); C100638:cytochrome P-450 CYP3A6 (rabbit); D009251:NADPH-Ferrihemoprotein Reductase; D004128:Dimethylnitrosamine