Inhibitory monoclonal antibodies to human cytochrome P450 1A2: analysis of phenacetin O-deethylation in human liver.
Human cytochrome P450 1A2 metabolizes a large number of common drugs and engages in carcinogen metabolism and activation. Baculovirus-expressed 1A2 was used to immunize mice producing hybridomas yielding monoclonal antibodies (MAbs). Three of 2050 clones assayed yielded the MAbs, MAb 26-7-5, MAb 951-5-1, MAb 1812-2-4, which were specific for 1A2 as assessed by enzyme-linked immunosorbent assay and immunoblots. The three MAbs inhibited 1A2-catalysed metabolism of phenacetin, 7-ethoxycoumarin, chlorzoxazone and phenanthrene by more than 85%. The MAbs were highly specific to 1A2 and did not inhibit 11 other human P450s. The phenancetin O-deethylation activity varied from 0.44-2.49 nmol/min/nmol P450 in eight human liver microsomes samples. MAb 26-7-5 inhibited 1A2-dependent phenacetin O-deethylation in these samples by 64-84% indicating the amount of 1A2 contribution to this reaction and in addition a role for other P450s in the O-deethylation. Independent analysis of recombinant human P450s showed that 1A1, 1A2, 2A6 and 2C19 exhibited phenacetin O-deethylation activity, with 1A1 and 1A2 being the most active followed by 2C19 and 2A6. Eight other P450s were inactive towards phenacetin O-deethylation. The role of different P450 in eight liver samples was analysed with specific individual inhibitory MAbs. Inhibitory antibodies to 1A2, 2C8/9/18/19, 2A6, 2D6, 2E1, and 1A1 were combinatorially added to the microsomes. The O-deethylation activity was inhibited by antibodies to 1A2 (64-84%), to 2C19 (4.6-20%) and to 2A6 (0-8.8%). The total activity inhibited by antibodies to P450 2E1, 2D6 and 1A1 was less than 4.5%, indicating a minor role for these P450s in phenancetin metabolism in human liver microsomes. Thus, 1A2, 2C 9 and 2A6 are the dominant P450s for phenacetin O-deethylation. These studies demonstrate the use of inhibitory MAbs to P450s for a simple and precise assessment of the quantitative role of each P450 in the metabolism of substrates, including drugs, carcinogens, mutagens, environmental chemicals and endobiotics.
Yang
T J
TJ
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Sai
Y
Y
Krausz
K W
KW
Gonzalez
F J
FJ
Gelboin
H V
HV
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000918:Antibody Specificity; D019388:Cytochrome P-450 CYP1A2 / Q000276:immunology; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000378:metabolism; D010615:Phenacetin / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D010616:Phenanthrenes; C031181:phenanthrene; D003577:Cytochrome P-450 Enzyme System; D019388:Cytochrome P-450 CYP1A2; D010615:Phenacetin