Bufuralol hydroxylation by cytochrome P450 2D6 and 1A2 enzymes in human liver microsomes.
Bufuralol 1'-hydroxylation is a prototypical reaction catalyzed by cytochrome P450 (P450) 2D6, an enzyme known to show debrisoquine/sparteine-type genetic polymorphism in humans. In the present study we further examined the roles of several human P450 enzymes, as well as P450 2D6, in the hydroxylation of (+/-)-bufuralol, using liver microsomes from several human samples and human P450 enzymes expressed in human lymphoblastoid cell lines or Escherichia coli. Kinetic analysis of bufuralol 1'-hydroxylation by liver microsomes showed that there were different Km and Vmax values in seven human samples examined; low Km values (approximately 0.05 mM) were observed in four samples (including sample HL-18), high Km values (approximately 0.25 mM) in two samples (including sample HL-67), and an intermediate Km value (approximately 0.1 mM) in one sample. Quinidine and anti-rat P450 2D1 antibody almost completely inhibited bufuralol 1'-hydroxylation in human sample HL-18 at a substrate concentration of 0.4 mM, whereas these effects were not so drastic when liver microsomes from human sample HL-67 were used. In contrast, a very low concentration (< 10 microM) of alpha-naphthoflavone or anti-human P450 1A2 antibody significantly inhibited bufuralol 1'-hydroxylation catalyzed by human sample HL-67, but not HL-18, with 0.4 mM bufuralol. When the relative contents of P450 2D6 and P450 1A2 in 20 human samples were determined, bufuralol 1'-hydroxylation in samples containing large amounts of P450 2D6 tended to be more sensitive to quinidine, whereas the P450 1A2-rich samples were highly susceptible to alpha-naphthoflavone. However, at low substrate concentrations bufuralol 1'-hydroxylation was shown to be catalyzed principally by P450 2D6, based on the inhibitory effects of anti-rat P450 2D1 antibody and quinidine, in both human samples HL-18 and HL-67. At least five other, minor, bufuralol products were formed by human liver microsomes, in addition to 1'-hydroxybufuralol. Two of them were identified as 4- and 6-hydroxybufuralol by 1H NMR spectroscopy and mass spectrometry. The formation of the 4- and 6-hydroxylated products was suggested to be catalyzed by P450 1A2, based on the results of correlation with P450 1A2 contents in 60 human samples and inhibition by anti-P450 1A2 and alpha-naphthoflavone. Purified recombinant P450 1A2 (expressed in E. coli) produced 1'-, 4-, and 6-hydroxybufuralol in a reconstituted system, although P450 2D6 (expressed in human lymphoblast cell lines) was found to catalyze only bufuralol 1'-hydroxylation.(ABSTRACT TRUNCATED AT 400 WORDS)
Yamazaki
H
H
Osaka Prefectural Institute of Public Health, Japan.
Guo
Z
Z
Persmark
M
M
Mimura
M
M
Inoue
K
K
Guengerich
F P
FP
Shimada
T
T
D000319:Adrenergic beta-Antagonists / Q000378:metabolism* / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000918:Antibody Specificity; D001571:Benzoflavones / Q000494:pharmacology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D004983:Ethanolamines / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000319:Adrenergic beta-Antagonists; D000911:Antibodies, Monoclonal; D001571:Benzoflavones; D004983:Ethanolamines; D011994:Recombinant Proteins; C011512:alpha-naphthoflavone; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6