Catalysis of the dealkylation/debenzylation of phenoxazone ethers by hemoglobin in the absence of peroxides: implications for investigations of embryonic biotransformation.
Investigations of catalysis of the O-dealkylation and O-debenzylation of phenoxazone (resorufin) ethers in human and rodent embryonic tissue homogenates indicated that, with few exceptions, each conceptal tissue investigated contained enzymes capable of catalyzing each of the reactions under study. All observable reactions exhibited NADPH dependence and strong inhibition by carbon monoxide, ketoconazole, alternate electron acceptors, and by hypoxic incubation conditions; but, they were not strongly inhibited by several other classical cytochrome P450 (P450) inhibitors. Cyanide, azide, superoxide dismutase/catalase, and glutathione/glutathione peroxidase each also failed to inhibit the reactions significantly. Subcellular fractionation experiments revealed that cytosolic fractions contained a preponderance of the observable monooxygenase activities. Attempts to identify components responsible for the cytosolic catalytic activity indicated that cytosolic nitric oxide synthases did not contribute significantly. Column fractionation of the cytosol indicated that significant catalytic activity coeluted with fractions containing hemoglobin (Hgb), and experiments with purified Hgb as enzyme source showed that Hgb would catalyze all reactions under study at very slow rates in the absence of added reductases or peroxides. Additions of either reductases or peroxides, however, resulted in marked increases in rates of Hgb-catalyzed reactions. Further investigations strongly suggested that virtually all dealkylation or debenzylation of phenoxazone ethers catalyzed by embryonic cytosolic fractions could be accounted for by the presence of Hgb in those fractions. Conceptal microsomal fractions, however, exhibited definitive, P450-dependent monooxygenase activities attributable to specific individual, identifiable P450 isoforms.
Juchau
M R
MR
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195, USA.
Chapman
D E
DE
Yang
H Y
HY
Lee
Q P
QP
Namkung
M J
MJ
Hinds
T R
TR
D000818:Animals; D001593:Benzyl Compounds / Q000378:metabolism; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis; D003640:Dealkylation; D004622:Embryo, Mammalian / Q000378:metabolism*; D004987:Ethers / Q000378:metabolism; D005260:Female; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D011247:Pregnancy; D051381:Rats
D001593:Benzyl Compounds; D004987:Ethers; D006454:Hemoglobins; D010078:Oxazines; D010545:Peroxides; C014180:resorufin; D003577:Cytochrome P-450 Enzyme System