Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted.
Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation.
Ingelman-Sundberg
M
M
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Kaur
H
H
Terelius
Y
Y
Persson
J O
JO
Halliwell
B
B
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D016166:Free Radical Scavengers; D005841:Gentisates; D006878:Hydroxides / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism*; D006900:Hydroxylation; D008051:Lipid Bilayers / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D012459:Salicylates / Q000378:metabolism*; D020156:Salicylic Acid; D013379:Substrate Specificity
D016166:Free Radical Scavengers; D005841:Gentisates; D006878:Hydroxides; D062385:Hydroxybenzoates; D008051:Lipid Bilayers; D012459:Salicylates; C009135:2,3-dihydroxybenzoic acid; D003577:Cytochrome P-450 Enzyme System; C031356:hydroxide ion; D020156:Salicylic Acid; C010925:2,5-dihydroxybenzoic acid