title: Study of the redox properties of naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) and its glutathionyl conjugate in biological reactions: one- and two-electron enzymatic reduction.
abstract: Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone), the basic unit of several tetracyclic antitumor antibiotics, and its glutathione conjugate were reduced by the one- and two-electron transfer flavoproteins NADPH-cytochrome P450 reductase and DT-diaphorase to their semi- and hydroquinone forms, respectively. Kinetic studies performed on purified DT-diaphorase showed the following results: KNADPHm = 68 microM, KQuinonem = 0.92 microM, and Vmax 1300 nmol X min-1 X microgram enzyme-1. Similar studies performed on purified NADPH-cytochrome P450 reductase indicated a lower KNADPHm (10.5 microM) and higher KQuinonem (2.3 microM). The Vmax values were 20-fold lower (46 nmol X min-1 X micrograms enzyme-1) than those observed with DT-diaphorase. DT-diaphorase reduced the naphthazarin-glutathione conjugate with an efficiency 5-fold lower than that observed with the parent quinone. The nucleophilic addition of GSH to naphthazarin proceeded with GSH consumption at rates slower than those observed with 1,4-naphthoquinone and its monohydroxy derivative, 5-hydroxy-1,4-naphthoquinone. The initial rate of GSH consumption during these reactions did not vary whether the assay was carried out under anaerobic or aerobic conditions. Autoxidation accompanied the DT-diaphorase and NADPH-cytochrome P450 reductase catalysis of naphthazarin and its glutathionyl adduct as well as the 1,4-reductive addition of GSH to naphthazarin. Superoxide dismutase at catalytic concentrations (nM range) enhanced slightly (1.1- to 1.6-fold) the autoxidation following the enzymatic catalysis of naphthazarin. Autoxidation during the GSH reductive addition to 1,4-naphthoquinones decreased with increasing number of -OH substituents, 1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone greater than 5,8-dihydroxy-1,4-naphthoquinone, thus revealing that the contribution of redox transitions other than autoxidation, e.g., cross-oxidation, to the decay of the primary product of nucleophilic addition increases with increasing number of -OH substituents. Superoxide dismutase enhanced substantially the autoxidation of glutathionyl-naphthohydroquinone adducts, thereby affecting only slightly the total GSH consumed and GSSG formed during the reaction. The present results are discussed in terms of the relative contribution of one- and two-electron transfer flavoproteins to the bioreductive activation of naphthazarin and its glutathionyl conjugate as well as the importance of autoxidation reactions in the mechanism(s) of quinone cytotoxicity.
authors.affiliation: Department of Pathology II, University of Linköping, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Ollinger
authors.forename: J
authors.initials: J
authors.lastname: Llopis
authors.forename: E
authors.initials: E
authors.lastname: Cadenas
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D004579:Electron Transport; D005397:Fish Venoms / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009285:Naphthoquinones / Q000378:metabolism*; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000378:metabolism*; D051381:Rats
chemical_list: D000903:Antibiotics, Antineoplastic; D005397:Fish Venoms; D009285:Naphthoquinones; C005503:naphthazarin; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase; D011808:Quinone Reductases; D005978:Glutathione