Evaluation of iron binding and peroxide-mediated toxicity in rat hepatocytes.
A novel assay was developed to determine subnanomolar amounts of Fenton-reactive iron (FRI) in biological tissues. FRI represents that pool of iron that is redox active and capable of participating in a model Fenton reaction. The FRI was used to identify a kinetically-defined cellular iron binding site. This site displays positive cooperativity, with apparent kinetic constants of Kd = 10.6 microM, Bmax = 20.7 nmol/mg protein, and the Hill coefficient = 1.4. After addition of exogenous ferrous ammonium sulfate to hepatocytes, binding occurred within a few seconds and was stable for at least an hour. Free extracellular iron, but not bound iron, stimulated lipid peroxidation in hepatocytes. In contrast, bound but not free iron produced a concentration-dependent increase in tert-butyl hydroperoxide (TBH)-mediated toxicity, suggesting the toxicological relevance of bound, rather than free iron. Furthermore, the hydroxyl radical scavengers mannitol and 2-deoxyribose inhibited Fe2/TBH-mediated lipid peroxidation, but not cell killing, suggesting that hydroxyl radical may not be involved in the critical toxic event. The divalent cations Mn2+ and Co2+ inhibited iron-mediated hepatocyte killing in the presence of TBH, but only if added prior to Fe2+. Mn2+, but not Co2+, inhibited Fe(2+)-mediated lipid peroxidation regardless of the order of addition. These results indicate the existence of a specific, kinetically-defined cellular iron binding site. Such binding is involved in peroxide-mediated toxicity, but independent of lipid peroxidation. The specific nature of this site and involvement with other forms of chemical intoxication or cellular iron homeostasis are unknown.
Shertzer
H G
HG
Department of Environmental Health, University of Cincinnati Medical Center, OH 45267-0056.
Bannenberg
G L
GL
Moldéus
P
P
D000818:Animals; D001665:Binding Sites; D003855:Deoxyribose / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D006861:Hydrogen Peroxide; D066298:In Vitro Techniques; D007501:Iron / Q000032:analysis* / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008353:Mannitol / Q000494:pharmacology; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010545:Peroxides / Q000633:toxicity*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D020122:tert-Butylhydroperoxide
C045076:Fenton's reagent; D005296:Ferrous Compounds; D010545:Peroxides; D000644:Quaternary Ammonium Compounds; D017392:Thiobarbituric Acid Reactive Substances; D008353:Mannitol; D003855:Deoxyribose; D020122:tert-Butylhydroperoxide; C038178:ammonium ferrous sulfate; D006861:Hydrogen Peroxide; D007501:Iron