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.