Ferrous ion autoxidation and its chelation in iron-loaded human liver HepG2 cells

Ferrous ion (Fe²⁺) is long thought to be the most likely active species, producing oxidants through interaction of Fe²⁺ with oxygen (O₂). Because current iron overload therapy uses only Fe³⁺ chelators, such as desferrioxamine (DFO), we have tested a hypothesis that addition of a Fe²⁺ chelator, 2,2′-dipyridyl (DP), may be more efficient and effective in preventing iron-induced oxidative damage in human liver HepG2 cells than DFO alone. Using ferrozine as an assay for iron measurement, levels of cellular iron in HepG2 cells treated with iron compounds correlated well with the extent of lipid peroxidation (r = 0.99 after log transformation). DP or DFO alone decreased levels of iron and lipid peroxidation in cells treated with iron. DFO + DP together had the most significant effect in preventing cells from lipid peroxidation but not as effective in decreasing overall iron levels in the cells. Using ESR spin trapping technique, we further tested factors that can affect oxidant-producing activity of Fe²⁺ with dissolved O₂ in a cell-free system. Oxidant formation enhanced with increasing Fe²⁺ concentrations and reached a maximum at 5 mM of Fe²⁺. When the concentration of Fe²⁺ was increased to 50 mM, the oxidant-producing activity of Fe²⁺ sharply decreased to zero. The initial ratio of Fe³⁺:Fe²⁺ did not affect the oxidant producing activity of Fe²⁺. However, an acidic pH (< 3.5) significantly slowed down the rate of the reaction. Our results suggest that reaction of Fe²⁺ with O₂ is an important one for oxidant formation in biological system, and therefore, drugs capable of inhibiting redox activity of Fe²⁺ should be considered in combination with a Fe³⁺ chelator for iron overload chelation therapy.