Humic acid mediates iron release from ferritin and promotes lipid peroxidation in vitro: a possible mechanism for humic acid-induced cytotoxicity

Humic acid (HA) has been shown to be a toxic factor for many mammalian cells, however the specific mechanism of the cytotoxicity induced by HA remains unclear. From the assessment of its redox properties, HA has been shown to be capable of reducing iron(III) to iron(II) in aqueous conditions over a broad range of pH values (from 4.0 to 9.0). By using thiobarbituric acid-reactive substances as an index, the presence of HA was noted to increase the extent of lipid peroxidation both for linoleic acids and within rat liver microsomes. In addition, the increase in HA-induced lipid peroxidation is partially inhibited by sodium azide (a singlet oxygen scavenger) or disodium 4,5-dihydroxy-1,3-benzene-disulfonic acid (a superoxide scavenger), reflecting the involvement of singlet oxygen and superoxide in the process of lipid peroxidation. The addition of HA into a reaction system has been shown to generate superoxide in a dose-dependent manner by the superoxide dismutase-inhibitable cytochrome c reduction assay. In addition, HA is able to reduce and release iron from ferritin, but this process is partially inhibited by superoxide scavengers. Subsequently, the iron released from ferritin was shown to accelerate the HA-induced lipid peroxidation. From our results we conclude that HA has the ability to reduce and release iron from ferritin storage as well as to promote lipid peroxidation. Therefore, HA coupled with released iron can disturb the redox balance and elicit oxidative stress within a biological system. This may be one of the most important mechanisms for HA-induced cytotoxicity.