Humic acid-induced echinocyte transformation in human erythrocytes: Characterization of morphological chances and determination of the mechanism underlying damage

Blackfoot disease (BFD) is a peripheral arterial occlusive disease found am ong human inhabitants along the southwest coast of Taiwan. Well water used for drinking and cooking contains humic acid (HA), which may be a possible etiological factor. In this study, HA toxicity was investigated in human er ythrocytes and was found to induce echinocytic formation. Morphological cha nges occurred in both a concentration- and time-dependent fashion. The pres ence of HA was also observed to facilitate the loading of erythrocytes with excess Ca2+ (1 mM), which may have occurred following permeability changes in cell membranes, leading to echinocytic transformations. Sodium dodecyl sulfate (SDS) gel electrophoresis indicated that echinocyte formation was d ue to the oxidation of normal membrane proteins that were replaced by high- molecular-weight proteins. Humic acid also induced hemoglobin oxidation in erythrocytes. Data show that oxidative stress generated by HA as well as di rect effects were exerted on the cytoskeleton of erythrocytes, and these ma y be significant factors in the etiology of BFD.