Abstract
Blackfoot disease (BFD) is a peripheral arterial occlusive disease found among the inhabitants of the southwest coast of Taiwan. Humic acid (HA) in well water used by the inhabitants for drinking is one possible etiological factors for BFD. In this study, the ability of natural HA, purified from the drinking well water of BFD endemic areas, and synthetic HA (SHA) to induce NO production and apoptosis, was studied in cultured human umbilical vein endothelial cells (HUVECs). Results show that HA could NO production and apoptosis in a dose-dependent manner. The characteristics of apoptosis in cultured endothelial cells include cell shrinkage, chromatin condensation, plasma membrane blebbing and DNA fragmentation. NO production and apoptosis of endothelial cells induced by HA were inhibited significantly by NOS inhibitor (L-NAME and L-NMA), free radical scavengers (SOD, vitamin C, and vitamin E), Ca2+ chelator (BATPA) and Protein kinase inhibitor (H7). These results suggest that Ca2+ and oxidative stress are mediators of apoptosis caused by HA. Destruction of endothelial cells has been implicated in the process of the onset of BFD. The induction of excessive NO and apoptosis in endothelial cells may contribute the etiology of HA-induced vascular disorder of BFD in humans.