Stimulation of endothelin production in cultured human endothelial cells by fluorescent compounds associated with blackfoot disease

The fluorescent compounds (FC) in humic substances isolated from the drinking water of endemic areas of blackfoot disease have been characterized as the causative factor of the disease. In this report we studied the effects of these fluorescent compounds on the synthesis and secretion of endothelin-1 by cultured human umbilical cord vein endothelial cells (HUVEC) in order to explore the possible pathological mechanism(s) of FC in the development of atherothrombotic vasculopathy in blackfoot disease. Incubation of confluent HUVEC with FC (100ug/ml) enhanced endothelin mRNA expression. A sensitive ELISA method was developed to quantitate secreted endothelin antigen levels in conditioned medium. Incubation of confluent HUVEC cultures with different concentrations of FC ( 0-200 ug/ml) resulted in concentration-dependent enhanced production of endothelin. No significant changes in endothelin production were observed when subconfluent HUVEC cultures were incubated with FC. FC dose-dependently reduced the growth rate of subconfluent cultured HUVEC and exerted cytotoxic effects on the viability and integrity of confluent HUVEC monolayers. Since endothelin is one of the most potent vasoconstrictors affecting blood pressure and blood flow, and is mitogenic for smooth muscle cell proliferation, these results imply that the enhanced endothelin production and the reduced cell viability and growth rate induced by FC may contribute to the atherothrombotic vasculopathy observed in blackfoot disease.