Humic acid in drinking well water induces inflammation through reactive oxygen species generation and activation of nuclear factor-κB/activator protein-1 signaling pathways: A possible role in atherosclerosis

Humic acid (HA) has been implicated as one of the etiological factors in the peripheral vasculopathy of blackfoot disease (BFD) in Taiwan. However, the underlying pathophysiological mechanisms of BFD are not well defined. In this study, we used an in vitro and in vivo model, in which HA (25-200 mu g/mL) activated macrophages to produce pro-inflammatory molecules by activating their transcriptional factors. HA exposure induced NO and PGE(2) production followed by induction of iNOS and COX-2 through NF-kappa B/AP-1 transactivation in macrophages. In addition, the production of INF-alpha and IL-1 beta was significantly increased by HA. Moreover, HA-induced iNOS and COX-2 expression were down-regulated by the NF-kappa B and AP-1 inhibitors pyrrolidine dithiocarbamate (PDTC) and Tanshinone, respectively. Furthermore, generations of ROS and nitrotyrosine, as well as activation of the AKT and MAPKs signaling cascades were observed after HA exposure. Specifically, HA-induced NF-kappa B activation was mediated by ROS and ART, and that HA-induced AP-1 activation was mediated by JNK and ERR. Notably, HA-mediated AKT, JNK, and ERR activation was ROS-independent. The inflammatory potential of HA was correlated with increased expression of HO-1 and Nrf2. Furthermore, an in vivo study confirms that mice exposed to HA, the serum levels of TNF-alpha and IL-1 beta was significantly increased in a dose-dependent manner. This report marks the first confirmation that environmental exposure of HA induces inflammation in macrophages, which may be one of the main causes of early atherogenesis in blackfoot disease. (C) 2013 Published by Elsevier Inc.