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 (25-200µg/mL) activated macrophages to produce pro-inflammatory molecules by activating their transcriptional factors. HA exposure induced NO and PGE2 production followed by induction of iNOS and COX-2 through NF-κB/AP-1 transactivation in macrophages. In addition, the production of TNF-α and IL-1ß was significantly increased by HA. Moreover, HA-induced iNOS and COX-2 expression were down-regulated by the NF-κB 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-κB activation was mediated by ROS and AKT, and that HA-induced AP-1 activation was mediated by JNK and ERK. Notably, HA-mediated AKT, JNK, and ERK 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-α and IL-1ß 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.

Aberrant ß-catenin expression in urothelial carcinomas in blackfoot disease-endemic areas.

Arsenic is a well-known toxic element and carcinogenic agent. The aim of this study was to investigate p63, E-cadherin, and ß-catenin proteins in urothelial carcinoma (UC) in both arsenic contaminated areas [so-called blackfoot disease (BFD) area] and non-BFD areas. The expressions of p63, E-cadherin, and ß-catenin proteins in 20 UC cases of blackfoot disease and 22 UC cases in non-BFD areas were detected using immunohistochemical methods. The results revealed a high p63 expression in 20 (47.6%) UC cases and high E-cadherin expression in six (14.3%) UC cases. Expressions of p63 and E-cadherin showed no significant correlations with clinicopathologic parameters. However, all 20 BFD cases and 12 of 22 (54.5%) non-BFD cases showed aberrant ß-catenin expression. Ten out of 22 (45.5%) non-BFD cases also had normal membranous immunoreactivity. The ß-catenin staining pattern significantly differed between cases in endemic and nonendemic areas of BFD (p=0.001). Tumor sites also significantly correlated with ß-catenin expression (p=0.044). In addition, membranous localization of ß-catenin was lower in UC from BFD-endemic areas compared with those from non-BFD endemic areas. In conclusion, it is suggested that relocalization of ß-catenin from membrane to cytoplasm may be involved in the tumorigenesis of UC from BFD-endemic areas.

Arsenic speciation in rice and risk assessment of inorganic arsenic in Taiwan population.

This study assessed the total arsenic content and arsenic speciation in rice to determine the health risks associated with rice consumption in various age-gender subgroups in Taiwan. The average total arsenic levels in white rice and brown rice were 116.6 ± 39.2 and 215.5 ± 63.5 ng/g weight (n = 51 and 13), respectively. The cumulative cancer risk among males was 10.4/100,000. The highest fraction of inorganic/total arsenic content in white rice ranged from 76.9 to 88.2 % and from 81.0 to 96.5 % in brown rice. The current study found different arsenic speciation of rice in southern Taiwan, where the famous blackfoot disease has been reported compared with arsenic speciation from other Taiwan areas. Therefore, rice and other grains should be further monitored in southern Taiwan to evaluate whether arsenic contamination is well controlled in this area.

Induction of macrophage cell-cycle arrest and apoptosis by humic acid.

Humic acid (HA) in well water is associated with Blackfoot disease and various cancers. Previously, we reported that acute humic acid exposure (25-200 µg/mL for 24 hr) induces inflammation in RAW264.7 macrophages. In this study, we observed that prolonged (72 hr) HA exposure (25-200 µg/mL) induces cell-cycle arrest and apoptosis in cultured RAW264.7 cells. We also observed that exposing macrophages to HA arrests cells in the G2 /M phase of the cell cycle by reducing cyclin A/B1 , Cdc2, and Cdc25C levels. Treating macrophages with HA triggers a sequence of events characteristic of apoptotic cell death including loss of cell viability, morphological changes, internucleosomal DNA fragmentation, sub-G1 accumulation. Molecular markers of apoptosis associated with mitochondrial dysfunction were similarly observed, including cytochrome c release, caspase-3 or caspase-9 activation, and Bcl-2/Bax dysregulation. In addition to the mitochondrial pathway, HA-induced apoptosis may also be mediated through the death receptor and ER stress pathways, as evidence by induction of Fas, caspase-8, caspase-4, and caspase-12 activity. HA also upregulates p53 expression and causes DNA damage as assessed by the comet assay. These findings yield new insight into the mechanisms by which HA exposure may trigger atherosclerosis through modulation of the macrophage-mediated immune system.