Induction of oxidative stress by humic acid through increasing intracellular iron: a possible mechanism leading to atherothrombotic vascular disorder in blackfoot disease.

Humic acid (HA), a potential toxin that has penetrated the drinking well water of blackfoot disease-endemic areas in Taiwan, has been implicated as an etiological factor of this disease. In this study, we investigated the effects of HA on the generation of reactive oxygen species (ROS) in cultured human umbilical vein endothelial cells (HUVECs). The generation of ROS was monitored by flow cytometry. Pretreatment of HUVECs with HA induced reactive oxygen species in a dose- and time-dependent manner. Xanthine oxidase inhibitor (Allopurinol), NADPH oxidase inhibitor (diphenylene iodomium) and calcium chelator (BAPTA) could not reduce the generation of ROS. Protein kinase C inhibitor (H7) could reduce the generation of ROS slightly, but the intracellular antioxidant glutathione monoethyl ester and the iron chelator desferrioxamine (DFO) could inhibit the generation of ROS completely. HA also enhanced the expression of ferritin and induced intracellular chelatable iron; however, HA reduced the expression of transferrin receptor. Pretreatment with DFO inhibited HA-mediated increases of ferritin synthesis and intracellular chelatable iron, but caused recovery of the inhibitory effect on transferrin receptor. Cotreatment with iron and HA induced more ROS and intracellular chelatable iron than iron or HA treatment alone. Furthermore, HA enhanced the accumulation of iron in endothelial cells. These data demonstrate that HA can increase the generation of ROS through enhancing the accumulation of intracellular iron. Taken together, our findings suggest that iron mediates HA-associated oxidative stress in endothelial cells, which may be a possible mechanism leading to atherothrombotic vascular injury observed for patients with blackfoot disease.

Humic acid suppresses the LPS-induced expression of cell-surface adhesion proteins through the inhibition of NF-kappaB activation.

Humic acid (HA), a potential toxin when penetrating the drinking well water of blackfoot disease-endemic areas in Taiwan, has been implicated as one of the etiological factors of this disease. In this study, we investigated the effects of HA on the expression of human vascular endothelial-leukocyte adhesion molecules and the activation of nuclear factor kappa B (NF-kappaB) in cultured human umbilical vein endothelial cells (HUVECs). The expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin was monitored by flow cytometry. Pretreatment of HUVECs with HA inhibited the lipopolysaccharide (LPS)-induced expression of these three adhesion molecules in a dose- and time-dependent manner. Since NF-kappaB can regulate the expression of these adhesion molecules, NF-kappaB activation was assessed by electrophoretic mobility shift assay (EMSA). Our results reveal that the activation of NF-kappaB by LPS is suppressed by HA in a dose- and time-dependent manner. Furthermore, HA reduces NF-kappaB binding to DNA slightly, but completely inhibits the degradation of IkappaBalpha at a concentration of 100 microg/ml. Thus, all our data demonstrate that HA can inhibit the LPS-induced expression of adhesion molecules through the inhibition of NF-kappaB activation. HA may also suppress the immune or inflammatory reaction of HUVECs responsible for endotoxin, which could be one possible explanation for the causes of the infection and inflammation observed for patients with blackfoot disease. Our results also suggest that immune or inflammatory disturbance occurs for patients with blackfoot disease and that NF-kappaB may be a critical molecule in the pathogenesis of this disease.

Parasites in grizzly bears from the central Canadian Arctic.

Standardized flotation techniques were used to survey 56 grizzly bear (Ursus arctos) fecal samples for parasites. The samples were collected during the spring and autumn of 1995 and 1996 in the central Arctic of the Northwest Territories (Canada). Parasites of the genera Nematodirus, gastrointestinal coccidia, and an unidentified first stage protostrongylid larva are reported for the first time from grizzly bear feces in North America. Parasites of the genera Diphyllobothrium and Baylisascaris also were collected. Prevalence of gastrointestinal parasites were significantly different between the spring and autumn seasons (31% and 58% respectively). Thus, we provide evidence supporting the theory that bears void gastrointestinal parasites before hibernation.

Free radical-scavenging properties of lignin.

Lignin is one of the major components of dietary fiber. It is a complex hydrophobic molecule that typically occurs in cell walls with heteroxylans. Our experimental data show that lignin is a free radical scavenger. When the NADH-phenazine methosulfate-nitro blue tetrazolium free radical-producing system is used, an alkali-lignin concentration of 46.29 micrograms/ml that causes 50% inhibition of uric acid production by xanthine oxidase (IC50) is a scavenger of superoxide anion radicals. Spectrophotometric assay has shown that alkali-lignin with an IC50 of 59.08 micrograms/ml inhibits the activity of xanthine oxidase, one of the enzymes related to the production of superoxide anion radicals, and presents a mixed-type noncompetitive inhibition pattern. Using the deoxyribose method, we have found that alkali-lignin is a hydroxyl radical scavenger with an IC50 of 250 micrograms/ml, and using the thiobarbituric acid method, we can see that alkali-lignin inhibits nonenzymatic and enzymatic lipid peroxidation with an IC50 of 72 and 100 micrograms/ml, respectively. Alkali-lignin also hinders the activity of glucose-6-phosphate dehydrogenase, another enzyme related to the generation of superoxide anion radicals, with an IC50 of 123.6 micrograms/ml, and obstructs the growth and viability of cancer (HeLa) cells in a dose-dependent manner. Our experimental results suggest another mechanism whereby the free radical-scavenging activity of lignin in dietary fiber may be involved in the fiber-colon cancer interaction. We also suggest that the ability of dietary fiber to protect against colon cancer may be partly determined by the amount of lignin in dietary fiber as well as the free radical-scavenging ability of lignin.