thiourea and hydroxide-ion

Reactive oxygen species (ROS) play an important but not yet fully defined role in the expression of inflammatory genes such as monocyte chemoattractant protein (MCP)-1. We used complementary molecular and biochemical approaches to explore the roles of specific ROS and their molecular linkage to inflammatory signaling in endothelial cells. Adenovirus-mediated expression of superoxide dismutase and catalase inhibited TNF-alpha-induced MCP-1 gene expression, suggesting important roles of superoxide (O(2)(-).) and H(2)O(2) in MCP-1 gene activation. In addition, the iron chelator 1,2-dimethyl-3-hydroxypyridin-4-one and the hydroxyl radical scavengers dimethylthiourea and dimethyl sulfoxide inhibited TNF-alpha-induced MCP-1 expression, suggesting important roles of iron and hydroxyl radicals in inflammatory signal activation. In contrast, scavenging of peroxynitrite with 5,10,15,20-tetrakis-(4-sulfonatophenyl)prophyrinato iron (III) chloride had no effect on TNF-alpha-induced MCP-1 expression. Inhibition of NADPH oxidase, the major oxidase responsible for O(2)(-). generation, with diphenylene iodonium suppressed TNF-alpha-induced MCP-1 mRNA accumulation. Rac1 is an upstream signaling molecule for the activation of NADPH oxidase and O(2)(-). generation. Expression of dominant negative N17Rac1 by adenovirus suppressed TNF-alpha-induced MCP-1 mRNA levels and MCP-1 protein secretion. Expression of N17Rac1 inhibited TNF-alpha-induced MCP-1 and NF-kappaB transcriptional activity. These data suggest that ROS such as superoxide and H(2)O(2) derived from Rac1-activated NADPH oxidase mediate TNF-alpha-induced MCP-1 expression in endothelial cells.

Topics: Antineoplastic Agents; Aorta; Catalase; Cells, Cultured; Chemokine CCL2; Deferiprone; Dimethyl Sulfoxide; Endothelium, Vascular; Free Radical Scavengers; Gene Expression; Humans; Hydrogen Peroxide; Hydroxides; Iron; Iron Chelating Agents; Metalloporphyrins; NADPH Oxidases; NF-kappa B; Promoter Regions, Genetic; Pyridones; rac1 GTP-Binding Protein; RNA, Messenger; Signal Transduction; Superoxide Dismutase; Superoxides; Thiourea; Tumor Necrosis Factor-alpha

The pathogenesis of toxic shock syndrome (TSS) remains unknown. On the basis of experimental data, it has been hypothesized that staphylococcal TSS Toxin 1 (TSST-1) may interact synergistically with low levels of endotoxin and give rise to many of the clinical findings. We have demonstrated previously that lipid A, the biologically active component of lipopolysaccharide (LPS), or endotoxin, induces dose-dependent necrosis of isolated rat renal tubular cells (RTCs). In the present studies, the authors investigated whether this injury could be augmented by TSST-1. The viability of RTCs was assessed by vital dye exclusion. Incubation of freshly isolated rat RTCs with either 1 ng/ml of TSST-1 or 0.1 ng/ml LPS or lipid A had minimal cytotoxicity (less than 6%). Exposure of RTCs to 1 ng/ml TSST-1 for 20 minutes, followed by washing, resulted in a significant enhancement of cytotoxicity when RTCs were exposed to 0.1 ng/ml LPS or lipid A. The sensitization of RTCs by TSST-1 to LPS- or lipid-A-induced injury was prevented by methylamine and chloroquine, two inhibitors of receptor-mediated endocytosis (RME). Chelation of extracellular calcium by 2 mM EGTA also blocked the TSST-1-induced sensitization of RTCs to LPS or lipid A. Inhibition of RTC arachidonic acid metabolism by methylprednisolone, indomethacin, ibuprofen, and piriprost significantly inhibited RTC necrosis induced by TSST-1 and LPS or lipid A by 33-62%. Thiourea and deferoxamine, agents which ameliorate oxidant injury, also inhibited this synergistic injury by 34-67%. Thus, TSST-1 enhanced the cytotoxic effects of LPS/lipid A, and the sensitization of RTCs appeared to involve RME or TSST-1. Oxidative metabolism of arachidonic acid and generation of reactive oxygen species appeared to participate in LPS/lipid-A-mediated RTC death.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bacterial Toxins; Cell Separation; Cell Survival; Chloroquine; Deferoxamine; Endocytosis; Endotoxins; Enterotoxins; Hydroxides; In Vitro Techniques; Kidney Tubules; Lipid A; Lipopolysaccharides; Male; Methylamines; Necrosis; Rats; Rats, Inbred Strains; Superantigens; Thiourea