Objective-Oxidative stress contributes to the pathogenesis of many diseases, including atherosclerosis and sepsis. We have previously described a novel class of therapeutic compounds with antioxidant and antiinflammatory properties. However, at present, the intracellular targets of these compounds have not been identified. The purpose of this study was to elucidate the mechanism by which 2 structurally-related antioxidants (AGI-1067 and AGI-1095) inhibit LPS induction of tissue factor (TF) expression in human monocytic cells and endothelial cells. Methods and Results-We found that succinobucol (AGI-1067) and AGI-1095 inhibited LPS induction of TF expression in both monocytic cells and endothelial cells. These compounds also reduced LPS induction of nuclear AP-1 and expression of Egr-1 without affecting nuclear translocation of NF-B. Importantly, these antioxidants inhibited LPS activation of the redox-sensitive kinase, apoptosis signal-regulating kinase-1 (ASK1) and the mitogen-activated protein kinases (MAPKs) p38, ERK1/2, and JNK1/2. Conclusions-AGI-1067 and AGI-1095 inhibit TF gene expression in both monocytic cells and endothelial cells through a mechanism that involves the inhibition of the redox-sensitive MAP3K, ASK1. These compounds selectively reduce the activation/induction of MAPK, AP-1, and Egr-1 without affecting NF-B nuclear translocation. (Arterioscler Thromb

The pathogenesis of chronic inflammatory diseases, including rheumatoid arthritis, is regulated, at least in part, by modulation of oxidation-reduction (redox) homeostasis and the expression of redox-sensitive inflammatory genes including adhesion molecules, chemokines, and cytokines. (1,1-dimethylethyl)-4-hydroxyphenyl]thio]-1-methylethyl]-thio]-2,6-bis(1,1-dimethylethyl)phenoxy]acetic acid] is a novel, orally active, phenolic antioxidant and anti-inflammatory compound with antirheumatic properties. To elucidate its anti-inflammatory mechanisms, we evaluated AGIX-4207 for a variety of cellular, biochemical, and molecular properties. AGIX-4207 exhibited potent antioxidant activity toward lipid peroxides in vitro and displayed enhanced cellular uptake relative to a structurally related drug, probucol. This resulted in potent inhibition of cellular levels of reactive oxygen species in multiple cell types. AGIX-4207 selectively inhibited tumor necrosis factor (TNF)-␣-inducible levels of the redox-sensitive genes, vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, with less inhibition of E-selectin, and no effect on intracellular adhesion molecule-1 expression in endothelial cells. In addition, AGIX-4207 inhibited cytokine-induced levels of monocyte chemoattractant protein-1, interleukin (IL)-6, and IL-8 from endothelial cells and human fibroblast-like synoviocytes as well as lipopolysaccharide-induced release of TNF-␣, IL-1␤, and IL-6 from human peripheral blood mononuclear cells. AGIX-4207 did not inhibit TNF-␣-induced nuclear translocation of nuclear factor of the -enhancer in B cells (NF-B), suggesting that the mechanism of action is independent of this redoxsensitive transcription factor. Taken together, these results provide a mechanistic framework for understanding the anti-inflammatory and antirheumatic activity of AGIX-4207 and provide further support for the view that inhibition of redoxsensitive inflammatory gene expression is an attractive approach for the treatment of chronic inflammatory diseases.Leukocyte trafficking to areas of tissue injury and infection is required for the initiation of an effective immune response aimed at tissue repair. This normally self-limiting process is mediated by a highly regulated series of cell-cell interactions between leukocytes and the vascular endothelium. In part, these interactions are controlled by inducible cellular adhesion molecules on the endothelium including vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion moleThis work was supported by AtheroGenics, Inc. Article, publication date, and citation information can be found at

The effect of dosing vehicle on toxicity and metabolism of unsaturated aliphatic nitriles was investigated in male Sprague-Dawley rats. Five unsaturated aliphatic nitriles--acrylonitrile, methacrylonitrile, allylnitrile, crotononitrile and fumaronitrile--were prepared in five different dosing vehicles--saline, corn oil, safflower oil, mineral oil, olive oil and Tween-20. Groups of six male rats were given 0.5 LD50 doses of the nitriles by gavage and they were observed for 12 It for cholinomimetic and central nervous system effects. Cyanide and glutathione levels were determined in blood and various organs at 1, 3 and 6 h after nitrile administration and thiocyanate levels were determined at 6 h after nitrile administration. The results indicate that all the vehicles studied potentiated the toxicity of all the nitriles compared to nitriles administered in saline and significantly increased their metabolism to cyanide and thiocyanate and nitrile-induced depletion of glutathione in rats. This behavior of vehicles illustrates the difficulty of identifying suitable vehicles for administration of lipophilic compounds in toxicology studies.

The use of moist smokeless tobacco products is associated with acute oral injuries such as leukoplakia. Unfortunately, there is a limited understanding of the mechanisms and signaling pathways associated with these injuries. The present studies investigate the effects of a Reference 1S3 moist smokeless tobacco extract (STE) on oral injury, oxidative stress and MAPK signaling in multiple human oral keratinocyte cell lines utilizing cytospin slide analysis to observe alterations in cell morphology, DCF‐DA fluorescence, and western blotting techniques. STE caused dose‐dependent keratinocyte cell death within 30 minutes to 3 h of exposure. STE enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 or p38 MAPK, but not ERK1/2, suppressed STE‐induced cell death. STE promoted the generation of reactive oxygen species (ROS). The anti‐oxidants, Trolox and L‐ascorbic acid, suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Thus, reference STE leads to cell death in human oral keratinocyte cell lines in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.Research supported by Altria Client Services, 601 East Jackson St. Richmond, VA 23219 U.S.A.