Randall L. Davis

Ethanol increases nuclear factor-κB activity in human astroglial cells

Abstract Alcohol abuse adversely affects essentially all the organs of the body, either directly or indirectly. Ethanol may contribute to brain damage via inflammation. Ethanol may also alter CNS immunocompetence and further the progression of certain CNS infections. Nuclear factor (NF)-κB helps regulate inflammatory gene expression in glia. It is possible that ethanol effects on CNS pathology are partly a consequence of ethanol modulation of NF-κB-associated pathways in glia. We have assessed the effects of 0.5–6xa0h ethanol exposure on cytokine (5xa0ng/ml interleukin-1β + 100xa0ng/ml interferon γ + 30xa0ng/ml tumor necrosis factor-α)-induced NF-κB activation in human A172 astroglial cells. Immunoblot analysis indicated that NF-κB p65 nuclear translocation occurred within 0.5xa0h after cytokine stimulation. Stimulation in the presence of ethanol resulted in increased nuclear p65 levels at 3xa0h, with 200xa0mM causing a greater increase than 50xa0mM ethanol. Gel shift assay data suggested that cytokine-induced NF-κB binding activity was greatest in cells exposed to 50xa0mM ethanol, followed by 200 and 0xa0mM ethanol exposed cells, respectively. Thus, in cytokine-stimulated cells, 200xa0mM ethanol resulted in greater nuclear p65 levels, yet, 50xa0mM ethanol exposure resulted in more pronounced DNA binding by NF-κB. These findings demonstrate that acute ethanol enhances p65 activity in human astroglia and further support the hypothesis that ethanol-mediated brain pathology involves modulation of NF-κB pathways. A better understanding of the mechanistic events involved in ethanol-induced CNS pathology should provide for therapeutic strategies to combat detrimental effects of alcohol on the CNS.

The effect of taurine supplementation on patients with type 2 diabetes mellitus.

Diabetes Mellitus (DM) is the fifth leading cause of death in the US. Nearly 80% of diabetic mortality is secondary to cardiovascular disease resulting from atherosclerosis. Current therapy is based upon control of blood glucose, cholesterol and triglycerides, primarily through insulin replacement in Type 1 diabetes or oral hypoglycemic agents and/or insulin replacement in Type 2 diabetes. The more intensive the control, the lower the incidence of diabetic complications such as atherosclerosis. The principal investigators were interested in finding safe and effective nutritional supplements that would reduce the need for insulin replacement therapy, provide tighter glucose control, and protect against oxidative stress and the vascular pathology associated with diabetes mellitus. One such supplement is taurine.

Inhibition of selenite-induced cytotoxicity and apoptosis in human colonic carcinoma (HT-29) cells by copper

Selenite catalytically oxidizes reduced glutathione (GSH) with subsequent generation of superoxide. Our laboratory has previously shown that this selenite-catalyzed generation of superoxide is strongly inhibited by copper [as copper(II) sulfate]. In the present study we have demonstrated that exposure of human colonic carcinoma cells (HT-29) to selenite resulted in the induction of apoptosis, DNA fragmentation, an increase in intracellular levels of the antioxidant GSH, and cytotoxicity. Selenite-induced apoptosis, DNA fragmentation, increases in GSH levels, and cytotoxicity were inhibited by copper(II) sulfate. Copper only protected cells from selenite cytotoxicity when cells were exposed to selenite and copper simultaneously, not when cells were pretreated with copper, then washed before selenite exposure. This suggests that copper elicits its protective effect extracellularly. Previous data reported by this laboratory clearly demonstrated that copper inhibited selenite-catalyzed superoxide generation. Collectively, these data suggest that reactive oxygen species may play a role in selenite-induced cytotoxicity, apoptosis, and DNA fragmentation.

Differential Indicators of Diabetes-Induced Oxidative Stress in New Zealand White Rabbits: Role of Dietary Vitamin E Supplementation

Determination of reliable bioindicators of diabetes-induced oxidative stress and the role of dietary vitamin E supplementation were investigated. Blood (plasma) chemistries, lipid peroxidation (LPO), and antioxidant enzyme activities were measured over 12 weeks in New Zealand White rabbits (control, diabetic, and diabetic + vitamin E). Cholesterol and triglyceride levels did not correlate with diabetic state. PlasmaLPOwas influenced by diabetes and positively correlated with glucose concentration only, not cholesterol or triglycerides. Liver glutathione peroxidase (GPX) activity negatively correlated with glucose and triglyceride levels. Plasma and erythrocyte GPX activities positively correlated with glucose, cholesterol, and triglyceride concentrations. Liver superoxide dismutase activity positively correlated with glucose and cholesterol concentration. Vitamin E reduced plasma LPO, but did not affect the diabetic state. Thus, plasmaLPOwas the most reliable indicator of diabetes-induced oxidative stress. Antioxidant enzyme activities and types of reactive oxygen species generated were tissue dependent. Diabetes-induced oxidative stress is diminished by vitamin E supplementation.

Chronic ethanol inhibits CXC chemokine ligand 10 production in human A172 astroglia and astroglial-mediated leukocyte chemotaxis

Astroglia are the most prevalent cell type in the human central nervous system (CNS) and perform important roles in normal tissue homeostasis, during pathological events and following trauma. Astroglial-derived chemokines have important neurotrophic effects and are important to CNS immunocompetence and response to injury, in part, due to their direct role in leukocyte and microglial cell recruitment. However, while ethanol is known to induce CNS pathologies and to be peripherally immunosuppressive, ethanol effects on chemokine expression in human astroglia are essentially unknown. We have demonstrated that chemotaxis of human U937 leukocytic cells, across a 0.5 microm pore polycarbonate transmembrane insert, is induced in response to culture media collected from 10 microg/ml lipopolysaccharide (LPS) + 10 ng/ml interleukin (IL)-1beta-stimulated A172 human astroglia cells. The involvement of the chemokine CXCL10 (also known as interferon-gamma inducible protein or IP-10) in astroglial-induced chemotaxis of U937 cells has been indicated, as chemotaxis can be reduced by an anti-CXCL10 neutralizing antibody. Interestingly, chemotaxis of U937 cells, in response to astroglial-exposed media, is reduced when astroglia are chronically (9 days) exposed to 50 mM ethanol before stimulation with LPS + IL-1beta. Furthermore, we observed that LPS + IL-1beta-stimulated CXCL10 production is inhibited in human A172 astroglia exposed to chronic 50 mM ethanol. Thus, alterations in astroglial CXCL10 expression may disrupt CNS immunocompetence and play an important role in ethanol-induced CNS pathologies.

Identification of cis-regulatory regions necessary for robust Nos2 promoter activity in glial cells: indirect role for NF-κB

Previous reports suggest the nitric‐oxide synthase 2 (Nos2) promoter contains negative and positive cis‐regulatory regions. This study identified such regions using rat C6 glial cells. Activity of the serially deleted rat Nos2 promoter fused to a luciferase reporter gene was found to vary with construct size independent of stimuli, decreasing markedly from 160 to 130u2003bp then increasing significantly from 110 to 94u2003bp. In contrast, time to peak activity was stimulus‐dependent but size‐independent; 4–8u2003h for a cytokine mixture or lipopolysaccharideu2003+u2003interferon‐γ, and 8–16u2003h for lipopolysaccharideu2003+u2003phorbol 12‐myristate 13‐acetate. Peak activity with heterologous promoters also varied; 4u2003h for 3.7u2003kb of the human Nos2A promoter, and 36u2003h for 1.8u2003kb of the murine promoter. Electrophoretic mobility shift assays and in vivo DNA footprinting data confirmed nuclear protein binding to promoter regions suspected of containing important regulatory sites based on reporter gene data. A binding site for NF‐κB was not required for Nos2 promoter activity. These findings provide significant new information on the relative importance of different regions of the rat Nos2 promoter for transcriptional activation and nitric oxide production by glial cells and support the existence of cell‐ and species‐specific mechanisms for transcriptional regulation of Nos2 activation.