Increased Heat Shock Protein 70 Expression Following Toxicant-mediated Cytotoxicity: A Ubiquitous Marker of Toxicant Exposure?
Parivash Farzaneh, Abdolamir Allameh, Steven Pratt, Nicholas Moore, Lucy Travis, Elke Gottschalg, Clive Kind and Jeffrey Fry
The up-regulation of heat shock protein (HSP) expression has been proposed as a general biomarker of cellular protection against various environmental stresses and chemicals. The present study investigated the possibility of using HSP70 up-regulation as a biomarker of toxicant exposure in vitro. Cells of a rat hepatoma cell line (FGC4) were exposed to concentrations of 1,3-dichloroacetone, duroquinone, diquat dibromide, menadione, hydrogen peroxide, cadmium chloride (CdCl2) and sodium (meta)arsenite (NaAsO2) that elicited 20–50% cytotoxicity over a 24-hour period, and HSP70 levels were measured by ELISA. Up-regulation of HSP70 expression was demonstrated following treatment with menadione, CdCl2 and NaAsO2, but not with the other chemicals tested. A shorter exposure time (6 hours) and/or the use of non-toxic concentrations reduced the level of HSP70 up-regulation with menadione, CdCl2 and NaAsO2, but did not uncover any up-regulation with the other chemicals. Although the toxicity of the majority of the chemicals tested is believed to involve an oxidative stress component, the results of this study clearly demonstrate that up-regulation of HSP70 expression cannot be used as a general biomarker of toxicant exposure in vitro.
In Vitro Effects of Certain Membrane-acting Agents on Superoxide and Hydrogen Peroxide Production, Protein Synthesis and Membrane ATPase Activity in Buffalo PMN Cells
Hemen Das, Golla Ramalinga Reddy, Tukaram More and Vineet Kumar Singh
Polymorphonuclear (PMN) cells play a key role in innate immunity, due to their ability to produce reactive oxidants such as superoxide (O2–) and hydrogen peroxide (H2O2), and to release antimicrobial proteins and peptides stored in their lysosomal granules. In the present study, the effects of the activation of buffalo PMN cells with various membrane-acting agents were evaluated in terms of O2– and H2O2 production, the activities of membrane ATPases, and protein synthesis. Studies involving the incorporation of 35S-methionine revealed significant protein-synthesising ability in resting PMN cells and in cells treated with lipopolysaccharide (LPS), as well as with opsonised zymosan (OZ). Protein synthesis, as judged by fluorography of the cytosolic fraction, showed more than 12 bands, whilst the cytoskeletal fraction showed 2–3 bands. PMN activation with concanavalin A (ConA), digitonin and sodium nitroprusside (SNP) resulted in increased O2– and H2O2 production. However, in the presence of anti-inflammatory agents such as indomethacin and cortisol, the production of O2– and H2O2 by these cells was found to decline. Studies pertaining to membrane ATPases revealed that verapamil hydrochloride (VpHCl) significantly increased total ATPase and Na+K+ATPase activity. ConA treatment yielded only a moderate level of activity. Similarly, digitonin up to 24μM also caused a significant increase in ATPase activity. Our bservations indicate that these membrane-acting agents influenced oxygen-dependent and oxygen-independent microbicidal mechanisms in buffalo PMN cells.