Title

Fish stress proteins as {\it in vitro\/} biomarkers

Date of Completion

January 1997

Keywords

Health Sciences, Toxicology|Chemistry, Biochemistry|Environmental Sciences

Degree

Ph.D.

Abstract

Biomarkers are currently used in environmental toxicology to indicate an organism's health status and thus help in monitoring the temporal and spatial extent of pollution, as well as provide important information for making hazard or risk assessments. Much of this current environmental interest is directed at developing useful biochemical or molecular biomarkers. The goal of this thesis was to develop a practical in vitro approach for selecting stress-inducible proteins that are candidates for these molecular biomarkers of effect and determine if they meet the basic requirements for biomarkers.^ These candidates were selected by using sodium dodecyl sulfate polyacrylamide gel electrophoresis to detect stressor-induced, dose-dependent changes in protein levels in a variety of cultured fish cells while simultaneous in vitro cytotoxicity assays measured the stressor's effect on cellular physiology and toxicity.^ Seven in vitro cytotoxicity assays were evaluated for their ability to accurately determined the doses at which various stressors have adverse, cytotoxic effects on cell proliferation, function, survival or viability in fish cell cultures. Three of the best performing assays (neutral red uptake to indicate cell viability, sulforhodamine B protein staining to indicate total cell number, and LDH release to indicate cell death) were selected and combined together in a single plate format to measure these cytotoxic effects. These effects were then correlated with corresponding stressor-induced changes in the levels of cellular stress proteins to determine their suitability as biomarkers. Three heat shock protein family members, Hsp90, Hsp70 and small HSPs showed stressor-inducible increases in their levels as a result of exposure to some of the stressors.^ Significant dose-dependent increases were detected in Hsp70 levels in each of the eight cell fish culture systems exposed to cadmium. Other stressors also induced similar effects in these cell lines. These increases were always induced at nonlethal stressor levels and satisfied the basic criteria for biomarkers of effect. A second HSP family, small HSPs, showed some potential as biomarkers in these experiments but testing with additional stressors and cell lines will be required to better understand their roles in cells during stress. ^