Effect of Chemical Toxins on Protein Expression and Degradation Machinery

Fatima C. Sequeira and John T. Elliott

A cell-based ribosome inhibition assay was used to investigate the effect fifty reference chemical toxins (MEIC library) had on protein expression and degradation machinery prior to cell death.  The assay relies on the use of automated quantitative fluorescence microscopy and engineered reporter Vero cell line that contains green fluorescent protein (GFP) under the control of a cytomegalovirus promoter as an indicator of global protein synthesis and degradation function.     To identify the toxicity of MEIC compounds to these cells, we exposed the cells high concentrations (600 µM) for 24-h, 48-h, 72-hours.  Twenty compounds that showed an effect on the cells at these concentrations were further evaluated by generating dose response curve.  Our results indicate that only 3 of the 20 toxic compounds (digoxin, copper sulfate and arsenic oxide) show changes in the intensity of the GFP level within cells before full cell death is observed.  This suggests that the components of the protein expression and degradation machinery are very robust and are not necessarily affected by many toxic chemicals.  The molecular mechanisms by which digoxin, copper sulfate, and arsenic oxide influence protein expression may provide insight into how similar compounds may affect cells in more complex organisms.