This is a well-written and clearly presented application from a junior investigator to study the effects of arsenite (As(III)) exposure on cultured porcine aortic endothelial cells. The specific focus of the study is to assess arsenite's effects on the AP-1 signal transduction complex, in particular on c-jun, c-jun phosphorylation, and c-fos interactions, using DNA gel shift, immunoprecipitation and cell transfection experiments. The research application is hypothesis driven and discussion is presented relating to expected results and some alternative strategies of experimental design. These are all strengths of the research application. One potential shortfall lies in the transfection scheme for the CAT reporter genes in the cultured endothelial cells. Primary cells such as this are after difficult to transfect with high efficiency. No plans are put forth to measure transfection efficiency directly, e.g., with the use of beta-galactosidase reporter based immunohistochemistry. Neither are any details discussed to normalize for transfection efficiency with a co-transfection reporter approach or to potentially assess other transfection efficiency with a co-transfection reporter approach or to potentially assess other transfection reagents to optimize assay efficiencies. Another potential shortfall of the experimental design is with respect to the AP-1 studies themselves. If initial tests end up disproving the working hypothesis that arsenite modulates AP-1 activities in the cells, then no fall-back position is discussed. Perhaps Nf-kappaB analyses should more directly included as a possibility in this case.
|Smith, K R; Klei, L R; Barchowsky, A (2001) Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 280:L442-9|