Heat shock protein 70 as an in vitro measure of toxicity

Abstract

The aim of this study was to assess the potential of the heat shock protein 70 as an in vitro measure of toxicity. The heat shock response was analysed by one-dimensional electrophoresis and a range of immunological methods. The two cell lines used, mouse connective tissue (L929) and normal rat kidney (NRK), were found to be extremely sensitive responding to a classic heat shock treatment. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) combined with silver staining revealed the induction of a 70kDa protein with only a 30 C increase in temperature. Western blotting identified this as the inducible isoform of heat shock protein 70 (hsp70). Exposure to three metals cadmium, mercury and copper showed induction of hsp70 coupled with a repression of normal protein synthesis. At the median lethal concentration (LC50), measurement of hsp70 revealed a more sensitive response than that of cell mortality as determined by the neutral red assay. Hsp70 induction was significant at the no effect level (NEL) determined by the neutral red assay as the concentration at which no cell mortality could be measured. Hsp70 induction was also shown at the lowest observed effective concentration (LOEC). Measurement of hsp70 revealed a more sensitive response for mixtures of the three metals than that of cell mortality. In addition, hsp70 induction revealed toxin interaction that resulted in an additive response. The use of ELISA in conjunction with hsp70 induction to measure in vitro toxicity revealed similar results to those found using one-dimensional SDS-PAGE. Below the NEL hsp70 induction was slightly higher for several of the metals suggesting that the ELISA assay was more sensitive. The use of immunocytochemistry in conjunction with hsp70 induction to measure in vitro toxicity also revealed similar results to those found using one-dimensional SDS-PAGE. The use of immunocytochemistry allows for qualitative and quantitative assessment of hsp70 induction. This appears more suited to low level toxicity as above the LC50 cell mortality becomes the greater response. Using this assay a heterogeneous response was observed at intermediate toxin concentrations. To assess the potential of hsp70 induction as an in vitro measure of toxicity a novel compound 2-Isobutyl piperidine was tested. Measuring the induction of hsp70 revealed a more sensitive response than cell mortality. Significant hsp70 induction was shown at the NEL and LOEC. Hsp70 induction showed a more sensitive response than the neutral red assay, which is considered a very sensitive measure of cell mortality. Induction of hsp70 at the NEL demonstrates the assays ability to identify sub lethal toxicity. The high level of induction at the LOEC demonstrates the sensitivity of the assay for measuring sub lethal toxicity. The repression of normal protein synthesis aids this assay by removing potentially interfering cellular proteins. Identifying toxin interaction suggests the assay could be applied to true environmental situations. The ability of hsp70 induction to be used in conjunction with ELISA and immunocytochemistry methods to measure in vitro toxicity provides the opportunity to develop a routine automated assay. The use of hsp70 induction to measure the toxicity of 2-Isobutyl piperidine demonstrates the potential to assess the toxicity of newly synthesised compounds.