Analysis of hsp70 mRNA levels in HepG2 cells exposed to various metals differing in toxicity

Abstract

Human hepatoma cells (HepG2) were exposed to several heavy metal salts and the induction of heat shock protein 70 (hsp70) mRNA was analysed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Metals were added to the cell medium at concentrations ranging from 0.1 to 100 μM and incubation was continued for 4 h. In addition we analysed the time dependence of hsp70 induction by adding each metal at a certain concentration followed by an incubation for 0.5 to 24 h. CdCl 2, NaAsO 2, AgNO 3 could be classified as very strong inducers (20-, 13- and 10-fold above control level) and they reached their maximum level of induction at 1–10 μM after 2 h. CuCl 2, MnCl 2, Pb(NO 3) 2, TlNO 3, CoCl 2 and NiCl 2 were also strong inducing agents, giving a 4–6 fold induction at 10–100 μM after 4–8 h. ZnSO 4, Hg(NO 3) 2 and AlCl 3 were only weak inducers (1.5–2 fold at 50–100 μM after 4–8 h) of hsp70 mRNA. Cytotoxic effects (measured by release of lactate dehydrogenase) could only be detected for 100 μM Hg 2+ after 4 h and when the cells were incubated with 5 μM Cd 2+ for more than 8 h. We also tested a few combinations of these heavy metal salts for their hsp70-inducing ability. Zn 2+ and Mn 2+ were able to diminish Cd 2+ induced hsp70 mRNA levels by 65%. Ag + mediated induction was reduced by 40% when combined with Cu 2+, whereas Hg 2+ increased induction by Ag + about 3-fold and led to a dramatic decrease in cell viability. In our study we were able to demonstrate that the analysis of hsp70 mRNA levels in chemically stressed HepG2 cells by RT-PCR can be a valuable tool for studying mechanisms of toxicity associated with elevated expression of hsp70.