title: 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 microM 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. CdCl2, NaAsO2, AgNO3 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 microM after 2 h. CuCl2, MnCl2, Pb(NO3)2, TlNO3, CoCl2 and NiCl2 were also strong inducing agents, giving a 4-6 fold induction at 10-100 microM after 4-8 h. ZnSO4, Hg(NO3)2 and AlCl3 were only weak inducers (1.5-2 fold at 50-100 microM after 4-8 h) of hsp70 mRNA. Cytotoxic effects (measured by release of lactate dehydrogenase) could only be detected for 100 microM Hg2+ after 4 h and when the cells were incubated with 5 microM Cd2+ for more than 8 h. We also tested a few combinations of these heavy metal salts for their hsp70-inducing ability. Zn2+ and Mn2+ were able to diminish Cd2+ induced hsp70 mRNA levels by 65%. Ag+ mediated induction was reduced by 40% when combined with Cu2+, whereas Hg2+ 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.
authors.affiliation: Institute of Hygiene, University of Graz, Austria.
authors.forename: E
authors.initials: E
authors.lastname: Steiner
authors.forename: B
authors.initials: B
authors.lastname: Kleinhappl
authors.forename: A
authors.initials: A
authors.lastname: Gutschi
authors.forename: E
authors.initials: E
authors.lastname: Marth
mesh_terms: D019256:Cadmium Chloride / Q000633:toxicity; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D019216:Metals, Heavy / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D018840:HSP70 Heat-Shock Proteins; D019216:Metals, Heavy; D012333:RNA, Messenger; D019256:Cadmium Chloride