Abstract
In the present study the free radical inducibility of the rainbow trout metallothionein-A (MT-A) gene was investigated. Earlier sequencing of the MT-A promoter has revealed four activator protein (AP) I sites as well as six metal responsive elements (MRE) in the MT-A promoter. To investigate the functionality of the identified elements, deletion mutants were introduced into the pGL-2 vector directly upstream from the luciferase reporter gene. Transfection of the pGL-2 vector constructs into the rainbow trout hepatoma (RTH-149) cell line showed that all MREs were needed for maximal inducibility. Exposure to hydrogen peroxide resulted in significantly increased activity of the luciferase reporter gene when the four AP I sites were included in the promoter compared with the expression level of the deletion mutant containing only MREs. In order to determine MTs ability to protect the cell from free radical damage, RTG-2 cells were pretreated with 150 μM Zn for 72 h to induce MT and thereafter exposed to various concentrations of hydrogen peroxide. Measuring cell survival in the pretreated groups compared with only hydrogen peroxide-exposed cells, showed that pretreated cells were more resistant than cells exposed to hydrogen peroxide alone.
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