METALLOTHIONEIN INDUCTION IN RAINBOW-TROUT GONADAL (RTG-2) CELLS DURING FREE-RADICAL EXPOSURE

In the present study the free radical inducibility of the rainbow trou t metallothionein-A (MT-A) gene was investigated. Earlier sequencing o f the MT-A promoter has revealed four activator protein (AP) I sites a s well as six metal responsive elements (MRE) in the MT-A promoter. To investigate the functionality of the identified elements, deletion mu tants 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 M REs were needed for maximal inducibility. Exposure to hydrogen peroxid e resulted in significantly increased activity of the luciferase repor ter gene when the four AP I sites were included in the promoter compar ed with the expression level of the deletion mutant containing only MR Es. In order to determine MTs ability to protect the cell from free ra dical damage, RTG-2 cells were pretreated with 150 mu M Zn for 72 h to induce MT and thereafter exposed to various concentrations of hydroge n peroxide. Measuring cell survival in the pretreated groups compared with only hydrogen peroxide-exposed cells, showed that pretreated cell s were more resistant than cells exposed to hydrogen peroxide alone. C opyright (C) 1996 Elsevier Science Ltd