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In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells within hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.

Exposure of a trout gonadal fibroblast (RTG-2) cell line to ZnCl2, CdCl2 and CuCl2 resulted in differential levels of accumulation of metallothionein (MT) mRNA. ZnCl2 being the most effective agent induced MT mRNA in 3 h, with 172-fold induction after 48 h and continued accumulation up to 144 h. Following CdCl2 treatment, mRNA could be detected after 24 h, reaching peak levels at 72 h. Furthermore, trout MT mRNA could be detected up to 8 days after withdrawal of extraneous ZnCl2. Using a novel technique of primer extension and DNA sequencing with total RNA as template, specificity of the trout MTa and MTb gene-specific primers was established. Primer extension studies revealed a higher response of MTa to ZnCl2 and CdCl2 compared to MTb. Insensitivity of MT mRNA induction to cycloheximide suggested that induction by the metals was independent of de novo protein synthesis. However, simultaneous exposure of cells to actinomycin D and metals completely inhibited MT mRNA synthesis implying control at the transcriptional level.

We have investigated the regulation of metallothionein gene expression in two fish cell lines. Rainbow trout hepatoma (RTH) cells synthesized metallothionein in response to heavy metal exposure. The maximum level of metallothionein synthesis detected during zinc exposure was much greater than during cadmium exposure. The time-courses of metallothionein synthesis were different for the different metal inducers, suggesting that metallothionein may be differentially regulated by cadmium and zinc in these cells. The metal-induced synthesis of metallothionein was correlated with increased translational activity and accumulation of metallothionein-mRNA, suggesting that metallothionein may be regulated at the transcriptional and post-transcriptional levels in RTH cells. Chinook salmon embryo (CHSE) cells, unlike RTH cells, did not synthesize metallothionein or metallothionein-mRNA in response to heavy metal exposure. However, when these cells were treated with 5-azacytidine prior to heavy metal exposure, the synthesis of metallothionein was induced, suggesting that DNA methylation may play a role in metallothionein gene expression in fish.