Effects of heavy metals and hydrogen peroxide on cell signaling in trout hepatoma cells

The effects of Cu super(2+) Hg super(2+) and H sub(2)O sub(2) on cytosolic Ca super(2+) and phosphorylation cascades have been studied by using trout hepatoma cells (RTH 149). Variations of cytosolic Ca super(2+) were evaluated by cell loading with fluo-3 and confocal microscopy, while activation of tyrosine kinases and of stress-activated MAP kinases were assessed by western immunoblotting. Cell exposure to both heavy metals and H sub(2)O sub(2) caused cytosolic Ca super(2+) rises, but in a Ca super(2+)-free medium the effect of H sub(2)O sub(2) almost disappeared, whereas that of Hg super(2+) was only reduced. Ca super(2+) waves were also detected in Hg super(2+)-exposed cells. These data suggest that H sub(2)O sub(2) acts by inducing Ca super(2+) entry, whereas Hg super(2+) would induce both Ca super(2+) entry and intracellular release. Heavy metals and H sub(2)O sub(2) also produced an increase in tyrosine phosphorylation. However, a putative homolog of P38 MAP kinase was activated by Hg super(2+), H sub(2)O sub(2), and by Cu super(2+) plus subliminal H sub(2)O sub(2), an oxyradical generating mixture, but not by Cu super(2+) alone. Moreover, a slight activation of a putative homolog of JNK MAP kinase was induced by heavy metals but not by H sub(2)O sub(2). In conclusion, different contaminants can interfere with signalling processes in these fish cells, but each agent shows peculiar cytotoxicity pathways that can involve both Ca super(2+)-dependent signalling and phosphorylation cascades.