EFFECT OF SELENIUM-COMPOUNDS ON MURINE B16 MELANOMA-CELLS AND PIGMENTED CLONED PB16 CELLS

The effects of selenium compounds such as sodium selenite, sodium selenate, seleno-DL-cystine and seleno-DL-methionine (100 mu M and 10 mu M) on B16 and pigmented cloned pB16 murine melanoma cells were investigated in vitro. At the tested concentrations, B16 cells showed a greater sensitivity to the toxic effects of sodium selenite and seleno-DL-cystine than pB16 cells, whereas no decrease of B16 and pB16 cell number was observed after incubation with sodium selenate or seleno-DL-methionine. Glutathione (GSH) percentages were strongly decreased only by selenite and seleno-DL-cystine; it was marked more in B16 than in pB16 cells. The pretreatment of B16 cells with a GSH depleting agent (10 mu M buthionine-[S,R]-sulfoximine) did not significantly influence the cytotoxic effects of selenite and seleno-DL-cystine. On both cell populations, GSH preincubation (50 mu M) enhanced the cytotoxicity of selenite whereas the survival of seleno-DL-cystine treated cells was increased. Glutathione peroxidase (GSH-Px) activity in B16 cells was more sensitive than in pB16 cells to the activating effect of selenite, and particularly of seleno-DL-cystine; however, cell-free controls indicated that activation was mainly due to glutathione reductase. The rate of Se-75 (as sodium selenite) uptake in both cell populations was maximal within the first hour of incubation, with a preferential accumulation in the cytosol; after 24 h of incubation, the amount of Se-75 in cytosol and pellet was approximately the same. Gel filtration chromatography of lysed cells after incubation for 6 h with 10 mu M Se-75-selenite showed that the radioactivity was eluted as two peaks corresponding to low (4-9 kDa) and high (280-320 kDa) molecular weights. Possible toxicological mechanisms are discussed at molecular level. For selenite, a major involvement of GSH is proposed, with production of selenodiglutathione and selenopersulfide, which should be directly responsible of the decrease in cell number, thiol oxidation and protein synthesis inhibition. For selenocystine, an active selenol species (Cy-Se-) is also hypothesized as being responsible for thiol oxidation and mutagenic effects. For both compounds oxygen active species could also be formed; however, a relevant role of GSH-Px was not apparent. The minor sensitivity of pB16 cells to the toxic effects of selenite and seleno-DL-cystine could be explained by the smaller depletion of GSH induced by those compounds in pB16 cells, a minor formation of selenium active species, the larger amount present of the oxyradical scavenger melanin, the secretion of some mitogenic factor by pB16 cells and/or a greater resistance to autocrine cytotoxic factors.