Brigitte Siwek

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 μM and 10 μM) 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 μM buthionine-[S,R]-sulfoximine) did not significantly influence the cytotoxic effects of selenite and seleno-Dl-cystine. On both cell populations, GSH preincubation (50 μM) 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 of75Se (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 of75Se in cytosol and pellet was approximately the same. Gel filtration chromatography of lysed cells after incubation for 6 h with 10 μM75Se-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.

Effects of trace metals on mouse B16 melanoma cells in culture

The effects of fourteen metal ions (As3+, As5+, Cd2+, Co2+, Cr3+, Cr6+, Hg2+, Li+, Mg2+, Mn2+, Ni2+, Se4+, V5+, VO2+) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10−4M, metal ions such as As3+, As5+, Cd2+, Cr6+, Se4+, V5+, VO2+, and, to a minor extent, Li+, Hg2+, and Co2+ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg2+>Cr6+=Cd2+>As3+, As5+>V5+, VO2+>Se4+=Ni2+=Co2+=Li+. An increased synthesis of melanin in B16 cells was noted after incubation with Co2+, Ni2+, Cd2+, and Li+, whereas Se4+ had, on the contrary, an inhibiting effect on melanogenesis.

Characterization of non pigmented B16 melanoma cell-derived cytotoxic factors.

We analyzed and tried to characterize substance(s) responsible for cytotoxic activities detected in culture media conditioned by non pigmented B16 melanoma cells (NPB16). The different cytological tests used showed that ultrafiltrated conditioned media (CM U1 fraction) contained several cytotoxic factors with a Mw lower than 1000 Da. These factors seemed to act either directly or indirectly on cell membranes, mitochondria, on the cell cycle and on protein and DNA synthesis. A cytotoxic activity could be found even after high dilution of CM U1. These cytotoxic factors were rapidly released by B16 cells in culture, independently of cell confluence. Their activities in the treated cells were also very fast and the cytotoxic effects were irreversible after only a few hours of treatment. These factors were not intermediate products during melanogenesis, neither polyamines, nor proteases. At least one of them seemed to be a small acidic and basic stable peptide without disulfide bounds but not heat stable. The synthesis of at least one of these cytotoxic factors was inhibited by cycloheximide and the cytotoxic activity was partially destroyed by pronase and trypsin, but not by pepsin. The cytotoxicity was not modified by copper complexants or free radical inhibitors (bovine serum albumin (BSA), tyrosine, superoxyde dismutase (SOD), catalase, vitamin E). Furthermore the levels of glutathione peroxydase activity and reduced glutathione did not change after treatment by CM U1 as compared to controls.