Lipid peroxidation induced by bolesatine, a toxin of Boletus satanas: Implication in m(5)dC variation in Vero cells related to inhibition of cell growth

Bolesatine, a glycoprotein from Boletus satanas Lent, has previously been shown to be mitogenic in rat and human lymphocytes at very low concentrations, whereas higher concentrations inhibited protein synthesis in vitro and in several in vivo systems. The low concentrations (1-10 ng/ml) of bolesatine were shown to activate protein kinase C (PKC) in vitro (cell-free system) and in Vero cells. In the same time, Vero cells significantly proliferated when incubated with bolesatine concentrations ranging from 1 to 10 ng/ml; the DNA synthesis increased by 27-59% as referred to the control, and InsP3 release increased in a concentration-dependent manner, up to 142%. At higher concentrations, 1-10 mu g in cell-free systems, bolesatine inhibits protein synthesis by hydrolyzing the nucleoside triphosphates GTP and ATP. In the present work, the implication of other toxic mechanisms, such as lipid peroxidation and active radical production, was investigated in relation to inhibition of cell growth, whereas possible modifications of the ratio m(5)dC/dC+m(5)dC were determined in order to correlate with the biphasic action of bolesatine in Vero cells. Low concentrations of bolesatine up to 10 ng/ml do not increase malonaldehyde (MDA) production, while they induce hypomethylation (5.2% as compared to 7.1%). Higher concentrations (above 20 ng/ml) increase MDA production, from 58 ng/mg of cellular proteins to 113 ng/mg at a concentration of 50 ng/ml, for example, and induce hypermethylation in Vero cell DNA. It is concluded that low concentrations of bolesatine that are proliferative induce hypomethylation, which could be one of the pathways whereby bolesatine induces cell proliferation. Higher concentrations which enhance lipid peroxidation also induce hypermethylation. These mechanisms could be at least partly implicated in the pathway whereby bolesatine induces cell death.