diethyl-maleate and Cell-Transformation--Neoplastic

Cytotoxicity and morphological transformation has been studied in BALB/3T3 Cl A31-1-1 mouse embryo cells for ammonium vanadate [vanadium(V)] and vanadyl sulphate [vanadium(IV)] alone or in combination with diethylmaleate (DEM), a cellular glutathione (GSH)-depleting agent. Cells exposed for 24 h to 10(-5) M vanadium(V) alone or in combination with 3 x 10(-6) M DEM showed the characteristic hyperfine EPR signal of vanadium(IV), which was more obvious in the case of exposure to vanadium(V) alone. This suggests that the amount of vanadium(V) reduced to vanadium(IV) decreased in GSH-depleted cells. While vanadium(IV) at concentrations of 3 x 10(-6) M and 10(-5) M was not transforming in the cells, vanadium(V) showed neoplastic transforming activity (P < 0.025 and P < 0.001 for the two doses, respectively) in comparison to controls (vanadium unexposed cells). Cytotoxicity and morphological transformation in cells exposed to vanadium(V) in combination with 3 x 10(-6) M DEM were significantly more intensive (P < 0.005 and P < 0.01 for the two doses of vanadate tested) compared to the corresponding values observed in cells exposed to vanadium(V) alone. This suggests that the final transforming activity response is dependent on the intracellular GSH-mediated mechanism of reduction of vanadium(V) to vanadium(IV): (i) the extent to which vanadium(V) should be bioreduced to less toxic vanadium(IV) via intracellular GSH is a key point in determining the intensity of the observed neoplastic action; (ii) the carcinogenic potential of vanadium(V) should be strictly dependent on its intracellular persistence which could lead to changes in normal metabolic patterns of vanadium(V) in the oxidized form due to lack of GSH-mediated reduction.

Topics: 3T3 Cells; Animals; Biotransformation; Cell Survival; Cell Transformation, Neoplastic; Electron Spin Resonance Spectroscopy; Glutathione; Maleates; Mice; Mice, Inbred BALB C; Oxidation-Reduction; Vanadium Compounds

The aim of this study has been to define cytotoxic mechanisms that may cause clonal expansion in the liver of pre-carcinogenic cells. An in vitro model, which has been described previously, was used. Hepatocytes were isolated from carcinogen-treated rats and a high proportion of the cells were gamma-glutamyltranspeptidase (GGT)-positive. The cells were incubated in suspension and exposed to toxic agents in concentrations that induced a moderate increase in cellular leakage within 3 h. Samples were withdrawn and sampled cells were then allowed to attach to collagen-coated plates. Attached cells were stained and the ratio of GGT-positive/GGT-negative cells (GGT-ratio) was determined. The initial GGT-ratio was 10.4 +/- 4.7% and an increased ratio was taken as a sign of toxicity that resulted in a selection of GGT-positive cells. In a first series of experiments it was shown that hydroquinone and menadione increase the GGT-ratio, while diquat, sodium selenite, diethyl maleate or phorone do not. However, diethyl maleate in combination with diquat increased the GGT-ratio. Hydrogen peroxide (5 mM) increased the GGT-ratio as effectively as hydroquinone (0.3 mM). Lower concentrations of H2O2 (0.05 mM) increased the GGT-ratio in GSH-depleted cells. The changes induced by hydroquinone and H2O2 in low concentration were reversible. In another series of experiments, plates coated with antibodies against beta 1-integrin were used. An increase in the GGT-ratio was obtained with anti beta 1-integrin, but not with broad spectrum anti-rat hepatocyte or anti-rat beta 2-microglobulin antibodies as substrata. These data suggested an involvement of the beta 1-integrin in the selection. Taken together, these data indicate that GGT-positive hepatocytes are protected against GSH depletion and oxidative stress that may result in reversible receptor alterations.

Topics: Animals; Biomarkers, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Collagen; Diethylnitrosamine; Diquat; Female; gamma-Glutamyltransferase; Glutathione; Hydrogen Peroxide; Hydroquinones; Ketones; Kinetics; Liver; Maleates; Phenobarbital; Rats; Receptors, Cell Surface; Receptors, Collagen; Selenium; Sodium Selenite; Vitamin K