Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation

Abstract

Cytotoxicity, morphological neoplastic transformation, cellular uptake and metabolic reduction were determined in BALB/3T3 CI A31-1-1 cells for trivalent arsenic (sodium arsenite, As³⁺) and for pentavalent arsenic (sodium arsenate, As⁵⁺). The levels of cellular uptake of⁷³As-labelled sodium arsenite and arsenate were dose-dependent and highest in the first hour. At equimolar concentration (3 × 10⁻⁶ M), cellular uptake was 4-fold higher for As³⁺ than for As⁵⁺. Cytotoxicity was higher for As³⁺ than for As⁵⁺, but when correlated to total As cell burden it showed no significant difference for the two forms. Morphological transformation focus assays showed transforming activity for both As³⁺ and As⁵⁺, with relative transformation frequencies also of ∼4:1. Recovery from the cytosol after exposure for 1–24 h was >90% for either form of absorbed As. Exposure to As³⁺ yielded 100% as As³⁺ in cytosol, but exposure to As⁵⁺ yielded >70% as As³⁺, showing a high rate of intracellular metabolic reduction. No methylated metabolites were detected by ion-exchange chromatography. After 24-h incubation in cell-free medium, oxidation of As³⁺ to As⁵⁺ occurred up to 30% of the dose, but incubation in the presence of cells lowered the oxidation level to 4%. As⁵⁺ was recovered unchanged from cell-free medium (24-h incubation), but in the presence of the cells it yielded up to 5% as As³⁺ within 24 h and the cumulative release of As³⁺ by cells exposed to As⁵⁺ was dose-dependent. Glutathione depletion by diethylmaleate inhibited reduction of As⁵⁺ to As³⁺ by these cells up to 25% of controls, showing that As⁵⁺ reduction is partly dependent on glutathione. These results suggest that As³⁺ is the form responsible for the cytotoxic and transforming effects, independently of the valence state of the inorganic arsenic in the culture medium.