Cytotoxicity of metals, metal-metal and metal-chelator combinations assayed in vitro.

A simple, rapid assay, based on the lysosomal incorporation of neutral red by cells, conveniently carried out in 96-well microtiter plates, was used to evaluate the cytotoxic effect of cationic and anionic metal salts on BALB/c mouse 3T3 fibroblasts. Ranking of the metals according to their decreasing potency was based on spectrophotometrically determined absorbance of the neutral red, extracted from surviving viable cells. The rank order was Cd greater than Hg greater than Ag greater than Zn greater than Mn greater than Cu greater than Co greater than Ni greater than Cr(III) for the cationic metals and Cr2O7 greater than CrO4 greater than AsO2 greater than AsO4 greater than SeO3 greater than SeO4 greater than MnO4 for the anionic metals tested. Cationic metals incubated with cultures in medium containing 1% fetal bovine serum (FBS) were 3-4 times more toxic than in medium with 10% FBS. Cadmium served as a representative metal for the use of this assay not only for concentration, but also for time dependent exposures. Thus a 10% cytotoxic effect after 1 h of incubation with 60 microM cadmium was increased to 90% after 6 h. Examination of the effect of metal-metal interaction on cytotoxicity showed a marked reduction of cadmium toxicity by zinc and to a lesser degree, by nickel. The neutral red assay was also effectively used to investigate the effect of the chelators ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA) and 2,3-dimercaptosuccinic acid (DMSA) on cadmium-induced injury. Cytotoxicity by cadmium was completely inhibited by EDTA, and partially by NTA, but DMSA was ineffective. Reduction of copper toxicity by chelation was less efficient than for cadmium. Use of a chelator as therapy against metal poisoning was only partially effective and limited to administration within 2 h after incubation of cells with cadmium. It is believed that the neutral red assay can be a valuable tool for the screening of cytotoxic and potentially therapeutic agents under controlled in vitro conditions.