In vitro cytotoxicity of the chlorinated naphthoquinone dichlone to human endothelial ECV304 cells

The cytotoxicity of the pro-oxidant fungicide dichlone (2,3-dichloro-1,4-naphthoquinone), to the human endothelial cell line, ECV304, was evaluated. The sensitivity of these cells to dichlone was intermediate between that of human hepatoblastoma HepG2 cells (least sensitive) and that of human GMO5757 fibroblasts. The midpoint cytotoxicity values for a 24-hr exposure to dichlone was about 0.02 mm when evaluated with the neutral red, acid phosphatase, and XTT tetrazolium assays. Lactic acid dehydrogenase leakage, after a 4-hr exposure, occurred initially at 0.05 mm dichlone. As with other naphthoquinones, cellular metabolism of dichlone presumably could proceed either by a one- or a two-electron reduction reaction. The enhancement of potency of dichlone towards ECV304 cells pretreated with the glutathione-depleting agents, dl-buthionine-[S,R]-sulfoximine, 1-chloro-2,4-dinitrobenzene, and 1,3-bis(chloroethyl)-1-nitrosourea; the reduction in potency of dichlone to cells pretreated with (-)-2-oxo-4-thiazolidine carboxylic acid; the decrease in intracellular glutathione on exposure to dichlone; the subtle damage to the plasma membrane of dichlone-treated cells (as detected by the leakage of lactate dehydrogenase from these cells); and the lack of potentiation of dichlone toxicity by pretreatment with dicoumarol, are all consistent with the one-electron reduction reaction as the dominant pathway and with the subsequent generation of reactive oxygen molecules. The ECV304 cell line proved to be a useful research tool to study cytotoxic injury to endothelial cells.