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 e valuated. The sensitivity of these cells to dichlone was intermediate between that of human hepatoblastoma HepG2 cells (least sensitive) and that of human GM05757 fibroblasts. The midpoint cytotoxicity values f or 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 metabolis m of dichlone presumably could proceed either by a one- or a two-elect ron reduction reaction. The enhancement of potency of dichlone towards ECV304 cells pretreated with the glutathione-depleting agents, DL-but hionine-[S,R]-sulfoximine, 1-chloro-2,4-dinitrobenzene, and 1,3-bis(ch loraethyl)-1-nitrosourea; the reduction in potency of dichlone to cell s pretreated with (-)-2-oxo-4-thiazolidine carboxylic acid; the decrea se in intracellular glutathione on exposure to dichlone; the subtle da mage to the plasma membrane of dichlone-treated cells (as detected by the leakage of lactate dehydrogenase from these cells); and the lack o f potentiation of dichlone toxicity by pretreatment with dicoumarol, a re all consistent with the one-electron reduction reaction as the domi nant pathway and with the subsequent generation of reactive oxygen mol ecules. The ECV304 cell line proved to be a useful research tool to st udy cytotoxic injury to endothelial cells.