Studies on the mechanism of cadmium-induced DNA single-strand breaks in HL-60 cells.

Abstract

Cadmium is an environmental contaminant and toxicant. It induces DNA single strand breaks but not double strand breaks in various cell cultures. Previous experiments suggested that cadmium induces DNA damage via an indirect mechanism instead of a direct interaction with DNA. Cadmium might induce DNA single str and breaks by (1) inhibiting DNA ligase activity, (2) inhibiting repair or replication of DNA synthesis or, (3) stimulating the production of reactive oxygen species such as superoxide radical, hydrogen peroxide, and hydroxyl radical. The objective of this research plan was to study the primary mechanism of cadmium-induced DNA single strand breaks by using a sublethal dose of cadmium. A dose-response study was undertaken to select the appropriate cadmium dose. Fifteen uM cadmium (cadmium sulfate) induced reversible cytotoxicity, i.e., HL-60 cells, a human leukemic cell line, could accommodate the toxicity of cadmium in the presence of the metal at this concentration and restore their capacity to grow in the absence of cadmium. Exposure to 21 or 30 uM cadmium treatment resulted in cell death. The cell number did not increase either in the presence or absence of cadmium. Fifteen uM cadmium was chosen as the cadmium dose to study the primary mechanism of cadmium-induced DNA damage. Fifteen uM cadmium induced repairable DNA single str and breaks in HL-60 cells. The str and breaks began to appear 3 hours after treatment and progressed to the maximum extent by 6 hours after treatment. Partial repair of this lesion occurred within 24 hours after treatment although DNA synthesis was still suppressed. Pretreatment of cells for four hours with 0.5, 2.5 and 5 mM deferoxamine, a specific chelator of ferric iron and a powerful inhibitor of the generation of hydroxyl radicals, protected against cadmium-induced DNA single str and breaks. Similar dose-dependent protection was found in cells pretreated with 2.5, 5 or 20 mM deferoxamine for 6 hours. Deferoxamine caused an increase in the amount of large molecules of DNA which appeared at the bottom of an alkaline sucrose gradient after centrifugation. This result suggested that iron ion-catalyzed formation of hydroxyl radical was involved in cadmium-induced single strand breaks in DNA.