TPEN

TPEN attenuates Fura-2 fluorescence changes induced by cadmium, mercury, and methylmercury. In cells stimulated with cadmium chloride (10/30 μM), the addition of TPEN at 3 h after exposure significantly decreases the elevated Fura-2 fluorescence ratio to the basal levels within 10 min (119.6±2.4% or 109±1.5% decrease in ΔRatio (F340/F380)). TPEN targets colon cancer cells through redox cycling of copper. TPEN dose- and time-dependently reduces cell viability. TPEN-induced cell death is also dependent on the redox cycling of copper since the copper chelator neocuproine inhibited DNA damage and reduced pChk1, γ-H2AX, and ATM protein expression.

DMSO:6 mg/mL (14.13 mM)

TPEN is dissolved in DMSO and then diluted with appropriate medium[1]. Human neuroblastoma cell line SH-SY5Y, are grown in Dulbecco's Modified Eagle's Medium (DMEM) mixed 1:1 with Ham's F-12 nutrient mixture containing 10% fetal bovine serum, 100 unit/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere. Two days before experimentation, cells are seeded at a density of 7×104 cells/cm2 in a 96-well plate. Cells in a 96-well plate are serum-starved for 4 hr; calcium indicator fura-2 is then loaded into the cells by using Calcium kit II fura-2. In brief, SH-SY5Ycells are incubated with 5 μM fura-2/AM in the presence of 0.04% Pluronic F-127, a dispersing agent to improve the efficiency of loading with fura-2, and 1.25 mM probenecid, a blocker of organic anion transport to prevent leakage of fura-2 from cells. After 1 hr incubation at 37°C, fura-2 fluorescence is measured at 500 nm emission after excitation at 340 nm (F340) or 380 nm (F380) using an Infinite M200 plate reader at 37°C.The change in [Ca2+]i is reflected by the ratio of F340 and F380. To determine the changes in fura-2 fluorescence ratio induced by heavy metal compounds, cells are treated with manganese chloride, lead acetate, cadmium chloride , mercuric chloride and MeHg chloride dissolved in distilled water. We confirmed that the cells adhered to the bottom of the plate after 6 hr exposure to heavy metal compounds. The cells are also treated with three Ca2+ channel blockers, lanthanum chloride dissolved in distilled water, verapamil and 2-APB dissolved in DMSO, 30 min before heavy metal exposure. The heavy metal chelator TPEN is dissolved in DMSO and added 3 hr after the stimulation with heavy metals to determine the contribution of endogenous and exogenous heavy metals on fura-2 fluorescence changes.We measured the effect of TPEN (20 μM) on the fura-2 fluorescence ratio after a 10 min treatment with TPEN, since our preliminary experiments showed that the effect of TPEN on fura-2 fluorescence reached maximum and stabilized within 10 min of the treatment[1].