Concentration-dependent cytotoxicity of copper ions on mouse fibroblasts in vitro: effects of copper ion release from TCu380A vs TCu220C intra-uterine devices.

Sustained release of copper (Cu) ions from Cu-containing intrauterine devices (CuIUD) is quite efficient for contraception. However, the tissue surrounding the CuIUD is exposed to toxic Cu ion levels. The objective for this study was to quantify the concentration dependent cytotoxic effects of Cu ions and correlate the toxicity due to Cu ion burst release for two popular T-shaped IUDs - TCu380A and TCu220C on L929 mouse fibroblasts. Fibroblasts were cultured in 98 well tissue culture plates and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphehyltetrazolium bromide (MTT) assay was used to determine their viability and proliferation as a function of time. For cell seeding numbers ranging from 10,000 to 100,000, a maximum culture time of 48 h was identified for fibroblasts without significant reduction in cell proliferation due to contact inhibition. Thus, for Cu cytotoxicity assays, a cell seeding density of 50,000 and a maximum culture time of 48 h in 96 well plates were used. 24 h after cell seeding, culture media were replaced with Cu ion containing media solutions of different concentrations, including 24 and 72 h extracts from TCuIUDs and incubated for a further 24 h. Cell viability decreased with increasing Cu ion concentration, with 30 % and 100 % reduction for 40 µg/ml and 100 µg/ml respectively at 24 h. The cytotoxic effects were further evaluated using light microscopy, apoptosis and cell cycle analysis assays. Fibroblasts became rounded and eventually detached from TCP surface due to Cu ion toxicity. A linear increase in apoptotic cell population with increasing Cu ion concentration was observed in the tested range of 0 to 50 µg/ml. Cell cycle analysis indicated the arrest of cell division for the tested 25 to 50 µg/ml Cu ion treatments. Among the TCuIUDs, TCu220C having 265 mm(2) Cu surface area released 9.08 ± 0.16 and 26.02 ± 0.25 µg/ml, while TCu380A having 400 mm(2) released 96.7 ± 0.11 and 159.3 ± 0.15 µg/ml respectively following 24 and 72 h extractions. The effects of TCuIUD extracts on viability, morphology, apoptosis and cell cycle assay on L929 mouse fibroblasts cells, were appropriate for their respective Cu ion concentrations. Thus, a concentration of about 46 µg/ml (~29 µM) was identified as the LD50 dose for L929 mouse fibroblasts when exposed for 24 h based on our MTT cell viability assay. The burst release of lethal concentration of Cu ions from TCu380A, especially at the implant site, is a cause of concern, and it is advisable to use TCuIUD designs that release Cu ions within cytotoxic limits yet therapeutic, similar to TCu220C.

Release behavior of cupric ions for TCu380A and TCu220C IUDs.

We immersed copper T380A and T220C IUDs in simulated uterine solution (SUS) and also in simulated body fluid (SBF). The release rate and corrosion products of copper ions after soaking in SUS and SBF for different time spans were studied by an atomic absorption spectrophotometer, scanning electron microscopy with energy dispersive x-ray microanalysis (SEM/EDX) and x-ray diffraction. The highest corrosion rate of the TCu380A IUD was obtained on the first day, while the highest corrosion rate of the TCu220C IUD was obtained on the third day. The copper corrosion rate of the TCu380A IUD was much faster than that of the TCu220C IUD in the first few days. The corrosion extent of copper wire was higher than that of a copper tube when first immersed in solution. For the same IUD in the same solution, the corrosion extent in SBF was higher than in SUS. The main compounds identified were cuprite (Cu(2)O). Copper wire and copper tubes were implanted in the uteruses of rats. In vivo experiments were also done. The corrosion rate in vivo was higher than that in vitro.