Space-temporal variation of copper ions in a CHO-K1 cell line and their cytotoxic effect.

NATHALIE ARNAL,1 MIGUEL REIGOSA2 MÓNICA FERNÁNDEZ LORENZO1,3

Amsterdam-Netherlands

Congreso; 8th World Biomaterials Congress; 2008

Copper ions are eluted from copper-based intrauterine devices (IUD) and increase systemic copper and local levels [1]. It has been previously reported that the dissolution of copper from a solid metal could generate a space-temporal variation of the copper ions concentration within an UMR106 cell line growing on a Petri dish [2].     The aim of this study was to investigate the cytotoxic effect of copper ions on CHO-K1 ovary cells. Two different sources of copper ions were used for comparison: metallic copper and extracts. CHO-K1 cells were seeded in 35 mm Petri dishes with 2,5x104cells/ml.  Experiments with extracts and disks were performed. Experiments with the Extracts: the extracts were obtained by the immersion of the copper disks (5 mm diameter, 1mm height) in Ham´s F-10 medium for periods between 3 h and 48 h. Then the cells were cultured into these extracts and were  incubated for periods between 3 and 48 h. Experiments with disks (ED) were performed using a   Petri disk that were divided in 3 ring areas (A, B and C) around the copper disk at 2.5 mm, 5 mm and 7.5 mm radial distance respectively. The cells were incubated during different exposure periods between 3 and 48 h. Subsequently, cell viability was evaluated by means of the Ethidium Bromide/Acridine orange assay. Figure 1 shows the results of ED experiments with cells corresponding to regions A and C respectively. In region  C (Fig. 1a) it could be observed that the number of cells increased to values higher than the control after 6 h exposure period, then decreased reaching values lower than the control and finally increased to reach a maximum after 30 h. A similar growth pattern with two cycles was shown in Figure 2 for 3 h extracts (low copper ions concentration). Accordingly it emerges that low concentrations of copper ions under short exposure periods seem to enhance the cell growth.  After a possible adaptation period the number of cells increased again during a second growth cycle. The number of survival cells of region B was markedly lower than the control. Again, a maximum is observed but shorter than that of region C. A very low number of surviving cells (zero in some places) were found in Region A (Figure 1b) with maxima for for 6-9 h. A direct relationship between the spatial-temporal variation of copper ion concentration and surviving cell numbers can be inferred from Figures 1a and 1b.  Thus, higher concentrations of copper ions in Region A, is associated to the lower number of surviving cells in this region. Additionally, the growth cycles are inhibited in region B in comparison with the control and are practically absent in region A. It can be concluded that low copper ions concentrations