The UVA Light Used during the Fluorescence Microscopy Assay Affects the Level of Intracellular Calcium Being Measured in Experiments with Electric-Field Exposure

Abstract

In the present paper, the induction of calcium signals in neuroblastoma cells, cells of T-cell leukemia, and osteogenic sarcoma cells were investigated in relation to the UVA irradiation used in fluorescence microscopy. Methods were developed to measure both the mean UVA irradiance and the intensity profile in the UVA-illuminated area of the microscope. This allowed us to calculate the applied UVA radiant exposure of the cells during each experiment. This investigation was undertaken because of the conflicting results in the literature on the effects of electromagnetic fields on the signals of the calcium-sensitive fluorescence probe FURA-2 in lymphocytes. Taking into account that each group used a different system with different optics and lamps, these conflicting results are now at least partially understandable. Our measurements indicate that in a typical experiment with FURA-2 the cells were irradiated with up to 776 kJ m(-2) during 25 min of exposure to UVA light. This causes changes in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Designating cells in which the [Ca(2+)](i) was distinctly increased during the experiment as "responding", we found Hill-type dependences on the irradiance. Jurkat cells showed a 50% response even at 10 kJ m(-2) and osteosarcoma cells at about 60 kJ m(-2), whereas neuroblastoma cells even at the maximum possible dose responded only minimally. In the case of neuroblastoma cells, we found a dependence of this effect on the CO(2) partial pressure during the preincubation. An electrical treatment with an a.c. field (5 kHz sinusoidal, amplitude modulation 16 Hz 100%, 800 V m(-1), 5 min) had a significant effect on intracellular calcium in neuroblastoma cells only in the case of cells that were not pretreated with CO(2) with high fluences of UVA irradiation. In conclusion, these results indicate that the possibility of UVA artifacts must be considered in all experiments using fluorescence microscopy. Furthermore, our results lead to the hypothesis that oxidative stress could be the link between UVA and electric-field effects.