Modifications of Cellular Autofluorescence Emission Spectra under Oxidative Stress Induced by 1 α,25dihydroxyvitamin D 3 and its Analog EB1089

Abstract

We attempted to characterize the cellular autofluorescence phenomenon of living HL-60 cells and to appraise its modifications under oxidative stress conditions induced by 1α,25(OH)₂D₃ (VD₃) and its analog EB1089. Autofluorescence emission spectra of human promyelocytic HL-60 leukemic cells were monitored using laser scanning confocal microspectrofluorometry under UV excitation. Evaluation of reactive oxygen species (ROS) release was performed using the 2′,7′-dichlorodihydrofluorescein diacetate (H₂-DCFDA) staining and fluorescence emission measurement. VD₃ (1, 10, 100 nM) or EB1089 (0.1, 1 and 10 nM) induces a decrease in autofluorescence emission intensity that can be attributed to the oxidation of the coenzyme nicotinamide adenine dinucleotide (phosphate) NAD(P)H into NAD(P)⁺. A dose-dependent increase (p<0.05) in ROS release is observed in VD₃- and EB1089-treated cells. As compared with VD₃- or EB1089-treated cells, doxorubicin-VD₃ or doxorubicin-EB1089 treatments strongly decrease the autofluorescence intensity and induce a higher release of ROS (p<0.05). The association of antioxidants (N-acetyl cysteine, superoxide dismutase, catalase) with VD₃ or EB1089 induce a more limited autofluorescence decrease and a weaker ROS generation, as compared with VD₃ and EB1089 treated cells. In conclusion, the free radicals release, generated by VD₃ and EB1089, was associated with the decrease in autofluorescence emission and can be modulated by doxorubicin and antioxidants.

Keywords

Laser scanning microspectrofluorometry Autofluorescence HL-60 cells EB1089 Oxidative stress

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