Ultraviolet action spectra for peroxide generation in human and pig epidermal keratinocytes loaded with dihydrorhodamine 123

Abstract

We developed a new and simple method for measuring peroxides in a single living cell, and the generation of peroxides upon ultraviolet (UV) irradiation was measured in human and pig epidermal keratinocytes. The method was based on the fact that the non-fluorescent dye, dihydrorhodamine 123, reacts in the presence of peroxides, such as H2O2, and changes into the fluorescent rhodamine 123, and hence the fluorescence intensity is proportional to the amount of reacted peroxide. The epidermal keratinocytes were loaded with the dihydrorhodamine under a fluorescence microscope and exposed to UV radiation. Taking C as the content of peroxides generated within the cell and I as the increase in fluence (radiation intensity x time = photons/cm2), the following empirical relationship was established: C = Cs (1-exp(-kI)), where Cs is the content of peroxides at the saturation state, and k is a kinetic parameter. The dependence of the two parameters on wavelength in the range 280-400 nm was studied. In human keratinocytes Cs had a peak at 310 nm and a small peak (shoulder) at 380 nm, while k increased gradually toward shorter wavelengths. In pig keratinocytes, on the other hand, k had a peak around 380 nm and a shoulder at 330 nm, while Cs remained unchanged. Aminotriazole, an inhibitor of catalase, and low temperatures increased the stationary levels of peroxide generation in pig keratinocytes upon UV irradiation, indicating that the reaction used for measuring intracellular peroxides is competitive with the intrinsic reactions in scavenging peroxides.