ULTRAVIOLET ACTION SPECTRA FOR PEROXIDE GENERATION IN HUMAN AND PIG EPIDERMAL-KERATINOCYTES LOADED WITH DIHYDRORHODAMINE-123

We developed a new and simple method for measuring peroxides in a sing le 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, dihydrorho damine 123, reacts in the presence of peroxides, such as H2O2, and cha nges into the fluorescent rhodamine 123, and hence the fluorescence in tensity is proportional to the amount of reacted peroxide. The epiderm al keratinocytes were loaded with the dihydrorhodamine under a fluores cence microscope and exposed to UV radiation. Taking C as the content of peroxides generated within the cell and I as the increase in fluenc e (radiation intensity x time = photons/cm(2)), the following empirica l relationship was established: C = C-s (1-exp(-kI)), where C-s is the content of peroxides at the saturation state, and k is a kinetic para meter. The dependence of the two parameters on wavelength in the range 280-400 nm was studied. In human keratinocytes C-s 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 C-s remained unchanged. Aminotriazole, an inhibitor of catalase, and low temperatu res increased the stationary levels of peroxide generation in pig kera tinocytes upon UV irradiation, indicating that the reaction used for m easuring intracellular peroxides is competitive with the intrinsic rea ctions in scavenging peroxides.