Following UV radiation, reactive oxygen species (ROS) are responsible for the generation of Comet assay (single-cell DNA gel electrophoresis) is widely used in photobiology and toxicology
oxidative stress in human skin. ROS are considered to play an important role in developing to determine the degree of DNA damage induced by ultraviolet (UV) radiation or other agents.
damage such as photoaging, immune suppression, cataract formation and carcinogenesis. The In principle, DNA fragments produced due to double-strand nicking by various mutagens or
putative antioxidant defense system which protects human skin cells against developing such indirectly during DNA repair, are separated by electrophoresis from intact DNA. During singledamage consists of two different types of protective molecules (enzymatic and nonenzymatic cell electrophoresis the DNA fragments form a ''tail'' originating from cell nucleus, producing a pathways). In this manner, we investigated the behavior of two enzymatic radical scavengers, comet-like figure. Usually, the amount of DNA in the tail is measured by image analysis and superoxide dismutase (SOD) and heme oxygenase I (HO-1), in addition to heat shock protein 70 employed to quantify the degree of DNA damage.
(Hsp 70) and ferritin following acute irradiation with UVA I, UVA I 1 II, and solar simulating We propose here an alternative method for DNA damage quantification that is based on measuring
light. Analysis of the content of these biomarkers in human skin after acute irradiation was of DNA loss from the nucleus. The cells (HaCaT line or human peripheral lymphocytes) are
performed by immunohistochemistry on biopsies from previously non-sun exposed sites of prepared and electrophoresed on microscope slides, as in the classic comet assay. DNA is stained
individuals with skin type II-III. In general we have found that their induction or depletion seems with propidium iodie, and fluorescence is measured by laser scanning cytometry (LSC) directly to be dependent on cell type, wavelength specificity and UV dose. The distribution of the first on microscope slides. In control, untreated cells a normal DNA histogram with low CV (,5%) three of these proteins showed a uniform presence throughout the epidermis and ferritin was was obtained with easily discernible G0/G1, S, and G2/M subpopulations. After induction of restricted to the basal keratinocytes of each volunteer before irradiation although heterogeneity DNA damage by UVA radiation, a sub-G1 peak appeared on the DNA histogram. By re-scanning was marked. Following acute UVA I (340-400 nm) irradiation with a dose of 1 or 2 MED a and digital imaging of the cells in the sub-G1 fraction we confirmed that they represented nuclei considerable dose-dependent decrease in antibody staining intensity was seen for SOD, HO-1, containing damaged DNA (''comets''). The fluorescence integral of the cells in the sub-G1 fraction and Hsp 70 whereas ferritin was shown to increase or diffuse into supra-basal keratinocytes. The correlated inversely with the tail moment (a measure of DNA content in the tail). Thus, LSC is same responses concerning the content of SOD, Hsp 70 and ferritin occurred, but to a lesser a method for objective and fully automated DNA damage analysis. LSC enables precise extent, with UVA I 1 II and solar simulating radiation, whereas HO-1 was nearly unaffected by quantification of the degree of damage, the proportion of damaged cells and correlating the degree these two wavebands. It is obvious that the pro-antioxidant balance can be overwhelmed by acute of damage with the position in cell cycle.
photo-oxidative stress and this is of importance to determine how these reactions can be avoided in order to provide protection against subsequent oxidative stress.