UVA-irradiated Cells Research Articles

Glutathione modulates the level of free radicals produced in UVA-irradiated cells

We have developed an assay to detect reactive oxygen species (ROS) generated by UVA radiation utilising chemical probes which become fluorescent upon oxidation. Using a human bladder carcinoma cell line (MGH-U1) and spontaneously immortalised keratinocytes (HaCaT), we have shown a UVA (narrow band 365±5 nm) dose-dependent increase in fluorescence by flow cytometry following loading of the cells with either dihydrorhodamine 123 (DHR) or 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The UVA response of both DHR and DCFH was enhanced by elevation of intracellular levels of the photosensitiser protoporphyrin IX by incubation for 2.5 h with 5-aminolaevulinic acid. Depletion of the antioxidant glutathione (GSH) using the inhibitor d,l-buthionine-sulphoximine (BSO), resulted in an increase in the UVA-induced fluorescence of DCF but not of rhodamine 123. Conversely, raising intracellular GSH levels with N-acetyl cysteine (NAC) had relatively little protective effect in terms of degree of induced fluorescence.

The use of fluorogenic probes to detect free radical production in UV-A irradiated cells

The use of fluorogenic probes to detect free radical production in UV-A irradiated cells

Direct Detection of 8-Oxodeoxyguanosine and 8-Oxoguanine by Avidin and Its Analogues

8-Oxodeoxyguanosine is present in DNA from many tissues. The direct demonstration of 8-oxodeoxyguanosine as a potential biomarker of oxidative DNA damage has implications for the study of mutagenesis, carcinogenesis, and free radical toxicity. Avidin is shown here to bind with high specificity to this potentially mutagenic oxidized nucleoside, 8-oxodeoxyguanosine, and to the oxidatively modified base, 8-oxoguanine. The serendipitous finding that avidin bound to the nuclei of UVA-irradiated cells has led to the development of a technique which allows detection of the damage product in a manner analogous to that of immunological techniques. The technique has been shown to be applicable to isolated DNA and to DNA in fixed cellular material and postmortem tissue. Statistically different levels of damage can be demonstrated in both isolated DNA and cultured cells exposed to free radical generating systems using a 96-well plate-based methodology. The sensitivity of this method allows the detection of 10−19mol of 8-oxodeoxyguanosine. This novel usage of avidin conjugates applies also to its bacterial analogue, streptavidin, and to a lesser extent to the monoclonal antibody to biotin (the ligand bound by the parent compound). This finding has tremendous potential as a simple method analogous to immunotechniques for the direct detection of 8-oxodeoxyguanosine. From structural considerations we speculate that avidin would also bind to 8-oxodeoxyadenosine.

GM-CSF production by epithelial cell line: upregulation by ultraviolet A.

It was demonstrated that UVB increases synthesis and expression of IL-1 alpha and GM-CSF by keratinocytes. Upregulation of GM-CSF by UVB is reported to be mediated by IL-1 alpha. However, regulation of IL-1 alpha and GM-CSF by UVA is not well-known. The purpose of the present study was to evaluate the effects of UVA on IL-1 alpha and GM-CSF production. Here we used a competitive RT-PCR for measuring cytokine gene expression in an epidermal cell line after UVA irradiation. IL-1 alpha and GM-CSF mRNA did not show any change at 1 h and 6 h following exposure to UVA. After UVA irradiation, however, IL-1 alpha mRNA decreased and GM-CSF mRNA increased at 24 h and the level of GM-CSF in culture supernatant increased at 24 h and 48 h. Addition of antihuman IL-1 alpha neutralizing antibody to UVA irradiated cells did not prevent the increase of GM-CSF mRNA expression. These results suggest that UVA radiation may induce GM-CSF production through an IL-1 alpha independent pathway.

Induction of 8-oxo-7,8-dihydro-2'-deoxyguanosine by ultraviolet radiation in calf thymus DNA and HeLa cells.

The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10(5) 2'-deoxyguanosine (dGuo) per kJ/m2 for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10(5) dGuo per kJ/m2, respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irradiated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.

Cytometric Analysis of the Proliferative Capacity of HUT 102 Lymphoblasts Exposed to Long-wave UV Light and Psoralen

Cultures of HUT 102 lymphoblasts, comprised of near-diploid and hyperdiploid cells (11 and 22 pg of DNA/nucleus), were irradiated for 2 h by long wave ultraviolet light (UV-A) (2.1 mW/cm2), reacted for the same time in the dark with suprapharmacologic concentrations of 8-methoxypsoralen (8-MOP) (15 micrograms/ml) and exposed to 8-MOP and UV-A (PUVA) under identical conditions. Flow cytometric determinations of cellular DNA content and IdUrd incorporation indicated that UV-A irradiated cells incorporated IdUrd at the control levels and that the number of cells in S and G2 + M phases of the cell cycle likewise were identical to controls. The dark reaction of HUT 102 cells with 8-MOP has, however, resulted in a transient increase of IdUrd incorporation, but it has not affected cellular proliferation rates. On the other hand, no IdUrd incorporation was detected after PUVA. The analysis of the DNA content of intact and PUVA-exposed cultures showed that the number of near-diploid S-phase cells was greater 24 h after PUVA, whereas the number of hyperdiploid cells in all phases of the cell cycle had decreased. Under stated conditions the viability of HUT 102 cells and their cell cycle disturbances by PUVA are determined in part by the amount of DNA in target lymphoblasts.