Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA‐irradiated cultured skin fibroblasts

Abstract

The damaging effect of long-wave ultraviolet radiation (UVA) on the plasma membranes of cultured human skin fibroblasts was evaluated by cytofluorometry performed after vital staining with fluorescein diacetate. The damage was associated with lipid peroxidation, as shown by accumulation of malondialdehyde and 4-hydroxyalkenals; such accumulation was induced by a UVA dose of only 8 J/cm2. Pretreatment with the effective membrane peroxidation inhibitor alpha-tocopherol (added in the form of alpha-tocopherol succinate) or the singlet oxygen quencher beta-carotene protected the cells from membrane damage. Further, depletion of intracellular glutathione by exposure of the cells to buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase aggravated the membrane-damaging effects. The results confirm the photodynamic effects of UVA, which presupposes the excitation of endogenous photosensitizer(s) and the production of reactive oxygen species. The present results indicate that this method of detection of alterations in plasma membrane stability may be highly suitable for studying various photobiological phenomena and for use as a model for testing substances that could protect skin from UVA damage. The trypan blue exclusion test proved to be too insensitive to detect these changes.

In vitro effects of ultraviolet A on histamine release from human basophils

“…The increase of the percentage of histamine release observed with the highest UVA doses could be ascribed to a marked dose‐dependent damage of basophils cell membranes starting from 7.5 J/cm 2 . This observation is in agreement with data reported from Skoog et al 16 . who, using the application of fluorescein diacetate as an indicator, demonstrated that UVA causes damage of cultured human fibroblast plasma membranes with doses between 4 and 8 J/cm 2 .…”

Systematic Study of Parameters Influencing the Action of Rose Bengal with Visible Light on Bacterial Cells: Comparison Between the Biological Effect and Singlet-Oxygen Production

“…In our experiment this UVB component was attenuated by the Pyrex glass. Cell killing by UVA, instead, is often associated with membranes as the target (47)(48)(49). If damage induced by RB with visible light were different from damage induced by UVA, then additivity could be expected for the action by both agents until some level of saturation were approached.…”

Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis

“…To evaluate the effect of trypsination on the FDA‐stained cells, we performed a microfluorometry test as described earlier (21). Cells were seeded at a density of 10 × 10 3 cells/cm 2 on cover slips in Petri dishes, radiated and incubated with FDA (1 µg/ml) as described for flow cytometry.…”