Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts.
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.
Skoog
M L
ML
Department of Dermatology, Linköping University, Sweden.
Ollinger
K
K
Skogh
M
M
D000447:Aldehydes / Q000032:analysis; D000975:Antioxidants / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002462:Cell Membrane / Q000528:radiation effects* / Q000648:ultrastructure; D002470:Cell Survival; D002478:Cells, Cultured / Q000187:drug effects; D004396:Coloring Agents; D003592:Cytophotometry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000528:radiation effects* / Q000648:ultrastructure; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects / Q000528:radiation effects; D008315:Malondialdehyde / Q000032:analysis; D011829:Radiation Dosage; D017382:Reactive Oxygen Species / Q000378:metabolism; D012867:Skin / Q000187:drug effects / Q000528:radiation effects* / Q000648:ultrastructure; D013473:Sunscreening Agents / Q000494:pharmacology; D014343:Trypan Blue; D014466:Ultraviolet Rays; D014810:Vitamin E / Q000494:pharmacology; D019207:beta Carotene / Q000494:pharmacology
D000447:Aldehydes; D000975:Antioxidants; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D013473:Sunscreening Agents; D019207:beta Carotene; D014810:Vitamin E; D008315:Malondialdehyde; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D014343:Trypan Blue; C018506:diacetylfluorescein