In Vitro UVB induced Cellular Damage Assessment Using Primary Human Skin Derived Fibroblasts

Abstract

Nowadays there is a great deal of concern resulting from the impact of UV light on human skin especially when skin damage levels are predicted to rise due to ozone layer depletion. The skin acts as an important biological barrier, and plays an important physiological and immunological role. This study reports the UVB-induced effect on skin derived primary fibroblasts one of the most abundant cells in the dermis. Two time points, immediately and 24 hour post UVB exposure were used to measure the magnitude of UVB-induced cytotoxicity. The induced cell membrane damage was assessed by neutral red (NR) dye uptake while cellular metabolic activity was assessed by the 5-(3-carboxy-methoxyphenyl)-2-(4.5-dimethylthiazolyl)-3-(4-sulphonyl) (MTS) cytotoxicity assay. A correlation was found between the MTS and neutral red (NR) assay in measuring UVB-induced cellular damage in exposed skin fibroblasts cultures immediately post exposure. These findings indicated that doses higher than 2.2 J/cm 2 were needed for the immediate expression of measurable cell membrane damage by the assays systems used. This should not indicate that no damage is triggered by lower irradiation levels, since our observations 24h post UVB-irradiation of the same cells showed cellular membrane damage for lower doses (0.7J/ cm 2 ) by comparison to immediately post exposure. The assessment of cellular metabolic activity indicated that MTS displayed a higher sensitivity in detecting early cell damage by comparison to the NR assay. The UVB induction follows a dose dependent response, and the cellular damage induced by exposure is best-measured 24h following exposure.

UVB damage onset and progression 24 h post exposure in human-derived skin cells

“…1 ). This is in agreement with published literature [20] , [14] on damages reported 24 h post exposure) highlighting the damage triggered by exposure of cell cultures to UVB irradiation. The cytotoxicity data also indicated different sensitivities to UVB induced damage between the two cell types used.…”

“…We have previously reported in [14] a good correlation between the MTS and NR assays in measuring UVB damages in exposed cells immediately post exposure. This correlation could not be observed 24 h post exposure hence the need for further investigations to elucidate the underlying mechanisms of UVB induced cellular damages.…”

“…The lamp output was measured by an IL-1700 research radiometer (International Light, Newburry Port, MA). Cells were irradiated with UVB doses ranging from 0 to 5.6 J/cm 2 [14] .

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Proteomic Alterations in Human Dermal Fibroblasts under Photo-Induced Pollution Caused by Excessive Solar Irradiations such as Infra-Red, Blue Light, UVA and UVB

“…HDF cells were seeded at 5 × 10 4 cells/ml in a 60 mm petri plate and grown in DMEM HG medium and incubated for 24 h at 37˚C. The cells were further exposed individually to UVA at 10 J/cm 2 for 5 minutes from 20 cm distance [8] [9] [10] [11], UVB at 200 mJ/cm 2 for 10 minutes from 20 cm distance [12] [13], BL at 50 mJ/cm 2 for 30 minutes from 40 cm distance [14] [15] [16] and Infrared at 750 J/cm 2 for 1 hour from 20 cm distance [17] [18]. Control cells without any irradiation also maintained separately.…”

Sternbergia Clusiana Bulb Ethanolic Extracts Induce Apoptosis in MCF-7 Breast Cancer Cells. (c2022)

“…Subsequently, the cells were treated the next day with SbBEE prepared in a fresh medium at increasing concentrations (0.15, 0.3, 0.5, 1 and 2.5 %v/v) for 24 h. MTS assay was implemented in order to assess the MCF-7 cell viability by measuring the conversion of the soluble tetrazolium salt into formazan by metabolically active cells. MTS (Promega) was prepared according to instructions set by the manufacturer by mixing a solution of PMS, MTS and DMEM at a ratio of 1:20:100 This mixture was incubated for 1 h and then the Varioskan™ LUX multimode microplate reader was allocated to measure the absorbance at 492 nm (Khalil, 2015).…”