Overexpression of selenoprotein H reduces Ht22 neuronal cell death after UVB irradiation by preventing superoxide formation

Abstract

Selenoproteins have been shown to exhibit a variety of biological functions, including antioxidant functions, maintaining cellular redox balance, and heavy metal detoxification. UV irradiation-induced damage is partially mediated by increased oxygen radical production. The present study is designed to examine the antioxidative effects of human selenoprotein H (hSelH) after brief period of UVB irradiation on the murine hippocampal neuronal cell line Ht22. Ht22 cells were stably transfected with the hSelH gene or with MSCV empty vector and exposed to UVB irradiation with or without the presence of serum. The results showed that cell viability was significantly higher in hSelH-transfected cells compared to the MSCV vector-transfected cells after 24 h of recovery with or without the presence of serum in the media. Further studies revealed that while the number of superoxide anion (O2*-) positive cells was increased following a 7 mJ/cm(2) of UVB irradiation and 5 h of recovery, overexpression of hSelH significantly reduced superoxide production. These results suggest that hSelH overexpression protects cells from UVB irradiation-induced cell death by reducing the O2*- formation.

Fig 1

Variable UVB irradiation exposure parameters on cell viability in MSCV (empty vector) transfected Ht22 cells. The irradiation doses given to the cultures were 0 mJ/cm², 7 mJ/cm², 11.5 mJ/cm², and 23 mJ/cm², in DMEM medium with serum, respectively. The percentage of cell viability was determined after 24 and 48 h of recovery using the Trypan blue exclusion assay. About 100%, 50%, 40% and 6% of cell viability were observed at 24 h of recovery 0 mJ/cm², 7 mJ/cm², 11.5 mJ/cm², and 23 mJ/cm² of UVB irradiation, respectively. Cell viabilities were further significantly decreased to 21 %, 4% and 0% at 48 h of recovery at the indicated UVB doses in the Figure. Experiments were performed in triplicate and presented as means ± SD. *Significantly different from non-radiated control at 24 h (P < 0.05) at the indicated UVB dose. #Significantly different from 24 h of recovery at 48 h of recovery (P < 0.05).

Fig 2

Photomicrographs showing effects of hSelH overexpression cell viability in Ht22 cells treated with 7 mJ/cm² of UVB irradiation followed by 24 h of recovery. Increased cell debris was present in UVB radiated MSCV-Ht22 cell compared to non-treatment control cells. More viable cells were observed when hSelH transfected Ht22 cells were exposed to UVB irradiation. Images were taken at 20X magnification with a Zeiss LSM5 laser-scanning confocal microscope. Bar = 10 μm.

Fig 3

Effects of hSelH overexpression on Ht22 cells exposed to 7 mJ/cm² of UVB irradiation with or without serum in media. Cell viability was examined after 24 h of recovery and presented as percentage (mean ± SD). Overexpression of hSelH protected cells from UVB-induced damage. *Significantly different from MSCV control, P < 0.0001 and #Significantly different from MSCV transfectant, P = 0.0004, in the presence or in the absence of serum, respectively.

Fig 4

Percentage of oxidized HEt positive cells in MSCV-Ht22 and hSelH-Ht22 cells after 5 h of recovery follow a 7 mJ/cm² of UVB irradiation. hSelH overexpression significantly reduced oxidized HEt positive cells compared with MSCV-Ht22 cells. *Significantly different from MSCV-Ht22, P = 0.0017.

Fig 5

Representative photomicrographs showing oxidized HEt positive staining in MSCV-Ht22 (A) and hSelH-Ht22 cells (B) after 5 h of recovery follow 7 mJ/cm² of UVB irradiation. Arrows denote oxidized HEt cells which indicating the formation of superoxide. Images were captured at 20X magnification with a Zeiss LSM5 laser-scanning confocal microscope. Bar = 10 μm.