Selenoprotein P regulates 1-(4-Chlorophenyl)-benzo-2,5-quinone-induced oxidative stress and toxicity in human keratinocytes

Abstract

Polychlorinated biphenyls and their metabolites are environmental pollutants that are believed to have adverse health effects presumably by inducing oxidative stress. To determine if 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ; metabolite of 4-monochlorobiphenyl, PCB3)-induced oxidative stress is associated with changes in the expression of specific antioxidant genes, mRNA levels of 92 oxidative stress-response genes were analyzed using TaqMan Array Human Antioxidant Mechanisms (Life Technologies), and results were verified by performing quantitative RT-PCR assays. The expression of selenoprotein P (sepp1) was significantly downregulated (8- to 10-fold) in 4-ClBQ-treated HaCaT human skin keratinocytes, which correlated with a significant increase in MitoSOX oxidation. Overexpression of Mn-superoxide dismutase or catalase or treatment with N-acetyl-l-cysteine suppressed 4-ClBQ-induced toxicity. Sodium selenite supplementation also suppressed 4-ClBQ-induced decrease in sepp1 expression, which was associated with a significant inhibition in cell death. Furthermore, HaCaT cells overexpressing sepp1 were resistant to 4-ClBQ-induced oxidative stress and toxicity. These results demonstrate that SEPP1 represents a previously unrecognized regulator of PCB-induced biological effects. These results support the speculation that selenoproteins can be an attractive countermeasure for PCB-induced adverse biological effects.

Keywords: 1-(4-Chlorophenyl)-benzo-2,5-quinone; 4-ClBQ; 4-monochlorobiphenyl; AdCAT; AdEmpty; AdMnSOD; Free radicals; HaCaT; MFI; N-acetyl-L-cysteine; NAC; Oxidative stress; PCB3; PEG-CAT; PEG-SOD; Polychlorinated biphenyls; ROS; SECIS; SEPP1; Selenoprotein P; adenovirus carrying a control vector without an insert; adenovirus carrying a human Mn-superoxide dismutase cDNA; adenovirus carrying a human catalase cDNA; mean fluorescence intensity; polyethylene glycol–catalase; polyethylene glycol–superoxide dismutase; reactive oxygen species; selenocysteine insertion sequence; selenoprotein P.

Fig. 1

4-ClBQ treatment perturbs cellular morphology and increases mitochondrial ROS levels. (A) Microscopic pictures of paraformaldehyde-fixed control and 4-ClBQ treated HaCaT cells that were stained with Phalloidin 488 and Hoechst; magnification: × 400; bars = 50 μm; n = 3. (B) Representative flow cytometry histograms of MitoSOX Red oxidation of control and 4-ClBQ treated HaCaT cells; PEG-SOD and PEG-catalase were used to determine the specificity of MitoSox Red oxidation for measurements of superoxide and hydrogen peroxide. (C) Flow cytometry measurements of MitoSOX Red oxidation and MitoTracker Green uptake in control and 4-ClBQ treated HaCaT cells at the end of 24 h treatment. MitoSOX Red oxidation was normalized to MitoTracker Green uptake in each sample, and the fold change in MFI was calculated relative to untreated cells. Asterisks represent statistical significance compared to untreated cells; P < 0.05, n = 3.

Fig. 2

4-ClBQ treatment perturbs cell cycle progression and induces cell death. (A) Cell numbers were counted at the end of 24 h treatments. (B) Representative histograms of DNA content that show an increase percentage of cells in the sub-G₁, S, and G₂ phases of 4-ClBQ treated cells. (C) Flow cytometry measurements of the percentage of cell cycle phases in control and 4-ClBQ treated cells. (D) Toxicity was measured using a clonogenic survival assay. Cell survival of 4-ClBQ treated cells was normalized to untreated cells. Asterisks represent statistical significance compared to untreated cells; P < 0.05, n = 3.

Fig. 3

Oxidative stress regulates 4-ClBQ induced toxicity. (A) Cell cultures were incubated with media containing 5.0 mM NAC 5 h prior or 5 h after the addition of 3.0 μM 4-ClBQ. Cell survival was determined by performing a clonogenic assay. Asterisks represent statistical significance compared to untreated cells; # represents statistical significance compared to 3.0 μM 4-ClBQ treated cells; P < 0.05; n = 3. (B) HaCaT cells were infected with adenoviruses AdEmpty (100 MOI), AdMnSOD (50 MOI), AdCAT (50 MOI) or AdMnSOD and AdCAT (50 MOI each). Transgene expression was measured using an immunoblotting assay. Cells were treated with 1.0 μM 4-ClBQ and survival was measured using a clonogenic assay. Surviving fraction was calculated relative to untreated AdEmpty infected cells. Asterisks represent statistical significance compared to AdEmpty infected untreated cells; # represents statistical significance compared to 1.0 μM 4-ClBQ treated AdEmpty infected cells; P < 0.05, n = 3.

Fig. 4

4-ClBQ treatment significantly inhibits sepp1 expression. (A) mRNA levels of sepp1, catalase, mnsod, cygb, and dusp1 that showed significant changes in the PCR-array were further verified by performing a quantitative RT-PCR assay; fold-change was calculated relative to individual mRNA levels in untreated cells. Quantitative RT-PCR measurements of sepp1 mRNA levels in (B) 24 h 4-ClBQ (0–3.0 μM) treated cells, and (C) 3.0 μM 4-ClBQ treated cells at the end of 4, 8, and 24 h of treatments. Fold change was calculated relative to time-matched untreated cells. Asterisks represent statistical significance compared to untreated cells; P < 0.05, n = 3. (D) Total proteins were precipitated from media collected from control and 4-ClBQ treated cells. SEPP1 protein levels were analyzed by immunoblotting. A Coomassie blue stained polypeptide band was used for loading correction.

Fig. 5

Sodium-selenite supplementation inhibits 4-ClBQ mediated down-regulation of sepp1 expression and protects HaCaT cells from 4-ClBQ induced toxicity. HaCaT cells were cultured in media supplemented with 100 nM of sodium selenite and then treated with 1.0 μM of 4-ClBQ for 24 h. (A) sepp1 mRNA levels were analyzed by performing a quantitative RT-PCR assay. Asterisks represent statistical significance compared to untreated cells; # represents statistical significance compared to 1.0 μM 4-ClBQ treated cells; P < 0.05; n = 3. (B) An immunoblotting assay was used to measure SEPP1 protein levels. A Coomassie blue stained polypeptide band was used for loading correction. (C) A clonogenic assay was used to measure cell survival. Asterisks represent statistical significance compared to untreated cells; # represents statistical significance compared to 1.0 μM 4-ClBQ treated cells; P < 0.05; n = 3.

Fig. 6

Overexpression of sepp1 abrogates 4-ClBQ induced oxidative stress and cytotoxicity in HaCaT cells. Cells were transfected with CMV-pShooter-sepp1 plasmid DNA that contains the ORF and two SECIS sequences of human sepp1 cDNA. Cells transfected with plasmid DNAs without an insert sequence (Empty) were included as controls. Transfected cells were cultured in 30 nM sodium-selenite supplemented media prior to and during the 4-ClBQ treatment. (A) Transgene expression was evaluated by measuring sepp1 mRNA and protein levels using quantitative RT-PCR and immunoblotting assays. Upper panel shows C_T plot of sepp1 amplification in Empty and CMV-pShooter-sepp1 plasmid DNA transfected cells. Asterisks represent statistical significance compared to untreated cells. P < 0.05; n = 3. (B) Flow cytometry measurements of MitoSOX Red oxidation in control and 1.0 μM 4-ClBQ treated cells transfected with Empty or CMV-pShooter-sepp1 plasmid DNA. Fold change was calculated relative to corresponding untreated cells. Asterisks represent statistical significance compared to untreated cells; # represents statistical significance compared to 1.0 μM 4-ClBQ treated empty-vector transfected cells. P < 0.05; n = 3. (C) A clonogenic assay was used to measure cell survival in 1.0 μM 4-ClBQ treated cells transfected with Empty or CMV-pShooter-sepp1 plasmid DNA. Surviving fraction was calculated relative to untreated cells transfected with Empty vector. Representative dishes showing a higher number of colonies in sepp1 overexpressing 4-ClBQ treated cells are included for comparison. Asterisks represent statistical significance compared to untreated empty-vector transfected cells; # represents statistical significance compared to 1.0 μM 4-ClBQ treated empty-vector transfected cells; P < 0.05; n = 3.