Norepinephrine Reduces Reactive Oxygen Species (ROS) and DNA Damage in Ovarian Surface Epithelial Cells - PubMed (original) (raw)

Norepinephrine Reduces Reactive Oxygen Species (ROS) and DNA Damage in Ovarian Surface Epithelial Cells

Pooja R Patel et al. J Bioanal Biomed. 2015.

Abstract

Objective: To determine the role of norepinephrine (NE) on DNA damage and reactive oxygen species (ROS) generation in ovarian surface epithelial cells.

Method: Non-tumorigenic, immortalized ovarian surface epithelial cells were treated with NE, bleomycin, and bleomycin followed by NE. The comet assay was performed on each treatment group to determine the amount of single and double-strand breaks induced by treatments. ROS levels for each treatment group were measured using the H2DCF-DA fluorescence assay. Finally, RNA transcripts were measured for each treatment group with regards to the expression of DNA repair and oxidative stress genes.

Results: The mean tail moment of untreated cells was significantly greater than that of cells treated with NE (p=0.02). The mean tail moment of cells treated with bleomycin was significantly greater than that of cells treated with bleomycin followed by NE (p<0.01). Treatment with NE resulted in significantly less ROS generation than in untreated cells (p<0.01). NE treatment after hydrogen peroxide treatment resulted in a noticeable decrease in ROS generation. Genes associated with oxidative stress were upregulated in cells treated with bleomycin, however this upregulation was blunted when bleomycin-treated cells were treated subsequently with NE.

Conclusion: NE is associated with decreased DNA damage and ROS production in ovarian surface epithelial cells. This effect is protective in the presence of the oxidative-damaging agent bleomycin. These results suggest an additional physiologic role for the stress hormone NE, in protecting ovarian surface epithelial cells from oxidative stress.

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Figures

Figure 1

Figure 1

Cell viability assay. Presence of 10 uM norepinephrine does not affect cell viability of IOSE-29 ovarian surface epithelial cells. NE: Norepinephrine.

Figure 2

Figure 2

Comet assay results. This graph represents the levels of DNA damage with respect to untreated ovarian surface epithelial cells as a reference. DNA damage in cells treated with norepinephrine is significantly less than DNA damage in untreated cells (p=0.02). DNA damage in cells treated with bleomycin followed by norepinephrine is significantly less than DNA damage in cells treated with only bleomycin (p=0.01). NE: Norepinephrine; Bleo: Bleomycin; Bleo->NE: 30 minute treatment with bleomycin followed by 30 minute treatment with norepinephrine.

Figure 3

Figure 3

Comet assay images. Images of comet assay results of IOSE-29 cells. As expected, treatment with bleomycin resulted in larger comet tails, signifying greater DNA damage. Treatment with norepinephrine resulted in less tail, and subsequent treatment with norepinephrine after treatment with DNA damaging agent resulted in smaller tail. Bleo: Bleomycin; NE: Norepinephrine; Bleo->NE: 30 minute treatment with bleomycin followed by 30 minute treatment with norepinephrine.

Figure 4

Figure 4

ROS generation measured by H2DCFDA. As expected, treatment with hydrogen peroxide resulted in greater ROS generation. Treatment with norepinephrine resulted in significantly less ROS generation than in untreated cells (p<0.01). Norepinephrine treatment after hydrogen peroxide treatment resulted in decreased ROS generation, however this different was not significant (p=0.14). H2O2: Hydrogen Peroxide; NE: Norepinephrine.

Figure 5

Figure 5

mRNA expression level of oxidative stress associated genes. All results are normalized to untreated cells. As expected, treatment with bleomycin resulted in increased expression of these genes when compared to untreated cells. Treatment with norepinephrine resulted in decreased expression of these genes when compared to untreated cells and treatment with norepinephrine after treatment with bleomycin resulted in decreased expression of these genes when compared with bleomycin treatment alone. Bleo: Bleomycin; NE: Norepinephrine; Bleo->NE: treatement with bleomycin for 30 minutes followed by treatment with norepinephrine for 30 minutes.

Figure 6

Figure 6

Proposed model of norepinephrine’s antioxidant effect in ovarian surface epithelial cells. Outlined above is the proposed pathway by which norepinephrine decreases ROS generation and subsequent DNA damage in ovarian surface epithelial cells. In this model, norepinephrine acts as a rapid ROS scavenger leading to a decrease in downstream DNA damage and reactive transcription of genes involved in the cellular response to oxidative stress.

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