Depletion of my protein of interest via siRNA mediated knock down increases oxidative stress in our basal keratinocyte cell line. I was able to show increased ROS via cellROX reagent and I was further able to show increased DNA damage (probably due to increased ROS) in these cells.
Now, I'd like to eliminate or decrease the increased oxidative stress levels in those cells (my protein of interest-depleted cells). Can I treat the cells with reduced glutathione (GSH)? What are my other options?
Thank you.
There are a lot of different antioxidants. I would recomend to use Trolox.
No you cannot treat cells with GSH, it does not permeate cell membranes.
I can suggest the GSH precursor NACET, it is trapped inside cells and transformed into GSH.
N-Acetylcysteine ethyl ester (NACET): a novel lipophilic cell-permeable cysteine derivative with an unusual pharmacokinetic feature and remarkable antioxidant potential. http://www.ncbi.nlm.nih.gov/pubmed/23000913
You can use Butylated hydroxytoluene (BHT) as antioxidant during any treatment.
I recommend to add N-acetylcysteine to prohibit free radical generation
In my opinion you can go for NAC/BHT to reduce the Oxidative stress.
Reactive oxygen species (ROS) are composed of 3 words Rezctive+oxygen+molecules. So, chemically ROS are reactive molecules which contain oxygen. For example, peroxides, superoxide, hydroxyl radical, and singlet oxygen.
ROS form physiologically as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis.
However, during any types of stress environmental stress (e.g., UV or heat exposure), acute exercise, metabolic disorder, infectious disease, toxicity ROS levels can increase dramatically. This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. ROS are also generated by exogenous sources such as ionizing radiation.
Antioxidants can counteract the ROS generation. So GSH is non enzymatic antioxidant, might be able to reduce to ROS generation Please read the following paper might be helpful for you.
http://toxsci.oxfordjournals.org/content/70/2/183.long
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2821140/
Superoxide dismutases (SOD) enzymatic antioxidant that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen. You could try with SOD too….thanks..
How is your siCTRL or reagent only experiment? Transfection reagents itself increase ROS a lot. Do not forget to do these controls.
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