I am doing ROS activity assay in renal carcinoma cell lines. I have treated three ROS scavengers (NAC, Trolex, and glutathione ethyl ester), surprisingly, all these are showing different activity when treated with the drug (not alone treatment group). For example, NAC has no effect on ROS production and cell death of the cell, however, trolex was found to reduce the production of ROS and cell death very less (not significant). On the other hand, GEE totally blocked the production of ROS and cell death induced by the drugs. Ideally, NAC is believed to produce glutathione in cells resulting in decrease or inhibition of ROS production, but in my case why NAC and GEE are showing the difference. If somebody has the idea about this kindly share with me.
Thank you
It's not unusual to see a difference in activity between the final product added exogenously and that of a preceding factor. Glutathione monoethyl ester is particularly good because it gets inside the cell. Maybe the cells just aren't making enough GSH from NAC, or quickly enough??
NAC is also an anti-oxidant by itself, but can interestinngly also act as a pro-oxidant:
http://www.ncbi.nlm.nih.gov/pubmed/12071352
Hi Anil Kumar!
Can you plz try with DCF (ROS Marker), which is called as dichloro Fluorescene di-acetate!!! I hope you will be able to get good result, by the emission of green fluorescence from the cells as a result of ROS production!!!
DCF is not recommended for use as a probe for OS, because it is non specific and prone to artefacts
Dear David, thanks for your answer I agree with you, the activity of NAC depends on the severity of the cell induced by the drug as you told it is not potent enough to produce GSH while GEE is directly supplying the desired GSH to overcome from oxidative stress. However, I detected the combined effect of NAC by using a standard anticancer drug and it is working well, that means my target drug has the severe effect on the cell that NAC is not able to instigate the cells to produce GSH I guess.
Dear Robert, thanks for enlighting me on this aspect of NAC activity, additionally, NAC is also reported to produce TNF alpha when combined with some drugs which even enhances the cytotoxicity instead of protection.
Dear Suresh, thanks for your suggestion but here the problem was different, NAC, a ROS inhibitor was not able to inhibit ROS production which means I am getting the results of ROS staining and FACS nicely but the inhibitor was showing the reverse result.
Actually, both NAC and et-GSH may be anti- as well as pro-oxidants depending on the dose (for et-GSH see e.g. http://www.ncbi.nlm.nih.gov/pubmed/16293684). The biggest difference is in regulatory functon of GSH. S-glutathionylation is a process that of interaction of GSH or GSSG (formed from GSH) with thiol groups of proteins http://www.ncbi.nlm.nih.gov/pubmed/17662654
http://www.ncbi.nlm.nih.gov/pubmed/18774901
and many more references...
S-glutathionylation is a powerful mechanism of coordinated regulation of multiple proteins that is involved in each biological process that is redox-dependent (you name it)
So, it is by far no the same what you use et-GSH or NAC
On my experience NAC is unable to enter the cells while some ethyl esters of cysteine does. It is possible that GSH is decomposed into its constituent aminoacids by the high gGT activity present in RCC cells. Thus may be the phenomenon is to bew ascribed to cys ethyl ester.
Here are some experiments we performed with NAC ethyl esther.
NAC is precursor of Glutathione but how much Glutathione will be formed that is not clear. Glutathione is tripeptide (cysteine, glycine & glutamate) molecule. Glutathione can not enter into cell but Glutathione mono ethyl ester is cell permeable derivative of Glutathione.
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