I'm trying to induce oxidative stress by using rotenone and SNP sodium nitroprusside in isolated mitochondria of rat brain. This is to study GSH assay from control (Mitochondria) group in comparison with { Mito + (SNP/Rotenone)}.
However, I'm not getting the promising results
could anyone suggest me why there is not decrease in GSH occurs significantly in (SNP or rotenone induced mitochondria comparision to control?
is there any matter of concentration?
or suggestions.
(doi : 10.14715/cmb/2014.60.2.6)
please fine protocol to perform GSH assay>
Hello Snehal Kadam
I will answer the question in parts -
(1) How to decide the concentration need to induce oxidative stress in isolated mitochondria of rat brain by using either rotenone & sodium nitroprusside?
The term “how much concentration…induce oxidative stress” is inappropriate. Any amount of R or SNP (or combination R, SNP, R+SNP) will induce oxidative stress (OS) and that amount can vary between negligible to quintals (hypothetically). Negligible concentrations will be outpowered by antioxidants (AOX) (including GSH as you have asked) but there would be a time when the antioxidants would be overpowered by OS. Thus the amount of toxic chemicals is proportional to stress. So there is something called a toxicity test to understand what concentration YOU want to evaluate.
(2) I'm trying to induce oxidative stress by using rotenone and SNP sodium nitroprusside in isolated mitochondria of rat brain. This is to study GSH assay from control (Mitochondria) group in comparison with { Mito + (SNP/Rotenone)}.
Agreed that you are trying to induce OS by R and SNP, but you need to have something considerable so that you can judge the response of GSH alterations (check overflowing online articles to understand and conduct the test). First conduct isolated tests or R and SNP then combos.
(3) However, I'm not getting the promising results
What according to you is a promising result? Results are results. There is nothing promising about anything and that's the gambit of every research. If we knew the results, then what's the need for any research. Agreed? The catch here is first conduct the test, decide the conc of R and SNP, expose MC to chemicals and see outcomes on ETC dysfunction. Also, could it be that the R+SNP combo is nullifying each other?
(4) could anyone suggest me why there is not decrease in GSH occurs significantly in (SNP or rotenone induced mitochondria comparision to control?
This question forms discussion part of your manuscript. That's what you need to reason in your publication as to why it has happened or what are probable reasons. For that you need to see what are the other AOX responses? It is not mandatory that GSH must decrease if there is exposure to R and SNP, and if that be then it is definitely a very strong response. There can also be a tandem act with other AOX that you are evaluating. GSH is a bifunctional AOX, at times it is conjugated by (GST-GSSH system) or at times it can also act alone (Srikanth et al. 2013 attached)
Thank you so much @Proteek Dasgupta, I'm very grateful to u for your response. I'll go through this information.
Clarified.
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