Do you know the level of antioxidant enzymes in your cells? I mean catalase, superoxide dismutase and glutathion peroxidase, The enzymes protect cells from ROS

There is not a direct relationship between cell viability and ROS production. There are many phisiologyical events modifying ROS amounts. Dying cells release ROS to the media and at the same time they decrease ROS production because of ETC impairment. It's gonna be hard to measure them and stablish a correlation, if that's what you want. 

I just can agree to Francisco. The behaviour of cell lines to ROS-mediated oxidative stress is strongly dependent on the scavenging enzymes, like SOD, Cat, GPx. But it also depends on the oxidative stress Regulators, like p38-MAPK. So propbably you check your Assay with your nanoparticles first with an apoptosis stain or Proliferation Assay, to get an idea, what concentration of your particles might be the best for your further investigation.

It is also known, that ROS and RNS play a role in cell cycle processes. So a certain level is needed for the expression of several cell cycle regulators, whereas to high concentrations of ROS and RNS can lead to cell damage.

Have you done viability assays with different time points? If you pick an early time point where cell death is still very low or none you could measure the ROS using DCF-DA in a more confident manner, to check if there is increase in ROS induced by the nanoparticles. A549 cells are lung cancer cells and in principle they should have a strong antioxidant system since lung cells are exposed to gas exchanges and you would expect them to have a good antioxidant/ redox regulatory system to cope with this.

Hi, just  to say that i'm agree with peter, and i would say that the response is much more complex, as you know that some ROS molecules have a pleiotropic functions in cell regulation and cell signalling, so the modulation of the redox equilibrum into the cell always followed by a cascading reactions leading to regulate the response, and the cell death is one from many scenario that could be played via activating mitochondrial or alternative pathway apoptosis, also inflammatory phenomenon could be exist, beside many other scenarios that could be happened, all of these reactions, as peter said, are time and dose dependant, but not only that as you working with nanoparticles, you have to bring in mind your nanoparticles size, physico-chemical characterizations of yours, and many other questions that you should respond when talking about nanotoxicity.....a hard work that you have to do,,, with all my best wishes

I have conducted cell viability analysis using MTT assay, from that i observe the viability decreases as the exposure time increases. Even at time points, 6 hours, at higher concentrations of the NPs, the viability is less than 40%. I have not tried analyzing the antioxidants in the A549 cell cultures. I am preparing a list of them to do that analysis.

Cell viability is affected mainly due to the following reasons:

1) Cell death by (a) Apoptosis (b) Necrosis (c) Autophagy (d) Senescence

2) Inhibition/mitigation of cell proliferation (slower proliferation gives you a smaller no. of viable cells in MTT/XTT etc.)

Coming to ROS, these species of free radicals are produced copiously in the cell, particularly in the mitochondria, where oxidative processes (such as OX-PHOS) occur. Several antioxidant enzymes (eg: superoxide dismutase, catalase etc.) present in the cell are meant to encounter and neutralize oxidative stress. However, in cellular compartments like the mitochondria, due to a vicious cycle of ROS production, the antioxidant machinery significantly falls short of its functional requirement. 7 out of the 13 polypeptides participating in the electron transport chain are encoded by the mitochondrial DNA. Extensive oxidative stress in the mitochondria damages mtDNA, along with mitochondrial proteins & lipids (lipid peroxidation forms more ROS!!). The mitochondrial SOD (Mn-SOD) often fails to quench the ROS thus formed. The damaged mtDNA often encodes defective ETC proteins, which in turn forms more ROS, and this continues in a cycle. ROS, thus formed, reduce mitochondrial membrane potential (ΔΨm), ie., depolarizes the mitochondrial membranes, thereby forming leak channenls in the mitochondrial membranes. Apoptotic proteins like cytochrome c and AIF come out into the cytosol through this channel and cause caspase-dependent as well as caspase-independent apoptosis. Similarly, ROS is also associated with senescence, necrosis and autophagy. The mode of cell death varies according to levels of ROS and the degree/target of ROS-induced oxidative damage. At the same time, oxidative stress can both stimulate and inhibit cellular proliferation, depending on the mode and site of ROS action. Interestingly, inhibition of cellular proliferation leads to senescence as a consequence.

As you can see, there is no single answer to your question. Effects of ROS on cell viability do not follow a thumb-rule; rather they are governed by a number of associated parameters to decide the fate of a cell.

I hope that helps. Cheers!

I think cell viability depend on the ratio of antioxidants to oxidants in cells.So I think you need measure antioxidants(SOD,CAT,GPX,...) and oxidants(MDA,Di tyrozin,Di hydroxyguanozin ...) and then find statistical correlation between these ratio and alive or death cells.Best wishes.

I am sorry to say that getting an authentic measure of oxidants and antioxidants for a given time Δt is not a simple linear practice. It's almost impossible to come up with authentic holistic values of either antioxidants or oxidants.

@ Gunjan Guha i think that i dont fully understand you. For instance i have MTT results and the TBARS results for meta complexes, do you have any ideia of how can i relate does results? Like, if one justify the other?