DNA double strand brakes can be detected by immuno fluorescent localization of histone gamma H2AX a Ser 139 phosphorylated version of H2AX which form foci that are atributed to DNA double strand brakes repair machinery. Another common method is to detect 8- hydroxyguanosine a marker of free radical DNA damage. The third method I would recommend is to test the proportion between the reduced and oxidated forms of gluthathion GSSG/GSH the main cellular small molecule antioxidant (There are various kits for those assays).

Good Luck

Hi Adam, there are a multitude of oxidative stress biomarkers... Perhaps you could let us know which model you are studying ? what radical species are being produced ? which antioxidants are being depleted? Answers to these questions are vital because certain radicals/oxidants have a preference to the molecules they oxidise... for example hypochlorus acid (produced by MPO, released from macrophages) oxidises proteins, forming chlorinated oxidation products (clorotyrosine, for example)... H2O2 can produce hydroxyl radicals which will oxidise almost anything (they are so reactive). If your oxidant induces lipid peroxidation, then you can measure malandialdehyde, 8-isoprostanes (gold standard), 4-hydroxynonenal... more info on your model, will help us guide you to a method... If protein oxidation is what you are after, I can suggest this excellent review, which includes a number of detailed standard operating procedures for a number of methods... http://193.146.160.29/gtb/sod/usu/$UBUG/repositorio/10320076_Hawkins.pdf

Hi Adam! I guess what you may first want to identify what is the outcome you are looking for in order to choose the most suitable marker. In other words, are you just looking for nuclear oxidative stress-related damage regardless of what may be causing it? or you are looking for oxidative damage caused by a particular injury? In the first case, I would go for something relatively unspecific, like 8-hydroxyguanosine, which is a classical marker of oxidative damage to DNA. However, if you want to look for damage caused by specific agents, you may want to look for some specific oxidative consequences on nuclear proteins (for instance, cysteine S-nitrosylation or tyrosine nitrosation if you are looking at RNS damage, or cysteine sulfenic acid formation or S-glutathionylation if you are looking at ROS damage).

Also, a word of caution for the GSSG/GSH ratio. GSH is a reducing agent that works as a ROS scavenger. However, GSH is also a nucleophilic agent that neutralizes electrophilic substances like 4-hydroxynonenal or toxic metabolites (quinones and such), that DO NOT INVOLVE GSH OXIDATION TO GSSG, but formation of GSH adducts. Therefore, you may get an increase in the GSSG/GSH ratio that is unrelated to oxidative stress but may be confused with one. If GSH is being consumed in an oxidative manner, then you should see a simultaneous increase in GSSG (due to GSH oxidation). On the other hand, if you measure a decrease GSH without a GSSG increase, then you may be looking at electrophilic -not oxidative- stress in the nucleus.

You can detect DNA DSBs in immunofluorescence using anti-gH2Ax antibodies.

You can detect DNA SSBs in immunofluorescence using anti-Poly(ADP-ribose) antibodies (that can be also used in western bloting)

You might also use commet assays or pulse field gel electrophoresis (with or without DNA denaturation ) to assess DNA damages.

I hate to brag, but have a look at my 2012 Thiomersal Paper and my 2010 Biotechniques paper

https://www.researchgate.net/publication/229326804_Thimerosal-Derived_Ethylmercury_Is_a_Mitochondrial_Toxin_in_Human_Astrocytes_Possible_Role_of_Fenton_Chemistry_in_the_Oxidation_and_Breakage_of_mtDNA?ev=prf_pub

https://www.researchgate.net/publication/45112747_Quantification_of_DNase_type_I_ends_DNase_type_II_ends_and_modified_bases_using_fluorescently_labeled_ddUTP_terminal_deoxynucleotidyl_transferase_and_formamidopyrimidine-DNA_glycosylase?fulltextDialog=true&ev=prf_pub_xaddfulltext

For DNA run ddTUNEL (TUNEL using 2', 3' ddUTP) to block all the 3'OH ends, then used the DNA repair enzyme Fpg to rip out all the damaged DNA bases, then add alkali phosphatase, then a second round of ddTUNEL. This shows the levels of Damaged DNA. Most is in the mitochondria.

Article Thimerosal-Derived Ethylmercury Is a Mitochondrial Toxin in ...

Article Quantification of DNase type I ends, DNase type II ends, and...

HI,

While all the suggestions listed above are standard methods, the most critical factor for success depends on finding the optimal condition right time point/concentration etc.

If you have access to flow cytometer then I would suggest using cellROX-Red or similar reagent (life tech) or dihydro ethidium or DCF-DA (is very tricky) for somewhat close to direct measure of ROS. Protein carbonylation is also fine, GSH is indirect measure for ROS, but standard as well. Don't rely on single method, always use more than one technique. Most importantly, you will need tie ROS to whatever effect that you are investigating, may be do a quick test with N-Acetyl Cysteine or non-thiol based butylated hydroxy inisole and determine whether ROS impacts your results. Proceed to measure ROS only if it makes sense after such experiments.

For DNA damage, Phospho-gamma-H2AX, p53-Ser15 and 7-OH-dG are early indicators, the first two by Western or fluorescent and the last one by fluoresence. Oxy-DNA assay kit for 7-OH-dG (EMD Bio/MERCK), works well because this is not an antibody based reagent. Depending on what you expect, use more than one method.

Having more than one method for ROS or DNA damage is important particularly when you opt for indirect methods, instead of HPLC or other direct assays.

All of the previous answers are applicable but if you want something quick and relatively simple why not use the good old TBARS assay. It measures products of lipid peroxidation such as malondialdehyde. Alternatively there are antibodies available which recognise MDA-derived protein modifications and can be used in an ELISA. Another type of modification also linked to oxidative stress is a dihydropyridine-type addict which are formed when acetaldehyde and MDA react together with cellular macromolecules.

Hi,

This is not a complete answer because most of those already given prvide correct information. However, most of those who referred to the markers of oxidized DNA, except. Ravi D. and Simon Worall, were incorrect referring to 8-hydroxyguano-

sine, while the correct names are 8-hydroxy (or 8-oxo)-2'-deoxyguanosine, which is a derivative of 2'-deoxyriboside, a component of DNA, while the former is a derivative of guanosine, a riboside that is a RNA component.

I think that detection of 8- hydroxy-2-deoxyguanosine as a marker of free radical DNA damage is good variant, there is ELISA kits/method for that. I'm not sure, but it is widespread method I think!? As I see you are biochemist so it will be easy to measure GSSG/GSH ratio (there is a kits for that, as Tomasz said).

One note for 8-OH-deoxyguanosine ELISA method, fallow accurately steps and keep out to not detach antibdies and other components in mixture while mixim them (use very low rpm on mixing plot).

I hope to be useful, GOOD LUCK!

Am I the only one under the impression, that this question turns into a general discussion on the methods to be used for directly or indirectly detecting and quantifying oxidative stress?

As nice and fruitful as such a discussion always is I offer the following suggestion:

Dear Adam Passmann, please tell us more about your actual experiment, hypothesis, idea and I am sure there will be several useful suggestions...

Thank you everyone, quite an overwhelming response! I have a lot to look into.

Andrew, I don't have the answer to a few of your questions but perhaps an explanation of what I'm doing will help.

To begin with I'm treating cells with H2O2 to induce an oxidative stress environment and see whether this continual insult affects the tumorigenicity of my cells. I have the tumorigenicity readouts well covered but I'm quite new to the oxidative stress world and need a readout to make sure I'm in fact creating the environment I believe I am and to see whether it is causing damage. Perhaps I'd be mostly looking for damage done to DNA as a result of the stress.

We also have a time-course of mouse tissue samples which are from mice placed on a carcinogenic diet. I'd like to be able to show an increase in oxidative stress/damage as a result of the diet as well.

I've had a go with a TBARS kit but I've run in to issues. I've also bought an antibody for 8OHG to stain paraffin slides but have had no luck getting it to work. Can anyone recommend a good one for mice? Perhaps I will try an ELIZA with it as the spec sheets states it is suitable for that too.

Many thanks,

Adam

Thanks for the detail Adam, welcome to the overwhelming arena of oxidative damage assessment ! So, if you are adding H2O2, it is likely that you will form a variety of radicals, by Fenton reaction kinetics and the action of peroxidases. Probably, however, you will form mostly hydroxyl radicals, which are highly reactive. Hydroxyl radicals will break DNA, so I would be looking at COMET assay and perhaps micronucleous assay. Both assays could be nicely matched to your experimental approach, since you are using cell culture.

You might want to have a look at protocols from the Wagner/Knasmueller group in Austria, who have done allot of this work.

Formation of micronuclei and other nuclear anomalies in exfoliated nasal and oral cells: Results of a human study with workers in a power plant processing poultry litter.

Georg G Wultsch, Armen A Nersesyan, ... Siegfried S Knasmueller

Int J Hyg Environ Health 216(1):82-7 (2013), PMID 22503715

Regarding your animal work, I would be targeting the liver (and lower intestine), since this is where the reactive forms of carcinogens are formed. Phase 1 detox of carcinogens will liberate reactive intermediates, which as others have indicated, would cause adduct formation and DNA damage. Perhaps the liver too would be a good site to assay GSH/GSSG ratio.

Hope this helps. We're over on the east coast, if you would like any more help, just find my address on the web (Griffith Uni).

Cheers,

Andrew

Thanks for your help Andrew and everyone else - time to go away and follow up on all these great suggestions.

you can also use the "Revlar" non invasive device that measures aldehydes in our breath, indication of the amount of oxidative stress...

http://revelartest.com/

hi,

the comet assay is a simple assay for detecting DNA damage .

for oxidative stress u can look into TBARS and important enzymes such as SOD and catalase

For checking the oxidative stress bio-markers in the mouse tissue (heart), we used the western blot to check 4-HNE, nitro-tyrosine level, they showed multiple bands. We also stained the tissue section with DCF,dihydroethidium (DHE). The results were very consistent with the functional data.

Adam,

As several researchers stated, depending on the direction you want to go next, you will need to choose the most appropriate technique combination.

The techniques i used and worked fine for me:

Proteins: Protein carbonylation status by DNPH antibody, S-Glutathionylation anti-GSSG immunoblotting.

Lipids: TBARS (MDA levels)

DNA and RNA: 8OHdG antibody (StressMarq) for ICC or FCM

also there are several techniques which are indirect but helpful for DNA compaction status such as Acridine Orange for FCM, mBBr (thiol status) for ICC or FCM.

Good luck

hi Adam

the best bio marker for detection of oxidative DNA damage is detection of 8 OHDG in biological fluids or in tissues by use of ELISA kit, but you must firstly make good idea about your research before starting the project.

best regards

i use HE and DCFH_DA to detect protein oxydation and superoxyde anion in the alive cells

Using comet assay method to determine DNA double strand breaks, 8-OHDG by ELISA method DNA methylation by PCR and FRAP assay for estimation of total antioxidant capacity should serve as good approaches for detection of DNA damage.