Dear Branko, I don't understand what you need to know about...

Season's Greetings

B

Dear Barbara,if you worked something similar you can sand me your protocol so I can compare with my. I have a problem with H2O2 because i make DNA damage with it. I order a new H2O2 and sometimes I again have a problem. I think in future I will use a adrenaline or tiroxine to couse a damage. What is your opinion about that problem? If I have a problem to make damage,I dont have a damage sample to compare my atioxidans effects and I waste money of my project.

Thank a lot !

Branko Kaitovic

Hi Branko, H2O2 is best known for its use as an oxidising agent but it decomposes, particularly under the influence of metal catalysts or in basic medium, into water and gaseous oxygen ( exothermic reaction), so you should take some precautions during handling and storage in order to preserve its stability, particularly:

- Storage at 4°C because a rise in temperature increases the decomposition rate of hydrogen peroxide.

- Metals catalyse its decomposition therefore the solution never has to come into contact with these materials, and impurities.

- Protect from light covering with aluminum film because light and solar radiations increase the decomposition rate of hydrogen peroxide solutions.

- the working solution should be freshly prepared from a stock H2O2 (8.8 M) the day of experiment.

I usually use a 200 μM H2O2 for 20’ a 4°C (atypical Comet assay on blood). Someone reports to treat cells with freshly prepared 10-100 μM hydrogen peroxide or 5-25 μM KMnO4 for 20 minutes at 4°C. Hydrogen peroxide or KMnO4 treatment will generate significant oxidative damage/DNA adducts in the majority of cells.

In order to verify if your solution has worked well you’d perform the FPg-Comet assay. When I use H2O2 for kinetic repair study I perform standard alkaline comet assay and I also note that the highest damage is observed 15’-20’ after H2O2 treatment (recovery time).

I don’t know your experimental model but you can find further information clicking on following links

http://www.sciencedirect.com/science/article/pii/S1383574207000385

http://cometassay.com/COmet Assay with DNA repair enzymes.pdf

Good luck

B

I agree with Barbara Tomasello. H2O2 can easily decompose itself without some care.

There is many ways to provoke DNA damage, an cheaper alternative to H2O2 is UV radiation, I found an article where's used natural sunlight (http://www.ncbi.nlm.nih.gov/pubmed/22998834).

In my lab, we have used UV-radiation (UV-B lamps), H2O2 and sodium nitroprusside (http://www.sciencedirect.com/science/article/pii/S0278691512008484), each one have a particular way to damage.

Furthermore, you can use ethyl methanesulfonate (EMS); ethyl nitrosourea (ENU); methyl methanesulfonate (MMS); N-nitrosodimethylamine (N-DMA); 1-nitrosopiperidine (http://mutage.oxfordjournals.org/content/18/1/45.full.pdf+html).

I hope I helped.

Good Luck!

Thaís.

Thanks a lot people. I think I will change my oxidative agens. I like KMnO4 because it is very chip. When I use H2O2 I put slides with it on ice for 15min. Maybe this is cause why H2O2 sometimes dont make a damage. If the recovery time is 20min and i put it on ice for 15min,repair mechanism can do something for that 5min. Am i correct ?

I have a full conditon of handling. My H2O2 is in refrigerator ( in dark ) ,with alu film and I prepare my working conc of 50microM every time before experiment so I dont know what can be problem... I will try with KMnO4... Which is the best conc of KMnO4 and how much I can put on slide ? I put 50microL of H2O2.

Thank a lot..!

Branko Kaitovic

Hi Branko, do you always use the same cellular model? Granted that the pre-analytical phase is performed well, the different level of DNA damage that you observe could depend on reducing environmental inside the cell responsible for neutralizing H2O2 (antioxidant molecules levels or activity of antioxidant enzymes such catalse,SOD, GPX ..). Moreover the level of DNA oxidation is ultimately controlled by the DNA repair process that in turn depends on repair systems efficiency of each cell line and/or on factors that inhibit or stimulate DNA repair.

Do you performe the cell treatment with H2O2 and antioxidant simultaneously?

Sorry I am inexperienced in KMnO4 treatment but I want to suggest you to make a titration in order to obtain the correct concentration that lets you to observe DNA damage.

... I'll reply to you about repair study soon

Every time I work pre/post treatment... For example for antioxidant conc C1 I do pre and post treatment. I first put cell sample mixet with 0,67% LMP on slides with 1% LMP. Than on one slide I put H2O2,and on second slide I put C1. Slide with H2O2 I put on ice for 15min,and side with C1 I put on incubation for 30min. After that on slide witch was in ice I put C1 and than this slide I put in incubation for 30 min,and on which first was on incubation i put H2O2 and I puth it than on ice for 15min. I hope you undestand me? I do two treatments but after first i change it. When I put cell sample and H2O2 i put that slide on ice to stop cellular repair mechanisms,than I can observe only an effects of my fungal extracts. And slides after fungal antioxidant I put on incubation that it can do effects like in bodies conditions. I hope you understand me.

Thank you !

Branko Kaitovic

Dear Branko, hoping I have understood you correctly I want to ask you: do you have a positive control (cells treated with H2O2 only)? This is very important to compare the damage among the different treatments. If you have problem with this sample, first you must resolve it and then you can continue. Thus you should : 1) do a titration by increasing H2O2 concentration, or 2) extend the H2O2 exposure time from 15 to 20’ for example. I tell you this since there isn’t a standard condition for all types of cells, so you have to find out it for your cellular model.

When I told you about my experience in assessing the ability of cells to repair H2O2 DNA damage I didn’t specify that I utillize both two approaches: the cellular repair assay and the in vitro assay. Obviously the choice depends on what I want to evaluate because the two approaches examine different aspects of repair.

With this experimental design you can't perform a common cellular repair assay to follow the rejoining of strand breaks through the cells’ own repair system because in this case comet assay is used to measure the damage remaining at each time-point after treating the cells with an appropriate genotoxic agent like H2O2. Instead for using in vitro assay you should modify everything and revolutionize the aim of your research.

In my opinion the best choice is the Comet modified version with repair enzyme (Fpf, Endo or both) in order to assess accurately the oxidative damage, even if this version is more expensive than standard alkaline one. As you know the oxidative lesions can be observed when the repair systems work, particularly Fpg/OGG1 or Endo III enzymes recognize and cut off the oxidized purines and pyrimidines respectively to produce apurinic sites. These, in turn, will be converted to strand breaks as a result of the endonuclease activity associated with these enzymes.

Considering that:

-you block their activity on ice (not for C1 post-treated sample, what’s the incubation temperature?)

-you analyze your samples immediately after H2O2 treatment (not for C1 post-treated sample, incubation for 30’)

the addition of these enzymes in vitro,after the initial lysis step, lets you to measure the total oxidative DNA damage, but if you want to use the classical version H2O2 must work well and then you could observe some differences.

Another idea could be to process all the samples at a recovery time of 30’ after H2O2 treatment:

1)Positive Control: 15’ H2O2 + 30’ incubation with 50 microlitres of PBS

2)C1 post-treatment: 15’ H2O2 + 30’ incubation with C1

3)C1 pre-treatment: 30’ incubation with C1+ 15’ H2O2 + 30’ incubation with 50 microlitres of PBS

In this case to perform one electrophoretic run you have to schedule the treatment: start with the treatment scheme n°3 and after 30’ with the other two

This is all…it’s your turn now

Good luck

B.

Ps: Sorry for my possible mistakes, I haven’t time to proofread it