Most of us use Ethidium Bromide in labs. Are there any microbes that are known to degrade EtBr? If not till now, is it possible to produce such an organism?
Hi
I saw this question quite late and hence my late response. It conists of 5 paragraphs:
1) EtBr is a salt! Salts are usuaily VERY stable (due to filling up the electronic demands of both participating ions)/
2. I have seen that many of the responses deal and use "I think"..."I believe"...
Well this is not a reliable or "good" science.
3. Indeed, EtBr absorbs UV light ( THIS IS its main virtue), but it cannot be used as a mean of "destroying" it. Bear in mind that a PROVEN and POTENT carcinogen as benzene also DOES absorb uv but it does NOT "destroy" it . Nevertheless, such a technique is so slow as to render such a "degragation " to impractical.
4. Here is "NEWS": EtBr wasNEVER proved to be carcinogenic but only "suspected" (!). There is NOT a SINGLE evidence that ANYBODY (from the tens of thousands ) scientists, laborarory workers, hospital/industrial doctors (most of the users!) showed to have EtBr originated cancer !
5. There is a "medium adsorbant" method in which the EtBr sokution is eluted through the adsorbant coloumn. Thus reducing the solution volume to 0.001% of the original solution volume.
6. All the above info reached my knowledge as a long proffesional carrear as a PhD (chemistry) & Hazardous Material Safety Officer and wrote a critical review (unpublished!) covering all known methods of "getting rid" of the "dangerous" EtBr.
when a mutant strain of Pseudomonas aeruginosa lacking its major multidrug efflux pump complex, MexAB-OprM, was incubated with 100 microM ethidium bromide, the fluorescence, caused by its binding to DNA following its entry into cells, decreased gradually. The intracellular ethidium bromide "induced" either its degradation or its efflux through the assembly of unknown efflux pumps.
Different strains of pseudomonoas are known to bioremediate the different toxic chemocals present in efluents of any industry. So it may be possible that some bacteria which have ability to remediate EtBr. We have to search them, no need to create a transgenic bacterium if we get some naturally occuring........
ethydium bromide is a one type of chemical which is used as a dye for agarose gel electrophoresis in PCR. it is a one type of dangerous dye that can cause the cancer. the microbes like pseudomonas species are known to degrade Etbr. but it does not produce any organism.
Sorry Bhumi I could not understand what are you trying to say..... specially the last line of your comment. please give me rferences of such paper showing that EtBr degreded by Pseudomonas..
I don't think such microbes is to be identified. But one can try Polcyclic aeromatic hydrocarbon degrading bacteria, it may have such capacity.
@Sanmugam, Babayan A, Nikaido H. (In Pseudomonas aeruginosa ethidium bromide does not induce its own degradation or the assembly of pumps involved in its efflux. Biochem Biophys Res Commun. 2004 Nov 19;324(3):1065-8.) showed, through quantitation of ethidium bromide by absorption spectroscopy, that the total amount of ethidium bromide in the system remained constant under those conditions, indicating the absence of its degradation. Furthermore, intracellular ethidium bromide kept increasing during the experiment, showing that the decrease of fluorescence was due to self-quenching, and that ethidium bromide is not pumped out by a newly assembled efflux system.
@ Bhumi, A proper reference would do great. EtBr has been used as plasmid curing agent beside its other known usage. I guess you meant that one cannot make EtBr-degrading bacteria but it can occur naturally (as you suggested Pseudomonas for example). Check out this link (http://en.wikipedia.org/wiki/Ethidium_bromide)
@Jaisingh, You are spot on saying that the solution may be available in the Nature. Pseudomonas have been known for doing wonders. But we cannot completely vote against engineered microbes. However, EtBr is not a threat that needs to be degraded so urgently and its disposal is quite achievable.
@ Devi Prasad, I think (might be wrong though) that there is still no such report on bacteria degrading EtBr. Answer to the second part of your question lies in itself. We do research to make things possible. So, there is always possibility to make such thing if that does not already exist. However, EtBr is not a threat that needs to be degraded so urgently and its disposal is quite achievable.
It is important to find out if there are organisms that can degrade ethidium bromide. There must be a distinction drawn between 'pumping out' and metabolic degradation of ethidium bromide. Although such lines of research are important, the data should be treated with utmost caution. A recent controversy about a bacteium that is thought to replace phosphate with arsenate is a good example of media 'hype' about scientific discoveries that are published in high impact journals without careful review. An article in EMBO Reports, (2012) 13 (4): 303 discusses this issue in detail.
Hi guys, I don't work with microorganisms, but I must to say that when I read the question I thought: "What the wreck is going on in his mind?" But after reading the answers I realized that that was a very good question and there were even nicer answers. Very helpful! I learned a lot! Congrats and thank you!
EtBr intercalates in DNA and this would arrest the activities involving the bacterial genome to a great extent. That is why EtBr arrests bacterial growth and cause their killing. Thus for plasmid curing one tries a number of different concentrations and from the highest concentration permitting the growth the cured strain is purified as plasmid replication is inhibitted at much lower concentration than what affects bacterial genome. One should look for a bacteria which produces an extracellularenzyme for easy degrdation of EtBr. The best available method is to treat with KMnO4 to oxidize EtBr to eliminate its genotoxicity.
Please treat ethidium bromide with sodium hypochlorite (bleach) before disposal
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Ethidium bromide may be a mutagen, carcinogen or teratogen although this depends on the organism and the conditions. ethidium bromide is aromatic. Its core heterocyclicmoiety is generically known as a phenanthridine, an isomer of which is the fluorescent dyeacridine. 3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide (IUPAC name). Ethidium bromide is thought to act as a mutagen because it intercalates double strandedDNA (i.e., inserts itself between the strands), deforming the DNA. Ethidium bromide can be degraded chemically, or collected and incinerated. It is common for ethidium bromide waste below a mandated concentration to be disposed of normally (e.g., pouring it down a drain). A common practice is to treat ethidium bromide with sodium hypochlorite (bleach) before disposal. EtBr can be removed from solutions with activated charcoal or amberlite ion exchange resin. Various commercial products are available for this use. Ethidium bromide in water, TBE buffer, Mops buffer, and cesium chloride solution may be completely degraded by reaction with sodium nitrite and hypophosphorous acid. Only nonmutagenic reaction mixtures were produced. Destruction was >99.8% in all cases; the limit of detection was 0.5 μg ethidium bromide per milliliter of solution. Ethidium bromide also may be removed completely from the above solutions by using Amberlite XAD-16 resin. The limit of detection was 0.05 μg ethidium bromide per milliliter of solution (0.27 μg/ml when cesium chloride solution was used).
My Cute, I think that indeed there is an organism that use carbons of EtBr as energy source but the men dont know it.
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EtBr is an interclating agent, may be used for mutation studies as well as curing studies. During cduring experiments, I found certain strains which were resistant to EtBr and could not be cured. So there may be a chance that some bacteria may also degrade EtBr, but since the quantity of EtBr is negligible in environment, so to my information no particular strain is found yet.
I am not sure whether it is possible to engineer a microbe with ability to metabolize EtBr if we take into account aforementioned problems (intercalation in DNA and its consequences). However, one alternative may be found amongst oxidative enzymes used in phenol and dye removal (isolated from various plants and microbes).
@Jai Ghosh..Please don't take away the credit from the original researchers (Xu et al. [Biochem. Biophys. Res. Commun. 305 (2003) 941]) who proposed that the intracellular ethidium bromide "induced" either its degradation or its efflux through the assembly of unknown efflux pumps. In 2004 this claim was further clarified (rather opposed and rightly so) by Babayan A, Nikaido H. [Biochem Biophys Res Commun. 2004 Nov 19;324(3):1065-8.]. Please read the original articles (or at least the abstracts).
I would consider metabolic degradation of EtBr over efflux pumps here since Mr. Prasad is talking about degradation, not just removal. Break the organic chemical down to its components/ to a lesser complex form or transform - that's degradation.
@Sundararajan Jayaraman, Very good suggestion and you are spot on in referring to the "arsenate eating bacteria".
@Jayasree, Its a very old paper from 1987 (?) that you have taken your material from. Mr. Prasad has been looking for information on bacteria that can degrade EtBr, not something else.
@Krishnan and Azra, nice inputs.
I guess Mr. Prasad can start with PAH-degraders/crude oil degraders. Try enrichment culture method to isolate EtBr degraders, if any. Another place to look for is laboratory/hospital/chemical/refinery waste. I once isolated a Pseudomonas from a port sediment to check Hg detoxification and ultimately found that bug to be a multi-resistant bacterium that could degrade PCBs, TBT, etc. You would probably need not engineer any microbe (unless you want to speed/scale up the degradation process) as you might find them in nature. Keep in mind that being chemical-resistant does not always mean chemical-degrader. All the very best!
i think that EtBr can be removed chemically from solutions with activated charcoal or amberlite ion exchange resin. Various commercial products are available for this use but i am not sure if there are any micrbes for this way
@Mr.shanmugam: Thats really a new piece of information. One more reason for the decrease in fluorescence could be because the bacteria could have dispersed EtBr. Could you privide any research article or substantiate the fact you mentioned?
@Mr.Jaysankar: Could you tell about self-quenching? and, regarding EtBr being a threat, may be it is not now. But even today we use chemicals to degrade it. I was wondering if a microbe could degrade it or use it as a source, it would be less of a strain finding out ways to dispose it, and also a benefit obtained from "waste".
@Mr.Krishnan Nair: I was about ask about EtBr's effect on the microbe. If we are looking at an extracellular enzyme, is it possible to restrict its entry into the cell?
@Ms.Hana Karimi: are you sure Etbr is degraded by UV light? I had been thinking the fluoroscence is only temporary.
And all those who have highlighted the chemicals used to degrade EtBr, can anyone of them be produced by bacteria?
Ethidium Bromide Degradation
(Kaufman, J.A., ed., 1990, Waste Disposal in Academic Institutions, Lewis Publishers, p. 127.)
If extraction is not possible the following EtBr degredation procedure can be used. This procedure is
more complicated and should be performed in a fume hood. Contact UOEHSM for additional information
regarding this procedure.
Procedure:
1) Dilute solutions of EtBr to a final concentration of less than or equal to 0.034% w/v (34mg EtBr/
100 ml solution).
2) Add 10 ml fresh bleach for every 1 mg EtBr (bleach deteriorates upon exposure to air).
3) Stir the mix continuously for four hours or overnight.
4) Test the final solution with a UV light to ascertain that the EtBr is destroyed.
5) Of the final solution, drain-dispose one part solution with 20 parts tap water
You can go through this side http://web.princeton.edu/sites/ehs/index.html it will provide alot of lab safty
I was able to get some information on the treatment of Etbr at this site www.northwestern.edu/research-safety/chem/ethid2.htm and followed the Lunn and Sansone 1987, Analytical Biochemistry 162,453-458 procedure.
Eukaryotic organisms can be attempted because the degrading ability (if available) resides in cytoplasm. The DNA will not be in direct contact during Interphase (synchronous culture of fungi in interphase can be tested for this ability). Prokaryotes may have problems of mutation as their DNA is not protected in membrane bound structure.
During 1950,s, This EtBr is Commonly used by veterinary doctors to treat trypanosomosis in cattle, a disease caused by Trypanaosomes. But, later this became impractical due to the high antibiotic resistance.
This shows that there will be some bacteria that degrade EtBr. If we observe the chemical structure of EtBr, it is most likely the phenanthrine ring with its attachments. So, it is possible to grow some cultures of bacteria that show resistance against the EtBr. This may solve the question of proper disposal and reducing the hazardous effects of EtBr.
All the Best for your work.
Yes it is possible, organisms can adapt and utilise Etbr. It is time consuming but efficient organisms can be developed with adaptations.
I think, Uv light only excite the molecules, it is non ionizing radiation so can not degrade ethBr. I am 100% sure on that.
Second, Bacteria can do transform or degrade or reduced or convert into salts and ppt it from the solution. Some body should start priliminary work on it. Microbes can do it.
@Mr Jaysankar: thank you for the valuable suggestion. i'll study more about what you said.
@Mr.Brijesh: i agree. but in that case, how is EtBr a mutagen or carcinogen for humans?
@Mr. Kesav: that was interesting. could you provide any references for the same?
Although out of the topic, I'd like to clear ; have studies confirmed if EtBr is mutagenic or carcinogenic for humans? It's found so for bacteria, but I doubt if its ' confirmed ' on human too.
prof. A.Mahasneh .Univ. of Jordan
yes, there would be some bacteria that is capable of degrading ETBr., I am sure a well designed screening program using soil samples from highly contaminated sites would yield some bacterial isolates that do the job.
@Heshani: yes, i agree. There is no such proof. I take back the words mutagen or carcinogen. But i believe it is toxic at high concentrations. Probably someone working with mammalian or other eukaryotic cell cultures can try it out and find out the effect of EtBr.
None that I know of but it is possible to isolate a microbe that can degrade EtBr. I learned from basic microbiology that there is no element in the crust of the earth which cannot be degraded by a some particular microbe so it is possible. And I perfectly agree with Prof Mahasneh, screening microbes from EtBr contaminated soil sites can be a useful lead. I need not mention the dangers associated with working with EtBr so careful consideration must taken before venturing into such studies, though I will write off the potential of such a study if it is successful.
EtBr is a potential carcinogen but we cannot overlook the potential of a study to find microbes that can degrade it. There has been instances of spills in labs which needed to be contained. I spilled EtBr on my lab coat once and never used the lab coat again. It is a carcinogen yes, but we can look at the bright side
To my knowledge, EtBr degrading microbes not reported. If there any organism degrading EtBr, definitely you can report it.
As per my knowledge such organisms are not reported and if any then kindly inform with reference
I stopped using Ethidium Bromide long time ago (15 years). Instead, fresh SYBR Green (Molecular Probes) diluted 100x times and added to each DNA sample before loading to electrophoresis gel works much better (higher sensitivity) and it is less toxic. By the way, SYBR Green degrades rapidly in water solutions. I guess, you may degrade EtBr by the use of bleach or UV or combination of both. However, I do not see a point to use dangerous substances if alternative approaches have been used for many years.
Hope you may not able to find a organism which can easily degrade the EtBr. But you can induce microbes to withstand some level of EtBr in their growing media.
Is it true that EtBr can be degraded by UV light?
@Prof. Zoran Vujcic: how can we ensure whether EtBr is degraded or not, if UV does not actually indicate its presence?
@Devi Prasad, Sorry for being late (courtesy, some boring meetings and discussions for the whole day) in replying to your question. Hope u get to read all of these for free (except the 2nd link).
1. http://www.prozyme.com/documents/tnpj100_20.pdf (self-quenching)
2. http://www.sciencedirect.com/science/article/pii/S0006291X04022041 (look into the figures available and get the full paper if possible).
3. Bacterial efflux pump inhibitors from natural sources (http://jac.oxfordjournals.org/content/59/6/1247.full.pdf+html)
4. Might be interesting to you: Ethidium bromide efflux by Salmonella: modulation by metabolic energy, pH, ions and phenothiazines. International Journal of Antimicrobial Agents
Volume 38, Issue 2, August 2011, Pages 140–145.
@Devi Prasad,
One more good paper (read this one and the reference #38 therein)
Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution (Photochemistry and Photobiology, 2001, 73(6): 585–599).
The paper submitted by seema T is beneficial for that study
I have seen this paper, it is more seutable
Thanks
Whatever you do, UV ligth is absorbed by EthBr and can destroy the molecule especially in presence of scavenger molecules, e.g. DNA when ii is intercalated with EthBr, BUT in older papers (there are a few, I remember working out a brief information about that for Biologists in the 80ies) the described degradation products where believed to be even more dangerous.
...and by the way, anything binding to DNA is hazardous, so is SYBR!
@Mr.Jaysankar: Thank you very much for the links. I will surely read them.
@Mr.Michael: what was EtBr degraded with, to produce more harmful products? only UV?
in some pleaces is used to degradation of EthBr the NaCl or commond salt and is exposed to UV light or directly on a sun (safe) place.
hello
EthBr is degraded with UV, light and some solutions.If we want to use microbes(it is good idea) they must be resistant to mutations
It is quite unlikely that there maybe some microbe that will degrade EtBr. I agree with Mojtaba Mohseni and Prof Vujcic that either the microbe has to be resistant to mutations which is very unlikely and also experimenting whether or not the microbe can degrade EtBr can be very dangerous. Decontamination procedures and biosafety issues of mutated microbes and microbes containing EtBr incorporated within their genome must be considered to avoid the mutated microbe causing problems within the community or soil after disposal.
However, your idea of looking out to use microbes is really good ! Since some of us use EtBr during gel preparation and some of us use it for staining the gel after the electrophoresis you can look at which stage you might want to experiment the microbe. Either one that acts on the gel which is most likely not practical and the only other option left would be to try decontaminate the EtBr staining solution. You must also figure out a way to check if the EtBr present in the solution of gel that you want to decontaminate is completely free of EtBr after treating with microbe.
EtBr a mutagen and polycyclic aromatic hudrocarbon, is commonly used here in Nepal by us and carefully handled. the query regarding the degradation of it is interesting. I went through some reviews and found the following papers are helpful. these papers are regarding the Polycyclic aromatic hydrocarbon degradation rather than EtBr. These papers are easily accesible.
http://aem.asm.org/content/67/6/2683.short
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1999.tb13510.x/full
http://aem.asm.org/content/71/7/3797.short
http://aem.asm.org/content/42/4/737.short
I still get amazed by the work of Prof. Chakroborty, who patented for Pseudomonas sp. for its capacity for degrading the petroleum products. He isolated the species and made some genetic changes. However i believe we need to explore ,more and go deep to unravel more microbes and their potential uses.
Good luck for the study
EtBr is a potent mutagen and carcinogen. Handle it carefully. It can cross blood-brain barrier and so if ingested there is chance that it can even go to brain. All gels and solutions containing EtBr should be treated with oxidizing agent to degrade it by oxidation. The best is to treat with KMnO4 not with hypochlorite. There were studies on the degradtion and efficacy of degradation products to act as mutagens/carcinogens in 80s. It was found that time that treating with KMnO4 is the best way to inactivate it.
Now, regarding microbial degradation. I feel it is hard as EtBr intercalates DNA and interfere with its functions. The extend of inhibition of DNA functions depends on the amount bound per total genome DNA . This in a plasmid carrying strain, the plasmid replication is first interfered as the quantity of EtBr per genome is higher for plasmid than for the bacterial genome and that is why EtBr is used for curing plasmids. A bacteria in which the genome exists in multiple compartmens or partitioned in several replicons could be ideal candidate to look for as this will have higher resistance to the intercalating dye. Apart fron this, it should have genes encoding enzymes for the degradation of polycyclic aromatic hydrocarbons like in psuedomonas. May be one can introduce this by gene manupulations.
Good luck in the study!.
Hi
I saw this question quite late and hence my late response. It conists of 5 paragraphs:
1) EtBr is a salt! Salts are usuaily VERY stable (due to filling up the electronic demands of both participating ions)/
2. I have seen that many of the responses deal and use "I think"..."I believe"...
Well this is not a reliable or "good" science.
3. Indeed, EtBr absorbs UV light ( THIS IS its main virtue), but it cannot be used as a mean of "destroying" it. Bear in mind that a PROVEN and POTENT carcinogen as benzene also DOES absorb uv but it does NOT "destroy" it . Nevertheless, such a technique is so slow as to render such a "degragation " to impractical.
4. Here is "NEWS": EtBr wasNEVER proved to be carcinogenic but only "suspected" (!). There is NOT a SINGLE evidence that ANYBODY (from the tens of thousands ) scientists, laborarory workers, hospital/industrial doctors (most of the users!) showed to have EtBr originated cancer !
5. There is a "medium adsorbant" method in which the EtBr sokution is eluted through the adsorbant coloumn. Thus reducing the solution volume to 0.001% of the original solution volume.
6. All the above info reached my knowledge as a long proffesional carrear as a PhD (chemistry) & Hazardous Material Safety Officer and wrote a critical review (unpublished!) covering all known methods of "getting rid" of the "dangerous" EtBr.
@Dr. Eliahu Nov, Thank you so much for your inputs. However, with due respect to your PhD (chemistry) & role as an HMSO, I beg to differ/disagree with some of your points (paragraphs...if you would prefer so).
1. A toxic chemical needs to be tackled irrespective of its nature....Tougher/ more complex the structure is; harder will be the effort to break it down or at least to reduce its toxic effect. So, EtBr might also cause enough trouble, if not already, unless it is handled carefully. We had enough lessons from DDT, PCBs, TBT, U, Hg, Pb, etc.
2. I don't get the idea of this paragraph. Scientists believe/trust/suppose/propose/think and it is based on certain principles/laws/facts/phenomenon etc. Hypothesis precedes theory and that also comes from thinking/belief, etc. If that is against so-called good/reliable science then how would we explain Higgs-Boson particle (today also they are not sure at the CERN)? But they believe that it exists there. This idea/thought came way back in 1964. Thankfully Prof. Higgs won't read this thread I guess (:-)).
3. It is not necessary that UV will degrade EtBr just because the later absorbs UV, Bioremediation (also biodegradation in case of organic chemicals) is indeed a slow process compared to its alternative chemical or physical remediation. But it is used because of its being less expensive and more eco-friendly. So, what seems to be so-called impractical, can make sense in the long run. Moreover, more than one type of treatments can be used hand in hand to get more effective result (though not always) at lesser cost.
4. The NEWS that EtBr is not a carcinogen...is not beyond debate. We can't discard this claim just because there have been not enough studies. It has been claimed to be potential carcinogen because of its (EtBr metabolites) capability to cause mutation and DNA-intercalating nature. Should we put a hold on its careful usage or research on its effective removal until it is PROVED/DISPROVED to be a carcinogen?? All potential toxic chemicals, especially carcinogens, should be paid special attention. Please have a look at some more literature, if you have time. I can't recall many at this time. Check out the followings for the time being.
a. Roy Chowdhury A, Bakshi R, Wang J, Yildirir G, Liu B, et al. (2010) The Killing of African Trypanosomes by Ethidium Bromide. PLoS Pathog 6(12): e1001226. doi:10.1371/journal.ppat.1001226.
b. Executive Summary Ethidium Bromide. http://ntp.niehs.nih.gov/?objectid=6F5F63F6-F1F6-975E-79965F7EE68AE7C0.
Several top class laboratories (Univ. of Hawaii, York, Woods Hole Oceanographic Institution, Uppsala University as well as the labs I have worked with, and too many to include here) indicate EtBr to be a potent carcinogen. So, it is wiser to take care against this chemical rather than using it casually. Moreover, we can't use human as guinea-pig to prove whether EtBr would cause cancer with 100% certainty. I, specially being younger and inexperienced, expect more cautious explanation from an experienced chemist and HMSO like you. Or else it sends wrong message.
5. There are several treatments available for EtBr. But they are not biological or inexpensive. Mr. Devi Prasad's inquiry to find EtBr-degrading bacteria or the answers/discussions offered by others, thus are not bad/unreliable science. Those adsorbent rasins also need to find a trash.
6. Your 6th paragraph (to support 1-5?) does not add much to our knowledge. I would appreciate if you kindly make your unpublished work available.
I apologize to other respected members/readers for this long reply. I am trying to help myself gain some more useful knowledge....:-)
Even though most people like myself do not respond to the questions but do like to know the answer if any one has it. Most of the times, the issues posed here are important. If anyone has a valid point to make, please make it known clearly. Take time to write it though. Like many, I had tough time following some of the responses since some times they are not clear. Most people do not have a lot of time to read extensive passages. So, make it brief and to the point. This is not a social media to casually answer/question back and forth. Let us also not address anyone personally. This way, we can listen to what anyone says if he/she knows for a fact. Also let us avoid media hypes such as bacteria eating arsenic, ethidium bromide etc. We have too many of those!
yes, many years ago my group found a filamentous fungus that could grow on high concentrations of EB . The work has not been published, but I am interested in following up the work further by developing a bioremediation program of research, and applying for a research grant. Is there any interest in collaborations? Funding agencies?
Would like to have your interest!
This fungal isolate was able to grow on EtBr up to 4 mg/mL concentrations. The work was done in 2003, and has not been further developed. This isolate has been used on a large scale to clean up of soil contaminated with high levels of polyaromatic hydrocarbons (PAH). It could have potential for bioremediation of EtBr !?
I would like to emphasize that my filamentous fungus could grown grew on 10 mM EtBr (4 g/L). This was the high concentration tested. Maybe it can utilize more? How many microbes are able to do this?
If you have a microbe capable of utilizing > 4g/L, then please let the scientific community know.
My response to Jaysankar De will be very brief. Introducing myself as PhD and safety officer was to emphesze my "humble" knowledge.
As for your response as to "believes" as regular tools for scientific knowledge I do Not think there is any need to respond as the toxicity of EB is not a matter of "assumption" but must be based on EXPERIENCE & KNOWLEDGE per facts. Your reference only solidified my answers.
Anyway It goes down to semantics and NOT facts...
Seems that some of our respected experienced seniors value experience too much over logic....Hope this cannot take away enthusiasm and curiosity from the young minds. Lets have respect for all..
I have never heard about a micro organism to degrade ethidium bromid (I'm also not a microbiologist)
As a biotechnologist, I would select diverse organism strain, and make them grow on EtBr medium in flask, with increased concentration on EtBr, to play with adaptive evolution.
after several runs, you would get at least EtBr tolerent organisms... test them for EtBr destruction... If there is no, at least you have a host for expressing an enzyme destructing EtBr. and then... we are out of my field.
Dear Gentleman, Sirs, Professors, ...
A very tough brainstorming is swingin out here.
To my litle knowladge and experience (forgive me/ or just inform me about the missing info that I write here, pls)
First I may say that EtBr & Hypochloride solution does not show any fluorescent under UV light; that is either EtBr might be degraded or hypochloride or any adduct generated through the reaction of both may act as a quencher.
Second issue is, there should be at least a combination of microbial strains or just one strain that can degrade this EtBr. thing. My porposed mechanism is first microorgansm should make an adduct of EtBr. most probably a Methyl adduct of EtBr. then this methyl adduct should be activated in to Methanol and then Acid form in order to break the stable cyclic nature of the ethidium bromide. I should note here that there exists enzymes called Mixed Function Oxidases, these enzyems probably can handle EtBr.
And the last thing, the mutation and carcinogenic effect of EtBr. should not be direct such as chemical modification of DNA but rather it should be indirect through DNA related enzymatic reactions inside the cell since the molecule is stated to be stable. So the target microorganisms degreding EtBr. should at least have these mixed function oxidasess , should have high rate of replication and should show least rate of mutation.
You should be screening many microorganisms not only bacteria or achea, and for screening you should be avare of what u are looking for; an enzyme, a tolerant phenotype, a couple of microorganisms workn in accordance, etc. for degrading EtBr.
Thanks in advance for your poroper feed-backs.
I am extremely thankful to all those who have posted to this question. I am a final year B.Tech student, and this EtBr idea just struck me from what I see in my labs everytime. I would like to try things out, but I do not have the facilities now. I will definitely consider all your suggestions and ideas and work it out in the future. I shall be happier if we can discuss about more inputs regarding this.
Many say that EtBr is not proven to be a mutagen. Why can't we try it out on animal/ mammalian cell cultures in the lab? Introduce EtBr in different concentrations to the cultures and observe changes (external/genetic)?
Lets return to the basic question of Devi Prasad, and that is to find a microbe that can degrade EtBr. Microbes normally won't do this job, but it has been observed that since it can intercalate with DNA, the organisms simply cleave the part of the DNA and carry out the damage-repair work, slowly throwing out the DNA-EtBr complex which can not be detected at times due to its small quantity which is outside the range of normal measurement. I am saying this because, all of whom who has claimed to have brought about microbial degradation of this compound have never showed the metabolic route or the intermediates of metabolism of degradation. In fact there are hardly any microbe known to possess that much energy to saturate the double bond of ethylene. I hope I have answered your question.
I have never heard before if their is any microbe that degrade EtBr. But if anyone knows if exists any potencial microbe with this activity...it will be very good to apply in the labs activities. But, I may agree with Jai Ghosh that microbes probally can not realize this kind of activity due to the amount of energy necessary to saturate the double band of ethylene.
Please check these 2 websites (just for example) if someone wants to know the pathway (s) of biodegradation;
1. http://umbbd.ethz.ch/ (Univ. of Minnesota database)
2. http://www.genome.jp/kegg/pathway.html (KEGG PATHWAY database)
^ sir, I think you have mistaken Ethylene Bromide for Ethidium Bromide. As far as microbes are concerned, I guess many cyclic aromatic compounds have been degraded, with their pathways elucidated.
Biodegradation/bioremediation studies (interdisciplinary science) involve at least microbiologist, biochemist/chemist, geologist/hydrologist, molecular biologist....So microbiology (fortunately I try to learn a little bit as a microbiologist) is an integral part of that kind of study....In a team environment we do not know everything alone...but the team as a whole knows how to explain the story. This applies to any inter/multi-disciplinary research.
Regards,
Jay
Devi Prasad, Please don't guess [I had an argument (which most of you considered probably as arrogance) with one of our respected seniors to justify using/not using this word] in this case. There are several reports on such biodegradation (just look at the two sites I provided in continuation to Jai Ghosh's comment). I wish you get enough opportunity in future to pursue research on this topic. I look forward to see your results (thesis/publication/report). All the very best!
Thank you very much sirs, for your wishes. I shall draw inspiration from all of you and pursue quality research.
And here, my question would be, if tried on animal/mammalian cell cultures, can the mutagenicity of EtBr be proved?
There is some models but I do not remember them.... Look for a publication written by Debian (unviversity of Calais) about mutagenicity of PAH (anthacene or benzopyrene) of mychorizal fungi (Glomus) ... If you cannot find a relevent paper try to ask her for her master thesis... there was several methods for that....
I would like to add something to Eliahus comment on the safety of EtBr.
A short research on Pubchem gives these toxicity excerpts (read more on http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=14710&loc=ec_rcs#x332)
/GENOTOXICITY/ The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2 mg/mL, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with gamma-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation. [Belyaev IY et al; Biochim Biophys Acta 1428 (2-3): 348-56 (1999)] PubMed Abstract
/GENOTOXICITY/ /Ethidium bromide/ appears to affect segregation in yeast during meiosis. Ethidium bromide forms a highly fluorescent complex with native DNA by intercalation between base-pairs.
/GENOTOXICITY/ Ethidium bromide, a frame-shift mutagen in Salmonella typhimurium strains with metabolic activation and ... is also mutagenic to yeast (Saccharomyces cerevisiae) producing respiratory deficient colonies (petite) by its effects on mitochondrial DNA.
Further, heating EtBr will release toxic hydrogen bromide and NO fumes.
Maybe there are no known people that have cancer from EtBr. But one should consider that the cancerogenous potenital of any novel pharmaceutical substance and all bulk chemicals (REACH) is, among other tests, estimated with the bacterial AMES test. As you can see from the PubChem entry, EtBr is a framshift mutagen in Salmonella, which are also used in the AMES test. Therefore I think it viable to consider EtBr mutagenic, if not cancerogenic. So even if there is no proof of cancer in human originating from EtBr, it should not be deposited in environmental waterways because no one can know what it will do in the wild. Further, EtBr is membrane permeable and a DNA intercalator. This alone should alert everybody.
Actually, here in Germany it is treated as toxic waste, which leads to high disposal costs. Among the safety concerns this was one of the main reasons we switched from EtBr to GelRed which is more expensive than EtBr at the first look (when comparing € / mg), but has a very long reach (the 10.000x stock can be diluted 1:1000 and added directly to the sample instead of the whole gel, gives beautiful black background and detects between 0.1 and 100 ng DNA / 3 mm lane).
Sorry for this long and a bit off-topic posting, but i couldn't let the post of Eliahu go uncommented.
Stephan, thanks for the posting. I learned some new facts. I tried to respond (July 4, 2012) to the same post you have referred to. Surprisingly, I found that many of the respected members quite just got carried away and it appeared to me suddenly that someone's experience/knowledge posed larger than the question itself. I also had the same experience with EtBr while working in Germany (at the GBF, Braunschweig; currently HZI) and one day I literally got scolded by my then supervisor for just mixing those blue (for EtBr work) with white ones:-)..Once again thanks for providing this information.
We were told for a long time (in my case >30 years) that ethidium bromide is a DNA binding dye, fluoresces brightly and a potential carcinogen. So, it is required to be handled with caution, such as wearing gloves when handling and be properly disposed. We do not even touch the computer key board hooked to the gel imager with bear hand. I am not clear why some think ethidium bromide is 'safe' to handle. Thanks to Stephen Lorenz for a detailed response and the info on an alternative for ethidium bromide-GelRed. We will explore that possibility. It is great that we get very useful information from this blog some times.
Dr. Jayaraman, glad to see your reply here. I have been following this thread since the beginning as this same question of Devi Prasad came in mind too when I started using EtBr (in India) some 10 yrs back. But I don't understand why your latest comments are contradictory to your last post (July 5). I had reacted to one of our members owing to of my so-called inexperience or enthusiasm to learn new things. Your previous post told us to avoid hypes like As or EtBr-degrading bacteria and today after Stephan's post you changed your stand. Could you explain this change since it makes sense for me to know due to your vast experience. I appreciate your time.
I stand by the point that I made earlier about 'hypes' in science. We should avoid claims such as bacteria eating toxic subtances without any solid evidence. I also stand by the point that I made recently about ethidium bromide is not safe to handle and should be avoided. I support the idea that we should use alternative safer methods of visualizing DNA on a gel. These two are distinct entities and should not be misunderstood. I hope it is clear that I am not contracting myself.
I (we all should) agree with the point on avoiding hypes or safe handling of toxic chemicals. EtBr has been known for being toxic since long time. Gel-red or Cyber green (for dsDNA) are in use for quite sometime now. My argument was on discarding of EtBr as a potential mutagen (carcinogen at extreme case) by one of our respected seniors since it somewhat diluted the discussion. Many of our members voted up that answer. We need more thoughtful reactions/replies/discussions from our seniors to teach ourselves. That has been my point beside the scientific aspects of the discussion. Thanks!
your question has nothing to do with EtBr - please open a new thread for your question.
The safety regulation in most countries require to filtrate any EtBr containing solution through activated carbon to remove it. Activated carbon has a high adsorption capacity. It later can safely disposed as solid toxic waste by the toxic waste department (most University and companies have this).
On the other hand, you asked about obtaining a microorganism able to degrade EtBr. I am not aware of one but you can spend some time on searching on the internet --a fast crude one did ot yield reliable INFO etc, But I believe in the omnipotence of microorganisms and there should be some around you can isolate. You could use sludge from your the aeration pond or anaerobic sludge from the anaerobic digestor of your local sewage plant. I would start with the later one and do anaerobic incubations at various pH and slowly increasing conc of EtBr in the presence of some YE (anaerobically; or acetate for aerobic incubations), since Br is usually a great leaving ligand for reductive dehalogenation (There is a huge literature out there on reductive dehalogenation of aliphatic and aromatic compounds, Anaerobes even get energy from the reductive dehalogenation (e.g., for a start: M. Mackiewicz and J. Wiegel. 1998. Comparison of energy and growth yields for Desulfitobacterium dehalogenans when utilizing chlorophenol and various traditional electron acceptors. Appl. Environ. Microbiol. 64:352-355). the other option is using relative UV resistant phototrophs. (e.g. we have some thermopiic halophilic alkaliphiles which are nearly totally resitant to highUV radiation. Other option is using the EtBr loaded carbon( after using the carbon for the adsorption of the EtBr) as a column fermenter for enrichments, eiher as batch or a semicontinous column fermenter. Such enrichments are easy to be done with relative little time and costs involved, i.e., as a side project. Testing would be done using aliquotes of your broth on standardized solution for quantification. Surely the final study of the degradation mechanisms and demonstration of effectiviity requires more involvement. I have no lab anymore, otherwise I would do it myself. good luck if I could entice you to try it (or anyone else reading this)
Hi there, I just observed bacterial growth on ethidium-br used staining solution. I must say they are rather abundant. So I plated them on H2O agar plates and I got bacterail colonies also here, also after 2nd plating. I am starting to believe that those bacteria might use Et.Br as carbon source. Does any of you have had a similar experience?
I would appreciate any response on this.
thanks.
Maria, congrats! I am glad to hear that at last someone reports bugs that grow on EtBr. This is probably why the question was raised by Devi at the first instance. Regular instinct says it could be there somewhere as microbes grow almost in every environment. Now you can look for the possible degradation product and pathway, if EtBr is being used as carbon source. Check whether the bug is chemolithotroph (it won't need EtBr then) or not. I would have loved to go back to the bench and work on it. Take the leads from the previous post by Juergen Wiegel and keep us updated.
Maria, I am glad someone is trying to get such a degrader. Please be extra cautious with your claim. So far-as you probably know yourself despite your exiting blog- you only can say you have a culture able to grow in the presence of EtBr. There are quite a lot of bacteria and fungi which are (facultative and oblicately) oligotrophs. More than 30 years ago I had cultures growing on the gas phase of beewax, which I used for greasing the lid of the desiccator (standard procedure at that time since the proper silicon grease. etc of today were not common neither very cheap,I realized it when there was no gas utilization of H2 /CO2 as it should have been for the desired chemolithoautotroph (Knallgas bacterium)..
But your observation are a greaat start (and as J.De I wish I could go back into the lab and elucidate the degradation path, but I closed my laqbs two years ago.. I hope you truly have indeed a degrader and like to encourage you to keep on going to do the mass bance of EtBr., I would have expected that the first EtBr-"degrader" / converter would be an anaerobic debrominator.
For the bacterial degraders of EtBr, you have to do more tests to verify that. For example, the growth of bacteria might be due to the TAE/TBE in staining buffer that provide carbon source for the bacteria. I agree with Juergen. The only conclusion so far is that those bacteria are resistant to EtBr or the mutation caused by EtBr, which might also be very interesting. I think it better to grow those bacteria on Etbr as a sole carbon source, both in liquid and agar plate. And keep autotrophic bacteria in mind as a possibility.
Ms.Maria, it is great to hear about a bacteria that is resistant to EtBr. Owing to the mutagenic effects of EtBr, you probably could try growing them for a few more generations, to ensure if they are actually resistant to the chemical. And you could check growth with higher concentrations of the same. Does the media after growth, show fluorescence in UV (could be an indicator of EtBr being "consumed")? Also, were you able to recognise the organism or its nature (gram + / gram -, etc.)?
Thank you all for your reaction on my EtBR-affaire!
Answ-to Daniel Deng: the EtBr I use for staining gel is H2O + EtBr up to 0,5ppm. The TAE/TBE is in the DNA-agarose gel therefore the buffer contribution as C-source might well be possible but is very little.
When I 1st saw them under the microscope I plated them out on H2O-agar plate (this medium contains only salts: NaCL/P/N). Bacterila colonies were visible meaning that they are alive. But definitely this is not yet an evidence. It only says that these bacteria whoever they are, seem to grow under this condition. Therefore I transferred them on a new H2O-agar plate and they showed up again.
Now I am setting up a more rigorous experiment in 3x w/ and w/o EtBr in the same medium w/o agar. I am planning to start with a concentration of 1ppm EtBr.
But I am still puzzled on finding a reliable method to measure EtBr degradation. Besides this, I am also a bit concerned on how to carry out the experiments safely for me and especially for the lab. At the moment I do the tests in the EtBr staining room.
Ms. Maria Briglia. I once tried using a protease bacteria named Serratia marcescens streak culture by using EtBr 50microL in 100mL of nutrient broth and i dono how it is possible that the bacteria started to grow on it unlike other bacteria i used E.coli as contol. But E.coli did not grow on EtBr medium. i used only 50microL did not check for other concentrations. if possible u can try out protease degrading bacteria. i did this experiment in 2011 to develop my microbiology skill. And the S.marcescens was flourishing in that medium. unfortunately i think i dont have any pictures of it. if possible ill check in ma folders and mail you. i have did that work for a week and discarded it. Bkoz ma proffs were saying it was carcinogenic and all. hope you can try it out.
Ms.Maria, you can monitor the UV absorbance of EtBr in a spectrophotometer.
if possible maria i can add up some points saying..prepare your own media in which it should not have EtBr chemical concentration in it. So u can easily come to the conclusion weather organism degrading and consuming the media and growing on it. I hope its lovely to these kind of experiments..All The Best.
Is it aerobic or anaerobic bugs ?.I will side with the colleague speculating that these bugs may have an excluding pump which can pump EtBr out . If so it will be a fascinating organism. Now you have a bug which says pursue me .
Hi Farid,
it is an aerobic bacterium. Tonight I checked the colonies from the H2O-agar plate under the UV light and I saw few of them blinking while the rest was not. In the liquid culture the whole cell suspension was blinking under the UV.
Well tomorrow I'll transfer them in a fresh medium, etc .
Thanks a lot for your feedback on my questions.
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