A real risk of lab science is accidental exposure to hazardous chemicals, especially ones that may be fatal if inhaled, harmful if swallowed or absorbed through skin, causing irritation to skin, eyes and the respiratory tract and causing heritable damage.This would be fine if ethidium bromide (EthBR) really was a serious hazard. But it's not. And it's actually less toxic than this alternative.
Ethidium bromide has just such characteristics and is frequently in use at many UBC labs. A safer alternative is available that is non-toxic, non-mutagenic and is not classified as cytotoxic waste when disposed. To alert the UBC research community to this safer alternative called SYBRSafe Gel Stain, the Department of Health Safety and Environment and the UBC Sustainability Office are sponsoring a seminar presentation by Invitrogen on the product comparison and practical application of SYBRSafe.
Ethidium (also called homidium) was developed as a treatment for trypanosomiasis (African sleeping sickness), and is still used in Africa where resistance to it is not a problem. The following quote is taken from the Encyclopedic Reference of Parasitology (2004): "...homidium is generally well tolerated at recommended dose and also at higher dose levels (no systemic toxicity)..." (source). The recommended dose for cattle is 1mg/kg body weight (up to 50mg/kg has been used in mice). Compare this with the 0.25 - 1 microgram/ml used in molecular biology (previous error corrected - thanks, anonymous commenter). A 50kg researcher would need to drink 50 liters of gel-staining solution to get even the non-toxic dose used in cattle.
Here's information from a materials safety data sheet (MSDS) provided by Fisher Scientific:
Ethidium Bromide: (inhalation, rat): 0.0118 - 0.1340 mg/L/6H. (oral,rat): 1503 mg/kg. sts determined to be toxic based on: LC50 (inhalation rat) 0.0472mg/L/1H * 100 (dilution rate)=approximately 4.72mg/L/1H.Translating as best I can, half of mice given 1.5g /kg body weight will die, as will half of mice that spend 6 hours breathing air containing about 0.12mg EthBr dust/liter. There is no evidence of neurotoxicity or of birth defects or reduced fertility even at the high doses routinely used in cattle.
Carcinogenicity:
CAS# 7732-18-5: Not listed by ACGIH, IARC, NTP, or CA Prop 65.
CAS# 1239-45-8: Not listed by ACGIH, IARC, NTP, or CA Prop 65.
Epidemiology: No information found
Teratogenicity: No information found
Reproductive Effects: No information found
Mutagenicity: Possible mutagenic effect in humans. The suspicion is based on proven damage to the genetic material in the somatic cells of man and animals and requires further clarification.
Neurotoxicity: No information found
Despite the evidence, the "EthBR is very dangerous" meme has become dogma in molecular biology. This is because EthBR can increase the frequency of mutations, a topic on which most people are very irrational.Ethidium is a flat molecule, just the right size to get in between the stacked base-pairs of double-stranded DNA. This, and its fluorescence, are what makes it a sensitive dye for detecting DNA. The presence of ethidium when DNA is being replicated can cause DNA polymerase to slip, creating short insertion or deletion mutations. Here's a 1999 reference. However there's no direct evidence that exposure to EthBr causes mutations, tumors or birth defects in any animal, and its routine use at high doses in cattle suggests that it doesn't.
Excessive concern about mutagenicity can make us overlook short-term toxic effects, and here EthBr is the safer dye. The reference above found that the SYBRsafe alternative was actually much more toxic than EthBr to the bacterial cells used in the mutagenicity tests. SYBRsafe was toxic at concentrations as low as 1 microgram/ml, whereas EthBr toxicity was not observed until 250micrograms/ml. The authors suggest that this is because living cells are much more permeable to SYBR green than to EthBr. But a MSDS for SYBR safe reports a LD50 for rats of >5g/kg, which is higher than that of EthBr (1.5g/kg). As both these LD50s are many orders of magnitude higher than the concentrations used in molecular biology, toxicity of gel staining solutions is trivial compared to the risks of of burns from melted agarose or slipping on spilled gel buffer.
Perhaps the largest real hazards associated with use of EthBr in molecular biology are the methods used to inactivate it. Some labs now incinerate all waste containing even a trace of EthBr, and others absorb it onto activated charcoal. Harsher methods involve use of bleach and sodium hydroxide, or hydrophosphorous acid and sodium nitrite, all much more dangerous than EthBr.
A final issue is cost, and here SYBR dyes may indeed be better, even though they cost a lot more per use than EthBr. This note compares the costs of different DNA stains used in agarose gels, and concludes that the new SYBR stains may save money because their greater sensitivity allows use of smaller amounts of the costly size standards. For example, this photo shows that SYBR gold can reveal bands containing as little as 100pg of DNA. (It also shows how big a problem fluorescent dust flecks can be when using SYBR stains.)
Despite the evidence of its safety, all the scientists I know continue to believe that EthBr is dangerous. It's not that we differ on how the evidence should be interpreted - they have never examined the evidence and see no point in doing so, even when I tell them that it contradicts their beliefs. I find this scary.
*I haven't been able to find out what SYBR safe is, nor the actual concentrations of the products on the market. It's all "proprietary information".
44 comments:
I would add to your comments that it's important to remember that Cybr-Gold doesn't have the same toxicity profile as Cybr-Safe - I am not even sure to what extent the toxicity of Cybr-Gold has been tested. The same goes for the other Cybr-products.
I think you should post this on the LSI newsletter and let everyone knows that EthBR isn't that dangerous to use. I can still remember the "horrified/end of the world" look on the person's face(upstair) when I "almost" placed my gel in a container on a bench. It was as if I had done something horribly wrong...
So I shouldn't have been so frustrated when an undergrad spilled a bottle of EtBr? That's good to know. Now if I could only find out about all that EMS I handled.
Dear Rosie:
A long time since grad school, hey? I have followed and enjoyed your work for a long time.
I LOVE the EtBr story. You warned me about this in graduate school, as I recall---ticking off relative risks compared to EtBr.
Do you mind if I send this around to people? I'll use a link so you get credit and all that. I just haven't seen this in one place before. You should consider what one of your commenter suggested and write an opinion piece on this subject?
Best wishes...
-Mark Martin
Your remarks are compelling, but what do you make of Invitrogen's claims?
www.invitrogen.com/sybrsafe
Deft marketing or scientifically justified?
Interesting, but main reason we use SYBR-safe in my laboratory is that it can be used in combination with blue light boxes, negating the need for UV boxes. Less damage to DNA samples and no risk of UV exposure to researchers.
Well this is encouraging news, especially after reading on wikipedia that the regular latex gloves I have been using in lab for the past year and a half don't protect against EthBr.
Here is the information to see the NTP data that caused the lastest RoC (Report on Carcinogens, 11th edition) to find "no additional testing"
http://ntp-server.niehs.nih.gov/index.cfm?objectid=6DE15C4C-F1F6-975E-73A5D57B3D6FC3C3
This link will save you one step at the NTP site
http://ntp.niehs.nih.gov/index.cfm?objectid=6F5EA06A-F1F6-975E-73079A5FE34F7E88
Propidium iodide, a related chemical, only stains the DNA in dead cells. I'd think it likely that ETBr doesn't get into living cells well either.
I just looked at the NTP report for EthBR, and one of the first characteristics under description is: "bitter-tasting dark red crystals from alcohol (Budavari, 1989)". We need to talk to this Budavari, and see if he/she is still alive, then we'll know. One assumes that there is a fair mass of EthBR in a crystal, and tasting it...blimey! Some sacrifices are made for science.
I was looking for information on possible carcinogenicity of ethidiumbromide for my master-thesis (environmental studies). So, thanks for the information
I FEEL REALLY STUPID. I LEFT MY BEAUTIFUL JOB, SOLD MY CAR, MOVED MY APARTMENT .... ALL BECAUSE I WANTED TO GET AWAY FROM THE EtBr. I TRIED TO RUN AWAY... I FELT THAT, IF I CAN DISTANCE MYSELF FROM THE THINGS I TOUCHED AFTER COMMING FROM LAB... I WOULD NOT DIE.
I USED TO WASH MY HANDS FOR OVER 15 MINUTES (45 MINUTE ON ONE OCCATION WHEN A DROP OF TAE BUFFER SPLASHED ON MY ARM). EVEN AFTER ALL THIS WASHING, I NEVER FELT "CONTENT".
READING THAT EtBr IS NOT REALLY TOXIC GIVES ME UNDESCRIBABLE AMOUNT OF JOY.
BUT YET, THE FEAR INSIDE ME IS NOT YET GONE. EVEN AS THIS WRITE THIS COMMENT... I STILL AM SCARED DEEP INSIDE.
BUT I HOPE THIS FINDING IS TRUE AND NO HARM IS DONE TO PEOPLE WORKING WITH EtBr. NO GENETIC DEFECTS...NO TERATOGENIC HAZARD.
THANK YOU FOR THIS GREAT NEWS.
Once a few drop of EthBR(10mg/ml) splashed on the skin of my chest. Though I washed immediately, I am scared to death. After 7 years of exposure, I am still alive. But I often think I will develop some skin cancer, breast cancer or lung cancer. This paper may give me a peace of mind.
Please write this article a little bit longer and send it to Biotechniques. More people have to read it!
thanks.
I have had problems with ethidium bromide hysteria in several labs. and been rebuked by junior "safety officers" for picking up my gels containing 0.5 micrograms/ml EB without gloves.
I pointed out that Masai and Samburu give gram quantities to their cattle while continuing to drink their blood and milk.
Also that EB binds to protein including my skin squames (which are dead) and are most unlikely to penetrate my skin.
Also that I used to analyse commercial EB for use as a trypanocide for 4 years in the 1950's. I do not say it was good practice in retrospect but my hands were stained red for prolonged periods and I ate sandwiches daily.
I am fit and well 50 years on.
None of this would convince so I took one person to the first aid kit and showed him the acriflavine cream that was there to put on burns and cuts. Nothing would suffice to calm the safety guru.
One last thing. There was until recently a safety website whose recommendation for handling sodium chloride included using gloves, a respirator, disposing of contaminated clothes and seeking medical help for spills. It did however recommend washing the offending chemical out of the eye with normal saline.
is there any methodology developed to remove the ethidium bromide from agarose gel and reuse the agarose?
Reverse the gel polarity
or else just boil the gel and reuse it ( 1min on normal setting
in a microwave)
Ethidium bromide induces mutations in cells and that is a fact. If there are not strong evidence that it is also a cancerogen it doesn't mean you should start drinking it with a coffee. I cannot understand that somebody who didn't present any experimental data about the safety of EtBr but only put together selected information gets such a positive response. I would suggest that Rosie Redfield and her bolivars should start administrating EtBr to themselves to proof who's right! Where is your common sense?
Why does she need to present experimental evidence, when there's already evidence out there, like people giving it to cows in significant quantities? That's just an extreme example, but it suffices to show the point. The paranoia about EtBr is over-blown. Just because something is a proven mutagen does not mean that in small quantities it is dangerous. That's a non-sequitar. It means it may be dangerous. But there can always be evidence to the contrary, which in this case there is. Furthermore, there is no direct evidence that it is actually dangerous.
There are also quite a few theories (with evidence) regarding hormesis that very small quantities of otherwise harmful things -- like radiation, which is mutagenic -- can be beneficial. There is evidence to suggest this. The explanatory theory is that small doses cause DNA mutations, which induces the DNA repair mechanisms, which "over-compensate" and end up correcting the new errors as well as others.
In fact, just about everything is hormetic. Vitamins normally healthy to you in moderate doses can prove lethal if given in extreme over-doses. (although the recommended daily values are extremely conservative, and there's much evidence to suggest that "megadoses" of various vitamins can be healthy, despite being 300-500% of recommended daily values).
In any event, no-one's saying that you should start drinking 10mg/ml concentrations of EtBr like Orange Juice. But just maybe, you shouldn't be so paranoid about it.
It is a common misconception that being "over-paranoid" or "overly cautious" isn't harmful. This is wrong. There is always opportunity cost, and who knows, maybe all the psychological stress people have over EtBr will shorten their lifespan much more than EtBr.
Hi, both of these molecules bind to DNA and it is this which prevents correct DNA replication (or RNA production) leading to mutations of DNA and damage to proteins. Obviously it is not always possible to, but I recommend evasion of contact with either.
Since it is non-base specific, however, there is low chance of problems occurring in adult life.
Hello. I am working on tagging/staining nanoparticles so that when I put them in agarose gel electrophoresis I can see them move. I can't use ethidium bromide and was wondering if you had any suggestions for an alternative. Thanks!
Rosie, I remember you once throwing up your arms and asking "where's the data?!" with respect to EtBr toxicity. This has stuck with me and I've since found that even the Molecular Cloning manual simply indicates that the breakdown products of ethidium bromide are 'mildly mutagenic'. Yet so many labs treat it like radioactivity and even dedicate rooms for the handling and post-staining of gels.
I read a note in "A field guide for
THE DIAGNOSIS, TREATMENT AND PREVENTION OF AFRICAN ANIMAL TRYPANOSOMOSIS" that "Ethidium bromide is a widely used compound in molecular biology, where it is handled with stringent security precautions because it is highly mutagenic and therefore potentially carcinogenic. The same compound has been used extensively in the treatment of bovine trypanosomosis since the 1950s. (The author has commonly seen the kettle, used in the morning to prepare the solution of ethidium bromide in hot water, become the tea-pot in the afternoon.)"
From one scientist to another, thank you for writing this article and dispelling the myth.
EtBr use in labs seems somewhat analogous to the potential danger of religion: so few data supporting so much certainty and emotion.
I agree with one of the other comments: this should be beefed up for Biotechniques.
Josh Neufeld.
I was interested in the critical comment from an anonymous correspondent, implying that Rosie Redfield is being irresponsible in criticising the orthodoxy concerning ethidium. Ironically the author warns against drinking ethidium bromide with one's coffee. Has that author seen what doses of greater than 200 micromolar caffeine can do to mitotic eukaryotic cells? [Schlegel and Pardee,
Science 232, 1264-1266 (1986)]. That concentration (~40mg/l) is not even a strong cup of coffee!
It is obvious that many things might be toxic in the absence of a protective skin/ mucosal barrier and in the absence of a very efficient system for detoxifying all of the poisons that even nature throws at us. But we have these barriers.
I threw out two t shirts this week because i got buffer from the gel on it. I still think that its better to be safe than sorry. And yeah i wash my hands 1 billion times a day to the point where my skin gets very dry.
Finally, thank goodness! I've ben telling people about the uses of EthBr as a drug for treating trypansomosis for years now and I am continually met with the same horrified looks (often from people who routinely handle their gels with latex gloves mind you!).
Personally, I am more afraid of the long-term effects of the pollution in the air we breath every day than almost any chemical in the lab!
Thank you, Maria Trainer.
•Ethidium bromide (From http://en.wikipedia.org/wiki/Staining_(biology)#Common_biological_stains)
Ethidium bromide intercalates and stains DNA, providing a fluorescent red-orange stain. Although it will not stain healthy cells, it can be used to identify cells that are in the final stages of apoptosis - such cells have much more permeable membranes. Consequently, ethidium bromide is often used as a marker for apoptosis in cells populations and to locate bands of DNA in gel electrophoresis. The stain may also be used in conjunction with acridine orange (AO) in viable cell counting. This EB/AO combined stain causes live cells to fluoresce green whilst apoptotic cells retain the distinctive red-orange fluorescence.
[edit]
"The recommended dose for cattle is 1mg/kg body weight (up to 50mg/kg has been used in mice). Compare this with the 0.25 - 1 microgram/liter used in molecular biology. A 50kg researcher would need to drink 50,000 liters of gel-staining solution to get even the non-toxic dose used in cattle."
Isn't the concentration used in gel staining typically 1 ug/mL, or 1 mg/L? For the 50kg researcher, "only" 50 liters of staining solution would be needed to get a veterinary bovine dose.
Brilliant brilliant brilliant and yes, as other people have said - please try to get someone to publish this as it is so very important. The reason I found this blog was because I was looking up EtBr due to paranoia and wondering what will become of my future children! Thank you so very much!
Indie
Dear Rosie:
I must thank you first. b/c i am an botany student doing my ph.D on molecular work. My tutor told that ethidium bromide is very dangerous. Even though i have not came in direct contact some times tearing of gloves occurs and I feel tense and i fear the total day that i may get something.
I also feared that as i compleate my ph.d i may also get canceralong with my degree due to this chemical. Please publish this information as such all people who are working with this chemical feel relax and happy.
maduri, India
Two years ago, I spilled some EthBr, in my hand and I did not wash it immediately. After few minutes I washed it. Since that day, I have become a paranoid person> thinking that I might develop a cancer. Actually this article helped me quite a lot to forget about. But still I am very worried. So, can some one please let me know, is their any chance of some one can develop a cancer due to EthBr. ?
i am a freshman coming from a lab in China.i am happy i have found and read it.do you have something new about EthBr or your latest investigation?if you have,could you tell me and send it to my E-mail:ouyang19860810@126.com?thank you for your article.
in our institute, most labs have been advised by the safety office to move from EtBr to SybrSafe. why? because they say it's safer and much easier to dispose.
i've forwarded this to our risk management officer and lab manager. i'll try to echo what they have to say...
thanks for the info!
Biochem Int. 1988 Aug;17(2):367-74. A simple quantitative method of estimation of cell-intactness based on ethidium bromide fluorescence. Dey CS, Majumder GC
I am a nephrologist in the UK, and I have just come across a patient who used EB for the Milk Marketting board to stain milk for bovine white blood cells. He used to make up large quantities of solution containing EB. He developed renal carcinoma in one kidney which was completely removed then a second primary in his other kidney three years later which was partially resected and now spread to his liver.
And how exactly have you established the causal relationship between ethidium bromide exposure and this person's recurring kidney cancer? Non-controlled experiments are often misleading.
Wouldn't this said patient have had to have drunk the solution for it to get anywhere near his kidneys?
I'm not about to drink it, what most lab users are worried about is it getting into our cells via the skin.
I see there being more chance that I'll get hereditary breast cancer than some skin cancer on my face beacause the tiny diluted droplet of stain solution that splashed onto my cheek somehow found it's way into my healthy skin cells!
I don't think anyone that is afraid of EtBr is thinking they are going to die from the initial dose.
Just because they are treating cattle with it doesn't really mean much. Do you not think these cattle are butchered a few years down the road and eaten? Or do you think they are aloud to live 60 years more like humans? Your logic is retarded. But yeah all you idiots can expose yourself to a mutagen just because some idiot said they do it to cows.
Oh hey I also heard about some guy who smoked a pack a day for 70 years. Lets all go do it!
I was only told that EtBr was 'toxic' from my supervisor when I was learning how to make agarose gels for PCR. I wasn't told about it being a mutagen or possible carcinogenic effects.
Today, I spilled some EtBr on my hand (though I had gloves on). As well, my bangs got in my way(as the fume hood fan was on) when I was pouring the gel to the cassette, leaving my hair to be wet in the agarose gel containing like, what, 1ul EtBr/100mL buffer solution.
As I got home, my friend told me that EtBr is rather a mutagen, and that a small amount can cause DNA mutation in my body and cause cancer. Of course I totally freaked out after that conversation with my friend. I ended up calling my hairdresser to book an appointment ASAP, shampoo-ed my hair three times, washed my hand a million times, etc.
Even though I'm sure I was exposed to less than 5ul of EtBr, the fact that it is a 'mutagen' just made me to freak out.
Thanks for the post, I feel relieved at least.
For more information please read information posted by the National Institute for Occupational Safety and HEalth (NIOSH), http://www.cdc.gov/niosh/rtecs/sf794eb0.html
There is no doubt that EtBr is a mutagen and carcinogen. The question is, why expose yourself to it when it's so easy to take percausion. The fact is that the effect - if cancer - take decades to develop. The husteria around it is there for a reason.
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