What is ethidium bromide why can it be dangerous

Ethidium bromide is the most commonly used dye for DNA and RNA detection in gels.

Inhaltsverzeichnis Show

Why this substance is hazardous
Recommended control measures
Health surveillance
Decontamination of Ethidium Bromide
Decontamination solution

Why this substance is hazardous

Ethidium bromide is a frameshift mutagen (causes changes to DNA) and intercalating agent (acts like insertion mutations). Although evidence shows that when applied in vitro it is a mutagen, contact with mammalian cells in other circumstance requires higher concentrations than that used in labs to cause an immediate risk to humans
It is a risk to the environment
It is toxic by inhalation

It is the most common and one of the cheaper nucleic acid stains but is also expensive to send into the waste stream.

Recommended control measures

Use gels to lower the risk of spills and splashes from liquid
Always use the minimum amount required
Use of ready-made stock minimises the time spent handling the substance so lowers the risk of exposure from spills
Only keep minimum stocks of higher concentrations of the substance as it is more difficult and therefore more expensive to send to waste
Always carry out the work in a fume cupboard if making up stock, to ensure that airborne particles of ethidium bromide are separated from users and diluted before allowed into the environment

Wash hands after use and immediately after a spill even if the gloves seem intact
Eye protection is required and if working on an open bench a full-face mask is recommended
Fasten the lab coat
Double gloving, first set inside the cuff, second set outside the cuff
Cover the workbench with containment trays or use similar coverings

Ethidium Bromide is classified as hazardous chemical waste
Solutions should be deactivated or filtered. Due to the hazardous nature of the chemicals required to deactivate the solutions, filtration is the preferred method. However, be aware that filtration will increase the concentration of the ethidium bromide

Health surveillance

Should there be concerns that the chemical has penetrated the glove (glove breakthrough), monitoring for ethidium bromide contamination can be undertaken by placing the operators' hands beneath a standard UV light, where ethidium bromide will show as a reddish/brown colour on the skin. Benchtops can be similarly monitored.

Decontamination of Ethidium Bromide

The following solution must be prepared immediately prior to use and can be used to decontaminate equipment and work areas. The solution should be prepared in a fume cupboard as a small amount of nitrogen dioxide may be given off when the solution is initially mixed. The solution is also strongly acidic (pH 1.8).

Decontamination solution

Mix 4.2 g of sodium nitrite (NaNO2, CAS # 7362-00-0) and 20 ml of hypophosphorous acid (50%) (H3PO2, CAS # 6303-21-5) in 300 ml of water
Wash the area once with a paper towel soaked in decontamination solution
Rinse the area five times with paper towels soaked in tap water, using a fresh towel each time
Using a UV light, check the area to ensure that all the EB has been removed (no reddish-orange fluorescence). Repeat decontamination procedure as necessary. If the acid could damage the contaminated surface, use additional rinses with paper towels soaked in tap water
Discard the decontamination solution, towels and gloves as hazardous waste

Cognisance must be taken of the specific hazards associated with using UV light sources; all persons in the area should wear appropriate Personal Protective Equipment (UV face shields, laboratory coat and long cuffed gloves fitted over the cuff of the lab coat).

There are alternatives available, however these are also hazardous chemicals so it will be substituting one risk for another rather than eliminating the risk.
A full risk assessment should be carried out to ensure that the best substance is used for your project.
Alternatives include but are not limited to MaestroSafe, SYBR Safe or GelRED and GelGreen.

> Read more about control measures for chemicals in our chemical safety library

Ethidium bromide is one of the most widely used nucleic acid stains in molecular biology laboratories and being both highly toxic and a mutagen, the one most likely to cause your health and safety officer headaches! Here Helen Baker examines some alternatives

Ethidium bromide is an intercalating agent commonly used in the laboratory to detect nucleic acids, in particular double stranded DNA. In agarose gel electrophoresis it labels DNA from PCR experiments or restriction digests with the aim of: sizing nucleic acid fragments; quantifying DNA; extracting DNA of a particular size for cloning purposes; or isolating full length PCR products from partial products and surplus nucleotides.

Although a highly sensitive stain, ethidium bromide is notoriously unsafe. Not only is it a very strong mutagen, it may also be a carcinogen or teratogenic. Its MSDS documents state that it is harmful if swallowed and very toxic by inhalation, as well as being irritating to the eyes, respiratory system and skin. Additionally it carries the risk of irreversible effects. Ethidium bromide can therefore pose a major safety problem for the researcher and be an environmental hazard during disposal.

A number of safer nucleic acid labels suitable for use in electrophoresis are available on the market. GelRed and GelGreen, for example, have improved safety over ethidium bromide. Initial mutagenicity tests on the dyes – conducted by both the manufacturer, Biotium, and externally by Litron Laboratories, a material toxicity test company – indicate that GelRed and GelGreen both have either undetectable or very weak mutagenic effect in the absence or presence of rat liver extracts S9.

These dyes were designed with safety in mind. It was reasoned that for a DNA dye to be mutagenic, it must be able to cross the cell membrane. Thus, as a first line of defence, an innovative structure was used for the gel stain molecule making it extremely difficult to cross cell membranes.

In addition, it was recognised that once a DNA dye enters a cell it might be subject to metabolism which could convert the dye into a chemical that could be either more mutagenic (as in the case of ethidium bromide) or less mutagenic than the unmetabolised original. Thus, as a second line of defence, chemical bonds were incorporated at strategic positions in the dye molecule so that on enzymatic cleavage the dyes will become very weak DNA-binding molecules. Using the standard Ames test, as measured in two bacterial strains, both GelRed and GelGreen were confirmed to be substantially safer than ethidium bromide (see box). It is believed these unique structural features are at least partially responsible for the observed low mutagenicity and low cytotoxicity of GelRed and GelGreen. But does improving safety mean compromising on results?

Figure 1: Agarose gels precast with GelRed or ethidium bromide

Sensitivity: as shown in Figure 1, GelRed is significantly more sensitive than ethidium bromide in precast gels for detection of low-level DNA, especially in the lower molecular weight area. Figure 2 shows GelRed also displaying consistently high sensitivity for post gel staining, regardless of the filter used (A vs C) and storage/handling conditions. An alternative dye, SYBR Gold (Invitrogen), showed comparable performance only when used fresh from the manufacturer and with a SYBR filter (B vs D). Following a few freeze-thaw cycles, SYBR Gold 10,000x solution degraded significantly, resulting in poor staining (E).

Stability: both GelGreen and, in particular, GelRed are so stable that the 10,000x stock solutions of the dyes can be routinely stored at room temperature. The dyes in TBE or a similar electrophoresis buffer can also be heated in the microwave oven, making them compatible with the common procedure used in making precast gels. Furthermore, precast gels made with the dyes can be produced in bulk and stored for later use.

Figure 2.

Flexibility: many researchers like the option to choose between precast gel staining and post gel staining. In general, post gel staining gives better sensitivity than precast, and eliminates the possibility of the dye interfering with the migration, and thus the separation, of the nucleic acid bands. On the other hand, precast gel staining is both simpler and more economical than post gel staining because it does not need an extra straining step and uses less dye. For this reason, precast gel staining is typically preferred over post gel staining, provided that sensitivity and/or band resolution is not a problem. GelRed, unlike some other ethidium bromide substitutes, can be used for both forms of staining.

There are many options now available that offer a safer alternative to ethidium bromide for agarose gel electrophoresis applications. Not only are these stains less hazardous to use but they are also easier and safer to dispose of. However, despite the fears of many potential users, using alternatives does not mean you have to compromise on results. In fact, many offer greater sensitivity than ethidium bromide. For further details, visit www.bioscience.co.uk.

Author: Helen Baker is technical product manager at Cambridge BioScience.