Ethidium bromide
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Ethidium bromide (sometimes abbreviated as "EtBr", the abbreviation also confusingly used for bromoethane) is an intercalating agent commonly used as a 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 color, intensifying almost 20-fold after binding to DNA. Under the name Homidium, it has been commonly used since the 1950s in veterinary to treat Trypanosomosis in cattle, a disease caused by trypanosomes. Ethidium bromide may be a strong mutagen. It is also widely assumed to be a carcinogen or teratogen although this has never been carefully tested.
Structure, chemistry, fluorescence
As with most fluorescent compounds, ethidium bromide is aromatic. Its core heterocyclic moiety is generically known as a phenanthridine, an isomer of which is the fluorescent dye acridine.
The reason for ethidium bromide's intense fluorescence after binding with DNA is probably not due to rigid stabilization of the phenyl moiety, because the phenyl ring has been shown to project outside the intercalated bases. In fact, the phenyl group is found to be almost perpendicular to the plane of the ring system, as it rotates about its single bond to find a position where it will abut the ring system minimally. Instead, the hydrophobic environment found between the base pairs is believed to be responsible. By moving into this hydrophobic environment and away from the solvent, the ethidium cation is forced to shed any water molecules that were associated with it. As water is a highly efficient fluorescent quencher, the removal of these water molecules allows the ethidium to fluoresce.
Applications
Ethidium bromide is commonly used to detect nucleic acids in the lab. In the case of DNA this is usually double-stranded DNA from PCRs, restriction digests, etc. Single-stranded RNA can also be detected, since it usually folds back onto itself and thus provides local base pairing for the dye to intercalate. Detection typically involves a gel containing nucleic acids placed on or under a UV lamp. Since ultraviolet light is harmful to eyes and skin, gels stained with ethidium bromide are usually viewed indirectly using an enclosed camera, with the fluorescent images recorded as photographs. Where direct viewing is needed, the viewer's eyes and exposed skin should be protected. In the laboratory the intercalating properties have long been utilized to minimize chromosomal condensation when a culture is exposed to mitotic arresting agents during harvest. The resulting slide preparations permit a higher degree of resolution, and thus more confidence in determining structural integrity of chromosomes upon microscopic analysis.
Alternatives
There are alternatives to ethidium bromide in the lab.[1] For example, several SYBR-based dyes are used by some researchers. SYBR dyes have been found to be less carcinogenic than EtBr and to give cleaner, higher powered staining. However, they are suspended in DMSO, which can rapidly pass through skin.[2] Despite the safety advantage of using SYBR dyes instead of EtBr for staining purposes, many researchers still prefer EtBr for difficult detections.
Health hazard
Ethidium bromide is a mutagen, suspected carcinogen and at high concentrations is irritating to the eyes, skin, mucous membranes and upper respiratory tract. The health effects of ethidium bromide exposure have not been thoroughly investigated. It is suspected to be carcinogenic and teratogenic because of its mutagenicity, although there is no direct evidence of either effect. The toxic effects of ethidium bromide may be experienced if swallowed, inhaled or absorbed through the skin. However, ethidium bromide is not easily absorbed through the skin because of positive charge and bulky structure.
The National Toxicological Program has published data [1] (this was current as of 08/08/2007) and an Executive Summary [2]. Both the data sheet and the executive summary note that ethidium bromide (under the name homodium bromide) has "some reported use as an antiparasitic & antiprotazoic drug in animals; has also been used as a drug or drug precursor." In the section Evidence for Possible Carcinogenic Activity of the Executive Summary it is stated that: "several early '70s studies reported that EB demonstrated antitumorigenic effects." In the Report on Carcinogens (11th Edition)[3], ethidium bromide is not listed in the body of the report but is listed in Table 1 with the testing status noted as "No additional testing".
Ethidium bromide is thought to act as a mutagen because it intercalates into double stranded DNA, thereby deforming the molecule. This is believed to block or trip biological processes occurring on DNA, like DNA replication and transcription.
Safe handling
Preparation of stock solutions and any operations capable of generating ethidium bromide dust or aerosols should be conducted in a fume hood to prevent inhalation. Nitrile gloves should be worn at all times. Latex gloves offer little protection from ethidium bromide – gloves made of nitrile rubber are much more effective (this from the manufacturers of nitrile rubber gloves).[3][4] When working with high concentrations (stocks or powder) or for a prolonged period of time, double gloving can further reduce the risk of exposure, especially if the outer glove is replaced whenever significantly contaminated.
Spills or personal exposure
As with most any chemical, if spilled on skin or eyes, rinse for 15 minutes using a safety shower or eyewash. Gloves should be worn at all times and changed if contaminated with the chemical. If inhaled or swallowed, seek medical attention immediately.
For spills, use a spill pillow or absorbent to soak up aqueous ethidium bromide. Carefully clean up solid ethidium bromide to avoid creating dusts. Place in a sealable container and dispose in a medical waste box.
Waste disposal
Ethidium bromide should be handled and disposed of as hazardous waste. This applies to gloves, pipette tips, test tubes, paper towels, etc., that are grossly contaminated with ethidium bromide as well. All ethidium bromide waste should be considered state regulated hazardous waste.
See also
References
- ↑ Dean Madden, Safer stains for DNA. accessed 2007-01-31.
- ↑ Huang Q, Fu WL. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin Chem Lab Med. 2005;43(8):841-2. PMID 16201894
- ↑ University of Edinburgh Health & Safety Department - Ethidium Bromide Hand Protection
- ↑ Ethidium Bromide
External links
- Borst P. Ethidium DNA agarose gel electrophoresis: how it started. IUBMB Life. 2005 Nov;57(11):745-7. PMID 16511967
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