Ethidium bromide

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. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomosis in cattle, a disease caused by trypanosomes. The high incidence of antibiotic resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Ethidium bromide may be a mutagen, carcinogen or teratogen although this depends on the organism and the conditions.

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 impinge upon 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 molecular biology laboratories. 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.

Ethidium bromide has also been used extensively to reduce mitochondrial DNA copy number in proliferating cells.

Alternatives
There are alternatives to ethidium bromide which are advertised as being less dangerous and having better performance. For example, several SYBR-based dyes are used by some researchers and there are other emerging stains such as Novel Juice. SYBR dyes are less mutagenic than EtBr by the Ames test with liver extract. However, SYBR Green I was actually found to be more mutagenic than EthBr to the bacterial cells exposed to UV (which is used to visualize either dye). This may be the case for other "safer" dyes, but whilst mutagenic and toxicity details are available these have not been published in peer reviewed journals. The above article does find that DAPI is a completely nonmutagenic stain. MSDS for SYBR Safe reports a LD50 for rats of >5 g/kg, which is higher than that of EtBr (1.5g/kg), but both are many orders of magnitude higher than the concentrations used in molecular biology. Also, many alternative dyes are suspended in DMSO, which has health implications of its own including increased skin absorption of organic compounds. Despite the performance advantage of using SYBR dyes instead of EtBr for staining purposes, many researchers still prefer EtBr since it is considerably less expensive.

Health risks
Ethidium bromide is thought to act as a mutagen because it intercalates double stranded DNA (i.e., inserts itself between the strands), deforming the DNA. This could affect DNA biological processes, like DNA replication and transcription. Ethidium bromide has been shown to be mutagenic to bacteria via the Ames test, but only after treatment with liver homogenate, which simulates the metabolic breakdown of the molecule being tested. The lack of detected mutagenicity without liver homogenate indicates that ethidium bromide is not directly mutagenic, but that its metabolites are. The identity of these mutagenic metabolites are unknown. The National Toxicology Program states it is nonmutagenic in rats and mice. These results are supported by a subchronic carcinogenicity study in mice conducted at the university of Düsseldorf where also no mutagenic effects could be detected. Ethidium bromide (Homidium brand) use in animals to treat trypanosome infection suggests that toxicity and mutagenicity are not high. Studies have been conducted in animals to evaluate EtBr as a potential antitumorigenic chemotherapeutic agent. Its chemotherapeutic use is due to its toxicity to mitochondria. A more recent study shows that EtBr acts as a topoisomerase I poison, just like several anticancer drugs used in humans. The above studies do not support the commonly held idea that ethidium bromide is a potent mutagen in humans, but they do indicate that it can be toxic at high concentrations.

Most use of ethidium bromide in the laboratory (0.25–1 microgram/ml) is below the level required for toxicity. The level is high enough that exposure may interfere with replication of mitochondrial DNA in some human cell lines, although the implications of that are not clear. Testing in humans and longer studies in any mammalian system would be required to fully understand the potential risk ethidium bromide poses to lab workers.

Ethidium bromide can be added to YPD media and used as an inhibitor for cell growth.

Ethi.Box
Ethi.Box is the first robot able to genetics to stain the gel containing DNA bands. It does this without human intervention and is safe. Ethi.box was designed and built in the Islamic Republic of Iran.

Handling and disposal
Ethidium bromide is not regulated as hazardous waste at low concentrations, but is treated as hazardous waste by many organizations. Material should be handled according to the material safety data sheet (MSDS). Wastes should always be treated in accordance with federal, state and local guidelines.

The disposal of laboratory ethidium bromide remains a controversial subject. 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. According to Lunn and Sansone, Chemical degradation using bleach yields compounds which are mutagenic by the Ames test. Data are lacking on the mutagenic effects of degradation products. Lunn and Sansone describe more effective methods for degradation. EtBr can be removed from solutions with activated charcoal or amberlite ion exchange resin. Various commercial products are available for this use.