Ethidium bromide

Ethidium bromide
File:Ethidium bromide.svg
File:Ethidium-bromide-from-monohydrate-xtal-1971-3D-balls-B.png
File:Ethidium-bromide-monohydrate-xtal-1971-3D-SF.png
IUPAC name 3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide
Other names 2,7-Diamino-10-ethyl-6-phenylphenanthridinium bromide, 2,7-Diamino-10-ethyl-9-phenylphenanthridinium bromide, 3,8-Diamino-1-ethyl-6-phenylphenantridinium bromide, 5-Ethyl-6-phenyl-phenanthridine-3,8-diamine bromide, Ethidium bromide, Homidium bromide, EtBr
style="background: #F8EABA; text-align: center;" colspan="2" | Identifiers
CAS number	1239-45-8 Y
PubChem	14710
EC number	214-984-6
KEGG	C11161
RTECS number	SF7950000
ATCvet code	QP51AX06
SMILES
style="background: #F8EABA; text-align: center;" colspan="2" | Properties
Molecular formula	C21H20BrN3
Molar mass	394.294 g/mol
Appearance	Purple-red solid
Melting point	260 - 262 °C
Solubility in water	~ 40 g/l
style="background: #F8EABA; text-align: center;" colspan="2" | Hazards
R-phrases	R36/37/38 R46
S-phrases	S22 S24/25 S26 S36/37/39 S45 S53
NFPA 704	75px 0 3 0
Flash point	> 100 °C
Y (what is this?)  (verify) Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)
Infobox references

File:Ethidium-bromide-abs.png

Absorption spectrum of 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 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 conditions employed.

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 about 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. SYBR dyes are less carcinogenic than EtBr by the Ames test with liver extract . However, SYBR green was actually found to be more mutagenic than EthBr to the bacterial cells exposed to UV (which is what a researcher typically does) . This may be the case for other "safer" dyes, but mutagenic and toxicity details are lacking. 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 EthBr (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, thereby deforming the molecule . This can affect DNA biological processes, like DNA replication and transcription. Ethidium bromide has been shown to be mutagenic to bacteria via the ames test (with liver homogenate) . The requirement of S9 liver homogenate indicates that ethidium bromide isn't mutagenic, but its metabolites are. The identity of these mutagenic metabolites are unknown. The National Toxicology Program states it is nonmutagenic in rats and mice . Ethidium bromide (Homidium) 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 . 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 are required .

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

Handling and disposal

Ethidium bromide is not technically 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). Waste should 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. 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 is 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 .

See also

• Phenanthridine
• Agarose gel electrophoresis

Further reading

• bg:Етидиев бромид