Ethidium bromide

Structure, chemistry, and 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. Absorption maxima of EtBr in aqueous solution are at 210 nm and 285 nm, which correspond to ultraviolet light. As a result of this excitation, EtBr emits orange light with wavelength 605 nm.

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 fluorescence 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 an ultraviolet 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 used 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 is also used during DNA fragment separation by agarose gel electrophoresis. It is added to running buffer and binds by intercalating between DNA base pairs. When the agarose gel is illuminated using UV light, DNA bands become visible. Intercalation of EtBr can alter properties of the DNA molecule, such as charge, weight, conformation, and flexibility. Since the mobilities of DNA molecules through the agarose gel are measured relative to a molecular weight standard, the effects of EtBr can be critical to determining the sizes of molecules.

Ethidium bromide has also been used extensively to reduce mitochondrial DNA copy number in proliferating cells. The effect of EtBr on mitochondrial DNA is used in veterinary medicine to treat trypanosomiasis in cattle, as EtBr binds molecules of kinetoplastid DNA and changes their conformation to the Z-DNA form. This form inhibits replication of kinetoplastid DNA, which is lethal for trypanosomes.

The chloride salt homidium chloride has the same applications.

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

The binding affinity of the cationic nanoparticles with DNA could be evaluated by competitive binding with ethidium bromide.

EtBr can stain proteins as well as nucleic acids.

Alternatives for gel

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 EtBr to the bacterial cells exposed to UV (which is used to visualize either dye). This may be the case for other "safer" dyes, but while mutagenic and toxicity details are available these have not been published in peer-reviewed journals. The MSDS for SYBR Safe reports an for rats of over 5 g/kg, which is higher than that of EtBr (1.5 g/kg). 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.

Possible carcinogenic activity

Most use of ethidium bromide in the laboratory (0.25–1 μg/mL) is below the LD50 dosage, making acute toxicity unlikely. Testing in humans and longer studies in a mammalian system would be required to fully understand the long-term risk ethidium bromide poses to lab workers, but it is clear that ethidium bromide can cause mutations in mammalian and bacterial cells.

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 manufacturer's safety data sheet (SDS).

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 (such as 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. Elsewhere, ethidium bromide removal from solutions with activated charcoal or ion exchange resin is recommended. Various commercial products are available for this use.

Drug resistance

Trypanosomes in the Gibe River Valley in southwest Ethiopia showed universal resistance between July 1989 and February 1993. This likely indicates a permanent loss of function in this area against the tested target, T. congolense isolated from Boran cattle.

References

References

1. "GESTIS-Stoffdatenbank".
2. (May 1995). "Comparison of isometamidium chloride and homidium bromide as prophylactic drugs for trypanosomiasis in cattle at Nguruman, Kenya". Acta Tropica.
3. (2016-04-18). "The Myth of Ethidium Bromide".
4. "Ethidium Bromide: Swap or Not {{!}} UCSB Sustainability".
5. (1981-03-15). "The hepatic metabolism of ethidium bromide to reactive mutagenic species: biochemical and structural requirements". Biochemical Pharmacology.
6. (2010). "Handbook of Biological Dyes and Stains: Synthesis and Industrial Application". Wiley.
7. "Application Note: Ethidium Bromide".
8. (November 2005). "Ethidium DNA agarose gel electrophoresis: how it started". IUBMB Life.
9. (October 1996). "The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis". Electrophoresis.
10. (November 2002). "Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control". Nucleic Acids Research.
11. (December 2010). "The killing of African trypanosomes by ethidium bromide". PLOS Pathogens.
12. (February 2006). "Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB". Eukaryotic Cell.
13. (October 2018). "Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation". Nature Communications.
14. (August 1977). "Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids". Biochemistry.
15. (2006-06-15). "Ethidium bromide is good not only for staining of nucleic acids but also for staining of proteins after polyacrylamide gel soaking in trichloroacetic acid solution". Analytical Biochemistry.
16. (2005). "Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis". Clinical Chemistry and Laboratory Medicine.
17. "Safer stains for DNA".
18. (February 1999). "Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test)". Mutation Research.
19. (May 2001). "Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli". Mutation Research.
20. "Novel Juice testing report". Newmarket Scientific.
21. National Toxicology Program. (2005-08-15). "Executive Summary Ethidium Bromide: Evidence for Possible Carcinogenic Activity".
22. (2005-08-15). "Executive Summary Ethidium Bromide". National Toxicology Program.
23. (June 1994). "Disposal of ethidium bromide". Trends in Biochemical Sciences.
24. (2003). "Hazardous Laboratory Chemicals Disposal Guide". CRC.
25. (May 1987). "Ethidium bromide: destruction and decontamination of solutions". Analytical Biochemistry.
26. (April 1988). "Ethidium bromide and safety--readers suggest alternative solutions". Trends in Genetics.
27. "Ethidium Bromide Disposal".
28. (April 1997). "Long-term occurrence of Trypanosoma congolense resistant to diminazene, isometamidium and homidium in cattle at Ghibe, Ethiopia". [[Elsevier]].
29. (September 1993). "Pharmacology of existing drugs for animal trypanosomiasis". [[Elsevier]].
30. "Homidium chloride". [[National Center for Biotechnology Information.
31. "Homidium bromide". [[National Center for Biotechnology Information.