Ethidium bromide

Ethidium bromide
Names
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
Identifiers
1239-45-8 YesY
3D model (Jmol) Interactive image
ChEBI CHEBI:4883 YesY
ChEMBL ChEMBL284328 YesY
ChemSpider 14034 YesY
ECHA InfoCard 100.013.622
EC Number 214-984-6
KEGG C11161 YesY
PubChem 14710
RTECS number SF7950000
UNII 059NUO2Z1L YesY
Properties
C21H20BrN3
Molar mass 394.294 g/mol
Appearance Purple-red solid
Melting point 260 to 262 °C (500 to 504 °F; 533 to 535 K)
~ 40 g/l
Pharmacology
QP51AX06 (WHO)
Hazards
R-phrases R25 R36/37/38 R46
S-phrases S22 S24/25 S26 S36/37/39 S45 S53
NFPA 704
Flash point > 100 °C (212 °F; 373 K)
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
YesY verify (what is YesYN ?)
Infobox references

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 trypanosomiasis in cattle, a disease caused by trypanosomes.[1] 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, although this depends on the organism exposed and the circumstances of exposure.

Structure, chemistry, fluorescence

Absorption spectrum of ethidium bromide

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.[2][3]

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

DNA sample separated using gel electrophoresis of nucleic acids and stained with ethidium bromide, that emits orange light after binding to DNA

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 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 has also been used extensively to reduce mitochondrial DNA copy number in proliferating cells.[4] Effect of EtBr on mitochondrial DNA is used in veterinary medicine to treat trypanosomosis in cattle, as EtBr binds molecules of kinetoplastid DNA and changes their conformation to Z-DNA form. This form inhibits replication of kinetoplastid DNA which is for trypanosomas lethal.[5]

Alternatives

There are alternatives to ethidium bromide which are advertised as being less dangerous and having better performance.[6][7] 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.[8] 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).[9] This may be the case for other "safer" dyes, but whilst mutagenic and toxicity details are available [10] these have not been published in peer reviewed journals. 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.[8] 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 intercalated between two adenine-thymine base pairs. The intercalation may cause mutation of DNA.

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.[11] 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.[12] 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.[13] These conclusions are supported by a subchronic carcinogenicity study in mice, whereby no mutagenic effects were detected.[14] 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.[15] Its chemotherapeutic use is due to its toxicity to mitochondria.[16] A more recent study shows that EtBr acts as a topoisomerase I poison, just like several anticancer drugs used in humans.[17] 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, and is 3 orders of magnitude lower than the therapeutic dosages used in cattle.[18] 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.[19]

Ethidium bromide can be added to YPD media and used as an inhibitor for cell growth.[20]

Handling and disposal

Ethidium bromide is not regulated as hazardous waste at low concentrations,[21] but is treated as hazardous waste by many organizations. Material should be handled according to the material safety data sheet (MSDS). The disposal of laboratory ethidium bromide remains a controversial subject.[22] 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.[23] 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.[24] However, ethidium bromide removal from solutions with activated charcoal or amberlite ion exchange resin is recommended.[25] Various commercial products are available for this use.[26]

See also

References

  1. ↑ Stevenson P, Sones KR, Gicheru MM, Mwangi EK (1995). "Comparison of isometamidium chloride and homidium bromide as prophylactic drugs for trypanosomiasis in cattle at Nguruman, Kenya.". Acta Trop. 59 (2): 257–258. doi:10.1016/0001-706X(94)00080-K. PMID 7676909.
  2. ↑ Sabnis, R. W. (Ram Wasudeo) (2010). Handbook of biological dyes and stains : synthesis and industrial application. Hoboken, N.J.: Wiley. ISBN 978-0-470-40753-0.
  3. ↑ "Application Note: Ethidium Bromide" (PDF). Retrieved 6 April 2014.
  4. ↑ Diaz F, Bayona-Bafaluy MP, Rana M, Mora M, Hao H, Moraes CT (November 2002). "Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control.". Nucleic Acids Res. 30 (21): 4626–33. doi:10.1093/nar/gkf602. PMC 135822Freely accessible. PMID 12409452.
  5. ↑ Ullu, Elisabetta; Roy Chowdhury, Arnab; Bakshi, Rahul; Wang, Jianyang; Yildirir, Gokben; Liu, Beiyu; Pappas-Brown, Valeria; Tolun, Gökhan; Griffith, Jack D.; Shapiro, Theresa A.; Jensen, Robert E.; Englund, Paul T. (2010). "The Killing of African Trypanosomes by Ethidium Bromide". PLoS Pathogens. 6 (12): e1001226. doi:10.1371/journal.ppat.1001226. ISSN 1553-7374.
  6. ↑ Huang Q, Fu WL (2005). "Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis". Clin. Chem. Lab. Med. 43 (8): 841–2. doi:10.1515/CCLM.2005.141. PMID 16201894.
  7. ↑ Dean Madden, Safer stains for DNA. accessed 2009-12-08.
  8. 1 2 Singer VL, Lawlor TE, Yue S (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).". Mutat Res. 439 (1): 37–47. doi:10.1016/s1383-5718(98)00172-7. PMID 10029672.
  9. ↑ Ohta T, Tokishita S, Yamagata H (2001). "Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli.". Mutat Res. 492 (1-2): 91–7. doi:10.1016/S1383-5718(01)00155-3. PMID 11377248.
  10. ↑ Novel Juice testing report
  11. ↑ M.J. Waring (1965). "Complex formation between ethidium bromide and nucleic acids.". Journal of Molecular Biology. 13 (1): 269–282. doi:10.1016/S0022-2836(65)80096-1. PMID 5859041.
  12. ↑ J McCann; B N Ames (1975). "Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals". PNAS. 72 (12): 5135–5139. doi:10.1073/pnas.72.12.5135. PMC 388891Freely accessible. PMID 1061098.
  13. ↑ National Toxicology Program (August 15, 2005). "Ethidium Bromide: Genetic Toxicity.". Retrieved September 30, 2009.
  14. ↑ Marossek V (December 18, 2001). "Identifizierung und Charakterisierung molekularbiologischer Veränderungen am Beispiel des Tumorsuppressors p53 in der Tamoxifen- bzw. Bromdeoxyuridin-induzierten Karzinogenese im Labornager.". Retrieved September 8, 2011.
  15. ↑ M.J. Kramer; E. Grunberg. (1973). "Effect of Ethidium Bromide against Transplantable Tumors in Mice and Rats.". Experimental Chemotherapy. 19 (4): 254–258. doi:10.1159/000221462.
  16. ↑ von Wurmb-Schwark N, Cavelier L, Cortopassi GA (2006). "A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line.". Mutat Res. 596 (1-2): 57–63. doi:10.1016/j.mrfmmm.2005.12.003. PMID 16488450.
  17. ↑ Gentry AC, Juul S, Veigaard C, Knudsen BR, Osheroff N (2011). "The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I.". NUCLEIC ACIDS RESEARCH. 39 (3): 1014–1022. doi:10.1093/nar/gkq822.
  18. ↑ Nick Oswald (September 26, 2007). "Ethidium Bromide: A Reality Check". Retrieved May 1, 2014.
  19. ↑ National Toxicology Program (August 15, 2005). "Executive Summary Ethidium Bromide: Evidence for Possible Carcinogenic Activity". Retrieved September 30, 2009.
  20. ↑ Caesar, Robert, Jonas Warringer, and Anders Blomberg. "Physiological Importance and Identification of Novel Targets for the N-Terminal Acetyltransferase NatB -- Caesar et al. 5 (2): 368 --." Eukaryotic Cell. 16 Dec. 2005. Web. 31 Jan. 2010. <http://ec.asm.org/cgi/content/full/5/2/368>.
  21. ↑ National Toxicology Program (August 15, 2005). "Executive Summary Ethidium Bromide: Table of Contents.". Retrieved September 30, 2009.
  22. ↑ HENGEN P. N. (1994). "Methods and Reagents: Disposal of Ethidium Bromide". Trends in Biochemical Sciences. 19 (6): 257–258. doi:10.1016/0968-0004(94)90152-X. PMID 8073504.
  23. ↑ Margaret-Ann Armour (2003). Hazardous laboratory chemicals disposal guide. CRC; 3 edition (February 27, 2003). pp. 222–223. ISBN 1-56670-567-3.
  24. ↑ Lunn G, Sansone EB (1987). "Ethidium bromide: destruction and decontamination of solutions". Anal. Biochem. 162: 453–8. doi:10.1016/0003-2697(87)90419-2.
  25. ↑ Bensaude O (1988). "Ethidium bromide and safety — readers suggest alternative solutions". Trends in Genetics. 4: 89–90. doi:10.1016/0168-9525(88)90092-3. PMID 3238760.
  26. ↑ Ethidium Bromide Disposal

Further reading

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