Ethidium bromide

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Not to be confused with Ethyl bromide.
Ethidium bromide
Names
Preferred IUPAC name
3,8-Diamino-5-ethyl-6-phenylphenanthridin-5-ium 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
  • EthBr
Identifiers
3D model (JSmol)
3642536
ChEBI
ChEMBL
ChemSpider
ECHA InfoCard 100.013.622 Edit this at Wikidata
EC Number
  • 214-984-6
KEGG
RTECS number
  • SF7950000
UNII
UN number 2811
  • InChI=1S/C21H19N3.BrH/c1-2-24-20-13-16(23)9-11-18(20)17-10-8-15(22)12-19(17)21(24)14-6-4-3-5-7-14;/h3-13,23H,2,22H2,1H3;1H checkY
    Key: ZMMJGEGLRURXTF-UHFFFAOYSA-N checkY
  • InChI=1/C21H19N3.BrH/c1-2-24-20-13-16(23)9-11-18(20)17-10-8-15(22)12-19(17)21(24)14-6-4-3-5-7-14;/h3-13,23H,2,22H2,1H3;1H
    Key: ZMMJGEGLRURXTF-UHFFFAOYAD
  • CC[n+]1c2cc(N)ccc2c3ccc(N)cc3c1c4ccccc4.[Br-]
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
QP51DX03 (WHO)
Hazards[1]
GHS labelling:
GHS06: ToxicGHS08: Health hazard
Danger
H302, H330, H341
P201, P202, P260, P284, P301+P312, P304+P340+P310
NFPA 704 (fire diamond)
NFPA 704 four-colored diamondHealth 4: Very short exposure could cause death or major residual injury. E.g. VX gasFlammability 1: Must be pre-heated before ignition can occur. Flash point over 93 °C (200 °F). E.g. canola oilInstability 0: Normally stable, even under fire exposure conditions, and is not reactive with water. E.g. liquid nitrogenSpecial hazards (white): no code
4
1
0
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).
checkY verify (what is checkY☒N ?)

Ethidium bromide (or homidium bromide,[2] chloride salt homidium chloride)[3][4] 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. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. 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.[5] The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Despite its reputation as a mutagen, tests have shown it to have low mutagenicity without metabolic activation.[6][7]

Structure, chemistry, and 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.[8][9]

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.[citation needed]

Applications

DNA sample separated using gel electrophoresis of nucleic acids and stained with ethidium bromide, which 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 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.[citation needed]

Ethidium bromide is also used during DNA fragment separation by agarose gel electrophoresis.[10] 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.[11]

Ethidium bromide has also been used extensively to reduce mitochondrial DNA copy number in proliferating cells.[12] 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.[13]

The chloride salt homidium chloride has the same applications.[3][4]

Ethidium bromide can be added to YPD media and used as an inhibitor for cell growth.[14]

The binding affinity of the cationic nanoparticles with DNA could be evaluated by competitive binding with ethidium bromide.[15][16]

Alternatives for gel

There are alternatives to ethidium bromide which are advertised as being less dangerous and having better performance.[17][18] 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.[19] 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).[20] This may be the case for other "safer" dyes, but while mutagenic and toxicity details are available[21] these have not been published in peer-reviewed journals. The MSDS for SYBR Safe reports an LD50 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.[19] 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.[citation needed]

Possible carcinogenic activity

Ethidium bromide intercalated between two adenine–thymine base pairs. The intercalation is said by some [by whom?] to motivate a high mutagenicity of DNA.[6]

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.[22]

Handling and disposal

Ethidium bromide is not regulated as hazardous waste at low concentrations,[23] but is treated as hazardous waste by many organizations. Material should be handled according to the manufacturer's safety data sheet (SDS).[citation needed]

The disposal of laboratory ethidium bromide remains a controversial subject.[24] 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.[25] 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.[26] Elsewhere, ethidium bromide removal from solutions with activated charcoal or ion exchange resin is recommended.[27] Various commercial products are available for this use.[28]

Drug resistance

Trypanosomes in the Gibe River Valley in southwest Ethiopia showed universal resistance between July 1989 and February 1993.[29] This likely indicates a permanent loss of function in this area against the tested target, T. congolense isolated from Boran cattle.[29]

See also

References

  1. ^ "GESTIS-Stoffdatenbank". gestis.dguv.de (in German). Retrieved 22 November 2021.
  2. ^ "Homidium bromide". PubChem. NCBI, NLM, US NIH. Retrieved 2020-09-08. CID 14710 from PubChem
  3. ^ a b Kinabo LD (September 1993). "Pharmacology of existing drugs for animal trypanosomiasis". Acta Tropica. 54 (3–4). Elsevier: 169–183. doi:10.1016/0001-706x(93)90091-o. PMID 7902656. S2CID 27564786.
  4. ^ a b "Homidium chloride". PubChem. NCBI, NLM, US NIH. Retrieved 2021-03-14. CID 11765 from PubChem
  5. ^ Stevenson P, Sones KR, Gicheru MM, Mwangi EK (May 1995). "Comparison of isometamidium chloride and homidium bromide as prophylactic drugs for trypanosomiasis in cattle at Nguruman, Kenya". Acta Tropica. 59 (2): 77–84. doi:10.1016/0001-706X(94)00080-K. PMID 7676909.
  6. ^ a b Lowe, Derek (2016-04-18). "The Myth of Ethidium Bromide". In the Pipeline. Retrieved 2019-02-28.
  7. ^ "Ethidium Bromide: Swap or Not | UCSB Sustainability". sustainability.ucsb.edu. Retrieved 2023-02-08.
  8. ^ Sabnis RW (2010). Handbook of Biological Dyes and Stains: Synthesis and Industrial Application. Hoboken, NJ: Wiley. ISBN 978-0-470-40753-0.
  9. ^ "Application Note: Ethidium Bromide" (PDF). Retrieved 6 April 2014.
  10. ^ Borst P (November 2005). "Ethidium DNA agarose gel electrophoresis: how it started". IUBMB Life. 57 (11): 745–747. doi:10.1080/15216540500380855. PMID 16511967.
  11. ^ Sigmon J, Larcom LL (October 1996). "The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis". Electrophoresis. 17 (10): 1524–1527. doi:10.1002/elps.1150171003. PMID 8957173. S2CID 10593378.
  12. ^ 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 Research. 30 (21): 4626–4633. doi:10.1093/nar/gkf602. PMC 135822. PMID 12409452.
  13. ^ Roy Chowdhury A, Bakshi R, Wang J, Yildirir G, Liu B, Pappas-Brown V, et al. (December 2010). "The killing of African trypanosomes by ethidium bromide". PLOS Pathogens. 6 (12): e1001226. doi:10.1371/journal.ppat.1001226. PMC 3002999. PMID 21187912.
  14. ^ Caesar R, Warringer J, Blomberg A (February 2006). "Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB". Eukaryotic Cell. 5 (2): 368–378. doi:10.1128/EC.5.2.368-378.2006. PMC 1405896. PMID 16467477.
  15. ^ Liang H, Peng B, Dong C, Liu L, Mao J, Wei S, et al. (October 2018). "Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation". Nature Communications. 9 (1): 4291. Bibcode:2018NatCo...9.4291L. doi:10.1038/s41467-018-06603-5. PMC 6191420. PMID 30327464.
  16. ^ Olmsted J, Kearns DR (August 1977). "Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids". Biochemistry. 16 (16): 3647–3654. doi:10.1021/bi00635a022. PMID 889813.
  17. ^ Huang Q, Fu WL (2005). "Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis". Clinical Chemistry and Laboratory Medicine. 43 (8): 841–842. doi:10.1515/CCLM.2005.141. PMID 16201894. S2CID 27423672.
  18. ^ Madden D. "Safer stains for DNA". Retrieved 2009-12-08.
  19. ^ a b Singer VL, Lawlor TE, Yue S (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. 439 (1): 37–47. Bibcode:1999MRGTE.439...37S. doi:10.1016/s1383-5718(98)00172-7. PMID 10029672.
  20. ^ Ohta T, Tokishita S, Yamagata H (May 2001). "Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli". Mutation Research. 492 (1–2): 91–97. Bibcode:2001MRGTE.492...91O. doi:10.1016/S1383-5718(01)00155-3. PMID 11377248.
  21. ^ "Novel Juice testing report" (PDF). Newmarket Scientific.
  22. ^ National Toxicology Program (2005-08-15). "Executive Summary Ethidium Bromide: Evidence for Possible Carcinogenic Activity" (PDF). Retrieved 2009-09-30.
  23. ^ "Executive Summary Ethidium Bromide" (PDF). National Toxicology Program. 2005-08-15. Retrieved 2009-09-30.
  24. ^ Hengen PN (June 1994). "Disposal of ethidium bromide". Trends in Biochemical Sciences. 19 (6): 257–258. doi:10.1016/0968-0004(94)90152-X. PMID 8073504.
  25. ^ Armour MA (2003). Hazardous Laboratory Chemicals Disposal Guide (3rd ed.). CRC. pp. 222–223. ISBN 1-56670-567-3.
  26. ^ Lunn G, Sansone EB (May 1987). "Ethidium bromide: destruction and decontamination of solutions". Analytical Biochemistry. 162 (2): 453–458. doi:10.1016/0003-2697(87)90419-2. PMID 3605608.
  27. ^ Quillardet, P.; Hofnung, M. (April 1988). "Ethidium bromide and safety--readers suggest alternative solutions". Trends in Genetics. 4 (4): 89–90. doi:10.1016/0168-9525(88)90092-3. PMID 3238760.
  28. ^ "Ethidium Bromide Disposal". Archived from the original on 2015-04-15. Retrieved 2006-10-03.
  29. ^ a b Mulugeta W, Wilkes J, Mulatu W, Majiwa PA, Masake R, Peregrine AS (April 1997). "Long-term occurrence of Trypanosoma congolense resistant to diminazene, isometamidium and homidium in cattle at Ghibe, Ethiopia". Acta Tropica. 64 (3–4). Elsevier: 205–217. doi:10.1016/s0001-706x(96)00645-6. PMID 9107367. S2CID 23878484.
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