Ethidium bromide: Difference between revisions

{{short description|DNA gel stain and veterinary drug}}

{{Distinguish|Ethyl bromide}}

| Watchedfields = changed

| verifiedrevid = 443740742

| Name =

<!-- also image EthidiumBromide.png exists -->| ImageSize =

| PIN = 3,8-Diamino-5-ethyl-6-phenylphenanthridin-5-ium bromide

| OtherNames = {{unbulleted list|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}}

| SystematicName =

| CASNo_Ref = {{cascite|correct|CAS}}

| CASNo = 1239-45-8

| Beilstein = 3642536

| ChEBI_Ref = {{ebicite|correct|EBI}}

| ChEBI = 4883

| ChEMBL_Ref = {{ebicite|correct|EBI}}

| ChEMBL = 284328

| ChemSpiderID_Ref = {{chemspidercite|correct|chemspider}}

| EINECS = 214-984-6

| KEGG_Ref = {{keggcite|correct|kegg}}

| KEGG = C11161

| PubChem = 14710

| RTECS = SF7950000

| UNNumber = 2811

| SMILES = CC[n+]1c2cc(N)ccc2c3ccc(N)cc3c1c4ccccc4.[Br-]

| Formula = C₂₁H₂₀BrN₃

| MolarMass = 394.294 g/mol

| Appearance = Purple-red solid

| Density =

| MeltingPtC = 260 to 262

| MeltingPt_notes =

| BoilingPt =

| Solubility = ~40 g/l

| Section3 =

| Section4 =

| Section5 =

| Section6 = {{Chembox Pharmacology

| ATCvet = yes

| ATCCode_prefix = P51

| ATCCode_suffix = DX03

}}

| Section7 = {{Chembox Hazards

| MainHazards =

| FlashPt = >

| FlashPtC = 100

| AutoignitionPt =

| NFPA-H = 4

| NFPA-F = 1

| NFPA-R = 0

| Hazards_ref = <ref>{{cite web |title=GESTIS-Stoffdatenbank |url=https://gestis.dguv.de/data?name=109233 |website=gestis.dguv.de |access-date=22 November 2021 |language=de}}</ref>

| GHSPictograms = {{GHS06}}{{GHS08}}

| GHSSignalWord = Danger

| HPhrases = {{H-phrases|302|330|341}}

| PPhrases = {{P-phrases|201|202|260|284|301+312|304+340+310}}

'''Ethidium bromide''' (or '''homidium bromide''',<ref name="Hom-Br-PubChem" /> chloride salt '''homidium chloride''')<ref name="Kinabo-1993" /><ref name="Hom-Cl-PubChem" /> is an [[intercalation (biochemistry)|intercalating]] agent commonly used as a [[fluorescent tag]] ([[nucleic acid]] [[staining (biology)|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 [[fluorescence|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.<ref>{{cite journal | vauthors = Stevenson P, Sones KR, Gicheru MM, Mwangi EK | title = Comparison of isometamidium chloride and homidium bromide as prophylactic drugs for trypanosomiasis in cattle at Nguruman, Kenya | journal = Acta Tropica | volume = 59 | issue = 2 | pages = 77–84 | date = May 1995 | pmid = 7676909 | doi = 10.1016/0001-706X(94)00080-K }}</ref> 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.<ref name=":0">{{Cite web|url=https://www.science.org/content/blog-post/myth-ethidium-bromide|title=The Myth of Ethidium Bromide| vauthors = Lowe, Derek |date=2016-04-18|website=In the Pipeline|language=en-US|access-date=2019-02-28}}</ref><ref>{{Cite web |title=Ethidium Bromide: Swap or Not {{!}} UCSB Sustainability |url=https://sustainability.ucsb.edu/blog/just-facts-labrats/ethidium-bromide-swap-or-not |access-date=2023-02-08 |website=sustainability.ucsb.edu}}</ref>

==Structure, chemistry, and fluorescence==

[[Image:Ethidium-bromide-abs.png|thumb|left|200px|Absorption spectrum of ethidium bromide]]

As with most [[fluorescent]] [[Chemical compound|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&nbsp;nm and 285&nbsp;nm, which correspond to [[ultraviolet light]]. As a result of this [[Excited state|excitation]], EtBr emits orange light with wavelength 605&nbsp;nm.<ref>{{Cite book | vauthors = Sabnis RW |title=Handbook of Biological Dyes and Stains: Synthesis and Industrial Application|year=2010|publisher=Wiley|location=Hoboken, NJ|isbn=978-0-470-40753-0}}</ref><ref>{{cite web|title=Application Note: Ethidium Bromide|url=http://www.berthold-jp.com/pdf/ethidium_bromide.pdf|access-date=6 April 2014}}</ref>

Ethidium bromide's intense fluorescence after binding with DNA is probably not due to rigid stabilization of the [[phenyl]] [[wikt:moiety|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 [[Fluorescence in the life sciences#Sensitivity to environment|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 [[Quenching (fluorescence)|quencher]], the removal of these water molecules allows the ethidium to fluoresce.{{Citation needed|date=September 2018}}

[[Image:AgarosegelUV.jpg|thumb|left|200px|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 [[Polymerase chain reaction|PCR]]s, [[restriction digest]]s, etc. Single-stranded [[RNA]] can also be detected, since it usually folds back onto itself and thus provides local [[base pair]]ing for the dye to intercalate. Detection typically involves a [[agarose gel electrophoresis|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.{{cn|date=February 2023}}

Ethidium bromide is also used during DNA fragment separation by [[agarose gel electrophoresis]].<ref>{{cite journal | vauthors = Borst P | title = Ethidium DNA agarose gel electrophoresis: how it started | journal = IUBMB Life | volume = 57 | issue = 11 | pages = 745–747 | date = November 2005 | pmid = 16511967 | doi = 10.1080/15216540500380855 | doi-access = free }}</ref> 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.<ref>{{cite journal | vauthors = Sigmon J, Larcom LL | title = The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis | journal = Electrophoresis | volume = 17 | issue = 10 | pages = 1524–1527 | date = October 1996 | pmid = 8957173 | doi = 10.1002/elps.1150171003 | s2cid = 10593378 }}</ref>

Ethidium bromide has also been used extensively to reduce [[mitochondrial DNA]] copy number in proliferating cells.<ref>{{cite journal | vauthors = Diaz F, Bayona-Bafaluy MP, Rana M, Mora M, Hao H, Moraes CT | title = Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control | journal = Nucleic Acids Research | volume = 30 | issue = 21 | pages = 4626–4633 | date = November 2002 | pmid = 12409452 | pmc = 135822 | doi = 10.1093/nar/gkf602 }}</ref> The effect of EtBr on mitochondrial DNA is used in veterinary medicine to treat [[trypanosomiasis]] in cattle, as EtBr binds molecules of [[Kinetoplastida|kinetoplastid]] DNA and changes their conformation to the [[Z-DNA]] form. This form inhibits replication of kinetoplastid DNA, which is lethal for trypanosomes.<ref name="UlluRoy Chowdhury2010">{{cite journal | vauthors = Roy Chowdhury A, Bakshi R, Wang J, Yildirir G, Liu B, Pappas-Brown V, Tolun G, Griffith JD, Shapiro TA, Jensen RE, Englund PT | display-authors = 6 | title = The killing of African trypanosomes by ethidium bromide | journal = PLOS Pathogens | volume = 6 | issue = 12 | pages = e1001226 | date = December 2010 | pmid = 21187912 | pmc = 3002999 | doi = 10.1371/journal.ppat.1001226 | doi-access = free }}</ref>

The chloride salt homidium chloride has the same applications.<ref name="Kinabo-1993" /><ref name="Hom-Cl-PubChem" />

Ethidium bromide can be added to [[YEPD|YPD]] media and used as an inhibitor for cell growth.<ref>{{cite journal | vauthors = Caesar R, Warringer J, Blomberg A | title = Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB | journal = Eukaryotic Cell | volume = 5 | issue = 2 | pages = 368–378 | date = February 2006 | pmid = 16467477 | pmc = 1405896 | doi = 10.1128/EC.5.2.368-378.2006 }}</ref>

The binding affinity of the cationic nanoparticles with DNA could be evaluated by competitive binding with ethidium bromide.<ref>{{cite journal | vauthors = Liang H, Peng B, Dong C, Liu L, Mao J, Wei S, Wang X, Xu H, Shen J, Mao HQ, Gao X, Leong KW, Chen Y | display-authors = 6 | title = Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation | journal = Nature Communications | volume = 9 | issue = 1 | pages = 4291 | date = October 2018 | pmid = 30327464 | pmc = 6191420 | doi = 10.1038/s41467-018-06603-5 | bibcode = 2018NatCo...9.4291L }}</ref><ref>{{cite journal | vauthors = Olmsted J, Kearns DR | title = Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids | journal = Biochemistry | volume = 16 | issue = 16 | pages = 3647–3654 | date = August 1977 | pmid = 889813 | doi = 10.1021/bi00635a022 }}</ref>

===Alternatives for gel===

There are alternatives to ethidium bromide which are advertised as being less dangerous and having better performance.<ref>{{cite journal | vauthors = Huang Q, Fu WL | title = Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis | journal = Clinical Chemistry and Laboratory Medicine | volume = 43 | issue = 8 | pages = 841–842 | year = 2005 | pmid = 16201894 | doi = 10.1515/CCLM.2005.141 | s2cid = 27423672 }}</ref><ref>{{cite web| vauthors = Madden D |url=http://www.bioscience-explained.org/ENvol1_2/index.html#schollar_test |title=Safer stains for DNA |access-date=2009-12-08}}</ref> For example, several [[SYBR Green|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.<ref name="Singer1999">{{cite journal | vauthors = Singer VL, Lawlor TE, Yue S | title = Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test) | journal = Mutation Research | volume = 439 | issue = 1 | pages = 37–47 | date = February 1999 | pmid = 10029672 | doi = 10.1016/s1383-5718(98)00172-7 | bibcode = 1999MRGTE.439...37S }}</ref> 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).<ref>{{cite journal | vauthors = Ohta T, Tokishita S, Yamagata H | title = Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli | journal = Mutation Research | volume = 492 | issue = 1–2 | pages = 91–97 | date = May 2001 | pmid = 11377248 | doi = 10.1016/S1383-5718(01)00155-3 | bibcode = 2001MRGTE.492...91O }}</ref> This may be the case for other "safer" dyes, but while mutagenic and toxicity details are available<ref>{{cite web|url=http://www.newmarketscientific.com/datasheets/Novel_Juice_Testing_Report_012011.pdf |title=Novel Juice testing report |publisher=Newmarket Scientific}}</ref> these have not been published in peer-reviewed journals. The [[Material safety data sheet|MSDS]] for SYBR Safe reports an {{LD50}} for rats of over 5&nbsp;g/kg, which is higher than that of EtBr (1.5&nbsp;g/kg). Many alternative dyes are suspended in [[Dimethyl sulfoxide|DMSO]], which has health implications of its own, including increased skin absorption of organic compounds.<ref name="Singer1999" /> 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.{{cn|date=February 2023}}

== Possible carcinogenic activity ==

[[Image:DNA intercalation2.jpg|thumb|left|200px|Ethidium bromide [[intercalation (biochemistry)|intercalated]] between two adenine–thymine base pairs. The intercalation is said by some {{By whom|date=February 2022}} to motivate a high mutagenicity of DNA.<ref name=":0" />]]

Most use of ethidium bromide in the laboratory (0.25–1&nbsp;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.<ref>{{Cite web | author = National Toxicology Program | title = Executive Summary Ethidium Bromide: Evidence for Possible Carcinogenic Activity | date = 2005-08-15 | url = https://ntp.niehs.nih.gov/ntp/htdocs/chem_background/exsumpdf/ethidiumbromide_508.pdf | access-date = 2009-09-30 }}</ref>

Ethidium bromide is not regulated as hazardous waste at low concentrations,<ref>{{Cite web | publisher = National Toxicology Program | title = Executive Summary Ethidium Bromide | date = 2005-08-15 | url = https://ntp.niehs.nih.gov/ntp/htdocs/chem_background/exsumpdf/ethidiumbromide_508.pdf | access-date = 2009-09-30 }}</ref> but is treated as hazardous waste by many organizations. Material should be handled according to the manufacturer's [[safety data sheet]] (SDS).{{cn|date=February 2023}}

The disposal of laboratory ethidium bromide remains a controversial subject.<ref>{{cite journal | vauthors = Hengen PN | title = Disposal of ethidium bromide | journal = Trends in Biochemical Sciences | volume = 19 | issue = 6 | pages = 257–258 | date = June 1994 | pmid = 8073504 | doi = 10.1016/0968-0004(94)90152-X }}</ref> 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.<ref>{{cite book |title=Hazardous Laboratory Chemicals Disposal Guide | vauthors = Armour MA |author-link1=Margaret-Ann Armour |year=2003 |publisher= CRC |edition=3rd |isbn= 1-56670-567-3 |pages=222–223}}</ref> 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.<ref>{{cite journal | vauthors = Lunn G, Sansone EB | title = Ethidium bromide: destruction and decontamination of solutions | journal = Analytical Biochemistry | volume = 162 | issue = 2 | pages = 453–458 | date = May 1987 | pmid = 3605608 | doi = 10.1016/0003-2697(87)90419-2 | url = https://zenodo.org/record/1253786 }}</ref> Elsewhere, ethidium bromide removal from solutions with [[activated carbon|activated charcoal]] or [[ion exchange resin]] is recommended.<ref>{{cite journal | title = Ethidium bromide and safety--readers suggest alternative solutions | journal = Trends in Genetics | volume = 4 | issue = 4 | pages = 89–90 | date = April 1988 | pmid = 3238760 | doi = 10.1016/0168-9525(88)90092-3 | last1 = Quillardet | first1 = P. | last2 = Hofnung | first2 = M. }}</ref> Various commercial products are available for this use.<ref>{{cite web |url=http://web.princeton.edu/sites/ehs/chemwaste/etbr.html |title=Ethidium Bromide Disposal |access-date=2006-10-03 |archive-url=https://web.archive.org/web/20150415220655/http://web.princeton.edu/sites/ehs/chemwaste/etbr.html |archive-date=2015-04-15 |url-status=dead }}</ref>

== Drug resistance ==

Trypanosomes in the [[Gibe River]] Valley in southwest [[Ethiopia]] showed universal resistance between July 1989 and February 1993.<ref name="Mulugeta-et-al-1997" /> This likely indicates a permanent loss of function in this area against the tested target, ''[[Trypanosoma congolense|T. congolense]]'' isolated from [[Boran cattle]].<ref name="Mulugeta-et-al-1997" />

* [[Gel electrophoresis|Agarose gel electrophoresis]] and [[gel electrophoresis of nucleic acids]]

* [[GelRed]] (itself derived from ethbr) and [[GelGreen]], marketed as safer and more intense DNA stains

* [[Propidium iodide]] and [[propidium monoazide]], related dyes

== References ==

{{reflist|30em|refs=

<ref name="Mulugeta-et-al-1997">{{cite journal | vauthors = Mulugeta W, Wilkes J, Mulatu W, Majiwa PA, Masake R, Peregrine AS | title = Long-term occurrence of Trypanosoma congolense resistant to diminazene, isometamidium and homidium in cattle at Ghibe, Ethiopia | journal = Acta Tropica | volume = 64 | issue = 3–4 | pages = 205–217 | date = April 1997 | pmid = 9107367 | doi = 10.1016/s0001-706x(96)00645-6 | publisher = [[Elsevier]] | s2cid = 23878484 }}</ref>

<ref name="Kinabo-1993">{{cite journal | vauthors = Kinabo LD | title = Pharmacology of existing drugs for animal trypanosomiasis | journal = Acta Tropica | volume = 54 | issue = 3–4 | pages = 169–183 | date = September 1993 | pmid = 7902656 | doi = 10.1016/0001-706x(93)90091-o | publisher = [[Elsevier]] | s2cid = 27564786 }}</ref>

<ref name="Hom-Cl-PubChem">{{cite web | title=Homidium chloride | website=[[PubChem]] | publisher=[[National Center for Biotechnology Information|NCBI]], [[United States National Library of Medicine|NLM]], US [[National Institutes of Health|NIH]] | url=http://pubchem.ncbi.nlm.nih.gov/compound/11765 | access-date=2021-03-14}} {{PubChem|11765}}</ref>

<ref name="Hom-Br-PubChem">{{cite web | title=Homidium bromide | website=[[PubChem]] | publisher=[[National Center for Biotechnology Information|NCBI]], [[United States National Library of Medicine|NLM]], US [[National Institutes of Health|NIH]] | url=http://pubchem.ncbi.nlm.nih.gov/compound/14710 | access-date=2020-09-08}} {{PubChem|14710}}</ref>

}}

== External links ==

* {{Commons category-inline}}

[[Category:Aromatic amines]]

[[Category:Bromides]]

[[Category:DNA intercalaters]]

[[Category:Phenanthridine dyes]]

[[Category:Staining dyes]]

[[Category:Embryotoxicants]]

[[Category:Experimental cancer drugs]]