Ethidium bromide targets nucleic acids by intercalating between the base pairs of double-stranded DNA and RNA. The fluorescence of EtBr increases 21-fold upon binding to double-stranded RNA and 25-fold upon binding to double-stranded DNA. It also binds to single-stranded and triple-stranded DNA, though with lower affinity. Due to its ability to bind DNA, EtBr is an inhibitor of DNA polymerase. It acts as a frameshift mutagen.

Ethidium Bromide (EtBr) is occasionally added to the running buffer when using agarose gel electrophoresis to separate DNA fragments. It is utilized because the DNA banding pattern can be seen when the molecule binds to the DNA and is illuminated with a UV light source. Intercalation between base pairs is how EtBr binds. The DNA molecule's weight, charge, conformation, and flexibility are all altered by this binding. DNA has always moved less freely in the gels containing EtBr[1].

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In vitro, ethidium bromide is not evaluated for traditional biological activity but is used as a nucleic acid stain. It intercalates double-stranded DNA and RNA, with fluorescence enhancement upon binding. The compound is used in fluorimetric assays for nucleic acids. It can also be used in conjunction with acridine orange to differentiate between viable, apoptotic, and necrotic cells. Its utility is based on its photophysical properties rather than pharmacological activity.

Ethidium bromide is not used for in vivo pharmacological evaluation. It is a laboratory reagent used for nucleic acid staining in electrophoresis and related applications. The compound is not administered to animals for therapeutic purposes. Its applications are limited to in vitro analytical and diagnostic uses. No in vivo activity data have been reported.

In vitro assays using ethidium bromide typically involve staining nucleic acids in agarose or polyacrylamide gels. Gels are incubated in a dilute EtBr solution (0.5 μg/mL) for 15-30 minutes, and DNA bands are visualized on a UV light box at 254 nm. EtBr can also be incorporated into the gel and running buffer at 0.5 μg/mL. The compound's fluorescence properties are characterized by spectrophotometry, with excitation and emission maxima around 518 nm and 605 nm respectively.

Ethidium bromide is not evaluated in traditional cellular assays as a therapeutic compound. However, it may be used in cell staining applications for fluorescence microscopy or flow cytometry to assess cell viability or nucleic acid content. When used with acridine orange, it can differentiate between viable, apoptotic, and necrotic cells. Cells are incubated with the dye and fluorescence is detected using appropriate excitation and emission filters.

Ethidium bromide is not used in in vivo animal experiments for pharmacological evaluation. It is a laboratory reagent used for nucleic acid staining in electrophoresis. The compound is not administered to animals for therapeutic or pharmacological assessment. Its applications are limited to in vitro laboratory settings.

Metabolism / Metabolites
Pretreatment with phenobarbital (PB) and 3-methylcholanthrene (3-MC) significantly enhanced the metabolism of ethidium bromide in isolated rat hepatocytes. Compared with untreated rat hepatocytes, PB and 3-MC pretreatment increased the content of the major metabolite ethidium bromide 8-N-glucuronide by 2.5-fold and 1.5-fold, respectively. Neither PB nor 3-MC pretreatment enhanced the formation of ethidium bromide 3-N-glucuronide. Two novel metabolites, hydroxyethidium bromide glucuronide and a transient unidentified substance, were detected by high-performance liquid chromatography (HPLC). These metabolites were generated exclusively by animal hepatocytes pretreated with 3-MC.

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Pharmacokinetic properties are not applicable to ethidium bromide as it is used as a laboratory reagent rather than a therapeutic agent. The compound has a molecular weight of 394.31 g/mol and a molecular formula of C₂₁H₂₀BrN₃. It is a solid with a melting point of 260-262°C (dec.). It is soluble in water and organic solvents. Storage: store at -20°C.

Toxicity Data
LC50 (Rat) = 11,000-134,000 mg/m³/1hr Interactions Bleomycin is an antitumor drug whose cytotoxicity is associated with the introduction of single-strand and double-strand breaks in cellular DNA. In experiments isolating cell nuclei, low concentrations of ethidium bromide significantly enhanced bleomycin-induced nuclear chromatin release. In vitro treatment of mouse L1210 leukemia cells with low concentrations of ethidium bromide followed by bleomycin treatment 1 hour later produced a synergistic effect, with an efficacy 8 times greater than that of either drug alone. Interestingly, when the order of drug addition was reversed, the synergistic effect was significantly reduced (2-fold). The combined use of DNA unwinding agents and strand-breaking agents may represent a novel and rational approach to cancer chemotherapy.

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Non-human toxicity values
Mouse subcutaneous injection LD50: 110 mg/kg
Mouse intraperitoneal injection LD50: 20 mg/kg
Rat intravenous injection LD50: 27 mg/kg
Rat subcutaneous injection LD50: 80 mg/kg
For more complete non-human toxicity data for ethidium bromide (6 types), please visit the HSDB record page.

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Ethidium bromide is considered a potential mutagen and must be handled with caution. It is classified as a frameshift mutagen. Hazard classifications include Acute Tox. 1 Inhalation, Acute Tox. 4 Oral, and Muta. 2. The compound is a known carcinogen and mutagen. Personal protective equipment including eyeshields, faceshields, gloves, and type P3 respirator cartridges should be used when handling.

[1]. Electrophoresis . 1996 Oct;17(10):1524-7.

Ethidium bromide is an organic bromide salt of ethidium. It acts as a trypanosome killer, anti-aging agent, and intercalating agent. It contains one ethidium atom. Ethidium bromide is a trypanosome killer and may also be an antiviral agent, widely used in experimental cell biology and biochemistry. Ethidium has various experimental application properties, including binding to nucleic acids, non-competitive inhibition of nicotinic acetylcholine receptors, and fluorescence. Its most commonly used form is the bromide. Mechanism of Action: Ethidium bromide (EB) is an intercalating agent that has been shown to prevent the in vitro interaction of estrogen receptor (R) with DNA. We now investigated the effect of this drug on R nuclear translocation to determine whether DNA integrity is necessary for this translocation. In a cell-free recombinant system consisting of purified nuclei and cytoplasm, pretreatment of nuclei with estradiol benzoate (EB) prevented approximately half of the estrogen receptor (R) nuclear translocation, but could not extract more than 17% of the previously translocated estradiol-R complex. A series of indirect evidences suggest that EB inhibits the nuclear translocation of R through interaction with nuclear DNA. The degree of inhibition correlates with the amount of drug bound to the nucleus and is consistent with the degree of chromatin ultrastructural modification. The drug did not cause irreversible changes to R. Inhibition of EB was observed only in DNA-containing particles and in estrogen receptors capable of binding to DNA. In viable uteruses, the drug also inhibited the nuclear translocation of R. These results indicate the existence of two types of nuclear translocation of R, one sensitive to EB and the other resistant to EB, suggesting that DNA is essential for the EB-sensitive translocation.

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Ethidium bromide (CAS# 1239-45-8, molecular formula C₂₁H₂₀BrN₃, molecular weight 394.31) is a nucleic acid stain and frameshift mutagen that intercalates double-stranded DNA and RNA. It is the most commonly used stain for PAGE or agarose gel electrophoresis. Due to its mutagenic properties, safer alternatives are increasingly favored in modern laboratory practice. No clinical trials or therapeutic approvals have been identified.

C21H20BRN3

394.31

393.084

C, 63.97; H, 5.11; Br, 20.26; N, 10.66

1239-45-8

1239-45-8 (Br); 3546-21-2; 602-52-8 (Cl)

14710

Brown to reddish brown solid powder

260-262 °C (dec.)(lit.)

>100°C

1.4575 (20ºC)

2.298

2

3

2

25

419

0

ZMMJGEGLRURXTF-UHFFFAOYSA-N

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

5-ethyl-6-phenylphenanthridin-5-ium-3,8-diamine;bromide

Dromilac; ETHIDIUM BROMIDE; EtBr; Homidium bromide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~78 mg/mL (~197.8 mM)
Ethanol : ~1 mg/mL (~2.5 mM)
Water : ~7 mg/mL (~17.8 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 9.09 mg/mL (23.05 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)