Anni Mai

Professor Grant
Chem 6310
December 1, 2011
General Toxico-Report on Ethidium Bromide
Background Information
​Ethidium Bromide (EtBr) is classified as aromatic amines, and more specifically
phenanthridine. In history, EtBr was used as a Trypanocidal agent, which kills
Trypanosome parasites. Depending on geography and economy, some countries or
areas still use EtBr for this purpose. In modern technology, EtBr is a common
fluorescent dye to visualize DNAs and RNAs in molecular biology related laboratories.
Other applications include chromosomal structure study and cell membrane
investigation.
​Information provided by the MSDS (Version from Sigma Aldrich) states that EtBr is a
carcinogen/mutagen and has acute toxicity in the severe case. The LD50 values in rat
range from 16mg/kg to 2177mg/kg depending on the way of introducing the substance.

Routes of Exposure (to human)

​In general, there are three routes of exposure to a toxin, inhalation, contact, and
ingestion. Commercial EtBr usually comes in the form of powder or solution. With the
powder form, dust and aerosols can simply be generated when transferring from the
stock container to another container. In the form of solution, ethidium’s organic property,
given by the three benzene rings and the hetero six-membered ring, would make it less
soluble in water. In other words, ethidium vapor is more likely to form. Heating of EtBr
can increase vapor effervescence. Also indicated in the MSDS, the ethidium vapor is
highly toxic. By the route of contact, it happens more frequently when the person
handling EtBr is not wearing protective gears, like gloves and lab coat. Because of
EtBr’s organic property, as well as its use in cell membrane study, it is more likely to be
absorbed by skin easily. Another way of contact may come from contaminated
equipments in the lab, or door knobs and other things a gloved person would have
touched. Base on the usage of EtBr, ingestion is the least possible exposure; containers
usually have warning labels and it would take intention ingestion to make this route
possible.

Partitioning and DNA

Intercalating
​Information listed in
the U.S. National Library
of Medicine database
states that the solubility in 2-methoxyethanol is 20mg/ml and in water is 20 parts. Again,
this reinforces that EtBr’s organic nature. The O/W partition coefficient, log Kow is
estimated to be -0.38. Therefore, ethidium would stay in the hydrophobic area more
than the hydrophilic, like the cell membrane lipid bilayer.
​Because of its partitioning behavior, ethidium is a good DNA intercalating agent. The
intercalating mechanism applies to double-stranded DNA, as well as double-stranded
RNA because a nice hydrophobic area is good for nesting ethidium. Figure 2 represents
the structure of intercalation. Although no define mechanism was described in literature,
the 3- and 8- primary amines seem to participate in hydrogen bonding to the double
helical backbone.
Frame shift mutation resulted from this intercalation, but unsure how and which
nucleotide was mis-incorporated. Mutation is the most responsible cause for cancer
development because the wrong transcripts lead to the wrong peptides that would not
fold into the proper conformation. Metabolisms that depend on this protein may not
proceed correctly.
In the chromatin structure study, the double-strand unwound but did not reach
complete unwound and then started to rewind in the opposite direction. In situation like
this, the major and minor grooves of the original double-strand are altered, and
polymerases binding that depend on groove fitting may fail to bind at the proper sites, or
bind to the wrong sites.

Figure 2. Ethidium intercalating between two AT pairs.

Ethidium Metabolism
​Literature didn’t have a great coverage on ethidium metabolism until some
researchers tried to investigate why certain pests developed resistance to ethidium
bromide which was used as pesticide. The metabolites study was done on rat model,
which used phenobarbitone and 3-methylcholanthrene separately as to induce the
metabolism of ethidium.
​For Phase I detoxification, ethidium underwent the Cytochrome P450 pathway. In
the research, Phase I was not discussed in detail, but the modification would not just
stop at the step after the insertion of O. Hydroxylation is possible on several places of
the benzene rings. Some cases of hydroxylation could lead to rearrangement and
forming quinones or quinoneimines depending on where hydroxylation happened on the
ring (see Fig. 7). Quinones would be the less problem of the two because it could be
utilized as a cofactor for redox reaction in other metabolic pathway. Quinoneimines on
the other hand is a very reactive species. This reinforced that some Phase I reaction
may indeed produce a more toxic/reactive metabolite.

​The major Phase II conjugation for ethidium is the glucuronidation. N-

glucuronidation and O-glucuronidation were the metabolites detected. For N-
glucouronidation, 3- and 8- nitrogen is more susceptible than the tertiary amine due to
steric hindrance. The O-glucuronide resulted from an intermediate, 2-hydroxy-ethidium.
Besides glucuronidation, acetyl- metabolites were also detected. The acetyl- group were
found on the 3- and 8- nitrogen again, a 3,8-diacetyl- metabolite, and the hydroxyl-
version of the 3-acetyl- were also detected.
​In this metabolite study article, the researchers had used two substances to induce
the ethidium metabolism. Phenobarbitone, or Phenobarbital sodium, is manufactured in
tablet or injection form. That means this might be a possible treatment for someone who
got high ethidium exposure; however, there is not in vivo examination ever trial on
human.
Personal Responsibility vs. Reality
​To laboratory researchers who handle ethidium bromide, no one else has greater
responsibility than they do. Reading the MSDS and understand the possible dangers
working with EtBr would be the first step for safety. The next would be making sure the
working environment is suitable, like having good ventilation and no flame around EtBr.
The most important part is wearing protective gears to minimize EtBr exposure, like
respirator mask and gloves. It also helps not to contaminate equipments or door knobs
so to reduce the exposure to other people who didn’t know.
​After talking about all the toxico-related areas of ethidium bromide, one has to look
at the reality. Although no study has done on the level of ethidium exposure and risk of
developing cancer, one can apply common sense to the situation. For example, the
ethidium bromide used in staining an agarose gel is usually 1ug/ml; the vapor produced
from such low concentration would not be that deadly. Of course, this hypothetical
situation is a short time exposure. The deal is, ethidium bromide is not that dangerous
at most of the low, working concentration, but just be aware that long time exposure
could possibly lead to accumulation and might not detoxify efficiently enough.

Works Cited
"Ethidium Bromide." TOXNET. U.S. National Library of Medicine. Web. 01 Dec. 2011.
<http://toxnet.nlm.nih.gov>.
File:DNA Intercalation2.jpg. Digital image. Wikipedia, the Free Encyclopedia. Web. 01
Dec. 2011. <http://en.wikipedia.org/wiki/File:DNA_intercalation2.jpg>.
"Phenobarbital." Wikipedia, the Free Encyclopedia. Web. 01 Dec. 2011.
<http://en.wikipedia.org/wiki/Phenobarbital>.
Sterz, R., M. Hermes, K. Peper, and R.j. Bradley. "Effects of Ethidium Bromide on the
Nicotinic Acetylcholine Receptor." European Journal of Pharmacology 80.4
(1982): 393-99. Print.
Tettey, J.n.a, G.g Skellern, J.m Midgley, M.h Grant, and A.r Pitt. "The Effect of Inducing
Agents on the Metabolism of Ethidium Bromide by Isolated Rat Hepatocytes."
Chemico-Biological Interactions 123.2 (1999): 105-15. Print.
Vergani, L. "Ethidium Bromide Intercalation and Chromatin Structure: A Thermal
Analysis." Thermochimica Acta 294.2 (1997): 193-204. Print.