Biomedicine & Pharmacotherapy 72 (2015) 7482

Available online at

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Review

Using the comet and micronucleus assays for genotoxicity studies:

A review
Rodrigo Pinheiro Araldi a,b,c, Thatiana Correa de Melo a,d, Thais Biude Mendes c,d,
Paulo Luiz de Sa Junior a, Bruno Heidi Nakano Nozima d, Eliana Tiemi Ito c,
Rodrigo Franco de Carvalho a, Edislane Barreiros de Souza c, Rita de Cassia Stocco a,*
a

Laboratorio de Genetica, Instituto Butantan, Sao Paulo, Sao Paulo, Brazil

Programa de Pos-graduacao Interunidades em Biotecnologia, Instituto de Ciencias Biomedicas (ICB), Universidade de Sao Paulo (USP), Sao Paulo, Sao Paulo,
Brazil
c
Laboratorio de Biologia Molecular, Genetica e Mutagenese, Faculdade de Ciencias e Letras de Assis, Universidade Estadual Paulista Julio de Mesquita Filho
(UNESP), Assis, Sao Paulo, Brazil
d
Programa de Pos-graduacao em Morfologia Funcional e Estrutural, Escola Paulista de Medicina, Universidade Federal de Sao Paulo (UNIFESP), Sao Paulo,
Sao Paulo, Brazil
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 11 March 2015
Accepted 3 April 2015

Physical, chemical and biological agents can act in the DNA, resulting in mutation involved in cancer.
Thus, genotoxic tests are required by regulatory agencies in order to evaluate potential risk of cancer.
Among these tests, the comet assay (CA) and micronucleus assay (MNA) are the most commonly used.
However, there are different protocols and recommendations already published. This is the rst review,
after the inclusion of CA in S2R1 guidance and OECD 489, which summarizes the main technical
recommendations of both CA and MNA.
2015 Published by Elsevier Masson SAS.

Keywords:
Genotoxicity tests
Comet assay
Micronucleus assay
Mutagenesis

1. Introduction
Even before the description of the DNA structure, it was already
evident that chemical, physical and biological agents could
interact with the genetic material, resulting in mutations [13],
which are associated to genomic instability and cancer [4].
Considering this, regulatory agencies such as Food and Drug
Administration (FDA), European Medicines Agency (EMA) and
Agencia Nacional de Vigilancia Sanitaria (ANVISA, Brazil) begun to
require tests of genotoxicity as essential part of drug validation
[5,6]. These tests include in vitro and in vivo assays to detect the
drug potential to induce genetic mutations and/or chromosomal
aberrations [7,8].
The Guideline S2 (R1) on Genotoxicity Testing and Data
Interpretation for Pharmaceuticals Intended for Human Use is
applied by FDA, EMA and ANVISA to test new drugs under
development. This Guideline suggests two options of battery tests:
Option 1 test of reverse mutation in bacteria, followed of one

* Corresponding author. Tel.: +55(11)2627 9701.

E-mail address: rita.stocco@butantan.gov.br (R. de Cassia Stocco).
http://dx.doi.org/10.1016/j.biopha.2015.04.004
0753-3322/ 2015 Published by Elsevier Masson SAS.

in vitro cytogenetic test to evaluate chromosomal damages

(chromosomal aberration or micronucleus assay) or genetic
mutation test in mice lymphoma TK cell and one in vivo test
(chromosomal aberration or micronucleus assay); Option 2 test
of reverse mutation in bacteria and in vivo genotoxicity evaluation
in two tissues: hematopoietic (micronucleus assay) and other in
vivo test [79], such as the comet assay [10]. However, the
guideline also allows the use different methods, since the
researcher/institution can prove the drug safety.
Among the available genotoxicity tests, comet assay (CA) and
micronucleus assay (MNA) are recognized due to their robustness,
sensitivity and statistical power to evaluate DNA breaks, which can
be considered hallmarks of mutagenicity [11]. Furthermore,
currently studies point out that the association of CA and MNA
is the best battery test to evaluate the mutagenic potential, since
both assays are highly sensitive, simple and allow to detect breaks
at chromatic and chromosomal levels, respectively [10]. However,
in function of great quantity of protocols published and the latest
discovery and recommendations of both CA and MNA, it is required
a review about these techniques. This review brings the latest
technical considerations and possible applications for CA and MNA
based on the literature and authors expertise. Moreover, this is

R.P. Araldi et al. / Biomedicine & Pharmacotherapy 72 (2015) 7482

rst review that brings technical aspects of these assays after the
CA and MNA had been included in the S2R1 and OECD 489 guidance
as battery option [12].

2. Comet assay (CA)

The CA, also known as single cell gel electrophoresis (SCGE) or
stling and
microgel electrophoresis (MGE), was introduced by O
Johanson [13] to detect DNA damages induced by radiation. Since
its development, several methodological modications were
proposed [14]. However, the alkaline method, developed by Singh
et al. [15], that allows the DNA denaturation, as well as the
detection of alkali-label sites, became the most used and
recommended due its broad-spectrum of detection of DNA damage
[1418].
CA has been used in different studies, such as: toxicology
genetics [1921], biomonitoring [2228], eco-genotoxicity
[2931], molecular epidemiology [32], nutrigenomics [33,34],
DNA repair system studies [3538], evaluation of nanomaterial
genotoxicity [39], evaluation of DNA integrity in mesenchymal
stem cell [40] and spermatozoids [4144]. CA was also proposed to
detect of bacteriophage mediated bacterial cell lysis [45] and
employed in plants [46].
Since currently works point out its versatility, CA has been
extensively employed in toxicological genetics studies [17,19,
4750], as it can be used as indicative of virus activity of both
human papillomavirus (HPV) [51] and bovine papillomavirus
(BPV) [52]. Studies involving the CA in virology have been
contributed with the elucidation of viral oncogenesis mechanisms.
Genotoxic action of measles virus [53] and bovine leukemia virus
[54] was also reported using the assay. Thus, CA can be considered
a gold standard method to study the oncogenic process associated
with virus infection [55]. Due the CA versatile, the technique was
currently included in the International Conference on Harmonization
of Technical Requirements for Registration of Pharmaceutical for
Human Use (ICH) S2R1 guidance [12].
After the standardization of the CA methodology in the
International Workshop on Genotoxicity Test Procedures [14] and
the establishment of technical recommendations on the 4th
International Workshop on Genotoxicity Testing [47], CA was
adopted as part of the battery of validation tests for new drugs
by pharmaceutical industries [5,56]. Thus, the in vivo rodent CA
was validated in 20062012 by the Japanese Center for the
Validation of Alternative Methods (JaCVAM) in conjunction with
the European Center for the Validation of Alternative Methods
(ECVAM), the Interagency of Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and the NTP
Interagency Center of Evaluation of Alternative Toxicological
Methods (NICEATM) [10].
A study with 838 drugs, analyzed by the CA, pointed out that
56.3% of them were genotoxic [56]. Other study, with 476 drugs,
also analyzed by the same methodology, showed that 43.5% of
them were genotoxic [6]. These data indicate the importance of
the mutagenic evaluation of pharmaceutical products, as well as
of new drugs candidates to enter in the market. Based on the
importance of results obtained by CA to predict possible
genotoxic risks, the assay was rstly proposed by the ICH and
recommended by FDA and EMA in the mutagenic analysis of
drugs [14,47,49]. Besides, CA is recommended a rst line
mutagenic test due its high sensitivity in relation to the
micronucleus assay (MNA) [49].
CA can be performed with any eukaryotic cell population in
vivo, in vitro or ex vivo, including vegetal tissue as Allium cepa
[57]. Other advantages of the technique include: simplicity and
low cost and time, since the protocol can be executed in less than

75

24 h [4,14,47,5861]. The CA allows to analyze the genotoxicity in

specic tissues, which are in direct contact with the tested
substance or in which occur the absorption, distribution,
metabolizing or excretion, allowing to detect the clastogenicity
in situ [5,14]. The technique could be associated to uorescent in
situ hybridization (FISH), brings new possible of its use to analyze
the DNA damage induction [62].
Due all advantages and applications of this technique, the
number of publications involving the CA has grown in the last
years consistently [59,63], making the comet assay a eld of great
interest [64,65]. PubMed registers more than 7600 citations of CA
between 1990 and 2013, reinforcing the importance of this
technique [65]. The database of PubMed registered 737 publications involving the CA in 2014 and 173 in 2015, since this date. In
function of the greater importance of the CA, some journal
dedicates special issues to the assay. The latest was published in
2015 by Mutagenesis [65].
CA also allows to detect breaks in DNA strands, which can be
visualized by the increased migration of free DNA segments,
resulting in images similar to comets, justifying the name of the
assay [37,60]. There are three CA techniques available: acid,
alkaline and neutral, based on the pH of the electrophoresis
buffer employed. At rst, it was established a paradigm that the
neutral technique allows to detect double strand breaks (DSBs),
whereas the alkaline technique, simple strand breaks (SSBs)
[58]. However, the CA indicates both SSBs and DSBs, independently of the used technique [14,60]. These SSBs and/or DSBs are
associated to chromosomal aberrations and genomic instability
[66]. The genomic instability is directly associated to malignancy [6773].
2.1. Technical principals and recommendations for the alkaline CA
The CA consists in the immobilization of a cellular suspension,
homogenized with low melting point (LMPA) agarose, in pretreated slides with normal melting point (NMPA) agarose
[52,60]. The material is covered with a coverslip in order to
ensure a homogeneous distribution. After the solidication, the
coverslip is removed and the slides are transferred to the lysis
solution [52]. This lysis solution contains cellular surfactants
(Triton X-100), which remove membranes [17,60]. The slides are
transferred to electrophoresis tank, being treated with a solution of
sodium chloride, in a concentration greater than 2.0 M and pH
>13.0 [19]. This solution promotes histone release and DNA
unfolding. Under electrophoretic eld, free DNA segments, product
of breaks (clastogenesis), migrate in direction of the cathode,
originating a comet tail [60]. After electrophoresis, the material is
neutralized, xated and stained. The slides are analyzed in
uorescent microscopy or optic microscopy, according to the
employed dye [60,74]. Although different methodologies have
been published, some recommendations were established to
guarantee the result quality. Among these recommendations are
as follow:
2.1.1. Choice of biological sample
CA can be performed in any tissue, including: whole blood [75],
peripheral blood mononuclear cells (PBMCs), isolated with FicollPaque or TrisEDTA buffer (TE) [61] or culture cells [76]. However,
the genotoxicity studies of chemical compounds require special
attention to the age of the biological material donor. Extensive
observations suggest that DNA damage accumulates with age
[11,77].
2.1.2. Material conservation
Studies point out that blood conservation of 4 8C induces DNA
damages, being recommended the conservation at 20 8C, 80 8C

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R.P. Araldi et al. / Biomedicine & Pharmacotherapy 72 (2015) 7482

or 196 8C [60,61,75,78,79]. Studies involving samples of fresh and

cryopreserved blood conrmed that the cryopreservation does not
induce DNA damage [78,79]. It is recommended that tissue
samples should be preserved at 20 8C in culture medium,
supplemented with 10% of fetal bovine serum and 10% of DMSO
(dimethyl sulfoxide) [60].

histones [60]. Electrophoresis may be performed in following

conditions: constant current of 300 mA, temperature between 4 8C
and 15 8C (to avoid DNA damages), for 20 min employing 1.15 V/
cm or 30 min at 0.8 V/cm [16,19,52,60]. After electrophoresis, it is
recommended to wash the slides in a neutralizing buffer for 5 min,
thrice [74].

2.1.3. Cell culture

Cell on cultivation at 37 8C promotes DNA damages [58]. So, cell
destined to the CA may be incubate for at a maximum of 72 h, to
avoid false-positive results [58].

2.1.8. Staining methods

Different dyes can be used in CA, including silver stain [8486],
ethidium bromide (EtBr) [19], propidium iodide (PI) [52], DAPI
(40 ,6-diamidino-2-phenylindole) [74,87], YOYO-1 [74] and SYBR
Gold [88]. Although it is known that DAPI and YOYO-1 increase the
sensitivity of DNA damage detection [74], EtBr and PI are the most
commonly used dyes [17,59].

2.1.4. Cellular isolation from tissue

Cellular isolation is required when sample tissues are
employed. Studies show that any form of cellular dissociation,
enzymatic, employing collagenase or trypsin, or mechanical is able
to induce DNA damage [4,14]. Some recommendations to avoid
DNA damages due the cellular dissociation are: use of trypsin at a
concentration of 0.01% for cellular dissociation [58], addiction of
EDTA to chelate the calcium and/or magnesium, avoiding
endonuclease activation and DMSO to prevent DNA damages
induced by the oxidation [14].
2.1.5. Lysis conditions
The lysis solution employed in the CA is based on Cook et al.
[80], which comprises a non-ionic detergent and a high molarity of
sodium chloride. The lysis removes the plasmatic membrane,
cytoplasm, nucleoplasm and more than 95% of all proteins,
allowing the migration of free duplex fragments [16,81]. Incubation
should be performed at 4 8C to avoid possible DNA damages for a
minimum of 1 h and a maximum of four weeks [60]. Addiction of
DMSO in lysis solution is also required when samples of whole
blood are employed to prevent DNA damages related to hemolysis
[14]. After lysis, it is recommended to wash the material in distilled
water to remove residues of salt and detergents that could affect
electrophoresis [14]. Although the lysis time could be between 1 h
to 1 mouth, it is important that each laboratory standardizes a time
of lysis [82].

2.1.9. Material analysis

The nucleoids can be analyzed by means of automated methods
or visual count and classication [37]. Automated methods are
based on densitometry parameters. They identify the emitted
uorescence intensity and geometrical aspects of the nucleoids,
such as tail length, head diameter and comet area [58]. The visual
method consists in analyzing 100 nucleoids per slide, which are
classied according to two systems: 02 [17,52] or 04 [60] (Fig. 1),
where nucleoids without DNA damage are classied in 0 and, those
with maximum damage in 2 or 4, depending on the system used.
Based on the number of comets observed per class in a total of
100 nucleoids, the score is obtained according to the following
formula: Score = Si  Ni, where i is the DNA damage class (02 or
04) and Ni, the number of nucleoids observed per class [89,90].
The visual method is preferably eligible than the automatized
method because overlapping nucleoids can be classied as a
unique comet [60]. Other problem associated to the automatized
systems is the recognition of hedgehog comets, cells that show
elevate DNA damage, since computer programs can analyze the
head as separate to the tail, classifying these comets as class zero
[60]. Based on these data, the visual method is adopted preferably
by most of laboratories [18,19,22,52].
2.2. Can comet assay be used to assess apoptosis?

2.1.6. Agarose concentration

The agarose concentration is considered a critic factor for the
technique [83]. It is recommended the concentration of 1.01.5% of
NMPA agarose at 60 8C for slide pre-coverage and, 0.60.8% of
LMPA agarose at 37 8C for homogenization of the biological
material [14,60].
2.1.7. Electrophoretic conditions
It is required the incubation of the material in an alkaline
electrophoresis buffer at 4 8C for 40 min to promote the release of

Although the CA has been used to predict apoptosis [17,9092],

the presence of hedgehog comets, characterized by a small head
and a long or absent tail, is not considered a apoptosis indicator by
some authors [14,18,60].
Apoptosis is a general term commonly used to describe a no
inammatory programmed cell death in contrast with frequently
non-programmed and highly inammatory necrosis. Several
features confer to apoptotic cells a peculiar signature allowing
them to be discriminated from cells compromised with others

Fig. 1. Classication of nucleoids according two systems: (A) 02, in which 0 represent cells without DNA damage and 2, cells with maximum DNA damage and (B) 04, in
which 0 represent cells without DNA damage and 4, cells with maximum DNA damage.

R.P. Araldi et al. / Biomedicine & Pharmacotherapy 72 (2015) 7482

non-apoptotic mechanisms [93]. Apoptosis is characterized for a

broad range of features that taken together constitute the
apoptosis signature. Among all apoptotic signatures, stands out
the morphological feature on which dying cells depict cytoplasm
retraction, detachment from surface and pyknosis. In addition, to
these morphologic rearrangements, several enzymes are concomitantly activated and act accurately in order to turn apoptosis an
irreversible process. The process starts with Flippases, which are
responsible for moving the phospholipid phosphatidylserine from
cytosolic (inner side) to out of leaet from cytoplasm membrane
[94]. Then, the cysteine-proteases caspases family (e.g. CASP3,
CASP6, CASP7) are responsible to deconstruct the cell framework
[95]. Finally, the caspase-activated-DNAse (CAD) promotes DNA
cleavage, generating fragments containing 180200 base pair (bp)
fragments. These DNA fragments are packaged into small vesicles
originated from cytoplasm membrane named apoptotic bodies
which, in turn, are cleaned through phagocytosis by neighboring
cells, concluding the apoptotic process [96].
Cellular tests aiming to decipher the cytotoxic mechanisms by
which molecules with therapeutic potential operate are widely
used in drug development [93]. Thus, the choice of the techniques
which must be applied will depend on the nature of the response
that is sought. For instance, light morphological determinations
are advantageous since is inexpensive, easy execution and rapid to
perform. Disadvantages are propensity to underestimation and
unsuitability for quantitative studies. Immunological methods
such as cytouorometry allows automated analyses on a per-cell
basis while requires high performance of primary antibodies.
Electron microscopy provides precise ultra-structural information
but is expensive and time-consuming. In addition, once the
methods applied to cell death characterization can be inuenced
by a wide range of interfering factors, results obtained by one
method must be conrmed by another one [93]. Thus, currently
several methods to identify DNA damages may be performed and
CA stands out as a potential alternative to evaluate DNA injuries
mainly due easy implementation and low costs. Many groups have
been applied CA in order to evaluate DNA damages in apoptotic
cells. However, albeit little is known about the mechanisms
involved in DNA fragmentation in necrotic cell, it is well
established that necrosis also leads to a DNA degradation
[97]. In this context, the result obtained through CA per se does
not allow characterize apoptosis since necrotic cells also presents
DNA fragmentation. Therefore, we suggest that studies aiming
elucidating therapeutics and cytotoxic mechanisms of chemical
compounds or even radiotherapy should also include tests for
determining the type of cell death (e.g. apoptosis, necrosis or
others) as well as assays for clastogenicity in order to avoid mutual
interference and misinterpretation. However, it is interesting to
stress that even performing these tests for cell death and
clastogenicity detection, this issue is still not totally solved since
not all DNA fragments are related to cell death process described
above. Dividing cells containing DNA strands breaks lacking
centromeres or even entire chromosomes that do not migrate to
spindle pole during cytokinesis can generate chromosomal
fragments that are packaged in nuclear envelope forming
structures that resembles small interphase nuclei, named micronuclei (MN) [98]. Apoptotic cells can form MN, however, the
relationship between MN and apoptosis is deeply complex and
new studies has to be developed in order to achieve better
understanding of this imbricate issue [99].

3. Micronucleus assay (MNA)

The MNA is an important in vivo and in vitro biomarker,
extensively used in the molecular epidemiology and cytogenetic

77

damages in populations exposed to genotoxic agents [100

104]. The micronucleus (MN) term, also known as HowellJolly
bodies [105], was introduced in 1951 related to acentric fragments
expelled from the main nucleus at late stages of anaphase
[106]. MNs can be formed through two mechanisms: chromosomal
breaks (clastogenesis) or disruption of the mitotic apparatus
(aneugenesis) [101,103]. Thus, using anti-kinetochore antibody it
is possible to verify if a particular drug induces MN formation via
clastogenesis or aneugenesis [105]. Absence of kinetochore in the
MNs indicates clastogenic action, whereas its presence, aneugenic
action [105].
MNs are formed along the erythropoiesis, which occurs in the
bone marrow or spleen of adult rodents [105]. Erythroblasts
excluded the nucleus after 6 h of the nal mitosis [107], originating
polychromatic erythrocytes (PCE), basophilic cells that contain
RNA in the cytoplasm detectable by Giemsa stain [105]. The PCEs
suffer a maturation process, originating normochromic erythrocytes (NCEs), acidophilic cells that stain orange or orange-pink
with Giemsa [105]. Thus, clastogenic and/or aneugenic agents are
able to originate chromosomal fragmentation or chromosomal
losses during the cellular division that are not integrated in the
nucleus of daughter cell, resulting in MNs [98,105]. These MNs are
enveloped by nuclear membrane during the telophase [98] and can
be visualized in the cytoplasm [105]. An elevate frequency of
micronucleated PCEs (MNPCEs) indicates chromosomal damage
[98,105,108].
However, the MNs not only represent chromosomal losses, but
also the result of DNA amplication [103]. DNA amplication is
commonly observed in oncogenic process, resulting in double
minute chromosomes (DM), which are removed from the main cell
nucleus, originating MNs [103,109]. The MNs expelling is
associated with the loss of allele dose, contributing to carcinogenesis [101,102]. A scheme of the micronucleus formation is shown
in Fig. 2.
Since 1959, the MNs have been proposed as marker of
cytogenetics damages [106]. However, the frequency analysis of
MNs as a cytogenetics test was only proposed in 1970 by Boller and
Schimid [110] and latter employed in polychromatic erythrocytes
of bone marrow [111] and lymphocytes [112]. The MNA has been
widely used in studies of genotoxicity [113115]. MNA can be
applied to any eukaryotic cell, being preferable used in substitution
of the chromosomal aberration test, because it does not require
karyotype analysis [108,114,116,117].
MNA, as well as the comet assay, has been used in virology eld.
Studies point out that the Tax protein of Human T-Leukemia Virus
type I (HTLV-I) and II (HTLV-II) induces the increase of MN
frequency [118,119]. Similar results were also observed with the
Epstein-Barr Virus (EBV) [120]. Currently studies have been
demonstrated that HPV is related with the increase frequency of
MN in both cytology and peripheral blood of women infected by
the virus [121,122]. Duensing and Munger [123,124] demonstrated that the E6 and E7 oncoproteins of HPV are directly associated
with the MNs induction. Other study developed for our group
pointed out that PBMCs and epithelial cells of bovine kidney (CRIB)
treated with 1 mg/ml of recombinant E6 oncoprotein of BPV-1
show elevated frequency of MN in relation to not treated cells (data
not published).
Some advantages of MNA over CA are: it only considers genetic
damages in mitotic cells, whereas CA detects DNA damages in both
interphase and mitotic cells [125], the MNA has a greater statistical
power, since it analyze over 1000 cells, whereas the CA analyze
100 cells [125]. Although the MNA has advantages in relation to the
chromosomal aberration test and CA, studies in toxicology genetic
reinforces the necessity of more than one mutagenic test
[19,89,126]. Thus, the association of MNA and CA can be
considered as a gold standard among mutagenic tests, because

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R.P. Araldi et al. / Biomedicine & Pharmacotherapy 72 (2015) 7482

Fig. 2. Different pathway of mutagenic drugs. Cytotoxic agents induce an early necrosis phase (1), which evolves to necrosis (2). Genotoxic agents induce DNA damages during
the G0 to S phase (3), which can induce apoptosis (5), resulting in apoptotic bodies formation, during the late apoptosis (6) or, leave to chromosomal damages, resulting in
micronucleus formation through the chromosome breaks and/or disruption of mitotic apparatus (4). Micronucleated cells can be destined to apoptosis (5 and 6).

they have high sensitivity, statistical power, being simple,

versatile, and demands low cost of time and investment. MNA
can be performed in polychromatic erythrocytes (in vivo) [19,104]
and cells culture of both lymphocyte [103] or cells lineages (in
vitro) [127]. Images of cells micronucleated obtained with both in
vivo and in vitro MNA are shown in Fig. 3. The in vitro technique
requires the cytokinesis blocking, being known as cytokinesisblock micronucleus assay (CBMNA) [108].
The MNAs protocol was standardized by means of an
international consortium between pharmaceutical companies F.
Hoffmann-La Roche, Novartis, Rhone-Poulenc Rorer and Biologie
Servier [117]. This consortium pointed the MNA as indicated to the
genotoxicity evaluation of drugs [117]. Furthermore, the MNA was
elect by the International Workshop on Genotoxicity Test Procedures
as gold standard test in mutagenesis [106]. The main recommendations for both in vivo and in vitro MNA are discussed in this
review.
3.1. Technical recommendations for the in vivo micronucleus assay
(MNA)
The in vivo MNA is preferably recommended in relation to the in
vitro MNA because it allows to evaluate the drug metabolizing,
pharmacokinetics and DNA repair [105,128]. The in vivo MNA is the
rst assay in the battery of genotoxicity tests accepted by agencies
such as FDA and EMA [105,129].
3.1.1. Species selection
Although any rodent can be used for the in vivo MNA, without
preference for strain, rats and mice are the most commonly species
employed [19,104,130]. However, studies pointed out that the
spleen of rats and humans are able to remove the MNPCEs of the

blood circulation [131,132]. For this reason, the MNPCEs frequency

analysis in peripheral blood is only accepted for mice [128].
It is recommended the use of animal aged to 68 weeks
[19,129], of both sexes, once different responses of metabolizing,
toxicity or pharmacokinetics can be observed according to the sex
[19,107,130]. The animal may be acclimated at a minimum period
of ve days in following conditions: food and water ad libitum,
lightdark cycle of 12:12 h and a temperature of 22  2 8C
[19,105,133].
3.1.2. Dose level and administration pathway
This is recommended a limit dose of 2 g/kg/day for treatments
of 14 days or less or, 1 g/kg/day for treatments period greater
than 14 days [105,130]. The tested drug(s) may be dissolved in
water or isotonic saline, for hydrophilic compounds, or vegetable oil for hydrophobic substances [130]. The drugs can be
administrated by oral gavage, subcutaneously or intraperitoneally [130,133].
3.1.3. Controls
The in vivo MNA requires a negative, positive and experimental groups, each one with a minimum of ve animals
[130]. As negative control, the drug solvent may be used
[19,130]. Positive control is required in all assays of toxicology
genetics [129]. Different drugs have been used as positive
control. Some examples of drugs and their respective concentrations used as positive control for mice are: 200 mg/kg ethylmethanesulfonate (CAS 62-50-0), 50 mg/kg N-ethyl-N-nitrosourea (CAS 759-73-9), 50 mg/kg mytomicin C (CAS 50-07-7)
and
4050 mg/kg
cyclophosphamide
(CAS
50-18-0)
[19,105,133]. Positive control may administrated by a different
pathway of the tested drug, if possible [105].

R.P. Araldi et al. / Biomedicine & Pharmacotherapy 72 (2015) 7482

79

Fig. 3. Possible results observed with both in vivo (AC) and in vitro (DM) micronucleus assay. Images of erythrocytes, obtained with total magnication of 1000:
normochromatic (A), polychromatic (B) and polychromatic with micronucleus (C). Images binucleated lymphocyte, obtained using Cyt-B in a total magnication of 1000:
normal, without chromosomal damage (D), with nucleoplasmatic bridge (E), showing two micronucleus (FI). Images of apoptotic bodies induce (JM).

3.1.4. Bone marrow obtainance

The bone marrow may be collected 2448 h after the single
dose administration or 1824 h after the last application of the
tested drug, for substances administrated more than once a day
[105]. A total of 2000 PCEs may be analyzed, being observed the
frequency of MNPCEs [130]. MNs can be stained with acridine
orange [134,135], Giemsa [136], Hoechst 33258 and pyronin-Y
[130].
3.1.5. Micronucleus analysis by ow cytometry
The use of ow cytometry was standardized in the 4th
International Workshop on Genotoxicity Test Procedures, representing an advance in the MNA [128]. The ow cytometry allows to
analyze a greater number of cells, which increase the statistical
power of the technique, as well as its sensitivity [128]. This
technique requires the use of anti-CD71 antibody and propidium
iodide for MNs staining [131,132]. The CD71 is a marker of
erythrocyte maturation, which is present is PCEs [131].

3.2. Technical principals and recommendations for the in vitro

micronucleus assay (CBMNA)
The CBMNA is an in vitro technique used to evaluate the
genotoxicity potential in lymphocytes or cell culture. The
technique requires the use of cytochalasin B (Cyt-B). The Cyt-B
is a metabolite, obtained from the fungal Drechslera dematioidea,
that inhibits both the rate of actin polymerization and the
interaction of actin [137], blocking the cytokinesis without causing
DNA damages [100,108]. The Cyt-B may be added after 24 h of
lymphocyte culture start or 1 h after the cells synchronization at a
maximum concentration of 0.01 M [138]. The cell synchronization
can be performed through the cell culture without fetal bovine
serum [139,140]. The use of Cyt-B in lineage cells is optional, once
genotoxic agents induce the formation of MNs per se [106]. Despite
considered optional, its use allows to evaluate the presence of
nucleoplasmatic bridges [106]. These bridges result from dicentric
chromosomes in which the two centromeres are pulled to opposite

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poles of the cell, indicating chromosomal rearrangements

[98]. However, the Cyt-B use is obligatorily required in lymphocyte
culture, being its use recommended in the following concentrations: 3 mg/ml for whole blood and 6 mg/ml for culture of
lymphocyte isolated with Ficoll-Paque or TE [106]. Cyt-B may
be dissolved in DMSO [98,141] and, applied after 44 h to
lymphocytes culture [108] or 1 h after the cell synchronization
of lineage cells [100,127,142].
For the CBMNA, the time of cell culture cannot exceed 72 h
[114] and the dose of tested drug cannot exceed 10 mM [106]. A
total of 1000 cells may be analyzed per slide [106]. Based on the
ratio between the micronucleated cells (A) observed and the total
of binucleated cells (B), it is possible to calculate the frequency of
MNs formation (MNr0) by the formula: MNr0 = A/B [127]. It is also
possible to calculate the index of proliferation with blocking
cytokinesis (IPBC). The IPBC can be obtained by the formula:
IPBC = [(1  N1)  (2  N2)  (3  N3)]/1000, where N1 is the
number of mononuclear cells, N2, number of binuclear cells and
N3, number of trinuclear cells.
4. Conclusion
In summary, the CA and MNA together allow to detect
aneugenic and clastogenic substances with high sensitivity and
statistical power. The use of both tests allows also evaluating the
systemic or in situ genotoxicity, once both assays can be employed
in specic tissues or cells. Thus, the introduction of the
recommendations summarized in this review, based on others
studies and reviews, by regulatory agencies, such as ANVISA,
should be considered. These recommendations are important to
guarantee the reliability of results.
Acknowledgements
The authors thanks to Fundacao de Amparo a Pesquisa do
Estado de Sao Paulo (FAPESP) process 2014/20617-5 by the
nancial support.
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