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Biomarkers, 2015; 20(1): 64–70

! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.992813

RESEARCH ARTICLE

Methy-sens Comet assay and DNMTs transcriptional analysis as a

combined approach in epigenotoxicology
Alessio Perotti1,2#, Valeria Rossi1, Antonio Mutti2, and Annamaria Buschini1#
1
Department of Life Sciences and 2Department of Clinical and Experimental Medicine, Università degli Studi di Parma, Parma, Italy
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Abstract Keywords
Epigenotoxicology needs simple and fast tools to assess xenobiotic epigenetic load. This work Comet assay, DNMT, epigenotoxicology,
proposes a comet assay modification designed to detect global methylation changes (Methy- methylation
sens Comet) through enzymatic digestion with two restriction enzymes (HpaII, MspI). In the
methylation-sensitive protocol tested for repeatability on A549 cells, nickel chloride induced History
hypermethylation and decitabine-induced hypomethylation. A concomitant assessment of DNA
methyltransferases (DNMTs) genes transcriptional levels has been performed, to implement a Received 7 October 2014
multifunctional approach to epigenotoxicology. Methy-sens Comet showed a general good Revised 24 November 2014
repeatability and sensitivity to methylation changes while DNMTs transcriptional levels granted Accepted 25 November 2014
additional proof of xenobiotic-induced impairment of methylome maintenance. Published online 17 December 2014

Introduction Data on xenobiotic epigenotoxic effects is increasing,

For personal use only.

while the need for tools to evaluate epigenotoxic activity of

The field of environmental epigenetics has been growing fast
compounds remains. Accordingly, we have chosen to focus on
in the past decade, generating awareness on the need of a new
the study of epigenotoxicity through the use of a relatively
approach in toxicology studies: epigenotoxicology. It is now
novel technique based on a solid, proven and broadly used
broadly accepted that environmental pollutants may express
system, such as the comet assay. The alkaline comet assay
part of their pathogenicity through epigenetic modifications.
(Singh et al., 1988) is a powerful, easy and versatile technique
Dynamic chromatin remodeling is a required step for the
that has been a landmark in DNA damage monitoring for over
initial phases of gene transcription and it can occur even after
two decades. This single cell approach gives the opportunity of
complete cell differentiation in response to environmental
a keen examination of genotoxic stress in a cell population. It
stimuli or xenobiotic interaction. Epigenetic factors, such as
has been applied to numerous types of cells ranging from
DNA methylation, histone modification and micro-RNAs,
animal and plant tissue (Brendler-Schwaab et al., 1994;
have a key role in these remodeling and alterations in these
Gichner et al., 2009; Sasaki et al., 1997) to specific human
mechanisms could lead to disease (Szyf, 2007).
tissue (Hughes et al., 1996; Øsnes-Ringen et al., 2013; Rojas
DNA methylation, in particular, is tightly linked to
et al., 2000; Szeto et al., 2005).
transcriptional silencing and fundamental in gene regulation,
Over the years, several modified protocols have arisen for
development and pathological events (Smith & Meissner,
the comet assay, allowing its users to identify different types of
2013). This epigenetic modification consists of an addition of a
damage. The use of lesion-specific enzymes has been a major
methyl group to the 50 position of the cytosine ring catalyzed by
contribution to the strength and potential of the comet assay,
a class of enzyme called DNA methyltransferase (DNMTs),
thus making it an optimal choice in biomonitoring. Good
whose primary role is the maintenance of the correct methy-
examples are FPG, ENDO III and OGG1-modified protocols
lation pattern in the genome. Epigenetic instability and,
for the detection of oxidative damage (Collins et al., 1993; Li
more specifically, uncontrolled variations in the global and
et al., 1997; Smith et al., 2006) and the UDG-modified protocol
local methylation profile and changes in DNMTs activity have
for the detection of misincorporated uracil (Duthie &
by now been recognized as a gateway to carcinogenesis
McMillan, 1997).
(Hatziapostolou & Iliopoulos, 2011).
Starting from 2006, two research teams have tried to develop
a comet assay version capable to detect global methylation
levels (Wasson et al., 2006; Wentzel et al., 2010). These
#Alessio Perotti and Annamaria Buschini are responsible for statistical methods showed promising results, but they were not followed
design and analysis. E-mail: alessio.perotti@nemo.unipr.it (A. Perotti); by further studies. Both methods were based on the utilization
annamaria.buschini@unipr.it (A. Buschini).
of two restriction endonucleases: HpaII and HhaI (Wasson
Address for correspondence: Prof. Annamaria Buschini, Department of
et al., 2006) or HpaI and MspI (Wentzel et al., 2010). In
Life Sciences, Università degli Studi di Parma, Parco Area delle Scienze
11/a, 43124 Parma, Italy. Tel: +39-0521905608. Fax: +39-0521905604. particular, Wentzel proposed the use of two isoschizomer
E-mail: annamaria.buschini@unipr.it endonucleases that both share the same restriction site (typical
 DOI: 10.3109/1354750X.2014.992813
of CpG islands) but one of them (HpaII) is blocked by
methylation in the restriction site; HpaII amount of digestion is
therefore inversely proportional to the cell global methylation
level. This allows the evaluation of global methylation changes
by the ratio between the two enzyme separate digestions,
bypassing, in fact, the contribution given to the tail intensity due
to an eventual genotoxic activity of the tested compounds.
In this work, we present our results on the potential use of
a new comet assay protocol, derived from Wentzel’s
approach, for the evaluation of changes in global methylation
levels (Methy-sens Comet) in epigenotoxical studies. We
evaluated the sensitivity of the new protocol using decitabine,
a well-known demethylating agent that blocks cell methy-
transferase (DNMTs) onto the DNA molecule (Stresemann &
Lyko, 2008), and nickel chloride (NiCl2), an environmental
toxicant that induces chromatine condensation and conse-
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quently global hypermethylation at sub-toxic concentrations
(Brocato & Costa, 2013; Hou et al., 2012). This procedure
allows to evaluate the capability of the modified protocol to
detect both hypermethylating and hypomethylating events. To
further validate our protocol we tested the transcriptional
activity of DNMTs in response to treatment, since it is known
that changes in DNA methylation profile results in altered
DNMTs activity and transcription (Li et al., 2012, 2014).
Materials and methods
Cell culture
For personal use only.
The A549 cells, a human lung carcinoma cell line, were
obtained from American Type Culture Collection (Rockville,
MD). The A549 cells were cultured in RPMI medium
(Euroclone, Milano, Italy), supplemented with 10% (vol/vol)
fetal bovine serum, 100 U/mL penicillin, 100 mg/mL strepto-
mycin, and 2 mmol/L L-glutamine. Cells were maintained at
37  C in a 5% CO2 humidified incubator and subcultured twice
a week.
Restriction enzymes and reagents
HpaII and MspI FastDigest restriction enzymes together with
their specific FD buffer were purchased from Fermentas
(Thermo Fisher Scientific, Waltham, MA). To develop the
new protocol, several enzymatic concentrations and pre-
treatment buffers were tested (data not shown). The working
concentration selected was 5 ml/mL of pure enzymatic solu-
tion in FD buffer. Decitabine and NiCl2 were purchased from
Sigma Aldrich (St. Louis, MO). They both were dissolved in
PBS at the desired concentration and used for cell treatment.
MTS assay
Cell viability was assessed by the MTS assay and by Trypan
blue exclusion. The CellTiter 96 AQueous Solution Reagent
kit (Promega, Madison, WI) was utilized for the MTS assay.
The reagent is composed of a tetrazolium compound [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium, inner salt; MTS] and an electron
coupling reagent. Cells were cultured at a density of 5  103
cells per well in a 96-well plate in RPMI-1640 supplemented
with 1% glutamine, 0.5% penicillin/streptomycin, and 5%
fetal bovine serum. After seeding, A549 cells were treated
with different concentrations of either Decitabine (0-10-20-
Comet assay and DNMTs in epigenotoxicology 65
40 mM) or NiCl2 (0-250-500-1000 mM), and incubated for
24 h. The MTS is reduced by cells into a formazan product
that is soluble in tissue culture medium. The quantity of
formazan produced, measured after 4 h at 450 nm(in a 96-well
plate reader MULTISKAN EX; Thermo Electron
Corporation, Vantaa, Finland), is directly proportional to the
number of living cells in culture.
Methy-sens Comet
Starting from the protocol developed by Wentzel et al. (2010),
we tested several modifications (pre-soaking buffer, enzym-
atic concentration, electrophoresis and unwinding time) to
maximize the sensitivity of the assay. Briefly, after treatment
cells were trypsinized and centrifuged (5 min, 800 g) to
remove culturing medium. Cells were therefore resuspended
in 90 mL Low Melting Agarose 0.7% (LMA), transferred onto
degreased microscope slides previously dipped in 1% normal
melting agarose (NMA) for the first layer. The agarose was
allowed to set for 15 min at 4  C before addition of a final
layer of low melting agarose (LMA). Cell lysis was carried
out at 4  C overnight by exposing cells to a buffer containing
2.5 M NaCl, 100 mM Na2EDTA, 8 mM Tris-HCl, 1% Triton
X-100 and 10% DMSO, pH ¼ 10.
After the lysis process, slides were incubated in PBS for
10 min RT to wash lysis buffer and to prepare them for the
enzymatic digestion. Slides were placed horizontally and
100 mL of the two enzymatic working solutions, containing
either MspI or HpaII, was distributed on the surface and then
immediately covered with a microscope coverslip, control
slides were incubated with 100 mL of FD buffer. After
incubation for 10 min at 37  C, slides were immediately
placed in the electrophoretic chamber and quickly submerged
with electrophoretic alkaline buffer, pH413 (1 mM Na2EDTA,
300 mM NaOH, 0  C) to stop enzymatic activity. The electro-
phoretic migration was performed with no DNA unwinding and
an electrophoresis time of 20 min (0.78 V cm1, 300 mA).
DNA was stained with 75 mL ethidium bromide (10 mg/mL)
before the examination at 400 magnification under a
Leica DMLS fluorescence microscope (Leica Microsystems
GmbH, Wetzlar, Germany) (excitation filter BP 515–560 nm,
barrier filter LP 580 nm), using an automatic image analysis
system (Comet Assay III–Perceptive Instruments Ltd, Stone,
Staffordshire, UK).
Total percentage of fluorescence in DNA fragmentation
tail (tail intensity - TI) provided representative data on the
epigenotoxic effects. For each sample, coded and evaluated
blind, 100 cells were analyzed. All steps of the comet assay
were conducted under yellow light.
Tail intensity % was chosen as our reference parameter as
it has been judged as the most useful in discriminating our
results. The fragmentation generated by the treatment with
MspI serves as a positive control as it is representative of the
maximum amount of fragmentation obtainable in the selected
reaction condition. Fragmentation generated by HpaII treat-
ment was, as previously said, inversely proportional to the
amount of methylation found in the cell (Figure 1).
Transcriptional activity
Primers used were derived from (Szemes et al., 2013). Total
RNA extraction was performed through RNeasy Mini Kit
 66 A. Perotti et al. Biomarkers, 2015; 20(1): 64–70
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Figure 1. Effects of enzymatic digestion (MspI or HpaII) on A549 cell nucleoids after electrophoresis. Undigested nucleoid (Control) is reported as
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comparison. HpaII displayed nucleoids come from cells untreated or treated with increasing dose of decitabine (24 h treatment).

(QIAGEN, Hilden, Germany), followed by retrotranscription

using Takara PrimeScript 1st strand cDNA synthesis kit
(Takara Bio Inc., Shiga, Japan). A quantitative real-time PCR
was conducted to evaluate the expression of genes involved in
DNA methylation (DNMT1, DNMT3a, DNMT3b) using
Thermo Scientific RT2 SYBR Green qPCR Master Mix
(Thermo Scientific, Waltham, MA). All the procedures were
conducted following the manufacturer’s protocols.

Statistical analysis
The SPSS 19 (SPSS Inc., Chicago, IL) statistical package was
used to analyze statistical differences among treatments
samples. The mean values from the repeated experiments
were used in a one-way analysis of variance. If significant Figure 2. Relative distribution of the mean TI% of HpaII and MspI
F-values (p50.05) were obtained, post hoc Student’s t-test treatments obtained through Methy-sens Comet on untreated A549 cells
(Bonferroni’s version) was conducted for pairwise comparison. and their percentage ratio (TI%[HpaII]/TI%[MspI]*100). Data reported
are the results of eight independent experiments.
Results
Our study focuses on the development of a new two-enzyme (MeanTI%HpaII ¼ 16 999; SDHpaII ¼ 2930; MeanTI%MspI
protocol that focuses on the rapidity of execution and on the
¼ 41 335; SDMspI ¼ 8980; MeanTI%HpaII/MspI ¼ 41 582;
repeatability for a future utilization in biomonitoring studies.
SDHpaII/MspI ¼ 3,498) (Figure 2). However, as final parameter
The first step in the validation of our method was the
used to compare global methylation levels amongst treatments
definition of an optimal protocol: we tested several enzymatic
has been chosen the ratio between TI% from the two separate
concentration and several unwinding and electrophoretic
enzyme treatments (TI%HpaII/TI%MspI*100). The two param-
times on A549 cell line to suit our laboratory condition (see eters are supposed to show similar behavior in response to
‘‘Materials and methods’’ section for final protocol). variable basal fragmentation in the cell population, this
implies that the use of the ratio between the two TI% offers a
Repeatability of Methy-sens protocol on control cells
parameter with a lowered coefficient of variation (CV)
From 10 individual experiments, we obtain mild intra- minimizing intra-experiment variability (CVHpaII ¼ 0.186;
experiment variations for both HpaII and MspI TI% parameter CVMspI ¼ 0.234; CVHpaII/MspI ¼ 0.091).
 DOI: 10.3109/1354750X.2014.992813 Comet assay and DNMTs in epigenotoxicology 67

After the definition of the protocol, we tested its sensibility response with the lowest cytotoxicity. Decitabine treatment
against induced macrovariations in global methylation levels. showed an increase in the transcription of DNMT1 and a
Substances known for their interaction with the methylome severe decrease in DNMT3a and DNMT3b transcription.
were tested on A549 cells. Decitabine, at the selected Nickel chloride treatment showed a response apparently
concentrations already reported by literature as sub-toxic
(Fojtova et al., 2007; Shin et al., 2013), did not showed
citotoxicity (Figure 3A). Nickel chloride, at 1000 mM con-
centration, showed a slight decrease in GI%. This decrease in
cell viability, however, did not generate an augmented
fragmentation in control cells (Figure 3B), and for this
reason it is not relevant with the test execution.Cells treated
with an increasing concentration of decitabine were then
tested with our modified protocol. Decitabine treatment
showed a significant increase in the fragmentation obtained
with HpaII digestion, supporting the hypothesis of an
induction of a global demethylation xenobiotic-mediated;
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moreover, HpaII/MspI TI% ratio showed a dose-dependent

increase (Figure 4A and B).
Similar results were obtained for nickel chloride treatment
where HpaII digestion generated a progressively decreasing
TI% along with the increase of NiCl2 concentration, starting
from 500 mM (Figure 5A and B). These results show the
capability of the modified protocol to detect both hyper-
methylating and hypomethylating events.

Inter-experimental variability
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To further validate the repeatability of our protocol, we

assessed the inter-experimental variability on at least four
independent experiments for decitabine and NiCl2. All the
experiments with decitabine treatment showed the expected
dose-response effect with a high reproducibility (Figure 6A).
We have obtained a very high reproducibility in the results for
NiCl2 (Figure 6B).

DNMTs transcriptional activity
The real-time PCR analysis of the transcriptional activity of
the DNMTs encoding genes was conducted at a concentration
of 20 mM for decitabine and 500 mM for nickel chloride for
24 h. The two concentrations were chosen as the highest
Figure 4. Methy-sens Comet on nucleoids from A549 cells treated for
24 h with increasing dose of decitabine. A: mean TI% from 100
individual nucleoids images (Contr.: non-digested nucleoids; HpaII:
nucleoids with 10 min HpaII digestion; MspI: nucleoids with 10 min
MspI digestion); B: combined percentage ratio of the mean TI% obtained
through enzymatic digestions (TI%[HpaII]/TI%[MspI]*100). *p50.05;
**p50.01.

Figure 3. Growth inhibition (GI%), measured through MTS assay, of A549 cells treated for 24 h with increasing dose of (A) Decitabine [0-10-20-
40 mM] and (B) NiCl2 [0-250-500-1000 mM].
 68 A. Perotti et al.
antithetical, with a decrease in DNMT1 transcriptional activ-
ity and an increase in DNMT3a transcription. DNMT3b,
however, seemed to be inhibited by the nickel treatment,
but not as much as by the decitabine treatment (Figure 7).
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For personal use only.
Figure 5. Methy-sens Comet on nucleoids from A549 cells treated for
24 h with increasing dose of NiCl2. (A) Mean TI% from 100 individual
nucleoids images (Contr.: non-digested nucleoids; HpaII: nucleoids with
10 min HpaII digestion; MspI: nucleoids with 10 min MspI digestion);
(B) Combined percentage ratio of the mean TI% obtained through
enzymatic digestions (TI%[HpaII]/TI%[MspI]*100). ***p50.001.
Figure 6. Inter-experiment reproducibility of Methy-sens Comet on A549 cells (n.4 independent experiments for each compound). Percentage ratio of
TI% obtained with HpaII and MspI enzymatic digestions with decitabine (A) and NiCl2 (B) treated cells. ***p50.001.
Biomarkers, 2015; 20(1): 64–70
Discussion
Epigenetic changes were first defined as a result of the
interaction between genes and environment, allowing
the organism to adapt to new environmental conditions. The
exponential growth of studies on the epigenetic effects of
environmental contaminants has broadened the range of
research on the etiology of environmental-related pathologies
including cancer (Hatziapostolou & Iliopoulos, 2011; Smith
& Meissner, 2013; Szyf, 2007). Epigenetic changes caused by
xenobiotics occur typically at concentrations previously
considered sub-toxic, thus, forcing the toxicological analysis
to gain a new outlook on the mechanisms of toxicity. Whilst
several tools had been developed to study this phenomenon,
the need for new simple and adaptable tools to characterize
the potential epigenetic damage, in particular at the single cell
level, remains. The comet assay is a technique, widely used in
biomonitoring studies, that has already proven its flexibility,
coupled with its simplicity. One of the various strengths of the
comet assay is that the analysis is carried out on single
isolated cells, allowing users to discriminate eventual
subpopulations with differential response within the cell
sample.
Figure 7. DNA methyltransferases (DNMTs) genes transcription of
A549 cells treated with decitabine 20 mM and NiCl2 500 mM for 24 h;
DNMTs mRNA quantification was carried out through Real Time PCR
analysis. *p50.05; **p50.01; ***p50.001.
 DOI: 10.3109/1354750X.2014.992813
The first comet methylation-sensitive protocol using the
two-enzyme approach by Wentzel et al. (2010) has shown its
capability of detecting differences in the amount of DNA
migration in cells exposed to demethylating agents. Our
improved protocol (Methy-sens comet) showed for the first
time that the two-enzyme approach is suitable to detect both
hypo- and hyper-methylation. Significant variations in DNA
methylation level were clearly stated performing Methy-sens
comet on A549 cells treated with a demethylating drug, such as
decitabine or a known hypermethylating agent like nickel
chloride. Decitabine is a cytosine analogue widely used for the
treatment of several type of leukemia, it has become a
reference drug in methylation studies for its capability of
inducing relevant global demethylation. Decitabine is incor-
porated in the double helix, mainly during the DNA replication
stage, and exerts its role blocking DNA methyl-transferases,
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particularly DNMT1, onto the DNA molecule when the
enzyme tries to methylate the incorporated cytosine analogue,
with a subsequent enzyme degradation. This degradation,
along with the decrease in cytoplasmatic DNMTs levels, leads
to a global demethylation that, in therapy, is used to reactivate
hypermethylated oncosuppressor genes in tumoral cells
(Stresemann & Lyko, 2008). Nickel chloride is a toxicant,
with well characterized toxic, genotoxic and carcinogenic
properties (Fletcher et al., 1994). However, in later years nickel
has gained a strong role as epigenotoxic contaminant.
Numerous studies have reported its ability in inducing
For personal use only.
heterochromatization alongside with strong hypermethylation
(Brocato & Costa, 2013; Fragou et al., 2011; Hou et al., 2012).
Ni2+ ions induce DNA hypermethylation with histone
deacetylation by displacing Mg2+ ions complexed with
DNA-phosphate backbone, hence tightening chromatin con-
densation and generating heterochromatin (Ellen et al., 2009);
this local condensation can subsequently induce local and
global hypermethylation (Sun et al., 2013).
Both substance effects have been detected by Methy-sens
comet and, interestingly, all these perceived variations were
observed at sub-toxic and sub-genotoxic concentrations
(Figure 3). Methy-sens Comet detected a dose-dependent
demethylation caused by decitabine in the analyzed A549
population (Figure 4) in agreement with literature data
(Kehrmann et al., 2014; Orta et al. 2009; Stresemann &
Lyko, 2008). Methy-sens Comet on nickel-treated A549 has
successfully detected nickel-induced hypermethylation start-
ing from a concentration of 500 mM (Figure 5). Furthermore,
the methylation-sensitive enzyme digestion provided repro-
ducible results. Among the evaluated parameters, the ratio
between the two TI% (HpaII/MspI) offers a parameter with a
low coefficient of variation, showing a strong intra-experi-
mental reproducibility (Figures 2–6).
Xenobiotic-induced changes in the cell methylation pattern
have a parallel influence on the main methylome maintenance
system as well, thus adding a possible new biomarker of the
xenobiotic-methylome interaction. To identify the presence of
concomitant effects of hypo- and hyper-methylating agents at
both structural level and transcriptional level we performed
real-time PCR analysis of DNMTs genes transcription. Both
treatments induced variations in DNMTs transcriptional
levels (Figure 7), showing a concurrent effect on the
methylation maintenance system and on the DNA structure
Comet assay and DNMTs in epigenotoxicology 69
revealed through Methy-sens Comet. Our data are in agree-
ment with previous studies that reported that decitabine
induces augmented transcription of DNMT1 as a result of the
depletion of cytoplasmatic DNMT1 (Alcazar et al., 2012;
Ghoshal et al., 2005). The severe reduction of mRNA
encoding DNMT3a and DNMT3b that we detected after
decitabine exposure, is not so easily explainable. Decitabine
affects particularly on de-novo DNMTs have shown to be cell-
specific (Kastl et al., 2010) with different behavior depending
on the type of cells used. In A549 few data are available: Kim
et al. (2012) observed a decrease in expression of DNMT3a
after 18-h treatment. To our knowledge, there are no data
available on DNMT3b. DNMT3b has been marked as
involved in cancer development and survival (Beaulieu
et al., 2002), but its role seems not to be essential for
cancer progression (Hagemann et al., 2012). In addition,
neither decitabine nor its parent compound 5-azacytidine
seemed to induce a depletion of DNMT3b. We can hypothe-
size that the down-regulation of the de novo DNMTs could be
related to an attempt to prevent erroneous novel methylations,
given the relatively high dose of decitabine we used. Anyhow,
since this data provide new insights in this context, we think
that the relationships among hypo-methylating agents treat-
ment, DNA epigenetic modifications and DNMTs transcrip-
tion need to be further evaluated.
As for decitabine treatment, hypermethylating agent
exposure resulted in altered DNMTs transcriptional patterns
(Figure 7). There are few information on nickel influence on
DNMT genes transcription: Ji et al. (2013) reported DNMT1
upregulation in NiS-tranformed human bronchial epithelial
cells. On the contrary, our data showed a decrease in DNMT1
and DNMT3b transcription with a parallel increase in
DNMT3a transcription induced by NiCl2 on a lung carcinoma
cell line (A549). This may suggest a scenario in which the
augmented heterochromatization caused by nickel, recalls
DNMT3a through the induction of chromatin condensation
and the recruitment of DNMT3a auxiliary factor DNMT3L
(Brocato & Costa, 2013; Deplus et al., 2002). The parallel
decrease in DNMT1 and DNMT3b could be explained by a
cellular response aimed at limiting the introduction of
excessive methylation or simply by a general cell response
to the induction of stress. However, additional data are
required to further characterize mechanisms beyond nickel
exposure. Anyway, the detected hypermethylation, along with
the disrupted DNMTs transcriptional activity, remains a
signal of a strong epigenotoxic stress.
Conclusions
Methy-sens Comet is a suitable and sensitive test to detect
global methylome variations in a cell population and its
intrinsic properties allow the user to discriminate changes up
to the single cell level. Its frequent use in biomonitoring
makes it a useful tool for future projects involving human
populations to assess exposures to xenobiotics with no or low
detectable toxic effects and with potential strong epigenetic
activities. The analysis of DNMTs transcription has high-
lighted that exogenous changes in the cell methylation pattern
have a parallel influence on the main methylome maintenance
system as well, thus adding possible new biomarkers of the
 70 A. Perotti et al.
xenobiote-methylome interaction. Moreover, the contempor-
ary assessment of the DNMTs transcriptional level and the
DNA methylation level through Methy-sens Comet could be a
new approach in environmental epi-genotoxicology.
Acknowledgements
We would like to thank Dr Mirca Lazzaretti and
Mrs Antonietta Cirasolo for technical support.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
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