Comet Methy-sens and DNMTs transcriptional analysis as a combined approach in epigenotoxicology
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1
Università degli Studi di Parma, Department of Life Sciences, Italy
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2
Università degli Studi di Parma, Department of Life Sciences, Italy
Epigenetic effects of environmental contaminants are currently becoming a major concern, considering their role in influencing development, adult life and etiology of disease. The field of environmental epigenetics has been growing fast in the past decade generating awareness on the need of a new approach in toxicology studies: epigenotoxicology. It is now broadly accepted that environmental pollutants may express part of their pathogenicity through epigenetic modification. Dynamic chromatin remodeling is a required step for the initial phases of gene transcription and it can occur even after complete cell differentiation in response to environmental stimuli or xenobiotic interaction. Epigenetic factors, such as DNA methylation, histone modification and micro-RNAs, have a key role in these remodeling and alterations in these mechanisms could lead to disease.
DNA methylation, in particular, is tightly linked to transcriptional silencing and fundamental in gene regulation, development and pathological events. This epigenetic modification consists in the addition of a methyl group to the 5’ position of the cytosine ring catalyzed by a class of enzyme called DNA methyltransferase (DNMTs), whose primary role is the maintenance of the correct methylation pattern in the genome. Epigenetic instability and, more specifically, uncontrolled variations in the global and local methylation profile and changes in DNMTs activity have by now been recognized as a gateway for carcinogenesis.
Given the need of simple and adaptive tools to assess these effects, this work proposes a revised version of a comet assay modification designed to detect global methylation changes through enzymatic digestion with two restriction enzyme (HpaII and MspI) (Perotti et al., 2015; Wentzel et al., 2010). These two enzyme are isoschizomer endonucleases that both share the same restriction site (typical 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 (figure 1).
We developed a new protocol of a methylation-sensitive (Comet Methy-sens) comet procedure and tested its repeatability on A549 cell line. We evaluated the sensitivity of the new protocol using decitabine, a well known demethylating agent that blocks cell methytransferase (DNMTs) onto the DNA molecule, and nickel chloride (NiCl2), an environmental toxicant that induces chromatine condensation and consequently global hypermethylation at sub-toxic concentrations. This procedure allows to evaluate the capability of the modified protocol to detect both hypermethylating and hypomethylating events.
Enzymatic digestion showed differential variation in response to exposure to global demethylating agent and hypermethylating agent thus proving Comet Methy-sens capability of detecting both positive and negative global methylation changes, showing at the same time a good repeatability in both controls and samples. Comet Methy-sens detected a dose-dependent demethylation caused by decitabine in the analyzed A549 population while on nickel-treated A549 the technique has successfully detected nickel induced hypermethylation starting from a concentration of 500 µM.
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, showing a concurrent effect on the methylation maintenance system and on the DNA structure revealed through Comet methy-sens.
In conclusion, our data demonstrate that Comet Methy-sens, in combination with the analysis of transcriptional levels of DNA methyl transferases, represents a simple and multifunctional approach to implement biomonitoring studies on epigenotoxicological effects of known and unknown xenobiotics.
References
Perotti, A., Rossi, V., Mutti, A., Buschini, A., 2015. Methy-sens Comet assay and DNMTs transcriptional analysis as a combined approach in epigenotoxicology. Biomarkers 20, 64–70. doi:10.3109/1354750X.2014.992813
Wentzel, J.F., Gouws, C., Huysamen, C., Dyk, E. Van, Koekemoer, G., Pretorius, P.J., 2010. Assessing the DNA methylation status of single cells with the comet assay. Anal. Biochem. 400, 190–4. doi:10.1016/j.ab.2010.02.008
Keywords:
epigenetics,
Epigenotoxicology,
Toxicology in vitro,
DNA Methylation,
DNA methyltransferases,
DNMT expression,
Comet Assay,
modification,
decitabine,
Nickel
Conference:
ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.
Presentation Type:
Oral Presentation
Topic:
New applications and technical improvements
Citation:
Perotti
A,
Galati
S,
Rossi
V,
Lazzaretti
M and
Buschini
A
(2015). Comet Methy-sens and DNMTs transcriptional analysis as a combined approach in epigenotoxicology.
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00055
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Received:
14 May 2015;
Published Online:
23 Jun 2015.
*
Correspondence:
PhD. Alessio Perotti, Università degli Studi di Parma, Department of Life Sciences, Parma, 43100, Italy, alessio.perotti@unipr.it