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. 2017 Sep 19;7:11872. doi: 10.1038/s41598-017-12148-2

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Methylation sensitive comet assay (MS-CA). The observed comet tails represent double-strand DNA breaks, introduced by the isoschizomeric enzymes MspI and HpaII; in the presence of an electric field, the HpaII- and MspI-digested DNA migrates faster, compared to the significantly slower migration of the undigested DNA. Digestion of the control (Chlamy) and treated (vitamin B₁₂ and MTX) samples resulted in a tail DNA increase for MTX and decrease for vitamin B₁₂, relative to the intact DNA in the comet head of the untreated controls – indicative of decreased DNA methylation levels in MTX-treated animals and increased DNA methylation in vitamin B₁₂-treated animals.