In this study, the level of DNA modifications was investigated in three developmental stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). Analysis was carried out using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. Our method made it possible, for the first time, to analyze a broad spectrum of DNA modifications in the three stages of Drosophila. Each stage was characterized by a specific modification pattern, and the levels of these compounds fluctuated throughout the D. melanogaster life cycle. The level of DNA modification was also compared between insects bred at 25 °C (optimal temperature) and at 18 °C, and the groups differed significantly. The profound changes in N6-methyladenine and 5-hydroxymethyluracil levels during the Drosophila life cycle and as a result of breeding temperature changes indicate that these DNA modifications can play important regulatory roles in response to environmental changes and/or biological conditions. Moreover, the supplementation of Schneider 2 cells with 1 mM L-ascorbic acid caused a time-dependent increase in the level of 5-(hydroxymethyl)-2′-deoxyuridine. These data suggest that a certain pool of this compound may arise from the enzymatic activity of the dTET protein.

Background: The experimental evidence has demonstrated that active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation residues in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. TETs’ activity may be upregulated by ascorbate. Given that aberrant DNA methylation of crucial genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in active DNA demethylation process. Moreover, we tried to verify if the expression level of genes involved in active DNA demethylation and products of this process may serve as predictive markers of breast cancer. Methods: The study included blood samples from breast cancer patients (n=74) and healthy subjects (n=71). We applied qRT-PCR with Universal Probe Library probes for measurement of expression of genes involved in active demethylation process, and two dimensional ultra performance liquid chromatography with tandem mass spectrometry detection (2D-UPLC MS/MS) for 5–mC and its derivatives assessment. Ascorbate level was determined using UPLC-MS. Results: Breast cancer patients exhibited significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3expression was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, ascorbate level in plasma of breast cancer patients was decreased with concomitant increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2) mRNA expression level. Conclusions: Presented study indicate particular role of TET3 in DNA demethylation in breast carcinogenesis. Furthermore, correlation between SLC23A2 and 5-mC level, may provide new path for future breast cancer research.

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