Introduction: Vitamin C (ascorbic acid) is a potent antioxidant and a crucial co-factor of many enzymes, including Fe2+ and alpha-ketoglutarate dependent dioxygenases such as TET (ten-eleven-translocation) proteins involved in active DNA demethylation. TET enzymes (TET1, TET2 and TET3) are able to oxidize 5-methylcytosine to 5-hydroxymethylcytosine and subsequently to 5-formylcytosine and 5-carboxycytosine - modifications involved in epigenetic regulation of gene expression. However, in order to maintain enzymatic function of those enzymes, after each step of oxidation, catalytic Fe ion in their active center must be restored to 2+ state by vitamin C. Therefore, in many studies involving epigenetic make-up, gene expression, cell maturation and differentiation supplementation of the vitamin C seems to be indispensable for obtaining most accurate and reliable data. However, vitamin C in aqueous solutions is unstable, quickly degrades and may change pH of culture medium. Moreover, in many cells the effect of vitamin C supplementation may be affected by abnormalities in the functioning of vitamin C transporters. Therefore, we have investigated effects of different forms of vitamin C on enzymatic activity of TET enzymes, in order to determine which of them can be an alternative to supplementation with crystalic ascorbic acid.
Methodology: HAP1 and MDA-MB-231 cells were cultured in accordance to manufacturer protocols and exposed for 24 hours to 100 µM of: ascorbic acid, sodium ascorbate, 6-O-palmitoyl-L-ascorbic acid, liposomal sodium ascorbate and “empty” liposomes (last two provided by Lipid Systems). Control cells were treated with volumetric equivalent of saline. DNA from harvested cells was isolated by phenolic extraction and subsequently enzymatically hydrolyzed to obtain nucleosides. Enzymatic activity of TET proteins characterized as the levels of their products in cellular DNA - was measured by two dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC MS/MS).
Results: In both cell lines we have observed significant increase in the levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine after incubation with each form of vitamin C when compared with untreated cells and cells exposed to “empty” liposomes. There were no significant differences between “empty” liposomes and control cells. Moreover, there were no significant differences in the levels of TET products between each form of vitamin C.
Conclusions: Liposomal form of vitamin C and 6-O-palmitoyl-L-ascorbic acid stimulate activity of TET proteins in the same manner as a free ascorbic acid, but are more stable in cell culture conditions. However, 6-O-palmitoyl-L-ascorbic acid is highly hydrophobic and must be dissolved in organic solvents that may potentially affect cell viability or their phenotype. Taking this into consideration, liposomal vitamin C seems to be a better alternative for vitamin C supplementation, as it is as efficient as ascorbic acid in stimulating enzymatic activity of TET proteins (and potentially other dioxygenases), is well soluble in aqueous solutions and does not change the pH of the culture medium. Moreover, due to passive transport of liposomes to the cells, effect of liposomal vitamin C supplementation is potentially unaffected by the expression patterns or mutational status of vitamin C transporters.
This work was supported by Polish National Science grant number: 2018/29/N/NZ1/00497