Vitamin C enhances substantially formation of 5-hydroxymethyluracil in cellular DNA.
The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells. The aim of this study was to determine the levels of the abovementioned TET-mediated oxidation products of 5-methylcytosine and thymine after addition of ascorbate, using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Intracellular concentration of ascorbate was determined by means of ultra-performance liquid chromatography with UV detection. Irrespective of its concentration in culture medium (10-100µM) and inside the cell, ascorbate stimulated a moderate (2- to 3-fold) albeit persistent (up to 96-h) increase in the level of 5-hydroxymethylcytosine. However, exposure of cells to higher concentrations of ascorbate (100µM or 1mM) stimulated a substantial increase in 5-formylcytosine and 5-carboxycytosine levels. Moreover, for the first time we demonstrated a spectacular (up to 18.5-fold) increase in 5-hydroxymethyluracil content what, in turn, suggests that TET enzymes contributed to the presence of the modification in cellular DNA. These findings suggest that physiological concentrations of ascorbate in human serum (10-100µM) are sufficient to maintain a stable level of 5-hydroxymethylcytosine in cellular DNA. However, markedly higher concentrations of ascorbate (ca. 100µM in the cell milieu or ca. 1mM inside the cell) were needed to obtain a sustained increase in 5-formylcytosine, 5-carboxycytosine and 5-hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions.
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Karlowicza 24, 85-092 Bydgoszcz, Poland. Electronic address: m.modrzejewska@onet.pl.
Martyna
M
Modrzejewska
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Karlowicza 24, 85-092 Bydgoszcz, Poland. Electronic address: m.gawronski@vp.pl.
Maciej
M
Gawronski
Biosystems Group, Institute of Automatic Control, Faculty of Automatics, Electronics, and Informatics, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland. Electronic address: Magdalena.Skonieczna@polsl.pl.
Magdalena
M
Skonieczna
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Karlowicza 24, 85-092 Bydgoszcz, Poland. Electronic address: ewelinaz@cm.umk.pl.
Ewelina
E
Zarakowska
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Karlowicza 24, 85-092 Bydgoszcz, Poland. Electronic address: marta_koltan@post.pl.
Marta
M
Starczak
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Karlowicza 24, 85-092 Bydgoszcz, Poland. Electronic address: marekf@cm.umk.pl.
Marek
M
Foksinski
Biosystems Group, Institute of Automatic Control, Faculty of Automatics, Electronics, and Informatics, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland. Electronic address: Joanna.Rzeszowska@polsl.pl.
Joanna
J
Rzeszowska-Wolny
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Karlowicza 24, 85-092 Bydgoszcz, Poland. Electronic address: danielg@cm.umk.pl.
Daniel
D
Gackowski
Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Karlowicza 24, 85-092 Bydgoszcz, Poland. Electronic address: ryszardo@cm.umk.pl.
Ryszard
R
Olinski
D044503:5-Methylcytosine / Q000819:agonists / Q000031:analogs & derivatives* / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology*; D003596:Cytosine / Q000819:agonists / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation / Q000187:drug effects; D045325:HCT116 Cells; D006801:Humans; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010084:Oxidation-Reduction; D010432:Pentoxyl / Q000819:agonists / Q000031:analogs & derivatives* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013941:Thymine / Q000819:agonists / Q000378:metabolism
C560973:5-formylcytosine; D011518:Proto-Oncogene Proteins; C011865:5-hydroxymethylcytosine; C008055:5-hydroxymethyluracil; D044503:5-Methylcytosine; C000618843:5-carboxycytosine; D010432:Pentoxyl; D003596:Cytosine; D004247:DNA; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D001205:Ascorbic Acid; D013941:Thymine
5-carboxycytosine; 5-formylcytosine; 5-hydroxymethylcytosine; 5-hydroxymethyluracil; 5-methylcytosine; Ascorbate