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Ryszard Olinski - One of the best experts on this subject based on the ideXlab platform.
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vitamin c enhances substantially formation of 5 Hydroxymethyluracil in cellular dna
Free Radical Biology and Medicine, 2016Co-Authors: Martyna Modrzejewska, Marta Starczak, Daniel Gackowski, Maciej Gawronski, Magdalena Skonieczna, Ewelina Zarakowska, Marek Foksinski, Joanna Rzeszowskawolny, Ryszard OlinskiAbstract: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.
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enigmatic 5 Hydroxymethyluracil oxidatively modified base epigenetic mark or both
Mutation Research-reviews in Mutation Research, 2016Co-Authors: Ryszard Olinski, Marta Starczak, Daniel GackowskiAbstract:The aim of this review is to describe the reactions which lead to generation of 5-Hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-Hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-Hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-Hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-Hydroxymethyluracil mispaired with guanine. Indeed, this mispair may be also formed as a result of deamination of 5-hydroxymethylcytosine, recently described "sixth" DNA base. It was demonstrated that 5-Hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5-Hydroxymethyluracil is epigenetic mark. The level of 5-Hydroxymethyluracil in various somatic tissues is relatively stable and resembles that observed in lymphocytes, about 0.5/10(6) dN in human colon, colorectal cancer as well as various rat and porcine tissues. Experimental evidence suggests that SMUG1 and TDG are main enzymes involved in removal of 5-Hydroxymethyluracil from DNA. 5-Hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5-Hydroxymethyluracil is with no doubt a product of both enzymatic and reactive oxygen species-induced reaction. This modification may probably serve as an epigenetic mark, providing additional layer of information encoded within the genome. However, the pool of 5-Hydroxymethyluracil generated as a result of oxidative stress is also likely to disturb physiological epigenetic processes, and as such may be defined as a lesion. Altogether this suggests that 5-Hydroxymethyluracil may be either a regulatory or erroneous compound.
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tissue specific differences in dna modifications 5 hydroxymethylcytosine 5 formylcytosine 5 carboxylcytosine and 5 Hydroxymethyluracil and their interrelationships
PLOS ONE, 2015Co-Authors: Daniel Gackowski, Marta Starczak, Martyna Modrzejewska, Ewelina Zarakowska, Ryszard OlinskiAbstract:Background Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-Hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions. Results Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively). Conclusions Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.
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urinary 5 Hydroxymethyluracil and 8 oxo 7 8 dihydroguanine as potential biomarkers in patients with colorectal cancer
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Zbigniew Banaszkiewicz, Agnieszka Siomekgorecka, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio).Conclusion: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.
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Urinary 5-Hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer.
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Agnieszka Siomek-gorecka, Zbigniew Banaszkiewicz, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was
Daniel Gackowski - One of the best experts on this subject based on the ideXlab platform.
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vitamin c enhances substantially formation of 5 Hydroxymethyluracil in cellular dna
Free Radical Biology and Medicine, 2016Co-Authors: Martyna Modrzejewska, Marta Starczak, Daniel Gackowski, Maciej Gawronski, Magdalena Skonieczna, Ewelina Zarakowska, Marek Foksinski, Joanna Rzeszowskawolny, Ryszard OlinskiAbstract: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.
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enigmatic 5 Hydroxymethyluracil oxidatively modified base epigenetic mark or both
Mutation Research-reviews in Mutation Research, 2016Co-Authors: Ryszard Olinski, Marta Starczak, Daniel GackowskiAbstract:The aim of this review is to describe the reactions which lead to generation of 5-Hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-Hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-Hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-Hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-Hydroxymethyluracil mispaired with guanine. Indeed, this mispair may be also formed as a result of deamination of 5-hydroxymethylcytosine, recently described "sixth" DNA base. It was demonstrated that 5-Hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5-Hydroxymethyluracil is epigenetic mark. The level of 5-Hydroxymethyluracil in various somatic tissues is relatively stable and resembles that observed in lymphocytes, about 0.5/10(6) dN in human colon, colorectal cancer as well as various rat and porcine tissues. Experimental evidence suggests that SMUG1 and TDG are main enzymes involved in removal of 5-Hydroxymethyluracil from DNA. 5-Hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5-Hydroxymethyluracil is with no doubt a product of both enzymatic and reactive oxygen species-induced reaction. This modification may probably serve as an epigenetic mark, providing additional layer of information encoded within the genome. However, the pool of 5-Hydroxymethyluracil generated as a result of oxidative stress is also likely to disturb physiological epigenetic processes, and as such may be defined as a lesion. Altogether this suggests that 5-Hydroxymethyluracil may be either a regulatory or erroneous compound.
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tissue specific differences in dna modifications 5 hydroxymethylcytosine 5 formylcytosine 5 carboxylcytosine and 5 Hydroxymethyluracil and their interrelationships
PLOS ONE, 2015Co-Authors: Daniel Gackowski, Marta Starczak, Martyna Modrzejewska, Ewelina Zarakowska, Ryszard OlinskiAbstract:Background Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-Hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions. Results Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively). Conclusions Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.
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urinary 5 Hydroxymethyluracil and 8 oxo 7 8 dihydroguanine as potential biomarkers in patients with colorectal cancer
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Zbigniew Banaszkiewicz, Agnieszka Siomekgorecka, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio).Conclusion: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.
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Urinary 5-Hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer.
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Agnieszka Siomek-gorecka, Zbigniew Banaszkiewicz, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was
George W Teebor - One of the best experts on this subject based on the ideXlab platform.
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definitive identification of mammalian 5 Hydroxymethyluracil dna n glycosylase activity as smug1
Journal of Biological Chemistry, 2001Co-Authors: Robert J Boorstein, Archie Cummings, Dina R Marenstein, Michael K Chan, Thomas A Neubert, Stuart M Brown, George W TeeborAbstract:Abstract Purification from calf thymus of a DNAN-glycosylase activity (HMUDG) that released 5-Hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20× the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein.
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excessive base excision repair of 5 Hydroxymethyluracil from dna induces apoptosis in chinese hamster v79 cells containing mutant p53
Carcinogenesis, 2001Co-Authors: Lijun Mi, George W Teebor, Wenren Chaung, Robert Horowitz, Robert J BoorsteinAbstract:We have demonstrated previously that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of Hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-Hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G(2) and that hmdUrd-treated cells underwent apoptosis after arrest in late S/G(2) phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.
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Oxidative damage to 5-methylcytosine in DNA
Nucleic Acids Research, 1995Co-Authors: Rober J. Boorstein, George W TeeborAbstract:Abstract Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-Hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-Hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.
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a mammalian cell line deficient in activity of the dna repair enzyme 5 Hydroxymethyluracil dna glycosylase is resistant to the toxic effects of the thymidine analog 5 hydroxymethyl 2 deoxyuridine
Molecular and Cellular Biology, 1992Co-Authors: Robert J Boorstein, Laino Chiu, George W TeeborAbstract:Abstract We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-Hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic repairability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine.
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site directed substitution of 5 Hydroxymethyluracil for thymine in replicating ox m4am3 dna via synthesis of 5 hydroxymethyl 2 deoxyuridine 5 triphosphate
Nucleic Acids Research, 1991Co-Authors: Dan D Levy, George W TeeborAbstract:Abstract 5-Hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of Thy. It is removed from DNA by HmUra-DNA glycosylase. To determine whether the replacement of Thy by HmUra is mutagenic, which might explain the repairability of HmUra, a HmUra residue was substituted for Thy in a target (amber) codon by in vitro extension of an oligonucleotide primer annealed to phi X-174am3 virion DNA. This was accomplished by synthesizing HmdUTP and using DNA polymerase to effect primer extension. E. coli spheroplasts were transfected with the HmUra-containing DNA and the yield of revertant phage determined following replication in the bacterial host. Since E. coli do not express HmUra-DNA glycosylase activity, mutagenesis could be assessed in the absence of repair. chi 2c analysis showed that replacing Thy with HmUra did not result in an increase in revertant phage. These data indicate that the oxidation of Thy to HmUra in cellular DNA probably does not result in substantial mutagenesis.
Marta Starczak - One of the best experts on this subject based on the ideXlab platform.
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vitamin c enhances substantially formation of 5 Hydroxymethyluracil in cellular dna
Free Radical Biology and Medicine, 2016Co-Authors: Martyna Modrzejewska, Marta Starczak, Daniel Gackowski, Maciej Gawronski, Magdalena Skonieczna, Ewelina Zarakowska, Marek Foksinski, Joanna Rzeszowskawolny, Ryszard OlinskiAbstract: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.
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enigmatic 5 Hydroxymethyluracil oxidatively modified base epigenetic mark or both
Mutation Research-reviews in Mutation Research, 2016Co-Authors: Ryszard Olinski, Marta Starczak, Daniel GackowskiAbstract:The aim of this review is to describe the reactions which lead to generation of 5-Hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-Hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-Hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-Hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-Hydroxymethyluracil mispaired with guanine. Indeed, this mispair may be also formed as a result of deamination of 5-hydroxymethylcytosine, recently described "sixth" DNA base. It was demonstrated that 5-Hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5-Hydroxymethyluracil is epigenetic mark. The level of 5-Hydroxymethyluracil in various somatic tissues is relatively stable and resembles that observed in lymphocytes, about 0.5/10(6) dN in human colon, colorectal cancer as well as various rat and porcine tissues. Experimental evidence suggests that SMUG1 and TDG are main enzymes involved in removal of 5-Hydroxymethyluracil from DNA. 5-Hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5-Hydroxymethyluracil is with no doubt a product of both enzymatic and reactive oxygen species-induced reaction. This modification may probably serve as an epigenetic mark, providing additional layer of information encoded within the genome. However, the pool of 5-Hydroxymethyluracil generated as a result of oxidative stress is also likely to disturb physiological epigenetic processes, and as such may be defined as a lesion. Altogether this suggests that 5-Hydroxymethyluracil may be either a regulatory or erroneous compound.
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tissue specific differences in dna modifications 5 hydroxymethylcytosine 5 formylcytosine 5 carboxylcytosine and 5 Hydroxymethyluracil and their interrelationships
PLOS ONE, 2015Co-Authors: Daniel Gackowski, Marta Starczak, Martyna Modrzejewska, Ewelina Zarakowska, Ryszard OlinskiAbstract:Background Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-Hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions. Results Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively). Conclusions Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.
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urinary 5 Hydroxymethyluracil and 8 oxo 7 8 dihydroguanine as potential biomarkers in patients with colorectal cancer
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Zbigniew Banaszkiewicz, Agnieszka Siomekgorecka, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio).Conclusion: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.
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Urinary 5-Hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer.
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Agnieszka Siomek-gorecka, Zbigniew Banaszkiewicz, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was
Martyna Modrzejewska - One of the best experts on this subject based on the ideXlab platform.
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vitamin c enhances substantially formation of 5 Hydroxymethyluracil in cellular dna
Free Radical Biology and Medicine, 2016Co-Authors: Martyna Modrzejewska, Marta Starczak, Daniel Gackowski, Maciej Gawronski, Magdalena Skonieczna, Ewelina Zarakowska, Marek Foksinski, Joanna Rzeszowskawolny, Ryszard OlinskiAbstract: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.
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tissue specific differences in dna modifications 5 hydroxymethylcytosine 5 formylcytosine 5 carboxylcytosine and 5 Hydroxymethyluracil and their interrelationships
PLOS ONE, 2015Co-Authors: Daniel Gackowski, Marta Starczak, Martyna Modrzejewska, Ewelina Zarakowska, Ryszard OlinskiAbstract:Background Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-Hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions. Results Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively). Conclusions Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.
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urinary 5 Hydroxymethyluracil and 8 oxo 7 8 dihydroguanine as potential biomarkers in patients with colorectal cancer
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Zbigniew Banaszkiewicz, Agnieszka Siomekgorecka, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio).Conclusion: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.
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Urinary 5-Hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer.
Biomarkers, 2015Co-Authors: Rafal Rozalski, Marta Starczak, Daniel Gackowski, Martyna Modrzejewska, Agnieszka Siomek-gorecka, Zbigniew Banaszkiewicz, Ryszard OlinskiAbstract:AbstractContext: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 5-Hydroxymethyluracil (5-hmUra).Materials and methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.Results: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was
