Accurate, Direct, and High-Throughput Analyses of a Broad Spectrum of Endogenously Generated DNA Base Modifications with Isotope-Dilution Two-Dimensional Ultraperformance Liquid Chromatography with Tandem Mass Spectrometry: Possible Clinical Implication

Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2′-deoxycytidine, 5-(hydroxymethyl)-2′-deoxycytidine, 5-formyl-2′-deoxycytidine, 5-carboxy-2′-deoxycytidine, 5-(hydroxymethyl)-2′-deoxyuridine, 2′-deoxyuridine, and 8-oxo-2′-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2′-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor’s margin; also 5-formyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2′-deoxyuridine and 5-(hydroxymethyl)-2′-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material containing a considerable amount of coeluting interferents with mass-spectrometry-based methods

Relationship between seminal plasma ascorbic acid concentration versus: A, level of 8-oxodG in sperm DNA; B, percentage of sperm with normal morphology.

<p>Relationship between seminal plasma ascorbic acid concentration versus: A, level of 8-oxodG in sperm DNA; B, percentage of sperm with normal morphology.</p

Ascorbic acid concentration in blood plasma and seminal plasma in samples from all studied subjects.

<p>Ascorbic acid concentration in blood plasma and seminal plasma in samples from all studied subjects.</p

Relationship between urinary excretion 8-oxoGua and the level of 8-oxodG in sperm DNA in all studied subjects.

<p>Relationship between urinary excretion 8-oxoGua and the level of 8-oxodG in sperm DNA in all studied subjects.</p

The effect of cigarette smoking on: A, ascorbic acid concentration in blood plasma; B, level of 8-oxodG in leukocyte DNA.

<p>The effect of cigarette smoking on: A, ascorbic acid concentration in blood plasma; B, level of 8-oxodG in leukocyte DNA.</p

Characteristic of subjects’ age and semen quality parameters of study groups.

<p>Values are expressed as median and interquartile range.</p

Comparison of the analytical parameters among the study groups.

<p>Values are expressed as median and interquartile range. Statistically significant differences (Mann-Whitney U test, p<0.05):</p>a<p>vs. control group,</p>b<p>vs. asthenozoospermic patients.</p><p>Statistically significant differences (Wilcoxon test, p<0.05):</p>1<p>vs. leukocytes,</p>2<p>vs. seminal plasma ascorbic acid.</p

Levels of 8-oxodG in spermatozoa and leukocytes.

<p>A, Levels of 8-oxodG in leukocytes DNA compared with sperm DNA in samples from all studied subjects. B, Levels of 8-oxodG in leukocytes DNA and sperm DNA in control group. C, Levels of 8-oxodG in sperm DNA in different studied groups. D, Levels of 8-oxodG in leukocytes DNA in different studied groups.</p

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2′-deoxycytidine level in cultured cancerous cell lines

<div><p>Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2′-deoxycytidine, 5-(hydroxymethyl)-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine as well as 5-(hydroxymethyl)-2′-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt’s lymphoma lymphoblasts (Raji), EBV-negative Burkitt’s lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of <i>TET1</i>, <i>TET2</i>, <i>TET3</i>, <i>SMUG1</i>, and <i>TDG</i> genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2′-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.</p></div

Doubling time of cells.

<p>Doubling time of cells.</p