Interrelationships between DNA modifications.

<p>Interrelationships between 5-methyl-2’-deoxycytidine, 5-hydroxymethyl-2’-deoxycytidine, 5-formyl-2’-deoxycytidine, 5-carboxyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine in DNA of various porcine and rat tissues. Coefficients of determination and p-values were calculated using Pearson’s analysis of correlation. Data is presented as mean values for biological replicates; error bars correspond to standard deviations.</p

Relationship between tissue-specific proliferation rate and epigenetic DNA modifications.

<p>Relationship between tissue-specific proliferation rate, determined in a murine model and expressed as the percentage of labelled 5-methylcytosine formed in DNA during cellular division (data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144859#pone.0144859.ref005" target="_blank">5</a>] kindly supplied by prof. Bachman) and the content of 5-methyl-2’-deoxycytidine and active demethylation products in DNA isolated from various porcine (red) and rat (green) tissues. Coefficients of determination were calculated using a linearized regression model with logarithmic fit for proliferation rate and linear fit for all other variables. Data presented as mean values for biological replicates; error bars correspond to standard deviations.</p

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

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 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

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

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

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