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

Abstract

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.

Endogenously generated DNA nucleobase modifications source, and significance as possible biomarkers of malignant transformation risk, and role in anticancer therapy

“…This proposed epigenetic regulation by 8-oxoGua would be in contrast to the role of 5-mCyt, which is responsible for transcription suppression. Interestingly, in our recently published study, we demonstrated a negative correlation between background levels of 8-oxoGua and 5-mCyt in DNA [27]. The mechanisms that underly site-specific DNA modifications, chromatin changes, and transcription, are highly complex and may depend on numerous factors, such as sequence context, tissue differences or/and specific proteins assembly, to name a few [48,50].…”

“…Recently, using LC-MS/MS-based methodology and after exclusion of the most likely factors responsible for potential artifactual formation of Ura in DNA (e.g. contamination of reagents with deaminases), the baseline level of Ura was estimated to be in the range of 0.056 to 4.03 dU/10 6 deoxuynucleosides (dN) in cultured cells lines [24,25], 1 to 9.6 dU/10 6 dN in human leukocytes [26], 11.41 dU/10 6 dN in human colorectal cancer tissue and 12.17 dU/10 6 dN normal colon tissue from the tumour's margin [27].…”

Chemoselective labeling and site‐specific mapping of 5‐formylcytosine as a cellular nucleic acid modification

“…1B). Sensitive chromatographic methods coupled with electrospray tandem mass spectrometry (HPLC-ESI-MS/ MS) [36,38,39], in combination with the use of stable isotope-labeled standards [18,40,41], enabled researchers to determine global levels of cytosine-related epigenetic bases in stem cells and in various mammalian tissues. Cytosine modifications can also be detected based on DNA digestion by modification-sensitive restriction enzymes.…”

Distinct pattern of epigenetic DNA modification in leukocytes from patients with colorectal carcinoma and individuals with precancerous conditions, benign adenoma and inflammatory bowel disease – a link to oxidative stress

“…Leukocytes were isolated from heparinized blood samples with Histopaque 1119 (Sigma, United Kingdom) solution, according to the manufacturer's instruction, and stored at -80°C until the analysis. Isolation of leukocyte DNA and its hydrolysis were carried out as previously described [27], but the cell pellet was immediately dispersed in ice-cold lysing buffer B, without homogenization and washing steps. Procedures used for the determination of 5-methyl-2'-deoxycytidine (5-mdC), 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC), 5-formyl-2'deoxycytidine (5-fdC), 5-carboxy-2'deoxycytidine (5-cadC), 5-(hydroxymethyl)-2'-deoxyuridine (5-hmdU) and 8-oxodG by 2D-UPLC-MS/MS have been described elsewhere [27].…”

“…Due to very low level of these modifications in mammalian genome (approximately 3-4 orders of magnitude lower than 5-hmCyt content), their accurate determination is highly challenging. In this study, we used our recently developed rapid, highly-sensitive and highly-specific isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) [26,27] to analyze global methylation and the levels of all the abovementioned TET-mediated oxidation products of 5-mCyt and thymine: 5-hmCyt, 5-fCyt, 5-caCyt and 5-hmUra. Moreover, we determined leukocyte content of the best characterized marker of oxidatively modified DNA, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), as well as the expression of TETs mRNA, and plasma concentrations of ascorbate and retinol.…”