Direct Analysis of 5-Methylcytosine and 5-Methyl-2′-deoxycytidine in Human Urine by Isotope Dilution LC-MS/MS: Correlations with N-Methylated Purines and Oxidized DNA Lesions

Abstract

Recent evidence suggests that active DNA demethylation involves base excision repair (BER) and nucleotide excision repair (NER) pathways. We hypothesized that the resulting excision products could be further excreted and present in urine. A highly specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was first developed for simultaneously measuring urinary 5-methylcytosine (5-meC) and 5-methyl-2'-deoxycytidine (5-medC). With the use of isotope internal standards and online solid-phase extraction (SPE), the detection limits of 5-meC and 5-medC were estimated to be 1.2 and 0.3 pg, respectively. This method was applied to measure urinary samples of 376 healthy males. Urinary samples were also measured for methylated and oxidized DNA lesions, namely, N7-methylguanine (N7-meG), N3-methyladenine (N3-meA), 8-oxo-7,8-dihydroguanine (8-oxoGua), and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using reported online SPE LC-MS/MS methods. Results showed that mean urinary levels of 5-meC and 5-medC were 28.4 ± 14.3 and 7.04 ± 7.2 ng/mg creatinine, respectively, supporting the possible presence of DNA demethylation through BER and NER mechanisms. Urinary levels of 5-meC were significantly positively correlated with N7-meG, N3-meA, and 8-oxodG. Good correlations between 5-meC and methylated and oxidized DNA lesions may have implied the underlying linkage between genetic (DNA lesions) and epigenetic (DNA methylation) alterations derived from exogenous exposure and/or from endogenous cellular processes in human and require further investigation.

Associations of urinary 5-methyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine with phthalate exposure and semen quality in 562 Chinese adult men

“…The deoxynucleoside of 5mC and 5hmC, namely 5-methyl-2′-deoxycytidine (5mdC) and 5-hydroxymethyl-2′-deoxycytidine (5hmdC) were successfully identified and quantified in urine in recent studies (Hu et al, 2012;Yin et al, 2015). Several biological processes, such as apoptosis, can cause DNA to degrade into single deoxynucleosides (Kawane et al, 2014;Nagata et al, 2003).…”

“…Several biological processes, such as apoptosis, can cause DNA to degrade into single deoxynucleosides (Kawane et al, 2014;Nagata et al, 2003). It is hypothesized that urinary 5mdC and 5hmdC molecules may result from metabolism of methylated DNA (e.g., DNA degradation or DNA excision repair) (Hu et al, 2012;Yin et al, 2015). It is likely that urinary 5mdC and 5hmdC contents may, to some extent, reflect metabolism status and function of tissues or organs in human body.…”

Detection of 1,N2-propano-2′-deoxyguanosine in human urine by stable isotope dilution UHPLC–MS/MS analysis

“…Various methods have been developed for the detection of urinary nucleosides, including electrochemical method [18], immunoassays [19,20], capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) [17,[21][22][23][24], matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [25,26], liquid chromatography tandem mass spectrometry (LC-MS) [27][28][29][30][31][32]. In comparison to other methods, the LC-MS based methodologies take the advantages of high sensitivity and direct analysis with structural information from MS measurements [33].…”

Citotoxicidade do corante dinitrofenilazo CI DB291: biotransformação, estresse oxidativo e alteração epigenética em células HepG2

“…SUZUKI et al, 1997;FACOMPRÉ et al, 2000;IACOMINO et al, 2006 Estudos cinéticos do ciclo celular demonstraram que o bloqueio da progressão do ciclo na fase G2/M é um requisito para a deflagração da apoptose (LIU et al, 2005). Considerando que a homeostase mitocondrial desempenha um papel importante nessa regulação, podemos dizer que os efeitos do corante DB291 sobre as mitocôndrias estão relacionados com a parada no ciclo celular, conforme o observado por outros autores em diferentes tipos de linhagens celulares (LI et al, 1999;FACOMPRÉ et al, 2000;LIU et al, 2005 (BIANCHINI et al, 2001;CADET et al, 2008) e esforços têm sido feitos nos últimos anos para minimizar sua formação artefatual durante o preparo do DNA para análise (RAVANAT et al, 2002;ESCODD, 2002;GEDIK et al, 2004;MANGAL et al, 2009 GEHRING et al, 2009;NEIHRS, 2009;HU et al, 2012), o que sugere a existência de uma correlação entre níveis de lesões em DNA e a perda de 5-metil-dC do genoma (HU et al, 2012).…”

“…A completa validação do método analítico utilizado está descrita abaixo. Estudos reportados na literatura mostram que a metionina, precursora da SAM e portanto molécula chave no processo de metilação enzimática do DNA, pode ser facilmente oxidada por ROS, formando metionina sulfóxido (MetO) (STADTMAN et al, 2005;HU et al, 2012). MetO pode, por sua vez, reagir com radicais hidroxila e gerar radicais metila que acabam levando à metilação não enzimática de outras bases do DNA, gerando lesões como O 6 -metil-2'-desoxiguanosina, N 7 -metil-2'-desoxiguanosina e N 3 -metil-2'-desoxiadenosina (STADTMAN et al, 2005;KAWAI et al, 2010;HU et al, 2012 …”