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Reactive nitrogen species (RNS) can modify proteins at tyrosine and tryptophan residues, and they are involved in the pathogenesis of various human diseases. In this study, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method that enables the simultaneous measurement of urinary 3-nitrotyrosine (3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA). After the addition of stable isotope-labeled internal standards, urine samples were purified and enriched using manual solid-phase extraction (SPE) and HPLC fractionation followed by online SPE LC-MS/MS analysis. The limits of quantification in urine were 3.1 and 2.5 pg/mL for 3-NTYR and NHPA, respectively. Inter- and intraday imprecision was <15%. The mean relative recoveries of 3-NTYR and NHPA in urine were 89-98% and 90-98%, respectively. We further applied this method to 65 urinary samples from healthy subjects. Urinary samples were also analyzed for N-nitrosodimethylamine (NDMA) as well as oxidative and methylated DNA lesions, namely, 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), N7-methylguanine (N7-MeG), and N3-methyladenine (N3-MeA), using reported LC-MS/MS methods. Urinary 3-NTYR and NHPA levels were measured at concentrations of 63.2 ± 51.5 and 77.4 ± 60.8 pg/mL, respectively. Urinary 3-NTYR and NHPA levels were highly correlated with each other and with 8-oxoGua and 8-oxodGuo. Our findings demonstrated that a relationship exists between oxidative and nitrative stress. However, 3-NTYR and NHPA were correlated with N7-MeG and N3-MeA but not with NDMA, suggesting that NDMA may not be a representative biomarker of N-nitroso compounds that are induced by RNS.

From 1986 to the present, the popular research model organism Caenorhabditis elegans has been thought to completely lack DNA methylation and seems to have lost DNA methylation enzymes from its genomes. In the present study, we report the development of a sensitive and selective assay based on LC-MS/MS to simultaneously measure 5-methyl-2'-deoxycytidine (5-mdC) and 5-hydroxymethyl-2'-deoxycytidine (5-hmdC) in DNA hydrolysates. With the use of isotope internal standards ([2H3]5-mdC and [2H3]5-hmdC) and online solid-phase extraction, the detection limits of 5-mdC and 5-hmdC were estimated to be 0.01 and 0.02 pg respectively, which correspond to a 0.000006% and 0.00001% methylation and hydroxymethylation level. This method was applied to investigate whether DNA methylation/hydroxymethylation exists in C. elegans. The present study for the first time demonstrates that 5-mdC is present in C. elegans genomic DNA (0.0019-0.0033% of cytosine methylated) using LC-MS/MS, whereas another epigenetic modification, 5-hmdC, is not detectable. Furthermore, we found that C. elegans DNA was hypo- or hyper-methylated in a dose-dependent manner by the DNA methyltransferase (DNMT)-inhibiting drug decitabine (5-aza-2'-deoxycytidine) or cadmium respectively. Our data support the possible existence of an active DNA-methylation mechanism in C. elegans, in which unidentified DNMTs could be involved. The present study highlights the importance of re-evaluating the evolutionary conservation of DNA-methylation machinery in nematodes which were traditionally considered to lack functional DNA methylation.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its urinary metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are the most investigated carcinogenic biomarkers of tobacco-specific nitrosamines. Here, we report the development of a sensitive and selective assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure urinary NNK and NNAL. With the use of isotope internal standards and online solid-phase extraction, urine samples were directly analyzed without prior sample purification. The detection limits of this method were 0.13 and 0.19 pg on column for NNK and NNAL, respectively. Inter- and intra-day imprecision was <10 %. Mean recovery of NNK and NNAL in urine was 99-100 %. This method was applied to measure urinary NNK and NNAL in 101 smokers and 40 nonsmokers to assess tobacco exposure. Urinary nicotine, cotinine, N3-methyladenine (N3-MeA), and N7-methylguanine (N7-MeG) were also measured by isotope-dilution LC-MS/MS methods. The results showed that urinary NNK was not observed in all smokers. Urinary free NNAL (0.10 ± 0.09 ng/mg creatinine) and total NNAL (0.17 ± 0.14 ng/mg creatinine) were detected in all smokers. Urinary concentrations of NNAL were significantly correlated with nicotine, cotinine, N3-MeA, and N7-MeG in smokers (P < 0.001). This method enables the direct and simultaneous measurement of NNK and NNAL in urine using only 50 μL of urine. This study first demonstrated in human that urinary tobacco-specific nitrosamines metabolite (NNAL) are highly correlated with their resulting methylated DNA lesions in urine, which may help to substantiate an increased cancer risk associated with tobacco smoke exposure.

Since methylation at the N-7 and O(6) positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N(7)-methylguanine (N(7)-MeG), O(6)-methylguanine (O(6)-MeG), and N(3)-methyladenine (N(3)-MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure N(7)-MeG, O(6)-MeG, and N(3)-MeA in DNA hydrolysates. With the use of isotope internal standards ((15)N(5)-N(7)-MeG, d(3)-O(6)-MeG, and d(3)-N(3)-MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N(7)-MeG, O(6)-MeG, and N(3)-MeA, respectively. Inter- and intraday imprecision (CV) were 3.6-9.6% and 2.7-13.6%, respectively. Mean recoveries were 96-109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N(7)-MeG, O(6)-MeG, and N(3)-MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N(7)-MeG, O(6)-MeG, and N(3)-MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents.

Background: Increasing scientific evidence shows the significant role of epigenetic mechanisms in drug use disorder, abstinence and relapse. Studies on human subjects are limited compared to those on animals, for various reasons such as poly-substance abuse, high drop-out rate and technical difficulties. Objectives: Our goal was to evaluate whether a monitored abstinence period of 21 days could induce changes in global DNA methylation in chronic heroin users. Method: In the current study, we present data on global DNA methylation on a set of 18 male patients with chronic heroin use disorder, carefully selected based on inclusion and exclusion criteria, who were hospitalized and closely monitored during a 21-day detoxification program, one of the few where no opioid agonist is administered. The participants were sampled twice, once upon enrolment to the program and once upon completion. Results: According to our results, no difference in global DNA methylation was detected between samples collected upon enrolment and samples collected upon completion of the program. Conclusion: The findings of this study do not rule out the possibility that the 21-day abstinence period was not long enough to observe changes in global DNA methylation, or that abstinence induced site-specific methylation changes (but not global changes), that certainly merit further evaluation.

Reactivenitrogen species (RNS) can modify proteins at tyrosineand tryptophan residues, and they are involved in the pathogenesisof various human diseases. In this study, we present the first liquidchromatography–tandem mass spectrometry (LC-MS/MS)-based methodthat enables the simultaneous measurement of urinary 3-nitrotyrosine(3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA).After the addition of stable isotope-labeled internal standards, urinesamples were purified and enriched using manual solid-phase extraction(SPE) and HPLC fractionation followed by online SPE LC-MS/MS analysis.The limits of quantification in urine were 3.1 and 2.5 pg/mL for 3-NTYRand NHPA, respectively. Inter- and intraday imprecision was <15%.The mean relative recoveries of 3-NTYR and NHPA in urine were 89–98%and 90–98%, respectively. We further applied this method to65 urinary samples from healthy subjects. Urinary samples were alsoanalyzed for N-nitrosodimethylamine (NDMA) as wellas oxidative and methylated DNA lesions, namely, 8-oxo-7,8-dihydroguanine(8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), N7-methylguanine (N7-MeG), and N3-methyladenine(N3-MeA), using reported LC-MS/MS methods. Urinary 3-NTYR and NHPAlevels were measured at concentrations of 63.2 ± 51.5 and 77.4± 60.8 pg/mL, respectively. Urinary 3-NTYR and NHPA levels werehighly correlated with each other and with 8-oxoGua and 8-oxodGuo.Our findings demonstrated that a relationship exists between oxidativeand nitrative stress. However, 3-NTYR and NHPA were correlated withN7-MeG and N3-MeA but not with NDMA, suggesting that NDMA may notbe a representative biomarker of N-nitroso compoundsthat are induced by RNS. [ABSTRACT FROM AUTHOR]

From 1986 to the present, the popular research model organism Caenorhabditis elegans has been thought to completely lack DNA methylation and seems to have lost DNA methylation enzymes from its genomes. In the present study, we report the development of a sensitive and selective assay based on LC-MS/MS to simultaneously measure 5-methyl-2'-deoxycytidine (5-mdC) and 5-hydroxymethyl-2'-deoxycytidine (5-hmdC) in DNA hydrolysates. With the use of isotope internal standards ([²H3]5-mdC and [²H3]5-hmdC) and online solid-phase extraction, the detection limits of 5-mdC and 5-hmdC were estimated to be 0.01 and 0.02 pg respectively, which correspond to a 0.000006% and 0.00001% methylation and hydroxymethylation level. This method was applied to investigate whether DNA methylation/hydroxymethylation exists in C. elegans. The present study for the first time demonstrates that 5-mdC is present in C. elegans genomic DNA (0.0019-0.0033% of cytosine methylated) using LC-MS/MS, whereas another epigenetic modification, 5-hmdC, is not detectable. Furthermore, we found that C. elegans DNA was hypo- or hyper-methylated in a dose-dependent manner by the DNA methyltransferase (DNMT)-inhibiting drug decitabine (5-aza-2'-deoxycytidine) or cadmium respectively. Our data support the possible existence of an active DNA-methylation mechanism in C. elegans, in which unidentified DNMTs could be involved. The present study highlights the importance of re-evaluating the evolutionary conservation of DNA-methylation machinery in nematodes which were traditionally considered to lack functional DNA methylation. [ABSTRACT FROM AUTHOR]

Epigenetic changes include the addition of a methyl group to the 5' carbon of the cytosine ring, known as DNA methylation, which results in the generation of the fifth DNA base, namely 5-methylcytosine. During active or passive demethylation, an intermediate modified base is formed, 5-hydroxymethylcytosine. We have currently quantified 5-methylcytosine and 5-hydroxymethylcytosine in the liver and brain of mice treated with cocaine or heroin, using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Our results show that global 5-methylcytosine levels are not affected by heroin or cocaine administration, neither in the liver nor in the brain. However, 5-hydroxymethylcytosine levels are reduced in the liver following cocaine administration, while they are not affected by cocaine in the brain or by heroin administration in the liver and the brain. Elucidation of the epigenetic phenomena that takes place with respect to drug abuse and addiction, via quantitative analysis of different modified bases, may enable a better understanding of the underlying mechanisms and may lead to more personalized and effective treatment options. [ABSTRACT FROM AUTHOR]