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Adductomics is expected to be useful in the characterization of the exposome, which is a new paradigm for studying the sum of environmental causes of diseases. DNA adductomics is emerging as a powerful method for detecting DNA adducts, but reliable assays for its widespread, routine use are currently lacking. We propose a novel integrated strategy for the establishment of a DNA adductomic approach, using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS), operating in constant neutral loss scan mode, screening for both known and unknown DNA adducts in a single injection. The LC-QqQ-MS/MS was optimized using a representative sample of 23 modified 2'-deoxyribonucleosides reflecting a range of biologically relevant DNA lesions. Six internal standards (ISTDs) were evaluated for their ability to normalize, and hence correct, possible variation in peak intensities arising from matrix effects, and the quantities of DNA injected. The results revealed that, with appropriate ISTDs adjustment, any bias can be dramatically reduced from 370 to 8.4%. Identification of the informative DNA adducts was achieved by triggering fragmentation spectra of target ions. The LC-QqQ-MS/MS method was successfully applied to in vitro and in vivo studies to screen for DNA adducts formed following representative environmental exposures: methyl methanesulfonate (MMS) and five N-nitrosamines. Interestingly, five new DNA adducts, induced by MMS, were discovered using our adductomic approach-an added strength. The proposed integrated strategy provides a path forward for DNA adductomics to become a standard method to discover differences in DNA adduct fingerprints between populations exposed to genotoxins, and facilitate the field of exposomics.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most investigated product of oxidatively damaged DNA lesion that has been associated with the development of aging, cancer and some degenerative diseases. Here, we present the first liquid chromatography-tandem mass spectrometry method that enables the simultaneous measurement of its repair products in plasma and saliva, namely 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxodGuo. Using this method, we investigated the underlying transport mechanism of the repair products of oxidatively damaged DNA between cellular compartments and biological matrices. Plasma, saliva and urine samples were collected concurrently from 57 healthy subjects. Various deproteinization methods were evaluated, and the precipitants acetonitrile and sodium hydroxide-methanol were, respectively, selected for plasma and saliva samples due to their effect on recovery efficiencies and chromatography. The mean baseline concentrations of 8-oxoGua and 8-oxodGuo in plasma were demonstrated to be 0.21 and 0.016 ng/mL, respectively, while in saliva they were 0.85 and 0.010 ng/mL, respectively. A relatively high concentration of 8-oxoGua was found in saliva with a concentration factor (CF, concentration ratio of saliva to plasma) of 4 as compared to that of 8-oxodGuo (CF: 0.6), implying that 8-oxoGua in plasma may be actively transported to saliva, whereas 8-oxodGuo was most dependent on a passive diffusion. Good correlations between urine and plasma concentrations were observed for 8-oxoGua and 8-oxodGuo, suggesting that blood was a suitable matrix in addition to urine. Significant correlation between 8-oxoGua and 8-oxodGuo in urine was only observed when the concentrations were not corrected for urinary creatinine, raising the issue of applicability of urinary creatinine to adjust 8-oxoGua concentrations.

A total sample-preparation and analysis time of 50 min is required for the high-throughput method of hair analysis proposed in this paper. The method is applicable to analysis of drugs commonly used in Asia, and their metabolites--methamphetamine (MA), amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), ketamine (K), norketamine (NK), dehydronorketamine (DHNK), 6-acetylmorphine (6-AM), morphine (MOR), and codeine (COD). Cut and weighed hair (10 mg) was incubated for 3 min with methanol-trifluoroacetic acid (TFA) during microwave-assisted extraction (MAE) at 700 W. The incubation solution was evaporated, the residue was reconstituted in deionized water-methanol, 99:1 (v/v), and 20 μL was injected on to a core-shell column (50 × 4.6 mm, 2.6 μm particle size) for liquid chromatographic-tandem mass spectrometric (LC-MS-MS) analysis. Gradient elution separation was performed in 8 min at a flow rate of 1 mL min(-1). No signal interfering with any of the analytes was found in fourteen blank hair samples from different sources. The limits of detection and quantification were 0.5 pg mg(-1) and 2.0 pg mg(-1), respectively, for MA, AMP, MDMA, MDA, K, NK, and DHNK, and 2.0 pg mg(-1) and 5.0 pg mg(-1), respectively, for 6-AM, MOR and COD. The linear range was between the LOQ and 1000 pg mg(-1), and the correlation coefficients were all greater than 0.999. Investigation of matrix effects revealed that all the analytes were suppressed by less than 20% and the standard deviation (SD) was always less than 7%. Recovery was always greater than 90% and the SD for each compound was less than 6%. Precision and accuracy for each analyte were within 15%. Eight authentic hair specimens from known drug abusers were successfully analyzed. Compared with traditional overnight incubation methods, the rapid 3-min extraction time achieved similar or greater extraction yields. Sample preparation by MAE was a reliable procedure for extraction of the analytes from hair but substantially simpler and faster than other methods.

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.

Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2'-deoxycytidine (5-medC) and 2'-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O (6)-methylguanine (O (6)-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O (6)-meG and N3-meA were however found to be undetectable in all lung tissue samples.

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.

Betel quid chewing has been associated with several human cancers. However, the role of betel quid in carcinogenesis remains uncertain. Piper betel contains high concentrations of safrole (an inducer of DNA oxidative damage). Safrole may be metabolized by hepatic sulfotransferase 1A1 (SULT1A1), or glutathione S-transferases (GSTM1, GSTT1, and GSTP1). Thus, we investigated the association of genetic polymorphisms of SULT1A1, GSTM1, GSTT1, and GSTP1 with DNA oxidative damage among betel quid chewers. A biomarker for oxidative stress, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) level, was analyzed using isotope-dilution LC-MS/MS in 64 betel quid chewers and 129 non-betel quid chewers. Data on demographics and habits (smoking, alcohol drinking, and betel quid chewing) were obtained from questionnaires. Our results revealed that urinary 8-OHdG level was higher in chewers with SULT1A1 Arg-His genotype than in chewers with SULT1A1 Arg-Arg genotype. Urinary 8-OHdG level was also higher in chewers with GSTP1 Ile-Ile genotype. Furthermore, the combined effect of SULT1A1 and GSTP1 genotypes on urinary 8-OHdG was evaluated. Non-chewers with both SULT1A1 Arg-Arg and GSTP1 Val-Val/Ile-Val (reference group) had the lowest mean level (3.6 ng/mg creatinine), whereas chewers with either SULT1A1 Arg-His or GSTP1 Ile-Ile had the highest 8-OHdG mean level (6.2 ng/mg creatinine; vs. reference group, P = 0.04). Chewers with both of SULT1A1 Arg-Arg and GSTP1 Val-Val/Ile-Val (4.6 ng/mg creatinine), and non-chewers with either SULT1A1 Arg-His or GSTP1 Ile-Ile (4.7 ng/mg creatinine) had a moderately increased 8-OHdG level. Thus, the susceptible SULT1A1 and GSTP1 genotypes may modulate increased DNA oxidative stress elicited by betel-quid chewing.