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Environmental exposure would cause DNA damage and epigenetic modification changes, potentially resulting in physiological dysfunction, thereby triggering diseases and even cancer. DNA damage and epigenetic modifications are thus promising biomarkers for environmental exposures and disease states. Benefiting from its high sensitivity and accuracy, high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is considered the "gold standard technique" for investigating epigenetic DNA modifications. This review summarizes the recent advancements of UHPLC-MS/MS-based technologies for DNA damage and epigenetic modifications analysis, mainly focusing on the innovative methods developed for UHPLC-MS/MS-related pretreatment technologies containing efficient genomic DNA digestion and effective removal of the inorganic salt matrix, and the new strategies for improving detection sensitivity of liquid chromatography-mass spectrometry. Moreover, we also summarized the novel hyphenated techniques of the advanced UHPLC-MS/MS coupled with other separation and analysis methods for the measurement of DNA damage and epigenetic modification changes in special regions and fragments of chromosomes. • Accelerated genomic DNA digestion strategies for LC-MS analysis. • Matrix desalination and salt interference mitigation. • New strategies for improving detection sensitivity of LC-MS. • Applications of DNA damage and epigenetic modification analysis via LC-MS and complementary techniques. [ABSTRACT FROM AUTHOR]

Abstract Ultraviolet (UV) radiation induces mutagenicity and cytotoxicity in human cells by the formation of DNA lesions, including cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), mainly on thymine-thymine (TpT) dinucleotides. Here, we firstly synthesized the two TpT dimeric lesions with satisfactory yields using a unique UV-irradiated water droplet approach followed by HPLC purification. By the use of purified TpT lesions as standards, we further developed and optimized a quantitative UHPLC-Q-TOF/MS method for the detection of CPDs and 6-4PPs. After the optimization of the enzyme composition and the pH values of hydrolysis solution, a combination of snake venom phosphodiesterase, nuclease P1, and calf intestine alkaline phosphatase can be used for one-step enzymatic digestion to efficiently release the dimeric lesions (CPDs and 6-4PPs) from the genomic DNA. By the use of the one-step digestion and UHPLC-Q-TOF/MS assay for scanning all dimeric lesions, we demonstrate that only are TpT dimeric lesions detectable in genomic DNA of HCT116 cells upon UVC irradiation. The estimated frequency of the CPD of TpT increases from 28.7 to 409 per 106 bases with increasing UVC dosage from 40 J/m2 to 1200 J/m2, while the 6-4PP of TpT increases from 3.7 to 54 per 106 bases. The proposed UHPLC-Q-TOF/MS method is promising for accurate identification and quantitative detection of UV-induced dimeric lesions in cellular DNA. Highlights • Unique UV-irradiated water droplet approach followed by HPLC purification was developed to prepare TpT dimer standards. • A simplified one-step enzymatic process to release TpT dimeric lesions from genomic DNA. • TT CPD and T(6-4)T levels were detected by UHPLC-Q-TOF/MS in HCT116 cells upon UVC-irradiation. [ABSTRACT FROM AUTHOR]

A sensitive and accurate stable isotope dilution UHPLC–MS/MS method was developed and validated for the detection and quantification of ProdG adducts in human urine, a surrogate for the ProdG adducts in genomic DNA of human. A specific solid phase extraction (SPE) approach was established for selective enrichment of urinary ProdG adducts and elimination of urinary matrix facilitating the coupled MS/MS detection. The recovery of the method is estimated about 84.8–107.2%, and the precision are about 0.8–3.6% for intraday and 2.8–10.0% for interday. Due to that the matrix effect is efficiently eliminated by SPE pretreatment, the limits of detection (LODs, S/N = 3) and quantification (LOQs, S/N = 10) are decreased to 100 and 300 amol for urinary ProdG adducts, respectively. By coupling the developed SPE pretreatment with the UHPLC–MS/MS analysis, ProdG adducts were accurately quantified in healthy human urine. [ABSTRACT FROM AUTHOR]

Crotonaldehyde (Cro) is one of widespread and genotoxic α,β-unsaturated aldehydes and can react with the exocyclic amino group of 2′-deoxyguanosine (dG) in genomic DNA to form 1, N 2 -propano-2′-deoxyguanosine (ProdG) adducts. In this study, two diastereomers of high purity were prepared, including non-isotope and stable isotope labeled ProdG adducts, and exploited stable isotope dilution-based calibration method. By taking advantage of synthesized ProdG standards, we developed a sensitive ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI–MS/MS) method for accurate quantification of two diastereomers of ProdG adducts. In addition to optimization of the UHPLC separation, ammonium bicarbonate (NH 4 HCO 3 ) was used as additive in the mobile phase for enhancing the ionization efficiency to ProdG adducts and facilitating MS detection. The limits of detection (LODs, S/N = 3) and the limits of quantification (LOQs, S/N = 10) are estimated about 50 amol and 150 amol, respectively. By the use of the developed method, both diastereomers of ProdG adducts can be detected in untreated human MRC5 cells with a frequency of 2.4–3.5 adducts per 10 8 nucleotides. Crotonaldehyde treatment dramatically increases the levels of ProdG adducts in human MRC5 in a concentration-dependent manner. [ABSTRACT FROM AUTHOR]

• A recyclable and reusable multi-enzymes cascade (SNSC-MNPs) for enzymatic digestion of genomic DNA was constructed. • SNSC-MNPs cascade enable fast digestion of genomic DNA into mono-nucleosides within 30 min. • Coupling SNSC-MNPs with HPLC-MS/MS, a sensitive and accurate method for quantitation of 5-hydroxymethylcytosine (5hmC) was developed. • TCBQ exposure induces elevated 5hmC levels in the genome of T24 cells. Long-term exposure to halobenzoquinones (HBQs) can induce genomic damages and abnormal epigenetic modifications. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has shown unique advantages in identification and sensitive analysis of these structurally modified DNA lesions. Prior to MS analysis, genomic DNA needs to be fully digested into mono-nucleosides. Here, we prepared Supernuclease (SN)-, snake venom phosphodiesterase (SVP)- and calf intestinal alkaline phosphatase (CIP)- individually immobilized magnetic nanoparticles (MNPs), and combined them according to certain formula to construct a recyclable SN-SVP-CIP magnetic nanoparticles (SNSC-MNPs) cascade for rapid and efficient DNA digestion. The SNSC-MNPs cascade can fully digest genomic DNA into mono-nucleosides within 30 min. The SNSC-MNPs cascade coupled with HPLC-MS/MS method can accurately and sensitively detect 5-hydroxymethylcytosine (5hmC) changes in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone. The immobilization of enzymes on MNPs can enhance the stability and enzymatic activity of the three enzymes, which guarantees the reusability and longtime preservation of the cascades. The relative digestive efficiencies are among 86% -106% up to ten times of reuse. The newly synthesized SNSC-MNPs cascade coupled with HPLC-MS/MS method is promising for fast identification and analysis of structural modifications in genomic DNA. [ABSTRACT FROM AUTHOR]

Abstract: An ultra-performance liquid chromatography tandem mass spectrometry with multiple reaction monitoring method (UPLC-MRM/MS) is developed for fast and sensitive analysis of four genotoxic stereoisomers of anti-benzo[a]pyrene diol epoxide (BPDE)–N 2dG adducts (trans-(+), trans-(−), cis-(+) and cis-(−)), which result from environmental exposure to ubiquitous pollutant benzo[a]pyrene (B[a]P). The developed method displays a low limit of detection of <0.7fmol (S/N=3) for the four stereoisomers of anti-BPDE–N 2dG, a dynamic range of 2 orders of magnitude (2.3–630fmol, R 2 ≥0.997), and one separation of 2–4min. The developed method enables us to use the stereoisomers of anti-BPDE–N 2dG as a biomarker and to study the stereoselectivity of metabolic activation of B[a]P in human lung A549 cells. The UPLC-MRM/MS analysis of cellular DNA exposed to B[a]P show that activation of B[a]P in A549 cells predominantly induces trans-(+)-anti-BPDE–N 2dG with cis-(+)-anti-BPDE–N 2dG and one syn-BPDE–N 2dG as two minorities, while trans-(−)-anti-BPDE–N 2dG and cis-(−)-anti-BPDE–N 2dG are absent. The observed preferential formation of trans-(+)-anti-BPDE–N 2dG in B[a]P treated A549 cells may result from combined stereoselectivity of the metabolic activation of B[a]P and the reaction of anti-BPDE with dsDNA. The results also suggest that a number of key optical intermediates are formed during activation of B[a]P in A549 cells, including trans-(+)-B[a]P-7,8-dihydrodiol and trans-(−)-B[a]P-7,8-dihydrodiol and their corresponding downstream metabolites (+)-anti-BPDE and (+)-syn-BPDE. [Copyright &y& Elsevier]