Novel biomarkers in amniotic fluid for early assessment of intraamniotic infection

Intra-amniotic infection/inflammation (IAI) is associated with preterm birth, short and long-term adverse clinical outcomes and oxidative stress. The diagnosis of IAI is based on histological and clinical findings; however, often these results are unspecific. Therefore, efforts have been directed towards validating reliable methods for patients lacking overt clinical symptoms. In this study, amniotic fluid (AF) samples were prospectively collected from 23 women grouped into two categories (with or without IAI) following clinical, microbiological and histological criteria. AFs were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of the following biomarkers: oxidized and nitrated tyrosines (Tyr), 8-hydroxy-2'-deoxyguanosine (8OHdG), oxidized glutathione (GSSG) and glutathione sulfonamide (GSA). 3-NO₂-Tyrosine (3NO₂-Tyr) and GSSG concentrations in AF were not identified as significantly relevant biomarkers in the presence of IAI. However, inflammatory biomarkers such as GSA (p=0.002) and 3-Chloro-Tyrosine [3Cl-Tyr (p=0.049)], and oxidative stress biomarker 8OHdG (p=0.021) were significantly increased in AF with IAI as compared to normal controls. Biomarkers of inflammation and oxidative stress determined in AF samples could represent a new approach towards an early diagnosis of IAI and subsequent chorioamnionitis in the clinical setting.     

Comparison of three oxidative stress biomarkers in a sample of healthy adults

Oxidative stress is a potentially important aetiological factor for many chronic diseases, including cardiovascular disease, neurodegenerative disease and cancer, yet studies often find inconsistent results. The associations between three of the most widely used biomarkers of oxidative stress, i.e. F₂-isoprostanes for lipid peroxidation and 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG) and the comet assay with FPG for oxidative DNA damage, were compared in a sample of 135 healthy African-American and white adults. Modest associations were observed between F₂-isoprostanes and the comet assay (r?=?0.22, p?=?0.01), but there were no significant correlations between 8-oxo-dG and the comet assay (r?=??0.09) or F₂-IsoP (r?=??0.04). These results are informative for researchers seeking to compare results pertaining to oxidative stress across studies and/or assessment methods in healthy disease-free populations. The development and use of oxidative stress biomarkers is a promising field; however, additional validation studies are necessary to establish accuracy and comparability across oxidative stress biomarkers.     

Quantification of the oxidative damage biomarker 2,3-dinor-8-isoprostaglandin-F2α in human urine using liquid chromatography-tandem mass spectrometry

F₂-isoprostanes are useful biomarkers of oxidative status in humans. We developed an ultraperformance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 2,3-dinor-8-iso prostaglandin F_2α, a urinary metabolite of 8-iso-prostaglandin F_2α. Urine was purified by solid-phase extraction and analyzed by UPLC-MS/MS with negative-ion electrospray ionization. The method was robust with a mean inaccuracy of 9%, interday and intraday imprecision of 7.5% or lower, and a lower limit of quantification of 0.5 μg/L, equivalent to 0.04 pmol injected onto the column. An analysis time of 6 min was shorter than previously published methods and amenable to large studies.     

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo⁻ fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo⁻.     

Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1–61) of p53 via an extended binding interface, with dissociation constant of 12.7 (± 0.7) μM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1–40) and TAD2 (residues 41–61) subdomains of p53. HmtSSB enhances intrinsic 3′-5′ exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) present at 3′-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.     

Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods.The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10⁶ dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.     

Concurrent quantification of multiple biomarkers indicative of oxidative stress status using liquid chromatography-tandem mass spectrometry

8-Hydroxy-2-deoxyguanosine (8-OHdG), 8-nitroguanine (8-NO₂Gua), 8-iso-prostaglandin F_2α (8-IsoPGF_2α), and N-acetyl-S-(tetrahydro-5-hydroxy-2-pentyl-3-furanyl)-L-cysteine (HNE-MA) are well-studied and representative biomarkers for oxidative DNA damage, inflammation, and lipid peroxidation; all of which have been associated with increases in risks of various diseases and cancers. A rapid and highly sensitive isotope-dilution liquid-chromatography tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantify the aforementioned biomarkers in urine. Upon validation, this method shows excellent feasibility, sensitivity (0.008–0.03 ng/mL) and satisfactory recoveries (88.7–95.4%); the calibration curves displayed excellent linearity with coefficients of determination (R²) greater than 0.998. Additionally, low variations were observed in the relative standard deviation for intra- and inter-day measurements for the four analytes. The relative matrix effects for all four analytes ranged from 2.04 to 3.27%, which signaled that interferences from endogenous levels of the analytes were deemed statistically insignificant. This study successfully developed an analytical method capable to simultaneously quantify urinary 8-OHdG, 8-NO₂Gua, 8-IsoPGF_2α, and HNE-MA. This analytical protocol can be applied towards conducting epidemiological studies to reveal the mechanisms related to disease development, and thus evaluate the associated risks of diseases.     

Oxidative stress-mediated arterial dysfunction in patients with metabolic syndrome: Effect of ascorbic acid

Arterial dysfunction is a hallmark of early atherosclerosis; however, its behavior in patients with metabolic syndrome (MS) is still unclear. We investigated the role of oxidative stress on ischemia-induced flow-mediated dilatation (FMD) in patients with MS. FMD and oxidative stress, as assessed by serum levels of 8-hydroxy-2-deoxy-2-deoxyguanosine (8-OHdG), were studied in 18 MS and 30 control subjects. Thereafter, in the 18 MS patients, FMD was assessed after iv infusion of 1 g vitamin C or placebo in a randomized, double-blind, crossover design; serial blood samples were taken in peripheral circulation before and after FMD to analyze 8-OHdG. Compared to controls, MS patients had higher 8-OHdG (p<0.001) and lower FMD (p<0.001); 8-OHdG and FMD were inversely correlated (R=-0.74; p<0.01). In MS patients, placebo administration did not change FMD, whereas vitamin C significantly enhanced it (p<0.001). After placebo, ischemia-induced FMD was associated with a significant increase in 8-OHdG (p<0.001), an effect that was counteracted by vitamin C. Vitamin C infusion was associated with an inverse correlation between the changes in FMD and oxidative stress (R=-0.67; p<0.01). The present study shows that arterial dilatation is impaired and that enhanced oxidative stress may play a key role in patients with MS.     

Analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine by ultra high pressure liquid chromatography–heat assisted electrospray ionization–tandem mass spectrometry

Increased amounts of reactive oxygen species (ROS), generally termed oxidative stress, are frequently hypothesized to be causally associated with many diseases. Analyses of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in DNA and urine are widely used biomarkers for oxidative stress. Over the years it became clear that analysis of 8-oxo-dG in DNA is challenging due to artifactual formation during sample work up. The present study demonstrates that 8-oxo-dG can be measured reliably and accurately when appropriate precautions are taken. First, the presence of an antioxidant, metal chelator, or free radical trapping agent during sample preparation improves reproducibility. Second, sample enrichment by HPLC fraction collection was used to optimize sensitivity. Third, heat assisted electrospray ionization (HESI) eliminated potential interferences and improved assay performance and sensitivity. Subsequently, the UPLC–HESI–MS/MS method was applied to show the biphasic dose response of 8-oxo-dG in H₂O₂-treated HeLa cells. Application of this method to human lymphocyte DNA (n = 156) gave a mean ± SD endogenous amount of 1.57 ± 0.88 adducts per 10⁶ dG, a value that is in agreement with the suggested amount previously estimated by European Standard Committee on Oxidative DNA Damage (ESCODD) and others. These results suggest that the present method is well suited for application to molecular toxicology and epidemiology studies investigating the role of oxidative stress.     

Oral Administration of Ginseng Ameliorates Cyclosporine-Induced Pancreatic Injury in an Experimental Mouse Model

Background

This study was performed to investigate whether ginseng has a protective effect in an experimental mouse model of cyclosporine-induced pancreatic injury.

Methods

Mice were treated with cyclosporine (30 mg/kg/day, subcutaneously) and Korean red ginseng extract (0.2 or 0.4 g/kg/day, oral gavage) for 4 weeks while on a 0.01% salt diet. The effect of ginseng on cyclosporine-induced pancreatic islet dysfunction was investigated by an intraperitoneal glucose tolerance test and measurements of serum insulin level, β cell area, macrophage infiltration, and apoptosis. Using an in vitro model, we further examined the effect of ginseng on a cyclosporine-treated insulin-secreting cell line. Oxidative stress was measured by the concentration of 8-hydroxy-2′-deoxyguanosine in serum, tissue sections, and culture media.

Results

Four weeks of cyclosporine treatment increased blood glucose levels and decreased insulin levels, but cotreatment with ginseng ameliorated the cyclosporine-induced glucose intolerance and hyperglycemia. Pancreatic β cell area was also greater with ginseng cotreatment compared with cyclosporine monotherapy. The production of proinflammatory molecules, such as induced nitric oxide synthase and cytokines, and the level of apoptotic cell death also decreased in pancreatic β cell with ginseng treatment. Consistent with the in vivo results, the in vitro study showed that the addition of ginseng protected against cyclosporine-induced cytotoxicity, inflammation, and apoptotic cell death. These in vivo and in vitro changes were accompanied by decreases in the levels of 8-hydroxy-2′-deoxyguanosine in pancreatic β cell in tissue section, serum, and culture media during cotreatment of ginseng with cyclosporine.

Conclusions

The results of our in vivo and in vitro studies demonstrate that ginseng has a protective effect against cyclosporine-induced pancreatic β cell injury via reducing oxidative stress.     

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of oxidative stress in rheumatoid arthritis and aging: effect of progressive resistance training

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured before and after 12 weeks of progressive resistance strength training in 8 healthy elderly (65–80 yr) and eight healthy young (22–30 yr) men and women, and in eight adults (25–65 yr) with rheumatoid arthritis (RA).Training subjects exercised at 80% of their one-repetition maximum and performed eight repetitions per set, three sets per session, on a twice-weekly basis. 8-OHdG was measured at baseline and follow-up (at least 24 hr after the last exercise session) in the RA and elderly subject groups, and at baseline only in young subjects.Baseline 8-OHdG levels were greater among subjects with RA compared to both healthy young (P < 0.001) and elderly (P < 0.05) subjects. There were no changes in 8-OHdG levels in either RA or elderly subjects as a result of the strength training intervention.These results suggest that subjects with RA have higher levels of oxidative stress than young and elderly healthy individuals. Furthermore, there is no change in oxidative stress, measured by urinary 8-OHdG, in elderly healthy individuals or in subjects with RA after a 12-week strength training intervention.     

Histones participate in base excision repair of 8-oxodGuo by transiently cross-linking with active repair intermediates in nucleosome core particles

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3′-phospho-α,β-unsaturated aldehyde (PUA) and 5′-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3′-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5′-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5′-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.     

Correlation between Serum Levels of 3,3?,5?-Triiodothyronine and Thyroid Hormones Measured by Liquid Chromatography-Tandem Mass Spectrometry and Immunoassay

ObjectiveFor measuring serum 3,3′,5′-triiodothyronine (rT3) levels, radioimmunoassay (RIA) has traditionally been used owing to the lack of other reliable methods; however, it has recently become difficult to perform. Meanwhile, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been attracting attention as a novel alternative method in clinical chemistry. To the best of our knowledge, there are no studies to date comparing results of the quantification of human serum rT3 between LC-MS/MS and RIA. We therefore examined the feasibility of LC-MS/MS as a novel alternative method for measuring serum rT3, thyroxine (T4), and 3,5,3′-triiodothyronine (T3) levels.MethodsAssay validation was performed by LC-MS/MS using quality control samples of rT3, T4, and T3 at 4 various concentrations which were prepared from reference compounds. Serum samples of 50 outpatients in our department were quantified both by LC-MS/MS and conventional immunoassay for rT3, T4, and T3. Correlation coefficients between the 2 measurement methods were statistically analyzed respectively.ResultsMatrix effects were not observed with our method. Intra-day and inter-day precisions were less than 10.8% and 9.6% for each analyte at each quality control level, respectively. Intra-day and inter-day accuracies were between 96.2% and 110%, and between 98.3% and 108.6%, respectively. The lower limit of quantification was 0.05 ng/mL. Strong correlations were observed between the 2 measurement methods (correlation coefficient, T4: 0.976, p < 0.001; T3: 0.912, p < 0.001; rT3: 0.928, p < 0.001).ConclusionsOur LC-MS/MS system requires no manual cleanup operation, and the process after application of a sample is fully automated; furthermore, it was found to be highly sensitive, and superior in both precision and accuracy. The correlation between the 2 methods over a wide range of concentrations was strong. LC-MS/MS is therefore expected to become a useful tool for clinical diagnosis and research.     

In Vitro Synergistic Antioxidant Activity and Identification of Antioxidant Components from Astragalus membranaceus and Paeonia lactiflora

Many traditionally used herbs demonstrate significantly better pharmacological effects when used in combination than when used alone. However, the mechanism underlying this synergism is still poorly understood. This study aimed to investigate the synergistic antioxidant activity of Astragalus membranaceus (AME) and Paeonia Lactiflora (PL), and identify the potential antioxidant components by 1,1-diphenyl-2-picrylhydrazine (DPPH) radical spiking test followed by a high performance liquid chromatography separation combined with diode array detection and tandem mass spectrometry analysis (DPPH-HPLC-DAD-MS/MS). Eight AME-PL combined extracts (E₁–E₈) were prepared based on bioactivity-guided fractionation. Among them, E₁ exhibited the strongest synergistic effect in scavenging DPPH radicals and reducing ferric ions (P<0.05). Moreover, E₁ presented strong cytoprotection against H₂O₂-induced oxidative damage in MRC-5 cells by suppressing the decrease of the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activities. A strong correlation between the increment of total phenolic/flavonoid and synergistic antioxidant activity, especially between the increment of total flavonoid and the increase in ferric reducing power was observed. Finally, seven antioxidant substances were identified in E₁ as oxypaeoniflora, catechin, calycosin-7-O-β-D-glucopyranoside, fomononetin-7-O-β-D-glucopyranoside, 9,10-dimethoxy-pterocarpan-3-O-β-D-glucopyranoside, quercetin and 2′-dihydroxy-3′,4′-dimethyl-isoflavan-7-O-β-D-glucopyranoside.     

Hydrolyzed camel whey protein alleviated heat stress-induced hepatocyte damage by activated Nrf2/HO-1 signaling pathway and inhibited NF-κB/NLRP3 axis

Liver damage is the most severe complication of heat stress (HS). Hydrolyzed camel whey protein (CWP) possesses bioactive peptides with obviously antioxidant and anti-inflammatory activities. The current study aims to investigate whether CWP that is hydrolyzed by a simulated gastrointestinal digestion process, named S-CWP, protects BRL-3A hepatocytes from HS-induced damage via antioxidant and anti-inflammatory mechanisms. BRL-3A cells were pretreated with S-CWP before being treated at 43 °C for 1 h, and the levels of the cellular oxidative stress, inflammation, apoptosis, biomarkers for liver function, the activities of several antioxidant enzymes, and the cell viability were analyzed. The expression level of pivotal proteins in correlative signaling pathways was evaluated by western blotting. We confirmed that S-CWP alleviated HS-induced hepatocytes oxidative stress by decreased reactive oxygen species (ROS), nitric oxide (NO), 8-Hydroxy-2′-deoxyguanosine (8-OHdG), lipid peroxidation (LPO), protein carbonylation (PCO), and the activities of NADPH oxidase while enhanced superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) activities, and GSH content. S-CWP suppressed HS-induced inflammatory response by reducing the phosphorylation of NF-κB p65, the expression of NLRP3, and caspase-1 and finally alleviated caspase-3-mediated apoptosis. S-CWP also alleviated HS-induced hepatocyte injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and restoring Heat Shock Protein 70 (HSP70) expression. Furthermore, S-CWP treatment significantly enhanced the expression of NF-E2-related nuclear factor erythroid-2 (Nrf2) and HO-1. The antioxidant and anti-inflammatory effects of S-CWP were weakened by ML385, a specific Nrf2 inhibitor. Additionally, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, significantly reversed S-CWP-induced reduction in the phosphorylation of NF-κB p65. Thus, our results revealed that S-CWP protected against HS-induced hepatocytes damage via activating the Nrf2/HO-1 signaling pathway and inhibiting NF-κB/NLRP3 axis.     

Effect of elite physical exercise by triathletes on seven catabolites of DNA oxidation

Abstract

The oxidized nucleoside 8-hydroxy-2’-deoxyguanosine has been widely studied as a marker of DNA oxidation; however, data on the occurrence of other metabolites in plasma that are related to DNA damage are scarce. We have applied an improved, sensitive, robust, and reliable method, involving solid phase extraction and ultrahigh-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS), to the precise quantitation of seven metabolites in the plasma of 15 elite triathletes after a 2-week training program. All compounds were eluted in the first 1.6 min, with limits of detection and quantification ranging between 0.001 and 0.3 ng.mL^?1 and 0.009 and 0.6 ng.mL^?1, respectively. Four compounds were detected in plasma: guanosine-3’-5’-cyclic monophosphate, 8-hydroxyguanine, 8-hydroxy-2’-deoxyguanosine, and 8-nitroguanosine. After two weeks of training, 8-hydroxyguanine exhibited the highest increase (from 0.031 ± 0.008 nM to 0.036 ± 0.012 nM) (p < 0.05), which could be related to the enhanced activity of DNA-repairing enzymes that excise this oxidized base. Increased levels of guanosine-3’-5’-cyclic monophosphate and 8-hydroxy-2’-deoxyguanosine were also observed. In contrast, levels of 8-nitroguanosine (p < 0.05) were significantly reduced, which might be a protective measure as this compound strongly stimulates the generation of superoxide radicals, and its excess is related to pathologies such as microbial (viral) infections and other inflammatory and degenerative disorders. The results obtained indicate an induced adaptive response to the increased oxidative stress related to elite training, and point to the benefits associated with regular exercise.     

Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms

Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNA methylation. The intra-day and inter-day precision values of this method were within 1.50-5.70%. By using 2-deoxyguanosine as an internal standard, UPLC-MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The average global DNA methylation level of sperm samples washed only by PBS (4.03%) was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%), indicating the possible aberrant DNA methylation level in abnormal sperm cells. Clinical application of UPLC-MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility.     

DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G₁-, G₂- or G₃-positions of the NarI site (5′-G₁G₂CG₃CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G₃ construct were mutants, largely G→T, compared to 18% and 24% from the G₁ and G₂ constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G₁ and G₃ constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G₂ construct. In vitro TLS using yeast pol ζ showed that it can extend G₃*:A pair more efficiently than G₃*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.