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15 ppm Rat Exposure Results.

Description of additional methods and procedures used in the study. Also includes Supplementary Reference.

DNA–protein crosslinks (DPC) arise from a wide range of endogenous and exogenous chemicals, such as chemotherapeutic drugs and formaldehyde. Importantly, recent identification of aldehydes as endogenous genotoxins in Fanconi anemia has provided new insight into disease causation. Because of their bulky nature, DPCs pose severe threats to genome stability, but previous methods to measure formaldehyde-induced DPCs were incapable of discriminating between endogenous and exogenous sources of chemical. In this study, we developed methods that provide accurate and distinct measurements of both exogenous and endogenous DPCs in a structurally specific manner. We exposed experimental animals to stable isotope–labeled formaldehyde ([13CD2]-formaldehyde) by inhalation and performed ultrasensitive mass spectrometry to measure endogenous (unlabeled) and exogenous (13CD2-labeled) DPCs. We found that exogenous DPCs readily accumulated in nasal respiratory tissues but were absent in tissues distant to the site of contact. This observation, together with the finding that endogenous formaldehyde–induced DPCs were present in all tissues examined, suggests that endogenous DPCs may be responsible for increased risks of bone marrow toxicity and leukemia. Furthermore, the slow rate of DPC repair provided evidence for the persistence of DPCs. In conclusion, our method for measuring endogenous and exogenous DPCs presents a new perspective for the potential health risks inflicted by endogenous formaldehyde and may inform improved disease prevention and treatment strategies. Cancer Res; 76(9); 2652–61. ©2016 AACR.

Typical calibration curve used for the quantification of dG-Me-Cys with the linearity from 0.15 to 6.0 fmol (S1); Schematic of rat nose only inhalation chamber (S2); Schematic of primate whole-body inhalation chamber (S3).

Hereditary angioedema (HAE) is a rare genetic disease caused by deficiency or dysfunction of C1 esterase inhibitor (C1-INH). Plasma C1-INH activity and concentrations of C1-INH and complement components 1q and 4 (C1q, C4) are critical to the HAE diagnosis. We describe a novel multiplexed assay to simultaneously measure C1-INH, C1q, and C4 levels in dried blood spot (DBS) of HAE patients. The blood proteins were extracted from 3 mm punches of DBS samples and were subsequently digested by trypsin. The signature peptide derived from each protein was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyte-depleted blood was generated as a surrogate matrix for the preparation of calibration curves to overcome the interference of endogenous proteins, and the assay reproducibility was further monitored by assessing the signal of plasma transferrin as a house-keeping protein. The assay was fully validated following regulatory guideline, with a quantification range of 12.5-800 μg/mL for C1-INH and C4 and 3.13-200 μg/mL for C1q. The precision and accuracy ranged from 3.3%-9.8% and -8.2%-12.6%, respectively. All the patient samples exhibited C1-INH levels lower than normal range except the Type II patient and the C4 and C1q concentrations were as expected. Results from the DBS-based LC-MS assay were highly correlated with the ELISA data measured in plasma of the same subjects. The method described here offers unique advantages such as less invasive sampling, minimal blood processing, and easy transportation and sample storage, allowing, for the first time, C1-INH, C4, and C1q levels to be simultaneously determined in a drop of dried blood.

Background Hereditary angioedema (HAE) is a rare genetic disease caused by deficiency or dysfunction of C1 esterase inhibitor (C1-INH). Timely and accurate diagnosis is an ongoing challenge. Measurement of plasma C1-INH activity is currently the critical standard test. We describe a novel and highly robust point-of-care assay to quantify C1-INH activity in dried blood spot (DBS). Methods C1-INH was extracted from 3 mm punches of DBS samples and incubated with excess amount of C1 esterase (C1s). The mixture was subsequentially incubated with C1s substrate, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitation of the enzyme reaction product. Results The assay was validated within a quantification range from 100 to 1500 mU/mL. The intra-day precision and accuracy ranged from 4.0% to 11.6% and −11.1% to −2.1%, and the inter-day precision and accuracy were 8.1–13.1% and −10.3% to 0.9%, respectively. Normal C1-INH activity (n = 103) ranged from 311 to 1090 mU/mL, whereas 23 out of 24 HAE patients exhibited C1-INH activity lower than 100 mU/mL. Conclusion DBS specimen collection for measurement of functional C1-INH activity in a physician’s office is straightforward and not limited by logistic considerations and therefore, appropriate for the diagnosis of HAE in high throughput diagnostic laboratories.

Energy storage can provide flexibility on both power generation and consumption sides when large renewables penetrate, leading to a low-carbon society development. Although the compressed air energy storage (CAES) system usually emerges as a grid-scale energy storage facility, the small-scale CAES system, especially isothermal or quasi-isothermal CAES, with constant or near constant power output may be effective to provide urgent power for special load in some other occasions. Meanwhile, the isobaric process can be enlightened by the phase change of condensable gas at its saturation pressure. From this viewpoint, the near constant discharge performance analysis of a dual hydraulic accumulator configuration quasi-isothermal compressed gas energy storage based on condensable gas R41 is proposed in this paper. The methods of liquid piston and spraying liquid are adopted to realize a quasi-isothermal process. The phase change of condensable gas at saturation pressure and dual accumulator configuration are two measures to maintain the gas pressure lay in a narrow range, implying a near constant discharge. Based on the system transient model, the transient analysis of system components, parametric analysis, off-design analysis and variant system performance analysis are conducted. Results show that the system round trip efficiency (RTE) and energy density (ED) are 63.98% and 0.1175 kWh/m3, respectively. The power output is located in the range from 0.93 kW to 1.11 kW, which is relatively stable and around the 1 kW rated power. Moreover, the initial quality of R41 and volume of accumulator have effect on system performance. Meanwhile, the Pelton turbine power level can be adjusted by altering jets number and nozzle effective area.

DNA–protein crosslinks (DPC) arise from a wide range of endogenous and exogenous chemicals, such as chemotherapeutic drugs and formaldehyde. Importantly, recent identification of aldehydes as endogenous genotoxins in Fanconi anemia has provided new insight into disease causation. Because of their bulky nature, DPCs pose severe threats to genome stability, but previous methods to measure formaldehyde-induced DPCs were incapable of discriminating between endogenous and exogenous sources of chemical. In this study, we developed methods that provide accurate and distinct measurements of both exogenous and endogenous DPCs in a structurally specific manner. We exposed experimental animals to stable isotope–labeled formaldehyde ([13CD2]-formaldehyde) by inhalation and performed ultrasensitive mass spectrometry to measure endogenous (unlabeled) and exogenous (13CD2-labeled) DPCs. We found that exogenous DPCs readily accumulated in nasal respiratory tissues but were absent in tissues distant to the site of contact. This observation, together with the finding that endogenous formaldehyde–induced DPCs were present in all tissues examined, suggests that endogenous DPCs may be responsible for increased risks of bone marrow toxicity and leukemia. Furthermore, the slow rate of DPC repair provided evidence for the persistence of DPCs. In conclusion, our method for measuring endogenous and exogenous DPCs presents a new perspective for the potential health risks inflicted by endogenous formaldehyde and may inform improved disease prevention and treatment strategies. Cancer Res; 76(9); 2652–61. ©2016 AACR.

As a widespread industrial chemical, formaldehyde carcinogenicity has been highly controversial. Meanwhile, formaldehyde is an essential metabolite in all living cells. Previously, we have demonstrated exogenous formaldehyde causes DNA adducts in a nonlinear manner between 0.7 ppm to 15.2 ppm using [(13)CD(2)]-formaldehyde for exposure coupled with the use of sensitive mass spectrometry. However, the responses from exposure to low doses of formaldehyde are still unknown. In this study, rats were exposed to 1, 30, and 300 ppb [(13)CD(2)]-formaldehyde for 28 days (6 hours/day) by nose-only inhalation, followed by measuring DNA mono-adduct (N(2)-HOMe-dG) and DNA-protein crosslinks (dG-Me-Cys) as formaldehyde specific biomarkers. Both exogenous and endogenous DNA mono-adducts and dG-Me-Cys were examined with ultrasensitive nano-liquid chromatography-tandem mass spectrometry. Our data clearly show that endogenous adducts are present in all tissues analyzed, but exogenous adducts were not detectable in any tissue samples, including the most susceptible nasal epithelium. Moreover, formaldehyde exposure at 1, 30 and 300 ppb did not alter the levels of endogenous formaldehyde induced DNA adducts or DNA-protein crosslinks. The novel findings from this study provide new data for risk assessment of exposure to low doses of formaldehyde.

The reactions of glutathione (GSH) with polybrominated diphenyl ethers (PBDEs) quinones with different degrees of bromination on the PBDEs ring were studied. Liquid chromatography coupled with mass spectrometric (LC-MS) analysis showed that four types of adducts were formed from the reaction of each PBDEs quinone (PBDE-Q) with GSH. The proposed reaction pathway was confirmed using ion trap-MS/MS technique. Our results demonstrate that adduct-1 was formed following the Michael Addition, a reaction of the sulfhydryl group from GSH with electron deficient carbon from PBDEs-Q ring. Compared with other carbons on the quinone ring, carbon in position 6 has a smaller steric hindrance, thus the addition reaction is likely to occur in that position. Hydrated quinone-GSH adduct-1 is easily oxidized to generate an adduct-2 in an aqueous solution. Substitution reaction from bromine atom on adduct-2 quinone ring with sulfhydryl group from GSH further generates adduct-3 that is unstable and can be readily hydrolyzed to adduct-4 in aqueous solution. Both adduct-1 and adduct-4 were unstable as well, immediately oxidized to adduct-2 and adduct-3 in the air, respectively. The results reveal that brominated quinones undergo a rapid non-enzymatic debromination upon reaction with GSH, and open a new view for our understanding on mechanism of metabolism and the toxicity of this class of compounds.

Biologically active peptides play a role in plant signaling and defense. Elderberry juice is known to contain a variety of anthocyanin compounds, a sub-set of polyphenols, which are responsible for the deep purple color of the juice. In this paper, we describe a method utilizing solid phase extraction (SPE) to remove anthocyanins from peptides. Liquid chromatography coupled with tandem mass spectrometry was used to separate and identify the peptides. The results showed that the use of SPE was an effective method to separate peptides from anthocyanins and other background compounds including high polyphenol content in the juice samples. More than 1000 peptides present in elderberry juice were successfully identified.

Introduction Pogostone possesses potent anti-bacterial and anti-fungal activities and has been used for the quality control of essential oil of Pogostemon cablin. Pogostone is easily absorbed after oral administration but its metabolism in mammals remains elusive. Objective To investigate the metabolic profile of pogostone in vitro and in vivo. Methods High-performance liquid chromatography coupled with mass spectrometry (LC–MS) techniques were employed. Orbitrap MS and ion trap tandem mass spectrometry (MS/MS) were utilised to analyse the metabolism of pogostone by virtue of the high sensitivity and high selectivity in the measurement. In vitro experiment was carried out using rat liver microsomes while the in vivo study was conducted on rats, which were orally administered with pogostone (80 mg/kg). Results In total, three mono-hydroxylated, one di-hydroxylated, one mono-oxygenated, one di-oxygenated metabolite, one hydrolysis and one hydroxy conjugated metabolites were found. In addition hydroxylation was demonstrated to be a major metabolic pathway of pogostone. Conclusion LC–MS was demonstrated to be a powerful tool for the metabolite identification of pogostone. The tentative identification of metabolites provides an insight for the metabolic clues of pogostone. Copyright © 2013 John Wiley & Sons, Ltd.

The role of endogenous versus exogenous DNA damage in genotoxicity and carcinogenesis is reviewed from recent publications on genotoxicity, focusing on low-dose effects and risk assessment. Data for aldehydes, alkylating agents, oxidative stress, and radiation are summarized, with particular emphasis on new approaches that provide quantitative data on endogenous DNA damage and mutagenesis. Such data have markedly improved with new analytical instrumentation, such as mass spectrometry. A major breakthrough in these techniques has been the incorporation of stable isotope exposures, so that exogenous DNA damage can be readily distinguished from endogenous DNA damage. This has greatly improved both the accuracy of the data and our knowledge. Such data lend themselves to the development of new approaches for science-based risk assessment. As these new approaches advance, it should be possible to make science-based risk assessments for genotoxic agents, rather than resorting to the default-driven approaches that have no scientific basis.

A liquid chromatography –electrospray ionization– ion trap mass spectrometry (LC –ESI–ITMS) method was developed for the simultaneous analysis of strychnine, brucine and their major metabolites. Strychnine and brucine were individually incubated with rat liver S9 fraction. The incubation samples were pooled together and analyzed with LC–ESI –ITMS in positive ion and full-scan detection mode. The calibration curves of strychnine and brucine in rat liver showed good linearity in ranges of 0.020 to 8.0 mg/mL for strychnine and 0.020 to 8.5 mg/mL for brucine. The limits of detections were both 0.008 mg/mL and the recoveries were 88.3 and 83.2% for strychnine and brucine, respectively. Two metabolites were identified as strychnine N-oxide and brucine N-oxide by comparing the molecular mass, retention time, full-scan mass spectra, tandem MS and MS 3 spectra with those of strychnine and brucine. The developed method provided high sensitivity and selectivity for the determination of poisonous alkaloids and their major metabolites and can be applied in the determination of samples in forensic and clinically toxicological cases.

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have emerged as contaminants of environmental concerns because they pose potential risks to human and animal health. The purpose of this study was to investigate the in vitro metabolism of OH-PBDEs and their potential inhibition against 17β-estradiol (E2) metabolism.Rat liver microsomes were used as a source of P450 enzymes in an in vitro metabolism study of OH-PBDEs. Inhibition of E2 metabolism and kinetic study were performed by incubating with rat liver microsomes in the presence of OH-PBDEs.The obtained data clearly demonstrated that OH-PBDEs, especially those congeners with lower bromination, could be metabolized to bromophenol and diOH-PBDEs. The less metabolic rate of OH-PBDEs was observed with the increasing number of bromine substituents. OH-PBDEs with hydroxyl group and bromine adjacent to the ether bridge showed faster metabolic rates. In addition, the results showed non-competitive inhibition of E2 metabolism by OH-PBDEs with IC(50) values in the range from 13.7 to 55.2 μM. The most potent OH-PBDE inhibitor was found to be 3'-OH-BDE-100. The inhibitory potencies for OH-PBDEs were significantly higher than those of parent PBDE and methoxylated metabolites, providing the evidence that PBDEs exerted estrogenic activity in part by their hydroxylated metabolites.OH-PBDEs exhibited large differences in their capacity to be metabolized and to inhibit E2 metabolism in rat liver microsomes. The finding might increase our understanding of healthy risk associated with PBDEs in human and wildlife.

Bromobenzoquinones (BBQs) represent a class of reactive metabolites of various aromatic contaminants with bromine-containing substituents, including bromobenzene, bromophenols, polybrominated diphenyl ethers (PBDEs). Recently, 2,6-dibromobenzoquinone also has been detected directly from drinking water. The alternation of the genome caused by covalent binding of chemicals or their metabolites to DNA provides a viable mechanism for carcinogenicity. In the present study, electrospray ionization coupled with ion trap mass spectrometry (ITMS), triple quadrupole MS or quadrupole time-of-flight MS was applied for the analysis of DNA adducts formed by BBQs. The study demonstrated 2-monobromobenzoquinone and 2,6-dibromobenzoquinone could covalently bind to deoxyguanosine (dG) and DNA in vitro. The chemical structures of the DNA adducts were confirmed by accurate mass values, collision-induced fragmentation tandem mass spectra as well as isotopic patterns. Generally, the reaction mechanism for the DNA adduction involved Michael addition between the electron-deficient carbon from the quinone and the nucleophilic exocyclic nitrogen from the dG followed by reductive cyclization with loss of a small molecule such as H2O, or HBrO. It was of particular interest to note that some adducts were generated from the reaction of one dG molecule with two BBQ molecules. The obtained results provided new information for assessing the potential cancer risk associated with bromobenzene, bromophenols, PBDEs and BBQs. Copyright © 2011 John Wiley & Sons, Ltd.

Perfluorooctane sulfonyl fluoride (PFOSF) is a main precursor of environmentally ubiquitous perfluorooctanesulfonate (PFOS), and the quantity released to the environment is substantial. Determination of PFOSF, particularly at low concentrations, presents significant challenges for high-performance liquid chromatography and liquid chromatography/mass spectrometry (LC/MS) analyses due to the lack of chromophore and ionizable functional group, respectively. In this study, a new method was developed by derivatizing PFOSF with benzylamine to allow rapid quantitative analysis by using LC/MS. The method demonstrated good linearity in the range from 2 to 80 ng mL(-1) with r(2)0.994 for the derivatization product while the absolute detection limit was 2.5 pg. Liquid-liquid and liquid-solid extraction procedures were established for analysis of water and soil samples, and recoveries were in the range of 51-128%. In addition, the derivatization was selective for PFOSF, whereas PFOS did not nearly react. The developed simple analytical method with good reproducibility might not only be applied for analysis of PFOSF in the environment but also be applicable for supporting investigations on environmental fate of PFOSF, particularly its environmental and biotransformation to PFOS.

Ultra performance liquid chromatography (UPLC) provides improved resolution, speed and sensitivity compared to conventional high performance liquid chromatography (HPLC). In this study, a robust UPLC–ESI–MS/MS method was developed for the rapid determination of nine hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in rat plasma. Under the optimized conditions, the OH-PBDE congeners were eluted within 7.0 min. The limits of quantification defined at the signal-to-noise ratio of 10 were 0.17–2.78 ng/mL in rat plasma. The method provided good linearity for the calibration curves with recoveries of 93.3–114.0% and repeatability with relative standard deviation (RSD) of 0.6–5.8% for intra-day and 3.2–10.4% for inter-day measurements. The developed method was applied for supporting the pharmacokinetics investigation of 6-OH-BDE-47 in two groups of Sprague–Dawley rats that received, respectively a single dose of 0.60 mg/kg (high dose) and 0.15 mg/kg (low dose) by intravenous injection. The results showed that plasma levels of 6-OH-BDE-47 declined bi-exponentially with elimination half-life of 71.7 and 85.6 min for lower and higher dose group, respectively. The obtained results of short elimination half-life suggested that 6-OH-BDE-47 might not accumulate significantly in rat.

A simple, rapid and sensitive CE-ESI-MS method for the simultaneous analysis of seven stimulants and narcotics (amphetamine, ephedrine, methadone, pethidine, tetracaine, codeine and heroin) was developed. The CE-ESI-MS experimental conditions were optimized as follows: 20 mmol/L ammonium acetate with pH 9.0 as running buffer, the separation voltage of 22 kV and the sheath liquid of isopropanol/water (1:1 v/v) containing 7.5 mmol/L acetic acid with 3.0 μL/min flow rate. Under the optimized conditions, the stimulants and narcotics were well separated within 4.6 min using a 70-cm length fused-silica capillary (50 μm id). The detection limits (S/N=3) of the CE-ESI-MS analysis were in the range of 0.40-1.0 ng/mL. Method repeatability of intra-day and inter-day was satisfactory. The recoveries obtained from the analysis of spiked urine samples were between 84.1 and 108%. The developed method was successfully applied for the simultaneous analysis of methadone, pethidine and codeine and their in vitro metabolites.

本文建立了一种基于液相色谱-电喷雾离子阱质谱(LC-ESI-ITMS)技术和“N in 1”模式的药物及其代谢物快速高通量分离与鉴定方法. 该方法首先利用体外代谢方法得到肝微粒体中药物与各自代谢产物混合体系, 然后采用“N in 1”模式对该混合体系进行处理,并利用LC-ESI-ITMS联用技术对各待测药物及其代谢物进行分析, 得到药物和代谢物的分子量信息, 进一步结合多级串联质谱(LC-MS n )分析结果获得结构信息, 对代谢物进行鉴定,从而快速的推测代谢物结构, 进而得到药物的生物转化规律. 本方法用于6 种药物及其4种代谢物的分析, 实验结果表明: 该方法具有快速、高效、灵敏, 选择性好等特点.

A new sonogenerated chemiluminescence (SCL) system has been developed to investigate the SCL behavior of acidinium ester (AE). The optimum parameters for this SCL system have also been investigated in detail. Under the optimum conditions, a typical SCL signal could be obtained in a mixture of DMF and alkaline water solutions with volume ratio of 3 : 2. According to electrospray ionization mass spectrometry and sonochemiluminescence emission spectra, SCL emission could be ascribed to the formation of N-methylacridone with a different mechanism from that in both the chemiluminescence and electrochemiluminescence reaction of acridinium ester. The acridinium ester has been used as a SCL probe for labeling of anti-human insulin guinea pig polyclonal antibody, and it was found that the concentration of AE-conjugated antibody was linear with the SCL intensity in the range of 0.15–0.75 ppb with a linear correlation coefficient of 0.9980.

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (1.5 million years old) but not old (6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N(6)-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.

Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N(2-)hydroxymethyl-dG (N(2)-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N(2)-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [(13)CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N(2)-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair.

Polybrominated diphenyl ethers (PBDEs) may be metabolized to form hydroxylated and quinone products. Study on the formation of DNA adducts altered by PBDEs quinones was conducted. Various types of DNA adducts generated from in vitro reaction of deoxyguanosine (dG), 2'-deoxyadenosine (dA), 2'-deoxycytidine (dC), thymidine (T) and DNA with a PBDE-quinone metabolite, namely 2-(2',4'-bromophenoxyl)-benzoquinone (2'4'BrPhO-BQ) were characterized. The results suggest that the quinone compound could form various DNA adducts with dG, dA and dC via Michael Addition, which was confirmed from analyses by electrospray ionization tandem mass spectrometry. Two adducts were respectively generated from the reactions of 2'4'BrPhO-BQ with dC and dG, while three adducts were produced with dA. The formation of adducts of 2'4'BrPhO-BQ-deoxynucleoside changed with different pH of reaction solution. The obtained results demonstrated that 2'4'BrPhO-BQ could covalently bind to DNA mediated by quinone group. The in vitro data of the formation of DNA adducts might be valuable to elucidate the mechanism of interaction between PBDEs and DNA in vivo.

Pogostone possesses potent anti-bacterial and anti-fungal activities and has been used for the quality control of essential oil of Pogostemon cablin. Pogostone is easily absorbed after oral administration but its metabolism in mammals remains elusive.To investigate the metabolic profile of pogostone in vitro and in vivo.High-performance liquid chromatography coupled with mass spectrometry (LC–MS) techniques were employed. Orbitrap MS and ion trap tandem mass spectrometry (MS/MS) were utilised to analyse the metabolism of pogostone by virtue of the high sensitivity and high selectivity in the measurement. In vitro experiment was carried out using rat liver microsomes while the in vivo study was conducted on rats, which were orally administered with pogostone (80 mg/kg).In total, three mono-hydroxylated, one di-hydroxylated, one mono-oxygenated, one di-oxygenated metabolite, one hydrolysis and one hydroxy conjugated metabolites were found. In addition hydroxylation was demonstrated to be a major metabolic pathway of pogostone.LC–MS was demonstrated to be a powerful tool for the metabolite identification of pogostone. The tentative identification of metabolites provides an insight for the metabolic clues of pogostone.

The concept of the Exposome, is a compilation of diseases and one’s lifetime exposure to chemicals, whether the exposure comes from environmental, dietary, or occupational exposures; or endogenous chemicals that are formed from normal metabolism, inflammation, oxidative stress, lipid peroxidation, infections, and other natural metabolic processes such as alteration of the gut microbiome. In this review, we have focused on the Endogenous Exposome, the DNA damage that arises from the production of endogenous electrophilic molecules in our cells. It provides quantitative data on endogenous DNA damage and its relationship to mutagenesis, with emphasis on when exogenous chemical exposures that produce identical DNA adducts to those arising from normal metabolism cause significant increases in total identical DNA adducts. We have utilized stable isotope labeled chemical exposures of animals and cells, so that accurate relationships between endogenous and exogenous exposures can be determined. Advances in mass spectrometry have vastly increased both the sensitivity and accuracy of such studies. Furthermore, we have clear evidence of which sources of exposure drive low dose biology that results in mutations and disease. These data provide much needed information to impact quantitative risk assessments, in the hope of moving towards the use of science, rather than default assumptions.

A homogeneous chemiluminescence (CL) reaction was initiated by ultrasound irradiation. Luminol sonochemiluminescence (SCL) reaction kinetics were determined under pseudo-first-order conditions, and the reaction followed the model for simple rise–fall kinetics. In addition, SCL quenching reactions induced by purines were also investigated in which the interactions between luminol and purines were analysed using the Stern–Volmer (S-V) mechanism. The results implied that the high rate constant of luminol CL quenched by purines may be attributed to ground state interactions originating from hydrogen bonding. Copyright © 2012 John Wiley & Sons, Ltd.

This study investigated the formation of DNA adducts of polybrominated diphenyl ethers (PBDEs) and the possible mechanisms. DNA adduction was conducted by in vitro reaction of deoxyguanosine (dG) and DNA with PBDE-quinone (PBDE-Q) metabolites, and DNA adducts were characterized by using electrospray ionization tandem mass spectrometry. The results suggested DNA adduction involved Michael Addition between the exocyclic NH(2) group at the N-2 position of dG and the electron-deficient carbon of quinone, followed by reductive cyclization with loss of (bromo-)1-hydroperoxy-benzene or water to form a type I or type II adduct. PBDE-Q with substituted bromine on the quinone ring was proven to be a favorable structure to form a type I adduct, while the absence of bromine on the quinone ring resulted in a type II adduct. Lower reactivity of adduction was also observed with increasing the number of bromine atoms on the phenoxyl ring. Our data clearly demonstrated PBDEs could covalently bind to DNA mediated by quinone metabolites, depending on the degree of bromine substitution. This study opened a new view on the mechanism of toxicity of PBDEs and reported the structure of PBDE-DNA adducts, which might be valuable for the evaluation on potential in vivo formation of PBDE-DNA adducts.

Bromobenzoquinones (BBQs) represent a class of reactive metabolites of various aromatic contaminants with bromine-containing substituents, including bromobenzene, bromophenols, polybrominated diphenyl ethers (PBDEs). Recently, 2,6-dibromobenzoquinone also has been detected directly from drinking water. The alternation of the genome caused by covalent binding of chemicals or their metabolites to DNA provides a viable mechanism for carcinogenicity. In the present study, electrospray ionization coupled with ion trap mass spectrometry (ITMS), triple quadrupole MS or quadrupole time-of-flight MS was applied for the analysis of DNA adducts formed by BBQs. The study demonstrated 2-monobromobenzoquinone and 2,6-dibromobenzoquinone could covalently bind to deoxyguanosine (dG) and DNA in vitro. The chemical structures of the DNA adducts were confirmed by accurate mass values, collision-induced fragmentation tandem mass spectra as well as isotopic patterns. Generally, the reaction mechanism for the DNA adduction involved Michael addition between the electron-deficient carbon from the quinone and the nucleophilic exocyclic nitrogen from the dG followed by reductive cyclization with loss of a small molecule such as H(2)O, or HBrO. It was of particular interest to note that some adducts were generated from the reaction of one dG molecule with two BBQ molecules. The obtained results provided new information for assessing the potential cancer risk associated with bromobenzene, bromophenols, PBDEs and BBQs.

Polybrominated diphenyl ethers (PBDEs) can be metabolically converted to their hydroxylated metabolites (OH-PBDEs). The estrogenic effects of PBDEs may be mediated by OH-PBDEs, but the mechanisms of which are still not understood. This study investigated the glucuronidation of 11 OH-PBDEs and their potential in modulating UDP-glucuronosyltransferases (UGTs) activity of 17β-estradiol (E2) in rat liver microsomes. The number of bromine atoms at phenolic ring was observed as the most influential factor of OH-PBDEs glucuronidation. 2'-OH-BDE-28 having one bromine atom at phenolic ring showed the fastest metabolic rates with t(1/2) value of 3.86 min, while 6-OH-BDE-137 having four bromine atoms at phenolic ring was the poorest substrate with t(1/2) value over 60 min. Regarding to the modulation of E2-UGTs activity, the phenolic hydroxyl group in OH-PBDEs played an essential role. Depending on the substitution patterns of bromine and hydroxyl group, OH-PBDEs inhibited or stimulated E2-UGTs activity. Ten of OH-PBDEs inhibited both 3-glucuronidation and 17-glucuronidation of E2 with IC(50) values varying from 3.80 to 129.38 μM, while 3'-OH-BDE-100 exhibited stimulating effects on 3-glucuronidation with EC(50) value of 35.95 μM. Kinetic analysis suggested noncompetitive inhibition mode of E2 glucuronidation by 3'-OH-BDE-7, 6-OH-BDE-47 and 2'-OH-BDE-68 with K(i) values varying from 11.95 to 67.22 μM. This study demonstrated OH-PBDEs exhibited large interindividual differences in glucuronidation and modulation of E2-UGTs activity. By inhibiting the formation of E2 glucuronidation, OH-PBDEs may increase E2 bioavailability in target tissue, thereby exerting an indirect estrogenic effect.

This work presents a new approach for the analysis of small molecules with direct negative ion laser desorption/ionization (LDI) on graphene flakes. A series of matrix interference-free mass spectra were obtained for the analysis of a wide range of small molecules including peptides, amino acids, fatty acids, as well as nucleosides and nucleotides. The mixture of analytes and graphene flakes suspension were directly pipetted onto a sample plate for LDI-time-of-flight mass spectrometry (TOFMS) analysis. Deprotonated monomeric species [M-H](-) ions were homogeneously obtained on uniform graphene flakes film when negative ion mode was applied. In positive ion mode, the analytes were detected in form of multiple adduct ions such as sodium adduct [M+Na](+), potassium adduct [M+K](+), double sodium adduct [M+2Na-H](+), double potassium adduct [M+2K-H](+), as well as sodium and potassium mixed adduct [M+Na+K-H](+). Better sensitivity and reproducibility were achieved in negative ion mode compared to positive ion mode. It is believed that the new method of matrix interference-free negative ion LDI on graphene flakes may be expanded for LDI-MS analysis of various small molecules.

Aristolochic acid nephropathy (AAN) is associated with the prolonged exposure to nephrotoxic and carcinogenic aristolochic acids (AAs). DNA adducts induced by AAs have been proven to be critical biomarkers for AAN. Therefore, accurate and specific quantification of AA–DNA adducts is important. In this study, a specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and applied for the determination of 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) in exfoliated urothelial cells of AA-dosed rats. After the isolation from urine samples, DNA in urothelial cells were subjected to enzymatic digestion and solid-phase extraction on a C18 Sep-Pak cartridge for the enrichment of DNA adducts. The sample extracts were analyzed by reverse-phase UPLC–MS/MS with electrospray ionization in positive ion mode. The quantification of the AA–DNA adduct was performed by using multiple reaction monitoring with reserpine as internal standard. The method provided good accuracy and precision with a detection limit of 1 ng/ml, which allowed the detection of trace of dA-AAI in exfoliated urothelial cells. After one-month oral dose of AAI at 10 mg/kg/day, 2.1 ± 0.3 dA-AAI per 109 normal dA was detected in exfoliated urothelial cells of rats. Compared to the traditional methods such as 32P-postlabelling and HPLC with fluorescence detection, the developed UPLC–MS/MS method is more specific and rapid with a retention time of 4 min. The outcome of this study may have clinical significance for diagnosing and monitoring AA-associated disease because detection of DNA adducts in exfoliated urothelial cells is non-invasive and convenient.

The arachidonic acid (AA) metabolic network produces key inflammatory mediators which have been considered as hallmark contributors in various inflammatory related diseases. Enzymes in this network, such as 5-lipoxygenase (5-LOX), cyclooxygenase (COX), leukotriene A(4) hydrolase (LTA4H) and prostaglandin E synthase (PGES), have been used as targets for anti-inflammatory drug discovery. Multi-target drugs and drug combinations have also been developed for this network. However, how the inhibitors alter the dynamics of metabolite production and which combinatorial target intervention solutions are better needs further exploration. We did a system based intervention analysis on the AA metabolic network. Using an LC-MS/MS method, we quantitatively studied the eicosanoid metabolites responses of AA metabolic network during stimulation of Sprague Dawley rat blood samples with the calcium ionophore. Our results indicate that inhibiting the upstream rather than the downstream target of 5-LOX pathway will simultaneously alter the AA metabolism to the COX pathway (and vice versa). Therefore, single-target inhibitors cannot control all the inflammatory mediators at the same time. We also suggest that in the case of multiple-target anti-inflammatory solutions, the combination of inhibitors of the downstream enzymes may have stronger inhibition efficiency and cause less side-effects compared to the other solutions. One therapeutic strategy, LTA4H/COX inhibition solution, was found promising for the intervention of inflammatory mediator biosynthesis and at the same time stimulating the production of anti-inflammatory agents.