DNA methylation on N(6)-adenine in mammalian embryonic stem cells.

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.

Evaluation of inhaled low-dose formaldehyde-induced DNA adducts and DNA-protein cross-links by liquid chromatography-tandem mass spectrometry.

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 and 15.2 ppm using [13CD2]-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 [13CD2]-formaldehyde for 28 days (6 h/day) by nose-only inhalation, followed by measuring DNA mono-adduct (N2-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.

Characterisation of the metabolism of pogostone in vitro and in vivo using liquid chromatography with mass spectrometry.

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 (LC­MS) 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: LC­MS 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.

Epigallocatechin-3-gallate inhibits homocysteine-induced apoptosis of endothelial cells by demethylation of the DDAH2 gene.

Our previous study showed that hypermethylation of dimethylarginine dimethylaminohydrolase 2 contributes to homocysteine-induced apoptosis of human umbilical vein endothelial cells. Epigallocatechin-3-gallate is a green tea-derived phenol which has been proved beneficial on atherosclerosis. It was demonstrated that epigallocatechin-3-gallate inhibits DNA methyltransferase activity and reactivates methylation-silenced genes in cancer cells. The aim of this study was to address whether epigallocatechin-3-gallate could induce DNA demethylation of the dimethylarginine dimethylaminohydrolase 2 gene, contributing to prevent endothelial cells from apoptosis induced by homocysteine. Human umbilical vein endothelial cells (ATCC, CRL-2480) were treated with homocysteine (1 mM) for 48 hours with or without epigallocatechin-3-gallate (20 µM) or 5-Aza (DNA methyltransferase inhibitor, 5 µM). Apoptosis rate of human umbilical vein endothelial cells was assayed by flow cytometry with an annexin V-FITC apoptosis detection kit. The mRNA and protein expression level of dimethylarginine dimethylaminohydrolase 2 and DNA methyltransferase 1 were detected by real-time PCR and Western blot, respectively. DNA methylation level of dimethylarginine dimethylaminohydrolase 2 was assayed by methylation specific PCR. The binding level of DNA methyltransferase 1 in the promoter of dimethylarginine dimethylaminohydrolase 2 was determined by chromatin immunoprecipitation-quantitative real-time PCR. It was shown that the apoptosis rate was decreased significantly in human umbilical vein endothelial cells treated with homocysteine compared with the control. Furthermore, the mRNA and protein level of dimethylarginine dimethylaminohydrolase 2 were downregulated, the dimethylarginine dimethylaminohydrolase 2 gene promoter was hypermethylated, and the DNA methyltransferase 1 mRNA and protein level were increased in human umbilical vein endothelial cells treated with homocysteine. Chromatin immunoprecipitation-quantitative real-time PCR revealed that homocysteine-induced binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter was increased. Pretreatment with epigallocatechin-3-gallate or 5-Aza inhibited such effects of homocysteine. In conclusion, epigallocatechin-3-gallate exerted protective effects on homocysteine-induced apoptosis in human umbilical vein endothelial cells by inhibiting promoter hypermethylation of the dimethylarginine dimethylaminohydrolase 2 gene and inducing dimethylarginine dimethylaminohydrolase 2 expression. These effects may be due to the decreased DNA methyltransferase 1 expression and binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter induced by epigallocatechin-3-gallate. This research suggests that modulating the epigenetic processes might be a novel plausible way for treatment of atherosclerosis.

Kinetics of luminol sonochemiluminescence quenched by purines.

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.

Homocysteine-induced hypermethylation of DDAH2 promoter contributes to apoptosis of endothelial cells.

Homocysteine (Hcy) could induce apoptosis of endothelial cells (ECs). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) is recognized as a protective factor to improve the endothelial function. Defect of DDAH2 has been confirmed to be involved in the Hcy-induced dysfunction of endothelial NO system. This study was to determine whether Hcy could inhibit DDAH2 expression and induce apoptosis of ECs via increasing DNA methylation level of DDAH2 promoter and whether DNA methylation inhibitor 5-azacytidine (5-aza) could attenuate the effect of Hcy on ECs. Firstly, human umbilical vein endothelial cells (HUVECs) were treated by Hcy with or without 5-aza for 48 h. MTT assay showed that Hcy reduced the viability of HUVECs in a dose-dependent manner. The level of asymmetric dimethylarginine (ADMA) and the apoptosis rate of HUVECs treated with Hcy at 1.0 mM were increased significantly compared with that of control. Moreover, we found that mRNA level of DDAH2 was down-regulated and DNA methylation level of DDAH2 promoter was increased significantly in HUVECs treated with Hcy, in concomitance with up-regulated protein level of DNA methyltransferase 1 (DNMT1). Furthermore, we also found that 5-aza could neutralize the effect of Hcy on ECs through up-regulating mRNA level of DDAH2 and inducing demethylation of DDAH2 promoter. In conclusion, hypermethylation of DDAH2 contributes to Hcy induced apoptosis of ECs. Modulating DNA methylation status of DDAH2 promoter may be a potential strategy for treatment of endothelial dysfunction.

Matrix interference-free method for the analysis of small molecules by using negative ion laser desorption/ionization on graphene flakes.

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.

Liquid chromatography/mass spectrometry method for determination of perfluorooctane sulfonyl fluoride upon derivatization with benzylamine.

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.

Determination of stimulants and narcotics as well as their in vitro metabolites by online CE-ESI-MS.

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.

Electrospray ionization tandem mass spectrometric characterization of DNA adducts formed by bromobenzoquinones.

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.

New evidence for toxicity of polybrominated diphenyl ethers: DNA adduct formation from quinone metabolites.

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.

3,4,5,6-Tetrahydroxyxanthone preserves intercellular communication by reduction of the endogenous nitric oxide synthase inhibitor level.

To observe the direct effects of 3,4,5,6-tetrahydroxyxanthone on connexin43 (Cx43) expression in cultured endothelial cells, cells were treated with lysophosphatidylcholine (LPC, 10 mg/l) for 24 h in the presence or absence of different concentrations of 3,4,5,6-tetrahydroxyxanthone (1, 3, or 10 µmol l(- 1)). The reactive oxygen species (ROS) production, cell viability, asymmetric dimethylarginine (ADMA) levels, and Cx43 expression were detected. 3,4,5,6-Tetrahydroxyxanthone significantly inhibited the increase in ROS production and ADMA level, increased cell viability and up-regulated Cx43 mRNA and protein expression induced by LPC. 3,4,5,6-Tetrahydroxyxanthone has protective effect in LPC-induced atherosclerotic lesions, which is at least partly related to the reduction of ADMA level and downregulation of Cx43 expression.

A robust multiplexed assay to quantify C1-inhibitor, C1q, and C4 proteins for in vitro diagnosis of hereditary angioedema from dried blood spot.

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.

A novel functional C1 inhibitor activity assay in dried blood spot for diagnosis of Hereditary angioedema.

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.

Using acridinium ester as the sonochemiluminescent probe for labeling of protein.

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.

Measurement of Endogenous versus Exogenous Formaldehyde-Induced DNA-Protein Crosslinks in Animal Tissues by Stable Isotope Labeling and Ultrasensitive Mass Spectrometry.

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 ([(13)CD2]-formaldehyde) by inhalation and performed ultrasensitive mass spectrometry to measure endogenous (unlabeled) and exogenous ((13)CD2-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.

Formation and characterization of glutathione adducts derived from polybrominated diphenyl ethers.

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.

Interaction of 2-(2',4'-bromophenoxyl)-benzoquinone with deoxynucleosides and DNA in vitro.

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.

Peptidomics study of anthocyanin-rich juice of elderberry.

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.

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage.

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.