It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N6-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N6-methyladenine. An increase of N6-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N6-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 N6-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, N6-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 N6-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 -HOMedG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicityinduced 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 CD 2 ]-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 (t 1/2 ) 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.
DNA-protein crosslinks (DPCs) 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. Due to 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 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.
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.
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.
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.
A novel method for the characterization of polymers by laser desorption/ionization on the layer of graphene nanoparticles coupled with time-of-flight mass spectrometry was demonstrated. Various polymers including polypropylene glycol, polystyrene and polymethyl methacrylate with average molecular weights from 425 to 3500 Da were analyzed.
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