Leonard B. Collins

Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine

Evidence for a possible route for DNA demethylation in animals is suggested. 5-methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and suppression of transposable elements. 5mC can be converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) proteins. Here, we show that, in addition to 5hmC, the Tet proteins can generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC in an enzymatic activity–dependent manner. Furthermore, we reveal the presence of 5fC and 5caC in genomic DNA of mouse embryonic stem cells and mouse organs. The genomic content of 5hmC, 5fC, and 5caC can be increased or reduced through overexpression or depletion of Tet proteins. Thus, we identify two previously unknown cytosine derivatives in genomic DNA as the products of Tet proteins. Our study raises the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation.

The Warburg Effect Dictates the Mechanism of Butyrate-Mediated Histone Acetylation and Cell Proliferation

Widespread changes in gene expression drive tumorigenesis, yet our knowledge of how aberrant epigenomic and transcriptome profiles arise in cancer cells is poorly understood. Here, we demonstrate that metabolic transformation plays an important role. Butyrate is the primary energy source of normal colonocytes and is metabolized to acetyl-CoA, which was shown to be important not only for energetics but also for HAT activity. Due to the Warburg effect, cancerous colonocytes rely on glucose as their primary energy source, so butyrate accumulated and functioned as an HDAC inhibitor. Although both mechanisms increased histone acetylation, different target genes were upregulated. Consequently, butyrate stimulated the proliferation of normal colonocytes and cancerous colonocytes when the Warburg effect was prevented from occurring, whereas it inhibited the proliferation of cancerous colonocytes undergoing the Warburg effect. These findings link a common metabolite to epigenetic mechanisms that are differentially utilized by normal and cancerous cells because of their inherent metabolic differences.

Evoked Extracellular Dopamine In Vivo in the Medial Prefrontal Cortex

Abstract: The measurement of evoked extracellular dopamine in the medial prefrontal cortex by using fast‐scan cyclic voltammetry with carbon‐fiber microelectrodes was established and release characteristics of mesoprefrontal dopamine neurons were examined in vivo in anesthetized rats. Despite the sparse dopaminergic innervation and the presence of more dense noradrenergic and serotonergic innervations overall in the medial prefrontal cortex, the measurement of extracellular dopamine was achieved by selective recording in dopamine‐rich terminal fields and selective activation of ascending dopamine neurons. This was confirmed by electrochemical, pharmacological, and anatomical evidence. An increased release capacity for mesoprefrontal dopamine neurons was also demonstrated by the slower decay of the evoked dopamine response after inhibition of catecholamine synthesis and the maintenance of the evoked dopamine response at higher levels in the medial prefrontal cortex compared with the striatum during supraphysiological stimulation.

Distribution of DNA Adducts Caused by Inhaled Formaldehyde Is Consistent with Induction of Nasal Carcinoma but Not Leukemia

Inhaled formaldehyde is classified as a known human and animal carcinogen, causing nasopharyngeal cancer. Additionally, limited epidemiological evidence for leukemia in humans is available; however, this is inconsistent across studies. Both genotoxicity and cytotoxicity are key events in formaldehyde nasal carcinogenicity in rats, but mechanistic data for leukemia are not well established. Formation of DNA adducts is a key event in initiating carcinogenesis. Formaldehyde can induce DNA monoadducts, DNA-DNA cross-links, and DNA protein cross-links. In this study, highly sensitive liquid chromatography-tandem mass spectrometry-selected reaction monitoringmethods were developed and [(13)CD(2)]-formaldehyde exposures utilized, allowing differentiation of DNA adducts and DNA-DNA cross-links originating from endogenous and inhalation-derived formaldehyde exposure. The results show that exogenous formaldehyde induced N(2)-hydroxymethyl-dG monoadducts and dG-dG cross-links in DNA from rat respiratory nasal mucosa but did not form [(13)CD(2)]-adducts in sites remote to the portal of entry, even when five times more DNA was analyzed. Furthermore, no N(6)-HO(13)CD(2)-dA adducts were detected in nasal DNA. In contrast, high amounts of endogenous formaldehyde dG and dA monoadducts were present in all tissues examined. The number of exogenous N(2)-HO(13)CD(2)-dG in 1- and 5-day nasal DNA samples from rats exposed to 10-ppm [(13)CD(2)]-formaldehyde was 1.28 +/- 0.49 and 2.43 +/- 0.78 adducts/10(7) dG, respectively, while 2.63 +/- 0.73 and 2.84 +/- 1.13 N(2)-HOCH(2)-dG adducts/10(7) dG and 3.95 +/- 0.26 and 3.61 +/- 0.95 N(6)-HOCH(2)-dA endogenous adducts/10(7) dA were present. This study provides strong evidence supporting a genotoxic and cytotoxic mode of action for the carcinogenesis of inhaled formaldehyde in respiratory nasal epithelium but does not support the biological plausibility that inhaled formaldehyde also causes leukemia.

A Gnotobiotic Mouse Model Demonstrates that Dietary Fiber Protects Against Colorectal Tumorigenesis in a Microbiota- and Butyrate-Dependent Manner

UNLABELLED Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues. SIGNIFICANCE These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy.

Structural Characterization of Formaldehyde-induced Cross-links Between Amino Acids and Deoxynucleosides and Their Oligomers

Exposure to formaldehyde results in the formation of DNA-protein cross-links (DPCs) as a primary genotoxic effect. Although DPCs are biologically important and eight amino acids have been reported to form stable adducts with formaldehyde, the structures of these cross-links have not yet been elucidated. We have characterized formaldehyde-induced cross-links of Lys, Cys, His, and Trp with dG, dA, and dC. dT formed no cross-links, nor did Arg, Gln, Tyr, or Asn. Reaction of formaldehyde with Lys and dG gave the highest yield of cross-linked products, followed by reaction with Cys and dG. Yields from the other coupling reactions were lower by a factor of 10 or more. Detailed structural examination by NMR and mass spectrometry established that the cross-links between amino acids and single nucleosides involve a formaldehyde-derived methylene bridge. Lys yielded two additional products with dG in which the linking structure is a 1,N(2)-fused triazino ring. The Lys cross-linked products were unstable at ambient temperature. Reactions between the reactive N(alpha)-Boc-protected amino acids and the trinucleotides d(T(1)B(2)T(3)) where B(2) is the target base G, A, or C and reactions between dG, dA and dC and 8-mer peptides containing a single reactive target residue at position 5 yielded cross-linked products with structures inferred from high resolution mass spectrometry and fragmentation patterns that are consistent with those between N(alpha)-Boc-protected amino acids and single nucleotides rigorously determined by NMR studies. These structures will provide a basis for investigation of the characteristics and properties of DPCs formed in vivo and will be helpful in identifying biomarkers for the evaluation of formaldehyde exposure both at the site of contact and at distant sites.

Mass spectrometric analysis of biomarkers and dilution markers in exhaled breath condensate reveals elevated purines in asthma and cystic fibrosis.

Exhaled breath condensate (EBC) analyses promise simple and noninvasive methods to measure airway biomarkers but pose considerable methodological challenges. We utilized mass spectrometry to measure EBC purine biomarkers adenosine and AMP plus urea to control for dilutional variability in two studies: 1) a cross-sectional analysis of 28 healthy, 40 cystic fibrosis (CF), and 11 asthmatic children; and 2) a longitudinal analysis of 26 CF children before and after treatment of a pulmonary exacerbation. EBC adenosine, AMP, and urea were readily detected and quantified by mass spectrometry, and analysis suggested significant dilutional variability. Using biomarker-to-urea ratios to control for dilution, the EBC AMP-to-urea ratio was elevated in CF [median 1.3, interquartile range (IQR) 0.7-2.3] vs. control (median 0.75, IQR 0.3-1.4; P < 0.05), and the adenosine-to-urea ratio was elevated in asthma (median 1.5, IQR 0.9-2.9) vs. control (median 0.4, IQR 0.2-1.6; P < 0.05). Changes in EBC purine-to-urea ratios correlated with changes in percent predicted forced expiratory volume in 1 s (FEV(1)) (r = -0.53 AMP/urea, r = -0.55 adenosine/urea; P < 0.01 for both) after CF exacerbation treatment. Similar results were observed using dilution factors calculated from serum-to-EBC urea ratios or EBC electrolytes, and the comparable ratios of EBC electrolytes to urea in CF and control (median 3.2, IQR 1.6-6.0 CF; median 5.5, IQR 1.4-7.7 control) validated use of airway urea as an EBC dilution marker. These results show that mass spectrometric analyses can be applied to measurement of purines in EBC and demonstrate that EBC adenosine-to-urea and AMP-to-urea ratios are potential noninvasive biomarkers of airways disease.

Interference by pH and Ca2+ ions during measurements of catecholamine release in slices of rat amygdala with fast-scan cyclic voltammetry

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to investigate catecholamine release and uptake induced by local electrical stimulation of rat brain slices containing the basolateral amygdaloid nucleus. The amygdala contains less catecholamine than the striatum, and the observed release is proportionately smaller. Stimulus trains of long duration were required to obtain a well-resolved concentration change in the basolateral amygdala. Voltammetric detection of catecholamines under these conditions was complicated by interference from two extracellular ions, H+ and Ca2+. Ion-selective microelectrodes were used in conjunction with carbon-fiber microelectrodes to monitor pH and Ca2+. The magnitude of the pH changes was correlated with stimulation length and followed the pattern of a brief alkaline shift followed by a longer acidic shift. Extracellular Ca2+ concentration decreased during stimulation and returned fairly rapidly to baseline after the stimulation was over. Because it was not possible to account for all of the ionic interferences using information in the voltammograms, other strategies were employed. Exposure of amygdala slices to L-DOPA or DA increased electrically evoked release of catecholamine, but the effect was transient, and uptake rates decreased during continued exposure to these agents. The most successful approach to remove the interferences was to subtract the response obtained after exposure of the slice to the catecholamine depleter, Ro 4-1284. This agent eliminates the catecholamine response but does not appear to alter the ionic changes.

Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site

Benzo[a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (Kings Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in Kings Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to Kings Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC-MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure.

A mass spectrometric method to simultaneously measure a biomarker and dilution marker in exhaled breath condensate

Exhaled breath condensate (EBC) collection is a simple and non-invasive method to sample airway secretions, but analysis is limited by extensive and variable dilution of airway secretions within the condensate. To overcome this limitation, we developed a sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to simultaneously detect adenyl purines as biomarkers of inflammation and urea as a dilution marker in EBC. Separation prior to mass spectrometry was achieved using a C18 column with methanol and formic acid as the mobile phase, and characteristic precursor to product ion transitions of m/z 268 to 136 (for adenosine), m/z 348 to 136 (for AMP), and m/z 61 to 44 (for urea) were monitored for quantification. To correct for matrix effects, isotopically labeled adenosine, AMP, and urea were used as internal standards. Using these methods, we detected urea and the adenyl purines adenosine and AMP in EBC from seven subjects with cystic fibrosis (CF) and seven healthy controls and found that the AMP/urea ratio was elevated in the CF samples. These results demonstrate that mass spectrometry can be used successfully in EBC analysis to simultaneously detect a biomarker for airway inflammation and control for variable dilution.