Peter W. Villalta

A study of FeCO− and the 3Σ− and 5Σ− states of FeCO by negative ion photoelectron spectroscopy

The 488 and 514 nm negative ion photoelectron spectra of FeCO−, obtained at an instrumental resolution of 5 meV (40 cm−1), show vibrationally resolved transitions from the anion ground state to the ground state and a low‐lying excited state of the neutral molecule. The ground state of FeCO is assigned as the 3Σ− state and the excited state, lying 1135±25 cm−1 higher in energy, as the 5Σ− state. The fundamental vibrational frequencies are νCO=1950±10, νFeC=530±10, and νbend=330±50 cm−1 in the 3Σ− state, and νCO=1990±15, νFeC=460±15, and νbend=180±60 cm−1 in the 5Σ− state. Principal force constants are estimated from these results. Based on a Franck–Condon analysis of the spectrum and other considerations, the Fe–C bond is determined to be 0.15±0.04 A shorter, and the C–O bond 0.05±0.02 A longer, in the 3Σ− state than in the 5Σ− state. These results demonstrate the importance of sdσ hybridization in reducing the σ repulsion between the metal 4s electron and the CO 5σ lone pair, a mechanism that is available...

Mass spectrometry of structurally modified DNA

Structural modifications of nucleobases within the deoxyribonucleic acid (DNA) of living cells can be induced as a result of actions of specialized DNA-modifying enzymes (creating epigenetic DNA modifications) or may result from exposure to reactive endogenous and exogenous electrophiles and oxidants (creating DNA adducts). Epigenetic DNA modifications such as 5-methylcytosine (MeC) are important regulators of cell function that influence chromatin structure and levels of gene expression. DNA methyltransferases (DMTs) catalyze the addition of the C-5 methyl group to cytosine nucleobases.1 DMTs preferentially recognize hypomethylated 5′-CG-3′ sequences, producing epigenetic modifications which preserve DNA methylation patterns. C-5 cytosine methylation controls gene expression by mediating the binding of specific proteins (methyl-CpG binding proteins) to MeCG sites, followed by the recruitment of histone-modifying enzymes that promote chromatin remodeling.2 Recent studies have discovered additional cytosine modifications, e.g. 5-hydroxymethyl-C, and 5-formyl-C, and 5-carboxyl-C; these modifications have been hypothesized to be demethylation intermediates or they may possess their own epigenetic functions within cells.3-5 In contrast to epigenetic modifications, chemical DNA damage including nucleobase alkylation, oxidation, deamination, and cross-linking occurs at a variety of sites, including the N-7, O-6, C-8, and N-2 of guanine; the N-1, N-3, and N-7 of adenine; the O-2 and O-4 of thymine; and the O-2 and N-4 of cytosine (Scheme 1 and Chart 1).6 Some carcinogens are inherently reactive towards DNA, while others must first be metabolically activated to electrophilic intermediates (e.g. epoxides, quinone methides, diazonium ions, and nitrenium ions), which subsequently bind to DNA producing nucleobase adducts (Figure 1).6 All living cells contain extensive DNA repair systems responsible for removing nucleobase lesions. If structurally modified DNA bases escape repair, they may induce base mispairing during DNA replication; thus, the chemical damage would be converted into permanent genetic damage (mutations).6 Accumulation of mutations in genes controlling cell growth, proliferation, programmed cell death, and cell differentiation is likely to cause cancer.7-10 Figure 1 Central role of DNA adducts in chemical carcinogenesis. Scheme 1 DNA sites frequently modified by carcinogens and their metabolites. Chart 1 Structures of representative DNA adducts Due to their central role in chemical carcinogenesis, DNA adducts are considered the true mechanism-based biomarkers of carcinogen exposure. The presence of DNA adducts within a given tissue can be correlated to the formation of reactive intermediates available for binding to DNA and other biomolecules.11 Unlike hemoglobin adducts that reflect a cumulative exposure to carcinogens over time, DNA adducts provide information on the burden of DNA damage within a given tissue at a specific time. Adducts can be used to quantify the capacity of DNA repair systems and to assess the potential for genetic damage as a result of faulty replication. By employing these measurements, DNA adduct levels have been utilized to set human exposure limits for industrial and environmental chemicals and also to identify individuals and populations at risk for developing cancer.11-14 The concentrations of epigenetically modified DNA bases in vivo are relatively high (e.g. four MeC per 100 of total nucleobases), however, the amounts of chemically induced DNA adducts in animal and human tissues can be quite low, in the range of 0.01 - 10 adducts per 108 normal nucleotides. Therefore, analytical methods used for quantifying carcinogen-DNA adducts must be ultra-sensitive, accurate, and specific, allowing the quantitation of low abundance DNA lesions in the presence of a large molar excess of normal nucleosides. Early studies of DNA damage utilized radiolabel-based assays such as 32P-postlabeling methods to measure adduct levels.15-18 Recent developments in mass spectrometry instrumentation have offered an alternative approach that provides both accurate and sensitive quantitation and structural information for the damaged bases, without the need for radioactivity.12 DNA adduct structure can be established using tandem mass spectrometry experiments, while mass spectrometry in combination with stable isotope labeled internal standards (isotope dilution HPLC-ESI-MS-MS or IDMS) is considered a golden standard for DNA adduct analysis due to its high specificity, sensitivity, and accurate quantification.14 Furthermore, mass spectrometry can be used for sequencing native and structurally modified DNA. The present review is devoted to the applications of mass spectrometry to DNA adduct and epigenetic DNA modification identification, screening, and quantitation. We will also discuss the use of MS based approaches to map the distribution of DNA modifications along DNA duplexes and to establish the biological consequences of DNA adduct formation in cells. Taken together, this article provides an overview of the contributions of mass spectrometry to the field of chemical carcinogenesis and epigenetics, with a primary focus on the new developments and recent advances in the field.

Analysis of 23 polycyclic aromatic hydrocarbons in smokeless tobacco by gas chromatography-mass spectrometry

Smokeless tobacco contains 28 known carcinogens and causes precancerous oral lesions and oral and pancreatic cancer. A recent study conducted by our research team identified eight different polycyclic aromatic hydrocarbons (PAHs) in U.S. moist snuff, encouraging further investigations of this group of toxicants and carcinogens in smokeless tobacco products. In this study, we developed a gas chromatography-mass spectrometry method that allows simultaneous analysis of 23 various PAHs in smokeless tobacco after a simple two-step extraction and purification procedure. The method produced coefficients of variation under 10% for most PAHs. The limits of quantitation for different PAHs varied between 0.3 and 11 ng/g tobacco, starting with a 300 mg sample. The recovery of the stable isotope-labeled internal standards averaged 87%. The method was applied to analysis of 23 moist snuff samples that included various flavors of the most popular U.S. moist snuff brands, as well as 17 samples representing the currently marketed brands of spit-free tobacco pouches, a relatively new type of smokeless tobacco. The sum of all detected PAHs in conventional moist snuff averaged 11.6 (+/-3.7) microg/g dry weight; 20% of this amount was comprised of carcinogenic PAHs. The levels of PAHs in new spit-free tobacco products were much lower than those in moist snuff; the sum of all detected PAHs averaged 1.3 (+/-0.28) microg/g dry weight. Our findings render PAHs one of the most prevalent groups of carcinogens in smokeless tobacco. Urgent measures are required from the U.S. tobacco industry to modify manufacturing processes so that the levels of these toxicants and carcinogens in U.S. moist snuff are greatly reduced.

Clear Differences in Levels of a Formaldehyde-DNA Adduct in Leukocytes of Smokers and Nonsmokers

Formaldehyde is considered carcinogenic to humans by the IARC, but there are no previous reports of formaldehyde-DNA adducts in humans. In this study, we used liquid chromatography-electrospray ionization-tandem mass spectrometry to quantify the formaldehyde-DNA adduct N(6)-hydroxymethyldeoxyadenosine (N(6)-HOMe-dAdo) in leukocyte DNA samples from 32 smokers of >or=10 cigarettes per day and 30 nonsmokers. Clear peaks coeluting with the internal standard in two different systems were seen in samples from smokers but rarely in nonsmokers. N(6)-HOMe-dAdo was detected in 29 of 32 smoker samples (mean +/- SD, 179 +/- 205 fmol/micromol dAdo). In contrast, it was detected in only 7 of 30 nonsmoker samples (15.5 +/- 33.8 fmol/micromol dAdo; P < 0.001). The results of this study show remarkable differences between smokers and nonsmokers in levels of a leukocyte formaldehyde-DNA adduct, suggesting a potentially important and previously unrecognized role for formaldehyde as a cause of cancer induced by cigarette smoking.

Application of a high-resolution mass-spectrometry-based DNA adductomics approach for identification of DNA adducts in complex mixtures

Liquid chromatography coupled with mass spectrometry (LC-MS) is the method of choice for analysis of covalent modification of DNA. DNA adductomics is an extension of this approach allowing for the screening for both known and unknown DNA adducts. In the research reported here, a new high-resolution/accurate mass MSn methodology has been developed representing an important advance for the investigation of in vivo biological samples and for the assessment of DNA damage from various human exposures. The methodology was tested and optimized using a mixture of 18 DNA adducts representing a range of biologically relevant modifications on all four bases and using DNA from liver tissue of mice exposed to the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In the latter experiment, previously characterized adducts, both expected and unexpected, were observed.

Analysis of Pyridyloxobutyl and Pyridylhydroxybutyl DNA Adducts in Extrahepatic Tissues of F344 Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are potent pulmonary carcinogens in rats. NNK and NNAL require metabolic activation to express their carcinogenicity. Cytochrome P450-catalyzed alpha-hydroxylation at the methyl position of NNK or NNAL generates reactive intermediates, which alkylate DNA to form pyridyloxobutyl (POB)-DNA adducts or pyridylhydroxybutyl (PHB)-DNA adducts. NNK is metabolized to NNAL in a reversible and stereoselective manner, and the tissue-specific retention of (S)-NNAL is believed to be important to the carcinogenicity of NNK. In the present study, we investigated the formation of POB- and PHB-DNA adducts in extrahepatic tissues of F344 rats treated chronically with NNK and (R)- and (S)-NNAL (10 ppm in the drinking water, 1-20 weeks). POB- and PHB-DNA adducts were quantified in nasal olfactory mucosa, nasal respiratory mucosa, oral mucosa, and pancreas of treated rats. Adduct formation in the nasal respiratory mucosa exceeded that in the other tissues. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) or O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (O(2)-PHB-dThd) was the major adduct, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua) or 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine (7-PHB-Gua). There was a remarkable similarity in adduct formation between the NNK and the (S)-NNAL groups, both of which were distinctively different from that in the (R)-NNAL group. For example, in the nasal olfactory mucosa, POB-DNA adduct levels in the NNK and (S)-NNAL groups were not significantly different from each other, while (R)-NNAL treatment generated 6-33 times lower amounts of POB-DNA adducts than did NNK treatment. In contrast, (R)-NNAL treatment produced significantly higher levels of PHB-DNA adducts than did NNK or (S)-NNAL treatment. Similar trends were observed in the nasal respiratory mucosa, oral mucosa, and pancreas. These results suggest extensive retention of (S)-NNAL in various tissues of NNK-treated rats and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis.

Analysis of total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in smokers' blood.

The sum of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL) is an excellent biomarker for uptake of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Although numerous studies have examined levels of total NNAL in the urine of people who use tobacco products, few have quantified this biomarker in blood, and the available methods used relatively large amounts of blood. A method is urgently needed for the analysis of total NNAL in blood, the fluid most commonly stored in molecular epidemiologic studies. We developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the analysis of total NNAL in 1-mL samples of plasma. LC-ESI-MS/MS provides both high-sensitivity and structural information supporting analyte identity. The method is practical and sensitive, with a detection limit of 8 fmol total NNAL/mL plasma. Levels of total NNAL averaged 42 ± 22 (SD) and ranged 1.7 to 88 fmol/mL plasma in 16 smokers; NNAL was not detected in the plasma of five nonsmokers. These results show that total NNAL can readily be quantified in 1-mL plasma samples.

1,2,3,4-Diepoxybutane-Induced DNA–Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

1,2,3,4-Diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds.

Synthesis of sequence-specific DNA-protein conjugates via a reductive amination strategy.

DNA-protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon exposure to bis-electrophiles, transition metals, UV light, and reactive oxygen species. Because of their superbulky, helix distorting nature, DPCs interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis and carcinogenesis. However, the biological implications of DPC lesions have not been fully elucidated due to the difficulty in generating site-specific DNA substrates representative of DPC lesions formed in vivo. In the present study, a novel approach involving postsynthetic reductive amination has been developed to prepare a range of hydrolytically stable lesions structurally mimicking the DPCs produced between the N7 position of guanine in DNA and basic lysine or arginine side chains of proteins and peptides.

Formation of formaldehyde adducts in the reactions of DNA and deoxyribonucleosides with α-acetates of 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N-nitrosodimethylamine (NDMA)

The cytochrome P450-mediated alpha-hydroxylation of the carcinogenic nitrosamines N-nitrosodimethylamine (NDMA, 1), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 6b) produces diazonium ions and formaldehyde. The DNA-binding properties of the diazonium ions have been thoroughly characterized, and there is no doubt that they are critical in cancer induction by these nitrosamines. However, the possibility of additional DNA damage via released formaldehyde has not been reported. In this study, we used acetoxymethylmethylnitrosamine (5), 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a), and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b) as stable precursors to the alpha-hydroxymethylnitrosamines that would be formed in the metabolism of NDMA, NNK, and NNAL. These alpha-acetates were incubated with calf thymus DNA in the presence of esterase at pH 7.0 and 37 degrees C. The DNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides, and the hydrolysates were analyzed by liquid chromatography-electrospray ionization-mass spectrometry-selected ion monitoring for formaldehyde DNA adducts. Convincing evidence for the formation of the formaldehyde adducts N6-hydroxymethyl-dAdo (11), N4-hydroxymethyl-dCyd (12), N2-hydroxymethyl-dGuo (13), and the cross-links di-(N6-deoxyadenosyl)methane (14), (N6-deoxyadenosyl- N2-deoxyguanosyl)methane (15), and di-(N2-deoxyguanosyl)methane (16) was obtained in these reactions. These results demonstrate that NDMA, NNK, and NNAL have the potential to be bident carcinogens, damaging DNA through the metabolic formation of both diazonium ions and formaldehyde.