Detection and quantitation of acrolein-derived 1,N2-propanodeoxyguanosine adducts in human lung by liquid chromatography-electrospray ionization-tandem mass spectrometry

Acrolein, a widely distributed environmental pollutant, reacts with dGuo in DNA to form two pairs of 1,N2-propano-dGuo adducts: (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (alpha-OH-Acr-dGuo) and (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (gamma-OH-Acr-dGuo). alpha-OH-Acr-dGuo is more mutagenic and mainly induces G --> T transversions. A recent study demonstrated that acrolein-DNA adducts are preferentially formed in p53 mutational hotspots in human lung cancer, but there are no reports on the presence of these adducts in the human lung. To directly investigate this question, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative analysis of Acr-dGuo adducts in DNA. Our method is based on the enzymatic hydrolysis of DNA isolated from the human lung in the presence of [13C10,15N5]Acr-dGuo as internal standards. Acr-dGuo adducts are enriched from the hydrolysates by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring. The method is accurate and precise, and the identity of the adducts was confirmed by monitoring different transitions from the same parent ion and by carrying out reactions with NaOH and NaBH4, which produced N2-(3-hydroxypropyl)-dGuo or 1,N2-(1,3-propano)-dGuo from gamma-OH-Acr-dGuo and alpha-OH-Acr-dGuo, respectively. Thirty DNA samples from lung tissue were analyzed, and Acr-dGuo adducts were detected in all samples. Both alpha-OH- and gamma-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16-209 adducts/109 nucleotides. These results demonstrate for the first time that both types of Acr-dGuo adducts are present in human lung DNA. There was no difference in adduct levels between current and ex-smokers. Collectively, the results support a plausible role for acrolein as one cause of p53 mutations in the human lung.     

Regioselective formation of acrolein-derived cyclic 1,N(2)-propanodeoxyguanosine adducts mediated by amino acids, proteins, and cell lysates

Acrolein (Acr) is a major component in cigarette smoke and a ubiquitous environmental pollutant. It is also formed as a product of lipid peroxidation. Following ring closure via the Michael addition, Acr modifies deoxyguanosine (dG) in DNA by forming cyclic 1,N(2)-propanodeoxyguanosine adducts (OHPdG). The reactions of Acr with dG yield, depending on the direction of ring closure, two regioisomers, α- and γ-OHPdG, in approximately equal amounts. However, previous (32)P-postlabeling studies showed that the γ isomers were detected predominantly in the DNA of rodent and human tissues. Because of the potential differential biological activity of the isomeric OHPdG adducts, it is important to confirm and study the chemical basis of the regioselective formation of γ isomers in vivo. In this study, it is confirmed that γ-OHPdG adducts are indeed the major isomers formed in vivo as evidenced by a LC-MS/MS method specifically developed for Acr-derived dG adducts. Furthermore, we have shown that the formation of γ-isomers is increased in the presence of amino-containing compounds, including amino acids, proteins, and cell lysates. A product of Acr and arginine that appears to mediate the regioselective formation of γ isomers was identified, but its structure was not fully characterized due to its instability. This study demonstrates that intracellular amino-containing compounds may influence the regiochemistry of the formation of OHPdG adducts and reveals a mechanism for the preferential formation of γ-OHPdG by Acr in vivo.     

1,N2-propanodeoxyguanosine adducts of the 1,3-butadiene metabolite, hydroxymethylvinyl ketone

1,3-Butadiene (BD) is a rodent and human carcinogen. While several epoxides formed during BD metabolism are mutagenic and may contribute to BD carcinogenicity, another proposed metabolite, hydroxymethylvinyl ketone (HMVK), could also be involved. A significant quantity of HMVK is likely to be formed since it is a proposed intermediate in the metabolism of 3-butene-1,2-diol (BD-diol) to 1,2-dihydroxy-4-(N-acetylcysteinyl)butane, the major mercapturic acid metabolite of BD in humans. In addition, BD-diol is a major BD metabolite in liver perfusion experiments in rodents. By analogy with other alpha,beta-unsaturated carbonyls, HMVK is likely to be mutagenic via formation of promutagenic 1,N(2)-propanodeoxyguanosine adducts. The objective of the current study was to investigate the formation of such adducts in vitro. The reaction between HMVK and dGuo yielded two major products shown to be identical by positive ion electrospray-MS, having protonated molecular ions with m/z consistent with HMVK-derived 1,N(2)-propanodeoxyguanosine (HMVK-dGuo). Rechromatography of each fraction yielded two fractions with retention times identical to those initially isolated, suggesting equilibration between two diastereomers. Two partially resolved sets of (1)H NMR signals were consistent with a 1:1 mixture of diastereomeric C-6-substituted adducts equilibrating slowly on an NMR time-scale. Following deglycosylation, C-6 substitution was verified by two-dimensional correlation NMR spectroscopy, indicating that the initial adducts were formed by Michael addition of dGuo-N1 to the terminal vinyl carbon followed by cyclization to the 1,N(2)-propano structure. Reactions with calf thymus DNA under physiological conditions yielded two sets of products. The first set had HPLC retention times and mass spectra identical to those of the previously characterized C-6-substituted HMVK-dGuo diastereomers. The second set had a molecular ion and fragmentation pattern identical to the C-6-substituted adducts and on this basis were assigned as the diastereomeric C-8 adducts. In addition to detecting HMVK-dGuo in treated DNA, the adducts were also present in control DNA. Overall, our research demonstrates that HMVK can form promutagenic DNA adducts and it therefore has the potential to play a role in BD-associated mutagenicity.     

Analysis of crotonaldehyde- and acetaldehyde-derived 1,n(2)-propanodeoxyguanosine adducts in DNA from human tissues using liquid chromatography electrospray ionization tandem mass spectrometry

Crotonaldehyde, a mutagen and carcinogen, reacts with deoxyguanosine (dGuo) in DNA to generate a pair of diastereomeric 1,N(2)()-propanodeoxyguanosine adducts (Cro-dGuo, 2), which occur in (6S,8S) and (6R,8R) configurations. They can also be formed through the consecutive reaction of two acetaldehyde molecules with dGuo. Cro-dGuo adducts inhibit DNA synthesis and induce miscoding in human cells. Considering their potential role in carcinogenesis, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to explore the presence of Cro-dGuo adducts in DNA from various human tissues, such as liver, lung, and blood. DNA was isolated from human tissues and enzymatically hydrolyzed to deoxyribonucleosides. [(15)N(5)]Cro-dGuo was synthesized and used as an internal standard. The Cro-dGuo adducts were enriched from the hydrolysate by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring (SRM). This method allows the quantitation of the Cro-dGuo adducts at a concentration of 4 fmol/micromol dGuo, corresponding to about 1 adduct per 10(9) normal nucleosides starting with 1 mg of DNA, with high accuracy and precision. DNA from human liver, lung, and blood was analyzed. The Cro-dGuo adducts were detected more frequently in human lung DNA than in liver DNA but were not detected in DNA from blood. The results of this study provide quantified data on Cro-dGuo adducts in human tissues. The higher frequency of Cro-dGuo in lung DNA than in the other tissues investigated is potentially important and deserves further study.     

Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducts in smokers and nonsmokers

A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay for tobacco-specific nitrosamine adducts of DNA is described. The assay is based on the observation that acid hydrolysis of DNA from animals treated with tobacco-specific nitrosamines releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB and the internal standard [4,4-D2]HPB are derivatized with pentafluorobenzoyl chloride and the resulting HPB-pentafluorobenzoate is purified by high-performance liquid chromatography prior to GC-NICI-MS analysis. DNA from human peripheral lung and tracheobronchial tissue, collected at autopsy, was analyzed for acid-released HPB. The mean HPB level (fmol/mg of DNA) for peripheral lung DNA was 11 +/- 16 (SD, n = 9) for smokers and 0.9 +/- 2.3 (n = 8) for nonsmokers. Mean adduct levels in tracheobronchus were 16 +/- 18 (n = 4) for smokers and 0.9 +/- 1.7 (n = 4) for nonsmokers. These are the first measurements of tobacco-specific nitrosamine-DNA adducts in humans. Further studies comparing the levels of DNA and globin adducts will provide a better understanding of the metabolic activation of tobacco-specific nitrosamines in humans and may provide a more accurate indication of an individual's risk of developing tobacco-related cancer.     

Quantitation of 7-ethylguanine in leukocyte DNA from smokers and nonsmokers by liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry

There is considerable evidence for the exposure of humans to an unknown ethylating agent, and some studies indicate that cigarette smoking may be one source of this exposure. Therefore, we have developed a liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry-selected reaction monitoring (LC-NSI-HRMS/MS-SRM) method for the analysis of 7-ethyl-Gua in human leukocyte DNA, a readily available source of DNA. [(15)N(5)]7-Ethyl-Gua was used as the internal standard. Leukocyte DNA was isolated and treated by thermal neutral hydrolysis. The hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-ethyl-Gua was analyzed by LC-NSI-HRMS/MS-SRM using the transition m/z 180 [M + H](+)→ m/z 152.05669 [Gua + H](+) for 7-ethyl-Gua and m/z 185 → m/z 157.04187 for the internal standard. The detection limit was approximately 10 amol on column, while the limit of quantitation was about 8 fmol/μmol Gua starting with 180 μg DNA (corresponding to 36 μg DNA on-column). Leukocyte DNA samples from 30 smokers and 30 nonsmokers were analyzed. Clear peaks for 7-ethyl-Gua and the internal standard were observed in most of the samples. The mean (±SD) level of 7-ethyl-Gua measured in leukocyte DNA from smokers was 49.6 ± 43.3 (range 14.6-181) fmol/μmol Gua, while that from nonsmokers was 41.3 ± 34.9 (range 9.64-157) fmol/μmol Gua. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the presence of 7-ethyl-Gua in human leukocyte DNA.     

Development of a 32P-postlabeling method for the detection of 1, N2-propanodeoxyguanosine adducts of 2-hexenal in vivo

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new 32P-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.     

Development of a 32P-postlabelling method for the detection of 1,N 2-propanodeoxyguanosine adducts of crotonaldehyde in vivo

Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsaturated carbonyl compound which forms 1,N2-propanodeoxyguanosine adducts. Humans are exposed to this compound at work places, and from tobacco smoke and air pollution, but also from food and beverages. Therefore crotonaldehyde can play a significant role in carcinogenesis. Since in vivo measurement of DNA adducts of crotonaldehyde can improve cancer risk assessment and contribute to the clarification of the role of crotonaldehyde in carcinogenicity, we developed, adapted and optimized a 32P-postlabelling technique for the adducts of crotonaldehyde based on nuclease P1 enrichment and on a polyethylene imine modified cellulose TLC to provide a detection sensitivity of three adducts per 10(9) nucleotides and a labelling efficiency of 80-90%. We also report a readily performable synthesis of adduct standards and demonstrated that DNA is completely digested to the 3'-monophosphate nucleotides under the conditions of our enzymatic DNA hydrolysis. We showed that the postlabelling method developed is appropriate for in vivo DNA-binding studies. Female Fischer 344 rats were treated by gavage with crotonaldehyde at doses of 200 and 300 mg/kg body weight, and 20 h after treatment adduct levels of 2.9 and 3.4 adducts per 10(8) nucleotides, respectively, were found in the liver DNA. Only 1.6 nucleotides per 10(8) nucleotides were found 12 h after treatment at 200 mg/kg body weight. Absolutely no adducts could be found in liver DNA of untreated rats with our method at the detection limit of three adducts per 10(9) nucleotides. In contrast to our group, the group of Chung have reported crotonaldehyde adduct levels in the range of 2.2 22 adducts per 10(8) nucleotides in DNA of untreated Fischer 344 rats. The clarification of this discrepancy is of importance for the elucidation of the role of crotonaldehyde in carcinogenicity, and both groups have decided to clarify this in cooperation in the near future.     

Modulation of DNA and protein adducts in smokers by genetic polymorphisms in GSTM1,GSTT1, NAT1 and NAT2

The formation of DNA and protein adducts by environmental pollutants is modulated by host polymorphisms in genes that encode metabolizing enzymes. In our study on 67 smokers, aromatic-DNA adduct levels were examined by nuclease P1 enriched 32P-postlabelling in mononuclear blood cells (MNC) and 4-aminobiphenyl-haemoglobin adducts (4-ABP-Hb) by gas chromatography-mass spectroscopy. Genetic polymorphisms in glutathione S-transferase M1 (GSTM1), T1 (GSTT1) and N-acetyl-transferase 1 (NAT1) and 2 (NAT2) were assessed by polymerase chain reaction-based methods. DNA adduct levels, adjusted for the amount of cigarettes smoked per day, were higher in GSTM1(-/-) individuals (1.30 +/- 0.57 adducts per 108 nucleotides) than in GSTM1(+) subjects (1.03 +/- 0.56, P = 0.05), higher in NAT1 slow acetylators (1.58 +/- 0.54) than in NAT1 fast acetylators (1.11 +/- 0.58, P = 0.05) and were also found to be associated with the NAT2 acetylator status (1.29 +/- 0.64 and 1.03 +/- 0.46, respectively, for slow and fast acetylators, P = 0.06). An effect of GSTT1 was only found in combination with the NAT2 genotype; individuals with the GSTT1(-/-) and NAT2-slow genotype contained higher adduct levels (1.80 +/- 0.68) compared to GSTT1(+)/NAT2 fast individuals (0.96 +/- 0.36). Highest DNA adduct levels were observed in slow acetylators for both NAT1 and NAT2 also lacking the GSTM1 gene (2.03 +/- 0.17), and lowest in GSTM1(+) subjects with the fast acetylator genotype for both NAT1 and NAT2 (0.91 +/- 0.45, P = 0.01). No overall effects of genotypes were observed on 4-ABP-Hb levels. However, in subjects smoking less than 25 cigarettes per day, 4-ABP-Hb levels were higher in NAT2 slow acetylators (0.23 +/- 0.10 ng/g Hb) compared to fast acetylators (0.15 +/- 0.07, P = 0.03). These results provide further evidence for the combined effects of genetic polymorphisms in GSTM1, GSTT1, NAT1 and NAT2 on DNA and protein adduct formation in smoking individuals and indicate that, due to the complex carcinogen exposure, simultaneous assessment of multiple genotypes may identify individuals at higher cancer risk.     

Attentional bias in active smokers, abstinent smokers, and nonsmokers

Attentional bias was studied with a modified version of the Stroop test in active smokers, abstinent smokers, and nonsmokers. The task was color-naming of incongruent color-words, smoking-related words, and neutral words. The results showed that the active smokers used longer verbal reaction time (VRT) to smoking-related words compared to abstinent smokers i.e., indicating stronger attentional bias in the active smokers. Furthermore, longer VRTs to the Stroop words compared to the smoking words and the neutral words were found only in nonsmokers and abstinent smokers. Finally, a significant negative correlation was found between attitudes against smoking and VRTs to the smoking-related words. Taken together the main finding was that the active smokers showed no differential response to the stimuli. This could be caused by a lack of ability to modulate attentional processes in active smokers.     

Regioisomeric synthesis and characteristics of the alpha-hydroxy-1,N(2)-propanodeoxyguanosine

Acrolein, a known mutagen, undergoes reaction in vitro under physiological conditions with both 2'-deoxyguanosine and native DNA to give rise to exocyclic adducts of the 5,6,7,8-tetrahydropyrimido[1,2-a]purine-10(3H)-one class having a hydroxyl group at either the 6 or the 8 position (these positions are respectively designated alpha and gamma when referring to the 1,N(2)-(propano-bridge). Previously, we have shown that the 8-hydroxy derivative has very low mutagenicity probably because, in double-stranded DNA, this residue exists in the open-chain aldehydic form [N(2)-(3-oxopropyl)-2'-deoxyguanosine] (5). To continue our investigation in this area, we needed ample supplies of the 6-hydroxy isomers. This current paper describes high-yield simple methods for the synthesis in bulk of the 6-hydroxy and the 6-methoxy exocyclic adducts 1 and 3 and a new efficient synthesis of 1,N(2)()-(prop-1,3-diyl)-2'-deoxyguanosine (4), previously used as a chemically stable model in studying the physico-biological implications of 1,N(2) exocyclic adduction to dG.     

An approach to cancer risk assessment for the food constituent 2-hexenal on the basis of 1,N 2-propanodeoxyguanosine adducts of 2-hexenal in vivo

2-Hexenal is formed by plants, and humans are regularly exposed to this mutagenic/genotoxic compound via vegetable foods. 2-Hexenal has not been tested for carcinogenicity, but it forms exocyclic 1,N2-propanodeoxyguanosine adducts like other carcinogenic alpha,beta-unsaturated carbonyl compounds. To quantify the respective DNA adducts as an approach to a theoretical cancer risk assessment, we used a newly developed 32P-postlabelling technique based on nuclease P1 enrichment, allowing a detection limit of 3 adducts per 10(8) nucleotides. Adduct levels were measured at different doses and the covalent binding index (CBI) was found to be dose-dependent. This can be explained by glutathione depletion at higher doses. The CBI at low doses was 0.06. A negligible cancer risk of 1-5 per 10(7) lives was estimated on the basis of TD50 values calculated from the correlation between CBI and TD50 of Lutz and on the daily intake of 2-hexenal via vegetable foods, fruit juices and black tea. A risk of 1.6-8.5 per 10(6) lives was estimated for the hypothetical case of glutathione depletion, e.g. due to consuming special medicaments. In every case, the benefit from eating fruit and vegetables is clearly higher than a possible low and unavoidable cancer risk. Utilization of 2-hexenal as a flavouring agent or as a fungicide, breeding fungus-resistant plants or technological gene construction of fungus resistance may lead to a high hypothetical cancer risk of 2-6 per 10(4) lives under certain circumstances which are avoidable and deserves special case-by-case consideration.     

LC/MS/MS method for the quantitation of trans-2-hexenal-derived exocyclic 1,N(2)-propanodeoxyguanosine in DNA

trans-2-Hexenal is an alpha,beta-unsaturated aldehyde to which humans are exposed daily in small amounts. Hexenal has demonstrated mutagenicity and genotoxicity in vitro and reacts with deoxyguanosine to form diastereomeric hexenal-derived exocyclic 1,N(2)-propanodeoxyguanosine (Hex-PdG) adducts. A highly sensitive and specific method for the measurement of Hex-PdG in DNA has not previously been available. An LC/MS/MS assay for the quantitation of Hex-PdG, using [(13)C4(15)N2]Hex-PdG as an internal standard, was developed, to assess binding of hexenal to DNA. Samples were purified prior to analysis by centrifuge filtration and solid phase extraction and analyzed by LC/MS/MS in the selected reaction monitoring (SRM) mode (SRM m/z 366.2 --> 250.2 for Hex-PdG; SRM m/z 372.2 --> 256.2 for [(13)C4(15)N2]Hex-PdG). Recovery of standards was 89% or greater, and quantitation was unaffected by the addition of increasing concentrations of calf thymus DNA (ctDNA). The limit of quantitation, determined in samples of 200 microg of ctDNA spiked with analyte standard, was 0.015 fmol/microg DNA, which corresponds to approximately 5 Hex-PdG/10(9) unmodified nucleotides. Hex-PdG was detected in ctDNA treated with 0.021 microM, 0.21 microM, or 2.1 mM hexenal but not in untreated DNA. Furthermore, Hex-PdG was not detected in DNA exposed to reactive oxygen species-mediated deoxyribose attack and lipid peroxidation, which resulted in a significant increase in the malondialdehyde-derived pyrimido[1,2-a]purin-10(3H)one. Hex-PdG was not detected in DNA of untreated rat liver, but Hex-PdG in hexenal-treated calf thymus DNA was quantifiable when spiked into the rat liver DNA at 0.035 or 0.35 fmol/microg DNA. These data indicate that Hex-PdG is formed following hexenal treatment and that this method is suitable for in vitro or in vivo assessment of Hex-PdG formation.     

Lung DNA adducts detected in human smokers are unrelated to typical polyaromatic carcinogens

Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other polyaromatics by 32P-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues of human smokers. However, our experimental studies in rodent models, which used modified chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines. In the present study, we have performed similar analysis of human lung tissues to study the chemical nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active, ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and n-butanol enrichment-mediated 32P-postlabeling assay. All DNA samples yielded low to highly intense adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea solvents. Adduct burden ranged from 6.6 to 2930 per 10(10) nucleotides. However, when adducts were resolved in a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms, this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by 32P-postlabeling resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-containing solvents, but no adducts were detected when the ammonium hydroxide-based solvent was used, suggesting that even nonpolyaromatic electrophiles can result in adduct DRZs on the chromatograms similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they are not related to PAHs and aromatic amines.     

Development of a (32)P-postlabeling method for the detection of 1,N(2)-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal in vivo

A (32)P-postlabeling method was developed for the sensitive detection of 1,N(2)-propanodeoxyguanosine adducts of the lipid peroxidation product trans-4-hydroxy-2-nonenal in vivo. The method development was based on the chemically synthesized HNE-1, N(2)-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by nuclease P1. They were subsequently reacted with [gamma-(32)P]ATP to give the respective 3'-5'-bisphosphates, which were two-directionally separated on PEI-cellulose TLC and quantitated by autoradiography. The medium labeling efficiency for the mixture of the two pairs of diastereomers was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. The method is applicable for the separation and quantitation of HNE-dGp-propano adducts in vivo. It was applied to DNA from colon and brain tissue of untreated Fischer 344 rats and humans. The determination of the limit of quantitation in DNA from rat colon by spiking of adduct standard revealed a sensitivity of <21 adducts/10(9) nucleotides. The analytical quantitation of 4-HNE-dGp-propano adducts resulted in adduct-levels per 10(9) normal nucleotides +/- the standard deviation of 223.32 +/- 79.84 in rat colon tissue, 90.37 +/- 11.94 in rat brain tissue, 378.44 +/- 52.42 in human colon tissue, and 185.15 +/- 6.48 in human brain tissue. The results clearly demonstrate the applicability of this method for the sensitive detection of endogenously formed 1,N(2)-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal, a specific marker for the lipid peroxidation process.     

Genome-wide DNA methylation differences in nucleus accumbens of smokers vs. nonsmokers

Numerous DNA methylation (DNAm) biomarkers of cigarette smoking have been identified in peripheral blood studies, but because of tissue specificity, blood-based studies may not detect brain-specific smoking-related DNAm differences that may provide greater insight as neurobiological indicators of smoking and its exposure effects. We report the first epigenome-wide association study (EWAS) of smoking in human postmortem brain, focusing on nucleus accumbens (NAc) as a key brain region in developing and reinforcing addiction. Illumina HumanMethylation EPIC array data from 221 decedents (120 European American [23% current smokers], 101 African American [26% current smokers]) were analyzed. DNAm by smoking (current vs. nonsmoking) was tested within each ancestry group using robust linear regression models adjusted for age, sex, cell-type proportion, DNAm-derived negative control principal components (PCs), and genotype-derived PCs. The resulting ancestry-specific results were combined via meta-analysis. We extended our NAc findings, using published smoking EWAS results in blood, to identify DNAm smoking effects that are unique (tissue-specific) vs. shared between tissues (tissue-shared). We identified seven CpGs (false discovery rate < 0.05), of which three CpGs are located near genes previously indicated with blood-based smoking DNAm biomarkers: ZIC1, ZCCHC24, and PRKDC. The other four CpGs are novel for smoking-related DNAm changes: ABLIM3, APCDD1L, MTMR6, and CTCF. None of the seven smoking-related CpGs in NAc are driven by genetic variants that share association signals with predisposing genetic risk variants for smoking, suggesting that the DNAm changes reflect consequences of smoking. Our results provide the first evidence for smoking-related DNAm changes in human NAc, highlighting CpGs that were undetected as peripheral biomarkers and may reflect brain-specific responses to smoking exposure.Subject terms: DNA methylation, Addiction     

Adolescents' value images of smokers, ex-smokers, and nonsmokers

Adolescents' value images of smokers, ex-smokers, and nonsmokers were investigated in a study of high school graduates. Overall, smokers were seen as being concerned with values related to personal enjoyment and autonomy. In contrast, nonsmokers were perceived as being more conventional, and more concerned with religious, interpersonal, and family values. Images of ex-smokers usually were intermediate, but resembled those of nonsmokers somewhat more than those of smokers. Interestingly, ex-smokers were perceived to place more importance on values relating to accomplishment and self-control than were either smokers or nonsmokers. In general, the value images were consistent among respondents who themselves were smokers, potential smokers, or nonsmokers. However, for a few values smokers and potential smokers had a more favorable image of smokers than did nonsmokers. Interestingly, males and females generally did not differ in their images of smokers, ex-smokers, and nonsmokers. Suggestions for prevention of adolescent smoking based on the value images are discussed.     

Replication of N2-ethyldeoxyguanosine DNA adducts in the human embryonic kidney cell line 293

N(2)-Ethyldeoxyguanosine (N(2)-ethyldGuo) is a DNA adduct formed by reaction of the exocyclic amine of dGuo with the ethanol metabolite acetaldehyde. Because ethanol is a human carcinogen, we assessed the biological consequences of replication of template N(2)-ethyldGuo, in comparison to the well-studied adduct O(6)-ethyldeoxyguanosine (O(6)-ethyldGuo). Single chemically synthesized N(2)-ethyldGuo or O(6)-ethyldGuo adducts were placed site specifically in the suppressor tRNA gene of the mutation reporting shuttle plasmid pLSX. N(2)-EthyldGuo and O(6)-ethyldGuo were both minimally mutagenic in double-stranded pLSX replicated in human 293 cells; however, the placement of deoxyuridines on the complementary strand at 5'- and 3'-positions flanking the adduct resulted in 5- and 22-fold enhancements of the N(2)-ethyldGuo- and O(6)-ethyldGuo-induced mutant fractions, respectively. The fold increase in the N(2)-ethyldGuo-induced mutant fraction in deoxyuridine-containing plasmids was similar after replication in 293T cells, a mismatch repair deficient variant of 293 cells, indicating that postreplication mismatch repair has little role in modulating N(2)-ethyldGuo-mediated mutagenesis. The mutation spectrum generated by N(2)-ethyldGuo consisted primarily of single base deletions and adduct site-targeted transversions, in contrast to the exclusive production of adduct site-targeted transitions by O(6)-ethyldGuo. The yield of progeny plasmids after replication in 293 cells was reduced by the presence of N(2)-ethyldGuo in parental plasmids with or without deoxyuridine to 39 or 19%, respectively. Taken together, these data indicate that N(2)-ethyldGuo in DNA exerts its principal biological activity by blocking translesion DNA synthesis in human cells, resulting in either failure of replication or frameshift deletion mutations.     

1,N 2-Cyclic deoxyguanosine adducts and guanine adducts of 2-haloacroleins. Isolation,characterization, isomerization and stability

The reaction of the mutagenic 2-haloacroleins, 2-fluoroacrolein, 2-chloroacrolein and 2-bromoacrolein, with nucleosides and 5′-mononucleotides was studied. We found two different regioisomers of 1,N ²-cyclic deoxyguanosine adducts of 2-chloroacrolein and 2-bromoacrolein: type A, the 6-hydroxy, 7-haloadduct in which the OH-substituent is vicinal to the N ²-atom of the guanine moiety and type B, the 8-hydroxy, 7-haloadduct in which the OH-group is adjacent to the N¹-atom of the guanine moiety. The major adducts were the trans pairs of diastereomers of type A and type B in which the 6,7-substituents as well as the 7,8-substituents are in the energetically favoured diaxial position of the newly formed tetrahydropyrimidine ring. In the case of the type A regioisomers, the cis pairs of diastereomers (traces with chloroacrolein and about 4% with bromoacrolein) were also found in which the halosubstituent probably takes the equatorial position. Due to the anomeric effect, the OH-group takes the axial position in both regioisomers. No cis isomers of the type B regioisomers could be isolated. Acid hydrolysis of the deoxyguanosine adducts released deoxyribose, and the respective guanine adducts were isolated and characterized. Besides the vicinal halo, hydroxy adducts, trace amounts of the corresponding dihydroxy adducts were formed by hydrolysis of the chlorine or bromine substituents. The dihydroxy compounds possess the same structures and conformations in the newly formed tetrahydropyrimidine ring as do the halo, hydroxy adducts. Under our conditions no adducts other than those with deoxyguanosine and guanine could be identified. We found, however, indication for the formation of deoxyadenosine adducts when using dimethylsulfoxide as a solvent. No adducts in substantical amounts could be isolated with fluoroacrolein due to its rapid polymerization. Received: 24 February 1994 / Accepted: 12 April 1994     

Comparative mood states and cognitive skills of cigarette smokers,deprived smokers and nonsmokers

Regular cigarette smokers (n=15), overnight deprived smokers (n=15) and nonsmokers (n=20), were assessed on a battery of mood questionnaires and cognitive performance tasks, before and after a cigarette/rest period. At the initial session, deprived smokers reported significantly greater feelings of stress, irritability, depression, poor concentration and low pleasure, than both nondeprived smokers and nonsmokers (all comparisons, p<0·01). After the rest/cigarette break, the mood states of all three groups became generally similar, although the previously deprived smokers still reported elevated depression. These findings suggest that mood gains after smoking reflect the simple reversal of abstinence effects. On the cognitive tasks, there were no significant differences in letter cancellation performance between subgroups, either before or after smoking. With mental arithmetic, abstinent and nonabstinent smokers attempted more problems than nonsmokers, both before and after the rest/cigarette break. This is suggestive of faster cognitive processing in smokers, irrespective of their nicotine status. However, the cognitive performance data were untypical in various ways and need replication. © 1998 John Wiley & Sons, Ltd.