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Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducts in smokers and nonsmokers
Abstract
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.
... In the largest study of 7-mG, the average level was 2.11 adduct per 10 7 nucleotides (67). In comparison, levels of HPB-releasing adducts were 0.1 per 10 7 nucleotides (42). Higher levels of 7-mG have been observed in smokers than non-smokers, suggesting NNK as one source (68,69). ...
... Dietary and endogenously formed nitroso compounds as well as environmental tobacco smoke could contribute to the adduct formation. HPB-releasing adducts have been found in the lung of both smokers and non-smokers (12,13,42,70). In a recent study, a seven-fold increase of HPB-releasing adduct levels was observed in the lung from smoking lung cancer patients compared to nonsmoking lung cancer patients (13). ...
... dCyd(42), N 2 -hydroxymethyl-dGuo(43), as well as the cross-links such as di-(N 6 -deoxyadenosyl)methane(44), (N 6 -deoxyadenosyl-N 2deoxyguanosyl)methane (45), and di-(N 2 -deoxyguanosyl)methane (46) (Figure 1.8) (80-84). These formaldehyde-DNA adducts have been identified in the reactions of NNKOAc with DNA (85). ...
... Although the structures of POB-DNA adducts were identified only recently, the pyridyloxobutylation pathway was established over 20 years ago and has been quantified indirectly by measuring HPB release as a reflection of DNA adduct levels Foiles et al., 1991). Using HPB release, the formation of POB-DNA adducts has been extensively studied in rodents (Nelson et al., 1996;Peterson et al., 1991b;Staretz et al., 1997a) and POB-DNA adducts have been detected in lung tissue from smokers (Foiles et al., 1991). ...
... Although the structures of POB-DNA adducts were identified only recently, the pyridyloxobutylation pathway was established over 20 years ago and has been quantified indirectly by measuring HPB release as a reflection of DNA adduct levels Foiles et al., 1991). Using HPB release, the formation of POB-DNA adducts has been extensively studied in rodents (Nelson et al., 1996;Peterson et al., 1991b;Staretz et al., 1997a) and POB-DNA adducts have been detected in lung tissue from smokers (Foiles et al., 1991). At low doses of NNK in the rat, the levels of HPB released from lung DNA are higher than those from liver (Murphy et al., 1990;Boysen et al., 2003;Morse et al., 1989;Peterson et al., 1991b), whereas in the A/J mouse, levels of HPB-releasing adducts are higher in liver than in lung (Hecht, 1998) (Table 1.2). ...
... metabolism has yet to be determined. (Staretz et al., 1997a;Trushin et al., 1994;Peterson et al., 2001) and is likely to be involved in tobacco-induced cancers in smokers (Foiles et al., 1991). Thus, elucidation of the processes involved in POB adduct repair may allow for the identification of susceptible individuals based on their ability to repair POB DNA damage and also may allow for the development of therapeutic strategies aimed at increasing the activity and/or efficiency of specific DNA repair pathways. ...
... Indeed, in 1991 our group demonstrated the presence of HPB-releasing adducts in lung DNA from smokers. 12 Levels of these adducts were higher than in non-smokers, as expected based on the tobaccospecificity of NNK and NNN. A much larger study examined levels of HPB-releasing adducts in human lung and clearly demonstrated significantly higher levels of HPBreleasing adducts in pulmonary DNA from lung cancer patients compared to controls. ...
... To each sample, 3.6 N HCl was added to bring the final concentration of the acid to 0.8 N, and the samples were hydrolyzed at 80 °C for 3 h to release HPB. 9,12,27 Additional experiments have demonstrated that no D-H exchange takes place in the internal standard under these conditions (data not shown). The hydrolysates were cooled down to room temperature, adjusted to neutral pH with 1 N NaOH, and 40 μL of each sample was transferred to clean microinsert vials for DNA quantitation by HPLC. ...
... The purpose of this study was to develop a simplified analytical procedure for the direct measurement of HPB by LC-ESI-MS/MS, thus excluding the derivatization step that was traditionally used for the analysis of HPB by gas chromatography-MS. 12 ...
Quantitation of DNA adducts could provide critical information on the relationship between exposure to tobacco smoke and cancer risk in smokers. In this study, we developed a robust and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB1)-releasing DNA adducts in human oral cells, a non-invasive source of DNA for biomarker studies. Isolated DNA undergoes acid hydrolysis, after which samples are purified by solid-phase extraction and analyzed by LC-ESI-MS/MS. The developed method was applied for analysis of samples obtained via collection with a commercial mouthwash from 30 smokers and 15 nonsmokers. In smokers, the levels of HPB-releasing DNA adducts averaged 12.0 pmol HPB/mg DNA (detected in 20 out of 28 samples with quantifiable DNA yield) and in nonsmokers, the levels of adducts averaged 0.23 pmol/mg DNA (detected in 3 out of 15 samples). For the 30 smoking subjects, matching buccal brushings were also analyzed and HPB-releasing DNA adducts were detected in 24 out of 27 samples with quantifiable DNA yield, averaging 44.7 pmol HPB/mg DNA. The levels of adducts in buccal brushings correlated with those in mouthwash samples of smokers (R = 0.73, p < 0.0001). Potentially the method can be applied in studies of individual susceptibility to tobacco-induced cancers in humans.
... As summarized in Table 1, the HPB released upon the acid hydrolysis of the peripheral lung and tracheobronchus DNA of cigarette smokers amounted to 11 ± 16 and 16 ± 18 fmol/mg DNA, respectively, in contrast to the low levels found in the DNA of the same tissues from nonsmokers (0.9 ± 2.3 fmol/mg peripheral lung DNA and 0.9 ± 1.7 fmol/mg tracheobronchus DNA) [80]. In the peripheral lung tissues of patients who had adenocarcinoma or squamous cell carcinoma or another diagnosis (1 patient), a significantly higher level of HPB-releasing DNA adducts was observed in self-reported smokers (404 ± 258 fmol/mg DNA) than in self-reported nonsmokers (59 ± 56 fmol/mg DNA) (p < 0.0001) [84]. ...
... The formation of HPB-releasing DNA adducts were readily detected in the liver, lung and nasal mucosa DNA of NNN-exposed mice and rats (Table 1) [75,[79][80][81]. The failure to detect HPB-releasing DNA adducts in other tissues was probably due to the detection limit of the analytical method used in those studies [75]. ...
The tobacco-specific N-nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) always occur together and exclusively in tobacco products or in environments contaminated by tobacco smoke. They have been classified as “carcinogenic to humans” by the International Agency for Research on Cancer. In 1998, we published a review of the biochemistry, biology and carcinogenicity of tobacco-specific nitrosamines. Over the past 20 years, considerable progress has been made in our understanding of the mechanisms of metabolism and DNA adduct formation by these two important carcinogens, along with progress on their carcinogenicity and mutagenicity. In this review, we aim to provide an update on the carcinogenicity and mechanisms of the metabolism and DNA interactions of NNK and NNN.
... Multiple in vitro and animal studies demonstrated the relationship of HPB-releasing DNA adducts to NNK and NNN dose as well as biomarkers of NNK exposure [15]. HPB-releasing DNA adducts were also detected by our group and others in human tissues including lung, tracheobronchus, esophagus, and cardia [23][24][25]. We demonstrated for the first time the presence of HPB-releasing DNA adducts in human lung, with adduct levels being higher in smokers than nonsmokers [25]. ...
... HPB-releasing DNA adducts were also detected by our group and others in human tissues including lung, tracheobronchus, esophagus, and cardia [23][24][25]. We demonstrated for the first time the presence of HPB-releasing DNA adducts in human lung, with adduct levels being higher in smokers than nonsmokers [25]. Similar results were observed by Schlobe, et al, with HPB levels in the lung being significantly higher in 21 self-reported smokers compared to that in 11 self-reported nonsmokers [24]. ...
DNA adducts are believed to play a central role in the induction of cancer in cigarette smokers and are proposed as being potential biomarkers of cancer risk. We have summarized research conducted since 2012 on DNA adduct formation in smokers. A variety of DNA adducts derived from various classes of carcinogens, including aromatic amines, polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, alkylating agents, aldehydes, volatile carcinogens, as well as oxidative damage have been reported. The results are discussed with particular attention to the analytical methods used in those studies. Mass spectrometry-based methods that have higher selectivity and specificity compared to 32P-postlabeling or immunochemical approaches are preferred. Multiple DNA adducts specific to tobacco constituents have also been characterized for the first time in vitro or detected in vivo since 2012, and descriptions of those adducts are included. We also discuss common issues related to measuring DNA adducts in humans, including the development and validation of analytical methods and prevention of artifact formation.
... NNN also generates adducts resulting from 5hydroxylation [51][52][53] . In contrast to experimental studies, evidence for the presence of NNN-and NNK-derived POB DNA adduct formation in human lung tissue, and its association with smoking, is limited and based on a small number of studies, which assessed 4-hydroxy-(3-pyridyl)-lbutanone (HPB)-releasing DNA adducts that can result from tobacco-specific nitrosamines 5,54,55 . ...
Tobacco usage is linked to multiple cancer types and accounts for a quarter of all cancer-related deaths. Tobacco smoke contains various carcinogenic compounds, including polycyclic aromatic hydrocarbons (PAH), though the mutagenic potential of many tobacco-related chemicals remains largely unexplored. In particular, the highly carcinogenic tobacco-specific nitrosamines NNN and NNK form pre-mutagenic pyridyloxobutyl (POB) DNA adducts. In the study presented here, we identified genome-scale POB-induced mutational signatures in cell lines and rat tumors, while also investigating their role in human cancer. These signatures are characterized by T greater than N and C greater than T mutations forming from specific POB adducts damaging dT and dC residues. Analysis of 2,780 cancer genomes uncovered POB signatures in approx 180 tumors; from cancer types distinct from the ones linked to smoking-related signatures SBS4 and SBS92. This suggests that, unlike PAH compounds, the POB pathway may contribute uniquely to the mutational landscapes of certain hematological malignancies and cancers of the kidney, breast, prostate and pancreas.
... Formation of DNA Adducts: Nitrosamines generate DNA adducts through their interaction with nucleophilic sites in DNA molecules.[16] These adducts can impede normal DNA replication and transcription processes, leading to the accumulation of genetic abnormalities and increased genomic instability. ...
The nitrosamines are a class of carcinogenic compounds that have garnered significant attention due to their association with an increased risk of various cancer types. More than thirty nitrosamines are on California's Proposition 65's list of substances that can cause cancer. Commonly used medications for the treatment of heartburn, hypertension, and type 2 diabetes have recently been contaminated with nitrosamine, which has led to multiple recalls by the US Food and Drug Administration (FDA). Among chemicals and within a particular species of laboratory animal, these nitrosamines share target tumor sites for their common genotoxic and tumorigenic properties. Sometimes the drug valsartan is used to estimate the additional cancer risks associated with NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) contamination. These estimates are based on nitrosamine levels reported by the US FDA, cancer potencies developed by California's Proposition 65 program and the US Environmental Protection Agency (EPA), and specific exposure scenarios. These estimates indicate that long-term drug use may raise the risk of cancer due to nitrosamine contamination, which is a major public health concern. The purpose of this thorough analysis is to clarify the connection between nitrosamine exposure and the onset of cancer.
... 21 NNN and NNK have shown carcinogenicity in animals and potential carcinogenicity in humans. 22 B[α]P, formed during the incomplete combustion of organic matter, has carcinogenic activity because it can form an adduct with deoxyguanosine to covalently modify DNA. 23 3.1. ...
The first certified reference cigarette, 1R6F, was produced by the Center for Tobacco Reference Products at the University of Kentucky in 2015 and certified in 2016. 1R6F reference cigarettes have been stored at -20 °C since they were manufactured. 1R6F has been widely used as a control cigarette or a monitor for nonclinical investigational purposes in tobacco product analysis and scientific research. However, there is little published data to demonstrate the stability of the 1R6F cigarette. In this paper, we report the results of a long-term storage study of the 1R6F cigarette tobacco filler and the resulting mainstream smoke. 1R6F cigarettes were stored under different conditions (room temperature, refrigerator (4 °C), and freezer (-20 °C)) for 3 years since April 2017. The constituents in the cigarette tobacco filler (oven volatiles, nicotine, N'-nitrosornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)) and the mainstream smoke (nicotine, NNN, NNK, benzo[α]pyrene, carbon monoxide, total particulate matter) were analyzed. Some physical parameters (resistance to draw and ventilation) were also measured. Analysis of our data showed that no significant differences in these major constituents were detected after storage of the 1R6F cigarette at -20 °C for 3 years.
... However, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) releasing DNA adducts (indicator of POB DNA adduct formation) have been reported in the lung and other tissues of smokers and non-smokers, and they were significantly higher in smokers than in nonsmokers. [43][44][45] Even though POB adducts were not detected, urinary NNAL was measured and it correlated with AP sites in smokers' leukocyte DNA ( Figure 6B). Even after removing an outlier (NNAL 2.29 pmol/mL and AP site 4.57 per 10 7 nts), a significant relationship was still observed (p=0.045, Figure 6D). ...
Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N⁷POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 10⁸ nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and non-smokers. Rats treated with 5 or 21 mg/kg bw NNK for four days by s.c. injection had 2-6 and 2-17 times more AP sites than controls in liver and lung DNA (p<0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks (p<0.05), but not for 50 and 70 weeks to 5 ppm NNK in their drinking water. Levels of N⁷POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm (S)-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA (p<0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to non-smokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals, and to evaluate AP sites in DNA of smokers and non-smokers.
... Moreover, human lung tissue metabolically activates NNK, although not as efficiently as rodent lung tissue [6]. Furthermore, Castonguay et al. and Foiles et al. showed that DNA adducts specific to NNK and the related nitrosamine N'-nitrosonornicotine (NNN) were detected in the lungs of smokers and metabolites of NNK were detected in their urine [6][7][8][9][10]. ...
Background
Cancer is caused by three factors: Nutrition, inflammation and cigarette smoke. This study in rat experimental models would enable us to understand the mechanism of lung cancer caused by NNK to which humans are continuously exposed, help us understand possible molecular targets, design drugs for humans against lung cancer.
Aim
A lung cancer model was developed by administering tobacco specific carcinogen: NNK [4-methylnitrosamino)-1-(3-pyridyl)-1-butanone] to male wistar rats in 24 weeks. Further, In silico approach was followed to screen the molecular targets.
Methods
A method was established in which subcutaneous and intraperitoneal injections of NNK were administered to male wistar rats simultaneously. For authentication of lung cancer in vivo we performed molecular docking simulations with protein biomarkers:Cox-2, p53, p38 MAPKs and EGFR using Hex-Discovery Studio, Schrödinger-maestro software.
Results
Lung morphology and histopathology indicated the initiation of bronchiolar epithelial hyperplasia, squamous dysplasia beginning in cancer 1 group after 16 weeks NNK exposure. 66.66% incidence of squamous cell carcinoma (SCC), 33.3% incidence of adenocarcinoma in cancer 2 group after being exposed to NNK. Results indicated that the SCC and adenocarcinoma gradually increased from 66.66% to 85.71% in cancer 2 group and 33.33% to 42.58% in cancer 3 groups respectively. Docking results indicate the total binding energy and glide energy of Cox-2, p53, p38 MAPKs, EGFR : 38.14, -211.58, -181.58, - 213.05 Kcal/mol and -39.25, -32.16,-36.49, -40.19 Kcal/mol, respectively.
Conclusion
Pulmonary adenocarcinoma developed in 24 weeks, in silico experiments confirm EGFR to be the most potential target for NNK induced lung Cancer.
... Measurement of DNA adducts offers a direct assessment of DNA damage. Recent studies have shown that the amount of NNN/NNK exposure, as assessed by urinary biomarkers, does not necessarily correlate to the level of HPB-releasing DNA adducts; therefore, there are likely to be other factors, besides the carcinogen doses, contributing to the variations in the levels of DNA damage among smokers (27)(28)(29)(30)(31)(32). The oral microbiome can disrupt host's defense mechanisms, inducing chronic inflammatory changes resulting in a cascade of events capable of causing extensive DNA damage (32)(33)(34)(35). ...
While smoking is inextricably linked to Oral/Head and Neck Cancer (HNSCC), only a small fraction of smokers develop HNSCC. Thus, we have sought to identify other factors which may influence the development of HNSCC in smokers including microbiology. To determine microbial associations with HNSCC among tobacco users, we characterized oral microbiome composition in smokers with and without HNSCC. 16S rRNA MiSeq sequencing was used to examine the oral mucosa microbiome of 27 smokers with (cases) and 24 without HNSCC (controls). In addition, we correlated previously reported levels of DNA damage with the microbiome data. Smokers with HNSCC showed lower microbiome richness compared to controls (p=0.012). Beta-diversity analyses assessed as UniFrac (weighted and unweighted) and Bray-Curtis distances, showed significant differences in oral mucosal microbiome signatures between cases and controls (r2=0.03, p=0.03) and higher inter-individual microbiome heterogeneity in the former (q<=0.01). Higher relative abundance of Stenotrophomonas and Comamonadaceae and predicted bacterial pathways mainly involved in xenobiotic and amine degradation were found in cases compared to controls. The latter, in contrast, exhibited higher abundance of common oral commensals and predicted sugar degradation pathways. Lastly, levels of DNA damage in the oral cavity were correlated with the microbiome profiles above. Oral microbiome traits differ in smokers with and without HNSCC, potentially informing the risk of eventual HNSCC and shedding light into possible microbially-mediated mechanisms of disease. These findings present data which may be useful in screening efforts for HNSCC among smokers who are unable to quit.
... The characterization of multiple adducts from DNA modified with high levels of carcinogens in vitro or the cell is readily accomplished by DNA adductomics approaches. However, in humans, the levels of DNA adducts formed with environmental genotoxicants or endogenously produced electrophiles are often at levels ranging from one adduct per 10 10 to 10 8 nts [35,36,95,96]. Thus, the screening of multiple DNA adducts and their identification poses great analytical challenges in human specimens, where the amounts of tissue are obtained in limited quantities. ...
The measurement of DNA adducts, the covalent modifications of DNA upon the exposure to the environmental and dietary genotoxicants and endogenously produced electrophiles, provides molecular evidence for DNA damage. With the recent improvements in the sensitivity and scanning speed of mass spectrometry (MS) instrumentation, particularly high-resolution MS, it is now feasible to screen for the totality of DNA damage in the human genome through DNA adductomics approaches. Several MS platforms have been used in DNA adductomic analysis, each of which has its strengths and limitations. The loss of 2′-deoxyribose from the modified nucleoside upon collision-induced dissociation is the main transition feature utilized in the screening of DNA adducts. Several advanced data-dependent and data-independent scanning techniques originated from proteomics and metabolomics have been tailored for DNA adductomics. The field of DNA adductomics is an emerging technology in human exposure assessment. As the analytical technology matures and bioinformatics tools become available for analysis of the MS data, DNA adductomics can advance our understanding about the role of chemical exposures in DNA damage and disease risk.
... (pp(6- (Ichihashi et al., 2003). iii) Les produits chimiques comme les drogues utilisées dans le traitement de cancers (les radiomimétiques comme la bléomycine ou le néocarzinostatine), les agents alkylants (diméthyle sulfate), les agents pontants (cisplatine, iv) Les polluants, tels que le Benzopyrène (fumée de diesel, incinérateurs) (Baird et al., 2005), les nitrosamines (fumée de cigarette) (Foiles et al., 1991), peuvent aussi causer des modifications chimiques des bases. v) Des agents génotoxiques divers: exposition prolongée au soleil, tabagisme, travailleurs de fours à charbon, travailleurs de fonderie. ...
La presbyacousie est une perte de l'audition liée au vieillissement qui représente la troisième maladie chronique la plus répandue chez les personnes âgées. À ce jour, il est admis que le stress oxydant peut causer des dommages irréversibles à l'ADN et entraîner une sénescence prématurée dans les cellules en cycle. Cependant, il n'existe aucune donnée concernant le rôle des dommages de l'ADN dans la dégénérescence des cellules cochléaires liée à l’âge. En plus le lien entre le stress oxydant, les dommages à l'ADN et le vieillissement des cellules cochléaires demeure obscure.Le premier objet de ce travail était d'élucider le rôle des dommages de l'ADN dans la dégénérescence des cellules cochléaires. Pour ce faire, nous avons utilisé des approches de biologie moléculaire et cellulaire pour identifier des voies de signalisation associées aux lésions de l'ADN dans des explants cochléaires issus de souris âgées de 3 jours traitées au cisplatine (CDDP), molécule connue pour son effet secondaire ototoxique. De plus, nous avons étudié l'implication de p53, un des effecteurs clés de la signalisation des dommages de l'ADN, in vivo en traitant avec le CDDP des souris dont le gène codant pour ce facteur de transcription a été invalidé. Les possibilités de protéger l’audition contre l’effet nocif du CDDP ont été également étudiées en utilisant des inhibiteurs spécifiques ciblant les étapes clés des voies de signalisation. Enfin, nous avons utilisé des modèles murins porteurs de xénogreffes de cancer du sein humain afin de vérifier si les co-traitements permettaient de préserver l’audition sans compromettre l’efficacité anti-tumorale du CDDP. Nos résultats montrent que le CDDP induit des cassures doubles brins de l'ADN dans les cellules ciliées qui sont à l'origine de l'activation de la voie ATM-Chk2-p53 et, in fine, de la mort de ces cellules par apoptose. Nous avons également montré que l'absence de p53 in vivo prévient la perte de l'audition et la dégénérescence des cellules ciliées externes après injection intrapéritonéale de CDDP. L’application systémique ou locale de PFT-α, un inhibiteur de p53 prévient efficacement la perte auditive sans compromettre l’efficacité anti tumorale du CDDP.Le deuxième objectif de ma thèse a reposé sur l’identification des liens existant entre la surproduction des espèces réactives de l'oxygène (ROS), les dommages de l’ADN et l’apparition précoce des marqueurs de la sénescence cellulaire et de la perte de l’audition liée à l’âge. Pour ce faire, j’ai utilisé des explants cochléaires de souris âgées de 3 jours traitées au peroxyde hydrogène (H2O2). L’apparition des dommages à l’ADN et des cellules en sénescence a été étudiée en utilisant des techniques d’immunomarquage et de Western blot. Les résultats ainsi obtenus in vitro ont été ensuite validés in vivo sur les cochlées provenant de souris SAMP8 et de souris SAMR1. Afin de confirmer le rôle du stress oxydant dans l’apparition précoce de la presbyacousie, nous avons utilisé une lignée de souris invalidées pour le gène P66Shc, qui est un gène d'adaptation au stress connu pour ses rôles dans la surproduction de ROS et dans l’inhibition des enzymes antioxydants. Enfin, nous avons évalué la possibilité de prévenir ou de ralentir la perte de l’audition liée à l’âge en traitant les souris SAMP8 avec un mimétique de SOD/catalase, le EUK207. Nos résultats ont montré que: i) la surproduction de ROS est l'un des principaux facteurs causaux de la dégénérescence des cellules sensorielles de la cochlée liée à l’âge ; ii) L’activation de la voie p53-p21 entraînant l’apparition précoce de la sénescence dans les cellules cochléaires post-mitotiques, peut expliquer le vieillissement prématuré de la cochlée; iii) Le EUK207, un piégeur du superoxyde et du peroxyde d'hydrogène peut atténuer la ARHL chez les souris SAMP8. L’ensemble de ces résultats mettent en évidence des stratégies novatrices et efficaces pour la protection de l'audition.
... Examples include but are not limited to the detection by electron-impact ionization (EI)-MS of various free radicalinduced DNA damages of the sugar moiety, bases, and DNA-protein crosslinks.176 The acid or base hydrolysis of DNA has been employed to recover PhIP, 4-ABP, o-toluidine, or 4-hydroxy-1-(3-pyridyl)-1butanone (HPB) from DNA adducts, followed by electron capture MS, which is also known as GC-negative ion chemical ionization (NICI)-MS.[183][184][185][186][187] Since GC/MS analysis is only compatible with volatile molecules, the products of carcinogens released from DNA, or the modified nucleobases or nucleosides require derivatization before characterization by GC/MS. ...
Hazardous chemicals in the environment and diet or their electrophilic metabolites can
form adducts with genomic DNA, which can lead to mutations and the initiation of
cancer. In addition, reactive intermediates can be generated in the body through
oxidative stress and damage the genome. The identification and measurement of DNA
adducts are required for understanding exposure and the causal role of a genotoxic
chemical in cancer risk. Over the past three decades, 32P-postlabeling, immunoassays,
gas chromatography/mass spectrometry, and liquid chromatography/mass spectrometry
(LC/MS) methods have been established to assess exposures to chemicals through
measurements of DNA adducts. It is now possible to measure some DNA adducts in
human biopsy samples, by LC/MS, with as little as several milligrams of tissue. In this
review article, we highlight the formation and biological effects of DNA adducts, and
highlight our advances in human biomonitoring by mass spectrometric analysis of
formalin-fixed paraffin-embedded tissues, untapped biospecimens for carcinogen
DNA adduct biomarker research.
... The presence of HPB-releasing DNA adducts in various human samples has been reported in prior publications (15)(16)(17)(18)(19), yet overall, the research in this field has been somewhat limited. In our efforts to provide a robust analytic methodology for the analysis of HPB-releasing adducts in studies of tobacco-induced cancer risk, we recently developed a highly sensitive liquid chromatography nanoelectrosprayhigh-resolution tandem mass spectrometry method for the analysis of these adducts using small amounts of DNA extracted from human oral cells (20). ...
Exposure to tobacco-specific nitrosamines (TSNA) and polycyclic aromatic hydrocarbons (PAH) is recognized to play an important role in the development of oral/head and neck squamous cell cancer (HNSCC). We recently reported higher levels of TSNA-associated DNA adducts in the oral cells of smokers with HNSCC as compared with cancer-free smokers. In this study, we further investigated the tobacco constituent exposures in the same smokers to better understand the potential causes for the elevated oral DNA damage in smokers with HNSCC. Subjects included cigarette smokers with HNSCC (cases, n = 30) and cancer-free smokers (controls, n = 35). At recruitment, tobacco/alcohol use questionnaires were completed, and urine and oral cell samples were obtained. Analysis of urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N'-Nitrosonornicotine (NNN; TSNA biomarkers), 1-hydroxypyrene (1-HOP, a PAH), cotinine, 3′-hydroxycotinine, and the nicotine metabolite ratio (NMR) were performed. Cases and controls differed in mean age, male preponderance, and frequency of alcohol consumption (but not total alcoholic drinks). Univariate analysis revealed similar levels of NNN, 1-HOP, and cotinine between groups but, as reported previously, significantly higher DNA adduct formation in the cases. Multiple regression adjusting for potential confounders showed persistent significant difference in DNA adduct levels between cases and controls [ratio of geometric means, 20.0; 95% CI, 2.7–148.6). Our cohort of smokers with HNSCC demonstrates higher levels of TSNA-derived oral DNA damage in the setting of similar exposure to nicotine and tobacco carcinogens. Among smokers, DNA adduct formation may act as a predictor of eventual development of HNSCC that is independent of carcinogen exposure indicators. Cancer Prev Res; 10(9); 507–13. ©2017 AACR.
See related editorial by Johnson and Bauman, p. 489
... [122][123][124] α-Hydroxylation is the major metabolic pathway in humans 125 Evidence for both methylation and pyridyloxobutylation of DNA in smokers is available. [126][127][128][129][130] It is likely that these adducts contribute to the overall tumorigenic properties of tobacco smoke in humans. Which NNK adducts contribute to the carcinogenic properties of NNK in humans is not known. ...
The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals. It is classified as a Group 1 human carcinogen by the International Agency for Cancer Research. NNK is bioactivated upon cytochrome P450 catalyzed hydroxylation of the carbon atoms adjacent to the nitrosamino group to both methylating and pyridyloxobutylating agents. Both pathways generate a spectrum of DNA damage that contributes to the overall mutagenic and toxic properties of this compound. NNK is also reduced to form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which is also carcinogenic. Like NNK, NNAL requires metabolic activation to DNA alkylating agents. Methyl hydroxylation of NNAL generates pyridylhydroxybutyl DNA adducts and methylene hydroxylation leads to DNA methyl adducts. The consequence of this complex metabolism is that NNK generates a vast spectrum of DNA damage, any of which can contribute to the overall carcinogenic properties of this potent pulmonary carcinogen. This article reviews the chemistry and the genotoxic properties of the collection of DNA adducts formed from NNK. In addition, it provides evidence that multiple adducts contribute to the overall carcinogenic properties of this chemical. Which adduct contributes to the genotoxic effects of NNK depends on the context, such as the relative amounts of each DNA alkylating pathway occurring in the model system, the levels and genetic variants of key repair enzymes and the gene targeted for mutation.
... Hydrochloric acid was added to each sample to bring the final concentration of the acid to 0.8 N, and the samples were incubated at 80°C for 3 h to release HPB. 14,16,20 The hydrolysates were adjusted to neutral pH with NaOH and loaded on HyperSep Hypercarb cartridges (25 mg, Thermo Scientific, Rockwood, TN) activated with 2 mL of MeOH and 2 mL of H 2 O. The cartridges were then washed with 2 mL of H 2 O. HPB was eluted with 1 mL of 65% MeOH, and guanine was eluted with 1 mL of MeOH (containing 1% TFA) sequentially. ...
Metabolic activation of the carcinogenic tobacco-specific N-nitrosamines leads to the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts. We recently developed a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method for the analysis of HPB-releasing DNA adducts in human oral cells. However, given the limited amounts of DNA that can be extracted from oral cells, higher sensitivity and selectivity are required for the reliable analysis of these adducts in future studies. We have developed a new sensitive LC-nanoelectrospray ionization-high resolution MS/MS method for the analysis of HPB-releasing DNA adducts in oral cells. A new procedure was also developed for guanine analysis by LC-MS/MS. The detection limit of the developed assay is 5 amol, and the limit of quantitation is 0.35 fmol HPB on-column, starting with 50 pg DNA. The method was tested by analyzing oral samples from 65 smokers, including 30 head and neck squamous cell carcinoma (HNSCC) patients and 35 cancer-free controls. In all smokers, the levels of HPB-releasing DNA adducts averaged 6.22±16.18 pmol/mg DNA, with significant inter-individual variation being consistent with previous reports. The median HPB-releasing DNA adduct level was 6.6 time greater for those with HNSCC than for smokers without HNSCC (p=0.002). The developed highly sensitive and selective method is valuable tool for future measurement of HPB-releasing DNA adducts in tobacco users, which can potentially provide critical insights for the identification of individuals at risk for cancer.
... Many different analytical methodologies have been developed for the determination of 7-mG (N7-guanine adducts), including chromatography [9], high pressure liquid chromatography (HPLC) [10], 32 P-postlabeling methods [11], gas chromatography [12] and liquid chromatography tandem mass spectrometry [13] as representative examples of such methods. Unfortunately, and despite their high sensitivity, they are time-consuming and expensive, which hinders their implementation for routine analyses. ...
The electrochemical determination of 7-methylguanine (7-mG) may result of interest because its presence can serve as probe of cytosine methylation of which is known as the most relevant epigenetic modification of DNA. This work explores the electrochemical response of 7-mG on different gold surfaces, both poly and single crystalline surfaces (Au (110), Au (111) and Au (100)). The results show that the adsorption-desorption process of the 7-mG is sensitive to the surface structure of the gold electrodes. Particularly, 7-mG adsorption-desorption profile on a Au (111) electrode exhibits some specific contributions which are found sensitive to the 7-mG concentration and, thereby could allow its quantification. These results may shed light on the future development of an electrochemical sensor for the diagnosis of the methylation degree in DNA.
... Tissue-specific carcinogenicity is observed in rodent tissues for NNN and NNK (5,(8)(9)(10)(11), with NNK much more potent than NNN in inducing lung adenocarcinomas in rodents (12). NNK is metabolized into intermediates which bind to human lung DNA (13), transform human epithelium in vitro (14), and induce nontumorigenic human bronchial epithelial cells to become neoplastically transformed in nude mice after subcutaneous transplantation (15). The lifetime dose of 1.6 mg NNK/kg body wt estimated for the average 2 pack-a-day U.S. smoker (4) is similar to the cumulative dose of 1.8 mg NNK/kg body wt which induced lung tumors in rats (8). ...
Background:
The most abundant and potent carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In vivo, NNK is rapidly metabolized to both the (R)- and (S)-enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which possesses similar carcinogenic properties as NNK. The major detoxification pathway for both NNAL enantiomers is glucuronidation by UDP-glucuronosyltransferase (UGT) enzymes including UGT2B10 and UGT2B17. The goal of the present study was to directly examine the role of UGT genotypes on urinary levels of NNAL glucuronides in smokers.
Methods:
NNAL-N-Gluc, (R)-NNAL-O-Gluc, (S)-NNAL-O-Gluc, and free NNAL were simultaneously and directly quantified in the urine of smokers by LC/MS analysis. Genotypes were determined by TaqMan assay using genomic DNA.
Results:
The functional knockout polymorphism in the UGT2B10 gene at codon 67 (Asp>Tyr) was significantly (P < 0.0001) associated with a 93% decrease in creatinine-adjusted NNAL-N-Gluc. The polymorphic whole-gene deletion of the UGT2B17 gene was associated with significant (P = 0.0048) decreases in the levels of creatinine-adjusted (R)-NNAL-O-Gluc, with a 32% decrease in the levels of urinary (R)-NNAL-O-Gluc/(S)-NNAL-O-Gluc among subjects with the UGT2B17 (*2/*2) genotype as compared to subjects with the UGT2B17 (*1/*1) genotype.
Conclusions:
These results suggest that functional polymorphisms in UGT2B10 and UGT2B17 are associated with a reduced detoxification capacity against NNAL and may therefore affect individual cancer risk upon exposure to tobacco.
Impact:
This is the first report to clearly demonstrate strong genotype-phenotype associations between both the UGT2B10 codon 67 Asp<Tyr genotype and urinary NNAL-N-Gluc levels and between the UGT2B17 copy number variant and urinary (R)-NNAL-O-Gluc levels in smokers. Cancer Epidemiol Biomarkers Prev; 25(7); 1175-84. ©2016 AACR.
... Consequently, the development of analytical procedures for its detection and quantification is a matter of outstanding significance. In this sense, and despite some methods are already available [7][8][9][10][11], most of them are time and DNA material consuming. Therefore, novel, fast, sensitive, simple, and economical methods for DNA methylation assays are still sought. ...
In this communication we report a voltammetric study of the adsorption–desorption of cytosine (C) and methylcytosine (mC) on well-defined gold (Au) electrodes. The voltammetric measurements clearly indicate that these processes are extremely sensitive to the Au surface structure and in particular to the presence of (111) surface domains. Interestingly, on Au(111) surfaces, a linear correlation between the C and mC concentrations (logarithm scale) and the peak potential of the main voltammetric feature is found. In addition, in the simultaneous presence of both molecules, mC governs the electrochemical response, which has allowed its accurate quantification in C-mC mixtures. In situ FTIR spectroscopic measurements have been carried out to deepen on this mC electrochemical sensitivity. This research may contribute to the future development of an electrochemical sensor for the determination of the degree of methylation in DNA.
... The procedure was performed essentially as previously described (21). Briefly, lung DNA was dissolved in H 2 O and the DNA concentration was determined by UV. ...
... Consequently it is important to improve the analytical methodologies for the 7mG determination, since it could be used as a cancer biomarker. Different technologies have been used for analysis of N7guanine adducts, such as, chromatography [13] , high Full Paper ELECTROANALYSIS pressure liquid chromatography (HPLC) [14] , 32Ppostlabeling methods [15] , gas chromatography [16] or liquid chromatography tandem mass spectrometry [17] . However, although these methods are sensitive enough, they are time-consuming and their costs made them economically unfeasible for routine implementation. ...
Despite 7-methylguanine (7mG) has a low biological relevance as a biomarker in DNA by itself, its increment may be correlated with the presence of methylating cytosine agents. In this work, screen-printed graphite electrodes (SPGE) were used to study the electrochemical characterization of 7mG by cyclic (CV) and square wave (SWV) voltammetry. The effect of the concentration of 7mG in solution and pH were systematically examined, as well as the repeatability of the measurements. The electrochemical response of 7mG in the presence of guanine and adenine was also investigated for the possibility of simultaneous detection of all three nucleic bases. The results aim to the future development of a portable and disposable electrochemical sensor based on SPGEs for the diagnosis of the methylation degree in DNA.
... GC-MS in the NICI mode was also employed for the measurement of 4-hydroxy-1-(3-pyridyl)-1-butanone (4-HPB)releasing DNA adducts formed from metabolites of the tobacco-specific nitrosamines 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). 8,87,203 Samples of human urinary bladder and lung DNA, hydrolyzed and subjected to GC-NICI-MS analysis, were shown to contain 4-ABP-DNA adducts at levels that are in keeping with 32 P-postlabeling analysis of the same samples using appropriate standards. 204,205 Additionally, the levels of MDA-induced M 1 dG were determined to be as low as 52 and 6.2 adducts per 10 8 nucleosides in rat liver 83 and human leukocyte DNA, 192 respectively, by the GC/EC-NICI-MS method. ...
Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may compromise cellular functions by blocking DNA replication and/or inducing mutations. Unambiguous identification of the structures and accurate measurements of the levels of DNA adducts in cellular and tissue DNA constitute the first and important step towards understanding the biological consequences of these adducts. The advances in mass spectrometry (MS) instrumentation in the past 2-3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of the biological consequences of DNA adducts. In this review, we summarized the development of MS techniques on these fronts for DNA adduct analysis. We placed our emphasis of discussion on sample preparation, the combination of MS with gas chromatography- or liquid chromatography (LC)-based separation techniques for the quantitative measurement of DNA adducts, and the use of LC-MS along with molecular biology tools for understanding the human health consequences of DNA adducts. The applications of mass spectrometry-based DNA adduct analysis for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies were also discussed.
... Acid hydrolysis of DNA. The procedure was performed essentially as described previously (20,31). Extractions. ...
A mixture of dietary benzyl isothiocyanate (BITC) and 2-phenethyl isothiocyanate (PEITC) inhibits lung tumori-genesis by a mixture of benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Previous studies indicated that inhibition of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) releasing DNA adducts of NNK by PEITC in the lung was responsible for inhibition of tumorigenicity. We have now extended these investigations to F-344 rats treated with 2 p.p.m. B[a]P in the diet and 2 p.p.m. NNK in the drinking water. The effects of BITC (1 µmol/g diet), PEITC (3 µmol/g diet), and a mixture of BITC plus PEITC (1 and 3 µmol/g diet) on DNA and hemoglobin (Hb) adducts of B[a]P and NNK, and on two urinary metabolites of NNK, were examined. DNA adducts were quantified after 8 and 16 weeks of treatment. Hb adducts were quantified in blood samples withdrawn every 2 weeks. 4-(Methylnitrosamino)-1-(3-pyri-dyl)-1-butanol (NNAL) and its glucuronide NNAL-Gluc were measured in urine every 4 weeks. PEITC or BITC plus PEITC significantly reduced levels of HPB releasing DNA adducts of NNK in lung at 8 and 16 weeks, but there was no effect of BITC. There were no effects of any of the treatments on levels of HPB releasing DNA adducts of NNK in liver, or on DNA adducts of B[a]P in either lung or liver. PEITC or BITC plus PEITC significantly inhibited the formation of Hb adducts of NNK from 2–12 weeks of treatment while there were no effects on Hb adducts of B[a]P. There was a significant increase in levels of NNAL and NNAL-Gluc in the urine of the rats treated with PEITC or BITC plus PEITC. These results demonstrate that dietary PEITC, or a mixture of BITC plus PEITC, inhibit the formation of HPB releasing adducts of NNK in the rodent lung, leading to inhibition of tumorigenesis.
... Diese Addukte wurden mit den in Tierversuchen durch das tabakspezifische Nitrosamin NNN erzeugten Ösophagustumoren in Verbindung gebracht[96]. Das Humanbiomonitoring hat allerdings für die HPB-Addukte weder am Hämoglobin noch an der DNA der Lunge die erwartete Spezifität für das Rauchen gezeigt [83,[103][104][105]107,108,158]. Bei Autopsien gewonnene Schleimhautproben des unteren Ösophagus und der Cardia waren die erstaunlich hohen Konzentrationen HPB abspaltender DNA-Addukte gänzlich unabhängig von der akuten Rauchbelastung[112]. Dies wurde in der vorliegenden Studie bestätigt, denn auch in den noch höher belasteten Biopsien der Ösophagusschleimhaut gab es keine Abhängigkeit der HPB-Addukte vom Rauchstatus (Tabellen 3-16 und[3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. ...
... HPB-releasing DNA adducts have been detected in the lungs of smokers and lung cancer patients and have been discussed as possible biomarkers of NNK and NNN metabolic activation (56,57). Our results do not support the use of pulmonary HPB-releasing DNA adducts as biomarkers because they vary with time, a consequence of their derivation from POB-DNA adducts. ...
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized to enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), found in the urine of virtually all people exposed to tobacco products. We assessed the carcinogenicity in male F-344 rats of (R)-NNAL (5 ppm in drinking water), (S)-NNAL (5 ppm), NNK (5 ppm), and racemic NNAL (10 ppm) and analyzed DNA adduct formation in lung and pancreas of these rats after 10, 30, 50, and 70 weeks of treatment. All test compounds induced a high incidence of lung tumors, both adenomas and carcinomas. NNK and racemic NNAL were most potent; (R)-NNAL and (S)-NNAL had equivalent activity. Metastasis was observed from primary pulmonary carcinomas to the pancreas, particularly in the racemic NNAL group. DNA adducts analyzed were O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd), 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua), O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]deoxyguanosine (O(6)-POB-dGuo), the 4-(3-pyridyl)-4-hydroxybut-1-yl (PHB) adducts O(2)-PHB-dThd and 7-PHB-Gua, O(6)-methylguanine (O(6)-Me-Gua) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts. Adduct levels significantly decreased with time in the lungs of rats treated with NNK. Pulmonary POB-DNA adducts and O(6)-Me-Gua were similar in rats treated with NNK and (S)-NNAL; both were significantly greater than in the (R)-NNAL rats. In contrast, pulmonary PHB-DNA adduct levels were greatest in the rats treated with (R)-NNAL. Total pulmonary DNA adduct levels were similar in (S)-NNAL and (R)-NNAL rats. Similar trends were observed for DNA adducts in the pancreas, but adduct levels were significantly lower than in the lung. The results of this study clearly demonstrate the potent pulmonary carcinogenicity of both enantiomers of NNAL in rats and provide important new information regarding DNA damage by these compounds in lung and pancreas.
... Methyl and POB DNA adducts have been detected in the lungs of human smokers. DNA isolated from peripheral lung and tracheobronchial tissue from smokers had significantly higher levels of POB adducts compared to non-smokers (Foiles et al., 1991;Holzle et al., 2007). The 7-mG adduct was found at higher levels in smokers compared to non-smokers (Mustonen et al., 1993). ...
Smokeless tobacco (SLT) has been reported to have deleterious effects on the health of its users. This study aims to analyze the constituents of locally collected SLT sample extracts (S1–S11) from Tabuk region of Saudi Arabia using GC-MS and investigate their cytotoxic effect on human gingival fibroblasts (hGFs), normal human fibroblasts (MRC5), and two cancer cell lines (HT29 and HepG2) using MTT assay. GC-MS results showed that pyridine, 3-(1-methyl-1H-pyrrol-2-yl)-, tetracyclo[4.4.1.1(7,10).0(2,5)]dodec-3-en-11-ol, and cotinine were found in S1, while ethyl iso-allocholate was traced in S2. Compounds 9,12-octadecadienoic acid, ethyl ester, 7-methyl-Z-tetradecen-1-ol acetate, cis-10-heptadecenoic acid and octadecanoic acid, ethyl ester, and nicotine traces were found in S4, while compound 3,7,11,15-tetramethyl-2-hexadecen-1-ol, tetradecamethyl-hexasiloxane, and phytol in S5. Additionally, octadecamethyl cyclononasiloxane, oleic acid, and trimethylsilyl ester were found in S6 and S9, respectively. Interestingly, extracts S4, S10, and S6 were the most cytotoxic to the normal fibroblasts (hGF and MRC5, with low selectivity index: <1), compared with doxorubicin and with their effect on the cancerous cells (HT29 and HepG2). Various components detected in SLT samples were carcinogenic, including nicotine and its derivatives, hexadecanoic acid, 1,2-benzenedicarboxylic acid, and octadecanoic acid. The present study showed that the cytotoxic and possibly carcinogenic effects of the SLT samples on gingiva and lung cells are attributed to many compounds and not only nicotine derivatives, all of which could create health threats for SLT users and lead to various types of cancers, including oral, lung, colon, and liver cancers.
s
Cancer is a leading factor of mortality globally. Cytochrome P450 (CYP) enzymes play a pivotal role in the biotransformation of both endogenous and exogenous compounds. Evidence from numerous epidemiological, animal, and clinical studies points to instrumental role of CYPs in cancer initiation, metastasis, and prevention. Substantial research has found that CYPs are involved in activating different carcinogenic chemicals in the environment, such as polycyclic aromatic hydrocarbons and tobacco-related nitrosamines. Electrophilic intermediates produced from these chemicals can covalently bind to DNA, inducing mutation and cellular transformation that collectively result in cancer development. While bioactivation of procarcinogens and promutagens by CYPs has long been established, the role of CYP-derived endobiotics in carcinogenesis has emerged in recent years. Eicosanoids derived from arachidonic acid via CYP oxidative pathways have been implicated in tumorigenesis, cancer progression and metastasis. The purpose of this review is to update on the current state of knowledge about the cancer molecular mechanism involving CYPs with focus on the biochemical and biotransformation mechanisms in the various CYP-mediated carcinogenesis, and the role of CYP-derived reactive metabolites, from both external and endogenous sources, on cancer growth and tumour formation.
Smokeless tobacco (ST) products are used worldwide, and consumption is increasing in the United States. Although ST products are considered to occupy a different position on the tobacco product continuum of risk compared to combusted tobacco products, they can still lead to health problems, including cancer, dental problems, and changes in heart rate and blood pressure. Therefore, the determination of harmful and potentially harmful constituents (HPHCs) released from ST products into human saliva is important. Four certified reference ST products were tested in this study: loose leaf chewing tobacco (3S1), Swedish-style snus (1S4), snus (1S5), and moist snuff (3S3). These certified reference ST products are manufactured for research purposes, not for human consumption. The reference ST products were used in this study because they have been well characterized and are intended and designed to represent commercial ST products. The reference ST products were incubated in human saliva at 37 °C with a range of incubation times for the evaluation of constituents released from these products into human saliva. In this study, alkaloids (nicotine and cotinine), tobacco-specific N-nitrosamines (TSNAs) (NNN and NNK), and benzo[α]pyrene (B[α]P) in the reference ST products and saliva samples were determined by GC-MS, GC-FID, or UPLC-MS-MS. Our results indicate that the amounts of each constituent released from the reference ST products were altered by the tobacco cut size and product format (pouched or unpouched). The constituents (TSNAs and alkaloids) in moist snuff and loose leaf chewing tobacco were released faster compared to those in Swedish-style snus and snus. B[α]P was only detected in reference moist snuff samples, and only 3.4% of the total B[α]P was released into human saliva after incubation for 60 min, whereas higher percentages of total TSNAs and alkaloids were released at different rates from the four reference ST products.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific nitrosamine (TSNA) that induces malignant tumors in rodent animals. High-risk human papillomavirus (hr-HPV) infection is an important cause of several human cancers. Epidemiological evidence has shown that HPV cooperatively induces carcinogenesis with tobacco smoke. In the present study, the synergistic carcinogenesis of NNK and HPV18 was investigated. Immortalized human esophageal epithelial SHEE cells containing the HPV18 E6E7 gene was constructed by lentiviral transfection. SHEE-E6E7 cells were exposed to NNK along with SHEE-V cells without HPV18 E6E7 as a negative control. The cooperation of NNK and HPV was examined by wound healing, transwell and colony-forming assays. The results showed that NNK exposure promoted the migration, invasion and proliferation abilities of both SHEE-E6E7 and SHEE-V cells, however the changes in these phenotypic features were remarkably stronger in SHEE-E6E7 cells than those in SHEE-V cells. Our findings indicate that NNK promotes malignant transformation of human esophageal epithelial cells and suggest a synergistic carcinogenesis with the HPV18 E6E7 oncogene. As reported previously, the formation of pyridyloxybutylated DNA adducts is a crucial step in NNK-mediated carcinogenesis. In order to clarify the influence of HPV on the formation of NNK-induced DNA adducts, the amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts were determined using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). We observed that the levels of HPB-releasing adducts in SHEE-E6E7 cells were significantly higher (p<0.01) than those of SHEE-V cells, which was in line with results of the phenotypic assays. In conclusion, this study provides direct evidence that NNK and HPV18 exhibit a synergistic effect on formation of DNA adducts and results in malignant transformation of esophageal epithelial cells. Such knowledge on interaction between infection and smoking habit in the development of cancers inform cancer prevention strategies. Further studies to delineate the molecular mechanism and to identify specific intervention targets are worthwhile.
The development of electroanalytical methods for the detection and quantification of nucleotides in DNA offers vital implications in assessing the degree of oxidation or epigenetic modification in DNA. Unfortunately, the electrochemical response of oligonucleotides is strongly influenced by the size, composition and nucleic base sequence. In this article, an optimized analytical procedure for the enzymatically breakdown of the oligonucleotides to their corresponding nucleotides for the evaluation of the electrochemical response through the use of square wave voltammetry (SWV) is presented. Enzymatic digestion of oligonucleotides has been optimized in terms of buffer composition, digestion time, strategy for stopping the enzymatic reaction and filtration requirement for enzyme removal, and then compared to an established protocol. Under the optimized protocol SWV response of a number of untreated and enzymatically digested six-mer oligonucleotides, namely 5′-GGGGGG-3′, 5′-AAAAAA-3′, 5′-CGCGCG-3′ and 5′-AAACGC-3′ have been analysed, providing a higher sensitivity for the determination of guanosine and adenosine monophosphate species under digestion conditions with a more facile and cost effective procedure. The novel strategy for the enzymatically treated oligonucleotides in combination with the SWV response provides a proof of principle for feasible applications in the diagnosis of methylated guanosine in DNA as a potential biomarker due to its relation with cancer.
DNA, deoxyribonucleic acid, is crucial to life. With its triplet coding, DNA serves as the template for messenger RNA, and is therefore responsible for the myriad proteins that ensure the ongoing health and life of the current cell or organism. Future generations are similarly dependent on the DNA code, which cellular mechanisms help propagate. DNA strands must remain intact and undamaged to guarantee that the code functions with high fidelity. If DNA becomes covalently damaged, repair mechanisms can excise the affected region and replace it with the correct sequence. Indications of this biochemical transformation can be found among the excretion products of the cell or organism. However, the repair processes do not always work perfectly, and certain modifications escape the repair system. These covalent modifications may be of critical importance to the causes of disease states, including carcino-genesis. In addition, certain medications, known to generate covalent modifications of DNA, are used therapeutically in an effort to combat an observed disease state. These deliberately produced DNA modifications also need to be studied. In this review, the term DNA modification includes not only DNA adducts (xenobiotic compounds covalently bound to DNA) but also other aberrant forms of DNA, such as ring-opened products and dimers.
Cigarette smoking is the major cause of lung cancer in the United States and worldwide. Tobacco products are estimated to cause approximately 90% of lung cancer cases. Experimental studies in laboratory animals have collectively demonstrated the carcinogenicity of cigarette smoke and cigarette smoke condensates (CSCs). There are over 5000 identified chemicals and more than 60 known carcinogens in cigarette smoke, with polycyclic aromatic hydrocarbons (PAHs) and the tobacco-specific nitrosamine NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) likely being the most important respiratory carcinogens. The carcinogenicity of cigarette smoke can also be enhanced by the presence of tumor promoters and cocarcinogens. The reaction of carcinogens with DNA can cause mutations and, if unrepaired, can lead to the activation of oncogenes or the inactivation of tumor suppressors. Epigenetic changes may also occur from exposure to tobacco carcinogens, leading to a change in gene expression. While the majority of lung cancer results from cigarette smoking, only 15% of smokers get lung cancer. Studies indicate that this may be due to genetic factors that contribute to a person's susceptibility to tobacco-induced cancers. Biomarkers have been developed to monitor uptake and activation of tobacco carcinogens, and perhaps could be used to predict susceptibility to lung cancer. Chemoprevention has developed as an approach to prevent or delay the onset of tobacco-induced lung cancer. However, cessation of tobacco products is the best way to prevent lung cancer.
Cigarette smoking is the major cause of lung cancer in the United States and worldwide. Tobacco products are estimated to cause approximately 90% of lung cancer cases. Experimental studies in laboratory animals have collectively demonstrated the carcinogenicity of cigarette smoke and cigarette smoke condensates (CSCs). There are over 5000 identified chemicals and more than 60 known carcinogens in cigarette smoke, with polycyclic aromatic hydrocarbons (PAHs) and the tobacco-specific nitrosamine NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) likely being the most important respiratory carcinogens. The carcinogenicity of cigarette smoke can also be enhanced by the presence of tumor promoters and cocarcinogens. The reaction of carcinogens with DNA can cause mutations and, if unrepaired, can lead to the activation of oncogenes or the inactivation of tumor suppressors. Epigenetic changes may also occur from exposure to tobacco carcinogens, leading to a change in gene expression. While the majority of lung cancer results from cigarette smoking, only 15% of smokers get lung cancer. Studies indicate that this may be due to genetic factors that contribute to a person's susceptibility to tobacco-induced cancers. Biomarkers have been developed to monitor uptake and activation of tobacco carcinogens, and perhaps could be used to predict susceptibility to lung cancer. Chemoprevention has developed as an approach to prevent or delay the onset of tobacco-induced lung caner. However, cessation of tobacco products is the best way to prevent lung cancer.
Most known chemical carcinogens are electrophilically reactive compounds (RX) or are formed in vivo from non-reactive precursors (A) (Miller and Miller, 1966). The electrophiles react with nucleophilic atoms (Y) of biomacromolecules giving rise to adducts (RY): $$A \to RX\xrightarrow[{({k_y})}]{{ + {Y^ - }}}RY + {X^ - }$$ (1) The measurement of adducts to sufficiently stable macromolecules may be used for identification of electrophiles in vivo and for dose monitoring as a basis for risk estimation. For reasons to be discussed below, the Stockholm group has used hemoglobin (Hb) for in vivo dose monitoring of chemical carcinogens in animals and humans.
Deoxyribonacleic acid (DNA) adduct is always one of the frontier and spotlight fields in the studies of environmental science, medicine and ecological toxicology in the recent decade. Analytical techniques of DNA adduct acting as research means for DNA adduct, which combines traditional instrumental analysis with techniques of molecular biology, can imply not only novel advances on the instrumental analysis but also the trends of research methods in the molecular biology. Focusing on studies of DNA adduct in Europe and America, the paper reviews the main analytical techniques of DNA adduct. Novel advances on analytical techniques of DNA adduct are fully reported.
The indisputable relationship between the world's most widespread exogenous chemical carcinogen exposure - tobacco smoke - and cancer, provides a framework for a better understanding of cancer mechanisms in general, particularly the interaction between carcinogen exposure and host factors, and for the development of novel preventive and therapeutic strategies. Furthermore, the biomarkers that are developed in this research can be applied in the approaching era of tobacco product regulation. This chapter discusses the mechanisms of tumour induction by tobacco carcinogens and the status of tobacco carcinogen biomarkers.
Introduction
Syntheses and Uses of Compounds Containing C C , C O or C N Groups Labelled with Stable Isotopes
Synthesis and Uses of Compounds Containing C C , C O or CN Groups Labelled with Tritium
Syntheses and Uses of Compounds Containing C C , C O or CN Groups Labelled with Radioactive Carbon
Syntheses and Uses of Compounds Containing C C , C O or CN Groups Labelled with Radioactive Halogen
Synthesis and Uses of Compounds Containing C C , C O , or CN Groups Labelled with Radioactive Sulphur
Isotope Effect Studies
Acknowledgements
DNA repair is a very important cellular mechanism involved in various genetic diseases and in carcinogenesis. DNA repair systems exhibit complementarities and interactions recently highlighted. However, this complexity is not taken into account by functional assays available nowadays.
Therefore we have designed a miniaturized in vitro assay allowing specific, functional and parallelized measurement of DNA repair activities. This test is an adaptation of an excision resynthesis assay to the microarray format.
We first setup and optimized the microarray fabrication, the data analysis and normalization, and the repair reaction. We then validated the assay and demonstrated that DNA repair activities measured were specific. Finally we demonstrated the potential of the assay in two different domains. We first phenotyped different cell types: the results showed the similarity of human fibroblasts repair profiles, and also the different repair abilities existing between fibroblast, keratinocyte, and peripheral blood mononuclear cells. Moreover we studied adaptation of cells to gamma irradiation. We demonstrated that DNA repair activities are involved in the radio-adaptation of cells .
Potential applications of this assay could be important in fundamental research as in applied one such as molecular screening, and in diagnosis.
While nicotine is not a carcinogen, several tobacco-specific nitrosamines derived from nicotine and other tobacco alkaloids are carcinogenic in laboratory animals; a property characteristic of over 200 nitrosamines 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), 4-(methylnitrosamino)-l-(3-pyridyl)- 1-butanol (NNAL), and N'-nitrosonornicotine (NNN) are strong rodent carcinogens, while N'-Nitrosoanabasine (NAB), N'-nitrosoanatabine (NAT), iso-NNAL, and 4-(methylnitrosamino)- l-(3-pyridyl)butyric acid (iso-NNAC) have little or no activity. The carcinogenicity of NNK, NNAL, and NNN leads to the hypothesis that they may play an important role in human cancer. In support of this hypothesis, numerous analytical studies summarized the presence of tobacco-specific nitrosamines in cured, unburned tobacco, as well as in tobacco smoke. Virtually, all marketed tobacco products contain these compounds. Numerous studies in rodents and primates, both in vitro and in vivo, demonstrate that NNK, NNAL, and NNN are extensively metabolized and form electrophilic intermediates that form covalent adducts with DNA and hemoglobin. These studies provide the mechanistic foundation for understanding the carcinogenic activities of tobacco-specific nitrosamines. The results of the carcinogenicity studies of NNK, NNAL, and NNN further support the hypothesis that these nitrosamines may be important in tobacco induced cancer. The structural similarities of NNK, NNAL, and NNN to nicotine indicate that these nitrosamines, like nicotine, should be extensively metabolized in humans; this has been difficult to demonstrate so far, due in part to the identical structures of nicotine and tobacco-specific nitrosamine metabolites.
N-Nitrosodialkylamines (nitrosamines) are a large group of substances containing the NNO group and various substituents on the amine nitrogen. They can be synthesized by reaction of the corresponding amines with nitrosating agents such as acidic nitrite, N2O3, N2O4, or inorganic nitrosyl compounds. They can be analyzed by gas or liquid chromatography with detection generally based on measurement of nitric oxide that is released from the nitrosamines by pyrolysis, photolysis, or acid hydrolysis. Many of the nitrosamines are agents which damage the liver in animals and humans. Most nitrosamines are known animal carcinogens, and some are suspected human carcinogens. Nitrosamines are metabolized by several enzyme systems, the most important being the cytochrome P450 family. Metabolic conversion to active electrophiles that react with DNA appears to be the initiating event in nitrosamine carcinogenesis. Humans may be exposed to nitrosamines by several routes including diet, commercial or industrial preparations such as cutting fluids, cosmetics, or sunscreens, tobacco products, and synthesis inside the body. Nitrosamines have not been unequivocally shown to be human carcinogens, but evidence relating oral cancer to the use of smokeless tobacco is increasingly persuasive.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) is a potent tobacco-specific lung carcinogen believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK to DNA damaging reactive intermediates proceeds via α-hydroxylation pathways. The end products of these pathways are excreted in urine of smokers as 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 3) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid, 4). The sum of these biomarkers (after NaBH4 treatment), referred to as total hydroxy acid, could potentially be used to measure the extent of NNK metabolic activation in smokers. However, the same metabolites are formed from nicotine; therefore, there is a need to distinguish the NNK- and nicotine-derived keto and hydroxy acid in smokers' urine. We previously developed a unique methodology based on the use of [pyridine-D4]NNK ([D4]1), which metabolizes to the correspondingly labeled biomarkers. In this study, we developed a sensitive and reproducible assay for the detection and quantitation of total [pyridine-D4]hydroxy acid ([D4]4) in human urine. A two-step derivatization approach was used to convert [D4]4 to [pyridine-D4]methyl 4-hexanoyl-4-(3-pyridyl)butanoate ([D4]6), and an LC-ESI-MS/MS method was developed for the analysis of this derivative with excellent sensitivity, accuracy, and precision. The robustness and reproducibility of the assay was further confirmed by its application for the analysis of urine samples from 87 smokers who smoked [D4]1-containing cigarettes for one week. The measured level averaged 130 fmol/mL urine. The developed assay can be used in future studies that may require evaluation of the relative efficiency of NNK metabolic activation in humans.
A gas chromatography/mass spectrometric assay method was developed for the simultaneous determination of neutral and bacis twenty-five disruptors in frog and fish. Afther homogenization and sonication of 5 g of sample, purification was achieves in one step with a solid phase extraction procedure using silica gelflorisl. Eluton was performed with 50mL of acetone : n-hexane (1 : 9) solution. The eluate was concentrated to approximately 10uL and dissolves with 100 uL of hexane and analyzed by GC-MS (SIM). The peaks had good chromatographic properties and the extraction of these compounds from sample also gave relatively high recoveries with small variatoins. Detection limits were 0.1 ng/g for 4-nitrotoluene, benzophenone, hexachlorobenzene, atrazine, malathion, o,p-DDT, o,p-DDT and permethrin, and 0.2 ng/g for heptachlor epoxide, γ-chlordane, α-chlordane, p,p'-DDE, p,p'-DDD, cypermethrin and fenvalerate, and 0.3 ng/g for trifluralin, metribuzin, alachlor, dieldrin and p,p'-DDT, and 0.5 ng/g for heptachlor, aldrin and parathion, and 0.7 ng/g for endrin, and 0.8 ng/g for nitrofen. The recoveries were between 33 and 109%. The method was used to analyze twenty-five frogs and forty-six fishes fishes samples caught from various regions in Korea. Benzophenone was detected at concentration of up to 17.2 ng/g in frog or fish. Heptachlor, aldrin, γ-chlordane, p,p'-DDE, p,p'-DDD, endrin and o,p-DDD were detected at concentrations of 0.7-12.5 ng/g in frog or fish. Also significant leveles of dieldrin (up to 22.5 ng/g) were observed. The developed method may be valuable to be used to the national monitoring project of EDS in biota samples.
Using modern technology, minute quantities of low-molecular-weight-proteins (LMWP), prostanoids, growth factors, and intrarenal and extrarenal enzymes can be measured in urine. Excretory patterns that are characteristic for the site and mechanism of renal injury often can be found. It is possible to recognize urinary biomarker patterns that suggest the putative environmental nephrotoxin. This fingerprinting approach has become an effective tool in recent years as urine from cohorts with known occupational nephrotoxin exposures has been analyzed for patterns of specific constituents in European cooperative studies. The authors' studies performed on subjects with occupational and environmental exposures in New Jersey confirm the pattern specificity and threshold effects for chromium, mercury and lead. In addition, they have been able to show that increased N-acetylglucosaminidase excretion following lead exposure correlates with current (blood lead) but not with cumulative (bone lead) exposure. The success of recent cooperative efforts has been in part due to the absence of clinical renal failure in study subjects. Urinary biomarkers indicate early renal injury. As renal failure progresses, excretory patterns become nonspecific. Moreover, renal injury that results in tubular proteinuria may not progress to renal failure. Nevertheless, biomarkers of renal injury can help establish acceptable exposure levels and identify the need for long-term surveillance to ascertain when clinical renal disease may result.
Modification of DNA bases in a hydrogen peroxide solution by UV irradiation of thymine was investigated. Twelve products resulting from interaction of radicals with thymine were identified by using gas chromatography/mass spectrometry (GC/MS) after trimethylsilyl derivatization. Some products were identified by comparing their retention times and mass spectra with those of synthetic compounds that are formed by treatment of thymine with OsO4. Major products were quantified by the GC/MS-selected ion monitoring mode. We also examined the effect of length of UV irradiation and amount of hydrogen peroxide on the quantitation of a number of DNA base-damaged products. The results indicate that the radical environment provided by the presence of hydrogen peroxide has a substantial effect on the types of products as well as their quantities. Moreover, the presence of hydrogen peroxide dramatically induced high yields of oxidative products.
Modification of cytosine in hydrogen peroxide solution by UV-irradiation was investigated. Hydroxyl radicals generated from H2O2 under UV irradiation oxidized cytosine to the monohydroxy and dihydroxy derivatives at the C5–C6 double bond. Several deamination products of modified cytosine derivative were also observed. Moreover, further transformation/rearrangement of some modified bases led to the formation of five-membered ring derivatives such as 5-hydroxyhydantoin and 3-amino-4,5-dihydroxypyrazole. Various products resulting from radical interaction with cytosine were identified by using gas chromatography/mass spectrometry (GC/MS) after trimethylsilylation derivatization. Some products were identified by comparing their retention time and mass spectra with those of synthetic compounds that were formed by the treatment of cytosine with OsO4. Major products were quantified by GC/MS-selected ion monitoring mode. Some of products including uracil glycol were significantly decomposed during formic acid hydrolysis.
A previously unknown urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was identified as 4-(methylnitrosamino)-1-[3-(6-hydroxypyridyl)]-1-butanone (6-hydroxyNNK). The metabolite was initially isolated from rat urine. On the basis of its MS and NMR, it was either a 4- or 6-hydroxypyridyl derivative of NNK. Model compounds were synthesized to distinguish between these possibilities; the results indicated that the metabolite was 6-hydroxyNNK. This was confirmed by independent synthesis; the spectral and chromatographic properties of 6-hydroxyNNK were the same as those of the metabolite. F-344 rats and A/J mice treated with 100 mg/kg NNK excreted approximately 1% of urinary metabolites as 6-hydroxyNNK; it was not detected as a sulfate or glucuronide conjugate. This is the first example of a pyridyl-hydroxylated metabolite of a tobacco-specific nitrosamine. On the basis of comparison to published data on other pyridine derivatives, 6-hydroxyNNK may be formed by bacterial metabolism. The potential utility of 6-hydroxyNNK as a dosimeter of human uptake of NNK is discussed.
The devastating link between tobacco products and human cancers results from a powerful alliance of two factors - nicotine and carcinogens. Without either one of these, tobacco would be just another commodity, instead of being the single greatest cause of death due to preventable cancer. Nicotine is addictive and toxic, but it is not carcinogenic. This addiction, however, causes people to use tobacco products continually, and these products contain many carcinogens. What are the mechanisms by which this deadly combination leads to 30% of cancer-related deaths in developed countries, and how can carcinogen biomarkers help to reveal these mechanisms?
Tobacco smoke contains a variety of genotoxic carcinogens that form adducts with DNA and protein in the tissues of smokers. Not only are these biochemical events relevant to the carcinogenic process, but the detection of adducts provides a means of monitoring exposure to tobacco smoke. Characterization of smoking-related adducts has shed light on the mechanisms of smoking-related diseases and many different types of smoking-derived DNA and protein adducts have been identified. Such approaches also reveal the potential harm of environmental tobacco smoke (ETS) to nonsmokers, infants and children. Because the majority of tobacco-smoke carcinogens are not exclusive to this source of exposure, studies comparing smokers and nonsmokers may be confounded by other environmental sources. Nevertheless, certain DNA and protein adducts have been validated as biomarkers of exposure to tobacco smoke, with continuing applications in the study of ETS exposures, cancer prevention and tobacco product legislation. Our article is a review of the literature on smoking-related adducts in human tissues published since 2002.
In an attempt to probe for polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human subjects resulting from smoking (or other chronic environmental exposure), lung tissue and lung tumours were obtained from patients hospitalized for lung cancer. DNA was isolated from the tissue samples and examined both in an ELISA using a polyclonal antibody against (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE)-DNA as well as by the nuclease P1-mediated modification of the 32P-post-labelling technique. The ELISA results showed BPDE-DNA antigenicity in lung DNA from 6 out of 21 patients, and adduct levels ranged from 2 to 134 adducts per 10(8) nucleotides. For all 21 patients, the autoradiographs of chromatograms of 32P-postlabelled digests of DNA from non-tumorous lung tissue showed a strong diagonal radioactive zone (DRZ). This DRZ was generally absent in tumorous tissue. DNA samples that were positive in the ELISA contained a dominant spot within the DRZ that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG). The quantities of the BPDE-dG spots ranged from 2.1 to 42 adducts in 10(9) nucleotides. These values were lower than the levels found in the ELISA but correlated well with the ELISA results (Kendall W = 0.97; P = 0.00). The levels of the DRZ adducts ranged from 1.9 to 34 adducts in 10(8) nucleotides. Correlations between smoking and DNA adduct levels were poor because of the small number of current smokers (n = 13). However, smokers of filter cigarettes had significantly lower DNA adduct levels compared with smokers of cigarettes without a filter (P = 0.02 by Fischer's exact test).
Human peripheral lung tissue samples were obtained at autopsy from 17 individuals of known occupational and smoking histories. A spectrum of different carcinogen-DNA adducts was detected using a variety of sensitive techniques. High-pressure liquid chromatography-linked synchronous fluorescent spectrophotometry and an ultrasensitive enzyme radioimmunoassay detected adducts derived from benzo[a]pyrene diol epoxide and other apparent polycyclic aromatic hydrocarbons. An amplified enzyme-linked immunosorbent assay demonstrated the presence of 4-aminobiphenyl-DNA adducts in many of these samples. A number of these specimens also contained O6-alkyldeoxyguanosine as measured by 32P-postlabeling techniques. Thus this pilot study indicates not only that human lung contains a spectrum of carcinogen-DNA adducts, but also that a full scale molecular dosimetry study of human exposure to both aryl and alkyl chemical carcinogens is warranted.
Covalent DNA addition products (adducts) formed by the reaction of chemical carcinogens or their metabolites with DNA are
critically involved in the initiation of chemical carcinogenesis and may serve as molecular markers and dosimeters for environmental
carcinogen exposures. Using a highly sensitive 32P-postlabeling assay for DNA adduct analysis, we studied DNA damage elicited by cigarette smoke in tissues of smokers. A multitude
of characteristic smoking-induced, presumably aromatic DNA adducts were found to occur in a dose-and time-dependent manner
in the lung, bronchus, and larynx of smokers with cancer of these organs and to decline only slowly after cessation of smoking.
Low levels of adducts appeared to persist for up to 14 years in the lungs of exsmokers with high previous exposures. These
results corroborate data of epidemiological studies showing that the lung cancer risk and mortality of smokers increase with
the intensity and duration of smoking and decline only slowly after cessation of smoking. Tissue distribution studies in autopsy
samples revealed the presence of smoking-associated DNA lesions also in the kidney, bladder, esophagus, heart, ascending aorta,
and liver. The most extensive DNA damage was found in lung and heart, i.e., 1 aromatic adduct in about 107 DNA nucleotides. Our results suggest that cigarette smoking-induced DNA adduct formation is causally related to cancer in
the target organs. [J Natl Cancer Inst 81:341–347, 1989]
Tobacco-specific nitrosamines are a group of carcinogens that are present in tobacco and tobacco smoke. They are formed from nicotine and related tobacco alkaloids. Two of the nicotine-derived nitrosamines, NNK and NNN, are strong carcinogens in laboratory animals. They can induce tumors both locally and systemically. The induction of oral cavity tumors by a mixture of NNK and NNN, and the organospecificity of NNK for the lung are particularly noteworthy. The amounts of NNK and NNN in tobacco and tobacco smoke are high enough that their total estimated doses to long-term snuff-dippers or smokers are similar in magnitude to the total doses required to produce cancer in laboratory animals. These exposures thus represent an unacceptable risk to tobacco consumers, and possibly to non-smokers exposed for years to environmental tobacco smoke. The permission of such high levels of carcinogens in consumer products used by millions of people represents a major legislative failure. Indeed, the levels of tobacco-specific nitrosamines in tobacco are thousands of times higher than the amounts of other nitrosamines in consumer products that are regulated by government authorities. Although the role of tobacco-specific nitrosamines as causative factors in tobacco-related human cancers cannot be assessed with certainty because of the complexity of tobacco and tobacco smoke, several lines of evidence strongly indicate that they have a major role, especially in the causation of oral cancer in snuff-dippers. Epidemiologic studies have demonstrated that snuff-dipping causes oral cancer. NNK and NNN are quantitatively the most prevalent known carcinogens in snuff, and they induce oral tumors when applied to the rat oral cavity. A role for NNK in the induction of lung cancer by tobacco smoke is likely because of its organospecificity for the lung. Tobacco-specific nitrosamines may also be involved in the etiology of tobacco-related cancers of the esophagus, nasal cavity, and pancreas. Because they are derived from nicotine, and therefore should be associated only with tobacco, tobacco smoke and other nicotine-containing products, tobacco-specific nitrosamines as well as their metabolites and macromolecular adducts should be ideal markers for assessing human exposure to, and metabolic activation of, tobacco smoke carcinogens. Ongoing research has demonstrated the formation of globin and DNA adducts of NNK and NNN in experimental animals. Sensitive methods for the detection and quantitation of these adducts in humans would provide an approach to assessing individual risk for tobacco-related cancers.(ABSTRACT TRUNCATED AT 400 WORDS)
DNA was isolated from tissues of K344 rats 24 h after treatment by s.c. injection with [5-3H]4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK) or [5-3H]N′-nitrosonor-nicotine ([5-3H]NNN) It was hydrolyzed with acid or at pH 7,100°C, and the hydrolysates were analyzed by HPLC. The major product in each
case was Identified as 4-hydroxy-1-(3-pyridyl)-1-butanone, formed by hydrolysis of a DNA adduct. It was detected in DNA from
the livers of rats treated with [5-3H]NNK or [5-3H]NNN, and in DNA from lungs of rats treated with [5-3H]NNK. These results demonstrate that 4-(3-pyridyl)-4-oxobutylation of DNA occurs in rats treated with NNK or NNN, and are
consistent with the hypothesis that these nitrosamines are metabolically activated by α-hydroxylation.
Alkylation of DNA and hemoglobin was compared in male F344 rats given a single s.c. injection of the tobacco-specific nitrosamine
4-(methyInitrosamino)-1-(3-pyridyl)-1-butanone (NNK), or its major metabolite formed by carbonyl reduction, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL).
In hepatic DNA, levels of 7-methylguanine and O6-methyl-guanine formed from NNK 1-48 h after treatment were similar to those formed from NNAL. In nasal mucosa and lung DNA,
levels of 7-methylguanine and O6Amethylguanine were somewhat higher after treatment with NNK than with NNAL. Acid hydrolysis of hepatric DNA, isolated from
rats treated with either [5-3H]NNK or [5-3H]NNAL, gave 180 ± 48 or 120 ± 23 μuno/mol guanine, respectively, of 4-hydroxy-1-(3-pyridyl)-1-butanone. Basic hydrolysis
of globin isolated from rats treated with either [5-3H]NNK of 5-3H]NNAL gave 4.1 ± 0.7 or 2.0 ± 0.1 pmol/mg, respectively of 4-hydroxy-1-(3-pyridyl)-1-butanone. These results indicate that
NNAL is not a detoxification product of NNK, since treatment of rats with NNAL results in modifications of DNA which are qualitativerly
and quantitatively similar to those observed upon treatment with NNK. Alkylation of DNA and globin by NNAL may result mainly
from its metabolic reconversion to NNK.
A highly-sensitive immunoassay utilizing antiserum specific for benzo[a]pyrene covalently bound to DNA has been employed to probe for adducts in the DNA of animal and human tissues and peripheral blood mononuclear cells. By enzyme-linked immunosorbent assay (ELISA) a dose-related increase in levels of benzo[a]pyrene-DNA adducts was observed in DNA from lung tissue of mice and rabbits injected i.p. with benzo[a]pyrene. Quantitation with ELISA was confirmed by i.p. injection of [3H]benzo[a]pyrene and determination of adduct levels in lung DNA by radioactivity. Thus, the ELISA assay was determined to be quantitative for benzo[a]pyrene-DNA adducts in vivo, and the lower limit of detectability established at 0.08-0.10 fmol/μg DNA. At this level of sensitivity no significant differences were observed between DNA from peripheral blood mononuclear cells of dogs on smoke inhalation machines and controls. In an attempt to probe for benzo[a]pyrene-DNA adducts in human subjects resulting from chronic environmental exposure, lung tissue, lung tumor and blood samples were obtained from patients hospitalized for lung cancer and other diseases. A detailed history of exposure to environmental sources of benzo[a]pyrene and to factors known to influence polycyclic aromatic hydrocarbon metabolism was attempted for 15 patients. DNA was extracted from the lung tissue of 27 patients and blood cells of several individuals and assayed by ELISA; 5 patients appeared to have low but measurable levels of benzo[a]pyrene-DNA adducts as determined by ELISA. All of these patients were in the lung cancer group. However, the number of subjects was too small to draw conclusions relating exposure history to the occurrence of hydrocarbon-DNA adducts. These preliminary results should encourage further studies on the utilization of immunoassays for carcinogen-DNA adducts as a potential tool in epidemiological studies attempting to relate biologically-effective dose of carcinogen to human cancer risk.