Silvia Balbo - Academia.edu (original) (raw)
Papers by Silvia Balbo
Molecular Carcinogenesis, 2016
Recently, we have shown that (S)-N'-Nitrosonornicotine [(S)-NNN], the maj... more Recently, we have shown that (S)-N'-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 wk and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate P-value < 0.05 and fold-change ≥2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4, and CD80), inflammation (Ephx2 and Inpp5d) and cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1, and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80, and Dgkg) are also altered in esophageal and head and neck tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and, therefore, could contribute to the development of biomarkers for the early detection and prevention of (S)-NNN-associated oral and esophageal cancers. © 2016 Wiley Periodicals, Inc.
Journal of Thoracic Oncology, 2016
Analytical chemistry, 2015
A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated ... more A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated and unknown DNA adducts induced by DNA alkylating agents in biological samples. Two new features were added to a previously developed approach to significantly broaden its scope, versatility, and selectivity. First, the neutral loss of a base (guanine, adenine, thymine, or cytosine) was added to the original methodology's neutral loss of the 2'-deoxyribose moiety to allow for the detection of all DNA base adducts. Second, targeted detection of anticipated DNA adducts based on the reactivity of the DNA alkylating agent was demonstrated by inclusion of an ion mass list for data dependent triggering of MS(2) fragmentation events and subsequent MS(3) fragmentation. Additionally, untargeted screening of the samples, based on triggering of an MS(2) fragmentation event for the most intense ions of the full scan, was included for detecting unknown DNA adducts. The approach was tested by ...
Methods in Pharmacology and Toxicology, 2013
Chemical research in toxicology, Jan 20, 2014
Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostag... more Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostaglandin biosynthesis, can react with DNA to form stable adducts. These adducts may contribute to the development of such inflammation-mediated diseases as cancer and cardiovascular and neurodegenerative diseases. The predominant MDA-derived DNA adduct formed under physiological conditions is 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG). In this study, we developed a novel liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high-resolution tandem mass spectrometry (HRMS/MS) method for the analysis of M1dG in human leukocyte DNA. After enzymatic hydrolysis of DNA, M1dG and the added internal standard [(13)C3]M1dG were reduced to their 5,6-dihydro derivatives by addition of sodium borohydride to the hydrolysate and purified by solid-phase extraction and column chromatography. The 5,6-dihydro derivatives in the purified samples were anal...
Mutagenesis, 2012
Alcohol consumption is an established risk factor for cancers of the head and neck, colorectum, l... more Alcohol consumption is an established risk factor for cancers of the head and neck, colorectum, liver and female breast. Acetaldehyde, the primary metabolite of ethanol, is suspected to play a major role in alcohol-related carcinogenesis. Acetaldehyde binds to DNA resulting in formation of adducts. DNA adducts are involved in mutagenesis and carcinogenesis. N (2)-Ethylidenedeoxyguanosine (N (2)-ethylidene-dGuo) is the major adduct formed in this reaction. Studies have shown an association between alcohol drinking and levels of this DNA adduct, suggesting its potential use as a biomarker for studying alcohol-related carcinogenesis. However, there are no reports on the kinetics of formation and repair of N (2)-ethylidene-dGuo after alcohol consumption. Therefore, we investigated levels of N (2)-ethylidene-dGuo in DNA from human peripheral blood cells at several time points after consumption of increasing doses of alcohol. Ten healthy non-smokers were recruited and asked to abstain fro...
IARC scientific publications, 2011
Biological monitoring is the analysis of human biological materials for a substance of interest a... more Biological monitoring is the analysis of human biological materials for a substance of interest and/or its metabolites (biomarkers) or a biochemical change that occurs as a result of an exposure to provide a quantitative measure of exposure or dose. These measures can be used in epidemiological studies either directly as estimates of exposure or indirectly in the calibration of other exposure assessment methods, such as questionnaires. This chapter will discuss important methodological considerations for the implementation of biomarkers of exogenous exposure in epidemiology by focusing on biomarker characteristics (e.g. variability, half-life) and their application in different study designs.
Alcohol and alcoholism (Oxford, Oxfordshire), Jan 24, 2015
Alcoholic liver disease (ALD) is linked to binge drinking and cigarette smoking. Heavy chronic ± ... more Alcoholic liver disease (ALD) is linked to binge drinking and cigarette smoking. Heavy chronic ± binge alcohol, or low-level exposures to dietary nitrosamines cause steatohepatitis with insulin resistance and oxidative stress in animal models. This study examines hepatotoxic effects of sub-mutagenic exposures to tobacco-specific nitrosamine (NNK) in relation to ALD. Long Evans rats were fed liquid diets containing 0 or 26% (caloric) ethanol (EtOH) for 8 weeks. In Weeks 3 through 8, rats were treated with NNK (2 mg/kg) or saline by i.p. injection, 3×/week, and in Weeks 7 and 8, EtOH-fed rats were binge-administered 2 g/kg EtOH 3×/week; controls were given saline. EtOH ± NNK caused steatohepatitis with necrosis, disruption of the hepatic cord architecture, ballooning degeneration, early fibrosis, mitochondrial cytopathy and ER disruption. Severity of lesions was highest in the EtOH+NNK group. EtOH and NNK inhibited insulin/IGF signaling through Akt and activated pro-inflammatory cytok...
Carcinogenesis, 2014
We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(meth... more We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in l...
Carcinogenesis, 2014
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized to enantiomers of 4-(methylni... more 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...
International journal of pharmaceutics, Jan 30, 2014
Indole-3-carbinol (I3C), a constituent of commonly consumed Brassica vegetables, has been shown t... more Indole-3-carbinol (I3C), a constituent of commonly consumed Brassica vegetables, has been shown to have anticancer effects in a variety of preclinical models of lung cancer. However, it has shown only limited efficacy in clinical trials, likely due to its poor oral bioavailability. Intranasal administration of I3C has the potential to enhance the pulmonary accumulation of the drug, thereby improving its availability at the target site of action. In this study, we developed a liposomal formulation of I3C and evaluated its lung delivery and chemopreventive potential in tobacco smoke carcinogen [4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK)]-treated mice. Intranasal administration of I3C liposomes led to a ∼100-fold higher lung exposure of I3C than the oral route of administration. Further, intranasal delivery of liposomal I3C led to a significant reduction (37%; p<0.05) in the levels of the DNA adduct formation induced by NNK treatment. Liposomal I3C also significantly incr...
![Research paper thumbnail of Preferential Glutathione Conjugation of a Reverse Diol Epoxide Compared with a Bay Region Diol Epoxide of Benzo[a]pyrene in Human Hepatocytes](https://attachments.academia-assets.com/42491480/thumbnails/1.jpg)
](Drug Metabolism and Disposition, 2010
Many studies have examined the relationship between polymorphisms in glutathione S-transferase ge... more Many studies have examined the relationship between polymorphisms in glutathione S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but the results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We incubated human hepatocytes from 10 donors with racemic anti-BaP-7,8-diol-9,10-epoxide (BPDE), believed to be a major ultimate carcinogen of BaP, or with the noncarcinogenic reverse diol epoxide, racemic anti-BaP-9,10-diol-7,8-epoxide (rev-BPDE). Incubations were carried out for 12 or 24 h. We used high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464 3 m/z 317 to analyze the incubation mixtures for the mercapturic acid products that would result from glutathione conjugation. The standard mercapturic acids were synthesized by reaction of BPDE or rev-BPDE with N-acetylcysteine. We obtained convincing evidence in human hepatocytes for mercapturic acid formation from rev-BPDE in all 10 samples, in amounts up to 17 pmol/ml. However, we could detect mercapturic acids from BPDE in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BPDE with glutathione is a minor pathway in humans, indicating that glutathione S-transferase genotyping is not an effective method for assessing risk of PAH-induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis.
Chemico-Biological Interactions, 2014
Benzene oxide, the initial metabolite of the human carcinogen benzene, reacts with DNA producing ... more Benzene oxide, the initial metabolite of the human carcinogen benzene, reacts with DNA producing 7-phenylguanine (7-PhG) and other products. We developed a highly sensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry-parallel reaction monitoring method for the analysis of 7-PhG in DNA. Accuracy and precision of the method were established and the detection limit was about 8amol of 7-PhG injected on the column and less than 1 adduct per 10(9) nucleotides in DNA. 7-PhG was detected in calf thymus DNA reacted with 1μM to 10mM benzene oxide. The method was applied for the analysis of DNA isolated from bone marrow, lung, and liver of B6C3F1 mice treated by gavage with 50mg/kg benzene in corn oil 5 times weekly for 4weeks. 7-PhG was not detected in any of these DNA samples. The method was applied to DNA from mouse hepatocytes exposed to 100μM benzene oxide and human TK-6 lymphoblasts exposed to 100μM, 1, and 10mM benzene oxide. 7-PhG was only detected in TK-6 cell DNA from the 10mM exposure. The method was also applied to leukocyte DNA from 10 smokers and 10 nonsmokers. 7-PhG was detected in only one DNA sample, from a nonsmoker. The results of this study do not support the hypothesis that the benzene oxide-DNA adduct 7-PhG is involved in carcinogenesis by benzene.
Chemico-Biological Interactions, 2013
An earlier study demonstrated that hydrolysates of all human liver DNA samples analyzed contain t... more An earlier study demonstrated that hydrolysates of all human liver DNA samples analyzed contain the DNA adduct 7-(2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-carboxyethyl)guanine (7-CEGua) with an average level of 74.6 adducts per 10(9) nucleotides. One possible source of this DNA adduct would be endogenous nitrosation of the normal pyrimidine metabolites dihydrouracil (DHU) and β-ureidopropionic acid (β-UPA), yielding the corresponding nitroso compounds N-nitrosodihydrouracil, a potent hepatocarcinogen, and N-nitroso-β-ureidopropionic acid. Another potential source would be reaction of endogenously formed acrylic acid with DNA. We tested these hypotheses in a study in which rats were treated with NaNO2 in the drinking water, alone, or in combination with dietary DHU or β-UPA, or with acrylic acid in the drinking water, for either 2 or 4 weeks. Hepatic DNA from these rats was analyzed for 7-CEGua, using liquid chromatography-tandem mass spectrometry-selected reaction monitoring with confirmation by high resolution mass spectrometry. The results demonstrated consistent statistically significant increases of 7-CEGua in hepatic DNA of the rats treated with the combination of NaNO2 and DHU compared to the corresponding controls, while the other treatments gave variable results. These results support the hypothesis that endogenous nitrosation of DHU could be a major source of 7-CEGua in human hepatic DNA. Development of methodology for analysis of 7-CEGua in human leukocyte DNA is also reported, which will allow testing of this hypothesis in epidemiologic and clinical studies.
Chemical Research in Toxicology, 2014
Systems toxicology is a broad-based approach to describe many of the toxicological features that ... more Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the 32 P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC−MS n ), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC−MS n instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology.
Chemical Research in Toxicology, 2013
Lung cancer is the leading cause of cancer death in the world. Evidence suggests that lung cancer... more Lung cancer is the leading cause of cancer death in the world. Evidence suggests that lung cancer could originate from mutations accumulating in a subpopulation of self-renewing cells, lung stem cells. Aldehyde dehydrogenase (ALDH) is a marker of stem cells. To investigate the presence of DNA modifications in these cells, we isolated ALDH-positive lung cells from A/J mice exposed to the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Using LC-NSI-HRMS/MS-PRM, O(6)-methyl-G, 7-POB-G, and O(2)-POB-dT were positively identified in ALDH-positive cell DNA. This is the first example of detection of carcinogen-DNA adducts in lung stem cells, supporting the hypothesis of their role in lung carcinogenesis.
Chemical Research in Toxicology, 2011
There is considerable evidence for the exposure of humans to an unknown ethylating agent, and som... more There is considerable evidence for the exposure of humans to an unknown ethylating agent, and some studies indicate that cigarette smoking may be one source of this exposure. Therefore, we have developed a liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry-selected reaction monitoring (LC-NSI-HRMS/MS-SRM) method for the analysis of 7-ethyl-Gua in human leukocyte DNA, a readily available source of DNA. [(15)N(5)]7-Ethyl-Gua was used as the internal standard. Leukocyte DNA was isolated and treated by thermal neutral hydrolysis. The hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-ethyl-Gua was analyzed by LC-NSI-HRMS/MS-SRM using the transition m/z 180 [M + H](+)→ m/z 152.05669 [Gua + H](+) for 7-ethyl-Gua and m/z 185 → m/z 157.04187 for the internal standard. The detection limit was approximately 10 amol on column, while the limit of quantitation was about 8 fmol/μmol Gua starting with 180 μg DNA (corresponding to 36 μg DNA on-column). Leukocyte DNA samples from 30 smokers and 30 nonsmokers were analyzed. Clear peaks for 7-ethyl-Gua and the internal standard were observed in most of the samples. The mean (±SD) level of 7-ethyl-Gua measured in leukocyte DNA from smokers was 49.6 ± 43.3 (range 14.6-181) fmol/μmol Gua, while that from nonsmokers was 41.3 ± 34.9 (range 9.64-157) fmol/μmol Gua. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the presence of 7-ethyl-Gua in human leukocyte DNA.
Chemical Research in Toxicology, 2011
Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pol... more Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pollutant and toxicant that is also formed endogenously through metabolism of amino acids and polyamines and lipid peroxidation. Acrolein reacts with DNA, producing two pairs of regioisomeric 1,N(2)-propanodeoxyguanosine adducts: (6R/S)-3-(2&amp;amp;amp;amp;amp;#39;-deoxyribos-1&amp;amp;amp;amp;amp;#39;-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2&amp;amp;amp;amp;amp;#39;-deoxyribos-1&amp;amp;amp;amp;amp;#39;-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). Previous studies indicate that these adducts might be involved in producing mutations in the p53 tumor suppressor gene, as observed in lung tumors in smokers, but there are only limited published data comparing acrolein-DNA adducts in smokers and nonsmokers. In this study, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to analyze Acr-dGuo adducts in human leukocyte DNA. The potential for artifactual formation was found in two steps of the assay: DNA isolation and DNA hydrolysis. This was eliminated by employing a Ficoll-Hypaque double density gradient to obtain leukocytes free of erythrocyte contamination and by adding glutathione to scavenge acrolein present in H(2)O. The accuracy and precision of the method were confirmed. Acr-dGuo adducts were analyzed in leukocyte DNA from 25 smokers and 25 nonsmokers. γ-OH-Acr-dGuo was the predominant isomer in all samples, while α-OH-Acr-dGuo was detected in only three subjects. There was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/10(9) nucleotides) and nonsmokers (9.8 ± 5.5 adducts/10(9) nucleotides). Although our study is limited in size, these results, together with the results of previous analyses of acrolein-derived mercapturic acids in the urine of smokers and nonsmokers, suggest that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.
![Research paper thumbnail of Analysis of r -7, t -8,9, c -10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene in Human Urine: A Biomarker for Directly Assessing Carcinogenic Polycyclic Aromatic Hydrocarbon Exposure Plus Metabolic Activation](https://attachments.academia-assets.com/42491478/thumbnails/1.jpg)
](Chemical Research in Toxicology, 2011
Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of c... more Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of cancers in humans. Benzo[a]pyrene (BaP) is a prototypic carcinogenic PAH, which requires metabolic activation to elicit its detrimental effects. The major end product of its diol epoxide metabolic activation pathway is r-7, t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans, anti-BaPT). Individual differences in exposure to, and metabolic activation of, carcinogenic PAH may influence cancer risk. Measurement of PAH metabolites in human urine could provide a direct way to assess individual differences in susceptibility to PAH-related cancer. In this paper, we describe a sensitive and reliable method for quantitation of trans, anti-BaPT in human urine using gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). [ 13 C 6 ] trans, anti-BaPT was used as the internal standard. The urine was treated with β-glucuronidase and sulfatase, and then trans, anti-BaPT was enriched by solid-phase extraction with polymeric reversed phase and phenylboronic acid cartridges. The sample was silylated and analyzed by GC-NICI-MS/MS with selected reaction monitoring (SRM) for the trimethylsilyl (TMS) derivatives of trans, anti-BaPT (m/z 446→ m/z 255) and [ 13 C 6 ]trans, anti-BaPT (m/z 452→ m/z 261). The mean assay recovery was 44%. The instrumental on-column detection limit was about 20 amol of trans, anti-BaPT (as BaPT-TMS). trans, anti-BaPT was readily detected in all urine samples analyzed including 30 smokers (0.71 ± 0.64 fmol/mg creatinine) and 30 non-smokers (0.34 ± 0.2 fmol/mg creatinine) (P = 0.0018). The results of this study demonstrate a highly sensitive and selective method for quantitation of trans, anti-BaPT in human urine. This is to our knowledge the first study to show that smokers have significantly higher levels of trans, anti-BaPT in their urine than do non-smokers. This method may be useful as a direct phenotyping approach to assess individual differences in uptake and metabolic activation of carcinogenic PAH.
Chemical Research in Toxicology, 2009
N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Nitrosonornicotine (NNN) is one of the... more N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Nitrosonornicotine (NNN) is one of the most important strong carcinogens in tobacco products and is believed to play a significant role in the induction of esophageal cancer in smokers and oral cavity cancer in snuff dippers. NNN is metabolically activated through cytochrome P450-catalyzed alpha-hydroxylation. 2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Hydroxylation produces a reactive intermediate 4-(3-pyridyl)-4-oxobutanediazohydroxide (7), which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. DNA pyridyloxobutylation from NNN treatment, as measured by released 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 8), has been observed in vitro and in vivo. In the present study, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to analyze specific POB-DNA adducts in the nasal olfactory, nasal respiratory, and oral mucosa of F344 rats treated chronically with (R)-NNN or (S)-NNN in the drinking water (10 ppm, 1-20 weeks). Adduct levels in the nasal respiratory mucosa exceeded those in the nasal olfactory and oral mucosa. (R)-NNN treatment generated 2-4 times more adducts in the nasal olfactory and respiratory mucosa than did (S)-NNN at most time points. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd, 11) predominated in the nasal olfactory and respiratory mucosa, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14). Levels of O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O(2)-POB-Cyt, 13) and O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deoxyguanosine (O(6)-POB-dGuo, 12) were significantly lower. In the oral mucosa, the opposite stereoselectivity was observed, with (S)-NNN treatment producing 3-5 times more POB-DNA adducts than did (R)-NNN. O(2)-POB-dThd and 7-POB-dGuo were the two major adducts, and their levels were similar. Overall, POB-DNA adduct formation in the nasal olfactory and nasal respiratory mucosa was similar to that previously observed in the lung, whereas in the oral mucosa, the trend resembled that in the esophagus. These results indicate that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues. NNN enters the nasal mucosa through the circulation, and tissue-specific metabolism is important, while in the oral mucosa, direct exposure and local activation both play significant roles. Our results also support the potential importance of NNN as an oral carcinogen in people who use smokeless tobacco products.
Molecular Carcinogenesis, 2016
Recently, we have shown that (S)-N&amp;amp;amp;#39;-Nitrosonornicotine [(S)-NNN], the maj... more Recently, we have shown that (S)-N&amp;amp;amp;#39;-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 wk and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate P-value &amp;amp;amp;lt; 0.05 and fold-change ≥2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4, and CD80), inflammation (Ephx2 and Inpp5d) and cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1, and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80, and Dgkg) are also altered in esophageal and head and neck tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and, therefore, could contribute to the development of biomarkers for the early detection and prevention of (S)-NNN-associated oral and esophageal cancers. © 2016 Wiley Periodicals, Inc.
Journal of Thoracic Oncology, 2016
Analytical chemistry, 2015
A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated ... more A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated and unknown DNA adducts induced by DNA alkylating agents in biological samples. Two new features were added to a previously developed approach to significantly broaden its scope, versatility, and selectivity. First, the neutral loss of a base (guanine, adenine, thymine, or cytosine) was added to the original methodology's neutral loss of the 2'-deoxyribose moiety to allow for the detection of all DNA base adducts. Second, targeted detection of anticipated DNA adducts based on the reactivity of the DNA alkylating agent was demonstrated by inclusion of an ion mass list for data dependent triggering of MS(2) fragmentation events and subsequent MS(3) fragmentation. Additionally, untargeted screening of the samples, based on triggering of an MS(2) fragmentation event for the most intense ions of the full scan, was included for detecting unknown DNA adducts. The approach was tested by ...
Methods in Pharmacology and Toxicology, 2013
Chemical research in toxicology, Jan 20, 2014
Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostag... more Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostaglandin biosynthesis, can react with DNA to form stable adducts. These adducts may contribute to the development of such inflammation-mediated diseases as cancer and cardiovascular and neurodegenerative diseases. The predominant MDA-derived DNA adduct formed under physiological conditions is 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG). In this study, we developed a novel liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high-resolution tandem mass spectrometry (HRMS/MS) method for the analysis of M1dG in human leukocyte DNA. After enzymatic hydrolysis of DNA, M1dG and the added internal standard [(13)C3]M1dG were reduced to their 5,6-dihydro derivatives by addition of sodium borohydride to the hydrolysate and purified by solid-phase extraction and column chromatography. The 5,6-dihydro derivatives in the purified samples were anal...
Mutagenesis, 2012
Alcohol consumption is an established risk factor for cancers of the head and neck, colorectum, l... more Alcohol consumption is an established risk factor for cancers of the head and neck, colorectum, liver and female breast. Acetaldehyde, the primary metabolite of ethanol, is suspected to play a major role in alcohol-related carcinogenesis. Acetaldehyde binds to DNA resulting in formation of adducts. DNA adducts are involved in mutagenesis and carcinogenesis. N (2)-Ethylidenedeoxyguanosine (N (2)-ethylidene-dGuo) is the major adduct formed in this reaction. Studies have shown an association between alcohol drinking and levels of this DNA adduct, suggesting its potential use as a biomarker for studying alcohol-related carcinogenesis. However, there are no reports on the kinetics of formation and repair of N (2)-ethylidene-dGuo after alcohol consumption. Therefore, we investigated levels of N (2)-ethylidene-dGuo in DNA from human peripheral blood cells at several time points after consumption of increasing doses of alcohol. Ten healthy non-smokers were recruited and asked to abstain fro...
IARC scientific publications, 2011
Biological monitoring is the analysis of human biological materials for a substance of interest a... more Biological monitoring is the analysis of human biological materials for a substance of interest and/or its metabolites (biomarkers) or a biochemical change that occurs as a result of an exposure to provide a quantitative measure of exposure or dose. These measures can be used in epidemiological studies either directly as estimates of exposure or indirectly in the calibration of other exposure assessment methods, such as questionnaires. This chapter will discuss important methodological considerations for the implementation of biomarkers of exogenous exposure in epidemiology by focusing on biomarker characteristics (e.g. variability, half-life) and their application in different study designs.
Alcohol and alcoholism (Oxford, Oxfordshire), Jan 24, 2015
Alcoholic liver disease (ALD) is linked to binge drinking and cigarette smoking. Heavy chronic ± ... more Alcoholic liver disease (ALD) is linked to binge drinking and cigarette smoking. Heavy chronic ± binge alcohol, or low-level exposures to dietary nitrosamines cause steatohepatitis with insulin resistance and oxidative stress in animal models. This study examines hepatotoxic effects of sub-mutagenic exposures to tobacco-specific nitrosamine (NNK) in relation to ALD. Long Evans rats were fed liquid diets containing 0 or 26% (caloric) ethanol (EtOH) for 8 weeks. In Weeks 3 through 8, rats were treated with NNK (2 mg/kg) or saline by i.p. injection, 3×/week, and in Weeks 7 and 8, EtOH-fed rats were binge-administered 2 g/kg EtOH 3×/week; controls were given saline. EtOH ± NNK caused steatohepatitis with necrosis, disruption of the hepatic cord architecture, ballooning degeneration, early fibrosis, mitochondrial cytopathy and ER disruption. Severity of lesions was highest in the EtOH+NNK group. EtOH and NNK inhibited insulin/IGF signaling through Akt and activated pro-inflammatory cytok...
Carcinogenesis, 2014
We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(meth... more We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in l...
Carcinogenesis, 2014
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized to enantiomers of 4-(methylni... more 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...
International journal of pharmaceutics, Jan 30, 2014
Indole-3-carbinol (I3C), a constituent of commonly consumed Brassica vegetables, has been shown t... more Indole-3-carbinol (I3C), a constituent of commonly consumed Brassica vegetables, has been shown to have anticancer effects in a variety of preclinical models of lung cancer. However, it has shown only limited efficacy in clinical trials, likely due to its poor oral bioavailability. Intranasal administration of I3C has the potential to enhance the pulmonary accumulation of the drug, thereby improving its availability at the target site of action. In this study, we developed a liposomal formulation of I3C and evaluated its lung delivery and chemopreventive potential in tobacco smoke carcinogen [4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK)]-treated mice. Intranasal administration of I3C liposomes led to a ∼100-fold higher lung exposure of I3C than the oral route of administration. Further, intranasal delivery of liposomal I3C led to a significant reduction (37%; p<0.05) in the levels of the DNA adduct formation induced by NNK treatment. Liposomal I3C also significantly incr...
Drug Metabolism and Disposition, 2010
Many studies have examined the relationship between polymorphisms in glutathione S-transferase ge... more Many studies have examined the relationship between polymorphisms in glutathione S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but the results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We incubated human hepatocytes from 10 donors with racemic anti-BaP-7,8-diol-9,10-epoxide (BPDE), believed to be a major ultimate carcinogen of BaP, or with the noncarcinogenic reverse diol epoxide, racemic anti-BaP-9,10-diol-7,8-epoxide (rev-BPDE). Incubations were carried out for 12 or 24 h. We used high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464 3 m/z 317 to analyze the incubation mixtures for the mercapturic acid products that would result from glutathione conjugation. The standard mercapturic acids were synthesized by reaction of BPDE or rev-BPDE with N-acetylcysteine. We obtained convincing evidence in human hepatocytes for mercapturic acid formation from rev-BPDE in all 10 samples, in amounts up to 17 pmol/ml. However, we could detect mercapturic acids from BPDE in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BPDE with glutathione is a minor pathway in humans, indicating that glutathione S-transferase genotyping is not an effective method for assessing risk of PAH-induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis.
Chemico-Biological Interactions, 2014
Benzene oxide, the initial metabolite of the human carcinogen benzene, reacts with DNA producing ... more Benzene oxide, the initial metabolite of the human carcinogen benzene, reacts with DNA producing 7-phenylguanine (7-PhG) and other products. We developed a highly sensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry-parallel reaction monitoring method for the analysis of 7-PhG in DNA. Accuracy and precision of the method were established and the detection limit was about 8amol of 7-PhG injected on the column and less than 1 adduct per 10(9) nucleotides in DNA. 7-PhG was detected in calf thymus DNA reacted with 1μM to 10mM benzene oxide. The method was applied for the analysis of DNA isolated from bone marrow, lung, and liver of B6C3F1 mice treated by gavage with 50mg/kg benzene in corn oil 5 times weekly for 4weeks. 7-PhG was not detected in any of these DNA samples. The method was applied to DNA from mouse hepatocytes exposed to 100μM benzene oxide and human TK-6 lymphoblasts exposed to 100μM, 1, and 10mM benzene oxide. 7-PhG was only detected in TK-6 cell DNA from the 10mM exposure. The method was also applied to leukocyte DNA from 10 smokers and 10 nonsmokers. 7-PhG was detected in only one DNA sample, from a nonsmoker. The results of this study do not support the hypothesis that the benzene oxide-DNA adduct 7-PhG is involved in carcinogenesis by benzene.
Chemico-Biological Interactions, 2013
An earlier study demonstrated that hydrolysates of all human liver DNA samples analyzed contain t... more An earlier study demonstrated that hydrolysates of all human liver DNA samples analyzed contain the DNA adduct 7-(2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-carboxyethyl)guanine (7-CEGua) with an average level of 74.6 adducts per 10(9) nucleotides. One possible source of this DNA adduct would be endogenous nitrosation of the normal pyrimidine metabolites dihydrouracil (DHU) and β-ureidopropionic acid (β-UPA), yielding the corresponding nitroso compounds N-nitrosodihydrouracil, a potent hepatocarcinogen, and N-nitroso-β-ureidopropionic acid. Another potential source would be reaction of endogenously formed acrylic acid with DNA. We tested these hypotheses in a study in which rats were treated with NaNO2 in the drinking water, alone, or in combination with dietary DHU or β-UPA, or with acrylic acid in the drinking water, for either 2 or 4 weeks. Hepatic DNA from these rats was analyzed for 7-CEGua, using liquid chromatography-tandem mass spectrometry-selected reaction monitoring with confirmation by high resolution mass spectrometry. The results demonstrated consistent statistically significant increases of 7-CEGua in hepatic DNA of the rats treated with the combination of NaNO2 and DHU compared to the corresponding controls, while the other treatments gave variable results. These results support the hypothesis that endogenous nitrosation of DHU could be a major source of 7-CEGua in human hepatic DNA. Development of methodology for analysis of 7-CEGua in human leukocyte DNA is also reported, which will allow testing of this hypothesis in epidemiologic and clinical studies.
Chemical Research in Toxicology, 2014
Systems toxicology is a broad-based approach to describe many of the toxicological features that ... more Systems toxicology is a broad-based approach to describe many of the toxicological features that occur within a living system under stress or subjected to exogenous or endogenous exposures. The ultimate goal is to capture an overview of all exposures and the ensuing biological responses of the body. The term exposome has been employed to refer to the totality of all exposures, and systems toxicology investigates how the exposome influences health effects and consequences of exposures over a lifetime. The tools to advance systems toxicology include high-throughput transcriptomics, proteomics, metabolomics, and adductomics, which is still in its infancy. A well-established methodology for the comprehensive measurement of DNA damage resulting from every day exposures is not fully developed. During the past several decades, the 32 P-postlabeling technique has been employed to screen the damage to DNA induced by multiple classes of genotoxicants; however, more robust, specific, and quantitative methods have been sought to identify and quantify DNA adducts. Although triple quadrupole and ion trap mass spectrometry, particularly when using multistage scanning (LC−MS n ), have shown promise in the field of DNA adductomics, it is anticipated that high-resolution and accurate-mass LC−MS n instrumentation will play a major role in assessing global DNA damage. Targeted adductomics should also benefit greatly from improved triple quadrupole technology. Once the analytical MS methods are fully mature, DNA adductomics along with other -omics tools will contribute greatly to the field of systems toxicology.
Chemical Research in Toxicology, 2013
Lung cancer is the leading cause of cancer death in the world. Evidence suggests that lung cancer... more Lung cancer is the leading cause of cancer death in the world. Evidence suggests that lung cancer could originate from mutations accumulating in a subpopulation of self-renewing cells, lung stem cells. Aldehyde dehydrogenase (ALDH) is a marker of stem cells. To investigate the presence of DNA modifications in these cells, we isolated ALDH-positive lung cells from A/J mice exposed to the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Using LC-NSI-HRMS/MS-PRM, O(6)-methyl-G, 7-POB-G, and O(2)-POB-dT were positively identified in ALDH-positive cell DNA. This is the first example of detection of carcinogen-DNA adducts in lung stem cells, supporting the hypothesis of their role in lung carcinogenesis.
Chemical Research in Toxicology, 2011
There is considerable evidence for the exposure of humans to an unknown ethylating agent, and som... more There is considerable evidence for the exposure of humans to an unknown ethylating agent, and some studies indicate that cigarette smoking may be one source of this exposure. Therefore, we have developed a liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry-selected reaction monitoring (LC-NSI-HRMS/MS-SRM) method for the analysis of 7-ethyl-Gua in human leukocyte DNA, a readily available source of DNA. [(15)N(5)]7-Ethyl-Gua was used as the internal standard. Leukocyte DNA was isolated and treated by thermal neutral hydrolysis. The hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-ethyl-Gua was analyzed by LC-NSI-HRMS/MS-SRM using the transition m/z 180 [M + H](+)→ m/z 152.05669 [Gua + H](+) for 7-ethyl-Gua and m/z 185 → m/z 157.04187 for the internal standard. The detection limit was approximately 10 amol on column, while the limit of quantitation was about 8 fmol/μmol Gua starting with 180 μg DNA (corresponding to 36 μg DNA on-column). Leukocyte DNA samples from 30 smokers and 30 nonsmokers were analyzed. Clear peaks for 7-ethyl-Gua and the internal standard were observed in most of the samples. The mean (±SD) level of 7-ethyl-Gua measured in leukocyte DNA from smokers was 49.6 ± 43.3 (range 14.6-181) fmol/μmol Gua, while that from nonsmokers was 41.3 ± 34.9 (range 9.64-157) fmol/μmol Gua. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the presence of 7-ethyl-Gua in human leukocyte DNA.
Chemical Research in Toxicology, 2011
Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pol... more Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pollutant and toxicant that is also formed endogenously through metabolism of amino acids and polyamines and lipid peroxidation. Acrolein reacts with DNA, producing two pairs of regioisomeric 1,N(2)-propanodeoxyguanosine adducts: (6R/S)-3-(2&amp;amp;amp;amp;amp;#39;-deoxyribos-1&amp;amp;amp;amp;amp;#39;-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2&amp;amp;amp;amp;amp;#39;-deoxyribos-1&amp;amp;amp;amp;amp;#39;-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). Previous studies indicate that these adducts might be involved in producing mutations in the p53 tumor suppressor gene, as observed in lung tumors in smokers, but there are only limited published data comparing acrolein-DNA adducts in smokers and nonsmokers. In this study, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to analyze Acr-dGuo adducts in human leukocyte DNA. The potential for artifactual formation was found in two steps of the assay: DNA isolation and DNA hydrolysis. This was eliminated by employing a Ficoll-Hypaque double density gradient to obtain leukocytes free of erythrocyte contamination and by adding glutathione to scavenge acrolein present in H(2)O. The accuracy and precision of the method were confirmed. Acr-dGuo adducts were analyzed in leukocyte DNA from 25 smokers and 25 nonsmokers. γ-OH-Acr-dGuo was the predominant isomer in all samples, while α-OH-Acr-dGuo was detected in only three subjects. There was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/10(9) nucleotides) and nonsmokers (9.8 ± 5.5 adducts/10(9) nucleotides). Although our study is limited in size, these results, together with the results of previous analyses of acrolein-derived mercapturic acids in the urine of smokers and nonsmokers, suggest that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.
Chemical Research in Toxicology, 2011
Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of c... more Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of cancers in humans. Benzo[a]pyrene (BaP) is a prototypic carcinogenic PAH, which requires metabolic activation to elicit its detrimental effects. The major end product of its diol epoxide metabolic activation pathway is r-7, t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans, anti-BaPT). Individual differences in exposure to, and metabolic activation of, carcinogenic PAH may influence cancer risk. Measurement of PAH metabolites in human urine could provide a direct way to assess individual differences in susceptibility to PAH-related cancer. In this paper, we describe a sensitive and reliable method for quantitation of trans, anti-BaPT in human urine using gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). [ 13 C 6 ] trans, anti-BaPT was used as the internal standard. The urine was treated with β-glucuronidase and sulfatase, and then trans, anti-BaPT was enriched by solid-phase extraction with polymeric reversed phase and phenylboronic acid cartridges. The sample was silylated and analyzed by GC-NICI-MS/MS with selected reaction monitoring (SRM) for the trimethylsilyl (TMS) derivatives of trans, anti-BaPT (m/z 446→ m/z 255) and [ 13 C 6 ]trans, anti-BaPT (m/z 452→ m/z 261). The mean assay recovery was 44%. The instrumental on-column detection limit was about 20 amol of trans, anti-BaPT (as BaPT-TMS). trans, anti-BaPT was readily detected in all urine samples analyzed including 30 smokers (0.71 ± 0.64 fmol/mg creatinine) and 30 non-smokers (0.34 ± 0.2 fmol/mg creatinine) (P = 0.0018). The results of this study demonstrate a highly sensitive and selective method for quantitation of trans, anti-BaPT in human urine. This is to our knowledge the first study to show that smokers have significantly higher levels of trans, anti-BaPT in their urine than do non-smokers. This method may be useful as a direct phenotyping approach to assess individual differences in uptake and metabolic activation of carcinogenic PAH.
Chemical Research in Toxicology, 2009
N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Nitrosonornicotine (NNN) is one of the... more N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Nitrosonornicotine (NNN) is one of the most important strong carcinogens in tobacco products and is believed to play a significant role in the induction of esophageal cancer in smokers and oral cavity cancer in snuff dippers. NNN is metabolically activated through cytochrome P450-catalyzed alpha-hydroxylation. 2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-Hydroxylation produces a reactive intermediate 4-(3-pyridyl)-4-oxobutanediazohydroxide (7), which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. DNA pyridyloxobutylation from NNN treatment, as measured by released 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 8), has been observed in vitro and in vivo. In the present study, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to analyze specific POB-DNA adducts in the nasal olfactory, nasal respiratory, and oral mucosa of F344 rats treated chronically with (R)-NNN or (S)-NNN in the drinking water (10 ppm, 1-20 weeks). Adduct levels in the nasal respiratory mucosa exceeded those in the nasal olfactory and oral mucosa. (R)-NNN treatment generated 2-4 times more adducts in the nasal olfactory and respiratory mucosa than did (S)-NNN at most time points. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd, 11) predominated in the nasal olfactory and respiratory mucosa, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14). Levels of O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O(2)-POB-Cyt, 13) and O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deoxyguanosine (O(6)-POB-dGuo, 12) were significantly lower. In the oral mucosa, the opposite stereoselectivity was observed, with (S)-NNN treatment producing 3-5 times more POB-DNA adducts than did (R)-NNN. O(2)-POB-dThd and 7-POB-dGuo were the two major adducts, and their levels were similar. Overall, POB-DNA adduct formation in the nasal olfactory and nasal respiratory mucosa was similar to that previously observed in the lung, whereas in the oral mucosa, the trend resembled that in the esophagus. These results indicate that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues. NNN enters the nasal mucosa through the circulation, and tissue-specific metabolism is important, while in the oral mucosa, direct exposure and local activation both play significant roles. Our results also support the potential importance of NNN as an oral carcinogen in people who use smokeless tobacco products.