Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in smokers in the united states: NHANES 2007–2008

The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of the tobacco-specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been measured in urine samples from all participants aged 6 years and older from the National Health and Nutrition Examination Survey 2007–2008. Participants with a serum cotinine concentration of ≥10?ng/mL were identified as tobacco users, primarily cigarette smokers. Regression models were developed to calculate geometric mean NNAL concentrations adjusted for serum cotinine, urinary creatinine, cigarettes per day, and Federal Trade Commission tar values of the cigarettes smoked. Significant differences were found by gender (p?=?0.003) and race/ethnicity (p?=?0.022 for non-Hispanic white versus non-Hispanic black smokers), but not by menthol type of the cigarettes. Females and non-Hispanic white smokers had the highest adjusted means for urinary NNAL (353 and 336 pg/mL, respectively). The results from this study demonstrated significant relationships between NNAL concentrations and serum cotinine (p?<?0.001) and urine creatinine (p?<?0.001). The joint effect of linear and quadratic terms for number of cigarettes smoked per day was also statistically significant (p?=?0.001). In addition to addressing current NNK exposure levels, these results will form a baseline for future estimates of tobacco users’ exposure to this carcinogen.     

Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). TSNA are suggested to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC–MS/MS method for the determination of total (free and conjugated) TSNA in human urine. The limits of detection (LOD) were 2.0, 0.8, 1.1 and 0.7 pg/ml for NNAL, NNN, NAB and NAT, respectively. Smokers were found to have significantly higher levels of TSNA in their urine than nonsmokers. In conclusion, the newly developed method is suitable for assessing the tobacco use-related exposure to NNK, NNN, NAB and NAT.     

Protective effect of hydroxychavicol, a phenolic component of betel leaf, against the tobacco-specific carcinogens

The phenolic compound, hydroxychavicol (HC), present in betel leaf, was synthesized and tested for its antimutagenic effect against the mutagenicity of the 2 tobacco-specific N-nitrosamines (TSNA), N′-nitrosonornicotine (NNN) and 4-(nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), in 2 different test systems, viz. the Ames Salmonella/microsome assay and the micronucleus test using Swiss male mice. We are reporting the synthesis of HC of a high degree of purity. We observed that HC suppressed the mutagenic effects of NNN and NNK in both test systems used. These results indicate that HC may have a role to play in reducing the risk of oral cancer in betel quid with tobacco chewers.     

DNA adduct formation from tobacco-specific N-nitrosamines

Tobacco-specific N-nitrosamines are a group of carcinogens derived from the tobacco alkaloids. They are likely causative factors for cancers of the lung, esophagus, pancreas, and oral cavity in people who use tobacco products. The most carcinogenic tobacco-specific nitrosamines in laboratory animals are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N'-nitrosonornicotine (NNN). DNA adduct formation from NNK and NNN has been studied extensively and is reviewed here. NNK is metabolically activated by cytochromes P450 to intermediates which methylate and pyridyloxobutylate DNA. The resulting adducts have been detected in cells and tissues susceptible to NNK carcinogenesis in rodents. The methylation and pyridyloxobutylation pathways are both important in carcinogenesis by NNK. NNK also induces single strand breaks and increases levels of 8-oxodeoxyguanosine in DNA of treated animals. NNAL, which like NNK is a potent pulmonary carcinogen, is also metabolically activated to methylating and pyridyloxobutylating intermediates. NNN pyridyloxobutylates DNA in its rat target tissues, esophagus and nasal mucosa. Methyl and pyridyloxobutyl DNA adducts are detected in human tissues. The methyl adducts most likely result in part from exposure of smokers to NNK, but these adducts are also detected in non-smokers. Some of the methyl adducts detected in non-smokers may be due to environmental tobacco smoke exposure. There are also potential dietary and endogenous sources of these adducts. Pyridyloxobutyl DNA adducts in human tissues result mainly from exposure to tobacco-specific N-nitrosamines. In laboratory animals, DNA adduct formation and carcinogenicity of tobacco-specific N-nitrosamines are closely correlated in many instances, and it is likely that similar relationships will hold in humans.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta™ Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas.To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta™ Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report [Cancer Res, 49 (1989) 5305]. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively.These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

Acute in vivo treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone does not alter base excision repair activities in murine lung and liver

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen found in unburned tobacco and tobacco smoke, and is believed to play an important role in human tobacco-induced cancers. In previous studies, NNK has been reported to induce oxidative DNA damage, and to alter DNA repair processes, effects that could contribute to pulmonary tumorigenesis in rodent models. The goal of this study was to determine the effects of NNK on levels of 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of DNA oxidation, and activity of base excision repair (BER), which repairs oxidative DNA damage. Female A/J mice were treated with a tumorigenic dose of NNK (10 μmol) i.p. At 1, 2 and 24 h post treatment, there were no statistically significant differences in lung or liver 8-OHdG levels between control and NNK-treated mice (P > 0.05). Furthermore, NNK did not alter lung or liver BER activity compared to control at any time point (P > 0.05). In summary, acute treatment with a tumorigenic dose of NNK did not stimulate oxidative DNA damage or significantly alter BER activity, and these effects may not be major mechanisms of action of NNK in mouse models.     

Gene expression profiles in HPV-immortalized human cervical cells treated with the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Human papillomavirus (HPV) infection is an established etiological factor for cervical cancer. Epidemiological studies suggest that smoking in combination with HPV infection plays a significant role in the etiology of this disease. We have previously shown that the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is present in human cervical mucus. Here, we hypothesized that treatment of HPV-16-immortalized human ectocervical cells (Ecto1/E6E7) with NNK would alter the expression of genes involved in cellular transformation. Ecto1/E6E7 cells were treated with water (vehicle control) alone or with 1 μM, 10 μM, and 100 μM of NNK in water for 12 weeks. The colony-forming efficiency increased following NNK treatment; the maximum effect was observed after 12 weeks with 100 μM NNK. Microarray analysis revealed that, independent of the dose of NNK, expression of 30 genes was significantly altered; 22 of these genes showed a dose-response pattern. Genes identified are categorized as immune response (LTB4R), RNA surveillance pathway (SMG1), metabolism (ALDH7A1), genes frequently expressed in later stages of neoplastic development (MT1F), DNA binding (HIST3H3 and CHD1L), and protein biosynthesis (UBA52). Selected genes were confirmed by qRT-PCR. Western blot analysis indicates that phosphorylation of histone 3 at serine 10, a marker of cellular transformation, was up-regulated in cells treated with NNK. This is the first study showing that NNK can alter gene expression that may, in part, account for transformation of HPV-immortalized human cervical cells. The results support previous epidemiological observations that, in addition to HPV, tobacco smoking also plays an important role in the development of cervical cancer.     

Biomonitoring of environmental tobacco smoke (ETS)-related exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The exposure of non-smokers to the tobacco-specific N-nitrosamine 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a rodent lung carcinogen, was determined in the air of various indoor environments as well as by biomonitoring of non-smokers exposed to environmental tobacco smoke (ETS) under real-life conditions using the urinary NNK metabolites 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-(N-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). NNK was not detectable (&lt;0.5 ng m^-3) in 11 rooms in which smoking did not occur. The mean NNK concentration in 19 rooms in which smoking took place was 17.5 (2.4-50.0) ng m^-3. The NNK levels significantly correlated with the nicotine levels (r=0.856; p&lt; 0.0001). Of the 29 non-smokers investigated, 12 exhibited no detectable NNAL and NNAL-Gluc excretion (&lt;3 pmol day) in their urine. The mean urinary excretion of NNAL and NNAL-Gluc of the 17 remaining non-smokers was 20.3 (&lt;3-63.2) and 22.9 (&lt;3-90.0) pmol day^-1, respectively. Total NNAL excretion (NNAL+NNAL-Gluc) in all non-smokers investigated significantly correlated with the amount of nicotine on personal samplers worn during the week prior to urine collection (r=0.88; &lt;0.0001) and with the urinary cotinine levels (r=0.40; p=0.038). No correlation was found between NNAL excretion and the reported extent of ETS exposure. Average total NNAL excretion in the non-smokers with detectable NNAL levels was 74 times less than in 20 smokers who were also investigated. The cotinine/total NNAL ratios in urine of smokers (9900) and non-smokers (9300) were similar. This appears to be at variance with the ratios of the corresponding precursors (nicotine/NNK) in mainstream smoke (16400) and ETS (1000). Possible reasons for this discrepancy are discussed. The possible role of NNK as a lung carcinogen in non-smokers is unclear, especially since NNK exposure in non-smokers is several orders of magnitude lower than the ordinary exposure to exogenous and endogenous N-nitrosamines and the role of NNK as a human lung carcinogen is not fully understood.     

Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco‐specific nitrosamine accumulation in cured leaves and cigarette smoke

Burley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.     

Bacterial mutagenicity of some tobacco aromatic nitrogen bases and their mixtures

The first aim was to compare the genotoxicities of two tobacco-specific nitrosamines (TSNA), 4-(methylnitrosamino)-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) in two types of tests, the Salmonella reverse mutation assay (250-2000 microg per plate) and the Mutatox test (up to 1000 microg/ml) using dark mutant M-169 of Vibrio fischeri. The second aim was to assess the effects of single other tobacco chemicals and metabolites (nicotine (NIC), cotinine (COT), trans-3-hydroxycotinine (3HC), cotinine-N-oxide (CNO) and nicotine-N-oxide (NNO)) on the mutagenic responses at relative concentrations observed physiologically. The Salmonella strains were TA100, TA7004, TA7005, and TA7006, all showing missense backmutations that are characteristic of the TSNA. NNN was a direct mutagen to strains TA100, TA7004, and in the Mutatox test, and was not mutagenic in the presence of rat or hamster S9. NNK was mutagenic only in strain TA7004 with rat and hamster S9, but not in TA100, but was directly mutagenic in the Mutatox test. While all the other tobacco chemicals were not mutagenic alone to strains TA100 and TA7004 in the presence and absence of rat or hamster S9, the Mutatox test produced direct mutagenicity for COT, 3HC, and NNO, but not CNO. The latter was mutagenic in the Mutatox test with rat or hamster S9, but only rat S9 was effective for COT, NNO and 3HC. Inhibitory potentiations of NNN by NIC and COT were observed on strain TA7004, and by NIC on strain TA100. There were no interactions on NNK in the presence of S9 for strain TA7004 or TA100. In contrast, a complex inhibition and enhancement behavior occurred in the Mutatox test for each interaction, but no effects were observed for CNO on NNK without S9, and few for NIC on NNK with hamster S9. Compounds which showed no activity alone modulated the genotoxicity of two potent TSNAs in both types of tests.     

The organoselenium compound 1,4-phenylenebis(methylene)selenocyanate inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorgenesis and enhances glutathione-related antioxidant levels in A/J mouse lung

Selenium, in the form of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) but not Se-enriched yeast (Se-yeast), was highly effective at inhibiting lung tumors induced by the tobacco specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice and at reducing NNK-induced DNA methylation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the lung. Our goal was to determine if p-XSC but not Se-yeast is effective at inducing levels of glutathione (GSH)-related antioxidants and reducing markers of GSH oxidation in the NNK-induced lung tumor model. In the first bioassay, 6-week-old mice were fed either control or experimental diets (containing 10 ppm as selenium from p-XSC or Se-yeast) and, beginning at 8 weeks of age, received NNK (3 micromol) by gavage once weekly for 8 weeks. After 18 weeks, p-XSC significantly reduced NNK-induced tumor burden by 74% (10.4 +/- 6.0 versus 2.7 +/- 1.5 tumors/mouse, P < 0.001) and tumor incidence from 96% to 68% (P < 0.01), whereas, Se-yeast had no effect. Lung GSH levels were unchanged by either NNK or Se-yeast, but were increased 70% in mice treated with both NNK and p-XSC (P < 0.01) and 41% in mice treated with p-XSC alone. In the second bioassay, the time course of effects of p-XSC was examined. As early as one week after initiation of p-XSC feeding lung and blood selenium levels were increased nearly six- and two-fold, respectively. Increases of 120% for GSH and 65% for Cys were observed in p-XSC groups compared to controls within one week after initiation of p-XSC feeding (P < 0.01). The levels of protein-bound:free GSH ratios and Cys ratios were significantly decreased in p-XSC-treated mice, regardless of NNK status, suggesting a decrease in the levels of oxidative stress. Altogether, these results indicate that p-XSC is a potent inducer of GSH and related thiol antioxidants in the lung leading to decreased levels of oxidative stress and suggest that p-XSC inhibits tumor formation, in part, by protecting against oxidative damage.     

Role of DNA methylation in the activation of proto-oncogenes and the induction of pulmonary neoplasia by nitrosamines

The relationships between DNA methylation and repair induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to the activation of proto-oncogenes and the induction of pulmonary neoplasia by this carcinogen is described. The formation of the O⁶-methylguanine (O⁶MG) adduct following metabolic activation of NNK appears to be a major factor in the induction of lung tumors in both rats and mice and in the activation of the K-ras oncogene in lung tumors from A/J mouse. The potent carcinogenicity of NNK in the rat lung correlated strongly with cell specificity for formation and persistence of the O⁶MG adduct in the Clara cells. This conclusion was supported by studies with nitrosodimethylamine (NDMA), a weak carcinogen in the rodent lung. Treatment with NDMA was not associated with any pulmonary cell specificity for DNA methylation. The high affinity for activation of NNK compared to NDMA was ascribed to a difference in cytochrome P-450 isozymes involved in the activation of these two nitrosamines. In the A/J mouse, the induction of pulmonary tumorigenesis involved direct genotoxic activation of the K-ras proto-oncogene as a result of the base mispairing produced by formation of the O⁶MG adduct. In contrast, the induction of pulmonary tumors in the rat by NNK does not appear to involve the ras pathway. It is apparent that different molecular mechanisms are involved in the development of pulmonary tumors by NNK in the mouse and rat. The studies described in this paper illustrate the utility of performing dose-response experiments and the quantitation of DNA methylation and repair in not only target tissues but also target cell types. The fundamental knowledge gained from unraveling the mechanism of carcinogenesis by NNK could lead ultimately to the identification of factors important in the development of human lung cancer.     

Inhibition of mutagenicity of N-nitrosamines by tobacco smoke and its constituents

Tobacco smoke is a complex chemical mixture including pyridine alkaloids and N-nitrosamines, with the concentration of the former several orders of magnitude higher than that of the N-nitrosamines. The major biologically important N-nitrosamines present in tobacco smoke are N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosonornicotine (NNN). These nitrosamines require metabolic activation by cytochrome P-450s for the expression of mutagenicity. Although nicotine, the major pyridine alkaloid in tobacco, has been shown to inhibit the metabolic activation of NNK, its effect on the mutagenicity of NNK and other N-nitrosamines has not been reported. In the present study, the ability of three pyridine alkaloids (nicotine, cotinine, nornicotine) and aqueous cigarette smoke condensate extract (ACE) to inhibit the mutagenicity of tobacco-related N-nitrosamines was tested on Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9). All three of the pyridine alkaloids tested, as well as ACE, inhibited the mutagenicity of NDMA and NNK, but not NNN, in a concentration-dependent manner. The induction of SCEs in mammalian cells (CHO) by NNK in the presence of metabolic activation was also significantly reduced by nicotine and cotinine. None of the observed reductions in mutagenicity could be explained by cytotoxicity. These results demonstrate that tobacco smoke contains chemicals, pyridine alkaloids and other unidentified constituent(s), which inhibit the mutagenicity of N-nitrosamines.     

Formamidopyrimidine adducts are detected using the comet assay in human cells treated with reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most lung-specific of the carcinogens present in tobacco smoke. Its bioactivation in cells leads to a small amount of methylation or pyridyloxobutylation DNA damage. Considering its great sensitivity, the comet assay seems a technique of choice to investigate NNK-related damage. Several strategies were used to impart some specificity to the assay: (1) using analogs that produce a limited variety of DNA lesions, as they mimic either the methylation or the pyridyloxobutylation pathway; (2) using cells with different bioactivation abilities; (3) using alkali conversion and/or enzymes specific for cleaving particular classes of damage; (4) using different lysis conditions to convert a specific class of DNA lesions into enzyme-sensitive lesions. We determined that several NNK-associated lesions can be detected with some specificity with the comet assay. For the methylation pathway, they are AP sites and the more frequent formamidopyrimidine (fapy) adducts. These fapy adducts correspond to N7-methylguanines generated in the cells that were ring-opened during the assay by the lysis solution at pH 10. For the pyridyloxobutylation pathway, alkylphosphotriesters and a roughly equal frequency of fapy sites were detected. By analogy to the methylation damage, these fapy adducts are thought to be the ring-opened form of N7-pyridyloxobutylguanines (N7-pobG). N7-pobG are unstable and this constitutes the first indirect demonstration of their formation in cells. But contrary to N7-m-fapy, the lysis time or pH did not influence the frequency of N7-pob-fapy adducts detected, suggesting that they already exist in the cells and are not related to the experimental conditions. These N7-pob-fapy have a strong mutagenic potential and we think that the comet assay, in spite of its limitations, is a good way to study them considering their low frequency and the inherent instability of the adduct from which they originate.     

Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N′-Nitrosonornicotine (NNN) by isolated rabbit lung cells

The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [³H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.Abbreviations NHMI N-nitrosoheptamethyleneimine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NNN N-nitrosonornicotine Supported by a grant and a fellowship (R.B.) from the Royal Norwegian Council for Scientific and Industrial Research.     

NNK诱发BEP2D细胞产生活性氧及其对DNA的损伤

通过测定细胞内和细胞上清中活性氧（ｒｅａｃｔｉｖｅ ｏｘｙｇｅｎ ｓｐｅｃｉｅｓ,ＲＯＳ）水平,以及ＤＮＡ 加合物——８－羟基脱氧鸟嘌呤核苷（８－ｈｙｄｒｏｘｙｄｅｏｘｙｇｕａｎｏｓｉｎｅ,ＯＨ８ｄＧ）含量,对烟草特异亚硝胺类化合物４－甲基亚硝胺－１（３－吡啶基）－１－丁酮（４－（ｍ ｅｔｈｙｌｎｉｔｒｏｓａｍ ｉｎｏ）－１－（３－ｐｙｒｉｄｙｌ）－１－ｂｕｔａｎｏｎｅ,ＮＮＫ）诱发人乳头状病毒永生化的人支气管上皮细胞（ｈｕｍ ａｎ ｐａｐｉｌｌｏｍ ａｖｉｒｕｓ－ｉｍ ｍ ｏｒｔａｌｉｚｅｄ ｈｕｍ ａｎｂｒｏｎｃｈｉａｌｅｐｉｔｈｅｌｉａｌｃｅｌｌｌｉｎｅ,ＢＥＰ２Ｄ）产生的ＲＯＳ及其对ＤＮＡ 的氧化损伤进行研究,并观察纳米硒的保护作用．结果表明,ＢＥＰ２Ｄ 细胞经不同浓度的ＮＮＫ 作用后,细胞内和细胞上清中ＲＯＳ以及ＯＨ８ｄＧ含量均显著增加,并有较好的剂量效应关系．１ μｍ ｏｌ·Ｌ－ １纳米硒（ｎａｎｏｓｅｌｅｎｕｉｍ ,ＮＳ）能明显抑制ＮＮＫ 诱发ＢＥＰ２Ｄ细胞产生的ＲＯＳ及ＯＨ８ｄＧ 水平．揭示ＮＮＫ 能造成细胞的氧化损伤,而ＮＳ对ＮＮＫ 所致细胞的氧化损伤有保护作用．     

Differential exposure biomarker levels among cigarette smokers and smokeless tobacco consumers in the National Health and Nutrition Examination Survey 1999–2008

Assessment of biomarkers is an appropriate way to estimate exposure to cigarette mainstream smoke and smokeless tobacco (SLT) constituents in tobacco consumers. Using the US National Health and Nutrition Examination Survey (NHANES, 1999–2008), biomarkers of volatile organic compounds, halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), acrylamide, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and metals were evaluated. In general, biomarker levels in SLT consumers were significantly lower than in smokers (excluding NNK and some HAHs) and were not significantly different compared with nonconsumers (excluding NNK and some PAHs). These results provide useful information for science-based risk assessment and regulation of tobacco products.     

High-performance liquid chromatographic analysis of metabolites of the nicotine-derived nitrosamines, N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

An improved high-performance liquid chromatographic system was developed for separation of 11 metabolites of the nicotine-derived nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The new system employed a 5-microns octadecylsilane bonded column eluted with aqueous sodium acetate-methanol gradients of varying pH. Analysis times were typically 30 min for NNN metabolites and 50 min for NNK metabolites, compared to 80 and 90 min, respectively, when 10-microns columns were used. The E and Z isomers of all nitrosamine-containing metabolites of NNK were separated. The chromatographic behavior of the 11 metabolites as well as NNN and NNK was studied between pH 4.0 and 7.5. The retention times of several metabolites were altered significantly as a function of pH. The results of the pH study provide valuable additional criteria for metabolite identification as well as optimized conditions for their separation. Applications of the system to the metabolism of [2'-14C]NNN in cultured rat esophagus and [carbonyl-14C]NNK in rat liver slices are presented.     

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone modulation of cytokine release in U937 human macrophages

 The nicotine-derived N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most abundant and potent carcinogens found in tobacco smoke. NNK induces lung tumors in rodents and is most likely involved in lung carcinogenesis in humans. Studies on the metabolism and carcinogenicity of NNK have been extensive. However, its effects on the immune system have not been investigated thoroughly. Considering that tobacco smoking partially suppresses the immune response in humans, and that immune surveillance plays a critical role in cancer development, we examined the effects of NNK on the production of selected cytokines. In a previous study, we observed an inhibition of NK cell activity and IgM secretory cell number in NNK-treated A/J mice [Rioux and Castonguay (1997) J Natl Cancer Inst 89: 874]. In this study, we demonstrate that U937 human macrophages activate NNK to alkylating intermediates by α-carbon hydroxylation and detoxify NNK by N-oxidation. We observed that NNK, following activation, induces the release of soluble tumor necrosis factor (TNF), but inhibits interleukin(IL)-10 synthesis. We also report that 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, and nitroso(acetoxymethyl)methylamine, which generate the same alkylating intermediates as NNK, have similar effects on TNF and IL-10. This suggests that pyridyloxobutylating and methylating intermediates generated from NNK are potent modulators of the immune response. The levels of IL-6, granulocyte/macrophage-colony-stimulating factor and macrophage chemotactic protein 1 were also decreased in supernatants of NNK-treated U937 macrophages. In contrast, IL-2 synthesis in Jurkat cells was inhibited by NNK treatment. This is the first study demonstrating that NNK, via its alkylating intermediates, alters the cytokine synthesis profile in human cells. Modulation of cytokine synthesis by NNK might partially explain the immunosuppresion observed in smokers. Inhibition of immune functions, resulting from NNK activation to alkylating agents, may facilitate lung tumor development. Received: 3 February 2000 / Accepted: 15 September 2000