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1.
Drug Metab Dispos ; 47(12): 1388-1396, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31578206

RESUMO

Menthol, which creates mint flavor and scent, is often added to tobacco in both menthol and nonmenthol cigarettes. A potent tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is extensively metabolized to its equally carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) as (R)- or (S)-NNAL enantiomers. NNAL is detoxified by UDP-glucuronosyltransferase (UGT) enzymes, with glucuronidation occurring on either NNAL's pyridine ring nitrogen (NNAL-N-Gluc) or the chiral alcohol [(R)- or (S)-NNAL-O-Gluc]. To characterize a potential effect by menthol on NNAL glucuronidation, in vitro menthol glucuronidation assays and menthol inhibition of NNAL-Gluc formation assays were performed. Additionally, NNAL and menthol glucuronides (MG) were measured in the urine of smokers (n = 100) from the Southern Community Cohort Study. UGTs 1A9, 1A10, 2A1, 2A2, 2A3, 2B4, 2B7, and 2B17 all exhibited glucuronidating activity against both l- and d-menthol. In human liver microsomes, both l- and d-menthol inhibited the formation of each NNAL-Gluc, with a stereospecific difference observed between the formation of (R)-NNAL-O-Gluc and (S)-NNAL-O-Gluc in the presence of d-menthol but not l-menthol. With the exception of three nonmenthol cigarette smokers, urinary MG was detected in all menthol and nonmenthol smokers, with l-MG comprising >98% of total urinary MG. Levels of urinary NNAL-N-Gluc were significantly (P < 0.05) lower among subjects with high levels of total urinary MG; no significant changes in free NNAL were observed. These data suggest that the presence of menthol could lead to increases in alternative, activating metabolic pathways of NNAL in tobacco target tissues, increasing the opportunity for NNAL to damage DNA and lead to the development of tobacco-related cancers. SIGNIFICANCE STATEMENT: High levels of the major menthol metabolite, menthol-glucuronide, was observed in the urine of smokers of either menthol or nonmenthol cigarettes. The fact that a significant inverse correlation was observed between the levels of urinary menthol-glucuronide and NNAL-N-glucuronide, a major detoxification metabolite of the tobacco carcinogen, NNK, suggests that menthol may inhibit clearance of this important tobacco carcinogen.


Assuntos
Carcinógenos/metabolismo , Glucuronídeos/urina , Mentol/urina , Microssomos Hepáticos/metabolismo , Nitrosaminas/metabolismo , Nitrosaminas/urina , Fumar/urina , Estudos de Coortes , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Células HEK293 , Humanos , Mentol/metabolismo , Fumar/metabolismo , Estereoisomerismo , Produtos do Tabaco , Transfecção
2.
Chem Res Toxicol ; 32(8): 1689-1698, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31307193

RESUMO

Tobacco specific nitrosamines (TSNAs) are among the most potent carcinogens found in cigarettes and smokeless tobacco products. Decreases in TSNA detoxification, particularly 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been associated with tobacco-related cancer incidence. NNK is metabolized by carbonyl reduction to its major carcinogenic metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is detoxified by glucuronidation at the nitrogen within the pyridine ring or at the chiral alcohol to form four glucuronide products: (R)-NNAL-O-Gluc, (S)-NNAL-O-Gluc, (R)-NNAL-N-Gluc, (S)-NNAL-N-Gluc. Stereoselective NNAL-Gluc formation and the relative expression of NNAL-glucuronidating UGTs (1A4, 1A9, 1A10, 2B7, 2B10, 2B17) were analyzed in 39 tissue specimens from the upper aerodigestive tract (esophagus (n = 13), floor of mouth (n = 4), larynx (n = 9), tongue (n = 7), and tonsil (n = 6)). All pooled tissue types preferentially formed (R)-NNAL-O-Gluc in the presence of racemic-NNAL; only esophagus exhibited any detectable formation of (S)-NNAL-O-Gluc. For every tissue type examined, UGT1A10 exhibited the highest relative expression levels among the NNAL-O-glucuronidating UGTs, ranging from 36% (tonsil) to 49% (esophagus), followed by UGT1A9 > UGT2B7 > UGT2B17. UGT1A10 also exhibited similar or higher levels of expression as compared to both NNAL-N-glucuronidating UGTs, 1A4 and 2B10. In a screening of cells expressing individual UGT enzymes, all NNAL glucuronidating UGTs exhibited some level of stereospecific preference for individual NNAL enantiomers, with UGTs 1A10 and 2B17 forming primarily (R)-NNAL-O-Gluc. These data suggest that UGTs 1A10 and 2B17 may be important enzymes in the detoxification of TSNAs like NNK in tissues of the upper aerodigestive tract.


Assuntos
Sistema Digestório/metabolismo , Glucuronídeos/metabolismo , Nitrosaminas/metabolismo , Sistema Digestório/química , Glucuronídeos/química , Glucuronosiltransferase/metabolismo , Células HEK293 , Humanos , Cinética , Estrutura Molecular , Nitrosaminas/química , Estereoisomerismo
3.
Toxicol Mech Methods ; 29(7): 499-510, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31050318

RESUMO

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is classified as a Group 1 human carcinogen. It is metabolically activated by P450 enzymes to intermediate methylate and pyridyloxobutylate DNA, resulting in the formation of DNA adduct that is critical for the carcinogenicity of NNK. To directly and objectively examine the DNA adduct formation profiles without the complexity of factors in vivo, in the present study, five kinds of methyl DNA adducts were first identified in the incubation model of NNK established with human lung epithelial cells (BEAS-2B). The level of methyl DNA adducts and metabolites of NNK were quantitatively analyzed, respectively. With the increase of exposure time and dose, the level of methyl DNA adducts and metabolites increased. Furthermore, with the changes of the activity of P450 enzymes, which is the main enzyme regulating the α-hydroxylation of NNK, we found the levels of both methyl adducts and metabolites formed via α-hydroxylation in experimental groups showed the same trend compared with those in control group, while the metabolites formed via other pathways changed in the opposite trend. The result proves that the methyl adducts induced by NNK generate via α-hydroxylation pathway in BEAS-2B cells.


Assuntos
Carcinógenos/toxicidade , Adutos de DNA/metabolismo , Metilação de DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nitrosaminas/toxicidade , Carcinógenos/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Sistema Enzimático do Citocromo P-450 , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Humanos , Hidroxilação , Pulmão/enzimologia , Pulmão/metabolismo , Nitrosaminas/metabolismo
4.
Toxicol Lett ; 311: 11-16, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31026483

RESUMO

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R2 = 0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R2 = 0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation.


Assuntos
Butanonas/sangue , Nitrosaminas/metabolismo , Piridinas/sangue , Albumina Sérica/metabolismo , Fumar/sangue , Ativação Metabólica , Biomarcadores/sangue , Biomarcadores/urina , Estudos de Casos e Controles , Feminino , Hemoglobinas/metabolismo , Humanos , Modelos Animais , Nicotina/urina , Ligação Proteica , Fumar/efeitos adversos , Fumar/urina , Espectrometria de Massas em Tandem , Fatores de Tempo
5.
Exp Eye Res ; 184: 135-145, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30885711

RESUMO

Retinitis pigmentosa (RP) is a degenerative disease of the retina that affects approximately 1 million people worldwide. There are multiple genetic causes of this disease, for which, at present, there are no effective therapeutic strategies. In the present report, we utilized broad spectrum metabolomics to identify perturbations in the metabolism of the rd10 mouse, a genetic model for RP that contains a mutation in Pde6ß. These data provide novel insights into mechanisms that are potentially critical for retinal degeneration. C57BL/6J and rd10 mice were raised in cyclic light followed by either light or dark adaptation at postnatal day (P) 18, an early stage in the degeneration process. Mice raised entirely in the dark until P18 were also evaluated. After euthanasia, retinas were removed and extracted for analysis by ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC-QTOF-MS). Compared to wild type mice, rd10 mice raised in cyclic light or in complete darkness demonstrate significant alterations in retinal pyrimidine and purine nucleotide metabolism, potentially disrupting deoxynucleotide pools necessary for mitochondrial DNA replication. Other metabolites that demonstrate significant increases are the Coenzyme A intermediate, 4'-phosphopantothenate, and acylcarnitines. The changes in these metabolites, identified for the first time in a model of RP, are highly likely to disrupt normal energy metabolism. High levels of nitrosoproline were also detected in rd10 retinas relative to those from wild type mice. These results suggest that nitrosative stress may be involved in retinal degeneration in this mouse model.


Assuntos
Modelos Animais de Doenças , Redes e Vias Metabólicas/fisiologia , Metaboloma/fisiologia , Nitrosaminas/metabolismo , Nucleotídeos de Purina/metabolismo , Pirimidinas/metabolismo , Retinite Pigmentosa/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL
6.
Occup Environ Med ; 76(4): 259-267, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30772817

RESUMO

OBJECTIVES: To develop a quantitative historical job-exposure matrix (JEM) for rubber dust, rubber fumes and n-Nitrosamines in the British rubber industry for 1915-2002 to estimate lifetime cumulative exposure (LCE) for a cohort of workers with 49 years follow-up. METHODS: Data from the EU-EXASRUB database-rubber dust (n=4157), rubber fumes (n=3803) and n-Nitrosamines (n=10 115) collected between 1977 and 2002-were modelled using linear mixed-effects models. Sample year, stationary/personal measurement, industry sector and measurement source were included as fixed explanatory variables and factory as random intercept. Model estimates and extrapolations were used to construct a JEM covering all departments in both sectors of the rubber manufacturing industries for the years 1915-2002. JEM-estimates were linked to all cohort members to calculate LCE. Sensitivity analyses related to assumptions about extrapolation of time trends were also conducted. RESULTS: Changes in rubber dust exposures ranged from -6.3 %/year (crude materials/mixing) to -1.0 %/year (curing) and -6.5 %/year (crude materials/mixing) to +0.5 %/year (finishing, assembly and miscellaneous) for rubber fumes. Declines in n-Nitrosamines ranged from -17.9 %/year (curing) to -1.3 %/year (crude materials and mixing). Mean LCEs were 61 mg/m3-years (rubber dust), 15.6 mg/ m3-years (rubber fumes), 2483.2 µg/m3-years (n-Nitrosamines sum score), 18.6 µg/m3-years (N-nitrosodimethylamine) and 15.0 µg/m3-years (N-itrosomorpholine). CONCLUSIONS: All exposures declined over time. Greatest declines in rubber dust and fumes were found in crude materials and mixing and for n-Nitrosamines in curing/vulcanising and preprocessing. This JEM and estimated LCEs will allow for evaluation of exposure-specific excess cancer risks in the British rubber industry.


Assuntos
Nitrosaminas/efeitos adversos , Exposição Ocupacional/efeitos adversos , Borracha/efeitos adversos , Adulto , Idoso , Estudos de Coortes , Poeira/análise , Feminino , Gases/análise , Humanos , Indústrias/métodos , Indústrias/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Nitrosaminas/metabolismo , Exposição Ocupacional/estatística & dados numéricos , Borracha/metabolismo , Reino Unido
7.
Occup Environ Med ; 76(4): 250-258, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30772818

RESUMO

OBJECTIVES: To quantitatively evaluate exposure-response associations between occupational exposures to rubber dust, fumes and N-nitrosamines and cancer mortality in the UK rubber industry. METHODS: Competing risk survival analyses were used to examine cancer mortality risk in a cohort of 36 441 males aged 35+ years employed in the British rubber industry in 1967, followed up to 2015 (94% mortality). Exposure measurements are based on a population-specific quantitative job-exposure matrix for rubber dust, rubber fumes and N-nitrosamines from the EU-EXASRUB project. RESULTS: Exposure (lifetime cumulative (LCE))-response associations were found for N-nitrosomorphiline and all cancers (subdistribution HR (SHR) 1.48, 95% CI 1.39 to 1.57) and cancers of the bladder, stomach, multiple myeloma, oesophagus, prostate and pancreas, as well as for N-nitrosodimethylamine and all cancers (SHR 2.08, 95% CI 1.96 to 2.21) and cancers of the bladder, stomach, leukaemia, multiple myeloma, prostate and liver. LCE to the N-nitrosamines sum were associated with increased risks from all cancers (SHR 1.89, 95% CI 1.78 to 2.01) and cancers of the lung, non-Hodgkin's lymphoma and brain. LCE to rubber dust and fumes are associated with increased mortality from all cancers (rubber dust SHR 1.67, 95% CI 1.58 to 1.78; rubber fumes SHR 1.91, 95% CI 1.80 to 2.03) and cancers of the bladder, lung, stomach, leukaemia, multiple myeloma, non-Hodgkin's lymphoma, oesophagus, prostate, pancreas and liver. CONCLUSIONS: Consistent with previous studies, N-nitrosamines exposures are associated with mortality from cancers of the bladder, lung, stomach, leukaemia, multiple myeloma, oesophagus, prostate, pancreas and liver. The long follow-up with nearly complete mortality enabled estimations of lifetime cancer mortality risk from occupational exposures in the rubber industry.


Assuntos
Exposição Ambiental/efeitos adversos , Neoplasias/mortalidade , Nitrosaminas/efeitos adversos , Adulto , Idoso , Estudos de Coortes , Poeira , Exposição Ambiental/estatística & dados numéricos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Neoplasias/etiologia , Nitrosaminas/metabolismo , Estudos Retrospectivos , Borracha/efeitos adversos , Borracha/metabolismo , Reino Unido
8.
Chem Res Toxicol ; 32(4): 773-783, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30740971

RESUMO

The tobacco-specific carcinogens N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) require metabolic activation to exert their carcinogenicity. NNN and NNK are metabolized to the same reactive diazonium ions, which alkylate DNA forming pyridyloxobutyl (POB) DNA base and phosphate adducts. We have characterized the formation of both POB DNA base and phosphate adducts in NNK-treated rats and the formation of POB DNA base adducts in NNN-treated rats. However, POB DNA phosphate adducts in NNN-treated rats are still uncharacterized. In this study, we quantified the levels of POB DNA phosphate adducts in tissues of rats chronically treated with ( S)-NNN or ( R)-NNN for 10, 30, 50, and 70 weeks during a carcinogenicity study. The highest amounts of POB DNA phosphate adducts were observed in the esophagus of the ( S)-NNN-treated rats, with a maximum level of 5400 ± 317 fmol/mg DNA at 50 weeks. The abundance of POB DNA phosphate adducts in the esophagus was consistent with the results of the carcinogenicity study showing that the esophagus was the primary site of tumor formation from treatment with ( S)-NNN. Compared to the ( R)-NNN group, the levels of POB DNA phosphate adducts were higher in the oral mucosa, esophagus, and liver, while lower in the nasal mucosa of the ( S)-NNN-treated rats. Among 10 combinations of all isomers of POB DNA phosphate adducts, Ap(POB)C and combinations with thymidine predominated across all the rat tissues examined. In the primary target tissue, esophageal mucosa, Ap(POB)C accounted for ∼20% of total phosphate adducts in the ( S)-NNN treatment group throughout the 70 weeks, with levels ranging from 780 ± 194 to 1010 ± 700 fmol/mg DNA. The results of this study showed that POB DNA phosphate adducts were present in high levels and persisted in target tissues of rats chronically treated with ( S)- or ( R)-NNN. These results improve our understanding of DNA damage during NNN-induced carcinogenesis. The predominant POB DNA phosphate isomers observed, such as Ap(POB)C, may serve as biomarkers for monitoring chronic exposure of tobacco-specific nitrosamines in humans.


Assuntos
Adutos de DNA/análise , Nitrosaminas/metabolismo , Fosfatos/análise , Piridinas/análise , Animais , Adutos de DNA/metabolismo , Hidrólise , Masculino , Espectrometria de Massas , Estrutura Molecular , Nitrosaminas/administração & dosagem , Nitrosaminas/química , Fosfatos/metabolismo , Piridinas/metabolismo , Ratos , Ratos Endogâmicos F344 , Estereoisomerismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-30634532

RESUMO

N'-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines (TSNAs) that exists widely in smoke and smokeless tobacco products. NNN can induce tumors in various laboratory animal models and has been identified by International Agency for Research on Cancer (IARC) as a human carcinogen. Metabolic activation of NNN is primarily initiated by cytochrome P450 enzymes (CYP450s) via 2'-hydroxylation or 5'-hydroxylation. Subsequently, the hydroxylating intermediates undergo spontaneous decomposition to generate diazohydroxides, which can be further converted to alkyldiazonium ions, followed by attacking DNA to form various DNA damages, such as pyridyloxobutyl (POB)-DNA adducts and pyridyl-N-pyrrolidinyl (py-py)-DNA adducts. If not repaired correctly, these lesions would lead to tumor formation. In the present study, we performed density functional theory (DFT) computations and molecular docking studies to understand the mechanism of metabolic activation and carcinogenesis of NNN. DFT calculations were performed to explore the 2'- or 5'- hydroxylation reaction of (R)-NNN and (S)-NNN. The results indicated that NNN catalyzed by the ferric porphyrin (Compound I, Cpd I) at the active center of CYP450 included two steps, hydrogen abstraction and rebound reactions. The free energy barriers of the 2'- and 5'-hydroxylation of NNN are 9.82/8.44 kcal/mol (R/S) and 7.99/9.19 kcal/mol (R/S), respectively, suggesting that the 2'-(S) and 5'-(R) pathways have a slight advantage. The free energy barriers of the decomposition occurred at the 2'-position and 5'-position of NNN are 18.04/18.02 kcal/mol (R/S) and 18.33/19.53 kcal/mol (R/S), respectively. Moreover, we calculated the alkylation reactions occurred at ten DNA base sites induced by the 2'-hydroxylation product of NNN, generating the free energy barriers ranging from 0.86 to 4.72 kcal/mol, which indicated that these reactions occurred easily. The docking study showed that (S)-NNN had better affinity with CYP450s than that of (R)-NNN, which was consistent with the experimental results. Overall, the combined results of the DFT calculations and the docking obtained in this study provide an insight into the understanding of the carcinogenesis of NNN and other TSNAs.


Assuntos
Carcinógenos/metabolismo , Nitrosaminas/metabolismo , Tabaco/química , Ativação Metabólica , Animais , Carcinógenos/química , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Adutos de DNA/metabolismo , Teoria da Densidade Funcional , Humanos , Hidroxilação , Simulação de Acoplamento Molecular , Nitrosaminas/química , Ratos
10.
Appl Biochem Biotechnol ; 188(2): 297-309, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30430346

RESUMO

6-(N-Hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose (6NSL) is a key intermediate in the synthesis of miglitol. Biotransformation of N-2-hydroxyethyl glucamine (NHEG) to 6NSL was performed by immobilized Gluconobacter oxydans, which was prepared by cultivating the cells in a home-made bubble column bioreactor where corn stover particles were loaded. The optimal carrier addition and aeration rate for 6NSL production by immobilized cells in the bioreactor were determined to be 25 g/L and 2.5 vvm respectively. The supplementation of NH4Cl was conducive to the biotransformation of NHEG and was performed by adding aqueous ammonia and HCl, which was taken as the pH controlling agents as well. An optimal pH control strategy using the mixture of aqueous ammonia and NaOH was applied, resulting in a 9.9% increased production of 6NSL, while repeated batches of biotransformation increased from three times to four times. Finally, the 6NSL concentration and the conversion rate of NHEG to 6NSLreached 44.2 ± 1.5 g/L and 88.4 ± 2.0%, respectively, in average after four cycles of biotransformation under the optimized condition.


Assuntos
Amino Açúcares/biossíntese , Reatores Biológicos/microbiologia , Gluconobacter oxydans/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/metabolismo , Biotecnologia , Biotransformação , Células Imobilizadas/metabolismo , Meios de Cultura , Concentração de Íons de Hidrogênio , Nitrosaminas/metabolismo , Zea mays
11.
Toxicol In Vitro ; 55: 185-194, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30552994

RESUMO

Many of the toxicants in tobacco smoke undergo biotransformation in the lungs of smokers, both to reactive and to detoxified derivatives. Human air-liquid-interface (ALI) airway tissue models have emerged as an advanced in vitro model for evaluating the toxicity of inhaled substances; however, the metabolic potential of these cultures has not been evaluated extensively. In this study, we compared the metabolic activities of an ALI tissue model to the undifferentiated normal human primary bronchial epithelial (NHBE) cells from which it was derived. Measurement of the basal levels of gene expression for 84 phase I drug metabolism enzymes indicated that most genes were upregulated in ALI cultures compared to NHBE cells. Furthermore, the enzymatic activities of three cytochrome P450s involved in the bioactivation of tobacco-specific nitrosamines were higher in the ALI cultures, and the bioactivation of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), as measured by the formation of two of its major metabolites, i.e., keto acid and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was significantly greater in the ALI cultures. Finally, NNK was a direct-acting genotoxicant in the ALI cultures, while the genotoxicity of NNK was detected in NHBE cells only in the presence of an exogenous liver S9 activation system. Taken together, our findings demonstrate the greater metabolic potential of well-differentiated ALI cultures than primary NHBE cells, supporting the potential use of ALI airway cultures as an alternative in vitro model for evaluating inhaled toxicants that require metabolic transformation.


Assuntos
Brônquios/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Nitrosaminas/farmacologia , Diferenciação Celular , Células Cultivadas , Humanos , Cetoácidos/metabolismo , Nitrosaminas/metabolismo , Testes de Toxicidade/métodos
12.
Sci Rep ; 8(1): 13300, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30185870

RESUMO

Burley tobacco (Nicotiana Tabacum) is a chlorophyll-deficiency mutant. Nitrate is one precursor of tobacco-specific nitrosamines (TSNAs) and is largely accumulated in burley tobacco. To decrease nitrate accumulation in burley tobacco, glycerol, a polyhydric alcohol compound and physiological regulating material, was sprayed and its effects were investigated based on metabolomic technology and molecular biology. The results showed that glucose, glutamine and glutamic acid increased by 2.6, 5.1 and 196, folds, respectively, in tobacco leaves after glycerol application. Nitrate content was significantly decreased by 12-16% and expression of eight genes responsible for carbon and nitrogen metabolism were up-regulated with glycerol applications under both normal and 20% reduced nitrogen levels (P < 0.01). Leaf biomass of plants sprayed with glycerol and 20% nitrogen reduction was equivalent to that of no glycerol control with normal nitrogen application. Carbohydrates biosynthesis, nitrate transport and nitrate assimilation were enhanced in glycerol sprayed burley tobacco seedlings which might contribute to reduced nitrate and increased carbohydrates contents. In conclusion, glyerol spray coupled with 20% nitrogen reduction would be an effective method to reduce nitrate accumulation in burley tobacco.


Assuntos
Glicerol/metabolismo , Nitratos/metabolismo , Tabaco/metabolismo , Metabolismo dos Carboidratos/fisiologia , Carboidratos/biossíntese , Carbono/metabolismo , Clorofila/metabolismo , Metaboloma/genética , Nitrato Redutase/metabolismo , Nitrato Redutases/metabolismo , Nitrogênio/metabolismo , Óxidos de Nitrogênio/metabolismo , Nitrosaminas/metabolismo , Folhas de Planta/metabolismo , Plântula/genética , Plântula/metabolismo , Ativação Transcricional , Regulação para Cima
13.
Toxicol Sci ; 166(1): 82-96, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30102407

RESUMO

The Chicken Egg Genotoxicity Assay (CEGA) demonstrated responsiveness to various DNA-reactive chemicals requiring metabolic activation, which implies broad bioactivation capability. To assess potential metabolic competence, expression profiles of metabolic genes in the embryo-chicken fetal liver were determined using microarray technology. Fertilized chicken eggs were injected under the CEGA protocol with vehicle (deionized water [DW]), the activation-dependent carcinogens, diethylnitrosamine (DEN), and N-nitrosodiethanolamine (NDELA) at doses producing no effect on survival. Previously in CEGA, DEN produced DNA damage, whereas NDELA did not. Expressions of 463 genes known to encode for phase I and II of endo- and xenobiotic metabolism were detected on the array. DW did not affect the expression of the selected genes, deregulating less than 1% of them. In contrast, DEN at 2 mg/egg and NDELA at 4 mg/egg produced significant transcriptomic alterations, up-regulating up to 41% and down-regulating over 31% of studied genes. Both nitrosamines modulated the majority of the genes in a similar manner, sharing 64 up-regulated and 93 down-regulated genes with respect to control group, indicating similarity in the regulation of their metabolism by avian liver. Differences in gene expression between DEN and NDELA were documented for several phase I CYP 450 genes that are responsible for nitrosamine biotransformation, as well as for phase II genes that regulate detoxication reactions. These findings could underlie the difference in genotoxicity of DEN and NDELA in CEGA. In conclusion, the analysis of gene expression profiles in embryo-chicken fetal liver dosed with dialkylnitrosamines demonstrated that avian species possess a complex array of inducible genes coding for biotransformation.


Assuntos
Alternativas aos Testes com Animais , Galinhas , Nitrosaminas/toxicidade , Óvulo/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Xenobióticos/toxicidade , Animais , Biotransformação , Técnicas In Vitro , Testes de Mutagenicidade , Nitrosaminas/química , Nitrosaminas/metabolismo , Óvulo/metabolismo , Xenobióticos/química , Xenobióticos/metabolismo
14.
Anal Chem ; 90(20): 11863-11872, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30086646

RESUMO

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen that exerts its carcinogenic effects upon metabolic activation. The identification and quantitation of NNK metabolites could identify potential biomarkers of bioactivation and detoxification of this potent carcinogen and may be used to predict lung cancer susceptibility among smokers. Here, we used in vivo isotope-labeling and high-resolution-mass-spectrometry-based methods for the comprehensive profiling of all known and unknown NNK metabolites. The sample-enrichment, LC-MS, and data-analysis workflow, including a custom script for automated d0- d4- m/ z-pair-peak detection, enabled unbiased identification of numerous NNK metabolites. The structures of the metabolites were confirmed using targeted LC-MS2 with retention-time ( tR) and MS2-fragmentation comparisons to those of standards when possible. Eleven known metabolites and unchanged NNK were identified simultaneously. More importantly, our workflow revealed novel NNK metabolites, including 1,3-Diol (13), α-OH-methyl-NNAL-Gluc (14), nitro-NK- N-oxide (15), nitro-NAL- N-oxide (16), γ-OH NNAL (17), and three N-acetylcysteine (NAC) metabolites (18a-c). We measured the differences in the relative distributions of a panel of nitroso-containing NNK-specific metabolites in rats before and after phenobarbital (PB) treatment, and this served as a demonstration of a general strategy for the detection of metabolic differences in animal and cell systems. Lastly, we generated a d4-labeled NNK-metabolite mixture to be used as internal standards ( d4-rat urine) for the relative quantitation of NNK metabolites in humans, and this new strategy will be used to assess carcinogen exposure and ultimately to evaluate lung-cancer risk and susceptibility in smokers.


Assuntos
Carcinógenos/análise , Carcinógenos/metabolismo , Metabolômica , Animais , Carcinógenos/administração & dosagem , Cromatografia Líquida , Injeções Intraperitoneais , Marcação por Isótopo , Espectrometria de Massas , Estrutura Molecular , Nitrosaminas/administração & dosagem , Nitrosaminas/metabolismo , Nitrosaminas/urina , Ratos , Ratos Endogâmicos F344
15.
Regul Toxicol Pharmacol ; 97: 103-109, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29928933

RESUMO

Tobacco-specific nitrosamines (TSNA) levels in tobacco cut filler and cigarette smoke were measured in more than 1000 commercially available cigarettes sampled between 2008 and 2014. Relative contributions to their transfer from tobacco to the mainstream smoke in terms of direct transfer by distillation, pyrorelease, and pyrosynthesis were evaluated on the basis of the comparison with the transfer of nicotine from tobacco to smoke. N'-nitrosonornicotine (NNN) was transferred essentially by distillation, while N'-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-bipyridyl)-1-butanone (NNK) and N'-nitrosoanabasine (NAB) were transferred by pyrorelease or pyrosynthesis as well. In the case of the Tobacco Heating System 2.2, the transfer of nicotine from tobacco to the aerosol was similar to that observed for cigarettes, while the % transfer of TSNAs from tobacco to THS 2.2 aerosol was 2-3 times lower than in cigarettes. This difference is due to the fact that the tobacco is heated instead of burnt resulting in a lower direct transfer by distillation and a lower if any contribution of pyrosynthesis or pyrorelease.


Assuntos
Temperatura Alta , Nitrosaminas/análise , Fumaça/análise , Produtos do Tabaco/análise , Tabaco/química , Aerossóis/química , Aerossóis/metabolismo , Nitrosaminas/metabolismo , Tabaco/metabolismo
16.
Drug Metab Lett ; 12(2): 117-124, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29886839

RESUMO

BACKGROUND: The high levels of blood alcohol achieved with chronic plus binge alcohol exposures are somewhat reduced by co-administration of tobacco-specific Nicotine-Derived Nitrosamine Ketone (NNK) suggesting that NNK may alter alcohol metabolism. OBJECTIVE: We examined ethanol and acetaldehyde-metabolizing enzyme activities and malondialdehyde adduct formation in rats exposed to ethanol (chronic + binge), NNK, or both. METHODS: 4-week old Long Evans rats were fed liquid diets containing 0% or 26% caloric ethanol for 8 weeks. Ethanol-fed rats were binge-administered ethanol (2 g/kg; on Mondays, Wednesdays, and Fridays) by intraperitoneal (i.p.) injection, while control group administered saline in weeks 7 and 8 (n=12/group). Six rats from each group were administered i.p. injections of NNK (2 mg/kg) or saline on Tuesdays, Thursdays, and Saturdays of weeks 3 through 8. Alcohol dehydrogenase, catalase, and aldehyde dehydrogenase activities were measured using commercial assays. Cytochrome P450 mRNA levels (17 isoforms) were measured by quantitative reverse transcription-polymerase chain reaction. Malondialdehyde immunoreactivity was measured by enzyme-linked immunosorbent assay. RESULTS: Dual exposures to ethanol and NNK significantly increased frontal lobe ADH activity relative to control (P=0.01) and ethanol only (P=0.04) treatments, and ALDH relative to control (P=0.02). In contrast, malondialdehyde-protein expression was not significantly altered by ethanol+NNK. Ethanol decreased CYP1A1 mRNA expression relative to control (P=0.02), and combined ethanol+NNK exposures decreased the expression of CYP1A1 (P=0.01) and CYP2C6 (P=0.03). CONCLUSION: Dual exposures to ethanol and NNK increase brain ethanol metabolism and inhibit the expression of CYP450s that regulate xenobiotic metabolism.


Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Família 2 do Citocromo P450/metabolismo , Etanol/metabolismo , Lobo Frontal/efeitos dos fármacos , Cetonas/farmacologia , Nicotina/farmacologia , Nitrosaminas/farmacologia , Acetaldeído/metabolismo , Consumo de Bebidas Alcoólicas , Animais , Lobo Frontal/enzimologia , Cetonas/metabolismo , Malondialdeído/metabolismo , Nicotina/metabolismo , Nitrosaminas/metabolismo , Ratos Long-Evans
17.
Carcinogenesis ; 39(8): 1079-1088, 2018 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-29788210

RESUMO

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of this study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant cytosolic reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL-formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17ß6, HSD17ß12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels ≥HSD11ß1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17ß12 the most highly expressed. Of these lung-expressing enzymes, only HSD17ß12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knock-down of HSD17ß12 resulted in significant decreases in (R)-NNAL-formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17ß12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Carcinógenos/metabolismo , Neoplasias Pulmonares/patologia , Nitrosaminas/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , Carcinogênese/induzido quimicamente , Carcinogênese/patologia , Carcinógenos/toxicidade , Citosol/efeitos dos fármacos , Citosol/enzimologia , Ensaios Enzimáticos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Pulmão/citologia , Pulmão/enzimologia , Pulmão/patologia , Neoplasias Pulmonares/etiologia , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Nitrosaminas/toxicidade , Oxirredução , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fumar/efeitos adversos , Estereoisomerismo , Tabaco/química , Tabaco/toxicidade
18.
Talanta ; 181: 132-141, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29426492

RESUMO

The predominant enantiomer of nicotine found in nature is (S)-nicotine and its pharmacology has been widely established. However, pharmacologic information concerning individual enantiomers of nicotine-related compounds is limited. Recently, a modified macrocyclic glycopeptide chiral selector was found to be highly stereoselective for most tobacco alkaloids and metabolites. This study examines the semi-synthetic and native known macrocyclic glycopeptides for chiral recognition, separation, and characterization of the largest group of nicotine-related compounds ever reported (tobacco alkaloids, nicotine metabolites and derivatives, and tobacco-specific nitrosamines). The enantioseparation of nicotine is accomplished in less than 20s for example. All liquid chromatography separations are mass spectrometry compatible for the tobacco alkaloids, as well as their metabolites. Ring-closed, cyclized structures were identified and separated from their ring-open, straight chain equilibrium structures. Also, E/Z-tobacco-specific nitrosamines and their enantiomers were directly separated. E/Z isomers also are known to have different physical and chemical properties and biological activities. This study provides optimal separation conditions for the analysis of nicotine-related isomers, which in the past have been reported to be ineffectively separated which can result in inaccurate results. The methodology of this study could be applied to cancer studies, and lead to more information about the role of these isomers in other diseases and as treatment for diseases.


Assuntos
Alcaloides/química , Carcinógenos/química , Nitrosaminas/química , Tabaco/química , Alcaloides/isolamento & purificação , Alcaloides/metabolismo , Carcinógenos/isolamento & purificação , Carcinógenos/metabolismo , Cromatografia Líquida/métodos , Glicopeptídeos/química , Espectrometria de Massas/métodos , Nicotina/química , Nicotina/isolamento & purificação , Nicotina/metabolismo , Nitrosaminas/isolamento & purificação , Nitrosaminas/metabolismo , Reprodutibilidade dos Testes , Estereoisomerismo
19.
Toxicol Pathol ; 46(2): 184-192, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29390940

RESUMO

Lung cancer is the most common cause of cancer-related deaths in humans worldwide. There is strong evidence that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play an important role in carcinogenesis caused by tobacco products. NNK and racemic NNAL are reported to induce lung and pancreatic tumors in rats. The carcinogenicity in Fischer 344 rats of NNK, NNAL, and its enantiomers ( R)-NNAL and ( S)-NNAL has been studied recently, and all test compounds induced significant numbers of lung tumors. We report here the detailed histopathological and immunohistochemical characterization of these tumors and their aggressive nature as shown by their metastasis locally and to the pancreas. The spectrum of treatment-related histopathological findings comprised pulmonary alveolar/bronchiolar (A/B) epithelial hyperplasia, A/B adenomas, and A/B carcinomas. A/B carcinomas frequently exhibited local invasion/metastasis within the mediastinum and thoracic cavity and distant metastasis to the pancreas that was confirmed by immunohistochemistry using the lung-specific markers prosurfactant protein-C and club (Clara) cell-10. Our observation regarding metastasis to the pancreas was an important, and unexpected, finding in this study both for the experimental animal model and potential human relevance.


Assuntos
Carcinógenos/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Nitrosaminas/toxicidade , Neoplasias Pancreáticas/secundário , Animais , Carcinógenos/metabolismo , Carcinoma/induzido quimicamente , Carcinoma/secundário , Neoplasias Pulmonares/patologia , Masculino , Nitrosaminas/metabolismo , Ratos , Ratos Endogâmicos F344 , Estereoisomerismo , Tabaco/química
20.
Chem Res Toxicol ; 31(1): 48-57, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29131934

RESUMO

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL, or (R)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290-4510, 872-1120, and 763-1430 fmol/mg DNA, accounting for 15-38%, 8%, and 5-9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents.


Assuntos
Adutos de DNA/química , Adutos de DNA/metabolismo , Nitrosaminas/química , Nitrosaminas/farmacologia , Fosfatos/química , Animais , Metilação de DNA , Hidrólise , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Estrutura Molecular , Nitrosaminas/metabolismo , Ratos , Ratos Endogâmicos F344 , Estereoisomerismo
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