RESUMEN

Para estabelecer medidas equivalentes para o ensaio de produtos de tabaco em escala mundial é necessário que haja métodos consensuais de mensuração do conteúdo e das emissões específicas dos cigarros. Nenhum regime de tragada obtido por máquinas é capaz de representar plenamente o comportamento humano de fumar: os ensaios realizados em máquinas de fumar são úteis para caracterizar as emissões de cigarro para fins de design e regulação, mas a divulgação aos fumantes das medições em máquinas pode resultar em interpretações equivocadas a respeito das diferenças de exposição e risco existentes entre as marcas. Os dados de emissão de fumaça obtidos por medições em máquinas podem ser usados como elementos para a avaliação do perigo do produto, mas não são e nem se destinam a ser medidas válidas de exposição ou risco para os seres humanos. A apresentação de diferenças nas medições em máquina como diferenças de exposição ou risco constitui uso indevido dos resultados do ensaio com métodos recomendados da TobLabNet da OMS. Este documento foi preparado por membros Rede de Laboratórios de Tabaco (TobLabNet) da Organização Mundial da Saúde (OMS) como um procedimento operacional padrão (POP) do método analítico para determinar nitrosaminas específicas do tabaco (TSNA) na corrente primária da fumaça sob o regime de tragada da Organização Internacional de Normalização (ISO) intenso.

Asunto(s)

Nitrosaminas , Productos de Tabaco , Fumar , Seguridad de Productos para el Consumidor , Ensayo de Materiales

RESUMEN

This study outlines the development and optimization of an analytical method using Disposable Pipette Extraction (DPX) followed by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis to determine NAs in medicines. HPLC-MS analysis utilized a reversed-phase and positive mode electrospray ion source. DPX parameters were optimized through univariate and multivariate analyses, including extraction phase, desorption solvent, sample pH, equilibrium time, and extraction/desorption cycles. The optimized conditions included a C18 extraction phase, methanol desorption solvent, pH at 7, an equilibrium time of 30 seconds, 2 extraction cycles, and 5 desorption cycles. Considering this method, it was possible to achieve a sample preparation step for the analysis of NAs in medicines using a minimal amount of extraction phase, sample, and desorption solvent. Furthermore, the total extraction procedure enables the extraction of NAs in around 4 minutes with NA recovery up to 98%. Analytical performance demonstrated precision and accuracy below 15% and a quantification limit of 1 ng mL-1, meeting validation requirements set by regulations worldwide. Thus, the DPX/HPLC-MS technique offers a faster and cost-effective method for analyzing NAs in medicines compared to traditional approaches. Besides, this method reduces solvent consumption and residue generation, enhancing environmental sustainability according to green chemistry principles.

Asunto(s)

Nitrosaminas , Cromatografía Líquida de Alta Presión/métodos , Nitrosaminas/análisis , Nitrosaminas/aislamiento & purificación , Límite de Detección , Espectrometría de Masas/métodos , Reproducibilidad de los Resultados , Extracción en Fase Sólida/métodos , Cromatografía Líquida con Espectrometría de Masas

RESUMEN

N-nitrosamines (NAs) are prevalent mutagenic impurities in various consumer products. Their discovery in valsartan-containing medicines in 2018 prompted global regulatory agencies to set guidelines on their presence and permissible levels in pharmaceuticals. In order to determine the NAs content in medicines, efficient and sensitive analytical methods have been developed based on mass spectrometry techniques. Direct analysis in real time-mass spectrometry (DART-MS) has emerged as a prominent ambient ionization technique for pharmaceutical analysis due to its high-throughput capability, simplicity, and minimal sample preparation requirements. Thus, in this study DART-MS was evaluated for the screening and quantification of NAs in medicines. DART-MS analyses were conducted in positive ion mode, for both direct tablet analysis and solution analysis. The analytical performance was evaluated regarding linearity, precision, accuracy, limits of detection, and quantification. The DART-MS proved to be suitable for the determination of NAs in medicines, whether through direct tablet analysis or solution analysis. The analytical performance demonstrated linearity in the range from 1.00 to 200.00 ng mL-1, limits of quantification about 1.00 ng mL-1, precision and accuracy lower than 15%, and no significant matrix effect for six drug-related NAs. In conclusion, the DART-MS technique demonstrated to be an alternative method to determine NAs in medicines, aligning with the principles of green chemistry.

Asunto(s)

Contaminación de Medicamentos , Límite de Detección , Espectrometría de Masas , Nitrosaminas , Nitrosaminas/análisis , Espectrometría de Masas/métodos , Comprimidos/análisis , Reproducibilidad de los Resultados

RESUMEN

The development of a fast, cost-effective, and efficient microextraction by packed sorbent setup was achieved by combining affordable laboratory-repackable devices of microextraction with a high-throughput cartesian robot. This setup was evaluated for the development of an analytical method to determine N-nitrosamines in losartan tablets. N-nitrosamines pose a significant concern in the pharmaceutical market due to their carcinogenic risk, necessitating their control and quantification in pharmaceutical products. The parameters influencing the performance of this sample preparation for N-nitrosamines were investigated through both univariate and multivariate experiments. Microextractions were performed using just 5.0 mg of carboxylic acid-modified polystyrene divinylbenzene copolymer as the extraction phase. Under the optimized conditions, the automated setup enabled the simultaneous treatment of six samples in less than 20 min, providing reliable analytical confidence for the proposed application. The analytical performance of the automated high-throughput microextraction by the packed sorbent method was evaluated using a matrix-matching calibration. Quantification was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry with chemical ionization at atmospheric pressure. The method exhibited limits of detection as low as 50 ng/g, good linearity, and satisfactory intra-day (1.38-18.76) and inter-day (2.66-20.08) precision. Additionally, the method showed accuracy ranging from 80% to 136% for these impurities in pharmaceutical formulations.

Asunto(s)

Nitrosaminas , Robótica , Nitrosaminas/análisis , Losartán/análisis , Espectrometría de Masas en Tándem/métodos , Límite de Detección , Microextracción en Fase Sólida/métodos , Cromatografía Liquida , Cromatografía Líquida de Alta Presión/métodos , Comprimidos

RESUMEN

N-nitrosamines (NA) impurities have unexpectedly been found in sartan products, angiotensin II receptor antagonists that are used to control hypertension, representing an urgent concern for industry, global regulators and for the patients. In this study, an HPLC-MS/MS method was developed and validated for the quantification of six NA (N-nitrosodimethylamine, N-Nitroso-N-methyl-4-aminobutyric acid, N-Nitrosodiethylamine, N-ethyl-N-nitroso-2-propanamine, N-nitroso-diisopropylamine and N-nitroso-di-n-butylamine) in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan products. The method was validated in terms of sensitivity, linearity, accuracy, precision, robustness and stability. The limits of quantification were 100, 31.25, 250, 33, 312.5 and 125 µg kg-1 in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan samples, respectively, which met the sensitivity requirements for the limits set by Food and Drug Administration of the United States. The standard curves showed good linearity. The recoveries ranged from 93.06 to 102.23% in losartan matrix, 83 to 85.9% in valsartan, 96.1 to 101.2% in olmesartan, 89.2 to 97.5% in irbesartan, 93.4 to 132.0% in candesartan and 62.3 to 106.2% in telmisartan matrix. The other parameters met the validation criteria, the good sensitivity and precision, high accuracy and simple and fast analysis provides a reliable method for quality control of NA in sartan pharmaceutical products. The developed method was successfully applied for the determination of N-nitrosamines in 71 sartan products marketed in Brazil.

Asunto(s)

Nitrosaminas , Humanos , Nitrosaminas/análisis , Bloqueadores del Receptor Tipo 1 de Angiotensina II , Cromatografía Líquida de Alta Presión , Espectrometría de Masas en Tándem/métodos , Losartán , Carcinógenos/análisis , Irbesartán/análisis , Preparaciones Farmacéuticas , Telmisartán , Brasil , Valsartán/análisis , Valsartán/química

RESUMEN

Chronic stress increases the systemic levels of stress hormones norepinephrine and cortisol. As well as tobacco-specific carcinogen NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone), they can induce expressive DNA damage contributing to the cancer development. However, it is unknown whether stress hormones have genotoxic effects in oral keratinocytes. This study investigated the effects of stress hormones on DNA damage in a human oral keratinocyte cell line (NOK-SI). NOK-SI cells stimulated with norepinephrine or cortisol showed higher DNA damage compared to untreated cells. Norepinephrine-induced DNA damage was reversed by pre-treatment with beta-adrenergic blocker propranolol. Cells treated with NNK combined to norepinephrine displayed reduced levels of caspases 3 and 7. Cortisol also reduced the activity of pro-apoptotic enzymes. NNK or norepinephrine promoted single-strand breaks and alkali-label side breaks in the DNA of NOK-SI cells. Pre-treatment of cells with propranolol abolished these effects. Carcinogen NNK in the presence or absence of cortisol also induced DNA damage of these cells. The genotoxic effects of cortisol alone and hormone combined with NNK were blocked partially and totally, respectively, by the glucocorticoid receptor antagonist RU486. DNA damage promoted by NNK or cortisol and carcinogen combined to the hormone led to intracellular γH2AX accumulation. The effects caused by NNK and cortisol were reversed by propranolol and glucocorticoid receptor antagonist RU486, respectively. Propranolol inhibited the oxidation of basis induced by NNK in the presence of DNA-formamidopyrimidine glycosylase. DNA breaks induced by norepinephrine in the presence or absence of NNK resulted in higher 8OHdG cellular levels. This effect was also induced through beta-adrenergic receptors. Together, these findings indicate that stress hormones induce DNA damage of oral keratinocytes and could contribute to oral carcinogenesis.

Asunto(s)

Daño del ADN , Hormonas/metabolismo , Queratinocitos/metabolismo , Mucosa Bucal/metabolismo , Estrés Fisiológico , 8-Hidroxi-2'-Desoxicoguanosina/metabolismo , Apoptosis , Roturas del ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Células Epiteliales , Histonas/metabolismo , Hormonas/farmacología , Humanos , Hidrocortisona/farmacología , Queratinocitos/efectos de los fármacos , Nitrosaminas/química , Nitrosaminas/farmacología , Norepinefrina/farmacología , Oxidación-Reducción , Nicotiana/química

RESUMEN

Tobacco smoke contains various cancer-causing toxic substances, including nicotine and nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). The cytochrome 2A13 is involved in nicotine metabolism and in the activation of the pro-carcinogenic agents NNK and NNN, by means of α-hydroxylation reactions. Despite the significance of cytochrome 2A13 in the biotransformation of these molecules, its conformational mechanism and the molecular basis involved in the process are not fully understood. In this study, we used molecular dynamics and principal component analysis simulations for an in-depth analysis of the essential protein motions involved in the interaction of cytochrome 2A13 with its substrates. We also evaluated the interaction of these substrates with the amino acid residues in the binding pocket of cytochrome 2A13. Furthermore, we quantified the nature of these chemical interactions from free energy calculations using the Molecular Mechanics/Generalized Born Surface Area method. The ligands remained favorably oriented toward compound I (cytochrome P450 O═FeIV state), to undergo α-hydroxylation. The hydrogen bond with asparagine 297 was essential to maintaining the substrates in a favorable catalytic orientation. The plot of first principal motion vs second principal motion revealed that the enzyme's interaction with nicotine and NNK involved different conformational subgroups, whereas the conformational subgroups in the interaction with NNN are more similar. These results provide new mechanistic insights into the mode of interaction of the substrates with the active site of cytochrome 2A13, in the presence of compound I, which is essential for α-hydroxylation.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/metabolismo , Simulación de Dinámica Molecular , Nicotina/metabolismo , Nitrosaminas/metabolismo , Hidrocarburo de Aril Hidroxilasas/química , Biocatálisis , Dominio Catalítico , Simulación del Acoplamiento Molecular

RESUMEN

BACKGROUND: Article 10 of the World Health Organization Framework Convention on Tobacco Control states the need for industry disclosure of tobacco contents and emissions. Currently, the profiles of key tobacco compounds in legal and illegal cigarettes are largely unknown. We aimed to analyze and compare concentrations of nicotine, nitrosamines, and humectants in legal and illegal cigarettes collected from a representative sample of smokers. METHODS: Participants of the International Tobacco Control cohort provided a cigarette pack of the brand they smoked during the 2014 wave. Brands were classified as legal or illegal according to the Mexican legislation. Nicotine, nitrosamines, glycerol, propylene glycol, and pH were quantified in seven randomly selected packs of each brand. All analyses were done blinded to legality status. Average concentrations per brand and global averages for legal and illegal brands were calculated. Comparisons between legal and illegal brands were conducted using t tests. RESULTS: Participants provided 76 different brands, from which 6.8% were illegal. Legal brands had higher nicotine (15.05 ± 1.89 mg/g vs 12.09 ± 2.69 mg/g; p < 0001), glycerol (12.98 ± 8.03 vs 2.93 ± 1.96 mg/g; p < 0.001), and N-nitrosanatabine (NAT) (1087.5 ± 127.0 vs 738.5 ± 338 ng/g; p = 0.006) concentrations compared to illegal brands. For all other compounds, legal and illegal brands had similar concentrations. CONCLUSION: Compared to illegal cigarettes, legal brands seem to have higher concentrations of nicotine, NAT, and glycerol. Efforts must be made to implement and enforce Article 10 of the Framework Convention on Tobacco Control to provide transparent information to consumers, regulators, and policy-makers; and to limit cigarette engineering from the tobacco industry.

Asunto(s)

Higroscópicos/análisis , Nicotina/análisis , Agonistas Nicotínicos/análisis , Nitrosaminas/análisis , Productos de Tabaco/análisis , Comercio/legislación & jurisprudencia , Crimen , México , Productos de Tabaco/legislación & jurisprudencia

RESUMEN

Tobacco-specific nitrosamines (TSNAs), mainly the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are known carcinogens. Part of the NNK found in smoke is provided from matrix-bound NNK, and its determination is extremely relevant. However, the reference extraction procedure of matrix-bound NNK is time-consuming and labor-intensive and has a limited analytical capacity. Three different methodologies were proposed to predict matrix-bound NNK: simple linear regression (LR) with soluble NNK; multiple linear regression (MLR) considering soluble NNK and characteristic parameters of the samples; and orthogonal partial least-squares (O-PLS) regression using high-throughput screening by flow injection analysis coupled to high-resolution mass spectrometry (HTS-FIA-HRMS) data. Simple linear regression showed a high influence of matrix and leaf origin. Although an existing linearity trend has been observed ( R2 = 0.62) for the global model, higher correlation values were achieved for matrix and country segregation models. Multiple linear regression predicted matrix-bound NNK with more satisfactory efficiency than simple linear regression models. The coefficients of determination were 0.87 and 0.94 for flue-cured Virginia and air-cured Burley, respectively. However, this method has a limited application, since previous information about the sample is required. The proposed method based on HTS-FIA-HRMS and O-PLS has shown the most suitable performance in the prediction of matrix-bound NNK, with errors comparable to the reference method, and a higher throughput. In addition, this approach allows to determine other soluble nitrosamines, namely N'-nitrosoanatabine, N'-nitrosoanabasine, and N-nitrosonornicotine, with relative percentage errors between 5.25 and 11.98%. Therefore, the third approach is the best method for a large number of cured tobacco for accuracy in determination of TSNAs.

Asunto(s)

Carcinógenos/análisis , Nicotiana/química , Nitrosaminas/análisis , Análisis de Inyección de Flujo/métodos , Análisis de los Mínimos Cuadrados , Espectrometría de Masas/métodos

RESUMEN

A simple, accessible and reproducible method was developed and validated as an alternative for the determination of nine volatile N-nitrosamines (NAs) in meat products, using a low volume of organic solvent and without requiring specific apparatus, offering the possibility of practical implementation in routine laboratories. The NAs were extracted with dichloromethane followed by a clean-up with phosphate buffer solution (pH 7.0). The extracts were analysed by gas chromatography-chemical ionisation/mass spectrometry (GC-CI/MS) in positive-ion mode using methanol as reagent. Limits of detection and quantification, recovery and reproducibility were determined for all NAs (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosodipropylamine, N-nitrosomorpholine, N-nitrosopiperidine, N-nitrosodibutylamine and N-nitrosodiphenylamine). Satisfactory sensitivity and selectivity were obtained even without concentrating the extract by solvent evaporation, avoiding the loss of the nine NAs studied. Limits of detection ranged from 0.15 to 0.37 µg kg(-1), whereas limits of quantification ranged from 0.50 to 1.24 µg kg(-1). Recoveries calculated in cooked ham that had been spiked at 10 and 100 µg kg(-1) were found to be between 70% and 114% with an average relative standard deviation of 13.2%. The method was successfully used to analyse five samples of processed meat products on the day of purchase and 7 days later (after storage at 4°C). The most abundant NAs found in the analysed products were N-nitrosodipropylamine and N-nitrosopiperidine, which ranged from 1.75 to 34.75 µg kg(-1) and from 1.50 to 4.26 µg kg(-1), respectively. In general, an increase in the level of NAs was observed after the storage period. The proposed method may therefore be a useful tool for food safety control once it allows assessing the profile and the dietary intake of NAs in food over time.

Asunto(s)

Aditivos Alimentarios/análisis , Cromatografía de Gases y Espectrometría de Masas , Productos de la Carne/análisis , Metanol/química , Nitrosaminas/análisis , Cloruro de Metileno/química , Fosfatos/química , Soluciones

RESUMEN

MR-CISD, MR-CISD+Q, and MR-AQCC calculations have been performed on the minima and transition states (corresponding to intramolecular proton transfer between the protonation sites) of the ground state of protonated nitrosamine and N,N-dimethylnitrosamine. Our highest level results (MR-AQCC/cc-pVTZ) for the smaller system indicate that protonation on the N amino (2a) is practically as favorable as the most favorable protonation on the O atom (1a). They also suggest that protonation on the nitroso N atom (2c) is ∼14.5 kcal/mol less favorable than 1a. Results obtained at the MR-CISD+Q/cc-pVTZ level indicate that the effect of methylation on the relative energies of the tautomers is, in order of importance, 2a > 2c and increases their energies by ∼17.5 and 4.8 kcal/mol, respectively. They also indicate that methylation alters significantly the intramolecular proton transfer barriers. The largest differences between the common geometric parameters of both systems have been found for 2a.

Asunto(s)

Modelos Químicos , Nitrosaminas/química , Protones , Metilación , Teoría Cuántica , Estereoisomerismo , Termodinámica

RESUMEN

Vegetable consumption is associated with a lower risk of suffering cardiovascular disease and cancer, traditionally ascribed to their content in antioxidants. Recently it has been proposed that this effect may be due to the presence of nitrates in vegetables. Cardiovascular diseases are usually associated with an impaired production of nitric oxide (NO) in blood vessels. One strategy aimed to correct this defective production is to generate NO from inorganic nitrates contained in vegetables. However, it has been traditionally thought that nitrites and nitrites, as those used in curing meats and as food preservers, could generate adducts that may increase cancer risk. This paradigm is now being revisited since the evidence that vegetables rich in nitrates may have a beneficial impact on human health, particularly on cardiovascular parameters, has showed promising results, although more complete clinical evidence is needed.

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El consumo de vegetales está asociado a un menor riesgo de desarrollar enfermedades cardiovasculares y cáncer, efecto que tradicionalmente se le ha atribuido a su contenido en antioxidantes. Recientemente se ha propuesto que parte de estos efectos se deberían al contenido de nitratos en los vegetales. La enfermedades cardiovasculares se asocian a una disminución en la producción del óxido nítrico (NO). Una estrategia para corregir esta producción defectuosa es generar NO a partir de nitratos inorgánicos contenidos en los alimentos. Sin embargo, se ha propuesto que los nitratos, como los usados en el proceso de curación de carnes y preservación de alimentos, podrían generar aductos cancerígenos. Este paradigma está siendo revisado y la evidencia que los alimentos ricos en nitratos pudieran tener efectos benéficos sobre la salud, particularmente sobre parámetros cardiovasculares, ha generado resultados alentadores, aunque aun se necesitan estudio clínicos más completos.

Asunto(s)

Humanos , Enfermedades Cardiovasculares/prevención & control , Hipertensión , Nitratos , Óxido Nítrico , Nitritos , Nitrosaminas

RESUMEN

Rapé, a diverse group of smokeless tobacco products indigenous to South America, is generally used as a nasal snuff and contains substantial amount of plant material with or without tobacco. Previously uncharacterized, rapé contains addictive and harmful chemicals that may have public health implications for users. Here we report % moisture, pH, and the levels of total nicotine, un-ionized nicotine, flavor-related compounds, tobacco-specific N-nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) for manufactured and hand-made rapé. Most rapé products were mildly acidic (pH 5.17-6.23) with total nicotine ranging from 6.32 to 47.6 milligram per gram of sample (mg/g). Calculated un-ionized nicotine ranged from 0.03 to 18.5 mg/g with the highest values associated with hand-made rapés (pH 9.75-10.2), which contain alkaline ashes. In tobacco-containing rapés, minor alkaloid levels and Fourier transform infrared spectra were used to confirm the presence of Nicotiana rustica, a high nicotine tobacco species. There was a wide concentration range of TSNAs and PAHs among the rapés analyzed. Several TSNAs and PAHs identified in the products are known or probable carcinogens according to the International Agency for Research on Cancer. Milligram quantities of some non-tobacco constituents, such as camphor, coumarin, and eugenol, warrant additional evaluation.

Asunto(s)

Nicotina/análisis , Nitrosaminas/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Tabaco sin Humo/análisis , Alcaloides/análisis , Brasil , Cinnamomum zeylanicum/química , Eugenol/análisis , Cromatografía de Gases y Espectrometría de Masas , Concentración de Iones de Hidrógeno , Mentol/análisis , Nicotina/química , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría de Masas en Tándem , Nicotiana/química

RESUMEN

The detection of an oxygen-atom photoexchange process of N-nitrosamines is reported. The photolysis of four nitrosamines (N-nitrosodiphenylamine 1, N-nitroso-N-methylaniline 2, N-butyl-N-(4-hydroxybutyl)nitrosamine 3, and N-nitrosodiethylamine 4) with ultraviolet light was examined in an (18)O2-enriched atmosphere in solution. HPLC/MS and HPLC-MS/MS data show that (18)O-labeled nitrosamines were generated for 1 and 2. In contrast, nitrosamines 3 and 4 do not exchange the (18)O label and instead decomposed to amines and/or imines under the conditions. For 1 and 2, the (18)O atom was found not to be introduced by moisture or by singlet oxygen [(18)((1)O2 (1)Δg)] produced thermally by (18)O-(18)O labeled endoperoxide of N,N'-di(2,3-hydroxypropyl)-1,4-naphthalene dipropanamide (DHPN(18)O2) or by visible-light sensitization. A density functional theory study of the structures and energetics of peroxy intermediates arising from reaction of nitrosamines with O2 is also presented. A reversible head-to-tail dimerization of the O-nitrooxide to the 1,2,3,5,6,7-hexaoxadiazocane (30 kcal/mol barrier) with extrusion of O═(18)O accounts for exchange of the oxygen atom label. The unimolecular cyclization of O-nitrooxide to 1,2,3,4-trioxazetidine (46 kcal/mol barrier) followed by a retro [2 + 2] reaction is an alternative, but higher energy process. Both pathways would require the photoexcitation of the nitrooxide.

Asunto(s)

Aminas/química , Nitrosaminas/química , Oxígeno Singlete/química , Estructura Molecular , Procesos Fotoquímicos , Espectrometría de Masas en Tándem , Rayos Ultravioleta

RESUMEN

Nuclear receptor subfamily 1, group I, member 3 (NR1I3) is reported to be a possible novel therapeutic target for some cancers, including lung, brain and hematopoietic tumors. Here, we characterized expression of NR1I3 in a mouse model of lung carcinogenesis induced by 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone (NNK), the most potent tobacco carcinogen. Lung tumors were collected from mice treated with NNK (400 mg/kg) and euthanized after 52 weeks. Benign and malignant lesions were formalin-fixed and paraffin-embedded for histology and immunohistochemistry, with samples snap-frozen for mRNA analysis. Immunohistochemically, we found that most macrophages and type I and II pneumocytes expressed NR1I3, whereas fibroblasts and endothelial cells were NR1I3-. Compared with benign lesions, malignant lesions had less NR1I3+ tumor cells. Gene expression analysis also showed an inverse correlation between NR1I3 mRNA expression and tumor size (P=0.0061), suggesting that bigger tumors expressed less NR1I3 transcripts, in accordance with our immunohistochemical NR1I3 tests. Our results indicate that NR1I3 expression decreased during progression of malignant lung tumors induced by NNK in mice.

Asunto(s)

Neoplasias Pulmonares/metabolismo , Nitrosaminas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Receptor de Androstano Constitutivo , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Ratones , Neoplasias Experimentales , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética

RESUMEN

Lung cancer is the leading cause of cancer-related mortality in both men and women throughout the world. This disease is strongly associated with tobacco smoking. The aim of this manuscript was to establish an in vitro model that mimics the chronic exposures of alveolar epithelial type II cells to the tobacco-specific nitrosamine carcinogen, NNK. Immortalized non-neoplastic alveolar epithelial cells type II, (E10 cells), from BALB/c mice were exposed to low concentration of NNK (100 pM) during 5, 10, 15, and 20 cycles of 48 h. NNK-transformed cells showed an increase of proliferation rate and motility. Moreover, these cells underwent epithelial-to-mesenchymal transition (EMT). Increased migratory capacity and EMT were correlated to the time of exposure to NNK. NNK-transformed cells were tested for their growth and metastatic capacity in vivo. Subcutaneous injection of cells exposed to NNK for 20 cycles (E10-NNK20 clone) into BALB/c mice led to the formation of subcutaneous tumors that arose after 40 ± 17 d in all animals, which died 95 ± 18 d after cell inoculation, with lymph nodes and lung metastasis. The morphological characteristics of tumors were compatible with metastatic undifferentiated carcinoma. Cells exposed to NNK for 5-10 cycles did not display metastatic capacity, while those exposed for 15 cycles displayed low capacity. Our results show that prolonged exposures to NNK led the cells to increasingly acquire malignant properties. The cellular model presented in this study is suitable for studying the molecular events involved in the different stages of malignant transformation.

Asunto(s)

Carcinógenos/toxicidad , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Pulmonares/patología , Nicotiana , Nitrosaminas/toxicidad , Alveolos Pulmonares/patología , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas para Inmunoenzimas , Técnicas In Vitro , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Cicatrización de Heridas

RESUMEN

Agaricus brasiliensis currently is one of the most studied fungi because of its nutritional and therapeutic properties as an anti-inflammatory agent and an adjuvant in cancer chemotherapy. The effects of orally administered aqueous A. brasiliensis extract (14.3- and 42.9-mg doses) on parenchymal lung damage induced by carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were observed in Wistar rats. NNK treatment induced pulmonary inflammation, but not lung cancer, in the rats. The lungs of animals treated with NNK showed a higher level of inflammation than those of the control group according to histopathologic examinations (P < 0.01) and kurtosis analysis (P < 0.001) of a global histogram generated from thoracic computed tomography scans. There was no significant difference in the alveolar and bronchial exudates between animals treated with a 14.3-mg dose of A. brasiliensis extract and the control without NNK. However, a significant difference was found between animals treated with NNK, received a 42.9-mg dose of A. brasiliensis (P < 0.05), and the controls not treated with NNK. We did not observe a significant difference between the kurtoses of the A. brasiliensis (14.3 mg) and control groups. However, a 42.9-mg dose of A. brasiliensis resulted in lower kurtosis values than those observed in the control group (P < 0.001). In conclusion, a low dose of A. brasiliensis was more effective in attenuating pulmonary inflammation. Similar to the histopathological results, the computed tomography scans also showed a protective effect of A. brasiliensis at the lower dose, which prevented gross pulmonary consolidation.

Asunto(s)

Agaricus/química , Inflamación/inducido químicamente , Enfermedades Pulmonares/inducido químicamente , Nitrosaminas/toxicidad , Animales , Antiinflamatorios no Esteroideos , Masculino , Ratas , Ratas Wistar

RESUMEN

Nitrosamines are well known for their carcinogenic potential. Recently, it was found that some of them may also interact with human nicotinic acetylcholine receptor (nAChR) subtypes. This work studied the effects of N-nitrosonornicotine (NNN) on recombinant rat α3ß4 nAChR in HEK cells as well as on nAChR endogenously expressed in PC12 pheochromocytoma cells and in BC3H1 muscle-type cells. Whole-cell recording in combination with the cell-flow technique for agonist and inhibitor application in the millisecond time region revealed that NNN inhibits the activity of neuronal nAChR expressed in HEK or PC12, whereas weak inhibitory effects on muscle-type nAChR were observed at NNN concentrations up to 3 mM. Pharmacological actions of NNN and the inhibition mechanism were studied in detail using recombinant α3ß4 nAChR expressed in HEK cells as a model. NNN-induced inhibition of nicotine-evoked α3ß4 nAChR activity was dose-dependent with an inhibitory constant (IC(50)) of 0.92 ± 0.05 mM. Analysis based on mathematical models indicated a noncompetitive inhibition mechanism of the rat α3ß4 nAChR by NNN. NNN's mechanism of action involves acceleration of conversion of the receptor from active to desensitized forms. In summary, this work shows that NNN inhibits rat α3ß4 nAChR in a noncompetitive way and interacts weakly with muscular nAChR.

Asunto(s)

Antagonistas Nicotínicos/farmacología , Nitrosaminas/farmacología , Receptores Nicotínicos/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Agonistas Nicotínicos/farmacología , Células PC12 , Ratas

RESUMEN

Connexins (Cxs) are proteins that form the communicating gap junctions, and reportedly have a role in carcinogenesis. Here, we evaluated the importance of Connexin43 (Cx43) in spontaneous and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis. Male wild-type (Cx43(+/+) ) and hemizygote (Cx43(+/-) ) CD1 × AJ F1 mice were injected with NNK or saline. After 60 weeks mice were euthanized; lung nodules were counted, measured, and fixed in formalin or snap frozen. Immunohistochemistry for Cx43 and Beta-catenin (ß-catenin) was performed and Cx43 mRNA expression was evaluated by real-time PCR. Cx43 deletion significantly increased the incidence and number of spontaneous nodules in the CD1 × AJ F1 mice and the number of gross lesions and the aggressiveness of lesions in NNK-treated mice. Cx43 mRNA increased significantly and was correlated with the aggressiveness of tumors, although lesions from Cx43(+/-) mice expressed less Cx43 RNAm than their counterparts. Lung parenchyma presented a Cx43 immunostaining pattern with points or plaques between cells. In hyperplasias and adenomas, Cx43 was found in the membrane and in cytoplasm. Malignant lesions presented increased Cx43 in cytoplasm and a few membrane spots of immunostaining. ß-catenin was weakly expressed in lung parenchyma. Though hyperplasias presented some cells with nuclear ß-catenin, NNK-induced tumors contained a higher number of this staining pattern. Also, no difference in ß-catenin occurred between both genotypes independently of the histological grade. In summary, our results indicate that Cx43 acts as a tumor suppressor gene in early lung tumorigenesis and loses this property in advanced carcinogenesis. Therefore, Cxs are better classified as conditional tumor suppressors.

Asunto(s)

Carcinógenos/toxicidad , Transformación Celular Neoplásica/patología , Conexina 43/fisiología , Neoplasias Pulmonares/etiología , Nitrosaminas/toxicidad , beta Catenina/metabolismo , Animales , Western Blotting , Susceptibilidad a Enfermedades , Femenino , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta Catenina/genética

RESUMEN

Neste trabalho foram avaliados os teores de nitratos e nitritos em queijos expostos à venda no Estado de Minas Gerais em 2009. As análises qualitativas e quantitativas foram realizadas em 77 amostras de queijos: Minas Frescal, Mussarela, Parmesão e Prato, coletadas pela Vigilância Sanitária por meio de PROMAC –Programa de Monitoramento de Aditivos e Contaminantes. Do total de amostras de queijo Minas Frescal, 7 apresentaram não conformidade com a legislação brasileira, em função da presença de nitrato. Os teores de nitrato estavam acima do limite estabelecido pela legislação em 18 das amostras de queijo Parmesão. No queijo Prato foram encontradas 10 das amostras com teor de nitrato acima do limite máximo permitido. Todas as amostras de queijo Mussarela avaliadas estavam em conformidade com a legislação. Em todas as amostras de queijo analisadas neste estudo não houve ocorrência de nitrito.(AU)

Asunto(s)

Legislación Alimentaria , Queso , Nitratos , Nitritos , Nitrosaminas