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Oral lesions, genotoxicity and nitrosamines in betel quid chewers with no obvious increase in oral cancer risk.

A link between the generation of areca nut-related N-nitrosamines in the saliva, the induction of genotoxic damage in the oral mucosa, as judged by an increase in micronucleated exfoliated cells (MEC), and a low incidence of oral cancer was studied in 2 population groups characterized by their habit of chewing quids without tobacco: Guamanians, who chew areca nuts (Areca catechu) with or without the addition of betel leaf (Piper betle); Taiwanese, who use areca nut, betel leaf or inference and slaked lime. The levels of N-nitrosoguvacoline (NG) in the saliva of chewers of fresh green areca nuts were very high (70.8 ng/ml) as compared to those reported for individuals using the more complex Indian betel quids (0.91 ng/ml or 5.6 ng/ml). None of the other areca nut-related nitrosamines (N-nitrosoguvacine (NGC), 3-(methylnitrosamino)propionitrile (MNPN) and 3-(methylnitrosamino)propionaldehyde (MNPA)) were detected in the saliva of Taiwanese betel quid chewers. The addition of slaked lime to the areca nut enhances the formation of NG during a chewing session. The frequency of MEC did not increase in the oral mucosa of areca nut chewers who do not use slaked lime, but showed a small but significant elevation in individuals using lime-containing quids. The elevation of MEC in Taiwanese, who are at low risk for oral cancer, is relatively small as compared to that found in chewers of Indian betel quids (pan), who show a highly elevated oral cancer risk. The results seem to suggest that NG may play only a minor role, if any, in the etiology of oral cancer among betel quid chewers.

Areca↗

Tobacco-specific nitrosamines in the saliva of Inuit snuff dippers in the Northwest Territories of Canada.

Levels of tobacco-specific nitrosamines (TSNA), nicotine and cotinine were estimated in the saliva of 20 snuff dippers (Inuit, Northwest Territories, Canada). Levels of N'-nitrosonornicotine (NNN), 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosoanatabine (NAT) plus N-nitrosoanabiasine (NAB) found in the saliva following a 15-min period of keeping 0.5-1.5 g of moist snuff in the gingival groove are considerable: 115-2610 ppb NNN, 123-4560 ppb NAT + NAB, and up to 201 ppb NNK. The amount of TSNA in the saliva increases with the length of time that the snuff is kept in the mouth. The estimated total amount of 444 micrograms TSNA, the largest part of which will be swallowed, exceeds by far the amounts of nitrosamines ingested through drinking beer (0.34 micrograms/day), eating cured meat products (0.17 micrograms/day), or using cosmetics (0.41 micrograms/day). The relatively high levels of potentially carcinogenic TSNA in the saliva, together with the current popularity of snuff usage by teenagers, is of particular concern.

Canada↗

Occurrence of volatile N-nitrosamines in dried chillies.

Twenty samples of dried chillies, fresh chillies and chilli sauces have been analysed for the presence of volatile N-nitrosamines. Over 75% of both dried whole chillies and chilli powder samples analysed contained N-nitrosodimethylamine and N-nitrosopyrrolidine (NPYR) at concentrations of 0.66-16.5 micrograms/kg and 0.48-6.0 micrograms/kg, respectively. Fresh chillies and chilli sauces were not found to contain volatile N-nitrosamines.

Condiments↗

A study of tobacco carcinogenesis. XLII. Bioassay in A/J mice of some structural analogues of tobacco-specific nitrosamines.

The tumorigenic activities in A/J mouse lung of the tobacco-specific nitrosamines, N'-nitrosonornicotine (NNN), 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and several structural analogues were evaluated. The analogues were N-nitrosopyrrolidine (NPYR), 5'-carboxy-N'-nitrosonornicotine (CNNN), N-nitrosoproline (NPRO) and 1-(3-pyridyl)-2-buten-1-one (PBO). The results were as follows (dose in mumol per mouse/lung tumors per mouse): NNN (100/1.8 +/- 1.4); NPYR (100/3.9 +/- 1.5); CNNN (200/0.3 +/- 0.5); CNNN (100/0.5 +/- 0.6); NPRO (100/0.6 +/- 0.7); NNK (20/7.2 +/- 3.4); PBO (20/0.7 +/- 1.0); saline control (0.0.5 +/- 0.7). Several conclusions were drawn from this assay. NNK and NPYR were more tumorigenic than NNN. CNNN was non-tumorigenic and thus appears to have potential as a monitor for endogenous formation of tobacco-specific nitrosamines. The alpha,beta-unsaturated ketone PBO does not appear to be an ultimate tumorigen of NNK or NNN.

Adenoma↗

A study of tobacco carcinogenesis XLIV. Bioassay in A/J mice of some N-nitrosamines.

The evaluation of the tumorigenic activity in A/J mouse lung of certain tobacco N-nitrosamines, namely 3-(methylnitrosamino)propionic acid (NMPA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-4-(3-pyridyl)-butyric acid (iso-NNAC), had the following results (total dose in micromol per mouse/lung tumors per mouse): NMPA (200/7.1 +/- 2.9); NNK (2/15.7 +/- 4.1); iso-NNAC (200/0.24 +/- 0.43); saline control (0.2 +/- 0.4). The tumorigenic activity of NMPA was not surprising since its lower homologue, N-nitrososarcosine, as well as its higher homologue, 4-(methylnitrosamino)-butyric acid, are known carcinogens. The high tumorigenic activity of NNK in strain A/J mice confirms earlier findings as to its carcinogenic potency in rats and hamsters. The lack of tumorigenic activity of iso-NNAC supports the observation that the pyridyl rest adjacent to the nitrosamino group inhibits the activity of some tobacco-specific N-nitrosamines (TSNA). Iso-NNAC is most likely formed endogenously from the nicotine metabolites cotinine and 4-(methylamino)-4-(3-pyridyl)butyric acid.

Animals↗

Involvement of lauric acid hydroxylase in the activation of beta-substituted nitrosamines.

The mutagenicity of N-nitrosobis (2-hydroxypropyl) amine (BHP), N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso-(2-hydroxy-propyl) (2-oxopropyl) amine (HPOP) was measured in V79 cells. Hepatocytes, used to metabolize (activate) the nitrosamines, were isolated from untreated Syrian hamsters (control) and hamsters treated with clofibrate (CLO) or dehydroepiandrosterone (DHEA) in vivo. BHP and HPOP mutagenicity increased 3- and 2-fold when hepatocytes from CLO- and DHEA-treated hamsters were used. BOP mutagenicity did not increase. 10-Undecynoic acid, a lauric acid hydroxylase inhibitor, inhibited the increase in BHP and HPOP mutagenicity by 80-90% but did not affect that of BOP. Antimycin A1, a fatty acyl coenzyme A beta-oxidase inhibitor did not affect the mutagenicity of these nitrosamines. Lauric acid hydroxylase, probably omega-1 hydroxylase (cytochrome P-450 IVA2), appears to be involved in the activation of BHP and HPOP.

Acyl-CoA Oxidase↗

N-nitrosamines and their effects on the level of glutathione, glutathione reductase and glutathione S-transferase activities in the liver of male mice.

The present study investigates the influence of different chemical structure of N-nitroso compounds on the hepatic level of reduced glutathione (GSH), glutathione reductase (GSH-R) and glutathione S-transferase (GST) activities in the liver of male Balb/C mice after treatment with 20 mg/kg body weight of each compound for 1 h as a single dose. The level of reduced glutathione decreased significantly between 37 and 70% after the treatment of male mice with ethylbutylnitrosamine (-37%), diphenylnitrosamine (-50%), propylbutylnitrosamine (-52%), diethylnitrosamine (-54%), ethylmethylnitrosamine (-55%), and dibutylnitrosamine (-70%), whereas, methylpropylnitrosamine increased the level of GSH by 71%. All the N-nitrosamine compounds tested increased the activity of glutathione reductase except ethylmethylnitrosamine had no effect. The activity of glutathione S-transferase activity was inhibited after treatment of the male mice with diphenylnitrosamine (-60%), dibutylnitrosamine (-60%), and methylpropylnitrosamine (-81%), while, ethylmethylnitrosamine and ethylbutylnitrosamine had no effect on such activity. On the other hand, diethylnitrosamine increased the activity of glutathione S-transferase by 50%. It can be postulated from this study that the chemical structure of N-nitrosamines plays a significant role in the alteration of GSH level and GSH metabolizing enzymes, since the substitution of different groups on the nitroso group was found to be capable of causing an alteration in such activities.

Animals↗

A critical look at N-nitrosamines in environmental tobacco smoke.

The amounts of cigarette smoke carcinogens released into the environment as sidestream smoke (SS) constituents are generally twice as high as the levels of the carcinogens that are inhaled as mainstream smoke (MS). However, certain carcinogens in undiluted SS, such as nitrosamines, exceed MS levels up to 50 times. Regardless of the fact that SS is usually substantially diluted before being inhaled, its constituents can be determined in environmental tobacco smoke (ETS) by modern analytical methods and levels of exposure of involuntary smokers can be assessed by the use of specific markers. Currently, the uptake of ETS by nonsmokers is determined by measuring nicotine and its metabolite cotinine in saliva, serum and/or urine. In on-going studies, we are now exploring the determination of the two highly carcinogenic nicotine-derived nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), respectively of their metabolites, in physiological fluids of smokers and nonsmokers.

Animals↗

The potential impact of a low-nitrosamine smokeless tobacco product on cigarette smoking in the United States: estimates of a panel of experts.

OBJECTIVE: To predict the impact on tobacco use in the US of a "harm reduction" policy that requires that the smokeless tobacco product meet low nitrosamine standards, but could be marketed with a warning label consistent with the evidence of relative health risks. METHODS: Low nitrosamine smokeless tobacco (LN-SLT) and cigarette use are predicted by a panel of experts using a modified Delphi approach. We specify a thought experiment to isolate the changes that would occur after the new LN-SLT policy was implemented. RESULTS: The panel predicted that the new policy would accelerate a decrease in smoking prevalence from 1.3 to 3.1 percentage points over 5 years compared to the current SLT product policy, with greater effects on males than females. Introduction of the new product was also predicted to result in modest additional use of SLT overall, with the greatest increases among males who initiated tobacco use under the new policy. CONCLUSION: An overall consensus was reached that the introduction of a new LN-SLT product under strict regulations would increase SLT use, but reduce overall smoking prevalence. This reduction would likely yield substantial health benefits, but uncertainties surround the role of marketing and other tobacco control policies.

Adolescent↗

Synthesis and properties of bifunctional chloroalkyl nitrosamines with an intercalating moiety.

Three N-nitroso-N-(arylcarbonyloxymethyl)-3-chloropropylamines were synthesized, and their chemical and biological properties were studied. All arylcarboxylates intercalated with double-stranded DNA, and their mutagenicity and DNA cross-linking activity were affected by their ring structure. The DNA interstrand cross-link formation increased dose dependently after treatment with the acridine analog. The anthraquinone analog showed the highest bacterial mutagenicity among the three nitrosamines in Salmonella typhimurium TA100, while in Salmonella typhimurium TA92, which can detect cross-linking agents, the acridine analog showed the highest mutagenicity. This agreed with the result of a cross-linking assay. These results suggest that the three-ring aromatic moiety gives DNA-intercalating ability to cross-linkable chloropropyl nitrosamine, and the acridine analog is considered as a possible new antitumor lead compound.

Intercalating Agents↗

No volatile N-nitrosamines detected in blood and urine from patients ingesting daily large amounts of ammonium nitrate.

Whole blood and urine from 23 patients ingesting ammonium nitrate daily in amounts varying from 2 . 5-9 g were analysed for volatile N-nitrosamines. By contrast with reports in the literature N-nitrosodimethylamine and N-nitrosodiethylamine were not found in blood (limit of detection 0 . 1 micrograms/kg) but a trace of N-nitrosopyrrolidine was found in the blood of one subject. Small amounts of N-nitrosomorpholine in some blood samples and in one blank were believed to be formed during the analysis since morpholine had been added to the samples to indicate artefactual nitrosamine formation. Traces of N-nitrosodimethylamine (0 . 1 microgram/kg) were found in six of the 23 urine samples.

Adolescent↗

Detection and determination of N-nitrosamines by thin-layer chromatography using fluorescamine.

A novel procedure is described for the detection and determination of N-nitrosamines (NAs) on thin-layer chromatographic plates. Ultraviolet irradiation of NAs on activated plates (silica gel or aluminum oxide) yields primary or secondary amines, which after spraying with fluorescamine reagent give fluorescent or non-fluorescent products, respectively. The majority of the 24 NAs examined afforded NAs, although the volatile dimethyl-, diethyl- and pyrrolidine-N-nitrosamines gave limits of 500, 40, and 40 ng, respectively. Spectrophotometric determinations of the relative fluorescence of 0.1-40 nmoles of NAs gave rise to nearly linear calibration curves when plotted on a log-log scale.

Chromatography, Thin Layer↗

Fast microwave-assisted dansylation of N-nitrosamines. Analysis by high-performance liquid chromatography with fluorescence detection.

A fast microwave-assisted dansylation procedure has been developed for the derivatization of N-nitrosamines prior to high-performance liquid chromatography determination. N-Nitrosomorpholine, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine and N-nitrosopiperidine are first denitrosated by hydrobromic acid-acetic acid to produce secondary amines, which are then quantitatively dansylated in 5 min using radiation power of 378 W and a maximum pressure of 1.4 bar inside the reactor. The reaction mixture is separated on a C18 column with acetonitrile-water (55:45, v/v) as mobile phase with fluorimetric detection at 531 nm (excitation at 339 nm). The detection limits range from 8 to 75 pg for N-nitrosomorpholine and N-nitrosodiethylamine, respectively. The method was applied to study the recoveries of N-nitrosamines in beer and their determination in cigarette smoke.

Chromatography, High Pressure Liquid↗

Dual column gas chromatographic system for use in mass spectral determination of nitrosamines.

A packed gas chromatographic column and a support coated open tubular (SCOT) column are connected in series. Between the columns are two micro-volume switching valves, one enabling solvent to be vented. Short retention nitrosamines are passed through both columns, whereas longer retention nitrosamines by-pass the SCOT column by means of the other switching valve.

Chromatography↗

The estimation of N-nitrosamines in tropical regions by reversed-phase paper and thin-layer chromatography.

A simple and convenient procedure based on reversed-phase paper and thin-layer chromatography is reported for the detection and estimation of N-nitrosamines in food products, particularly cured meat. The method can easily be applied in tropical regions when plenty of sunshine is available. It is particularly useful in food-testing laboratories where sophisticated analysical instruments such as the gas chromatograph, mass spectrometer, etc., are not available. By this procedure, it is possible to detect up to 10 mug and to estimated up to 75-mug (reversed phase) and 50-mug (thin layer) amounts of N-nitrosamines.

Chromatography, Paper↗

Determination of N-nitrosamines in latex by sequential supercritical fluid extraction and derivatization.

A new method to determine N-nitrosamines in latex products has been developed by combination of supercritical fluids and chemical derivatization. A new design for a liquid trap has been introduced. A factorial fractional design was used in order to evaluate the influence of the different factors affecting the process. Factors such as pressure, temperature, static and dynamic time, restrictor temperature and volume of an hydrobromic acid-acetic anhydride mixture (1:10, v/v) were included in the design. CO2 was used as the extraction fluid. Gas chromatography with nitrogen and phosphorus sensitive detection was employed to achieve good sensitivity attending to the molecular structure of these compounds (N-nitrosamines and their corresponding secondary amines). The obtained results have shown to be useful to increase selectivity and reduce sample handling.

Chromatography, Gas↗

Analysis of tobacco-specific N-nitrosamines in snuff by ethyl acetate extraction and liquid chromatography-tandem mass spectrometry.

A rapid, selective and sensitive method for routine analysis of the four tobacco-specific N-nitrosamines, N'-nitrosonornicotine, N'-nitrosoanatabine, N'-nitrosoanabasine and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone in snuff has been developed. The nitrosamines were isolated by ethyl acetate extraction and analysed by LC-MS-MS. Except for evaporation and filtration, no additional clean-up steps are needed in the proposed method. The detection limits for standard in solvent are between 0.0005 and 0.001 microg/ml (0.005 and 0.01 microg/g).

Acetates↗

Nitrosamine excretion in patients with continent ileal reservoirs for urinary diversion.

Adenocarcinomas are a recognized complication following ureterosigmoidostomy for which the endogenous formation of N-nitroso compounds may be a risk factor. As an alternative means of urinary diversion, the continent ileal reservoir has recently been developed. Microbiological and chemical investigations on the urine of patients with an ileal reservoir showed the presence of bacteria, nitrate, nitrite and N-nitrosamines formed endogenously in the ileal pouch. The role of nitrosamines in carcinogenesis in these patients as a late stage complication resulting from the use of a continent ileal reservoir is discussed.

Adult↗