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Structural requirements, synthesis and interaction of secondary nitrosamine inhibitors with acetylcholinesterase from Torpedo fuscomaculata.

A series of secondary diaryl and dialkyl nitrosamines have been synthesised and tested as substrates and/or inhibitors of highly purified acetyl- cholinesterase from Torpedo fuscomaculata. None were found to act as substrate, but many could selectively inhibit the enzyme. Kinetic analysis has shown that all the nitrosamines act as reversible competitive inhibitors with respect to the substrate, acetylthiocholine chloride; with time they act as irreversible covalent inhibitors. Scatchard analysis indicates that aliphatic nitrosamines have a weaker affinity for the enzyme compared to the aromatic and heterocyclic nitrosamines. In all cases the number of binding sites was four. Pseudo first-order kinetics are observed with the rate constant being proportional to the concentration of the nitrosamine and the order of reaction being equal to one.

Acetylcholinesterase↗

[Nitrosamine formation from vegetables produced in Japan].

Many vegetables produced in Japan contain large amounts of nitrate that may be converted into nitrite in the oral cavity and afford carcinogenic nitrosamines in the stomach. On the other hand, vegetables contain ascorbate and other components that may affect the formation of nitrosamines. In this study, nitrosamine formation from vegetables with high nitrate content produced in Japan was examined under simulated oral cavity and stomach conditions. Extracts of chingensai, komatsuna and itomitsuba were digested with nitrate reductase and subsequently treated with an excess of morpholine at pH 3.0. The amount of N-nitrosomorpholine produced from each of the vegetable extracts was not affected by the vegetable components in the extracts. Addition of a large amount of ascorbate was required to decrease nitrosamine formation from the extract. The results indicated that nitrosamine formation from these vegetables could not be prevented by other components in the vegetables.

Ascorbic Acid↗

Effect of modified atmosphere packaging and irradiation in combination on content of nitrosamines in cooked pork sausage.

The effect of modified atmosphere packaging and irradiation in combination on nitrosodimethylamine (NDMA) and nitrosopyrrolidine (NPYR) levels in pork sausage was studied. Emulsion-type cooked pork sausage was manufactured and packaged in aerobic, CO2 (100%), N2 (100%), and CO2/N2 (25%/75%) environments, respectively, and irradiated at 0, 5, 10, and 20 kGy with gamma irradiation. The nitrosamine contents were significantly reduced by irradiation, and the reduction of nitrosamines was more extensive with modified atmosphere packaging than with aerobic packaging. The correlation coefficient between irradiation dose and nitrosamine content indicated that irradiation can reduce the levels of nitrosamines. The combination of irradiation and modified atmosphere packaging is effective in enhancing the chemical safety of sausage by reducing nitrosamines, if present, as well as enhancing the microbial safety of cooked pork sausage.

Aerobiosis↗

[Investigation on nitrosamines in the diets of the inhabitants of high-risk area for esophageal cancer in the southern China and analysis of the correlation factors].

Nan'ao County in Guangdong Province was an esophageal cancer high-risk area in the southern China. Of the suspected etiological factors in the environment, N-nitrosamines and their precursors have been received the most attention. 55 samples of the diets ingested by the inhabitants were collected and tested for volatile N-nitroso compounds and for their precursors by GC-TEA. Five kinds of N-nitrosamines (NDMA, NDEA, NPYR, NPIP and NMBzA) were detected in the samples. The average level of them was 312.0 micrograms/kg(median). The day intake of the nitrosamines was 286.5 micrograms/head/day. By the computerized stepwise regression analysis and curve fitting, we studied the correlation among the nitrosamines, the precursors and the major food items. It demonstrated that a relatively high content of volatile N-nitrosamines was present in the diets collected in the area.

Carcinogens↗

Determination of eight volatile nitrosamines in thirty cured meat products with capillary gas chromatography-high-resolution mass spectrometry: the presence of nitrosodiethylamine and the absence of nitrosopyrrolidine.

Five samples each of six cured meat products were analysed for the presence of eight volatile nitrosamines. A combined steam distillation-extraction step at pH 8-9 plus a concentration step were used for preparation of extracts. These were analysed, without further clean-up, using a coupled capillary GC-HRMS system at a resolution of 4 000. The MS was used in the fixed mass mode, but after the retention time of each nitrosamine the mass adjustment was tuned to the mass of the next nitrosamine. This combination allowed not only an unambiguous identification of the nitrosamines but also their direct quantitative determination. The products investigated were cooked ham, fermented sausage, raw bacon, mildly fried bacon, fried minced mean and smoked meat. Only one nitrosamine, NDEA, was sometimes found to be present in relatively large amounts. It occurred in two of the bacon samples; the concentration increased during mild frying from 4 and 13 mug/kg to 16 and 43 mug/kg, respectively. It was also present in two of the smoked meat samples in amounts of 7 and 91 mug/kg, respectively. Its identity was further confirmed on a second and third capillary column also coupled to the MS, and by direct introduction of the extracts into the MS at a resolution of 15 000. A silicon compound with practically the same retention time and mass as NDMA, which originates from the antifoam agents used as shown by other investigators, was found in the extracts in amounts corresponding to abuot 2 mug/kg of product. Nevertheless, seven samples were found positively to contain NDMA in amounts of 2-6 mug/kg. NMEA, NMPA, NMBA and NDPA could not be detected in any of the samples, nor could NPip or NPy. That no NPy (i.e., less than 50 mug/kg) was found in the fried bacon samples may be due to the mild frying method used. The use only of capillary GC with a nitrogen-selective detector, without further sample clean up, would have led to many false-positive results due to the presence of other nitrogen-containing compounds.

Animals↗

Nitrosamines in cured meat products.

One hundred samples of specially selected spiced meat products (sausages, salami, bologna, wieners, meat loaf, canned luncheon mean, etc) were analysed for nitrate, nitrite and volatile nitrosamines. None of the samples contained high levels of nitrosamines, but many contained traces, generally in the range from 2-50 mug/kg. Some contained as many as four nitrosamines, namely, NDMA, NDEA, NPip and NPy. In a few cases the samples were reanalysed after two weeks' storage at 4 or -20 degrees C, but no significant change in the nitrosamine levels could be detected. The identity of the nitrosamines was confirmed by GLC-high-resolution-MS.

Animals↗

N-Nitrosamines.

Nitrosamines are a large group of chemical compounds that have been found to be carcinogenic in all species of animals tested. They are classified as suspect human carcinogens even though direct causal evidence is lacking. This chapter reviews the nomenclature, chemistry, physical properties, exposure sources, toxicology, epidemiology, analytical methods, risk assessment, and regulatory aspects of various nitrosamine compounds. Occupational exposure to nitrosamines within the rubber industry is discussed in detail. Problems concerning total nitrosamine exposure in humans and the determination of cancer risks are highlighted. Additionally, industrial hygiene exposure control measures and medical surveillance pertaining to nitrosamines are discussed.

Animals↗

Role of vitamin E as nitrite scavenger and N-nitrosamine inhibitor: a review.

This review covers the following three aspects: 1. In vivo and in vitro inhibition of N-Nitrosamine formation. 2. Inhibition of N-Nitrosamines in food model systems by vitamin E. 3. Synergistic influence of vitamin E and C, and vitamin E and selenium on N-Nitrosamine synthesis. Vitamin E is effective in preventing the nitrosation of amino substrates under physiological conditions. Both vitamins (E and C) together have a stronger inhibiting effect on the formation of N-Nitrosamine. Data of this review strongly suggest that alpha-tocopherol, when ingested simultaneously with food, may reduce human exposure to carcinogenic N-Nitrosamine.

Animals↗

[An experimental study on bladder carcinogenesis in dogs by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and its urinary metabolites].

N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was administered to beagle dogs to examine the urinary metabolites. After extraction and purification of the urine sample, the urinary metabolites were identified by gas-chromatography mass-spectrometry and measured using a gas chromatograph equipped with a thermal energy analyzer (GC-TEA). Five N-nitroso-compounds (N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), N-butyl-N-(2-hydroxy-3-carboxypropyl)-nitrosamine (BHCPN), N-butyl-N-(carboxymethyl)-nitrosamine (BCMN), N-butyl-N-(2-oxopropyl) nitrosamine (BOPN) and BBN-glucuronide) were detected and identified by comparison with mass spectra of synthetic specimens. In the quantitative analysis by GC-TEA, BCMN greater than BHCPN greater than BCPN were larger part than BOPN and BBN-glucuronide. In early experimental period (0-25 wks) administered BBN (600 mg/day) to two dogs, small tumor was recognized in the urinary bladder of one dog earlier than the other. The former dog excreted larger amounts of BCPN in the urine than the latter. In late experimental period (100 wks approximately) administered BBN (400 mg/day) to five dogs, the dog that was found initial tumor earliest (103 wk) excreted the largest amounts of BCPN in the urine. Therefore, the excretion of BCPN had correlated with the carcinogenesis.

Animals↗

Gas chromatographic-thermal energy analysis method for determination of volatile N-nitrosamines in baby bottle rubber nipples: collaborative study.

A collaborative study was conducted on the U.S. Food and Drug Administration (FDA) dichloromethane extraction method for determining volatile N-nitrosamines in baby bottle rubber nipples. Following dichloromethane extraction, N-nitrosamines were determined by gas chromatography-thermal energy analysis. Six pairs of blind duplicate rubber nipple samples representing 6 lots were analyzed by 11 collaborating laboratories. All samples were portions taken from equilibrated composites of cut-up rubber nipples obtained from manufacturers in the United States. Recoveries of the internal standard (N-nitrosodipropylamine) at approximately 20 ppb ranged from 10 to 120%. Reproducibility relative standard deviations (RSDx) were between 35 and 45% for N-nitrosamine levels from 10 to 20 ppb. However, when data from laboratories with recoveries less than 75% were excluded (this is now specified in the method), RSDx values were between 11 and 32% for N-nitrosamine levels from 6 to 26 ppb. Values were consistent with or better than those reported for other analytical techniques designed to quantitate trace contaminants at the low ppb level, e.g., aflatoxin in foods. The method has been adopted official first action for the quantitation of volatile N-nitrosamines in baby bottle rubber nipples.

Bottle Feeding↗

Structure-activity relationships in metabolism and mutagenicities of N-nitrosamines.

The metabolism of a series of nitrosamines in vitro was monitored by measuring nitrogen production and was compared with mutagenesis by the same compounds, allowing separation of mutagenic potencies into metabolic and postmetabolic terms. The rate of nitrogen production from symmetrical di-n-alkyl and methylalkyl nitrosamines increased with increasing molecular weight. The cyclic nitrosamines N-nitrosopiperidine and N-nitrosopyrrolidine were metabolized slightly less rapidly than the most hydrophobic compounds, and N-nitrosomorpholine was metabolized at about half this rate. N-Nitrosomethylaniline was metabolized to nitrogen relatively slowly. Branching at the alpha-carbons reduced alpha-oxidative metabolism several fold. Substitution at the beta-carbon of N-nitrosodiethylamine or N-nitrosodi-n-propylamine with hydroxyl, cyano, oxo and methoxyl groups reduced metabolism to an even greater extent. Carboxyl substitution at the 4-position of N-nitrosopiperidine greatly reduced nitrogen formation, but 4-tert-butyl substitution had little effect. Effects of structure on mutagenic activities in Salmonella followed a different pattern. Higher homologue di-n-alkyl nitrosamines were more potent than lower homologues at lower doses, when potencies were taken from slopes of dose-response curves. However, when mutagenic potencies were expressed as 'mutagenic efficiencies' (revertants/mumol nitrogen), regardless of dose, the order of potency was N-nitrosodimethylamine greater than N-nitrosodiethylamine greater than N-nitrosodi-n-propylamine greater than N-nitrosodibutylamine. For the series of methylalkyl nitrosamines, mutagenic potencies were greatest for the higher molecular weight compounds, but they were all similar to that of N-nitrosodimethylamine when expressed as mutagenic efficiencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Weight↗

In vivo studies in Syrian golden hamsters: a transplacental bioassay of ten nitrosamines.

The carcinogenic effects of low doses of 10 nitrosamines were determined in pregnant Syrian golden hamsters and their offspring. Compounds studied included dimethylnitrosamine, di-n-propylnitrosamine, di-n-butylnitrosamine, nitrosopiperidine, nitrosohexamethyleneimine, 2-dydroxypropyl-propyl-nitrosamine, 2-oxopropyl-propyl-nitrosamine, methylpropylnitrosamine, di(2-hydroxypropyl) nitrosamine, and 4-hydroxybutyl-butyl-nitrosamine. Tumor incidences of all organ systems were almost always higher and latencies shorter in the mothers than in the offspring. Exceptions occurred in the respiratory system in which several compounds induced a low incidence of tumors in the offspring but none in the mothers. Fetal susceptibility appeared greatest toward the end of gestation. For purposes of bioassay, transplacental exposure was less efficient than conventional adult treatment.

Animals↗

Mutagenicity of alpha-hydroxy N-nitrosamines in V79 Chinese hamster cells.

N-Nitrosodialkylamines are activated metabolically by alpha-hydroxylation. Chemical properties and bacterial mutagenicity of alpha-hydroxy N-nitrosamines have been reported previously. This paper describes potent and direct mutagenicity of four N-nitroso-N-(hydroxymethyl)alkylamines in V79 Chinese hamster cells, using ouabain resistance as an indicator. The mutagenic potency depended on the alkyl group, decreasing in the following order: methyl greater than ethyl greater than propyl, butyl. A similar order was observed for cytotoxicity. Mutagenic and cytotoxic potencies of these alpha-hydroxy N-nitrosamines in V79 cells were well correlated not only with those of model compounds (alpha-acetoxy and alpha-hydroperoxy N-nitrosamines), but also with their alkylating ability, measured by alkylation of thiophenol. The mutagenic activity of the alpha-hydroxy N-nitrosamines in V79 cells was shown to be parallel to that in Salmonella typhimurium TA1535 and to that of N-nitrosodialkylamines in V79 cells, after metabolic activation by rat hepatocytes. The results obtained here further support the conclusion that the alpha-hydroxy N-nitrosamine is the active species in the metabolic activation of carcinogenic and mutagenic N-nitrosodialkylamines.

Animals↗

Ester-mediated nitrosamine formation from nitrite and secondary or tertiary amines.

N-Nitrosamines are formed from the heating of either a secondary or a tertiary amine with sodium nitrite in the presence of a high-boiling ester such as 2-acetoxyethanol in ethylene glycol. The four secondary and six tertiary amines examined were found to produce N-nitrosamines in yields ranging from 4% to 80% when equimolar amounts of amine and ester were heated at 120 degrees C with one- to ten-fold equivalents of sodium nitrite in ethylene glycol. Secondary amines competitively produced acetamides at a rate slightly greater than N-nitrosamine formation. Preincubation of a large excess of sodium nitrite and ester led to the rapid formation of N-nitrosamines in high yield. The reaction of tribenzylamine resulted in the formation of both benzaldehyde and dibenzylnitrosamine. N,N-Dimethylbenzylamine reacted to give nearly equimolar amounts of N-nitrosodimethylamine and N-nitroso-N-methylbenzylamine. It is proposed that the nitrosating agent is a nitrous ester, and it is shown that 2-benzoxyethyl nitrite rapidly nitrosates secondary and tertiary amines under these reaction conditions. It is also proposed that these transformations are good models for the environmental formation of N-nitrosamines in foods and commercial products.

Amines↗

Photochemistry of N-nitrosamines in neutral media.

N-Nitrosamines have long been held to be photostable in solution in absence of acid, although a few reports to the contrary have been published. This paper reports a study of the photolysis of N-nitrosopiperidine and N-nitrosodiisopropylamine in aprotic and protic solvents. It is shown that the cleavage of the N-N bond is a very facile process. In the absence of radical scavengers, however, reformation of the nitrosamines is very rapid, giving little or no net photolysis. In the presence of scavengers, photolysis proceeds with high efficiency to give non-nitrosamine products. When the trapping agent is oxygen, the net reaction is photo-oxygenation to the corresponding nitramine. The nitramine, in turn, is photolabile and undergoes photolysis to give cleavage products. The formation of the nitramine proceeds through the initial photochemical cleavage of the nitrosamine to the aminyl radical and nitric oxide, followed by the oxidation of the nitric oxide to nitrogen dioxide and the recombination of the latter with the aminyl radical. These results have some important implications for the destruction of nitrosamine wastes.

Hydrogen-Ion Concentration↗

Volatile N-nitrosamines in baby bottle rubber nipples and pacifiers. Analysis, occurrence and migration.

A simple direct extraction method has been developed for rapid determination of volatile N-nitrosamines in rubber nipples and pacifiers. It consists of overnight extraction of the sample with dichloromethane in the presence of ascorbyl palmitate for N-nitrosation inhibition, filtration of the extract and rinsing of the samples with dichloromethane, concentration of the extract using a Kuderna-Danish concentrator, and final analysis by gas-liquid chromatography-Thermal Energy Analyser. The method gave excellent (80-100%) recoveries of various volatile N-nitrosamines that had been added to cut nipples or pacifiers at 20-80 micrograms/kg levels, and gave comparable or lower values than those obtained with another published method. The method is recommended for rapid screening purposes. A survey of 30 samples of various nipples and pacifiers indicated the presence of the following N-nitrosamines: N-nitrosodimethylamine (up to 70 micrograms/kg), N-nitrosodiethylamine (up to 88 micrograms/kg), N-nitrosodi-n-butylamine (up to 2796 micrograms/kg), N-nitrosopiperidine (up to 180 micrograms/kg), and N-nitrosomorpholine (up to 86 micrograms/kg). A more recent study, however, indicated a general decline in the levels of various volatile N-nitrosamines in these products. These N-nitrosamines were shown to migrate easily from the rubber products to liquid infant formula, orange juice and simulated human saliva.

Humans↗

Quantitative measurement of the exhalation rate of volatile N-nitrosamines in inhalation experiments with anaesthetized Sprague-Dawley rats.

Volatile N-nitrosamines have been detected in the human environment--in work places, such as the rubber, leather and chemical industries, in tobacco smoke and also inside new cars. In order to make risk assessments on the basis of inhalation experiments with animals at dose levels relevant to the human situation, it is important to know the actual absorption rate in the respiratory tract. In this study, 63 female Sprague-Dawley rats were exposed to four nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine and N-nitrosomorpholine) in air. Respiratory parameters were monitored by pneumotachography. After 10 min of inhalation, the exhaled air was collected for 10 min in steps of 2 min intervals and analysed for its content of nitrosamines with a Thermal Energy Analyzer. Inhalation and exhalation were maintained by endotracheal intubation under narcosis with Thalamonal. The influence of this anaesthetic on the urinary excretion of N-nitrosodimethylamine after gavage and inhalation was tested: in comparison with unanaesthetized animals and with a group under ether narcosis, the excretion of the nitrosamine by the Thalamonal-anaesthetized animals was drastically reduced (factor of 20 to 60). Independent of the concentration of inhaled nitrosamine, ranging from 1-450 micrograms/L in air, the relative amounts of exhaled substance 10 min after inhalation were 0.9% N-nitrosodimethylamine, 0.4% N-nitrosodiethylamine, 6% N-nitrosopyrrolidine and 5% N-nitrosomorpholine. Extrapolation of these data back to the first exhalation (i.e., following the last inhalation) revealed that a remarkable amount of substance may be exhaled (up to 30% N-nitrosodimethylamine).

Air Pollutants↗

Formation of volatile nitrosamines by drug-nitrite interactions under physiological conditions.

Twenty-eight drugs, most of which are tertiary amines, were tested for the formation of volatile nitrosamines by reaction with nitrite under physiological conditions; the drugs (10mM) were incubated with nitrite ( 40mM ) at pH 3.0, 37 degrees for 1 and 4 hr. The volatile nitrosamines formed were determined by gas chromatography-thermal energy analysis. Of the 28 drugs, 24 formed measurable amounts of volatile nitrosamines that are known carcinogens. The yields of nitrosodimethylamine (NDMA) from aminopyrine (55-65%) and minocycline (11%) were higher than that from dimethylamine under the same conditions. This result suggests that there may be a pathway not involving the secondary amine (dimethylamine) as an intermediate in the formation of NDMA from minocycline as well as from aminopyrine, Tolazamide gave rise to nitrosopiperidine ( NPIP ) in addition to nitrosohexamethyleneimine ( NHXI ), formation of which was expected from the chemical structure of tolazamide, and the yield of NPIP (2-7%) was higher than that of NHXI (0.2-1.2%). Ascorbic acid ( 40mM ) was effective in decreasing the formation of nitrosamines from drugs by reaction with nitrite, although the blocking effects varied between 88 and 100% depending on the drugs tested or on the nitrosamines formed.

Amines↗