Long-Term Stability of Volatile Nitrosamines in Human Urine.

Volatile nitrosamines (VNAs) are established teratogens and carcinogens in animals and classified as probable (group 2A) and possible (group 2B) carcinogens in humans by the IARC. High levels of VNAs have been detected in tobacco products and in both mainstream and sidestream smoke. VNA exposure may lead to lipid peroxidation and oxidative stress (e.g., inflammation), chronic diseases (e.g., diabetes) and neurodegenerative diseases (e.g., Alzheimer's disease). To conduct epidemiological studies on the effects of VNA exposure, short-term and long-term stabilities of VNAs in the urine matrix are needed. In this report, the stability of six VNAs (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine and N-nitrosomorpholine) in human urine is analyzed for the first time using in vitro blank urine pools fortified with a standard mixture of all six VNAs. Over a 24-day period, analytes were monitored in samples stored at ∼20°C (collection temperature), 4-10°C (transit temperature) and -20 and -70°C (long-term storage temperatures). All six analytes were stable for 24 days at all temperatures (n = 15). The analytes were then analyzed over a longer time period at -70°C; all analytes were stable for up to 1 year (n = 62). A subset of 44 samples was prepared as a single batch and stored at -20°C, the temperature at which prepared samples are stored. These prepared samples were run in duplicate weekly over 10 weeks, and all six analytes were stable over the entire period (n = 22).

[The elaboration of gas chromatographic method of the determination of N-nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine) in biological samples (urine)].

The issues of the elaboration of a method for the determination of N-nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine) in urine by means of the method of capillary gas chromatography with the use of a thermionic detector are considered. There were performed investigations on the study of the efficacy of the extraction of N-nitrosamines from the urine by steam distillation and gas chromatographic detection of headspace. With the aim of the maximal recovery of N-nitrosamines from the urine and setting parameters of the extraction two method were used to prepare the bioassay for the analysis the alkalization with potassium hydroxide and the addition of salting out reagent--neutral salts of alkali and alkaline earth metals. During the process of performed studies there was found that the greatest degree of extraction of N-nitrosamines from the urine by the method of headspace analysis is achieved if using the salting-out agent in an amount of 16 g of sodium sulfate and for N-nitrosodimethylamine is 99%, for N-nitrosodiethylamine--100%.

Quantitation of urinary volatile nitrosamines from exposure to tobacco smoke.

A sensitive and selective method was developed and validated to detect six volatile nitrosamines (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine and N-nitrosomorpholine) in human urine. This method uses a liquid-liquid extraction cartridge followed by analysis with gas chromatography-tandem mass spectrometry (GC-MS-MS) and quantification based on isotopic dilution. This is the first GC-MS-MS method reported for measuring volatile nitrosamines in human urine. This method reduces the sample volume required in other methods from 5-25 to 2 mL. The limits of detection (2.62, 1.99, 2.73, 0.65, 0.25, 3.66 pg/mL, respectively) were better than existing methods, largely because of improved positive chemical ionization achieved by using ammonia gas and reducing background noise. Using nitrogen as the collision gas allowed the confirmation transition in the low mass region to be monitored. The analysis of human urine using this validated method is accurate (relative bias of 0-19%) and precise (relative standard deviation of 0.2-18% over two months of analyses). The validated method was applied to 100 urine samples and the levels of all six volatile nitrosamines were reported for the first time in urine specimens collected from smokers and nonsmokers, with smoking status determined by urinary cotinine measurement. Among 100 smokers and nonsmokers, the levels of three analytes (N-nitrosodimethylamine, N-nitrosomethylethylamine and N-nitrosopiperidine) were significantly higher in smokers than nonsmokers (p < 0.05).

Investigation of the formation of N-nitrosodiethanolamine in B6C3F1 mice following topical administration of triethanolamine.

To determine potential nitrosation of triethanolamine (TEA) to N-nitrosodiethanolamine (NDELA) at different physiological conditions of the GI tract, in vitro NDELA formation was examined in aqueous reaction mixtures at several pHs (2-10) adjusted with acetic, sulphuric or hydrochloric acids or in cultures of mouse cecal microflora incubated. In vivo NDELA formation was also determined in blood, ingesta, and urine of female B6C3F1 mice after repeated dermal, most relevant human route, or single oral exposure to 1000 mg/kg TEA in the presence of high oral dosages of NaNO(2). Appropriate diethanolamine (DEA) controls were included to account for this impurity in the TEA used. Samples were analyzed for NDELA using GC/MS. The highest degree of nitrosation of TEA to NDELA ( approximately 3%) was observed in the in vitro cultures at pH 4 and acetic acid with lower amounts obtained using sulphuric acid ( approximately 1.3%) and hydrochloric acid ( approximately 1.2%). At pH 7, <1% of the TEA was nitrosated to NDELA and at pH 2 (HCl) or pH 10 (NaOH) no NDELA was found above the limit of detection. In incubated cultures containing cecal microflora and nutrient broth, only 0.68% of TEA was nitrosated to NDELA. No NDELA was formed in rats repeatedly dermally dosed with TEA at the limits of detection in blood (0.001 microg/ml, ppm), ingesta (0.006 microg/ml, ppm), and urine (0.47 microg/ml, ppm). Levels of NDELA measured in blood and ingesta after a single oral dose of TEA and NaNO(2) were less than those in DEA controls. These findings in toto confirm the lack of any significant formation of NDELA from TEA in vivo.

N-nitrosodiethanolamine urinary excretion in workers exposed to aqueous metalworking fluids.

OBJECTIVE: This work was intended to clarify the extent of exposure of workers occupationally exposed to N-nitrosodiethanolamine (NDELA), a carcinogenic nitrosamine, while working with aqueous metalworking fluids (MWFs) formulated with ("nitrite-formulated") or without ("nitrite-free") nitrite and to study the relationships between the nitrite and NDELA content of the MWFs as well as between the concentration of NDELA in MWFs and in urine. METHOD: Pre-shift and post-shift urine samples from 100 workers directly exposed to MWFs in 15 factories were analysed for NDELA with chemiluminescent detection (TEA) according to a previously described analytical procedure. The method was also applied to eight indirectly exposed workers and to 48 unexposed subjects. The NDELA and concentrations in 84 fluids used by the workers were also determined. RESULTS: No detectable NDELA could be observed in the control group. The mean post-shift NDELA excretion in workers exposed to "nitrite-formulated" and "nitrite-free" MWFs were 44.6 and 0.4 microg/l, with maxima of 277 and 2.7 microg/l, respectively. According to the correlation between the nitrite and NDELA concentrations in "nitrite-free" MWFs, there is a low probability of fluids exceeding 5 mg/l NDELA when the nitrite content does not exceed 20 mg/l. The NDELA concentrations in the fluids and urine were found to be highly correlated, particularly after correction for creatinine (r=0.917 in post-shift samples). Cutaneous contact probably contributes, at least in part, to the overall body uptake of NDELA: CONCLUSION: Due to clear evidence of urinary NDELA excretion in workers exposed to contaminated MWFs, and despite a lack of knowledge of the human risk following NDELA exposure, levels of NDELA in MWFs should be kept as low as possible. NDELA fluid concentrations of less than 1 mg/l must be considered as the objective to be attained, even if the limit of 5 mg/l is temporarily satisfactory and consistent with a nitrite limit of 20 mg/l that is easy to verify with inexpensive colorimetric tests. "Nitrite-formulated" fluids, still sometimes used, should be prohibited. Meanwhile, the material safety data sheets (MSDS) of commercially available products should be clearly labelled to indicate their nitrite content.

Dietary exposure and urinary excretion of total N-nitroso compounds, nitrosamino acids and volatile nitrosamine in inhabitants of high- and low-risk areas for esophageal cancer in southern China.

We assessed the exposure of total N-nitroso compounds (TNOCs) in the inhabitants of high- and low-risk areas for esophageal cancer in southern China. Samples of 24 hr diet and 12 hr overnight urine were collected from 120 male adults in each of the 2 areas, a high-risk area (Nan'ao County) and a low-risk area (Lufeng County) for esophageal cancer. Annual standardized mortality rates of esophageal cancer in Nan'ao and Lufeng are 110/10(6) and 10/10(6) respectively. The 240 healthy male subjects (35-64 years old) were selected by a 3-stage random cluster sample procedure. Levels of TNOCs, NAAs and volatile nitrosamines in the samples were measured. The TNOC detection rate (95%) in the diet, the TNOC daily intake (4.25 +/- 0.84 micromol), TNOC excretion levels (0.04 +/- 0.01 nmol/12 hr) and daily intake of volatile nitrosamines (5.84 +/- 0.71 micromol) in the high-risk area were significantly greater than values in the low-risk area (A +/- B = mean +/- SE). The TNOC detection rate in the diet, the TNOC daily intake, TNOC excretion levels and daily intake of volatile nitrosamines in the low-risk area were 70%, 0.25 +/- 0.06 micromol, 0.02 +/- 0.01 nmol/12 hr and 3.18 +/- 0.31 micromol, respectively. NAA excretion levels showed no difference between the 2 areas (16.3 +/- 7.18 micromol/12 hr for Nan'ao and 31.2 +/- 26.4 micromol/12 hr for Lufeng). Thus, TNOCs are implicated in the etiology of esophageal cancer in southern China.

Determination of N-nitrosodiethanolamine in urine by gas chromatography thermal energy analysis: application in workers exposed to aqueous metalworking fluids.

OBJECTIVE: The aim of this study was to describe a detailed and validated methodology designed for the analysis of carcinogenic N-nitrosodiethanolamine (NDELA) down to sub-microgram/l levels in urine and its application to a number of workers exposed to NDELA-contaminated aqueous metalworking fluids (MWF). METHODS: Following a work-up procedure based on solid-phase extraction of NDELA, the urinary extracts were analysed without derivatization by gas chromatography on a polar wide-bore column with chemiluminescent detection using a thermal energy analyser (TEA). N-Nitroso-(2-hydroxypropyl)amine was used as an internal standard. The method was applied to 12 workers using "nitrite-free" or "nitrite-formulated" MWF and to 15 unexposed subjects. The NDELA content of the MWF was also determined using a similar, but simpler method able to easily quantify NDELA down to at least 0.1 mg/l. RESULTS: Contamination by NDELA traces of some chemicals used for the sample preparation, particularly ethyl formate, must be carefully checked since it can give rise to false-positive results of up to 1 or 2 micrograms/l. The response was linear in the range of 0-500 micrograms/l. Between 0.5 and 10 micrograms/l, the recovery rate was close to 95%, while repeatability ranged from 12.5 to 6.4% (n = 5). The detection limit was 0.3 microgram/l (Signal/noise = 3). No detectable NDELA could be observed in the control workers. There was no significant increase in NDELA levels at the end of shift spot samples from an exposed worker over 1 week. Higher NDELA concentrations were found in two workers (4.3 and 10.7 micrograms/l) exposed to "nitrite-formulated" fluids (contaminated with 65 and 18 mg NDELA per 1, respectively) than in nine workers (range, 0.4-1.3 micrograms/l exposed to "nitrite-free" fluids with lower levels of NDELA (range, 0.5-6.6 mg/l). CONCLUSION: The detailed methodology described in this work and applied to a limited industrial situation was found to be suitable for monitoring NDELA in the urine of workers exposed to aqueous MWF. A much larger screening has been undertaken with the aim of obtaining better information on the real exposure of workers sometimes exposed to "nitrite-formulated" fluids that are still used.

Biological monitoring of workers exposed to N-nitrosodiethanolamine in the metal industry.

Biological monitoring of occupational hazards was performed in workers using cutting fluids containing N-nitrosodiethanolamine (NDELA). The study involved a group of 25 male subjects from some metal factories in central Italy who used cutting fluids with an NDELA content of > or = 5 mg/l (high-exposure group) and a group of 37 males exposed to cutting fluids with an NDELA content < 5 mg/l (low-exposure group). For comparison, we recruited a control group consisting of 37 subjects living in the same area. For all subjects, internal dose (urinary excretion of NDELA, mutagens, and thioethers), early biological effects (sister chromatid exchanges in blood peripheral lymphocytes), and urinary excretion of D-glucaric acid (DGA) as an endpoint product in the glucuronidation pathway were assessed. The results showed that only the workers using cutting fluids with NDELA concentrations of > or = 5 mg/l excreted trace amounts of NDELA in their urine. Urine excretion of mutagens was similar in the two exposure groups and in the controls. High-exposure subjects had a higher mean value of urinary thioethers than low-exposure and control subjects, but no differences were found in urinary DGA or lymphocyte sister chromatid exchange among the three groups. Smoking status increased the mean values of all the biomarkers, and coffee drinking was associated with urinary DGA excretion.

Combined effects of L-ascorbic acid, citric acid or their sodium salts on tumor induction by N-butyl-N-(4-hydroxybutyl)nitrosamine or N-ethyl-N-(4-hydroxybutyl)nitrosamine in the rat urinary bladder.

L-Ascorbic acid, citric acid or their sodium salts (at levels equivalent to 5% sodium L-ascorbate) were fed in the diet simultaneously with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) or N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) (0.025% BBN or 0.021% EHBN) in the drinking water to male F344 rats for 20 weeks to determine whether urinary pH changes affect the carcinogenicity of BBN or EHBN. In the urine, pH was decreased in rats fed the acidic chemicals and increased in rats fed their corresponding sodium salts. Histopathologically, the incidences and numbers of preneoplastic and neoplastic lesions in groups treated with each test chemical were not different from those in control groups except for sodium citrate-treated groups in which induction of carcinomas was higher, resulting from increased intake of either carcinogen and also from increased urinary excretion of main carcinogenic metabolites. These results show that the test chemicals do not affect the carcinogenicity of BBN or EHBN on the rat urinary bladder when simultaneously administered despite significant differences in urinary pH.

N-nitrosodiethanolamine excretion in metal grinders.

Grinding fluids usually contain ethanolamines and nitrite as anticorrosive agents; these are known precursors of N-nitrosodiethanolamine (NDELA). In a preliminary study, it was demonstrated that workers' exposure to NDELA can be monitored by urine analysis. In order to estimate total daily exposure, 12 workers in a grinding shop were investigated by a three-step biological monitoring programme, giving the following results: (1) after exposure-free weekends, no NDELA was found in urine; (ii) urine collected during working shifts contained NDELA in up to microgram/kg concentrations; (iii) total daily NDELA excretion in 24-h urines was up to 40 micrograms; (iv) the amount of excreted NDELA correlated with the amount of NDELA contamination in the grinding fluid; (v) NDELA seems to be accumulated in the body during the week; (vi) other workers in machine shops, like maintenance and transport workers, are also heavily exposed to NDELA.

Effects of disulfiram on the metabolism of nitrosodiethylamine during liver carcinogenesis.

We have studied the effects of disulfiram (DSF) administration on the metabolism of nitrosodiethylamine (NDEA) in rats during acute and chronic administration. DSF was found to have the following effects during the course of carcinogenesis: (a) marked decrease in the exhalation of 14CO2 derived from 14C-NDEA; (b) reduction of the total levels of DNA and RNA ethylation in the liver. In acute experiments DSF caused an increase in the amount of NDEA in organs and in the urine. We suggest that inhibition of NDEA biotransformation and the subsequent decrease in the total level of DNA ethylation may prevent specific chemical interactions relevant to carcinogenesis.

Biological monitoring in the metal working industry.

N-Nitrosodiethanolamine (NDELA) is a strong carcinogen in animal experiments. Its occurrence in cutting and grinding fluids represents a major risk for workers who come into contact with those compounds. But until now it was not possible to describe the extent of N-nitrosodiethanolamine exposure at the workplace. Since 60-90% of N-nitrosodiethanolamine given by oral, intravenous, epicutaneous or intratracheal application in rat experiments is excreted unchanged in the urine, N-nitrosodiethanolamine should be found in the urine of workers in the metal working industry. Analyses of grinding fluids containing di- and triethanolamine in combination with up to 30% nitrite showed concentrations of up to 593 mg/kg N-nitrosodiethanolamine in the original, concentrated fluid and up to 90 mg/kg in ready-to-use emulsions. In preliminary investigations, it was also found in the urines of metal grinders: of 264 urines analysed, 166 showed positive results (greater than 0.5 micrograms/kg) with levels up to 103 micrograms/kg N-nitrosodiethanolamine. These results indicate that workers' exposure to NDELA can be monitored by urine analysis.

Effects of route of administration and dose on the carcinogenicity of N-nitrosodiethanolamine in the Syrian golden hamster.

N-Nitrosodiethanolamine was assayed for carcinogenicity in Syrian golden hamsters by s.c. injection, topical application, and oral cavity swabbing. Three groups of 30 hamsters each received 27 weekly s.c. injections of either 500, 170, or 58 mg of N-nitrosodiethanolamine per kg in 0.9% NaCl solution. In the group treated with 500 mg/kg, 19 of 30 animals developed nasal cavity tumors, 7 of 30 had tracheal tumors, and 2 of 30 had tumors of the larynx. Among the animals treated with 170 mg/kg, 7 of 29 presented with nasal cavity tumors and 4 of 29 presented with tracheal tumors. In the group treated with 58 mg/kg, only two tracheal tumors were observed. Acetone solutions of N-nitrosodiethanolamine were applied to the shaved backs of three groups of 30 hamsters, each three times weekly for 36 weeks, at doses of 25, 8, or 2.5 mg; the total doses were the same as in the groups treated by s.c. injection. At the 25-mg dose level, 5 of 30 animals developed nasal cavity tumors and 4 of 30 animals had tumors of the trachea. No skin tumors were observed. The incidence of respiratory tract tumors in the groups treated with 8 or 2.5 mg was not significant compared to controls. The oral cavities of 40 hamsters were swabbed three times weekly for 45 weeks with 20 mg of N-nitrosodiethanolamine; the total dose was the same as the highest doses given by s.c. or topical administration. Seventeen of 38 hamsters had nasal cavity tumors, 6 of 38 developed tracheal tumors, and 1 of 38 presented with a tumor of the larynx. No tumors were observed in the oral cavity. The results of this study demonstrate that N-nitrosodiethanolamine is organospecific for the Syrian golden hamster nasal cavity and trachea and that it induces tumors in these sites at doses lower than previously reported.

The fate of N-nitrosodiethanolamine after oral and topical administration to rats.

14C-labelled N-nitrosodiethanolamine ([14C]NDELA) was given to Osborne-Mendel rats at two dose levels, 0.5 or 50 mg/kg, by oral or topical administration. The excreta and tissues were analysed at various times from 4 hr to 1 wk after administration to determine the distribution of radioactivity. After oral administration, [14C]NDELA was rapidly absorbed from the gastro-intestinal tract, distributed throughout all organs and tissues, and then excreted, mainly via the kidneys. The tissue concentration reached a peak at 8 hr, but some activity remained after 1 wk. After topical application NDELA was slowly absorbed percutaneously, but once absorbed was distributed as in the orally dosed rats. Metabolic profiles of urine and bile samples from both the orally and topically dosed rats were identical, although the quantities varied. In addition to unchanged NDELA, one metabolite was present. The dose level had little effect on the quantities of unchanged NDELA or the metabolite present.

Urinary excretion of N-nitrosamines in rats and humans.

Various sources may contribute to the total human exposure to nitrosamines (foods, drugs, cosmetics, polluted air and endogenous formation of N-nitroso compounds). The average intake of nitrosamines can be calculated using analytical data. The more relevant method of biological monitoring can be used, however, to estimate individual exposure, in which case possible in vivo formation may also be detected. Since blood measurements can be carried out only under great difficulty and reflect only the momentary situation, the urinary excretion of nitrosamines was studied in animal experiments. The urinary excretion of nitrosodimethylamine (NDMA), nitrosomorpholine (NMOR) and nitrosodiethanolamine (NDELA) in SD-rats was measured within 24 h after epicutaneous, intratracheal, oral and intravenous application. The dose range covered was 5, 50 and 500 micrograms/animal for NDMA, 4, 44 and 440 micrograms/animal for NMOR and 0.03-300 mg/animal for NDELA. Under the influence of diethylether, a 7- to 20-fold increase of excretion was observed. Biological monitoring in humans at low doses (10-100 micrograms NDMA) is possible only if the excretion rate is increased by administration of ethanol. By reducing the activity of metabolizing enzymes with ethanol or other suitable compounds, possible in vivo formation of nitrosamines might also be more easily detectable. Excretion rates do not seem to be dose-dependent in the ranges investigated.

Penetration of rat skin by N-nitrosodiethanolamine and N-nitrosomorpholine.

N-Nitrosomorpholine (NMOR) and N-nitrosodiethanolamine (NDELA) were painted on the clipped upper dorsal skin of male F344 rats. NDELA was applied undiluted, dissolved in water, and dissolved in cutting oil; NMOR was applied dissolved in water and in ethyl acetate. Aqueous solutions of the nitrosamines were used for gavage. Rats were housed individually. Blood and urine samples were analyzed for nitrosamines by chromatography combined with a Thermal Energy Analyzer. Maximum penetration of NMOR was approximately equal to 34% 2 hours after application of 5 mg to the skin or by gavage; less than 1% appeared in the urine in 24 hours. Skin painting with NDELA in water (20 mg/100 microliters) and in cutting oil (25 mg/25 microliters) yielded small concentrations of NDELA (always < 25 micrograms/ml blood). When 50 mg of undiluted NDELA was painted on the skin, 130 to 220 micrograms/ml of blood was recovered after 1 hour. Administering 50 mg NDELA in water by gavage yielded similar blood concentrations. Maximum skin penetration observed with NDELA was 78% 1 hour after application of 50 mg. From 20 to 30% of the NDELA applied undiluted and by gavage appeared in the urine in 24 hours. Although animals and humans differ, skin exposure to NMOR or NDELA represents a risk due to absorption.

Urinary excretion of N-nitrosodiethanolamine administered orally to rats.

N-Nitrosodiethanolamine (NDE1A) was administered by gavage to male rats in single doses of 1000, 500 and 100 mg/kg body wt. More than 70% of a given dose was excreted unchanged in the urine, essentially within the first 24 h after exposure. This high excretion rate might explain the relatively low carcinogenic potential of NDE1A, and also offers a possible method of monitoring exposure to this compound under occupational and/or environmental conditions.