Detection of carcinogenic aromatic amines in the urine of non-smokers

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Abstract

Smoking is thought to be one of the most important anthropogenic risk factors involved in the development of urinary bladder cancer in humans. Tobacco smoke contains a complex mixture of chemicals including potent carcinogens such as aromatic amines. In the present study the amounts of several freebase aromatic amines including the potent carcinogens 2-aminonaphthalene and 4-aminobiphenyl have been analyzed in the urine of 48 German urban living smokers and non-smokers. The results indicate that (i) both groups excrete the identical set of four aromatic amines; (ii) smokers excrete approximately twice the total amount of these amines, but similar amounts of 2-aminonaphthalene and 4-aminobiphenyl are found in non-smokers; and (iii) the excreted aromatic amines are decomposed in the urine within a few hours thus, explaining why aromatic amines are difficult to detect in this matrix. Their decomposition could be prevented by adding small amounts of p-toluidine to the freshly collected urine. Unlike smokers the origin of aromatic amines detected in the urine of non-smokers is at present unknown. Based on the cotinine levels found in the urine of non-smokers environmental tobacco smoke can be excluded as a major source of aromatic amines. In addition, neither diesel exhaust-related nitroarenes nor the corresponding amino-derivatives, to which they may be metabolically converted, were found. The detected urinary levels of aromatic amines arising from sources other than tobacco smoke or diesel exhaust may play a role in the bladder cancer etiology of non-smokers.

Introduction

Tobacco smoking has been implicated as a major risk factor for the development of cardiovascular diseases and different types of cancer in humans including lung and urinary bladder cancer. Specifically, many cohort- and case-control studies indicate that up to 50% of the male and 25% of the female bladder cancer cases could be attributed to cigarette smoking (IARC, 1986). Several classes of chemical carcinogens such as polycyclic aromatic hydrocarbons and aromatic amines are thought to be important carcinogenic agents in tobacco smoke. Among the aromatic amines 2-aminonaphthalene (2-AN) and 4-aminobiphenyl (4-ABP) have been detected in the mainstream of cigarette smoke (Patrianakos and Hoffmann, 1979). Epidemiological studies together with case reports have shown that occupational exposure to 2-AN and 4-ABP is causally associated with the occurrence of human bladder cancer (IARC, 1982, IARC, 1987, Gehrke et al., 1996, Vineis and Pirastu, 1997). More recently Ward et al. (1996) presented data which support the assumption that occupational exposure to o-toluidine also contributes to bladder cancer excess among workers in the rubber industry. Furthermore, after oral administration 4-ABP has been demonstrated to be a bladder carcinogen in various animals including dogs, rabbits, mice and rats (IARC, 1982, IARC, 1987). Similarly, 2-AN has been identified as an urinary bladder carcinogen in hamsters, dogs and non-human primates after its oral administration (IARC, 1982, IARC, 1987).

The carcinogenic activity of aromatic amines in mammals is mediated by N-hydroxy-amines and N-hydroxyamides and involves N-oxidation catalyzed predominantly in the liver by cytochrome P450 1A2 or other CYP isoforms (Kimura et al., 1999) followed by transport of these metabolites or their more stable conjugates to the bladder. The acidic conditions of the urine facilitate the formation of electrophilically reactive nitrenium ions which are thought to covalently bind to DNA in urothelial cells to form stable DNA adducts (Kadlubar et al., 1977, Poupko et al., 1979). In addition to humans, in dogs a species-specific one-electron activation of aromatic amines in the urinary bladder epithelium by prostaglandin H synthase to DNA-binding intermediates has been described (Eling et al., 1990).

Measurements of hemoglobin adduct levels of aromatic amines have been used to monitor human exposure to this class of carcinogens and elevated hemoglobin adduct levels have been detected in red blood cells of smokers compared to non-smokers (Tannenbaum et al., 1986, Bryant et al., 1987, Bartsch et al., 1990, Maclure et al., 1990, Ronco et al., 1990, Hammond et al., 1993, Neumann et al., 1993, Falter et al., 1994, Riffelmann et al., 1995, Ward et al., 1996). Environmental tobacco smoke has been discussed as a source of aromatic amines in the case of non-smokers because these compounds have been found in the sidestream of tobacco smoke in much higher concentrations than in mainstream smoke (IARC, 1986).

The exact determination of aromatic amine concentrations in an environmental matrix or in body fluids is complicated by their tendency to undergo further reactions. For example, in tobacco smoke, within a few seconds, some of them react with other constituents of smoke formed during the combustion process. Recently, in our laboratory it has been discovered that addition of an excess of p-toluidine to the collecting solution of mainstream smoke obtained from mechanically smoked cigarettes can significantly (by a factor of 3 and more) reduce this process. p-Toluidine acts as an intercept reactant immediately stabilizing the amount of amines originally formed and resulting in high recovery rates (Grimmer et al., 1995).

In the present study the burden of smokers and non-smokers by aromatic amines has been investigated by measuring the urinary excretion of four aromatic amines as free bases in 48 individuals. Nitroarenes have been detected in a number of combustion products including diesel condensate (Wei and Shu, 1983, Gibson, 1983). Therefore, environmental automotive nitroarenes which may be metabolically converted to aromatic amines in mammals (Beland et al., 1985) have been discussed as a source of urinary aromatic amines in non-smokers. To obtain information on the relevance of this source, the urine of the above individuals has also been analyzed for several nitroarenes as well as for their phenolic metabolites.

Section snippets

Chemicals

All chemicals of analytical grade were purchased either from Boehringer Mannheim (Mannheim, Germany) or Sigma-Aldrich Chemie (Deisenhofen, Germany). The reference materials, 2- and 4-nitrobiphenyl, 1- and 2-nitronaphthalene, 1-nitropyrene, 2-nitrofluorene, 1- and 2-aminonaphthalene (2-AN), 2- and 4-aminobiphenyl (4-ABP), d5-aniline, d7-2-AN and d9-4-ABP were purchased from Promochem GmbH (Wesel, Germany). The isomeric 3-, 6-, 8- and 10-hydroxy-1-nitropyrenes were kindly provided by Dr L.M.

Results

Individual aromatic amines were quantified by GC/MS-coupling and a representative chromatogram is illustrated in Fig. 1. The concentrations of aromatic amines, cotinine and creatinine found in the 24-h urine of smokers (12 individuals), non-smokers (14 individuals) and passive smokers (22 individuals) are presented in Table 2.

The urinary excretion of cotinine, reflecting the exposure to nicotine was found to be approximately 100 times higher in the case of smokers (634–6519 μg/24 h) compared to

Discussion

The ability of cigarette smoke to cause urinary bladder cancer in humans has been attributed to the presence of potent carcinogens such as polycyclic aromatic hydrocarbons (IARC, 1982, IARC, 1986, IARC, 1987, Gustavsson et al., 1988) and/or aromatic amines (IARC, 1982, IARC, 1986, IARC, 1987). The latter class of carcinogens is in particular suspected of contributing to this type of cancer and a dose-dependent higher bladder cancer risk for smokers compared to non-smokers has been reported (

Conclusions

The findings of the present study demonstrate that in urine non-smokers excrete similar amounts of carcinogenic aromatic amines compared to smokers. Furthermore, large variations in the excreted amounts of aromatic amines have been observed for both smokers and non-smokers. This may suggest that the well known increased bladder cancer incidence of smokers is predominantly related to other tobacco smoke constituents than aromatic amines. On the other hand, environmental tobacco smoke may be

Acknowledgements

The authors are greatly indebted to PD. Dr G. Scherer (ABF München, Germany) for organizing the sample collection and delivering the urinary cotinine and creatinine concentrations as well as to Dr L.M. Ball (University of North Carolina at Chapel Hill, USA) who kindly supplied us with the hydroxy-1-nitropyrene standards.

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