Journal of Analytical Toxicology, Vol. 30, April 2006
Determination of Three CarcinogenicAromatic Amines in Urine of Smokersand Nonsmokers Kirsten Riedel 1, Gerhard Schererl, *, Johannes EngP, Heinz-Werner Hagedorn 1, and Anthony R. Tricker 2
7Analytisch-Biologisches ForschungslaborGmbH, Goethestrasse20, 80336 MOnchen, Germanyand 2philip Morris Products S.A., PMI Researchand Development, Quai Jeanrenaud56, 2000 Neuch$tel, Switzerland
Abstract Aromatic amines (arylamines) such as o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl occur in the environment and are constituents of tobacco smoke. Human exposure to these aromatic amines has long been associatedwith an elevated risk of bladder cancer. A validated, specific, and sensitive method for measuring o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in cigarette smokersand nonsmokerswas developed. The method uses acid hydrolysisof the arylamine conjugates in urine, extraction with n-hexane, derivatization with penlafluoropropionic anhydride, and subsequent analysis with gas chromatography combined with mass spectrometry using negative ion chemical ionization. The limits of detection were 4 ng/L for o-toluidine and 1 ng/L for 2-aminonaphthalene and 4-aminobiphenyl. Smokers (N = 10) excreted significantly higher amounts of o-toluidine (204 versus 104 ng/24 h), 2-aminonaphthalene (20.8 versus 10.7 ng/24 h), and 4-aminobiphenyl (15.3 versus 9.6 ng/24 h) than nonsmokers (N = 10). Urinaryarylamine excretion in smokers was associated with the extent of smoking as assessed by daily cigarette consumption, urinaryexcretion of nicotine equivalents (nicotine plus its fivemajor metabolites), cotinine in saliva, and carbon monoxide in exhaled breath. All nonsmokers investigated had quantifiable amounts of o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in their urine, confirming that other environmental sources of exposure to these compounds also occur. In conclusion, the analytical method is suitable for measuring short-term exposure to arylamines in urine of non-occupationally exposed smokersand nonsmokers.
Introduction
Tobacco smoking is a major cause of bladder cancer in humans without known occupational exposure to bladder carcinogens (1). Aromatic amines (arylamines) in tobacco smoke, in particular o-toluidine, 2-aminonaphthalene ([3-naphthylamine), and 4-aminobiphenyl are hypothesized to be the
causative agents. The chemical structures of these three aromatic amines are shown in Figure 1. o-Toluidine has been classifted as "probably carcinogenic to humans" (Class n di2A) by the International Agency for Research on Cancer (IARC) (2) and "to be regarded as carcinogenic for humans" (Class 2) for workplace carcinogens by the MAK-Commission of the Deutsche Forschungsgemeinschaft (DFG) (3). 2-Aminonaphthalene and 4-aminobiphenyl have both been classified as "carcinogenic to humans" by IARC (2) and the DFG (3). Matsuda and Hoffmann (4) first reported a method for the determination of aromatic amines in mainstream cigarette smoke. The same working group later published a method for the determination of aromatic amines in both mainstream and sidestream smoke of cigarettes (5). Since then, several analytical methods have been developed using various techniques (6-8). Mainstream cigarette smoke of 18 leading U.S. commercial cigarette brands shows median yields of 15.5 ng/cigarette (range 5.7-28.6 ng/cigarette) 2-aminonaphthalene and 4.5 ng/cigarette (1.8-7.8 ng/cigarette) for 4-aminobiphenyl (9). Stabbert et al. (10) investigated eight U.S. commercial cigarette brands and reported mainstream smoke yields of 8.6-144.3 ng/cigarette o-toluidine, 1.5-14.1 ng/ cigarette 2-aminonaphthylaminer and 0.3-2.3 ng/cigarette 4-aminobiphenyl. More recently, Counts et al. (11) reported mainstream smoke yields of 2.3-17.2 ng/cigarette 2-aminonaphthylamine and 0.5-3.3 ng/cigarette 4-aminobiphenyl in 48 commercial cigarette brands. A simplified metabolic scheme for 4-aminobiphenyl (as an example for arylamines) is shown in Figure 2 (12). The metabolic conversion mainly takes place in the liver where the aromatic amine is N-acetylated, N-glucuronidated, or oxidized to the N-hydroxyarylamine (13,14). The N-acetylarylamine is a detoxification product and is excreted in the urine. The CH3
o-Toluidine
2-Aminonaphthalene
4-Aminobiphenyl
Figure1. Chemicalstructuresthe carcinogenicarylamineso-toluidine, * Author to whom correspondenceshould be
[email protected].
2-aminonaphthalene,and 4-aminobiphenyl.
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187
Journal of Analytical Toxicology, Vol. 30, April 2006
opsies, or exfoliated urothelial cells. In a large number of studies, smoking was found to increase the levels of arylamine hemoglobin adducts (12,23,24). 4-Aminobiphenyland 3-aminobiphenyl hemoglobin adducts have been most frequently investigated and are about 5-10-fold higher in smokers compared with nonsmokers. In bladder biopsies and exfoliated urothelial cells, DNA adducts that co-chromatographed with N-(deoxyguanosine-8-yl)-4-ABP are found, and adducts levels are generally higher in smokers compared with nonsmokers and correlated with the number of cigarettes smoked (15). Several studies report biological monitoring based on urinary excretion for occupational exposure to o-toluidine (21,25,26) and 2-aminonaphthylamine (27,28). The effect of smoking on the urinary excretion of o-toluidine (21,26,29), 2-aminonaphthalene (21,28,30), and 4-aminobiphenyl (28) has also been investigated in several studies. Both o-toluidine and 2-aminonaphthylamine excretion have been reported to be significantly increased in smokers compared to nonsmokers in one study (21). The purpose of this study was to develop a specific and sensitive method for determination of o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl excretion in smokers and nonsmokers without known occupational exposure to arylamines. The analytical O II method was required to be sensitive "N~I{ enough to allow quantification of the background exposure to these arylamines Gh~ Liver in the urine of nonsmokers. The devel~; :::: oped method was applied to a small series of urine samples from smokers and nonsmokers, and the relationship between 4-ABP-H~ ~dducl smoking dose, determined as daily Blood cigarette consumption, urinary excretion of nicotine equivalents (nicotine + 5 major metabolites), cotinine in saliva, Urinary bladder and carbon monoxide in exhaled breath and the amount ofo-toluidine, 2-amino0 naphthalene, and 4-aminobiphenyl in urine of smokers was investigated.
N-glucuronide may be transferred to the urinary bladder, where it can be hydrolyzed to the parent compound at pH < 7. A significant amount of the N-hydroxyarylamine can enter the blood stream where it is further oxidized to form the nitroso compound. During this reaction, methemoglobin is formed, particularly with single ring aromatic amines. The nitroso compound may react with cysteine residues in hemoglobin to form hemoglobin adducts. The N- and O-glucuronides of the N-hydroxyarylamine can reach the urinary bladder where formation of highly electrophilic nitrenium ions can occur, which may form DNAadducts (15). In the rat, ring-hydroxylated products and their conjugates are the major urinary metabolites of o-toluidine (16,17). Biomarkers for human exposure to aromatic amines have been extensively discussed in two recent reviews (12,18). Biomarkers of the internal dose mainly comprise the parent arylamines in urine, which can be transported in unmetabolized form into the urinary bladder or released from the N-glucuronide by acid hydrolysis in the bladder (Figure 2). The effective dose can be assessed by determining hemoglobin or DNA adducts (19-22). The latter type of biomarkers can be measured in DNA isolated from peripheral blood, bladder bi-
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Figure2. Metabolic routesof 4-aminobiphenyl (4-ABP)according to Talaskaand AI Zoughool (12) with
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Chemicals Table I. Quantifier Ions and Retention Times for the Three Analytes and the Internal Standards Analyte/InternalStandard o-Toluidine o-Toluidine-d9 2-Aminonaphthalene 2-Aminonaphthalene-d7 4-Aminobiphenyl 4-Aminobiphenyl-d9
188
Quantifier Ion (m/z)
Retention Time (min)
233 240 269 276 295 304
7.84 7.79 12.81 12.80 14.60 14.57
o-Toluidine,2-aminonaphthalene, and 4-aminobiphenyl used for method calibration were purchased from Dr. Ehrensdorfer (Augsburg, Germany). o-Toluidine-d9,2-aminonaphthalene-d7, and 4-aminobiphenyl-d9 used as internal standards for quanitification were purchased from CDN Isotopes (Pointe-Claire, QC, Canada). All other chemicals were of analytical grade or better.
Urine sample preparation The analytical procedure for the determination ofo-toluidine, 2-aminonaphthalene, and 4-aminobiphenyt in human urine followed the method described by Weiss and Angerer (31), with modifications. Human urine (5 mL) was spiked with the in-
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