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Chem. Res. Toxicol. 1999, 12, 78-82
Detection of Monocyclic Aromatic Amines, Possible Mammary Carcinogens, in Human Milk Lillian S. DeBruin,† Janusz B. Pawliszyn,‡ and P. David Josephy*,† Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry (GWC2), Department of Chemistry & Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1, and Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 Received July 13, 1998
Environmental chemicals may play a role in the etiology of human breast cancer. Aromatic amines, industrial chemicals found as environmental pollutants, have been identified as rat mammary carcinogens. We have detected the monocyclic aromatic amines, aniline, o-toluidine, and N-methylaniline at parts per billion levels in human milk samples, by applying solidphase microextraction coupled with gas chromatography/mass spectrometry. Our findings indicate that human breast ductal epithelial cells are directly exposed to aromatic amines, including o-toluidine, which is a mammary carcinogen in female rats.
Introduction Identified hormonal and genetic risk factors account for fewer than half of breast cancer cases, and many researchers have proposed that environmental chemical exposures may also be important (1-3). The late onset of postmenopausal breast cancer is consistent with the hypothesis that chemical carcinogenesis is involved in its etiology. There are large geographic variations in breast cancer incidence, with particularly high rates in the industrial northeastern United States. Residence in a heavily industrialized and/or high-vehicular traffic area has been associated with increased breast cancer (4). Molecular analysis also indicates a possible role for genotoxic chemicals in breast carcinogenesis. Breast tissue samples from breast cancer patients have significantly higher levels of aromatic DNA adducts than samples from healthy controls (5, 6). Some samples of human nipple aspirate fluid and lipid extracts of breast tissue are positive in the Ames mutagenicity assay (7-9). Bioassays have identified several classes of chemicals as rat mammary carcinogens, including chlorinated compounds, polycyclic aromatic hydrocarbons (PAHs1), aromatic amines (AAs), and nitroaromatic compounds, which are widespread in the environment. AAs are used in the manufacturing of plastics (such as polyurethane foams), dyes, pesticides, and pharmaceuticals. Environmental sources of AAs include industrial waste, air and water pollution, tobacco smoke, and diet. The polycyclic AAs 2-naphthylamine, benzidine, and 4-aminobiphenyl, * To whom correspondence should be addressed: Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext 3833. Fax: (519) 7661499. E-mail:
[email protected]. † University of Guelph. ‡ University of Waterloo. 1 Abbreviations: AAs, aromatic amines; Hb, hemoglobin; MDL, method detection limit; PAHs, polycyclic aromatic hydrocarbons; PCBs, polychlorinated biphenyls; ppb, parts per billion; PDMS/DVB, poly(dimethyl)siloxane/divinylbenzene; ppt, parts per trillion; SIM, selected ion monitoring; SPME, solid-phase microextraction; TCDD, 2,3,7,8tetrachlorodibenzo-p-dioxin.
and possibly also the monocyclic AAs o-toluidine and 4-chloro-o-toluidine, are human bladder carcinogens (10, 11). These AAs were linked to human bladder cancer by epidemiological studies of occupationally exposed (male) workers. Women exposed to AAs in a dye factory in Moscow (formerly the USSR) may have a higher incidence of breast cancer (12). Analysis of human milk is a noninvasive method for detecting chemicals which have passed through the breast ductal epithelium, the target tissue for breast carcinogenesis. Several classes of xenobiotic chemicals have been detected in human milk (13). Milk levels of globally distributed environmental organochlorine pollutants, such as pesticides and polychlorinated biphenyls (PCBs), have been surveyed for decades (14, 15). However, few of these compounds are rodent mammary carcinogens, and their significance for human breast cancer has not been established (16). PAHs, some of which are rodent mammary carcinogens, have been detected in human milk (13). AAs are rat mammary carcinogens, and AAs fed to lactating rats are excreted in milk (17-19). If women are exposed to environmental AAs, elimination via breast milk can be anticipated. Nevertheless, the possible presence of AAs in human milk has not previously been studied. Our study was designed to test this possibility. We have established an analytical method for the detection and quantification of monocyclic AAs in biological fluids, including human milk (20). The solid-phase microextraction (SPME) procedure is a rapid, one-step, solvent-free extraction and concentration system, and affords high specificity and reproducibility. We have now quantitated levels of aniline, o-toluidine, and N-methylaniline in human milk samples from smoking and nonsmoking mothers. This is the first report of the detection of environmental AAs in human milk and indicates that the ductal epithelial cells of the breast are exposed to AAs. o-Toluidine (21) is a mammary carcinogen (inducing fibroadenomas and adenomas in the female rat), and its presence in human milk warrants further attention.
10.1021/tx980168m CCC: $18.00 © 1999 American Chemical Society Published on Web 12/08/1998
Aromatic Amines in Human Milk
Chem. Res. Toxicol., Vol. 12, No. 1, 1999 79
Experimental Procedures Chemicals and Materials. Aniline, o-toluidine, and Nmethylaniline were purchased from Aldrich (Sigma-Aldrich Canada, Oakville, ON). Caution: Aromatic amines are known or suspected carcinogens and should be handled in a fume hood. The internal standards were o-toluidine-d9 (CDN Isotopes Inc., Point-Claire, PQ) and aniline-d5 (Supelco, Sigma-Aldrich Canada). A stock solution containing aniline-d5 (1000 µg/mL) and otoluidine-d9 (500 µg/mL) was prepared in methanol and then diluted 100-fold to a working solution. All SPME holders and poly(dimethyl)siloxane/divinylbenzene (PDMS/DVB) fibers were obtained from Supelco. Instrumentation. All analyses were carried out on a HewlettPackard gas chromatograph (HP 5890 series II) coupled to a quadrupole mass spectrometer (HP5971 mass selective detector). Midmass autotunes with perfluorotributylamine were carried out on a daily basis. The electron energy was 70 eV, and the source temperature was 200 °C. The carrier gas was helium (18 cm/s measured at 45 °C). The injector (with a silanized 0.75 mm insert) was maintained at 210 °C, and all injections were made in the spitless mode. The column [5% diphenyl-95% dimethylsiloxane copolymer, 30 m × 0.25 mm i.d., 0.25 µm film (DB-5MS, J&W Scientific, Folsom, CA)] with a retention gap (1 m × 0.25 mm i.d., intermediate polarity) was held at 45 °C for 1.0 min and then ramped to 130 °C (at 5 °C/ min) and to 250 °C (25 °C/min) and held at 250 °C for 2 min. For the SIM analyses, the following ions were selected: 66, 93, 98, 106, 107, and 114. The base ion for aniline was 93, with 66 as the qualifying ion. The m/z 98 ion was used to monitor aniline-d5. The base ion for o-, m-, and p-toluidine and Nmethylaniline was 106, with 107 as the qualifying ion. The m/z 114 ion was used to monitor the level of o-toluidine-d7 (deuterium exchange of the amino group of o-toluidine-d9 occurs in aqueous solution). Calibration curves were constructed for the quantitation of aniline with aniline-d5, and for o-toluidine and N-methylaniline with o-toluidine-d9. Samples. From healthy lactating volunteers, milk samples (10-60 mL) were collected, immediately frozen, and then stored at -70 °C. A total of 31 mothers donated a single milk sample. The stage of lactation ranged from 2 days to 134 weeks. None of the donors reported occupational exposure to AAs. Twentythree percent (n ) 7) of the donors were smokers; four of these were light smokers (
