Anal. Chem. 2006, 78, 6422-6432
Quantification of N-(Deoxyguanosin-8-yl)-4-aminobiphenyl Adducts in Human Lymphoblastoid TK6 Cells Dosed with N-hydroxy-4-acetylaminobiphenyl and Their Relationship to Mutation, Toxicity, and Gene Expression Profiling Elaine M. Ricicki,† Wen Luo,†,‡ Wenhong Fan,‡ Lue Ping Zhao,‡ Helmut Zarbl,*,‡ and Paul Vouros*,† 1The
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, and 2Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
Gene expression profiles that are anchored to phenotypic endpoints may lead to the identification of signatures that predict mutagenicity or carcinogenicity. The study presented here describes the analysis of DNA adducts in the human TK6 lymphoblastoid cell line after exposure to N-hydroxy-4-aminobiphenyl, a mutagenic metabolite of 4-aminobiphenyl. A validated nano-LC microelectrospray mass spectrometry assay is reported for the detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the principal DNA adduct of 4-aminobiphenyl. Limits of quantification, based on a signal-to-noise ratio of 10:1, are determined to correspond to ∼27 fg of dG-C8-ABP injected on-column. The assay has been used to measure the steady-state levels of the adduct in the human TK6 lymphoblastoid cell line as a function of dose (0.5, 1.0, and 10.0 µM) and time (2, 6, and 27 h) after exposure to N-hydroxy-4-aminobiphenyl. The levels of dGC8-ABP adducts in the cells, ranging from 18 to 500 adducts in 109 nucleotides, were then correlated to cell toxicity, induced mutation at the TK (thymidine kinase) and HPRT loci, and gene expression profiling through microarray analysis. Cell cultures were evaluated for toxicity by growth curve extrapolation, mutation assays were performed on the HPRT and TK loci, and gene expression profiles were generated by analyses using microarray technology. In the mutation assay analysis, as the toxicant concentration increased, there was an increase in mutation fraction, indicating a direct correlation to metabolite dosing level and mutations occurring at these two loci. Statistical analysis of the gene expression data determined that a total of 2250 genes exhibited statistically significant changes in expression after treatment with N-OH-AABP (P < 0.05). Among the genes identified, 2245 were up-regulated, whereas 5 genes that had functions in cell survival and cell growth and, hence, could be indicators of toxicity, were down-regulated relative to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic mark6422 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
ers, such as DNA adduct levels, toxicity, and mutagenicity. Many aromatic amines, in particular, arylamines and nitroamines, are potent mutagens and have been implicated in chemical carcinogenesis. Some of the chemicals in this classification include 2-chloroaniline (2-CA), 4-chloroaniline (4-CA), 2-methylaniline (2MA), 4-methylaniline (4-MA), 2,4-dimethylaniline (2,4-DMA), 2,6dimethylaniline (2,6-DMA), 2-aminobiphenyl (2-ABP), 3-aminobiphenyl (3-ABP), and 4-aminobiphenyl (4-ABP), the latter being the primary focus of several investigations of mutagenesis and tumorigenesis.1-3 4-Aminobiphenyl is an environmental contaminant found in cigarette smoke, paints, food colors, hair dyes, and fumes from heated oils and fuels.4-7 The mutagenic activity of aromatic amines has been demonstrated using a variety of approaches. Mutation assays in both cell lines and animal models have concluded that these compounds, especially 4-aminobiphenyl, have significant mutagenic activity. For example, Phillipson and Ioannides first investigated hepatic microsomal preparations derived from mice, hamsters, rats, pigs, and humans for the metabolic activation of the aromatic amines to mutagens.8 More recently, Lasko et al. investigated the mutagenesis of a reactive * Corresponding author address: Department of Chemistry and Chemical Biology, Northeastern University, 120 Hurtig Hall, 360 Huntington Ave., Boston, MA 02115. Phone: (617) 373-2840. Fax: (617) 373-2693. E-mail:
[email protected]. † Northeastern University. ‡ Fred Hutchinson Cancer Research Center. (1) Flamini, G.; Romano, G.; Curigliano, G.; Chiominto, A.; Capelli, G.; Boninsegna, A.; Signorelli, C.; Ventura, L.; Santella, R. M.; Sgambato, A.; Cittadini, A. Carcinogenesis 1998, 19, 353-357. (2) Otteneder, M.; Lutz, W. K. Mutat. Res. 1999, 424, 237-247. (3) Schieferstein, G. J.; Littlefield, N. A.; Gaylor, D. W.; Sheldon, W. G.; Burger, G. T. Eur. J. Cancer Clin. Oncol. 1985, 21, 865-873. (4) Oh, S. W.; Kang, M. N.; Cho, C. W.; Lee, M. W. Dyes Pigm. 1997, 33, 119-135. (5) Garrigos, M. C.; Reche, F.; Pernias, K.; Sanchez, A.; Jimenez, A. J. Chromatogr., A 1998, 819, 259-266. (6) Tokiwa, H.; Nakagawa, R.; Horikawa, K. Mutat. Res. 1985, 157, 39-47. (7) Turesky, R. J.; Freeman, J. P.; Holland, R. D.; Nestorick, D. M.; Miller, D. W.; Ratnasinghe, D. L.; Kadlubar, F. F. Chem. Res. Toxicol. 2003, 16, 11621173. (8) Phillipson, C. E.; Ioannides, C. Mutat. Res. 1983, 124, 325-336. 10.1021/ac0607360 CCC: $33.50
© 2006 American Chemical Society Published on Web 07/22/2006
form of 4-aminobiphenyl in the Escherichia coli virus, M13mp10, and Levine et al. researched frameshift mutations in 4-aminobiphenyl-induced mutations in Salmonella.9,10 Studies of DNA binding spectra and biopsies of human tissues have also implicated 4-aminobiphenyl in human bladder carcinogenesis.11,12 Aromatic amines, including 4-aminobiphenyl, can bind covalently to DNA bases, including various sites on guanosine and adenosine bases; however, 4-aminobiphenyl has shown preferential adduction to deoxyguanosine at the C8 position forming N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The latter adducts have been shown to be premutagenic in both in vitro and in vivo assays, suggesting that these adducts are also the precursors for carcinogenic lesions.13,14 As a result, the detection and measurement of 4-aminobiphenyl adduct levels in tissues of exposed individuals has significant implications for risk assessment. Mutagenic adducts of 4-aminobiphenyl, including dG-C8-ABP, have been detected in human urinary bladder and lung tissue at levels ranging from
