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* ,† 1 The Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, and 2 Fred 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 pre- sented 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-aminobi- phenyl. 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 dG- C8-ABP adducts in the cells, ranging from 18 to 500 adducts in 10 9 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 in- crease 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 treat- ment 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 rela- tive to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic mark- ers, such as DNA adduct levels, toxicity, and mutagenic- ity. Many aromatic amines, in particular, arylamines and nitro- amines, 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 (2- MA), 4-methylaniline (4-MA), 2,4-dimethylaniline (2,4-DMA), 2,6- dimethylaniline (2,6-DMA), 2-aminobiphenyl (2-ABP), 3-aminobi- phenyl (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 contami- nant 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: p.vouros@neu.edu. † Northeastern University. ‡ Fred Hutchinson Cancer Research Center. (1) Flamini, G.; Romano, G.; Curigliano, G.; Chiominto, A.; Capelli, G.; Bonin- segna, 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, 1162- 1173. (8) Phillipson, C. E.; Ioannides, C. Mutat. Res. 1983, 124, 325-336. Anal. Chem. 2006, 78, 6422-6432 6422 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006 10.1021/ac0607360 CCC: $33.50 © 2006 American Chemical Society Published on Web 07/22/2006