Abstract Introduction: Bladder cancer (BCa) is the second most prevalent urological malignancy and the fourth highest cause of cancer-related death in the United States. Earlier studies have linked BCa development to alterations in metabolic pathways. Significant among these are decreased activity of N-acetyl transferases causing a slow-acetylator phenotype leading to inefficient detoxification of aromatic hydrocarbons causal to onset of BCa. Interestingly, Afro-American patients inherently exhibit such slow acetylator phenotype and are known to have a more aggressive form of the tumor compared to Caucasians. This indicates existence of a metabolic niche that governs the racial disparity in BCa, which is not well understood. Also, there is an imminent need to develop non-invasive markers for early detection and prognosis of BCa, since urine cytology which is the current clinical standard is not specific to the tumor. Using mass spectrometry we report metabolic alterations in BCa and delineate bioprocesses that are altered during its progression. Our data for the first-time demonstrates role of methylation in attenuation of xenotbiotic metabolism in BCa. Furthermore, the metabolic profiles seed further analysis to examine the racial disparity in these tumors. Methods: Total metabolome from flash frozen clinically annotated bladder-derived tissues (n=58,31 benign adjacent and 27 BCa, 26 matched pairs) were examined using a combination of Q-TOF (unbiased) and triple-quadrupole (targeted) mass spectrometry. Panel of well-defined standards were used to ensure reproducibility of the profiling process. The metabolites were pre-fractionated using liquid chromatography prior to mass spectrometry in both the positive and negative ionization mode. The unbiased mass spectral data was searched using Metlin library to identify the compounds. The metabolomic profiles thus generated were analyzed to delineate class-specific signatures which were interrogated for altered bioprocesses using Molecular Concept Map (OCM, www.oncomine.org). The altered bioprocesses were validated in cell line models using a combination of Q-PCR, immunoblot analysis and functional assays. Results and discussion: A total of 2019 compounds were detected across the 58 bladder-derived specimens of which, 423 compounds were significantly altered in BCa compared to adjacent benign. 50 of the differential compounds were named and used for developing a classificatory signature and bioprocess mapping. Included among these were polycylic compounds like aniline, catechols, aromatic amino acids, polyamines and S-adenosyl methionine (SAM). Interestingly this BCa-specific metabolic signature in tissues was able to delineate tumor from benign with an accuracy of 75 %. Importantly the functional mapping of the metabolic data revealed enhanced methylation potential in tumors as being one of the factors de-regulating the xenobiotic metabolism. In vitro experiments using bisulfite sequencing and methyltransferase inhibitor 5-Aza-cytidine confirmed this methylation-induced attenuation of phase I/II metabolic genes namely CYP1A1, CYP1B1, EPHX1 and GSTT1 in BCa. In summary, using unbiased metabolomic profiling report metabolic fingerprint for bladder cancer. Importantly our data for the first time reveals methylation-induced silencing of xenobiotic metabolism in bladder tumors. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A52.