Bladder cancer (BC) is the sixth most common cancer affecting approximately 79,000 adults annually in the United States. Tobacco smoking and occupational exposures to carcinogens, such as aromatic amines, are the most important risk factors of BC. Several epidemiological studies have reported that the consumption of well-done meat or nitrite-cured meats is risk factors of BC; however, the data are inconsistent. Endogenous by-products of lipid peroxidation are other possible agents that damage DNA of the bladder. However, the identities of chemical agents that damage DNA and initiate BC remain largely unknown. Exfoliated urinary cells are a promising, noninvasive biospecimen to screen for DNA adducts formed by genotoxic chemicals in the genitourinary system. We have established an in vitro model system of exfoliated urinary cells to investigate the stability of DNA adducts in the urine matrix and to develop a quantitative method of retrieval, identification, and measurement of DNA adducts, using aristolochic acid (AA-I and 4-aminobiphenyl (4-ABP) as prototypical genitourinary human carcinogens. The RT4, a well-differentiated human bladder cancer cell line that largely retains transitional epithelial structure, has been reported to be a reliable model cell line to study aromatic amine metabolism, DNA adduct formation, and cytotoxicity. Thus, this cell line is ideal for our in vitro model system. RT4 cells were treated with 4-ABP and AA-I for 24 hours. Afterward, one million treated cells were spiked into 40 mL of freshly voided human urine and incubated for up to 24 hours at either 4 °C or ambient temperature. The cell viability exceeded 80% over time and the recovery of DNA was comparable at both temperatures. The DNA adducts of 4-ABP and AA-I formed in RT4 cells were stable in the urine of three subjects up to 24 hours of incubation at both temperatures. The quantitation of DNA adducts was performed by UPLC-ion trap MS3. Our preliminary data are highly promising and suggest that DNA adducts of other genitourinary carcinogens can be screened in human urinary cells. With further optimization of the bioanalytical method, we will examine for DNA adducts in exfoliated urinary cells of tobacco smokers to identify major DNA damaging agents in tobacco smoke.

Noninvasive measurement of bladder carcinogen DNA adducts in human urinary cells by liquid chromatography-tandem mass spectrometry

256th National Meeting (2018)

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