DNA adducts are a measure of internal exposure to environmental and endogenous genotoxicants and are useful for risk assessment. Unfortunately, in molecular epidemiologic studies, the measurement of DNA adducts is frequently precluded by the unavailability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues are often accessible and represent a rich and largely untapped source for DNA adduct biomarker research. FFPE tissue has not been employed for quantitative measurement of DNA adducts because of the technical challenges in retrieval of high quality DNA that is fully digestible by nucleases. We report here that DNA adducts of aristolochic acids (AAs) can be measured in FFPE tissues at a level of sensitivity comparable to freshly frozen tissue. AAs are nephrotoxic and carcinogenic compounds found in Aristolochia plant species, many of which have been used worldwide for medicinal remedies. AAs are implicated in the etiology of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). 8-Methoxy-6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a major component of Aristolochia herbs and a potent human urothelial carcinogen. We established a method to quantitatively retrieve the aristolactam-DNA adduct 7-(deoxyadenosin-N6-yl) aristolactam I (dA-AL-I) from FFPE tissue. Adducts were measured, using ultra performance liquid chromatography/multistage scan mass spectrometry (UPLC-MS/MSn), in liver and kidney tissues of mice exposed to AA-I, at doses ranging from 0.001 to 1 mg/kg body weight. dA-AL-I was then measured in 10 �m thick tissue-sections of FFPE kidney from patients with upper urinary tract cancers. dA-AL-I adduct levels in FFPE were comparable to those levels measured in fresh frozen samples. The limit of quantification of dA-AL-I was 3 adducts per 109 DNA bases per 3 �g of DNA. The ability to retrospectively analyze FFPE tissues for DNA adducts may provide clues to the origin of human cancers for which an environmental cause is suspected.

Human formalin-fixed paraffin-embedded tissues: An untapped specimen for biomonitoring carcinogen DNA adducts by mass spectrometry

246th National Meeting (2013)

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