The measurement of DNA adducts of carcinogens in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues frequently are accessible, although challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts by mass spectrometric methods. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. Reactive metabolites of AA covalently bind to DNA, generating persistent aristolactam (AL)-DNA adducts. These lesions are responsible for mutations (A ? T transversions) in the human TP53 gene, resulting in urothelial cancer. We established a method to measure AL-DNA adducts in FFPE tissue by ultra-performance liquid chromatography-multi stage scan mass spectrometry (UPLC-MSn). In this study, we report additional features of the formalin fixation process that could impact the quantity and quality of DNA, and the recovery of AL-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues preserved with formalin decreased during 1 week of fixation; however, the levels of AL-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification yielding sequence data of comparable quality to DNA of fresh frozen tissue. The DNA retrieval methodology was applied to human biospecimens. The estimates of AL-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for up to 9 years showed good concordance. AL-DNA adducts were measured at level of 3 adducts per 109 DNA bases using only 2 �g of DNA. Thus, DNA from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues about the origin of human cancers for which an environmental cause is suspected.

Formalin-fixed paraffin embedded tissue: An underutilized biospecimen for quantitation of carcinogen DNA adducts

248th National Meeting (2014)

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