Mass Spectrometry-Based Method to Measure Aflatoxin B 1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B 1 (AFB 1 ) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus , which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB 1 undergoes bioactivation in the liver to produce AFB 1 - exo -8,9-epoxide, which forms the covalently bound cationic AFB 1 - N 7-guanine (AFB 1 -N 7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B 1 (AFB 1 -FapyGua) adducts. The AFB 1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB 1 . Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB 1 - N 7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB 1 -FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB 1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB 1 -FapyGua adducts formed in AFB 1 -exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB 1 -FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS 2 measurements can detect AFB 1 -FapyGua at a quantification limit of 4.0 adducts per 10 8 bases when only 0.8 μg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB 1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.