Mass Spectrometry-Based Method to Measure Aflatoxin B₁ DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues

Aflatoxin B₁ (AFB₁) 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. AFB₁ undergoes bioactivation in the liver to produce AFB₁-exo-8,9-epoxide, which forms the covalently bound cationic AFB₁-N7-guanine (AFB₁-N7-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 B₁ (AFB₁-FapyGua) adducts. The AFB₁ formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB₁. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB₁-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB₁-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB₁ 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 AFB₁-FapyGua adducts formed in AFB₁-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB₁-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS² measurements can detect AFB₁-FapyGua at a quantification limit of 4.0 adducts per 10⁸ 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 AFB₁ DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.