posted on 2024-03-18, 15:38authored byMedjda Bellamri, Lihua Yao, Rachana Tomar, Vladimir Vartanian, Carmelo J. Rizzo, Michael P. Stone, John D. Groopman, R. Stephen Lloyd, Robert J. Turesky
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.