Synthesis and Characterization of ¹⁵N₅-Labeled Aflatoxin B₁–Formamidopyrimidines and Aflatoxin B₁–N7-Guanine from a Partial Double-Stranded Oligodeoxynucleotide as Internal Standards for Mass Spectrometric Measurements

Abstract

Aflatoxin B1 (AFB1) exposure through contaminated food is a primary contributor to hepatocellular carcinogenesis worldwide. Hepatitis B viral infections in livers dramatically increase the carcinogenic potency of AFB1 exposures. Liver cytochrome P450 oxidizes AFB1 to the epoxide, which in turn reacts with N7-guanine in DNA, producing the cationic trans-8,9-dihydro-8-(N7-guanyl)–9-hydroxyaflatoxin B1 adduct (AFB1–N7-Gua). The opening of the imidazole ring of AFB1–N7-Gua under physiological conditions causes the formation of the cis- and trans-diastereomers of 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)–9-hydroxyaflatoxin B1 (AFB1–FapyGua). These adducts primarily lead to G → T mutations, with AFB1–FapyGua being significantly more mutagenic than AFB1–N7-Gua. The unequivocal identification and accurate quantification of these AFB1–Gua adducts as biomarkers are essential for a fundamental understanding and prevention of AFB1-induced hepatocellular carcinogenesis. Among a variety of analytical techniques used for this purpose, liquid chromatography–tandem mass spectrometry, with the use of the stable isotope-labeled analogues of AFB1–FapyGua and AFB1–N7-Gua as internal standards, provides the greatest accuracy and sensitivity. cis-AFB1–FapyGua-15N5, trans-AFB1–FapyGua-15N5, and AFB1–N7-Gua-15N5 have been synthesized and used successfully as internal standards. However, the availability of these standards from either academic institutions or commercial sources ceased to exist. Thus, quantitative genomic data regarding AFB1-induced DNA damage in animal models and humans remain challenging to obtain. Previously, AFB1–N7-Gua-15N5 was prepared by reacting AFB1-exo-8,9-epoxide with the uniformly 15N5-labeled DNA isolated from algae grown in a pure 15N-environment, followed by alkali treatment, resulting in the conversion of AFB1–N7-Gua-15N5 to AFB1–FapyGua-15N5. In the present work, we used a different and simpler approach to synthesize cis-AFB1–FapyGua-15N5, trans-AFB1–FapyGua-15N5, and AFB1–N7-Gua-15N5 from a partial double-stranded 11-mer Gua-15N5-labeled oligodeoxynucleotide, followed by isolation and purification. We also show the validation of these 15N5-labeled standards for the measurement of cis-AFB1–FapyGua, trans-AFB1–FapyGua, and AFB1–N7-Gua in DNA of livers of AFB1-treated mice.

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

“…The measured AFB 1 –FapyGua adduct levels in the liver of 7 newborn mice employing the Velos ion trap with multistage MS 3 scanning and Orbitrap at the MS 2 scan stage are reported in Figure . Similar to previous studies, the a R -AFB 1 –FapyGua was the predominant isomer, occurring at ∼5 to 7-fold higher levels than the a S -AFB 1 –FapyGua. , The levels of adducts recovered from fresh frozen and FFPE specimens were generally within 20% for most liver samples. Some minor differences in adduct levels between the Velos ion trap and Orbitrap MS were observed, particularly for a S -AFB 1 –FapyGua in mouse livers ML2 and ML3, which were about 3-fold above LOQ values on the Velos.…”

“…We recently established a method to efficiently reverse formalin-mediated cross-links of DNA in FFPE tissues under mild conditions, allowing for quantitative measurements of DNA adducts formed with aristolochic acid-I, aromatic amines, heterocyclic aromatic amines, nicotine-derived nitrosamine ketone (NNK), and polycyclic aromatic hydrocarbon carcinogens. , This breakthrough technology paves the way for advancing DNA adduct biomarker research with human FFPE tissues. , In this study, we show that ring-opened AFB 1 –FapyGua adducts are stable toward the FFPE and DNA retrieval processes in the newborn mouse model . We employed isotopically labeled AFB 1 -[ 15 N 5 ]-FapyGua that were site-specifically synthesized in an 11-mer oligodeoxynucleotide as the internal standards. , …”

“…The AFB 1 epoxide was reacted with unlabeled and 15 N-labeled oligodeoxynucleotides to form the AFB 1 –FapyGua adducts. The purification, UV spectroscopy, and mass spectral characterization of the unlabeled and 15 N 5 -labeled AFB 1 –FapyGua oligodeoxynucleotides were reported. , A molar extinction coefficient of 18,000 at 368 nm was used to determine the concentrations of the AFB 1 –Fapy-dG 11-mer oligodeoxynucleotides. , …”

“…The AFB 1 DNA adduct measurements in rodent liver were performed following isolation and acid treatment of DNA, recovering AFB 1 – N 7-Gua and the diastereomeric ring-opened AFB 1 –FapyGua adducts (Scheme ). , Recent studies reported the simultaneous quantification of AFB 1 – N 7-Gua, a S -AFB 1 –FapyGua, and a R -AFB 1 –FapyGua in mouse liver DNA by triple quadrupole mass spectrometry employing each isotopically 15 N-labeled internal standard. , …”