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

Pawel Jaruga, Rachana Tomar, Melis Kant, Vladimir Vartanian, Benjamin Sexton, Carmelo J. Rizzo, Robert J. Turesky, Michael P. Stone, R. Stephen Lloyd, Miral Dizdaroglu

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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.

Original languageEnglish (US)
Pages (from-to)14841-14854
Number of pages14
JournalACS Omega
Issue number16
StatePublished - Apr 25 2023

Bibliographical note

Funding Information:
This work was partially supported by the following sources: National Institute of Standards and Technology (P.J., M.K., and M.D.) and National Institutes of Health (NIH) grants R01 CA-55678, R01 ES-029357, and P01 CA-160032 (M.P.S., C.J.R, R.J.T., and R.S.L.). R.S.L. acknowledges support from the National Institute of Environmental Health Sciences (R01 ES-031086) and from the Oregon Institute of Occupational Health Sciences at Oregon Health & Science University via funds from the Division of Consumer and Business Services of the State of Oregon (ORS 656.630). The Vanderbilt-Ingram Cancer Center was funded by NIH grant P30 CA-068485.

Funding Information:
Certain equipment, instruments, software, or materials, commercial or non-commercial, are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement of any product or service by NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose. This work with the Project no. MML-16-0016 and with the Research Collaboration Agreement no. RCA-23-001 between Oregon Health & Science University (OHSU) and NIST was reviewed and approved by the Office of Technology Transfer of OHSU and the Technology Partnership Office of NIST. We thank Dr. Michelle Reyzer at Vanderbilt Mass Spectrometry facility for her assistance in the measurements with MALDI-TOF mass spectrometry. We thank Dr. John Groopman of Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, for very helpful discussions.

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

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