Production, purification and characterization of 15N5-labeled cis- and trans-aflatoxin B1-formamidopyrimidines, and aflatoxin B1-N7-guanine as internal standards for mass spectrometric measurements

Published

April 11, 2023

Author(s)

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

Abstract

Exposure to aflatoxin B1 (AFB1) through contaminated food is a primary contributor to the occurrence of 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 in the past, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the use of the stable isotope-labeled analogues of AFB1-N7-Gua and AFB1-FapyGua as internal standards afforded the greatest accuracy and sensitivity for measurements. AFB1-N7-Gua-15N5, cis-AFB1-FapyGua-15N5 and trans-AFB1-FapyGua-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. Herein, we report the synthesis, isolation, purification, and validation of AFB1-N7-Gua-15N5, cis-AFB1-FapyGua-15N5 and trans-AFB1-FapyGua-15N5 for use as internal standards for mass spectrometric measurements of the AFB1-Gua adducts in biological samples.

Citation

ACS Ω

Pub Type

Journals

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https://doi.org/10.1021/acsomega.3c01328

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Keywords

aflatoxin B1, carcinogenesis, AFB1-N7-Gua, cis-AFB1-FapyGua, trans-AFB1-FapyGua, LC-MS/MS, AFB1 adducts' standards

Citation

Jaruga, P. , Kant, M. , Dizdar, M. , Tomar, R. , Vartanian, V. , Sexton, B. , Rizzo, C. , Turesky, R. , Stone, M. and Lloyd, R. (2023), Production, purification and characterization of 15N5-labeled cis- and trans-aflatoxin B1-formamidopyrimidines, and aflatoxin B1-N7-guanine as internal standards for mass spectrometric measurements, ACS Ω, [online], https://doi.org/10.1021/acsomega.3c01328, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=935378 (Accessed April 20, 2024)