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

Aflatoxin B-1 (AFB(1)) exposure through contaminated food is a primary contributor to hepatocellular carcinogenesis worldwide. Hepatitis B viral infections in livers dramatically increase the carcinogenic potency of AFB(1) exposures. Liver cytochrome P450 oxidizes AFB(1) 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 (AFB(1)-N7-Gua). The opening of the imidazole ring of AFB(1)-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-ylformamido)-9-hydroxyaflatoxin B-1 (AFB(1)-FapyGua). These adducts primarily lead to G -> T mutations, with AFB1-FapyGua being significantly more mutagenic than AFB(1)-N7-Gua. The unequivocal identification and accurate quantification of these AFB(1)-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 AFB(1)-FapyGua and AFB(1)-N7-Gua as internal standards, provides the greatest accuracy and sensitivity. cis-AFB(1)-FapyGua-N-15(5), trans-AFB(1)-FapyGua-N-15(5), and AFB(1)-N7-Gua-(1)5N(5) 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 AFB(1)-induced DNA damage in animal models and humans remain challenging to obtain. Previously, AFB(1)-N7-Gua-N-15(5) was prepared by reacting AFB(1)-exo-8,9-epoxide with the uniformly N-15(5)-labeled DNA isolated from algae grown in a pure N-15-environment, followed by alkali treatment, resulting in the conversion of AFB(1)-N7-Gua-N-15(5) to AFB(1)-FapyGua-N-15(5). In the present work, we used a different and simpler approach to synthesize cis-AFB(1)-FapyGua-N-15(5), trans-AFB(1)-FapyGua-N-15(5), and AFB(1)-N7-Gua-N-15(5) from a partial double-stranded 11-mer Gua-N-15(5)-labeled oligodeoxynucleotide, followed by isolation and purification. We also show the validation of these N-15(5)-labeled standards for the measurement of cis-AFB(1)-FapyGua, trans-AFB(1)-FapyGua, and AFB(1)-N7-Gua in DNA of livers of AFB(1)-treated mice.

Automated summary

Exposure to Aflatoxin B-1 (AFB(1)) through contaminated food is a major cause of liver cancer worldwide. Hepatitis B viral infections in the liver enhance the carcinogenic effect of AFB(1) exposure. The formation of AFB(1)-N7-Gua and AFB(1)-FapyGua adducts in DNA, resulting from the reaction between AFB(1) and N7-guanine, is critically important for the development and prevention of AFB1-induced liver cancer. Liquid chromatography-tandem mass spectrometry with stable isotope-labeled analogues of AFB(1)-N7-Gua and AFB(1)-FapyGua as internal standards is the most accurate and sensitive method for the identification and quantification of these biomarkers.