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

doi:10.1021/acsomega.3c01328

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

Medicinal Chemistry

Research output: Contribution to journal › Article › peer-review

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.

Original language	English (US)
Pages (from-to)	14841-14854
Number of pages	14
Journal	ACS Omega
Volume	8
Issue number	16
DOIs	https://doi.org/10.1021/acsomega.3c01328
State	Published - 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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Jaruga, P., Tomar, R., Kant, M., Vartanian, V., Sexton, B., Rizzo, C. J., Turesky, R. J., Stone, M. P., Lloyd, R. S., & Dizdaroglu, M. (2023). 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. ACS Omega, 8(16), 14841-14854. https://doi.org/10.1021/acsomega.3c01328

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. / Jaruga, Pawel; Tomar, Rachana; Kant, Melis et al.
In: ACS Omega, Vol. 8, No. 16, 25.04.2023, p. 14841-14854.

Research output: Contribution to journal › Article › peer-review

Jaruga, P, Tomar, R, Kant, M, Vartanian, V, Sexton, B, Rizzo, CJ, Turesky, RJ, Stone, MP, Lloyd, RS & Dizdaroglu, M 2023, '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', ACS Omega, vol. 8, no. 16, pp. 14841-14854. https://doi.org/10.1021/acsomega.3c01328

@article{ead060518af641fc958b534823e0eaac,

title = "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.",

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

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: {\textcopyright} 2023 The Authors. Published by American Chemical Society.",

year = "2023",

month = apr,

day = "25",

doi = "10.1021/acsomega.3c01328",

language = "English (US)",

volume = "8",

pages = "14841--14854",

journal = "ACS Omega",

issn = "2470-1343",

publisher = "American Chemical Society",

number = "16",

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TY - JOUR

T1 - 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

AU - Jaruga, Pawel

AU - Tomar, Rachana

AU - Kant, Melis

AU - Vartanian, Vladimir

AU - Sexton, Benjamin

AU - Rizzo, Carmelo J.

AU - Turesky, Robert J.

AU - Stone, Michael P.

AU - Lloyd, R. Stephen

AU - Dizdaroglu, Miral

N1 - 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.

PY - 2023/4/25

Y1 - 2023/4/25

N2 - 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.

AB - 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.

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