Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard

Frederick A. Beland, Daniel R. Doerge, Mona I. Churchwell, Miriam C. Poirier, B. Schoket, M. Matilde Marques

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P- postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N- Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 ± 0.8 adducts/108 nucleotides (mean ± SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 ± 1.7 dG-C8-4- ABP/108 nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG- C8-4-ABP/108 nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 ± 26 and 63 ± 20 dG-C8- 4-ABP/108 nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.

Original languageEnglish
Pages (from-to)68-77
Number of pages10
JournalChemical Research in Toxicology
Volume12
Issue number1
DOIs
Publication statusPublished - Jan 1999

Fingerprint

DNA Adducts
Nucleotides
Fluoroimmunoassay
Lanthanoid Series Elements
Electrospray ionization
Hydrolysis
Enzymatic hydrolysis
Electrospray Ionization Mass Spectrometry
High Pressure Liquid Chromatography
Nucleosides
Mass spectrometry
DNA
Liver
Labeling
4-biphenylamine

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard. / Beland, Frederick A.; Doerge, Daniel R.; Churchwell, Mona I.; Poirier, Miriam C.; Schoket, B.; Marques, M. Matilde.

In: Chemical Research in Toxicology, Vol. 12, No. 1, 01.1999, p. 68-77.

Research output: Contribution to journalArticle

Beland, Frederick A. ; Doerge, Daniel R. ; Churchwell, Mona I. ; Poirier, Miriam C. ; Schoket, B. ; Marques, M. Matilde. / Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard. In: Chemical Research in Toxicology. 1999 ; Vol. 12, No. 1. pp. 68-77.
@article{3ee7a8fa83e4464e88a58aa41115d1f4,
title = "Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard",
abstract = "32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P- postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N- Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 ± 0.8 adducts/108 nucleotides (mean ± SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 ± 1.7 dG-C8-4- ABP/108 nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG- C8-4-ABP/108 nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 ± 26 and 63 ± 20 dG-C8- 4-ABP/108 nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.",
author = "Beland, {Frederick A.} and Doerge, {Daniel R.} and Churchwell, {Mona I.} and Poirier, {Miriam C.} and B. Schoket and Marques, {M. Matilde}",
year = "1999",
month = "1",
doi = "10.1021/tx980172y",
language = "English",
volume = "12",
pages = "68--77",
journal = "Chemical Research in Toxicology",
issn = "0893-228X",
publisher = "American Chemical Society",
number = "1",

}

TY - JOUR

T1 - Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard

AU - Beland, Frederick A.

AU - Doerge, Daniel R.

AU - Churchwell, Mona I.

AU - Poirier, Miriam C.

AU - Schoket, B.

AU - Marques, M. Matilde

PY - 1999/1

Y1 - 1999/1

N2 - 32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P- postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N- Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 ± 0.8 adducts/108 nucleotides (mean ± SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 ± 1.7 dG-C8-4- ABP/108 nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG- C8-4-ABP/108 nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 ± 26 and 63 ± 20 dG-C8- 4-ABP/108 nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.

AB - 32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P- postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N- Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 ± 0.8 adducts/108 nucleotides (mean ± SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 ± 1.7 dG-C8-4- ABP/108 nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG- C8-4-ABP/108 nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 ± 26 and 63 ± 20 dG-C8- 4-ABP/108 nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.

UR - http://www.scopus.com/inward/record.url?scp=0032943363&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032943363&partnerID=8YFLogxK

U2 - 10.1021/tx980172y

DO - 10.1021/tx980172y

M3 - Article

VL - 12

SP - 68

EP - 77

JO - Chemical Research in Toxicology

JF - Chemical Research in Toxicology

SN - 0893-228X

IS - 1

ER -