High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts

Frederick A. Beland, Mona I. Churchwell, Linda S. Von Tungeln, Shoujun Chen, Peter P. Fu, Sandra J. Culp, B. Schoket, Erika Gyorffy, J. Mináróvits, Miriam C. Poirier, Elise D. Bowman, Ainsley Weston, Daniel R. Doerge

Research output: Contribution to journalArticle

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Abstract

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/ MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8, 9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-2H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 μg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10 8 nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3- 3H]BPDE, the method gave a value very similar to those obtained by radiolabeling, 32P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-3H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 ± 0.04 dG-BPDE adducts/108 nucleotides. This value is much lower than the 102 ± 14 total DNA adducts/ 108 nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/108 nucleotides, when using 100 μg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

Original languageEnglish
Pages (from-to)1306-1315
Number of pages10
JournalChemical Research in Toxicology
Volume18
Issue number8
DOIs
Publication statusPublished - Aug 2005

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Electrospray ionization
Electrospray Ionization Mass Spectrometry
High performance liquid chromatography
Tandem Mass Spectrometry
Mass spectrometry
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
DNA Adducts
High Pressure Liquid Chromatography
Coal Tar
DNA
Nucleotides
Immunoassay
Lung
7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene-10-deoxyguanosine
benzo(a)pyrene-DNA adduct
Salmon
Benzo(a)pyrene
Polycyclic Aromatic Hydrocarbons
Polycyclic aromatic hydrocarbons
Nutrition

ASJC Scopus subject areas

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

Cite this

High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts. / Beland, Frederick A.; Churchwell, Mona I.; Von Tungeln, Linda S.; Chen, Shoujun; Fu, Peter P.; Culp, Sandra J.; Schoket, B.; Gyorffy, Erika; Mináróvits, J.; Poirier, Miriam C.; Bowman, Elise D.; Weston, Ainsley; Doerge, Daniel R.

In: Chemical Research in Toxicology, Vol. 18, No. 8, 08.2005, p. 1306-1315.

Research output: Contribution to journalArticle

Beland, FA, Churchwell, MI, Von Tungeln, LS, Chen, S, Fu, PP, Culp, SJ, Schoket, B, Gyorffy, E, Mináróvits, J, Poirier, MC, Bowman, ED, Weston, A & Doerge, DR 2005, 'High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts', Chemical Research in Toxicology, vol. 18, no. 8, pp. 1306-1315. https://doi.org/10.1021/tx050068y
Beland, Frederick A. ; Churchwell, Mona I. ; Von Tungeln, Linda S. ; Chen, Shoujun ; Fu, Peter P. ; Culp, Sandra J. ; Schoket, B. ; Gyorffy, Erika ; Mináróvits, J. ; Poirier, Miriam C. ; Bowman, Elise D. ; Weston, Ainsley ; Doerge, Daniel R. / High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts. In: Chemical Research in Toxicology. 2005 ; Vol. 18, No. 8. pp. 1306-1315.
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abstract = "A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/ MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8, 9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-2H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 μg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10 8 nucleotides). The results indicated an inter- and intraday accuracy of 99-100{\%} and precision of 1.6-1.7{\%} (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3- 3H]BPDE, the method gave a value very similar to those obtained by radiolabeling, 32P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-3H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3{\%} coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 ± 0.04 dG-BPDE adducts/108 nucleotides. This value is much lower than the 102 ± 14 total DNA adducts/ 108 nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/108 nucleotides, when using 100 μg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.",
author = "Beland, {Frederick A.} and Churchwell, {Mona I.} and {Von Tungeln}, {Linda S.} and Shoujun Chen and Fu, {Peter P.} and Culp, {Sandra J.} and B. Schoket and Erika Gyorffy and J. Min{\'a}r{\'o}vits and Poirier, {Miriam C.} and Bowman, {Elise D.} and Ainsley Weston and Doerge, {Daniel R.}",
year = "2005",
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language = "English",
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TY - JOUR

T1 - High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts

AU - Beland, Frederick A.

AU - Churchwell, Mona I.

AU - Von Tungeln, Linda S.

AU - Chen, Shoujun

AU - Fu, Peter P.

AU - Culp, Sandra J.

AU - Schoket, B.

AU - Gyorffy, Erika

AU - Mináróvits, J.

AU - Poirier, Miriam C.

AU - Bowman, Elise D.

AU - Weston, Ainsley

AU - Doerge, Daniel R.

PY - 2005/8

Y1 - 2005/8

N2 - A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/ MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8, 9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-2H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 μg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10 8 nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3- 3H]BPDE, the method gave a value very similar to those obtained by radiolabeling, 32P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-3H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 ± 0.04 dG-BPDE adducts/108 nucleotides. This value is much lower than the 102 ± 14 total DNA adducts/ 108 nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/108 nucleotides, when using 100 μg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

AB - A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/ MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8, 9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-2H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 μg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10 8 nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3- 3H]BPDE, the method gave a value very similar to those obtained by radiolabeling, 32P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-3H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 ± 0.04 dG-BPDE adducts/108 nucleotides. This value is much lower than the 102 ± 14 total DNA adducts/ 108 nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/108 nucleotides, when using 100 μg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

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