Identification and Quantitation of Dibenzo[a,l]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro: Preponderance of Depurinating Adducts

K. M. Li, R. Todorovic, Eleanor G Rogan, Ercole Cavalieri, F. Ariese, M. Suh, R. Jankowiak, G. J. Small

Research output: Contribution to journalArticle

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Abstract

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen known among aromatic hydrocarbons. DB [a,l]P-11,12-dihydrodiol, precursor to the bay-region diol epoxide, is slightly less carcinogenic than the parent compound. DB[a,l]P and its 11,12-dihydrodiol were covalently bound to DNA by cytochrome P-450 in 3-methylcholanthrene-induced rat liver microsomes, and DB[a,l]P was also bound to DNA by horseradish peroxidase. The “stable” (remaining intact in DNA under normal conditions of purification) and “depurinating” (released from DNA by cleavage of the glycosidic link between the purine base and deoxyribose) adducts were identified and quantified. Stable adducts were analyzed by the 32P-postlabeling technique. Depurinating adducts were identified by comparison of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by means of fluorescence line-narrowing spectroscopy. When DB[a,l]P was activated by horseradish peroxidase, the depurinating adducts 3-(DB[a,l]P-10-yl)adenine (DB[a, l]P-10-N3Ade, 33%), 7-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N7Ade, 27%), and 7-DB[a,l]P-10-yl)guanine (DB[a,l]P-10-N7Gua, 5%) were formed. Unidentified stable adducts comprised the remaining 35% of the detected adducts. When DB[a,l]P was activated by microsomes, the one-electron oxidation depurinating adducts DB[a,l]P- 10-N3Ade (28%), DB[a,l]P-10-N7Ade (14%), DB[a,l]P-10-N7Gua (2%), and DB[a,l]P-10-C8Gua (6%), as well as the diol epoxide depurinating adducts (±)-anti-DB[a,l]P-diol epoxide (DE)-14-N7Ade (31%) and (±)-an-DB[a,l]PDE-14-N7Gua (3%), were formed. Stable adducts predominantly formed via the DB[a,l]PDE pathway represented 16% of the adducts detected. When DB fa,l]P-11,12-dihydrodiol was activated by microsomes, the same two depurinating adducts arising from DB[a,l]PDE were found, but they constituted only 19% of the adducts because the amount of stable adducts was much higher than with DB[a,l]P. Analysis of stable DNA adducts by the 32P-postlabeling method indicates that the profiles formed from DB[a,l]P and its 11,12-dihydrodiol were qualitatively similar. These results demonstrate that the major depurinating adducts formed by both DB[n,l]P and its 11,12-dihydrodiol are at the N-3 and N-7 of adenine, resulting in apurinic sites in the DNA.

Original languageEnglish (US)
Pages (from-to)8043-8049
Number of pages7
JournalBiochemistry
Volume34
Issue number25
DOIs
StatePublished - Jun 1995

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DNA Adducts
Liver Microsomes
Liver
Rats
Epoxy Compounds
Adenine
DNA
Horseradish Peroxidase
Microsomes
Deoxyribose
Aromatic Hydrocarbons
DNA Cleavage
Methylcholanthrene
Guanine
Carcinogens
Cytochrome P-450 Enzyme System
Spectrum Analysis
Fluorescence
High Pressure Liquid Chromatography
Purification

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification and Quantitation of Dibenzo[a,l]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro : Preponderance of Depurinating Adducts. / Li, K. M.; Todorovic, R.; Rogan, Eleanor G; Cavalieri, Ercole; Ariese, F.; Suh, M.; Jankowiak, R.; Small, G. J.

In: Biochemistry, Vol. 34, No. 25, 06.1995, p. 8043-8049.

Research output: Contribution to journalArticle

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title = "Identification and Quantitation of Dibenzo[a,l]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro: Preponderance of Depurinating Adducts",
abstract = "Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen known among aromatic hydrocarbons. DB [a,l]P-11,12-dihydrodiol, precursor to the bay-region diol epoxide, is slightly less carcinogenic than the parent compound. DB[a,l]P and its 11,12-dihydrodiol were covalently bound to DNA by cytochrome P-450 in 3-methylcholanthrene-induced rat liver microsomes, and DB[a,l]P was also bound to DNA by horseradish peroxidase. The “stable” (remaining intact in DNA under normal conditions of purification) and “depurinating” (released from DNA by cleavage of the glycosidic link between the purine base and deoxyribose) adducts were identified and quantified. Stable adducts were analyzed by the 32P-postlabeling technique. Depurinating adducts were identified by comparison of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by means of fluorescence line-narrowing spectroscopy. When DB[a,l]P was activated by horseradish peroxidase, the depurinating adducts 3-(DB[a,l]P-10-yl)adenine (DB[a, l]P-10-N3Ade, 33{\%}), 7-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N7Ade, 27{\%}), and 7-DB[a,l]P-10-yl)guanine (DB[a,l]P-10-N7Gua, 5{\%}) were formed. Unidentified stable adducts comprised the remaining 35{\%} of the detected adducts. When DB[a,l]P was activated by microsomes, the one-electron oxidation depurinating adducts DB[a,l]P- 10-N3Ade (28{\%}), DB[a,l]P-10-N7Ade (14{\%}), DB[a,l]P-10-N7Gua (2{\%}), and DB[a,l]P-10-C8Gua (6{\%}), as well as the diol epoxide depurinating adducts (±)-anti-DB[a,l]P-diol epoxide (DE)-14-N7Ade (31{\%}) and (±)-an-DB[a,l]PDE-14-N7Gua (3{\%}), were formed. Stable adducts predominantly formed via the DB[a,l]PDE pathway represented 16{\%} of the adducts detected. When DB fa,l]P-11,12-dihydrodiol was activated by microsomes, the same two depurinating adducts arising from DB[a,l]PDE were found, but they constituted only 19{\%} of the adducts because the amount of stable adducts was much higher than with DB[a,l]P. Analysis of stable DNA adducts by the 32P-postlabeling method indicates that the profiles formed from DB[a,l]P and its 11,12-dihydrodiol were qualitatively similar. These results demonstrate that the major depurinating adducts formed by both DB[n,l]P and its 11,12-dihydrodiol are at the N-3 and N-7 of adenine, resulting in apurinic sites in the DNA.",
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TY - JOUR

T1 - Identification and Quantitation of Dibenzo[a,l]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro

T2 - Preponderance of Depurinating Adducts

AU - Li, K. M.

AU - Todorovic, R.

AU - Rogan, Eleanor G

AU - Cavalieri, Ercole

AU - Ariese, F.

AU - Suh, M.

AU - Jankowiak, R.

AU - Small, G. J.

PY - 1995/6

Y1 - 1995/6

N2 - Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen known among aromatic hydrocarbons. DB [a,l]P-11,12-dihydrodiol, precursor to the bay-region diol epoxide, is slightly less carcinogenic than the parent compound. DB[a,l]P and its 11,12-dihydrodiol were covalently bound to DNA by cytochrome P-450 in 3-methylcholanthrene-induced rat liver microsomes, and DB[a,l]P was also bound to DNA by horseradish peroxidase. The “stable” (remaining intact in DNA under normal conditions of purification) and “depurinating” (released from DNA by cleavage of the glycosidic link between the purine base and deoxyribose) adducts were identified and quantified. Stable adducts were analyzed by the 32P-postlabeling technique. Depurinating adducts were identified by comparison of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by means of fluorescence line-narrowing spectroscopy. When DB[a,l]P was activated by horseradish peroxidase, the depurinating adducts 3-(DB[a,l]P-10-yl)adenine (DB[a, l]P-10-N3Ade, 33%), 7-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N7Ade, 27%), and 7-DB[a,l]P-10-yl)guanine (DB[a,l]P-10-N7Gua, 5%) were formed. Unidentified stable adducts comprised the remaining 35% of the detected adducts. When DB[a,l]P was activated by microsomes, the one-electron oxidation depurinating adducts DB[a,l]P- 10-N3Ade (28%), DB[a,l]P-10-N7Ade (14%), DB[a,l]P-10-N7Gua (2%), and DB[a,l]P-10-C8Gua (6%), as well as the diol epoxide depurinating adducts (±)-anti-DB[a,l]P-diol epoxide (DE)-14-N7Ade (31%) and (±)-an-DB[a,l]PDE-14-N7Gua (3%), were formed. Stable adducts predominantly formed via the DB[a,l]PDE pathway represented 16% of the adducts detected. When DB fa,l]P-11,12-dihydrodiol was activated by microsomes, the same two depurinating adducts arising from DB[a,l]PDE were found, but they constituted only 19% of the adducts because the amount of stable adducts was much higher than with DB[a,l]P. Analysis of stable DNA adducts by the 32P-postlabeling method indicates that the profiles formed from DB[a,l]P and its 11,12-dihydrodiol were qualitatively similar. These results demonstrate that the major depurinating adducts formed by both DB[n,l]P and its 11,12-dihydrodiol are at the N-3 and N-7 of adenine, resulting in apurinic sites in the DNA.

AB - Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen known among aromatic hydrocarbons. DB [a,l]P-11,12-dihydrodiol, precursor to the bay-region diol epoxide, is slightly less carcinogenic than the parent compound. DB[a,l]P and its 11,12-dihydrodiol were covalently bound to DNA by cytochrome P-450 in 3-methylcholanthrene-induced rat liver microsomes, and DB[a,l]P was also bound to DNA by horseradish peroxidase. The “stable” (remaining intact in DNA under normal conditions of purification) and “depurinating” (released from DNA by cleavage of the glycosidic link between the purine base and deoxyribose) adducts were identified and quantified. Stable adducts were analyzed by the 32P-postlabeling technique. Depurinating adducts were identified by comparison of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by means of fluorescence line-narrowing spectroscopy. When DB[a,l]P was activated by horseradish peroxidase, the depurinating adducts 3-(DB[a,l]P-10-yl)adenine (DB[a, l]P-10-N3Ade, 33%), 7-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N7Ade, 27%), and 7-DB[a,l]P-10-yl)guanine (DB[a,l]P-10-N7Gua, 5%) were formed. Unidentified stable adducts comprised the remaining 35% of the detected adducts. When DB[a,l]P was activated by microsomes, the one-electron oxidation depurinating adducts DB[a,l]P- 10-N3Ade (28%), DB[a,l]P-10-N7Ade (14%), DB[a,l]P-10-N7Gua (2%), and DB[a,l]P-10-C8Gua (6%), as well as the diol epoxide depurinating adducts (±)-anti-DB[a,l]P-diol epoxide (DE)-14-N7Ade (31%) and (±)-an-DB[a,l]PDE-14-N7Gua (3%), were formed. Stable adducts predominantly formed via the DB[a,l]PDE pathway represented 16% of the adducts detected. When DB fa,l]P-11,12-dihydrodiol was activated by microsomes, the same two depurinating adducts arising from DB[a,l]PDE were found, but they constituted only 19% of the adducts because the amount of stable adducts was much higher than with DB[a,l]P. Analysis of stable DNA adducts by the 32P-postlabeling method indicates that the profiles formed from DB[a,l]P and its 11,12-dihydrodiol were qualitatively similar. These results demonstrate that the major depurinating adducts formed by both DB[n,l]P and its 11,12-dihydrodiol are at the N-3 and N-7 of adenine, resulting in apurinic sites in the DNA.

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