Identification and Quantitation of Benzo[a]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro

P. D. Devanesan, N. V S RamaKrishna, R. Todorovic, Eleanor G Rogan, Ercole Cavalieri, H. Jeong, R. Jankowiak, G. J. Small

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Abstract

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6- C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BPDNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained. Activation of BP by horseradish peroxidase afforded 32% of stable unidentified adducts and 68% of depurination adducts, with 48% of BP-6-N7Ade, 9% of BP-6-N7Gua, and 11% of BP-6-C8Gua. These results show that with activation by cytochrome P-450 the BP-DNA adducts are predominantly formed by one-electron oxidation and lost from DNA by depurination.

Original languageEnglish (US)
Pages (from-to)302-309
Number of pages8
JournalChemical Research in Toxicology
Volume5
Issue number2
DOIs
StatePublished - Mar 1 1992

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7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
Liver Microsomes
Liver
Rats
DNA Adducts
Chemical activation
DNA
Epoxy Compounds
Horseradish Peroxidase
benzo(a)pyrene-DNA adduct
In Vitro Techniques
High pressure liquid chromatography
Electrons
Oxidation
Methylcholanthrene
Microsomes
Cytochrome P-450 Enzyme System
Cations

ASJC Scopus subject areas

  • Toxicology

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Identification and Quantitation of Benzo[a]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro. / Devanesan, P. D.; RamaKrishna, N. V S; Todorovic, R.; Rogan, Eleanor G; Cavalieri, Ercole; Jeong, H.; Jankowiak, R.; Small, G. J.

In: Chemical Research in Toxicology, Vol. 5, No. 2, 01.03.1992, p. 302-309.

Research output: Contribution to journalArticle

Devanesan, P. D. ; RamaKrishna, N. V S ; Todorovic, R. ; Rogan, Eleanor G ; Cavalieri, Ercole ; Jeong, H. ; Jankowiak, R. ; Small, G. J. / Identification and Quantitation of Benzo[a]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro. In: Chemical Research in Toxicology. 1992 ; Vol. 5, No. 2. pp. 302-309.
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abstract = "The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6- C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BPDNA adducts formed by rat liver microsomes showed 81{\%} as depurination adducts: BP-6-N7Ade (58{\%}), BP-6-N7Gua (10{\%}), BP-6-C8Gua (12{\%}), and BPDE-10-N7Ade (0.5{\%}). Stable adducts (19{\%} of total) included BPDE-10-N2dG (15{\%}) and unidentified adducts (4{\%}). Microsomal activation of BP 7,8-dihydrodiol yielded 80{\%} stable adducts, with 77{\%} as BPDE-10-N2dG and 20{\%} of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94{\%}) was higher when BPDE was reacted with DNA, and only 1.8{\%} of BPDE-10-N7Ade was obtained. Activation of BP by horseradish peroxidase afforded 32{\%} of stable unidentified adducts and 68{\%} of depurination adducts, with 48{\%} of BP-6-N7Ade, 9{\%} of BP-6-N7Gua, and 11{\%} of BP-6-C8Gua. These results show that with activation by cytochrome P-450 the BP-DNA adducts are predominantly formed by one-electron oxidation and lost from DNA by depurination.",
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T1 - Identification and Quantitation of Benzo[a]pyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro

AU - Devanesan, P. D.

AU - RamaKrishna, N. V S

AU - Todorovic, R.

AU - Rogan, Eleanor G

AU - Cavalieri, Ercole

AU - Jeong, H.

AU - Jankowiak, R.

AU - Small, G. J.

PY - 1992/3/1

Y1 - 1992/3/1

N2 - The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6- C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BPDNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained. Activation of BP by horseradish peroxidase afforded 32% of stable unidentified adducts and 68% of depurination adducts, with 48% of BP-6-N7Ade, 9% of BP-6-N7Gua, and 11% of BP-6-C8Gua. These results show that with activation by cytochrome P-450 the BP-DNA adducts are predominantly formed by one-electron oxidation and lost from DNA by depurination.

AB - The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6- C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BPDNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained. Activation of BP by horseradish peroxidase afforded 32% of stable unidentified adducts and 68% of depurination adducts, with 48% of BP-6-N7Ade, 9% of BP-6-N7Gua, and 11% of BP-6-C8Gua. These results show that with activation by cytochrome P-450 the BP-DNA adducts are predominantly formed by one-electron oxidation and lost from DNA by depurination.

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