Identification and Quantitation of 7,12-Dimethylbenz[a]anthracene-DNA Adducts Formed in Mouse Skin

P. D. Devanesan, N. V S RamaKrishna, N. S. Padmavathi, S. Higginbotham, Eleanor G Rogan, Ercole Cavalieri, G. A. Marsch, R. Jankowiak, G. J. Small

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Abstract

Identification and quantitation of the depurination and stable DNA adducts of 7,12-dimethylbenz [a] anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99% of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CH2-N7Ade (79%) was formed compared to 7-MBA-12-CH2-N7Gua (20%). The stable adducts accounted for only 1% of all the adducts detected and 80% of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3 group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70% lower in the HRP system and 30% lower in the microsomal system compared to native DNA. In the microsomal system, just the two major adduct spots decreased, whereas the amounts of all of the adducts decreased with HRP. These results demonstrate that in mouse skin, DMBA-DNA adducts are predominantly formed via one-electron oxidation and that the adducts formed by the diolepoxide pathway are minor. The double helical structure of DNA is necessary for formation of adducts by one-electron oxidation but is not a critical factor in the diolepoxide pathway.

Original languageEnglish (US)
Pages (from-to)364-371
Number of pages8
JournalChemical Research in Toxicology
Volume6
Issue number3
DOIs
StatePublished - Jan 1 1993

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9,10-Dimethyl-1,2-benzanthracene
Skin
DNA
Horseradish Peroxidase
DNA Adducts
Electrons
Oxidation
Guanine
Liver Microsomes
Adenine
Microsomes
Liver
Cytochrome P-450 Enzyme System
7,12-dimethylbenz(a)anthracene-DNA adduct
Rats
Spectrum Analysis
Fluorescence
Chemical activation
High Pressure Liquid Chromatography
Spectroscopy

ASJC Scopus subject areas

  • Toxicology

Cite this

Identification and Quantitation of 7,12-Dimethylbenz[a]anthracene-DNA Adducts Formed in Mouse Skin. / Devanesan, P. D.; RamaKrishna, N. V S; Padmavathi, N. S.; Higginbotham, S.; Rogan, Eleanor G; Cavalieri, Ercole; Marsch, G. A.; Jankowiak, R.; Small, G. J.

In: Chemical Research in Toxicology, Vol. 6, No. 3, 01.01.1993, p. 364-371.

Research output: Contribution to journalArticle

Devanesan, PD, RamaKrishna, NVS, Padmavathi, NS, Higginbotham, S, Rogan, EG, Cavalieri, E, Marsch, GA, Jankowiak, R & Small, GJ 1993, 'Identification and Quantitation of 7,12-Dimethylbenz[a]anthracene-DNA Adducts Formed in Mouse Skin', Chemical Research in Toxicology, vol. 6, no. 3, pp. 364-371. https://doi.org/10.1021/tx00033a018
Devanesan, P. D. ; RamaKrishna, N. V S ; Padmavathi, N. S. ; Higginbotham, S. ; Rogan, Eleanor G ; Cavalieri, Ercole ; Marsch, G. A. ; Jankowiak, R. ; Small, G. J. / Identification and Quantitation of 7,12-Dimethylbenz[a]anthracene-DNA Adducts Formed in Mouse Skin. In: Chemical Research in Toxicology. 1993 ; Vol. 6, No. 3. pp. 364-371.
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abstract = "Identification and quantitation of the depurination and stable DNA adducts of 7,12-dimethylbenz [a] anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99{\%} of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CH2-N7Ade (79{\%}) was formed compared to 7-MBA-12-CH2-N7Gua (20{\%}). The stable adducts accounted for only 1{\%} of all the adducts detected and 80{\%} of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3 group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70{\%} lower in the HRP system and 30{\%} lower in the microsomal system compared to native DNA. In the microsomal system, just the two major adduct spots decreased, whereas the amounts of all of the adducts decreased with HRP. These results demonstrate that in mouse skin, DMBA-DNA adducts are predominantly formed via one-electron oxidation and that the adducts formed by the diolepoxide pathway are minor. The double helical structure of DNA is necessary for formation of adducts by one-electron oxidation but is not a critical factor in the diolepoxide pathway.",
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AU - Padmavathi, N. S.

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AU - Rogan, Eleanor G

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N2 - Identification and quantitation of the depurination and stable DNA adducts of 7,12-dimethylbenz [a] anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99% of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CH2-N7Ade (79%) was formed compared to 7-MBA-12-CH2-N7Gua (20%). The stable adducts accounted for only 1% of all the adducts detected and 80% of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3 group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70% lower in the HRP system and 30% lower in the microsomal system compared to native DNA. In the microsomal system, just the two major adduct spots decreased, whereas the amounts of all of the adducts decreased with HRP. These results demonstrate that in mouse skin, DMBA-DNA adducts are predominantly formed via one-electron oxidation and that the adducts formed by the diolepoxide pathway are minor. The double helical structure of DNA is necessary for formation of adducts by one-electron oxidation but is not a critical factor in the diolepoxide pathway.

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