Metabolism of 6-Substituted Benzo[a]Pyrene Derivatives: O-Dealkylation and Regiospecificity in Aromatic Hydroxylation

Andrew J. Alpert, Ercole Cavalieri

Research output: Contribution to journalArticle

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Abstract

The carcinogen benzo[a]pyrene (BP) is metabolized by monooxygenase enzymes to phenols, dihydrodiols, and diones. The major product, 3-hydroxy-BP, is measured in the fluorescence assay for aryl hydrocarbon hydroxylase (AHH). In this assay, the BP phenols, which are readily oxidized to nonfluorescent diones, are not detected. Blocking the 6 position with various substituents prevents dione formation and stabilizes the phenolic metabolites. These substituted BP derivatives were used as possible alternative substrates to BP in the AHH assay and to investigate positional specificities of monooxygenase enzymes in aromatic hydroxylation, as well as O-dealkylation when the substrates were BP alkoxy derivatives. Phenolate anions at positions 1 and 12 could be distinguished from 3-phenolate anions of BP derivatives on the basis of the fluorescence excitation spectra. This permitted identification of phenolic metabolites of BP derivatives obtained after incubation with rat liver and lung microsomes. In some cases other metabolites were identified by high-pressure liquid chromatography. No worthy substitute for BP in the AHH assay was found, since the derivatives were hydroxylated at a lesser rate, hydroxylated at positions other than 3, metabolized by O-dealkylation, or yielded products less fluorescent than the 3-hydroxy-BP. AHH was found to have positional specificity for 3-hydroxylation of BP and some of its derivatives. The minor role of 1-hydroxylation is presumably due to a steric constraint of the enzyme. Three different enzyme activities were observed: (1) an uninduced 3-hydroxylase with some 1-hydroxylase activity; (2) a 3-methylcholanthrene-induced 3-hydroxylase, which has more 1-hydroxylase activity and is more sensitive to the structure of the substrate; (3) an uninducible O-dealkylase. Hydroxylase and O-dealkylase have the same regiospecificity: the hydroxylase can attack positions 1, 3, and 6, but not 12, while the O-dealkylase can dealkylate groups at positions 1, 3, and 6, but not 12. 6-Methoxy-BP was metabolized both by hydroxylation and O-dealkylation. Compounds metabolized mainly by hydroxylation were spectral type I. These included BP, 6-methoxy-BP, and 6-(methoxymethyl)-BP. Compounds metabolized by O-dealkylation, the 1, 6- and 3, 6-dialkoxy derivatives of BP, were spectral type II compounds, the first reported which do not contain a basic nitrogen atom.

Original languageEnglish (US)
Pages (from-to)919-927
Number of pages9
JournalJournal of Medicinal Chemistry
Volume23
Issue number8
DOIs
StatePublished - Jan 1 1980

Fingerprint

Dealkylation
Benzo(a)pyrene
Hydroxylation
Mixed Function Oxygenases
Aryl Hydrocarbon Hydroxylases
Phenols
Enzymes
Anions
Fluorescence
Methylcholanthrene
Liver Microsomes
Carcinogens
Nitrogen
High Pressure Liquid Chromatography
Lung

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

Cite this

Metabolism of 6-Substituted Benzo[a]Pyrene Derivatives : O-Dealkylation and Regiospecificity in Aromatic Hydroxylation. / Alpert, Andrew J.; Cavalieri, Ercole.

In: Journal of Medicinal Chemistry, Vol. 23, No. 8, 01.01.1980, p. 919-927.

Research output: Contribution to journalArticle

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title = "Metabolism of 6-Substituted Benzo[a]Pyrene Derivatives: O-Dealkylation and Regiospecificity in Aromatic Hydroxylation",
abstract = "The carcinogen benzo[a]pyrene (BP) is metabolized by monooxygenase enzymes to phenols, dihydrodiols, and diones. The major product, 3-hydroxy-BP, is measured in the fluorescence assay for aryl hydrocarbon hydroxylase (AHH). In this assay, the BP phenols, which are readily oxidized to nonfluorescent diones, are not detected. Blocking the 6 position with various substituents prevents dione formation and stabilizes the phenolic metabolites. These substituted BP derivatives were used as possible alternative substrates to BP in the AHH assay and to investigate positional specificities of monooxygenase enzymes in aromatic hydroxylation, as well as O-dealkylation when the substrates were BP alkoxy derivatives. Phenolate anions at positions 1 and 12 could be distinguished from 3-phenolate anions of BP derivatives on the basis of the fluorescence excitation spectra. This permitted identification of phenolic metabolites of BP derivatives obtained after incubation with rat liver and lung microsomes. In some cases other metabolites were identified by high-pressure liquid chromatography. No worthy substitute for BP in the AHH assay was found, since the derivatives were hydroxylated at a lesser rate, hydroxylated at positions other than 3, metabolized by O-dealkylation, or yielded products less fluorescent than the 3-hydroxy-BP. AHH was found to have positional specificity for 3-hydroxylation of BP and some of its derivatives. The minor role of 1-hydroxylation is presumably due to a steric constraint of the enzyme. Three different enzyme activities were observed: (1) an uninduced 3-hydroxylase with some 1-hydroxylase activity; (2) a 3-methylcholanthrene-induced 3-hydroxylase, which has more 1-hydroxylase activity and is more sensitive to the structure of the substrate; (3) an uninducible O-dealkylase. Hydroxylase and O-dealkylase have the same regiospecificity: the hydroxylase can attack positions 1, 3, and 6, but not 12, while the O-dealkylase can dealkylate groups at positions 1, 3, and 6, but not 12. 6-Methoxy-BP was metabolized both by hydroxylation and O-dealkylation. Compounds metabolized mainly by hydroxylation were spectral type I. These included BP, 6-methoxy-BP, and 6-(methoxymethyl)-BP. Compounds metabolized by O-dealkylation, the 1, 6- and 3, 6-dialkoxy derivatives of BP, were spectral type II compounds, the first reported which do not contain a basic nitrogen atom.",
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N2 - The carcinogen benzo[a]pyrene (BP) is metabolized by monooxygenase enzymes to phenols, dihydrodiols, and diones. The major product, 3-hydroxy-BP, is measured in the fluorescence assay for aryl hydrocarbon hydroxylase (AHH). In this assay, the BP phenols, which are readily oxidized to nonfluorescent diones, are not detected. Blocking the 6 position with various substituents prevents dione formation and stabilizes the phenolic metabolites. These substituted BP derivatives were used as possible alternative substrates to BP in the AHH assay and to investigate positional specificities of monooxygenase enzymes in aromatic hydroxylation, as well as O-dealkylation when the substrates were BP alkoxy derivatives. Phenolate anions at positions 1 and 12 could be distinguished from 3-phenolate anions of BP derivatives on the basis of the fluorescence excitation spectra. This permitted identification of phenolic metabolites of BP derivatives obtained after incubation with rat liver and lung microsomes. In some cases other metabolites were identified by high-pressure liquid chromatography. No worthy substitute for BP in the AHH assay was found, since the derivatives were hydroxylated at a lesser rate, hydroxylated at positions other than 3, metabolized by O-dealkylation, or yielded products less fluorescent than the 3-hydroxy-BP. AHH was found to have positional specificity for 3-hydroxylation of BP and some of its derivatives. The minor role of 1-hydroxylation is presumably due to a steric constraint of the enzyme. Three different enzyme activities were observed: (1) an uninduced 3-hydroxylase with some 1-hydroxylase activity; (2) a 3-methylcholanthrene-induced 3-hydroxylase, which has more 1-hydroxylase activity and is more sensitive to the structure of the substrate; (3) an uninducible O-dealkylase. Hydroxylase and O-dealkylase have the same regiospecificity: the hydroxylase can attack positions 1, 3, and 6, but not 12, while the O-dealkylase can dealkylate groups at positions 1, 3, and 6, but not 12. 6-Methoxy-BP was metabolized both by hydroxylation and O-dealkylation. Compounds metabolized mainly by hydroxylation were spectral type I. These included BP, 6-methoxy-BP, and 6-(methoxymethyl)-BP. Compounds metabolized by O-dealkylation, the 1, 6- and 3, 6-dialkoxy derivatives of BP, were spectral type II compounds, the first reported which do not contain a basic nitrogen atom.

AB - The carcinogen benzo[a]pyrene (BP) is metabolized by monooxygenase enzymes to phenols, dihydrodiols, and diones. The major product, 3-hydroxy-BP, is measured in the fluorescence assay for aryl hydrocarbon hydroxylase (AHH). In this assay, the BP phenols, which are readily oxidized to nonfluorescent diones, are not detected. Blocking the 6 position with various substituents prevents dione formation and stabilizes the phenolic metabolites. These substituted BP derivatives were used as possible alternative substrates to BP in the AHH assay and to investigate positional specificities of monooxygenase enzymes in aromatic hydroxylation, as well as O-dealkylation when the substrates were BP alkoxy derivatives. Phenolate anions at positions 1 and 12 could be distinguished from 3-phenolate anions of BP derivatives on the basis of the fluorescence excitation spectra. This permitted identification of phenolic metabolites of BP derivatives obtained after incubation with rat liver and lung microsomes. In some cases other metabolites were identified by high-pressure liquid chromatography. No worthy substitute for BP in the AHH assay was found, since the derivatives were hydroxylated at a lesser rate, hydroxylated at positions other than 3, metabolized by O-dealkylation, or yielded products less fluorescent than the 3-hydroxy-BP. AHH was found to have positional specificity for 3-hydroxylation of BP and some of its derivatives. The minor role of 1-hydroxylation is presumably due to a steric constraint of the enzyme. Three different enzyme activities were observed: (1) an uninduced 3-hydroxylase with some 1-hydroxylase activity; (2) a 3-methylcholanthrene-induced 3-hydroxylase, which has more 1-hydroxylase activity and is more sensitive to the structure of the substrate; (3) an uninducible O-dealkylase. Hydroxylase and O-dealkylase have the same regiospecificity: the hydroxylase can attack positions 1, 3, and 6, but not 12, while the O-dealkylase can dealkylate groups at positions 1, 3, and 6, but not 12. 6-Methoxy-BP was metabolized both by hydroxylation and O-dealkylation. Compounds metabolized mainly by hydroxylation were spectral type I. These included BP, 6-methoxy-BP, and 6-(methoxymethyl)-BP. Compounds metabolized by O-dealkylation, the 1, 6- and 3, 6-dialkoxy derivatives of BP, were spectral type II compounds, the first reported which do not contain a basic nitrogen atom.

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