17β-Estradiol metabolism by hamster hepatic microsomes: Comparison of catechol estrogen O-methylation with catechol estrogen oxidation and glutathione conjugation

Michael Butterworth, Serrine Lau, Terrence Monks

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49 Citations (Scopus)

Abstract

Catechol estrogens, the cytochromes P450 mediated metabolites of 17β- estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17β-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17β-estradiol, in hamster hepatic microsomes. 17β-Estradiol was readily converted to 2- and 4-hydroxy- 17β-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 μM-1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy-17μ-estradiol were identified: 2-hydroxy-1,4- bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1-glutathion-S-yl-17β- estradiol, and 2-hydroxy-4-glutathion-S-yl-17β-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17β-estradiol was identified: 4-hydroxy-1- glutathion-S-yl-17β-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation linked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17β-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17β-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by ~50% when compared to the value obtained when GSH is used to trap the o- quinones in the absence of ascorbate. A reassessment of 17β-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogen- induced carcinogenesis.

Original languageEnglish (US)
Pages (from-to)793-799
Number of pages7
JournalChemical Research in Toxicology
Volume9
Issue number4
DOIs
StatePublished - Jun 1996
Externally publishedYes

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Catechol Estrogens
Methylation
Microsomes
Metabolism
Cricetinae
Glutathione
Estradiol
Oxidation
Catechol O-Methyltransferase
Liver
Mixed Function Oxygenases
Metabolic Networks and Pathways
Metabolites
Quinones
Sulfides
Cytochrome P-450 Enzyme System
Ascorbic Acid
Enzyme activity
Estrogens
Carcinogenesis

ASJC Scopus subject areas

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

Cite this

@article{d5fb395a7088482cb70a67c76545d28f,
title = "17β-Estradiol metabolism by hamster hepatic microsomes: Comparison of catechol estrogen O-methylation with catechol estrogen oxidation and glutathione conjugation",
abstract = "Catechol estrogens, the cytochromes P450 mediated metabolites of 17β- estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17β-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17β-estradiol, in hamster hepatic microsomes. 17β-Estradiol was readily converted to 2- and 4-hydroxy- 17β-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 μM-1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy-17μ-estradiol were identified: 2-hydroxy-1,4- bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1-glutathion-S-yl-17β- estradiol, and 2-hydroxy-4-glutathion-S-yl-17β-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17β-estradiol was identified: 4-hydroxy-1- glutathion-S-yl-17β-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation linked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17β-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17β-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by ~50{\%} when compared to the value obtained when GSH is used to trap the o- quinones in the absence of ascorbate. A reassessment of 17β-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogen- induced carcinogenesis.",
author = "Michael Butterworth and Serrine Lau and Terrence Monks",
year = "1996",
month = "6",
doi = "10.1021/tx9501952",
language = "English (US)",
volume = "9",
pages = "793--799",
journal = "Chemical Research in Toxicology",
issn = "0893-228X",
publisher = "American Chemical Society",
number = "4",

}

TY - JOUR

T1 - 17β-Estradiol metabolism by hamster hepatic microsomes

T2 - Comparison of catechol estrogen O-methylation with catechol estrogen oxidation and glutathione conjugation

AU - Butterworth, Michael

AU - Lau, Serrine

AU - Monks, Terrence

PY - 1996/6

Y1 - 1996/6

N2 - Catechol estrogens, the cytochromes P450 mediated metabolites of 17β- estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17β-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17β-estradiol, in hamster hepatic microsomes. 17β-Estradiol was readily converted to 2- and 4-hydroxy- 17β-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 μM-1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy-17μ-estradiol were identified: 2-hydroxy-1,4- bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1-glutathion-S-yl-17β- estradiol, and 2-hydroxy-4-glutathion-S-yl-17β-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17β-estradiol was identified: 4-hydroxy-1- glutathion-S-yl-17β-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation linked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17β-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17β-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by ~50% when compared to the value obtained when GSH is used to trap the o- quinones in the absence of ascorbate. A reassessment of 17β-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogen- induced carcinogenesis.

AB - Catechol estrogens, the cytochromes P450 mediated metabolites of 17β- estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17β-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17β-estradiol, in hamster hepatic microsomes. 17β-Estradiol was readily converted to 2- and 4-hydroxy- 17β-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 μM-1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy-17μ-estradiol were identified: 2-hydroxy-1,4- bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1-glutathion-S-yl-17β- estradiol, and 2-hydroxy-4-glutathion-S-yl-17β-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17β-estradiol was identified: 4-hydroxy-1- glutathion-S-yl-17β-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation linked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17β-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17β-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by ~50% when compared to the value obtained when GSH is used to trap the o- quinones in the absence of ascorbate. A reassessment of 17β-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogen- induced carcinogenesis.

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