Role of murine cytochrome P-450 2F2 in metabolic activation of naphthalene and metabolism of other xenobiotics

Michael A. Shultz, Prabhakara V Choudary, Alan R Buckpitt

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

Despite their substantially lower levels relative to hepatic tissue, pulmonary cytochrome P-450 (CYP) monooxygenases play an important role in the metabolic activation of substrates that cause lung injury. The target- and species-selective toxicity of a number of pulmonary toxicants has been attributed to the presence and distribution of activating enzymes with high k(cat) in target airways of susceptible species. However, experimental demonstration of these concepts and quantitative assessment of the contribution of individual CYP isoforms is lacking. This study was undertaken to characterize the catalytic activities of CYP2F2 with naphthalene, a murine Clara cell toxicant, as well as with other xenobiotics that either undergo metabolic activation to cytotoxic intermediates or that function as 'isoform- selective' substrates. Recombinant CYP2F2 was produced using the baculovirus expression vector system in Spodoptera frugiperda and Trichoplusia ni cells, accounting up to ~20% of the total cellular protein. Incubations containing naphthalene, recombinant CYP2F2, NADPH-cytochrome P-450 oxidoreductase, and NADPH-regenerating system metabolized naphthalene with a high degree of stereoselectivity to 1R,2S-naphthalene oxide (66:1 enantiomeric ratio). The K(m) and k(cat) values, along with the specificity constant, for naphthalene metabolism by recombinant CYP2F2 were 3 μM, 104 min-1, and 5.8 x 105 M- 1 s-1, respectively. Recombinant CYP2F2 also metabolized ethoxyresorufin, pentoxyresorufin, p-nitrophenol, and 1-nitronaphthalene at easily detectable levels. The results from this work suggest that CYP2F2 1) plays a key role in the species- and cell-selective toxicity of naphthalene and 2) efficiently metabolizes a number of other substrates, including the lung toxicant 1- nitronaphthalene.

Original languageEnglish (US)
Pages (from-to)281-288
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume290
Issue number1
StatePublished - Jul 1999

Fingerprint

Xenobiotics
Cytochrome P-450 Enzyme System
Lung
Protein Isoforms
Spodoptera
NADPH-Ferrihemoprotein Reductase
Baculoviridae
Lung Injury
NADP
Metabolic Activation
naphthalene
Liver
Enzymes
Proteins
1-nitronaphthalene

ASJC Scopus subject areas

  • Pharmacology

Cite this

Role of murine cytochrome P-450 2F2 in metabolic activation of naphthalene and metabolism of other xenobiotics. / Shultz, Michael A.; Choudary, Prabhakara V; Buckpitt, Alan R.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 290, No. 1, 07.1999, p. 281-288.

Research output: Contribution to journalArticle

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abstract = "Despite their substantially lower levels relative to hepatic tissue, pulmonary cytochrome P-450 (CYP) monooxygenases play an important role in the metabolic activation of substrates that cause lung injury. The target- and species-selective toxicity of a number of pulmonary toxicants has been attributed to the presence and distribution of activating enzymes with high k(cat) in target airways of susceptible species. However, experimental demonstration of these concepts and quantitative assessment of the contribution of individual CYP isoforms is lacking. This study was undertaken to characterize the catalytic activities of CYP2F2 with naphthalene, a murine Clara cell toxicant, as well as with other xenobiotics that either undergo metabolic activation to cytotoxic intermediates or that function as 'isoform- selective' substrates. Recombinant CYP2F2 was produced using the baculovirus expression vector system in Spodoptera frugiperda and Trichoplusia ni cells, accounting up to ~20{\%} of the total cellular protein. Incubations containing naphthalene, recombinant CYP2F2, NADPH-cytochrome P-450 oxidoreductase, and NADPH-regenerating system metabolized naphthalene with a high degree of stereoselectivity to 1R,2S-naphthalene oxide (66:1 enantiomeric ratio). The K(m) and k(cat) values, along with the specificity constant, for naphthalene metabolism by recombinant CYP2F2 were 3 μM, 104 min-1, and 5.8 x 105 M- 1 s-1, respectively. Recombinant CYP2F2 also metabolized ethoxyresorufin, pentoxyresorufin, p-nitrophenol, and 1-nitronaphthalene at easily detectable levels. The results from this work suggest that CYP2F2 1) plays a key role in the species- and cell-selective toxicity of naphthalene and 2) efficiently metabolizes a number of other substrates, including the lung toxicant 1- nitronaphthalene.",
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