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 highkcat 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 andTrichoplusia 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 Km andkcat values, along with the specificity constant, for naphthalene metabolism by recombinant CYP2F2 were 3 μM, 104 min−1, and 5.8 × 105M−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.
Footnotes
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Send reprint requests to: Dr. Michael Shultz, Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616. E-mail: mashultz{at}ucdavis.edu
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↵1 This work was supported by National Institute of Environmental Health Sciences Grants ES08408, ES04699, and ES04311. University of California, Davis is a Center for Environmental Health Sciences (Grant ES05711), and support for core facilities used in this work is gratefully acknowledged. M.A.S. is supported by NIEHS Predoctoral Fellowship ES05707.
- Abbreviations:
- Sf9
- Spodoptera frugiperda
- Tn5
- Trichoplusia ni
- pfu
- plaque-forming units
- GST
- glutathione-S-transferase
- AcNPV
- Autographa californica nuclear polyhedrosis virus
- PAGE
- polyacrylamide gel electrophoresis
- p.i.
- postinfection
- m.o.i.
- multiplicity of infection
- GSH
- glutathione
- ALA
- 5-aminolevulinic acid
- FC
- ferric citrate
- PCR
- polymerase chain reaction
- Received November 30, 1998.
- Accepted March 16, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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