Molecular Pharmacology, Vol 12, 476-482, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Electron Spin Resonance Study of ¹⁷O-Enriched Oxybenzo[a]pyrene Radical

¹ Laboratory of Chemical Physics, National Institute of Arthritis, Metabolism, and Digestive Diseases
² Developmental Pharmacology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014

Benzo[a]pyrene was incubated in vitro with cofactors and liver microsomes from rats previously treated with -naphthoflavone, and the polycyclic hydrocarbon and its metabolites were extracted with a benzene-acetone mixture. Following evaporation of the solvent, the residue was dissolved in toluene or in ethanol-potassium phosphate buffer and examined by room-temperature electron spin resonance spectroscopy. Contrary to reports from other laboratories, the ESR signal respresenting an oxybenzo[a]pyrene free radical is initially very small or undetectable. Under various experimental conditions, we could not measure directly the free radical signal in aliquots from the microsomal incubation mixture. The signal rapidly increases in intensity during examination by ESR, and reaches a maximum in 2-3 hr, and lasts for at least 12 hr. The appearance and growth of the signal do not occur when the solvent used for ESR analysis is deoxygenated. Incubations with benzo[a]pyrene, cofactors, and microsomes under an atmosphere of ¹⁷O₂ demonstrate that the source of oxygen in the radical is atmospheric and the reaction is mediated by cytochrome P-448 or P-450, but that the free radical we observed occurs nonenzymatically, presumably via abstraction of a hydrogen atom (1-electron air oxidation) by molecular oxygen in the solvent used for ESR analysis. Whether this reaction is important in the binding of benzo[a]pyrene metabolites to DNA and in the etiology of chemical carcinogenesis remains to be determined.

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ACKNOWLEDGMENTS We are indebted to Dr. Herman Ziffer for his useful discussions and help in the experimental work. We are also grateful to Mr. William H. Jennings for his skillful spectral simulation and Ms. Nancy M. Jensen for technical assistance.