ENZYMATIC OXIDATION OF ETHYL CARBAMATE TO VINYL CARBAMATE AND ITS ROLE AS AN INTERMEDIATE IN THE FORMATION OF 1, N-6-ETHENOADENOSINE

The carcinogen ethyl carbamate has been postulated to be activated by oxidation to vinyl carbamate and then to an epoxide which can react with nucleic acids [Dahl, G. A., Miller, J. A., and Miller, E. C. (1978) Cancer Res. 38, 3793-3804]. To date, the enzymatic conversion of ethyl carbamate to vinyl carbamate had not been demonstrated. Recently, we obtained evidence that the same cytochrome P-450 enzyme (P-450 2E1) is involved in the oxidation of both ethyl carbamate and vinyl carbamate [Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991) Chem. Res. Toxicol. 4, 168-1791. When human liver microsomes were incubated with NADPH and ethyl carbamate, the products vinyl carbamate, 2-hydroxyethyl carbamate, and ethyl N-hydroxycarbamate were detected by use of (a) combined capillary gas chromatography/chemical ionization mass spectrometry or (b) high-performance liquid chromatography of radioactive materials. A K(m) of approximately 54-mu-M was estimated for the conversion of vinyl carbamate to 1,N6-ethenoadenosine (in the presence of adenosine), but when the reaction was done with ethyl carbamate as the substrate, the rate of product formation was nearly first order in ethyl carbamate concentration (K(m) > 2 mM) and the rate was considerably slower than in the case of vinyl carbamate. The model derived with these parameters predicts a steady-state level of 0.22-mu-M vinyl carbamate, consonant with the value of approximately 0.2-mu-M estimated experimentally. A large kinetic deuterium isotope effect (> 7) was observed for the formation of 1,N6-ethenoadenosine from ethyl carbamate, and high isotope effects (6-8) were also noted for the formation of vinyl carbamate and 2-hydroxyethyl carbamate. These results are consistent with the view that the enzyme cytochrome P-450 2E1 abstracts a hydrogen atom from the terminal carbon of ethyl carbamate: subsequent oxygen rebound yields 2-hydroxyethyl carbamate, while abstraction of an electron/proton pair from the radical yields vinyl carbamate. The rapid oxidation of vinyl carbamate to its epoxide is then catalyzed by the same enzyme. Such a reaction may be expected to be more general for ethyl compounds containing good leaving groups, and 1,N6-ethenoadenosine was formed from ethyl bromide.