Chem. Res. Toxicol. 1991,4, 413-421 (24) Farrelly, J. G.(1980)A new assay for the microsomal metabolism of nitrosamines. Cancer Res. 40,3241-3244. (25) Reinke, L. A,, and Moyer, M. J. (1985)p-Nitrophenol hydroxylation: a microsomal oxidation which is highly inducible by ethanol. Drug Metab. Dispos. 13, 548-552. (26) Segel, I. W. (1975)Enzyme kinetics, pp 57-59,John Wiley & Sons, New York. (27) Koop, D. R. (1986)Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450isozyme 3a. Mol. Phurmacol. 29,399-404. (28) Keefer, L.K., Kroeger-Koepke, M. B., Ishizaki, H., Michejda, C. J., Saavedra, J. E., Hrabie, J. A., Yang, C. S., and Roller, P.P. (1990) Stereoselectivity in the microsomal conversion of Nnitrosodimethylamine to formaldehyde. Chem. Res. Toxicol. 3, 540-544. (29) Ekstrom, G.,Norsten, C., Cronholm, T., and Ingelman-Sundberg, M. (1987)Cytochrome P-450dependent ethanol oxidation.
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Kinetic isotope effects and absence of stereoselectivity. Biochemistry 26,7348-7354. (30) Gillette, J. R., and Korzekwa, K. (1990)Predicted deuterium effects on the formation of metabolites by various P-450mechanisms. In Drug metabolizing enzymes: genetics, regulation and toxicology. Proceedings in the VZZZth International Symposium on Microsomes and Drug Oxidations (Ingelman-Sundberg, M., Gustafsson, J.-A., and Orrenius, S., Ede.) p 422,Karolinska Institute, Stockholm, Sweden. (31) Northrop, D. B. (1978)Determining the absolute magnitude of hydrogen isotope effecta. In Isotope effects on enzyme catalyzed reactions (Cleland, W. W., O'Leary, M. H., and Northrop, D. B., Eds.) pp 122-148,University Park Press, Baltimore, MD. (32) Gorsky, L. D., Koop, D. R., and Coon, M. J. (1984)On the stoichiometry of the oxidase and monooxygenaae reactions catalyzed by liver microsomal cytochrome P-450.J. Biol. Chem. 259, 6812-6817.
Enzymatic Oxidation of Ethyl Carbamate to Vinyl Carbamate and Its Role as an Intermediate in the Formation of 1,N'-Ethenoadenosine F. Peter Guengerich* and Dong-Hyun Kim? Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received December 21, 1990 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. 38,3793-38041. To date, the enzymatic conversion of ethyl carbamate to vinyl carbamate had not been demonstrated. Recently, we obtained evidence that the same cytochrome P-450enzyme (P-4502E1) is involved in the oxidation of both ethyl carbamate and vinyl carbamate [Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991)Chen. 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 Nhydroxycarbamate 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, of -54 r M was estimated for the conversion of vinyl carbamate to 1,Methenoadenosine (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, > 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 pM vinyl carbamate, consonant with the value of -0.2 pM estimated experimentally. A large kinetic deuterium isotope effect (>7)was observed for the formation of 1,I@-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-4502E1 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,M-ethenoadenosine was formed from ethyl bromide. Ethyl carbamate (urethan) has long been known to be carcinogenic at multiple sites in rodents, particularly in the lung (I). This cancer suspect is encountered in the chemical and tanning industries and-potentially of even greater concern-is present at low levels in fermented foods and beverages from the reaction of ethanol with carbamyl Present address: Korea Institute of Science and Technology, P.O. Box 131, Chongyangni, Seoul, South Korea.
phosphate. A striking structure-activity relationship is seen for tumorigenesis-neither methyl nor propyl carbamate is nearly as carcinogenic (2). The chemistry involved in the activation of ethyl carbamate t~ its ultimate carcinogenic form has been difficult to elucidate. Ethyl carbamate is not mutagenic itself, even in the presence of a microsomal activation system. A known metabolite, ethyl N-hydroxycarbamate, was viewed as a reactive form in the earlier literature (2),but it is now
0 1991 American Chemical Society OS93-228~/91/2704-0413~02.50/0
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Chem. Res. Toxicol., Vol. 4, No. 4, 1991
Scheme I. Postulated Mechanism of Ethyl Carbamate Bioactivation (3-6) 0
II
CHaCH2-0-CNH2
RNA, DNA, or (d)Adenosine
-(d)Ribose-
clear that this product is not mutagenic and is less tumorigenic than its precursor (3). The Millers postulated the pathway shown in Scheme I and have shown that vinyl carbamate is mutagenic upon microsomal oxidation and is considerably more carcinogenic than ethyl carbamate ( 4 ) . Further, the spectrum of tumors produced by vinyl carbamate is similar to that seen with ethyl carbamate. More recently, the epoxide has been synthesized and shown to form a potentially mutagenic DNA adduct, 1JP-ethenoadenosine (q-this electrophile is now being tested as a carcinogen. A key deficiency in this hypothesis has been the inability to detect vinyl carbamate as an oxidation product of ethyl carbamate in vitro or in vivo (3,4). Some of the enzymology relevant to the activation of ethyl carbamate has also been unclear. The Millers demonstrated the role of mixed-function oxidases (and presumably P-450)'in the activation of ethyl carbamate and vinyl carbamate ( 3 , 4 , 6 , 1 3 ) .Waddell and his associates have demonstrated the roles of several compounds in the inhibition of ethyl carbamate metabolism and considered roles for alcohol and aldehyde dehydrogenases (14-16). These findings and the inhibition of metabolism by diethyldithiocarbamate and its oxidized form disulfiram (6) may now be interpreted in light of the preferential inhibition of a particular form of P-450,P-4502E1, by all of these compounds (12, 17). We previously provided evidence that the oxidation of both ethyl carbamate and vinyl carbamate to 1P-ethenoadenosine was catalyzed by P-4502E1 (12). With this background, we investigated the role of vinyl carbamate as an intermediate in the oxidation of ethyl carbamate to more proximate carcinogens.
Experlmental Procedures Chemicals. Ethyl carbamate and methyl carbamate were obtained from Aldrich Chemical Co. (Milwaukee, WI) and further purified by sublimation at 15 mm Hg. Ethyl N-hydroxycarbamate and Nfl-dimethylnitrosamine were purchased from Aldrich and used without further purification. 2-Hydroxyethyl carbamate (originally purchaeed from Pfaltz and Bauer, Stanford, CT), vinyl carbamate (6),and [ethyl-2H6]ethylcarbamate ( 4 ) (checked and Abbreviations: P-450,liver microsomal cytochrome P-450;G O 2E1,a P-450enzyme induced by ethanol, isoniazid, and several other compounds (8-12); HPLC,high-performance liquid chromatography; GC/MS, combined capillary gas chromatography/chemical ionization mass spectrometry.
Guengerich a n d Kim found to be >99% enriched by GC/MS, vide infra) were all generous gifts of Professor J. A. Miller, University of Wisconsin (Madison, WI). Adenosine and chlorzoxazone were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. [ethyl-1,2-3H]Ethylcarbamate (20 Ci/mmol) was prepared by ChemSyn Laboratories (Lenexa, KS) and provided through Dr. F. F. Kadlubar (National Center for Toxicological Research, Jefferson, AFt). The material, dissolved in CHZCl2,was found to contain 4-5% of an apparently more hydrophobic impurity when analyzed by HPLC on an octadecylsilyl (C18) column (0.1% aqueous H3P0,, v/v, with a gradient of CH&N applied after 15 min) or a silica column (20% tetrahydrofuran in hexane, v/v). In the latter system this impurity eluted near the positions of N-(hydroxyethy1)carbamate and vinyl carbamate and needed to be removed. I t must be emphasized that ethyl carbamate and vinyl carbamate are volatile (sublime) and must be handled carefully to prevent loss and exposure (4). The CHZClzsolution (2 mCi, 0.1 rmol) was reduced from 2 mL to -0.1 mL under an Nz stream at 23 OC and mixed with 0.2 mL of HzO-the residual CHzClzwas evaporated under the Nzstream, and the entire sample was loaded onto a 10 X 250 mm Beckman octadecasilyl HPLC column (5 pm, Beckman, San Ramon, CA) and eluted with HzO a t a flow rate of 4 mL mi&. The fractions eluting in a peak a t t R 13 min were found to contain ethyl carbamate, as judged by determination of radioactivity in aliquots. The material was judged to be >99.9% radiochemically pure when an aliquot of the CHzClzextract was analyzed by using the above silica HPLC system (vide infra). The pooled fractions (55% yield) were used directly from the aqueous solution for incubations with microsomes. Enzymes. Human liver samples were obtained from organ donors through Tennessee Donor Services (Nashville, TN) and denoted with the initials "HL" (human liver) and a number assigned in chronological order. Samples were stored at -70 "C, and microsomes were prepared and stored as described elsewhere ( 1 4 1 9 ) . In a previous study (12),sample HL 114 was found to have the highest catalytic activities for the conversion of both ethyl carbamate and vinyl carbamate to l,I@-ethenoadenosine and was used in the studies presented here. In all incubations with microsomes the glycerol concentration was kept si
Bg
s!
d
20
0
0
0.10
0.20
Rate of ethyl carbamate oxidation to ethenoadenosine Figure 1. Correlation of rates of formation of 1P-ethenoadenosine from ethyl carbamate and vinyl carbamate in different human liver samples. Resulta from a previous study are plotted here, with the unita of pmol of 1p-ethenoadenosineformed min-' mg of microsomal protein-' (12).
-
with care being taken to avoid concentration to dryness (when indicated, the products were then extracted into 1mL of H20 and subsequently into 2 mL of CH2C12;this organic extract was then concentrated under an Nzstream). An aliquot (1-2 pL) was injected onto a 0.2 mm X 9 m Carbowax 20 M column with the effluent directed into a Ribermag R-10-1OC mass spectrometer operating in the chemical ionization mode with CH, as the carrier gas (the selectivity and sensitivity for the products were found to be enhanced in the chemical ionization mode relative to eledron impact). The expected (M+ H)+ions were monitored. In some cases scans were obtained for selected peaks. Quantitation was done by using computer-generated integrals of peaks of the selected ions, utilizing ratios to the internal standard methyl carbamate.
Results Initial Characterization of Products Formed from Ethyl Carbamate. In the course of earlier studies evidence was obtained suggesting that human P-450 2E1 oxidizes both ethyl carbamate and vinyl carbamate t o a product that reacts with adenosine to form 1P-ethenoadenosine (12). In these studies the activities measured in different liver samples were highly correlated with each other (Figure 1). These rates are comparable to those measured in mouse liver microsomes (6). These findings suggested that the paradigm depicted in Scheme I might be correct, with both oxidation reactions catalyzed by P-450 2El-however, since the rate measured with vinyl carbamate was -400-fold faster than that with ethyl carbamate [asalready demonstrated in in vitro and in vivo experimental animal systems by Leithauser et al. (611,then the steady-state level of vinyl carbamate might be extremely low and have gone undetected in previous work (3,4). Radioactive ethyl carbamate was obtained in highly purified form, and the products of microsomal incubation were analyzed with modifications of a silica HPLC system used previously by Dahl et al. (4) (tetrahydrofuran replaced ethyl acetate in the solvent system in order to allow for detection of the weak response of the standards under consideration at 190 nm). A CH2C12extract of a 4-h incubation of [eth~l-l,2-~H]ethyl carbamate with human liver microsomes indicated the NADPH-dependent formation of small amounts of three products tentatively identified as ethyl N-hydroxycarbamate, vinyl carbamate, and 2-hydroxyethyl carbamate (Figure 2). Attempts to
.-C
. €
0
i B *Oo0
1000
0
5
10
15
20
25
30
Time, min Figure 2. Formation of oxidation products of ethyl carbamate. Human liver microsomes (3 mg of protein mL-') were incubated with 0.1 M potassium phosphate and 0.3 mM [ethyl-1,2-SH]ethyl carbamate (100 mCi mmol-') in the presence (A) or absence (B) of an NADPH-generating system (18)(incubation volume 0.5 mL). After 4 h (at 37 "C) products were extracted into 0.5 mL of CHzClz and 50-pL aliquots were injected onto a 4.6 X 250 mm Alltech silica HPLC column (5 pm, Applied Science Labs, Avondale,PA). The column was eluted with a mixture of 20% tetrahydrofuran in hexane (v/v) for 15 min, and then the tetrahydrofuran concentration was increased to 40% (v/v) in a linear manner over a period of 3 min. The flow rate was 2 mL min-I and the column effluent was mixed with Floscint I cocktail (flow rate 2 mL min-') in a Flo-one on-line detector equipped with a 2.5-mL flow cell (RadiomaticInstruments, Tampa,FL). The inset in part A shows the same chromatogram with the scale contracted 150-fold. Although the ethyl carbamate peak slopes and retention times may appear different in parts A and B, the respective recoveries were 1.79 X lo7and 1.86 X 10' cpm (in the ethyl carbamate peak). The tR values of the standards corresponded to the indicated radioactive peaks: ethyl N-hydroxycarbamate, 4.0 min; vinyl carbamate,6.5min; ethyl carbamate, 7.5 miq and 2-hydroxyethyl carbamate, 18.5 min.
separate all three of these products and ethyl carbamate using reverse-phase HPLC were unsuccessful. The reproducibility of the tR values and resolution were not accurate enough to allow unambiguous assignment of the structures in the silica HPLC, however, and recovery was difficult to estimate because of the polar nature of the compounds (and high solubility in H20) and difficulty in measuring the absorbance of the material. Characterization of Microsomal Oxidation Products of Ethyl Carbamate by GC/MS. GC/MS was considered as a more definitive means of further charac-
Guengerich and Kim
416 Chem. Res. Toricol., Vol. 4, No. 4, 1991
8 U a m
4
Figure 3. GC/MS of standard vinyl carbamate, ethyl Nhydroxycarbamate, and 2-hydroxyethyl carbamate. Selected ion monitoring was at m/z 88 and 106 in the two charta. The t R of ethyl carbamate was 5 min 36 s (m/z 90,not shown). See Experimental Procedures for details. t
p"
1
X
.01 r
fl
B .001
8
c 0 Q) c
:
A
O
B [Product], mM
Figure 4. Standard curves for the estimation of concentrations of oxidation products of ethyl carbamate by GC/MS. The internal standard was methyl carbamate (1.0 mM; t R 2 min 18 s under the conditions of Figure 3). Relative responses at m/z 88 (vinyl carbamate, 0 ) and m/z 106 (2-hydroxyethylcarbamate, A;ethyl N-hydroxycarbamate,0 )to methyl carbamate (m/z 76) are based upon integration of the peaks ( m / z vs time). terizing the products of ethyl carbamate oxidation. The suspected products vinyl carbamate, ethyl N-hydroxycarbamate, and 2-hydroxyethyl carbamate were readily separated from ethyl carbamate (Figure 3), and amounts of each could be estimated in extracts with the use of methyl carbamate as an internal standard (Figure 4). Products of the microsomal oxidation were extracted into CH2C12and analyzed by GC/MS (Figure 5). Selected ion monitoring revealed the presence of peaks with the expected retention times and m l z values corresponding to (M + H)+ ions for vinyl carbamate and 2-hydroxyethyl carbamate and one corresponding to N-hydroxyethyl carbamate (m/z 106, results not shown). The presence of each of these two peaks was dependent on the addition of NADPH. The concentrations of these products were estimated by comparisons of peak areas with those of the internal standard methyl carbamate-the levels of vinyl carbamate, ethyl N-hydroxycarbamate, and 2-hydroxyethyl carbamate present were estimated to be 0.2, 1.5, and 1.0 pM, respectively. Further verification of the identities was difficult because of the low concentrations present and the interfer-
4 *-.. .
. .i r. . . . .6a. . . . .;a' . . . . ...il .. . . . . . . . . i..... . . i.r. . &I
h.
..-
Time, min Figure 5. GC/MS of products of a human liver microsomal incubation with ethyl carbamate. (A) Incubation in the presence of an NADPH-generating system. (B)Incubation devoid of NADPH.
ence of other materials in the low m/z range under consideration. Some of the interfering materials could be removed by extracting the hydrophilic carbamates into H20 (and then back into CH2C12for analysis). GC/MS showed more definitive peaks (Figure 6A,B), and scanning measurementa revealed the major ions seen for each of the standard compounds under consideration (Figure 6C-E). Kinetics of 1,W-Ethenoadenosine Formation. The formation of 1,Wethenoadenosine from vinyl carbamate was saturable with regard to substrate concentration and was characterized by a K, of 54 ctM and V, of 55 pmol of 1,M-ethenoadenosine formed min-l (mg of protein)-' (Figure 7A). In contrast, the rate of formation of 1flethenoadenosine from ethyl carbamate appeared to be first order in substrate. Attempts to fit data to linear plots yielded a K, estimate of at least 2.3 mM (inset to Figure 7B). The rate of 1,M-ethenoadenosine formation is 2 orders of magnitude faster from vinyl carbamate than from ethyl carbamate. Two other substrates for P-450 2E1, N,Ndimethylnitrosamine (11) and chlorzoxazone (N), inhibited the conversion of vinyl carbamate to 1Jvs-ethenoadenosine, but ethyl carbamate did not at the concentrations examined (Figure 8). When either 2-hydroxyethyl carbamate or ethyl Nhydroxycarbamate (1 mM) was incubated with microsomes, NADPH, and adenosine, no 1,Wethenoadenosine was detected (> u1 (Figures 1and 7). Since vinyl carbamate can be considered an intermediate in the overall sequence, then in the steady state (presumed t o be reached after 2 h)
and the collective rate constants are described by a single rate constant in each overall step, and K2 is the apparent K, for the conversion of vinyl carbamate to 1,V-ethenoadenosine (and V2 is the V,, for this reaction). In this model the yield of 1JP-ethenoadenosine from the epoxide
V2[W O2 = K 2+ [VC] because even with [EC] = 1mM and [VC] = 20 pM there is no inhibition of the oxidation of vinyl carbamate (Figure
-4VCl dt
u1
-0
= u2
Guengerich and Kim
420 Chem. Res. Toxicol., Vol. 4, No. 4, 1991
8). Indeed, the lack of inhibition is apparently due to the
high K , for ethyl carbamate. The simplified expression can be used (without inclusion of correction for the inhibitor). Thus
VZ[VCl = Kz+ [VC] and since [VC]
