Chem. Res. Toxicol. 1992, 5 , 2-5
2
Communications Roles of the Vinyl Chloride Oxidation Products 2-Chlorooxirane and 2-Chloroacetaldehyde in the in Vitro Formation of Etheno Adducts of Nucleic Acid Bases F. Peter Guengerich Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received August 12,1991 Vinyl chloride is of interest because of its involvement in the etiology of liver hemangiosarcoma and possibly other tumors in industrial workers (1). Similar tumors can be produced in animal models, and the vinyl halides are considered classic chemical carcinogens. Tumor initiation by the vinyl halides and other vinyl monomers such as acrylonitrile and vinyl carbamate appears to involve initial oxidation to epoxides (2). In the case of the vinyl halides these halooxiranes rearrange rapidly to 2-haloacetaldehydes (Scheme I). Both of these entities are reactive electrophilic species and can react with nucleic acid bases. The question of which is most important has been considered. Zajdela et al. (3) found that 1-chlorooxirane caused skin tumors but 2-chloroacetaldehyde did not. Gwinner et al. (4)reported that bis(2-chloroethyl) ether, which is metabolized to 2-chloroacetaldehyde, was not tumorigenic. In a series of in vitro experiments we utilized purified epoxide hydrolase and alcohol dehydrogenase as specific quenching reagents for 1-halooxiranes and 2haloacetaldehydes, respectively-these studies provided evidence that 1-halooxiranes are the major electrophiles involved in DNA alkylation and that 2-haloacetaldehydes are the electrophiles most responsible for protein alkylation (5,6). The conclusions reached in the previous studies need to be reconsidered in light of the evidence that the major nucleic acid adduct formed from vinyl halides is "42oxoethy1)guanine (7). In early studies it was difficult to demonstrate the formation of the etheno adducts in vivo (8). However, there is now a considerable body of evidence indicating that these minor etheno adducts are probably much more biologically important than "-(2-oxoethyl)guanine because of their in vivo persistence (9)and their ability to miscode in vitro (10-14).The question of how these minor adducts are formed from vinyl halide metabolites is still open, for 2-haloacetaldehydes are often used in the synthesis of these adducts (15)and as surrogates for vinyl halides in DNA modification studies (2,11, 12,16,17). The kinetic and inhibition approaches used earlier were applied specifically to the question of how etheno adduds are generated under conditions resembling those encountered in cells.
Experimental Procedures Caution! The following chemicals are hazardous and should be handled carefully: 1-chlorooxiraneand 2-chloroacetaldehyde. These should be handled with protective clothing in a wellventilated fume hood. Chemicals. Calf thymus DNA, adenosine, 1,M-ethenoadenosine, and 1,M-ethenodeoxyadenosinewere purchased from Sigma Chemical Co. (St. Louis, MO). 2-Chloroacetddehyde was
0893-228x/92/2705-02$03.00/0
Scheme I. Enzymatic Oxidation of Vinyl Chloride to 1-Chlorooxirane and Rearrangement to 2-Chloroacetaldehyde DNA adducts
4''P-450
~
u'
1
epoxide hydrolase
H
O
W
0
.c1O -
1
alcohol dehydrogenase
C 1 W O H
obtained as a 50% (w/v) aqueous solution from Aldrich Chemical Co. (Milwaukee, WI). Vinyl chloride was purchased from MG Industries (Valley Forge, PA) as a 5OOO ppm mixture in air, under pressure. 2-Chlorooxirane was synthesized by photochemical chlorination of ethylene oxide in the presence of tert-butyl hypochlorite as described elsewhere (51,and the concentration was estimated using 4-@-nitrobenzyl)pyridinereagent (5,18)-this assay was also used in the estimation of the half-life. "420xoethyl)guanine was prepared by treatment of guanosine with epichlorohydrin, IO4- treatment, acid hydrolysis, and preparative HPLC (19, 20); NL,3-ethenoguanine was prepared by Osmethylation of guanine, reaction with 2-~hloroacetaldehyde,acid hydrolysis to remove the @-methyl group, and preparative HPLC (21). N3,4-Ethenodeoxycytidinewas prepared by reaction of deoxycytidine with 2-chloroacetaldehyde and preparative HPLC (22). These latter three compounds were >98% pure as judged by HPLC in the analytical systems and by reference of their fluorescence, 'H NMR, and mass spectra with those in the literature. Enzymes. Epoxide hydrolase was purified from rat liver microsomes to electrophoretic homogeneity as deacribed elsewhere (23). Horse liver alcohol dehydrogenase was purchased from Sigma and dialyzed before we. When liver microsomes were used as a source of P-4501 to oxidize vinyl chloride, the rata (male Sprague-Dawley, 150 g, from Harlan Industries, Indianapolis IN) were treated with isoniazid to induce P-4502E1 (24). Incubations and Assays. Buffers containing chloride ion were avoided to prevent the possibility of nucleophilic attack on 1chlorooxirane (18). Calf thymus DNA (2.5 mg mL-') was incubated with 1-chlorooxirane or 2-chloroacetaldehyde in 0.10 M potassium phosphate buffer (pH 7.4) for 15 min at 23 "C. DNA was precipitated by the addition of 5 volumes of cold C2H50H, recovered by centrifugation at 3000g for 10 min, dried under an Nz stream, dissolved in HzO, and digested with 0.1 N HCl (70 "C, 45 min) or enzymatically [in 50 mM Ria-HC1 buffer (pH 6.8) containing 10 mM MgCl,] with bovine pancreas DNase I [Sigma, 50 pg (mg of DNA)-', 1 h at 37 "C] and then a mixture of snake Abbreviations: GSH,L-glutathione; P-450, microsomal cytochrome P-450.
0 1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 3
Communications
30
I
/
'
1,NCEtheno- . deoxyadenosine, nmol 0
loo
lollX 0
0
50
20
40 60 80 100 1-Chlorooxirane,pmol
1 120
Figure 1. Analysis of adducts formed by treatment of DNA with 1-chlorooxirane. Calf thymus DNA (10 mg) was treated with the indicated amount of 1-chlorooxiranefor 15min in a total volume of 2.0 mL of 0.10 M potassium phosphate buffer (pH 7.4) at 23 "C. The DNA was precipitated by the addition of 10 volumes of cold C2H50H,and adducts were measured as described under Experimental Procedures. The inset shows the same information with an expanded scale. All oints presented are means from duplicate experiments: hR(2-oxoethyl)guanine, 0 ; 1,pethenodeoxyadenosine, A; N2,3-ethenoguanine,0.
venom phosphodiesterase [Boehringer-Mannheim,Indianapolis, IN; 5 pg (mgof DNA)-'] and Escherichia coli alkaline phosphatase [Sigma, 3 units (mg of DNA)-'] for 12 h at 37 O C (25). In some of the incubations vinyl chloride (5000 ppm in head space) was oxidized by rat liver P-450 in the presence of adenosine (30 mM) or calf thymus DNA (2.5 mg mI-'). DNA was separated by extraction with butanol and phenol solutions and recovered by C2H50Hprecipitation prior to digestion (26). Recovery and enzymatic digestion were estimated by quantitation of deoxyadenosine by HPLC (A,,, vide infra). ~-(2-Oxoethyl)guanjne and M,%ethenoguaninewere separated and quantified by HPLC and fluorescence measurements as described elsewhere (27). The procedure for the separation and estimation of 1p-ethenoadenosine used in this laboratory has been described previously (28,29). The same HPLC procedure was utilized for the analysis of 1fl-ethenodeoxyadenosine, with reduction of the CHJN concentration to 6% (v/v), and this separation system was also used to estimate deoxyadenosine recovery [the gradient system of Cirroussel et al. (25) was used in preliminary studies but did not appear to offer any advantage].
Results 1-Chlorooxirane was prepared and reacted with calf thymus DNA. Analysis of the products indicated formation in the order N7-(2-oxoethyl)guanine > 1,W-ethenoadenosine > N2,3-ethenoguanine (Figure 1). Two other etheno products, 3,N4-ethenocytosine and 1,W-ethenoguanine, have only weak fluorescent properties and were not measured in these assays. 1-Chlorooxirane was shown to react with calf thymus DNA to form both 1,Pethenoadenosine and N2,3-ethenoguanine considerably more effectively than did 2-chloroacetaldehyde in experiments where the reaction was quenched after 10 half-lives of the epoxide (5) (Figure 2). The lack of formation of N7-(2-oxoethyl)guanine from 2-chloroacetaldehyde is consistent with the reported absence of its in vivo formation with chemicals that generate this electrophile through metabolism (4, 30). Rat liver microsomes were incubated with vinyl chloride and NADPH in the presence of either adenosine (Figure 3A) or calf thymus DNA (Figure 3B), and 1,P-ethenoadenosine or 1,IP-ethenodeoxyadenosinewas measured. In both cases the effects of increasing concentrations of enzymes known to specifically destroy 1-chlorooxirane
20
0
N *,3-Etheno-
1.0
40
60
40
60
tB '
guanine, nmol
/
0.51
05 .& 0
0
.
20
Reactant, pmol
Figure 2. Comparison of levels of formation of etheno adducts formed in DNA from 1-chlorooxiraneand 2-chloroacetaldehyde. Calf thymus DNA (10 mg) was incubated with the indicated amount of reactant [1-chlorooxirane( 0 )or 2-chloroacetaldehyde (o)] for 15 min at 23 "C in a total volume of 2.0 mL of 0.10 M potassium phosphate buffer (pH 7.4). The DNA was precipitated by the addition of 10 mL of cold CzH50H,and adducts were measured as described under Ex erimental Procedures. (A) l,I@-Ethenodeoxyadenosine. (B)$,3-Ethenoguanine. All points presented are means from duplicate experiments.
(epoxide hydrolase) or 2-chloroacetaldehyde (alcohol dehydrogenase, plus NADH) were examined (5,s). Epoxide hydrolase almost completely blocked lP-ethenoadenosine formation in both cases, but alcohol dehydrogenase had no effect. It was also found that the addition of 10 mM GSH to the incubations containing adenosine and DNA (without added epoxide hydrolase or alcohol dehydrogenase) reduced 1,IP-ethenoadenosine formation by 76 and 61%, respectively.2
Discussion The oxidation of vinyl chloride to 1-chlorooxiraneand its subsequent transformation to 2-chloroacetaldehyde have been known for some time (for review see ref 2). 1-Chlorooxiraneand 2-chloroacetaldehyde can show similar mutagenicities in some bacterial and mammalian cell systems (2, 31), and the question of which of these two electrophilic halogen species is most important in formation of biologically relevant DNA adducts has arisen. We originally utilized two in vitro approaches to examine the question of whether 1-halooxiranes or 2-haloacetaldehydes are most important ( 5 , 6 ) : reaction kinetics can be measured directly with the chemicals, and epoxide hydrolase and alcohol dehydrogenase [or aldehyde dehydrogenase (5)] can be used in situ in microsomal incubations containing vinyl halides (Scheme I). On the basis of such experiments it is concluded that 1-chlorooxirane (and not 2-chloroacetaldehyde) is the main entity giving rise to the etheno adducta (Figures 1-3). Our previous work indicates
* It is unclear why more dramatic inhibition of DNA binding was not observed in our earlier studies (6);clearly, GSH can react with 1chlorooxirane to block etheno adduct formation, although not as effectively as high concentrations of epoxide hydrolase (Figure 3).
4 Chem. Res. Toxicol., Vol. 5, No. 1, 1992
1,N'-Ethenoadenosine, pmoi 6o 40
Communications
R
t\
LI
l,N'-Ethenodeoxy. adenosine, pmol 10
5
'0
I\ L2L-I 0.1
0.2
0.3
0.4
0.5
Enzyme, mg mL"
Figure 3. Effects of the addition of purified epoxide hydrolase and alcohol dehydrogenase on the formation of 1JVB-ethanoadenosine adducts in systems containing rat liver microsomes and vinyl chloride. The indicated concentrations of purified rat liver epoxide hydrolase (0)or horse liver alcohol dehydrogenase ( 0 ) (plus 1mM NADH) were added to systems containing 0.5% (v/v) vinyl chloride gas in the head space, 0.1 M potaesium phosphate buffer (pH 7.4), an NADPH-generating system, and either (A) 0.10 mg of liver microsomal protein prepared from isoniazidtreated rata and 30 mM adenosine (totalliquid volume 0.12 mL and head space 3.9 mL) or (B) 1.0 mg of liver microsomal protein prepared from isoniazid-treated rata and 2.5 mg of calf thymus DNA mL-' (total liquid volume 2.0 mL and head space 5.0 mL). In both cases reactions proceeded for 45 min at 37 "C and either (A) 1P-ethenoadenosine or (B) 1p-ethenodeoxyadenosine was measured as described under Experimental Prooedurea. All points are presented as means of duplicate experiments.
that formation of N7-(2-oxoethyl)guanie is probably also due to 1-chlorooxirane(Figure l),and we previously concluded that protein modification is the result of modification by 2-haloacetaldehydes ( 5 , 6 ) . The reaction of 2haloacetaldehydes with nucleic acid bases is known to be relatively slow (6,22),and the preferential reaction of the bases with 1-halooxiranes can probably be attributed simply to their greater electrophilicity. For further consideration of the mechanism see Guengerich and Raney (32). These conclusions regarding the origin of the etheno derivatives are consistent with the lack of tumorigenicity of 2-chloroacetaldehyde (3) and compounds such as bis(2chloroethyl) ether which generate 2-chloroacetaldehyde (4), if indeed the etheno adducts are responsible for the biological effects. 1,2-Dihdoahnesare tumorigenic, but the mechanism involves GSH conjugation (30); indeed, inhibitors of P-450 oxidation actually are potent cocarcinogens in rat liver (33). We considered the possibility that other vinyl compounds bearing good leaving groups might also be substrates for P-450 2E1 and give rise to etheno adducts. However, neither vinyl acetate nor vinyl ethyl ether did at levels >0.2% that seen with vinyl chloride [nor did diethyl ether, which could conceivably be dehydrogenated to vinyl ethyl ether (34)]. Thus, rat P-450 2E1 appears to be somewhat selective among olefins and ethyl com-
pounds. It is known that the enzyme catalyzes the 0deethylation of diethyl ether (35),and vinyl acetate might be readily hydrolyzed by esterases. It is unclear which of the etheno bases is most likely to be responsible for the tumorigenic effects of vinyl halides and other vinyl monomers. 1,IVj-Ethenoadenine,M,4ethenocytosine, and iV,3-ethenoguanine all appear to be formed at roughly similar levels in vivo (25,36). Differing results have been obtained with regard to miscoding in various in vitro polymerase fidelity experiments (10,11, 13, 14). While lfl-ethenoadenosine has not been as dramatic as other etheno adducts in its miscoding properties in some of these assays, six of seven hepatomas raised in B6C3 F1 mice treated with vinyl carbamate (which yields the same etheno adducts as vinyl chloride) showed an AT to TA transversion at the second position in the 61st codon of the rasH gene, which is probably consistent only with lp-ethenoadenine as the mutagenic lesion (37). Most of the work done here focused on the use of lfl-ethenoadenine as a prototypic adduct. However, the results & appear to apply to W,ðenoguanine (Figure l ) , and selective 13C-labelingexperiments indicate that the mechanisms of formation of 1p-ethenoadenosine and 3,N4-ethenocytosineboth involve attack of the basic endocyclic nitrogen of the pyrimidine ring (N' of adenine or N4of cytosine) on the unsubstituted methylene of the 1-halooxirane (32). Thus, the conclusions regarding the roles of 1-halooxiranes probably apply to all of the etheno adducts in DNA.
Acknowledgment. This work was supported in part by NIH Grants CA 44353 and ES 00267. I thank K. A. Atkins for synthesis of N7-(2-oxoethyl)guanineand W,3ethenoguanine and M. V. Martin for technical assistance in the purification of epoxide hydrolase. Registry No. 1-Chlorooxirane, 7763-77-1; 2-chloroacetaldehyde, 107-20-0; fl-(2-oxoethyl)guanine, 73100-87-5; 1pethenodeoxyadenosine, 68498-25-9; P,3-ethenoguanine, 5628713-9; vinyl chloride, 75-01-4; epoxide hydrolase, 904863-9; alcohol dehydrogenase, 9031-72-5; 1fl-ethenoadenosine, 39007-51-7.
References (1) Forman, D., Bennett, B., Stafford, J., and Doll, R. (1985) Exposure to vinyl chloride and angiosarcoma of the liver: a report of the register of cases. Br. J. Znd. Med. 42, 750-753. (2) Bolt, H. M. (1988) Roles of etheno-DNA adducts in tumorigenicity of olefins. CRC Crit. Rev. Toxicol. 18, 299-309. (3) Zajdela, F., Croisy, A., Barbin, A., Malaveille, C., Tomatis, L., and Bartsch, H. (1980) Carcinogenicity of chloroethylene oxide, an ultimate reactive metabolite of vinyl chloride, and bis(chloromethy1)ether after subcutaneous administration and in initiation-promotion experiments in mice. Cancer Res. 40, 352-356. (4) Gwinner, L. M., Laib, R. J., Filser, J. G., and Bolt, H. M. (1983) Evidence of chloroethylene oxide being the reactive metabolite of vinyl chloride towards DNA: comparative studies with 2,2'-dichlorodiethylether. Carcinogenesis 4, 1483-1486. (5) Guengerich, F. P., Crawford, W. M., Jr., and Watanabe, P. G. (1979) Activation of vinyl chloride to covalently bound metabolites: roles of 2-chloroethylene oxide and 2-chloroacetaldehyde. Biochemistry 18, 5177-5182. (6) Guengerich, F. P., Mason, P. S., Stott, W. T., Fox, T. R., and Watanabe, P. G. (1981) Roles of 2-haloethylene oxides and 2haloacetaldehydes derived from vinyl bromide and vinyl chloride in irreversible binding to protein and DNA. Cancer Res. 41, 4391-4398. (7) Laib, R. J., Gwinner, L. M., and Bolt, H. M. (1981) DNA alkylation by vinyl chloride metabolites: etheno derivatives or 7alkylation of guanine? Chem.-Biol. Interact. 37, 219-231. (8) Singer, B., and Bartach, H. (1986) The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis, IARC Scientific Publications, Lyon. (9) Fedtke, N., Boucheron, J. A., Walker, V. E., and Swenberg, J. A. (1990) Vinyl chloride-induced DNA adducts. 11. Formation
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Chem. Res. Toxicol. 1992, 5, 5-7 and persistence of 7-(2’-oxoethyl)guanineand W,3-ethenoguanine in rat tissue DNA. Carcinogenesis 11,1287-1292. (10) Barbin, A., Laib, R. J., and Bartsch, H. (1985) Lack of miscoding properties of 7-(2-oxoethyl)guanine, the major vinyl chloride-DNA adduct. Cancer Res. 45, 2440-2444. (11) Singer, B., Abbott, L. G., and Spengler, S. J. (1984)Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,P-ethenodeoxyadenosineand 04-methyldeoxythymidine,using polymerases of varying fidelity. Carcinogenesis 5, 1165-1171. (12) Kusmierek, J. T., and Singer, B. (1982) Chloroacetaldehydetreated ribo- and deoxyribopolynucleotides. 2. Errors in transcription by different polymerases resulting from ethenocytosine and its hydrated intermediate. Biochemistry 21, 5723-5728. (13) Singer, B., Spengler, S. J., Chavez, F., and Kusmierek, J. T. (1987) The vinyl chloride-derived nucleoside, W,3-ethenoguanosine, is a highly eficient mutagen in transcription. Carcinogenesis 8, 745-747. (14) Singer, B., Kusmierek, J. T., Folkman, W., Chavez, F., and Dosanjh, M. K. (1991) Evidence for the mutagenic potential of the vinyl chloride induced adduct, W,3-etheno-deoxyguanosine, using a site-directed kinetic assay. Carcinogenesis 12, 745-747. (15) Leonard, N. J. (1984) Etheno-substituted nucleotides and coenzymes: fluorescence and biological activity. CRC Crit. Reu. Biochem. 15, 125-199. (16) Barbin, A., Friesen, M., O’Neill, I. K., Croisy, A., and Bartsch, H. (1986) New adducts of chloroethylene oxide and chloroacetaldehyde with pyrimidine nucleosides. Chem.-Bid. Interact. 59, 43-54. (17) Bedell, M. A., Dyroff, M. C., Doerjer, G., and Swenberg, J. A. (1986) Quantitation of etheno adducts by fluorescence detection. In The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis (Singer, B., and Bartach, H., Eds.) pp 425-436, IARC Scientific Publications, Lyon. (18) Barbin, A., &r&iat, J. C., Croisy, A., ONeill, I. K., and Bartsch, H. (1990) Nucleophilic selectivity and reaction kinetics of chloroethylene oxide assessed by the 4-(p-nitrobenzyl)pyridineassay and proton nuclear magnetic resonance spectroscopy. Chem.-Biol. Interact. 73, 261-277. (19) Roe, J., Jr., Paul, J. S., and Montgomery, P. O., Jr. (1973) Synthesis and PMR spectra of 7-hydroxyalkylguanosiniumacetates. J. Heterocycl. Chem. 10, 849-857. (20) Piper, J. R., Laseter, A. G., and Montgomery, J. A. (1980) Synthesis of potential inhibitors of hypoxanthine-guanine phosphoribosyltransferase for testing as antiprotozoal agents. 1. 7Substituted 6-oxopurines. J. Med. Chem. 23, 357-364. (21) Sattsangi, P. D., Leonard, N. J., and Frihart, C. R. (1977) 1JP-Ethenoguanine and N2,3-ethenoguainine. Synthesis and comparison of the electronic spectral properties of these linear and angular triheterocycles related to the Y bases. J. Org. Chem. 42, 3292-3296. (22) Barrio, J. R., Secriot, J. A., 111, and Leonard, N. J. (1972) Fluorescent adenosine and cytidine derivatives. Biochem. Biophys. Res. Commmun. 46, 597-604. (23) Guengerich, F. P., Wang, P., Mitchell, M. B., and Mason, P. S. (1979) Rat and human liver microsomal epoxide hydratase. Purification and evidence for the existence of multiple forms. J. Biol. Chem. 254, 12248-12254.
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(24) Thomas, P. E., Bandiera, S., Maines, S. L., Ryan, D. E., and Levin, W. (1987) Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes. Biochemistry 26, 2280-2289. (25) Ciroussel, F., Barbin, A., Eberle, G., and Bartach, H. (1990) Investigations on the relationship between DNA ethenobase adduct levels in several organs of vinyl chloride-exposed rats and cancer susceptibility. Biochem. Pharmacol. 39, 1109-1113. (26) Kadlubar, F. F., Miller, J. A., and Miller, E. C. (1976) Microsomal N-oxidation of the hepatocarcinogen N-methyl-4-aminoazobenzene and the reactivity of N-hydroxy-N-methyl-4-aminoazobenzene. Cancer Res. 36, 1196-1206. (27) Fedtke, N., Walker, V. E., and Swenberg, J. A. (1989) Determination of 7-(2’-oxoethyl)guanine and W,3-ethenoguanine in DNA hydrolysates by HPLC. Arch. Toxicol. Suppl. 13,214-218. (28) Leithauser, M. T., Liem, A., Stewart, B. C., Miller, E. C., and Miller, J. A. (1990) 1,WEthenoadenosine formation, mutagenicity and murine tumor induction as indicators of the generation of an electrophilic epoxide metabolite of the closely related carcinogens ethyl carbamate (urethane) and vinyl carbamate. Carcinogenesis 11,463-473. (29) Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991) Role of human cytochrome P-450 IIEl in the oxidation of several low molecular weight cancer suspects. Chem. Res. Toxicol. 4,168-179. (30) Kim, D.-H., and Guengerich, F. P. (1990) Formation of the DNA adduct S-[2- (N-guanyl)ethyl]glutathione from ethylene dibromide: effects of modulation of glutathione and glutathione S-transferase levels and the lack of a role for sulfation. Carcinogenesis 11,419-424. (31) Malaveille, C., Bartach, H., Barbin, A., Camus, A. M., and Montesano, R. (1975) Mutagenicity of vinyl chloride, chloroethyleneoxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Commun. 63, 363-370. (32) Guengerich, F. P., and h e y , V. M. (1991) Formation of etheno adducts of adenosine and cytidine from l-Mooxiranee. Evidence for a mechanism involving initial reaction with the endocyclic nitrogen atoms. J. Am. Chem. SOC. (in press). (33) Wong, L. C. K., Winston, J. M., Hong, C. B., and Plotnick, H. (1982) Carcinogenicity and toxicity of l,2-dibromomethane in the rat. Toxicol. Appl. Pharmacol. 63, 155-165. (34) Guengerich, F. P., and Kim, D.-H. (1991) Enzymatic oxidation of ethyl carbamate to vinyl carbamate and its role as an intermediate in the formation of 1$P-ethenoadenosine. Chem. Res. Toxicol. 4, 413-421. (35) Brady, J. F., Lee, M. J., Li, M., Ishizaki, H., and Yang, C. S. (1988) Diethyl ether as a substrate for acetone/ethanol-inducible cytochrome P-450 and as an inducer for cytochrome(s) P-450. Mol. Pharmacol. 33, 148-154. (36) Eberle, G., Barbin, A., Laib, R. J., Ciroussel, F., Thomale, J., Bartsch, H., and Rajewsky, M. F. (1989) l$P-ethen0-2’-deoxyadenosine and 3,N4-etheno-2’-deoxycytidinedetected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride. Carcinogenesis 10, 209-212. (37) Wiseman, R, W., Stowers, S. J., Miller, E. C., Anderson, M. W., and Miller, J. A. (1986) Activating mutations of the c-Hams protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse. Proc. Natl. Acad. Sci. U.S.A. 83, 5825-5829.
Semiempirical Self-Consistent Field (CNDO) Calculations of Arsenical-Antidote Adducts Dennis W. Bennett,+Lihua Huang,* and Kilian Dill*J Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin 53201, and Department of Chemistry, Clemson University, Clemson, South Carolina 29634 Received August 8, 1991 Introduction A requisite feature of antidotes for heavy metals such as arsenic is the ability of the antidote to sequester the
* Author to whom correspondence should be addressed. t University
of Wisconsin.
* Clemson University.
metal and eventually excrete the adduct. Thus, the pharmacokinetics of the antidote and the stability of the adduct fOrmed are of utmost importance 88 the first step in the detoxification process. In recent years, we have published extensively on the reactions of organic arsenicals with simple thiol-containing compounds in order to develop a strategy in the search for
0893-228x/92/2705-0005$03.00/00 1992 American Chemical Society
