Chem. Res. Toxicol. 1989,2, 280-281
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L'ommuntcattons Origin of Tetrahydrotetrols Derived from Human Hemoglobin Adducts of Benzo[a Ipyrene labeled tetrols and subjecting it to the same procedure. Sir: The ultimate carcinogen anti-BPDE (l),l derived from metabolic activation of benzo[a]pyrene (BP), reacts with DNA by nucleophilic attack of a DNA base at the (2-10 position with concomitant opening of the oxirane ring ( 1 , Z ) . Analogous reactions would be expected to occur between nucleophilic sites of amino acids in proteins with 1. Despite several recent studies (3-7) of the interaction of hemoglobin with BP in vivo or 1 in vitro, no definitive evidence for the product structures has been reported. We now report l80incorporation experiments which clearly demonstrate that esterification of one or more carboxylate sites of human hemoglobin occurs in the presence of 1. We also present evidence that esters are the most abundant adducts formed by 1 with human hemoglobin in vitro as well as in vivo.
1
Carboxylic esters of benzylic alcohols may undergo hydrolysis via a BAL1 mechanism (8). When this mechanism is operative (7 < pH < 8.5), oxygen from solvent water is incorporated into the resulting alcohol. However, increasing pH to >9 results in a change of mechanism to the more common BAc2mode of hydrolysis, in which case no cleavage of the alkyl-oxygen bond occurs. Therefore, the model ester 2 was synthesized2 to ascertain if 1-derived esters at (2-10 are hydrolyzed in a similar manner at different pH values. In Hz180at pH 8.1 (12 h), 2 underwent hydrolysis with >95% incorporation of l80into the resulting tetrols 3 and 4, as determined by GC-MS of the tetra-TMS derivatives. The ratio of stereoisomer 3 to 4 approached unity (3:2), consistent with formation of an intermediate with pronounced carbocation character. As expected, hydrolysis of 2 at pH 10.8 resulted in predominant formation of 3 (ratio of 3 to 4 = 601) in which no l80label could be detected. Freshly isolated human erythrocytes were treated with 1 (1pmo1/20 wmol of Hb tetramer) after resuspension in phosphate-buffered saline. Adducted globin was prepared from cell lysates by Sephadex G-25 chromatography, precipitation in 0.015% HCl/acetone, and extraction of the redissolved globin with water-saturated n-butanol. Complete removal of noncovalently bound tetrols was demonstrated by incubating native hemoglobin with 14C-
When adducted globin was digested with a proteolytic enzyme, tetrols 3 and 4 were the major products. This was determined by subjecting a Pronase digest of globin that had been adducted by [l4C]-1to reverse-phase HPLC using conditions that eluted all applied radioactivity. The radiogram generated by scintillation counting of collected fractions indicated that the ratio of (3 + 4) to all other radioactive components was ca. 4:1.3 The isolation of tetrols under the conditions just described implies that they arise from adducts in which the covalent bond between the BP residue and the protein does not survive even enzymatic proteolysis. To test whether the presumed adducts are carboxylic esters, adducted globin was treated under two sets of conditions designed to reproduce closely the conditions under which a change of mechanism for the hydrolysis of 2 was observed. Trypsin4 digestion was performed in H2180(8 h at pH 8.3). A mixture of 3 and 4 (in a ratio of 3:2) was observed with 60% incorporation of l80label into each alcohoL5 Denaturation of the adducted protein in 8 M urea/H;*O (24 h; room temperature) at pH 10.9, neutralization, and extraction with ethyl acetate afforded tetrols 3 and 4 in a ratio of 8:l with no detectable l80 incorporation. This specific pH-related change in label incorporation and product ratios is consistent only with a change in the mechanism of hydrolysis of an ester and indicates that adduct formation occurred either at the C-termini or the side chain of Asp/Glu in human hemoglobin. Pooled hemoglobin from human donors was also analyzed for BP-hemoglobin adducts. Globin was prepared as above and then subjected to Pronase digestion (pH 7.3) and immunoaffinity chromatography (7,9), using a monoclonal antibody (8Ell) (IO)which was raised against the W-guanosine adduct of 1 and which recognizes a variety of other C-10 analogues of 3 and 4. The fraction recognized by the monoclonal antibody6 was subjected to microbore C-18 HPLC, and collected fractions were analyzed by synchronous fluorescence spectroscopy (11,12). The major products detected that could have been formed from 1'
A similar product ratio was established by monitoring the absorbance of the eluent at 343 nm. The HPLC was equipped with a HewlettPackard 1040 diode-array detector which allowed full UV spectra of each peak to be acquired. The spectra confirmed the presence of the pyrene chromophore in each peak presumed to be derived from adducts. ' Enzymatic proteolysis was chosen as the mildest method of exposure of adduct bonds to water by destruction of the globin structure. Urea denaturation could not be used because globin was insufficiently soluble at near-neutral pH. Trypsinolysis generates significant concentration8 of nucleophiles, in particular, amino groups, which would be acylated by esters. To the extent that this reaction competes with B a l solvolysis, it reduces the amount of l80incorporated. Any ester that was produced b trypsincatalyzed hydrolysis also would contribute to the 40% of 0-labeled The complete name is 7~,8a-dihydroxy-9a,lOa-epoxy-7,8,9,lO-tetra- tetrols. Presumably, all of the products derived from 1; synthetic ethylenehydrobenzo[a]pyrene. For in vitro reactions racemic 1 was used. diamine, cysteamine, and glutathione adducts of 1 are all bound effecN-t-BOC-L-Alaninewas dissolved in 10 mM NaHC08, pH 7:3, and tively by this antibody and should be representative of peptide adducts. 0.1 equiv of racemic 1 in THF was added dropwise over several minutes. The determination of the origin of the products was based on their The reaction proceeded for 2 h at room temperature before the products synchronous fluorescence spectra, which are highly specific. syn-BPDEwere separated by preparative C18HPLC. Final purification was accomderived tetrols would exhibit identical spectra but can be distinguished plished by HPLC using an analyticalC18Nova-Pak column, yielding 1.5% chromatographically. It is assumed that fluorescence quantum yields are product based on 1. The 1:l mixture of diastereomers gave satisfactory generally the same for all fluorescent peaks. H NMR spectrum, consistent with the assigned structure, 2.
2
0893-228~/89/2702-0280$01.50/0 0 1989 American Chemical Society
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 281
Communications were 3 and 4 in a ratio of ca. 2.51. The ratio observed is very close to that observed in experiments using in vitro adducted hemoglobin and is consistent with the formation of a carbocation during hydrolysis of an ester. When human hemoglobin was subjected to urea denaturation at pH 10.2, the ratio of tetrol3 to 4 increased to 5.41. While the increase in the ratio of 3 to 4 is not as great as it is for in vitro adducted hemoglobin, it is qualitatively the expected change. The identity of the tetrols was confirmed by their emission-excitation fluorescence spectra, which were identical with those of standards, and GC/NICI and GC/EI mass spectra of the tetra-TMS derivatives, which afforded characteristic fragment ions at 446 amu, corresponding to [M - (TMS-O-TMS)]*-and 404 amu, corresponding to [M - (TMS-OCH=CHO-TMS)]' +,respectively. The spectroscopicevidence and hydrolysis product ratios clearly indicate that the major human hemoglobin adducts formed by reaction with 1 in vitro are carboxylic esters. Available evidence suggests that the same is true in vivo. While the protein retains its native conformation, these esters, which would be unstable in solution, are resistant to hydrolysis. A similar situation has been observed previously by us. The sulfinamide bond formed by 4aminobiphenyl with the 938 cysteine residue of hemoglobin is inaccessible to water (13) and is hydrolytically stable (14);upon digestion of the protein with Pronase, however, only 4-aminobiphenyl could be isolated. Presumably, hemoglobin and its bound benzo[a]pyrene residue likewise adopt a conformation in which the ester is situated in a region of the protein from which water is excluded. Acknowledgment. We gratefully acknowledge financial support from the National Institutes of Health (Grants ES01640, ES02109, and ES04675) and the Italian Association for Cancer Research (AIRC) for a fellowship to R.P. B.W.D. is supported by a fellowship from NIH (Grant ES07020) and the MIT Hazardous Substances Management Program (Dow Chemical-MONT.EC0.). We are also indebted to Regina Santella for her generous gift of monoclonal antibody.
References (1) Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland, F. A., Harvey, R. G., Kasai, H., Miura, T., and Nakanishi, K. (1976) Benzo[a]pyrene-nucleic acid derivative found in vivo: structure of a benzo[a]pyrenetetrahydrodiol epoxideguanosine adduct. J. Am. Chem. SOC. 98,5714-5715. (2) Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., Conney,
A. H., Yagi, H., and Jerina, D. M. (1978) Binding of benzo[a]pyrene 7,8-diol-9,10-epoxideto DNA, RNA, and protein of mouse skin occurs with high stereoselectivity. Science 199, 778-781. (3) Shugart, L. (1985) Quantitating exposure to chemical carcinogens: In vivo alkylation of hemoglobin by benzo[a]pyrene. Toricology 34,211-220. (4) Shugart, L. (1986),Quantifying adductive modification of hemoglobin from mice exposed to benzo[a]pyrene. Anal. Biochem. 152,365-369. (5) Wallin, H., Jeffrey, A. M., and Santella, R. M. (1987) Investigation of benzo[a]pyrene-globin adducts. Cancer Lett. 35, 139-146. (6) Weston, A., Rowe, M. L., Manchester, D. K., Farmer, P. B., Mann, D. L., and Harris, C. C. (1989) Fluorescence and mass spectral evidence for the formation of benzo[a]pyrene anti-diol epoxide-DNA and -hemoglobin adducts in human. Carcinogenesis 10, 251-257. (7) Lee, B. M., and Santella, R. M. (1988) Quantitation of protein adducts as a marker of genotoxic exposure: Immunologic detection of benzo[a]pyrene-globin adducts in mice. Carcinogenesis 9, 1773-1777. (8) Ingold, C. K. (1969) Structure and Mechanism in Organic Chemistry; 2nd ed., pp 1137-1142 and 1157-1163, Cornel1 University Press, Ithaca, NY. (9) Groopman, J. D., Trudel, L. J., Donahue, P. R., MarshakRothestein, A., and Wogan, G. N. (1984) High affinity monoclonal antibodies for aflatoxins and their application to solid phase immunoassays. Roc. Natl. Acad. Sci. U.S.A. 81,7728-7731. (10) Santella, R. M., Lin, C. D., Cleveland, W. L., and Weinstein, I. B. (1984) Monoclonal antibodies to DNA modified by benzo[alpyrene diol epoxide. Carcinogenesis 5, 373-377. (11) Gan, LA.,Otteson, M. S., Doxtader, M. M., Skipper, P. L., Dasari, R. R., and Tannenbaum, S. R. (1989) Quantitation of carcinogen bound protein adducts by fluorescence measurements. Spectrochim. Acta 45A, 81-86. (12) Vahakangas, K., Haugen, A., and Harris, C. C. (1985) An applied synchronous fluorescence spectrophotometric assay to study benzo[a]pyrene diol epoxide-DNA adducts. Carcinogenesis 6, 1109-1115. (13) Ringe, D., Turesky, R. J., Skipper, P. L., and Tannenbaum, S. R. (1988) Structure of the single stable hemoglobin adduct formed by 4-aminobiphenyl in vivo. Chem. Res. Toricol. 1, 22-24. (14) Green, L. C., Skipper, P. L., Turesky, R. J., Bryant, M. S., and Tannenbaum, S. R. (1984) In vivo dosimetry of 4-aminobiphenyl in rats via a cysteine adduct in hemoglobin. Cancer Res. 44, 4254-4259.
Paul L. Skipper, Stephen Naylor, Liang-Shang Gan Billy W. Day, Roberta Pastorelli Steven R. Tannenbaum* Room 56-309, Department of Chemistry Division of Toxicology, Whitaker College Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Received July 18, 1989
