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Articles Characterization of Benzo[a]pyrene anti-Diol Epoxide Adducts to Human Histones Carlton K. SooHoo,+Kuldip Singh,t Paul L. Skipper,? Steven R. Tannenbaum,*lt*sand Ramachandra R. Dasarill Department of Chemistry, Division of Toxicology, and George R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received August 5, 1993 Nuclei from human lymphoblast cells grown in culture were treated with [7-14C]-(f)-r-7,t-
8-dihydroxy-t-9,t-10-epoxy-7,8,9,l0-tetrahydrobenzo[alpyrene (anti-BPDE), and the nucleosomal core histones were isolated for adduct studies by cryogenic fluorescence line narrowing spectroscopy. The four core histones H2A, H2B, H3, and H4 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which yielded each histone free of contamination by the others. Further purification of histones H2A, H2B, and H4 by reversed-phase HPLC also yielded a tetrahydrotetrol of benzo[alpyrene, indicating that these three histones had some labile adducts. No tetrol was observed upon purification of histone H3.Fluorescence emission spectra of the HPLC-purified histones recorded at 4 K after vibronic excitation into the SIstate were generally similar. Fluorescence line-narrowed spectra of model compounds formed by reaction of anti-BPDE with acetic acid, ethylenediamine, cysteamine, and histidine were also recorded. Only the spectra of the ethylenediamine adduct model matched consistently, at different excitation wavelengths, the spectra of the adducted histones. From this it is concluded that the stable human histone adducts of anti-BPDE are formed by reaction with lysine residues and/or the amino groups of the N-termini.
Introduction The histones are a class of small positively charged DNA binding proteins that are found in the eukaryotic nucleus. Traditionally, a structural role for these proteins has been assigned,but it is now clear that a gene regulatory role can also be shown (I, 2). Due to their intimate contact with DNA, it is reasonable to expect that carcinogens would form adducts to the histone side chains in direct proportion to the amount of DNA adducts formed. In the absence of repair mechanisms for them, histone adducts would be an accurate and specificdosimeter for tissue-specificDNA damage, which is frequently repaired with great efficiency. There have been several studies that have demonstrated adduct formation to this class of small proteins (3-7), including one in which the relationship of histone to DNA adducts formed by metabolites of benzo[alpyrene was examined (7).To the best of our knowledge, however, no direct structural information concerning carcinogenhistone adducts has been reported. Benzo[al pyrene is a ubiquitous carcinogen that has fluorescentproperties which make it convenientfor study. Its 7,8-diol9,10-epoxidemetabolites react with side-chain carboxyl and imidazole groups in hemoglobin and serum *Author to whom correspondence should be addressed at the Massachueette Institute of Technology, Room 56-309,77Massachusetts Ave., Cambridge, MA 02139. t Division of Toxicology, Whitaker College. t Present address: Department of Chemistry, University of Missouri, St. Louis, MO 63121. 1 Department of Chemistry. fl George R. Harrison Spectroscopy Laboratory. Abstract published in Advance ACS Abstracts, January 15, 1994.
albumin (8-13). It has previously been reported that the specificmetabolite r-7,t-&dihydroxy-t-9,t-lO-epoxy-7,8,9,10-tetrahydrobenzo[alpyrene(anti-BPDE)' binds to histidines in rat liver and chicken erythrocyte histones (5). Quantitative analysis of anti-BPDE-protein adducts can be performed conveniently by GC-MS if the adducts are esters, because esters hydrolyze cleanly to yield tetrols. We were thus interested in determining whether the reaction of anti-BPDE with human histones yields ester adducts and whether other, possibly more stable, adducts are formed as well, in which case it would be appropriate to develop methods for analysis other than GC-MS. The approach taken was cryogenic fluorescenceline-narrowing spectroscopy (FLN),which is a highly sensitive technique that has been used previously to identify the atoms involved in the covalent bond formation between antiBPDE and proteins. FLN analysis of purified adducted histones indicated that the predominant interaction of anti-BPDE yielding stable adducts with the protein was through lysine residues or, possibly, the N-terminal amino groups. In the course of purifying the histones, we also found evidence for unstable ester adducts, especially to histone H4.
Materials And Methods Chemicals. Caution: a n t i - B P D E is carcinogenic; all contact with this material should be avoided. Abbreviations: FLN, f l u o " e line narrowing;TFA, trifluoroacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophesis; anti-BPDE, r-7,t-8-dihydroxy-t-9,t-lO-epoxy-7,8,9,lO-tetr~~ drobenzo[alpyrene.
oa93-22a~19412101-0~34~04.5010 o 1994 American Chemical Society
BAP Adducts in Human Histones
Chem. Res. Toxicol., Vol. 7, No. 2, 1994 135
I I\ 4 l Y
500 [7-14C]-anti-B[a]PDE (55mCi/mmol)was purchased from the H2A NCI Chemical Repository maintained by ChemSyn Science 400 * Laboratories (Lenexa, KS). The synthesis of the model com300 pounds S-(7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[alpyren200 10-yl)cysteamine,N-(2-aminoethyl)-N-(7,8,9-trihydroxy-7,8,9,10-0 tetrahydrobenzo[alpyren-10-yl)amine,and N*-(7,8,9-trihyd 100 ; droxy-7,8,9,10-tetrahydrobenzo[a]pyren-l0-yl)histidine has been 0 previously described (9,IO). 7,8,9-Trihydroxy-lO-acetoxy-7,8,9,c 10-tetrahydrobenzo[a]pyrene was prepared by the same procea dure described for synthesis of the propionate ester (10). The zn 800 trans-acetate was isolated by preparative HPLC using a C180 pBondpack column (7.8 mm X 300 mm). 600 Instrumentation. Cryogenic laser-induced fluorescence emis400 . H4 sion spectra were recorded with a system that has been described 200 previously (11). Briefly, the excitation source was a Nd3+/YAG 0 pumped dye laser (Spectra-Physics DCR-ll/PDL 3) with a 0 10 20 30 40 50 60 resolution of
