Anal. Chem. 1992, 84, 3030-3044
a030
Separation and Identification of DNA-Carcinogen Adduct Conformers by Polyacrylamide Gel Electrophoresis with Laser-Induced Fluorescence Detection Glenn A. Marsch, Ryszard Jankowiak, John H. Farhat, and Gerald J. Small’ Ames Laboratory- USDOE and the Department of Chemistry, Iowa State University] Ames, Iowa 50011
We have developed a separation p r o t o d utlklng h b b r e s o i u t k n p d y a ~ g . l ~ ( P A Gt Eo )W e stable a&benz@abyrene dlol epoxide adducts of digodeoxynuckotkh. Both enantiomers producedmultiple adduct species. The dktrlbuth of adduct type8 couM be quantitated by densitometry of autoradiogram or Coronkov counting of eluted o i b o m ~modlfkd ~ by anlcBPDE konnn. L m Induced fluoresconce (LIF) spectra of eluted adducts at 4.2 K (fluorescence ilnanarrowlng 8pectroscopy) and 77 K revealedthat bands to pure”eraol pyrene chromophore. CarcinogmwwdM 0llgod.Oxynuckotkk. were dnglaolranded, but there were often codderabie stacking interadlonr between the pyrenyl rosiduos and the ollgonuckotlde bases, indkathg that .kctrophore8edollaomen were singbstranded but In a natlvo, vemw randomcdl,contormakn. TlncrblltytoId.nWyadquaHtcrt.cldducb by PAGE-LIF, coupled with the high rcsrdutlon and sen8ltiVny d both techniques, makes PAGE and LIF In tandem a p o t m t b l y p o w atool In t h e d u d y o f c h . m l c d c ~ or other Ilgand-ONA Interactions.
INTRODUCTION It is generally believed that chemical carcinogenesis is initiated by covalent binding of the chemical compound or ita metabolites to DNA.lJ Typically,a heterogeneous mixture of adducts is generated for a given carcinogen. For polycyclic aromatic compounds such as benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) it has been established that the two major metabolic pathways which lead to DNA damage are monooxygenation (or diol epoxide)3-6 and one-electron oxidation (or radical Furthermore, a given metabolite may bind to different or different
* To whom correspondence should be addressed.
(1)Miller, E. C.; Miller, J. A. Cancer 1981,47,2327-2345. (2)Miller, J. A. Cancer Res. 1970,30,558-576. (3)Phillip, D. H. Nature 1983,303,468-472. (4)Sime,P.; Grover, P. L. In Polycyclic Hydrocarbons and Cancer; Gelboin, N. V., Ts’o, P.0. P., Eda.;Academic Presa: New York, 1981. (5)Conney, A. H. Cancer Res. 1982,42,4875-4917. (6)Weinstein, I. B.; Jeffrey, A. M.; Jennette, K. W.; Blobstein, S. H.; Harvey, R. G.; Harris, C.; Autrup, H.; Kaaai, H.; Naknnishi, K. Science 1976,193,592-595. (7)Cavalieri, E.;Rogan,E. Enuiron. Health Perspect. 1986,64,69-84. (8)Cavalieri, E.;Rogan, E. In Polycyclic Hydrocarbons and Carcinogenesis;Harvey, R. G.,Ed.;American Chemical Society: Washingtan, DC, 1985. (9)Jeffrey, A. M.; Grzeskowiak, K.; Weinstein, I. B.; Nakanishi, K.; Roller, P.; Harvey, R. G. Science 1979,206,1309-1311. (10)Sage, E.;Hazeltine, W. A. J. Biol. Chem. 1984,259,11098-11102. (11)Devauesan, P. D.; RamaKriihna,N. V. 5.; Tcdorovic, R.; Rogan, E. G.; Cavalieri, E. L.; Jeong, H.; Jankowiak, R.; Small, G. J. Chem. Res. Toxicol. 1992,5,302-309. (12)Ram-, N. V. S.; Gao, F.;Padmavathi, N. S.; Cavalieri, E. L.; Rogan, R. G.; Cerny, R. L.; Gross, M. L. Chem. Res. Toxicol. 1992,5, 293-302. 0w52700/92/038c303~03. W/O
nucleophilic centers of a and exist in distinctly different adduct-DNA conformations. In addition, metab olism may lead to a distribution of stereoisomeric metabolitee and, as a consequence, stereospecific additions to DNA, e.g. cis versus tram additionl‘j-18 of anti-benzo[a]pyrene-7,8dihydrodiol9,lO-epoxides(anti-BPDEs). Complex heterogeneity makes the identification of the type(@)of chemical lesion responsible for tumorigenesis a formidable problem. For example, (+)-anti- and (-)-antiBPDE, whoee structures are given in refs 3-5, etc., produce the same stable chemical adducts and yet the former is significantly more c a r c i n o g e n i ~ . ~ ~ ~Furthermore, J”~~ the enhanced reactivity of the forme#J6@@ cannot entirely account for the disparity in carcinogenic activity. However, a variety of techniques have been usedto show that22*2b32 the distribution of adducts formed by the two diastereomers is significantly different, with (-)-anti-BPDE forming more quaai-intercalatedadductathan (+)-anti-BPDE,whoee major adduct is an external type. For both isomers adduction is primarily at the exocyclic- N H 2 group of guanine.6*13”I’hus, the disparity in carcinogenic activity is probably due to a combination of factors including reactivity and the repair (13)Jeffrey, A. M.;Jennette, K.W.; Blobetein, 5.H.;Weinstein, I. B.; Belaud, F.A.; Harvey, R. G.; Kasal, H.; Miura, I.; Nakaniehi,K. J. Am. Chem. SOC.1976,98,5714-5715. (14)&borne, M. R.;Harvey, R. G.; Brookes, P. Chem. Biol. Interact. 1978,20,123-130. (15)&borne, M. R.;Jacobs, S.; Harvey, R. G.; Brookes, P. Carcinogenesis 1981.2.553-558. (16)Cosmau; C.; Ibanez, V.; Geacintov, N. E.; Harvey, R. G. Carcinogenesis 1990,11,1667-1672. (17)Chew, S.C.; Hilton, B. D.; Roman, J. M.; Dipple, _ _ A. Chem. Res. Toxicol. 1989,2,334-340. (18)Small, G. J.; Jankowiak, R.; Jeong, H.; Marsch, G. A. Polycyclic Aromatic Compounds (in press). (19)Thakker, D. R.;Yagi, H.; Lu, A. Y.H.; Lewin, W.; Conney, A. H.; Jerina, D. M. R o c . Natl. Acad. Sci. U.S.A. 1976,73,3381-3386. (20) Buening, M. K.; Wislocki, P. G.; Lewin,W.; Yagi, H.; Thakker, D. R.; Akagi, H.; Koreeda, M.; Jerina, D. M.; Conney, A. H. R o c . Natl. Acad. Sci. U.S.A. 1978,75,53565361. (21)Slaga, T. J.; Bracken, W. J.; Gleason, G.; Lewin, W.; Yagi, H.; Jerina, D. M.; Conney, A. H. Cancer Res. 1979,39,67-71. (22)Grblund, A.;JernstrBm, B. Quart. Reu. Biophys. 1989,22,1-37. (23)Meehan, T.; Straub, K. Nature 1977,277,410-412. (24)Brookes, P.;Osborne, M. R. Carcinogenesis 1982,3,1223-1226. (25)Jankowiak, R.;Small, G. J. Chem. Res. Toxicol. 1991,4,266-269. (26)Jankowiak,R.;Small, G. J. Anal. Chem. 1989, 61,1023A-1034A. (27)Geacintov,N.E.;Ibanez,V.;Gagliano,A.G.;Jacobe,S.A.;Harvey, R. G. J. Biomol. Struct. Dyn. 1984,1,1473-1484. (28)J e m W m , B.; Lycksell, P.-0.; Grblund, A.; Nordh, B. Carcinogenesis 1984,5,1129-1135. (29) Geacintov, N. E.; Yoehida, H.; Ibanez,V.; Jacobs, S. A.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1984,122,33-39. (30)Zinger, D.; Geacintov, N.E.;Harvey, R. G. Biophys. Chem. 1987, 27,131-138. (31)Erilrseon, M.;NordBn,B.; Jernstrtim, B.; Griialund,A.Biochemistry 1988,7,1213-1221. (32)Kim, S. K.;Geacintov, N. E.; Brenner, H. C.; Harvey, R. G. Carcinogenesis 1989,10,1333-1335. (33)Jeffrey, A. M.; Weinstein, I. B.; Jennette, K. W.; Grzeekowiak, K.; Nakaniehi, K.; Harvey, R. G.; Autrup, H.; Harris, C. Nature 1977,269, 348-360. -. - - - -.
(34)Khg,H.W.;&bome,M.R.;Belaud,F.A.;Harvey,R.G.;Brookea,
P. Proc. Natl. Acad. Sei. U.S.A. 1976,73,2679-2681. (B 1992 A m d c a n
Chemical Socle@
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1002
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spectra under non-line-narrowed and line-narrowed (FLN) conditions is described in detail in refs 25 and 26. We mention only that the excitation source is a Lambda-Physik FL-2002 pulsed dye laeer pumped by a Lambda-Physik EMG 102 MSC excimer (XeCl) laser and that the gateable diode-array and 1-m McPherson scanning monochromator provided a spectral resolution of =4 cm-l at 400 nm. Preparation of Oligodeoxynucleotides and Denaturation Profiles. Two G-containingoligodeoxynucleotide, d(TTAAG GAATT) and d(AATTGGTTAA), and their complementswere synthesized by the phosphotriester method on an Applied Biosystems,Inc. PCR Mate DNA synthesizer at the Iowa State University Nucleic Acids Facility. The central 5'-NGGd' sequences are sites of preferred binding by anti-BPDEe,'W1* and the AT flanks were sites of least preferred covalent binding by the isomers." Oligomers were purified there by revereedphase liquid chromatography on a Vydac C4 column using a linear gradient with acetonitrile and 50 mM triethylemmonium acetate (pH 7.0). Oligomer denaturation curves were obtained with a Hewletb Packard 8452A W/vis spectrophotometer measuring abeorbances at 260 nm. The temperature was increased in 6-deg increments from 5 to 60 "C by a Fisher Scientific Model 9OOO isotemp refrigerated circulator. Duplex oligomers were 300 and 450pM ford(TTAAGGAA'IT).d(AAATM=CTTAA) andd(AAITGGITAA).dVI"ITCCAATT),respctively, in 20mM Na2HPO4, 100 mM NaC1, and 1mM Na2EDTA; pH 7.0. Oligodeoxynucleotide-BPDE Reactions. The appropriate strand was labeled on the 5'-terminue by [y32P]ATPfrom New England Nuclear and T4 polynucleotidekinase from Sigma, Inc. The radiolabeled DNA oligomers were repurified by electrophoresis through 20 % polyacrylamide gels under denaturing conditions (T = 40 O C and 7 M urea). To extract radiolabeled and purified oligomer, autoradiography of the gel was done by Kodak XOMAT-AFt5 X-ray film, and the bands corresponding to whole 10-mer8 were excised. The polyacrylamide fragments imbedded with DNA were placed in 2 mL of doubly dietilled water and eluted out overnight. Eluted DNA oligomers were desalted by Sep-Pak cartridges from Waters Associaias, and the resultingmethanoVwaterelutionswere dried in a Savant SpeedVac concentrator. Oligonucleotides labeled at the 5' terminus with 81p and a 10-fold excess of complement unlabeled by STwere mixed together in a buffer solution composed of 20 mM dibasic phosphate (pH 7.01, 100 mM NaC1, and 1 mM NaaDTA. Oligomer solutions were placed in an 80 OC heating block and slowlycooled to 20 O C over 2 h to induce slow error-freeannealing of the strands. anti-BPDE enantiomerswere addedat a ratio of one carcinogen molecule per 25 oligonucleotidebases. BPDEswere r e a d with DNA oligomers in the dark (to prevent photolysis) for 2 h at 2 O C (d(8lpAATTGGITAA)+ complement) or 20 "C (d(swITAAGGAATT) + complement). BPDEs hydrolyze to benzo[a]pyrenetetrols (BPT), and these were eliminated by five extractions with 1-butanol,being certain not to pellet DNA oligomer. After the last extraction,the reaction mixtures were dried under a vacuum with the SpeedVac concentrator and resuspended in 10pL of gel sample buffercomposed of l/lOX TBE buffer, xylene cyanole FF tracking dye, and 50% glycerol. Separation and Purification of BPDE-Modified DNA Oligomers. DNA oligomer samples were applied to a 50-cm x 80-cm Bio-Rad Sequi-Gen nucleic acids sequencing system EXPERIMENTAL SECTION powered by a Pharmacia ECPS 3000/150 power supply. Gels Laser-Excited Fluorescence Spectroscopy. The apparatus were 20 % acrylamide (19:l acrylamide:bis(acrylamide) w/v with used for obtaining low-temperature laser-excited fluorescence 1X TBE buffer and no urea. Gels polymerized overnight; then preelectrophoresiswas performed, -2 h at 2000 V. The samples (35)Geacintov, N.E.In Polycyclic Aromatic Hydrocarbon Carcinowere then added to the gel and electrophoresed at 1500 V and genesis; Harvey, R. G., Ed.;American Chemical Society, Washington, -15 mA for 12 h; with the temperature of the gel at =25 OC. DC, 1985. (36)Lu,P.;Jeong,H.;Jankowialr,R.;Small,G.J.;Kim,S.K.;Cosman, Autoradiography of gels using many exposure times was done at M.; Geacintov, N. E. Chem. Res. Toricol. 1991,4,59-68. ambient temperature with Kodak XOMAT-AR6 X-ray fii, in (37)Jankowialr, R.;Lu,P.; Small,G. J.; Geacintov, N. E. Chem. Res.
efficiencies of different conformers. We have reported extensively18~26,26~38-38~ on the application of LIF at 77 K and fluorescence line narrowing (FLN) at 4.2 K to the analysisof macromolecular DNA and oligonucleotide damage from BP and DMBA. Germane to this paper are the results which led to the identification of four DNA adducts from both (+)-and (-)-anti-BPDE, which we will refer to as (+)-j and (-1-j = 1-41 in what follows. The non-linenarrowed fluorescence origins of the j = 1-4 adducts are An = 377.5-378.5, 378.5-380.0, 380.0-381.0, and =382.0 nm, respectively. For both isomers the first three of these arise from binding to the exocyclicNH2 group of guanine. The j = 4 adducts were ascribed to binding at the NLNH2 position of adenine.% Three diagnostic approaches were used to assign the j = 1-3 adducts to external, base-stacked, and quasiintercalated conformations in double-stranded DNA and polynu~leotides.'~~2~~~%~3' It was found, for example, that fluorescence quenching by acrylamide decreased with increasing X(o,o,a (or j ) and that the linear electron-phonon coupling strength ranged from weak to strong in going from j = 1to j = 3. The strong coupling for the j = 3 adducts, which is consistent with a quasi-intercalated conformation for which the base-fluorophore interaction endows the fluorescent statewith charge-transfer character, led to barely discernible zero-phonon lines (ZPL) in the FLN and linenarrowed fluorescenceexcitation spectra at 4.2 K. In contrast, pronounced ZPL with weak phonon sidebands were observed for the j = 1 adducts. To improve our understanding of the initial phases of chemical carcinogenesis, it will be important to continue to develop new bioanalytical methodologies which have improved separation and/or spectral selectivity characteristics. To this end, we have been exploring the utility of polyacrylamide gel electrophoresis(PAGE)-laser induced fluorescence (LIF) in the detailed study of BPDE-oligodeoxynucleotide conformers. High-pressure liquid chromatographic (HPLC) methodologies have recently been used to separate singlestranded (8s) oligodeoxynucleotides modified by carcinogens.16*sBRill and Vealqofound that 20% polyacrylamidegels under denaturing conditions provide extremely high resolution and sensitivity as a probe for ligand-DNA reactions. The longer experimental run times for PAGE (compared to HPLC) are offset by the capability to electrophorese many samples simultaneously. Thus, the availability of DNA sequencing equipment at relatively low expense, combined with the high-resolution PAGE provides, gave the impetus for using PAGE, rather than HPLC methodologies, to isolate pure carcinogen-modified oligomers. Polyacrylamide gel electrophoresishas long been used to separate DNA molecules according to size (since the charge-to-mass ratio is essentially the same for all unmodified DNA molecules), but this is the first time, to our knowledge, that DNA molecules modified by carcinogen are separated according to conformation of fluorophore-oligomercomplexby PAGE, and unambiguously identified by LIF.
Toxicol. 19W,3!39-46. (41)Rill,R.L.;Marsch, G. A. Biochemistry l990,29,606(w058. (38)Ra"hua,N.V.S.;Devaneaan,P.D.;Rogan,E.G.;Cavalieri, (42)Lobanenkov, V. V.; Plumb, M.; Goodwin, G. H.; Grover, P. L. E. L.; Jeong, H.; Jankowiak, R.;Small, G. J. Chem. Res. Toricol. 1992, Carcinogenesis 1986,7 , 1689-1695. 5,220-226. (43)Marsch, G.A. Ph.D. Dissertation,The Florida State University, (39)Huang, G.;Krugh, T. R. A d . Biochem. 1990,190,21-25. 1990. (40)Veal,J. M.; Rill,R. L. Biochemistry 1989,28,3243-3250.
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ANALYTICAL CHEMISTRY, VOL.
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1.4
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T (OC) Denaturation curves of d(lTAAGGAAlT)d(AAlTCCTTAA) and d(AATTGGlTAA)d(TTAACCAATT) ( 0 ) in 20 mM dibasic phosphate, 100 mM NaCI, and 1 mM Na2EDTA;pH 7.0. Absorbances at 260 nm are all plotted with respect to Am at 5 "C (A,,). The leftand righthand ordinates refer to the metting of d(TTAAGGAAlT)=d(AATTCCTTAA) and d(AATTGGlTAA)d(lTAACCAAlT), respectively. F w e 1.
(X)
order to locate the BPDE-modified oligomers. Quantitation of BPDE adducts was by densitometry of autoradiograms with an SL-504-XL scanning densitometer from Biomed Instruments, Inc. or by scintillation counting of eluted radiolabeled BPDEoligonucleotide covalent complexes with a Beta-Trac _liquid scintillation counter from Tm Analytic. Bands corresponding to carcinogen-modified and unmodified oligomers were excised from the gel and placed in Eppendorf tubes with 1 mL of glass-distilled H2O. Oligomers eluted overnight at 4 O C . Sampleswere centrifuged,and the supernatant was dried down under a vacuum. Samples were resuspended in a standard glass, composed of 50% glycerol and 50 % water (v/v), commonly used for low-temperature spectroscopy. Safety Considerations. The preparation and electrophoresis of BPDE-modified DNA oligomers involves using carcinogens and ionizing radiation in liquid form. For both carcinogens and radiolabel, contamination must be eliminated by wearing latex gloves, laboratory coats, and splash-proof goggles. Hazardous materials should be sequestered from other areas of the laboratory. Radiolabel exposure is diminished by working with Plexiglas shielding and the proper dosimeters (Geiger counters and body dosimeters). Wastes must be placed only in approved waste containers and disposed of by the proper Environmental Safety and Health personnel.
RESULTS AND DISCUSSION OligonucleotideDenaturationCurves. Melting curves for the two duplex DNA oligomers used in this study are shown in Figure 1. The oligomers d(AATTGGTTAA)* d(TTAACCAATT) and d(TTAAGGAATT)=d(AATTCCTTAA) exhibited hyperchromicities of =10 % and 37 % , respectively, with corresponding melting temperatures of =22 and =35 "C. The large differences in denaturation temperature and degree of hyperchromicity between two duplexes of identical length and base composition (80% A-T and 20 % G-C) underscores the strong effect of sequence on T,. Electrophoresisof BPDEModified Oligonucleotides. Electrophoresis of single- and double-stranded oligomers (Figure 2) through 20 % gels was conducted under conditions usually considered nondenaturing (T=25 "C, no urea). Under the conditions of single-hit kinetics employed in the carcinogen-oligonucleotidereactions, most oligonucleotidespossess no BPDE adduct. Only the bands corresponding to these unreacted molecules are shown in Figure 2, in order to ascertain whether oligonucleotide duplexes with no adduct denature during electrophoresis under the above conditions. The ss oligodeoxynucleotide d(32pTTAACCAATT) (lane 1) possesses a faster electrophoretic mobility than ita complement, d(32pAA'M'GGTI'AA) (lane 3). d(32pAATI'GGTTAA) initially annealed to its complement (kept a t 2 "C prior to electrophoresis) comigrated with single-stranded d(32pAATTGGTI'AA), suggesting that under the electrophoresis conditions employed in this study, the duplex denatured into
Flgure 2. Autoradiogram (5-min exposure, no Intensifying screen) showing the migration of d(3pAATTGGTTAA) and q3,TTAACcAArr) mdeculesodginaityin duplex or In singhtranded form. Electrophoresis was performed under classically "native" conditions: Tof gel =25 O C and no urea. The anode is toward the bottom of the figure. Only the section of the gel containing DNA oligomers not covalently bound by carcinogen is shown. Lanes 1 and 3 show the electrophoresis of single-strandedd(3pTTAACCAAlT) and d(3pAAT'fGGlTAA),respectively, and lane 2 showsthe electrophoresisof botholigomersannealed to one another prior to electrophoresis. Lanes 4, 6, and 8 show the migration of oligomers Originally in duplex form and bound by (-)-antiBPDE, (+)-antkBPDE, and (-)-cIs-BPT, respectively, wfth the 32P radiolabel on theGcontaining strand. Lanes 5 and 7 depict themlgratbn of oligomers bound by (-)-antbBPDE and (+FantCBPDE, respectively, with the label on the C-containing strand. The nomenclature 1'3plTGGTT" and "3pAACCAA" on Figures 2 and 3 refer to d(3pAATTGGTTAA) and d(3plTAACCAAlT), respectively.
single strands (lane 2). Lanes 4-8 depict the electrophoresis of oligonucleotidesoriginally in duplex form (2' = 2 "C) and modified by (-)-anti-BPDE, (+)-anti-BPDE, or (-)-cis-BPT. Only the bands corresponding to oligomer unmodified by BPDE are shown. In all cases the oligonucleotidesthat were originally in duplex form and labeled on the 5' terminus of one strand by 32Pcomigrated with whichever single strand was labeled (G-containing strand, lanes 4,6, and 8, compare with lane 3; C-containing strand, lanes 5 and 7,compare with lane 1). However, if the duplexes remained intact during electrophoresis,the migration of these duplex oligonucleotides would be a t the same rate regardlessof which strand possessed the 32Pradionuclide. Therefore, under the electrophoresis conditions used in this study, oligomer with no adduct will not assume a duplex conformation. Since the Tm of d(AATTGGTI'AA)-d(TTAACCAATT) is higher than the Tm of d(TTAAGGAATT)=d(AATTCCTTAA) (Figure l), the electrophoresis of the latter duplex will also denature the strands. A much longer exposure of the 20 % polyacrylamide gel of Figure 2 revealed the formation of stable adducts by antiBPDE enantiomers (Figure 3). The figure illustrates the resolutionpossible with the electrophoresisof BPDEmodified oligomers through highly polymerized acrylamide gels. There were severalbands correspondingto adducted DNA oligomers, and these all migrated slower than oligonucleotides with no adduct. There was discrimination between adduct species, with (-)-anti-BPDE forming one major and one minor adduct (lane 1)and (+)-anti-BPDE forming two slowly migrating major adducts and several faster migrating minor adducts (lane 3). Densitometry of autoradiogram lanes 1and 3 (not shown) indicates that
