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(CANCER RESEARCH 43, 1851-1860, April 1983]0008-5472/83/0043-OOOOS02.00
Covalent Complexes of DMA and Two Stereoisomers of Benzo(a)pyrene 7,8-Dihydrodiol-9,10-epoxide Studied by Fluorescence and Linear Dichroism1
Ola Undeman, Per-Olof Lycksell, Astrid Gräslund,TorbjörnAstlind, Anders Ehrenberg, Bengt Jernström,Folke Tjerneld2, and Bengt Norden
Department ol Biophysics, University of Stockholm. ArrhenìusLaboratory, S-106 91 Stockholm [O. U., P-O. L, A G.. T. A., A. E.¡:Department of Forensic Medicine,Karolinska Institutet, Box 60400, S-104 01 Stockholm ¡BJ.]: and Department of Physical Chemistry, Chalmers University of Technology, S-41296 Göteborg¡F.T.,
B. N.¡,Sweden
ABSTRACT
Fluorescence and absorption spectroscopy and linear dichro-
ism have been used to study the covalent adducts of calf thymusDNA with the two Stereoisomers of benzo(a)pyrene 7,8-dihydro-diol-9,10-oxide, (±)-7£i,8a-dihydroxy-9tt,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anf/-BPDE; a strong carcinogen)and (±)-7/3,8a-dihydroxy-9j3,10/i-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene (syn-BPDE; a weak carcinogen). Each stereoiso-
mer gives rise to two types of complexes, I and II, with characteristic spectral properties: type I with light absorption and fluorescence excitation maxima at 322, 337, and 354 nm; and typeII with the corresponding maxima at 316, 330, and 345 nm. Inanf/-BPDE-DNA, the type II component dominates and the typeI component amounts to <10%. In syn-BPDE-DNA, approxi
mately 35%, of the adduci is of type II and approximately 65%of type I. From linear dichroism, it is concluded that the type IIcomponent of a/if/'-BPDE-DNA has the plane of the chromophore
molecule nearly parallel to the helix axis. The type I componentof syn-BPDE-DNA has a very different geometry with the chromophore probably intercalated between the DNA bases. This isalso in accord with the large wavelength shift of the light absorption and the weak quenching of the fluorescence by 02 for thetype I complex. The properties of complex type II of anf/-BPDE-DNA are in agreement with a wedge-type geometry at the bindingsite. The Stern-Volmer quenching curves are bent, and the
fluorescence decays are not monoexponential, which demonstrates that there is heterogeneity in the microenvironment ofthe chromophore. From the dynamic quenching constants with02, it is found that different subcategories of the chromophoreare differently exposed to the medium. Addition of Ag+ to anti-
BPDE-DNA (type II complexes) leads to increased fluorescence
and longer decay times. The AgNnduced effects are probablydue to a conformational change of DNA when Ag+ is bound,
causing the anti-BPDE adducts to interact less strongly with theDNA. Component type I of syn-BPDE-DNA is not affected byAg+ in a similar way. Instead, a weak quenching is observed.
Upon denaturation, both anti- and syn-BPDE-DNA give a typeof single-stranded complex with light absorption and fluorescence excitation maxima at 316, 332, and 351 nm. The chro-
mophores are probably sandwiched between the DNA bases.
INTRODUCTION
BP,3 a ubiquitous environmental contaminant, is a potent
mutagen and carcinogen in various experimental systems and isalso believed to be involved in the etiology of certain humantumors (14, 40).
BP is not carcinogenic per se but must undergo biologicaltransformation to specific electrophilic intermediates (11) thatsubsequently interact with cellular constituents, such as DNA(45).
Of particular importance for BP carcinogenesis is the formationof BP 7,8-oxide, the epoxide hydrolase catalyzed hydrolysis tofrans-7,8-dihydroxy-7,8-dihydro-BP and the subsequent activation to syn and anti isomers of f/-ans-7,8-dihydroxy-9,10-epoxy-
7,8,9,10-tetrahydro-BP (11, 45). Both isomers bind to DNA andexhibit biological activity. The anti isomer and in particular its (+)-
enantiomer has, however, been shown to be the most activeform(1, 17,39,41,46).
The (+)-anf/-BPDE has been shown to preferentially bind tonative DNA when incubated with racemic anti-BPDE (22). The
selectivity is abolished when native DNA is replaced by denaturedDNA (22, 27). Accordingly, the structure and conformations ofDNA and the stereochemistry of the reacting electrophile seemto be factors of fundamental importance in determining the finalpattern of BP intermediates bound to DNA, and of their abilitiesto undergo biochemical repair reactions (30).
Several nucleophilic sites in DNA have been shown to reactwith BPDE (21, 28, 31). Quantitatively, the major binding site isthe 2-amino group of guanine, which reacts with BPDE throughitsC-10 position (45).
The structure of the DNA carcinogen complex(es) seems tobe crucial for the carcinogenic effect, and structural featureshave therefore been studied widely, with varying results (5, 7-9,15,19,20,24,34-37,42,46,47). Some studies have suggested
that the BPDE molecule binds on the outside of the DNA helixwith the bulky pyrene moiety in the minor groove of the DNA (7-
9, 24, 34, 35). Others have proposed an intercalated complex,e.g., Drinkwater ef al. (5), who based their proposal on theunwinding of supercoiled SV40 DNA modified with BPDE. Mee-
han and Sträub(27) argued that the specific binding of BPDE to
1This work was supported by grants from the Swedish Cancer Society, the
Swedish Council for Coordination and Planning of Research, the Swedish NaturalScience Research Council. NIH (Grant 1RC1CA26261 -01 ), and the Magn. BergvallsStiftelse.
2 Present address: Department of Biochemistry, Kemicentrum, University ofLund, S-220 07 Lund, Sweden.
Received May 12, 1982; accepted December 29, 1982.
3The abbreviations used are: BP. benzo(a)pyrene; BP 7,8-oxide. benzo(a)pyrene7.8-oxide; frans-7,8-dihydroxy-7.8-dihydro-BP. frans-7,8-dihydroxy-7,S-dihydro-benzo(a)pyrene; (rans-7.8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-BP. trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene; ant/-BPDE, (±)-7,i,8n-dihydroxy-9a,10<>-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene; BPDE, BP-diol-epoxide.7,8-dihydroxy-9,10-epoxy-7.8,9,10-tetrahydrobenzo(a)pyrene; syn-BPDE, (±)-7¿,8<i-dihydroxy-9,i,10.i-epoxy-7,8.9,10-tetrahydrobenzo(a)pyrene;LD. linear dichroism: BPT. (±)-7¿,8<i.9..,10¿-tetrahydroxy-7.8.9.10-tetrahydro-
benzo(a)pyrene: CD. circular dichroism.
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O. Undeman et al.
DNA could reflect a specific interaction, as would be expectedfor intercalation. A wedge-shaped intercalated carcinogen DNA
complex has been proposed by Hogan ef al. (15) in a study usingDNA fragments of defined length. The heterogeneity of thebinding has been stressed in studies using normal DNA, anddifferent classes of bound BPDE have been characterized (15,20, 42).
In this investigation, we have used physical techniques tostudy the complexes formed when DNA reacted with eitherracemic anf;-BPDE or racemic syn-BPDE, a diastereomer of anti-BPDE. The 2 diastereomeric BP diol-epoxides are depicted in
Chart 1. In spite of the structural similarity between the 2 BPDEisomers, the syn-BPDE isomer is a weak carcinogen compared
to the anti isomer (2). The aim of this study was to elucidate apossible structural difference between the DNA complexes ofthese 2 BP derivatives.
The studies reported here include absorbance and fluorescence spectroscopic measurements on the native and denaturedforms of BPDE-modified calf thymus DNA, as well as LD meas
urements on the native modified DNA oriented by flow. Fluorescence quenching agents have been used to test the accessibilityof the DNA-bound chromophores. We also report results from
fluorescence decay measurements.
MATERIALS AND METHODS
Materials. DNA (Sigma calf thymus type I) was purchased from SigmaChemical Co. (St. Louis, Mo.) and used without further purification, anti-BPDE and syn-BPDE were obtained through the Cancer Research
Program, Division of Cancer Cause and Prevention, National CancerInstitute (Bethesda, Md.). The purity of the diol-epoxides was checkedby thin-layer chromatography (tetrahydrofuran:hexan:methanol:tri-ethylamine, 75:25:5:1; 0.2-mm silica gel prespotted with triethylamine,
RI 0.59). No fluorescent impurities were detected. BPT was prepared byhydrolysis of anf/-BPDE and separation of the products by high-pressure
liquid chromatography. All other chemicals were of analytical grade.Preparation of BPDE-reacted DNA. The BPDE-DNA adducts were
prepared by direct addition of either racemic anf/-BPDE or racemic syn-
BPDE dissolved in a small amount of dimethylsulfoxide to an aqueousDNA solution and incubation at 37°for 30 min. One ml of the reaction
mixture contained 1 mg of DNA, 200 nmol of BPDE, 25 n\ of dimethylsulfoxide, and 10 ¿Triolof sodium cacodylate (pH 7.0 adjusted withHN03). DNA samples without the BP derivatives were always preparedin parallel.
In order to remove noncovalently bound hydrocarbons, the reactedDNA solutions were extracted 7 to 8 times with 4 volumes of ethylacetate previously equilibrated with cacodylate buffer. The doublestrandedness of the DNA samples was checked by measuring thehyperchromicity of the DNA denatured with formaldehyde and heat.Samples with an hyperchromicity lower than 40% were discarded (13).DNA concentrations in nucleotide equivalents were determined by the
OH
syn- onti-BPDE
Chart 1. Structure of syn-BPDE (syn-) and an(/-BPDE. Bipolar arrows, approximate orientation of the transition moments of the L, <—A (approximately 345 nm)and L0 «—A (370 nm) transitions.
UV absorption using <260nm=6600 w~1cm~1. The extent of modification,based on light absorption at 345 or 353 nm using e = 29,000 M"1 cm"1,
was approximately 1 BPDE residue per 250 DNA bases (37) for bothanti- and syn-BPDE adducts used in the present study.
In some preparations, the DNA was precipitated with NaCI-saturatedethanol and stored at -20°.
Denatured DNA samples were prepared by heating for 5 min to 100°
and rapid cooling in ice water. To prevent reassociation when cooling,formaldehyde was added to 1%. After the heating procedure, it wasnecessary to make 1 or 2 extractions with ethyl acetate to removeadducts which were released during heating.
Spectroscopic Measurements. All spectroscopic measurementswere made on BPDE-DNA in 10 ITIMsodium cacodylate buffer adjustedwith HNO3 to pH 7.0 at 20°.
Absorption spectra were recorded on a Cary 219 spectrophotometer.The bandwidth used was 1 nm. The fluorescence excitation spectrawere recorded on a Shimadzu RF510 spectrofluorometer equipped witha quantum counting compensator. The bandwidth used was 3 nm. Thefluorescence quenching studies were carried out on a spectrofluorometerdescribed elsewhere (18) but now equipped with a Jobin-Yvon double
monochromator on the excitation side. The excitation bandwidth usedwas 2 nm, with the emission monochromator set at 420 and 40 nmspectral bandwidth. All quenching studies were performed at 20 ±1°.
The fluorescence quencher AgNO3 (Merck & Co., Inc.) was added inaliquots to 1.5 ml DNA solution. The solution was bubbled with nitrogengas saturated with water (O2 concentration, <10 ppm) for 15 min priorto each measurement.
The fluorescence quenching by oxygen was measured in a pressurizedcell, as described by Lakowicz ef a/. (23). The solution was slowlybubbled with the oxygen at the desired pressure to speed up theequilibrium. Oxygen concentrations were calculated using Henry's law,
and the oxygen solubility was 1.4 mw per atm of oxygen pressure at20°(23).
Appropriate background spectra, recorded with unmodified DNA,were always subtracted point by point in a computer from the samplefluorescence spectra.
LD of Flow-oriented BPDE-DNA. The LD technique makes it possible
to measure the orientation of transition dipole moments of orientedchromophores. Using polarized light, the absorbance of light-polarized
parallel A, or perpendicular A±to an orientation direction is measuredand compared by forming LD = A ¡—A±.
In this study, the DNA molecules were oriented by flow in a Couettecell. The DNA solution is introduced into the narrow space (0.5 mm)between 2 concentric quartz cylinders. By rotating the inner cylinder, avelocity gradient is generated in the solution. Hence, the DNA moleculesare affected by shear forces which will tend to stretch and align the DNAmolecules along the flow direction. The LD measurements were madealong a radial direction through the cylinders with A ( parallel to the flowdirection (43).
DNA is a flexible chain molecule. It has been shown by statisticaltheory that short segments of the long DNA molecule are oriented asuniaxial particles in the flow field (29). The flow dichroism can be analyzedby the equation
— = - (3 cos2«- 1)-S (A)A 2
where A is the absorbance of the isotropie sample (no flow), «is theangle which the transition dipole moment forms with the local helix axisof the DNA segment, and S is an orientation function. For perfectorientation with all DNA segments parallel to the flow direction, S = 1;for an isotropie sample, S = 0. The reduced LD, LD/A, factorizes into anoptical part % (3 cos2«- 1), depending only on the orientation of the
chromophores relative to the local DNA helix axis, and S, which relatesonly to the properties of the DNA in the flow field.
If n absorbing chromophores with different absorbance A¡(\) andtransition dipole moments forming angles »,with the DNA helix axis arepresent, then Equation A becomes
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Fluorescence and Linear Dichroism of BPDE-DNA
. m = YA (> 2 AA, (X)
2A (A)(3 cos2«,- 1)-S (B)
The estimation of S is necessary for the calculation of the «,'s.From
the Watson-Crick picture of the DNA helix, the transition dipole momentsof the DNA bases serve as an inner standard for «= 90°, since theabsorption around 260 nm corresponds to JT%—*•transitions with their
transition dipole moments in the base planes.The LD spectra were measured on a Jasco J500 CD spectrometer
according to the method of Davidsson and Norden (4). The bandwidthused was 2 nm. The factor S was estimated for each sample by usingEquation A with «= 90°and the reduced LD around 260 nm. The sheargradient in the solution was 2050 sec"1. To obtain final spectra, proper
backgrounds were subtracted.Fluorescence Lifetime Measurements. The fluorescence lifetime
measurements were performed using a single photon-counting instru
ment constructed at the Biophysics Department. The instrument is setup in a 90° optical configuration and contains as essential optical
components a free-running flash lamp (N2 at 1 atm) monitored by a 1P28photomultiplier tube, a monochromator (Jobin-Yvon H-20), a Gian prismpolarizer, and a 1-cm square cuvet. The fluorescence photons wereanalyzed with a film polarizer (Polarex KS-W78), an interference filter
(Schott Optical Glass, Inc., Duryea, Pa.), a cutoff filter (Schott) and aphotomultiplier tube (RCA 8850. RCA Electro-Optics, Lancaster, Pa.).For the time measurements, a time-to-amplitude converter (Ortec TAC
457; Ortec, Inc., Oak Ridge, Tenn.) was used. The reference time pathconsisted of a discriminator (Ortec 436) and a delay (Ortec 425A). Thefluorescence time path consisted of an amplifier (Ortec 454) and adiscriminator (Ortec 473A). The single-photon condition was assured bytaking the pulse appearing on the last dynode of the RCA 8850 photo-multiplier through 2 amplifiers (Ortec 113 and 471) and a single-channelanalyzer (Ortec 551 ) gating the output of the time-to-amplitude converter.
A Nuclear Data ND 4410 (Nuclear Data, Inc., Schaumburg, III.) was usedfor data acquisition and spectrometer control. All of the decay curveswere recorded at 20°. The possible artifacts due to fluorescence ani-
sotropy were checked and found to be negligible (38). The fluorescencedecay of the DNA-bound chromophore was obtained after correction for
the background emission from DNA. The decay of a DNA blank samplewas recorded with a measuring time about 4 times longer than for theBPDE-DNA sample, divided by the ratio of the measuring times, andsubtracted channel by channel from the BPDE-DNA decay.
The numerical analysis of the decay curves was performed on a DEC-10 computer, using a nonlinear least-squares fitting procedure. The
mathematical model used to describe the fluorescence decay was a sumof exponential terms in which each term corresponds to a population ofDNA-bound chromophores with a similar fluorescence lifetime. Since theshape of the true response function ("lamp pulse") of the fluorescence
decay instrument is dependent on the wavelength (44), we have usedPOPOP in benzene (decay time, 1.25 nsec) for the generation of theresponse function according to the method of Wahl ef al. (44). The modelwas convoluted with the response function thus calculated, and thenonlinear parameters in the model were estimated in the "least-squares"
sense with a Marquardt algorithm (26), modified according to the methodof Grinvald and Steinberg (12). The goodness of the fit between theexperimental and theoretical decays was evaluated by inspection of theweighed residuals and the reduced x2 value. Confidence intervals were
calculated separately for each parameter without considering the correlation among the parameters. When 2 parameters correlate strongly,their calculated confidence intervals will be underestimated (12).
RESULTS
Stability of Carcinogen DNA Adducts. DNA modified mainlyat the exocyclic amino group of guanine was prepared by reaction with either racemic (±)anf/-BPDE or the diastereomer (±)syn-BPDE.
When BPDE is reacted with DNA, in addition to the covalentBPDE-DNA complexes, the BP tetrol BPT is formed (16). BPT
binds noncovalently to DNA. The intercalative binding is accompanied by a 10-nm shift toward longer wavelengths in the light
absorption of BPT (16). The DNA samples were extracted severaltimes with ethyl acetate to remove noncovalently bound hydrocarbons.
The unbound BPDE derivatives were readily removed fromthe reaction mixture of anf/'-BPDE and DNA. The DNA adducts
of anf;-BPDE thus prepared were apparently stable for 7 to 8 hr
at room temperature.The DNA adducts of syn-BPDE were much less stable. About
1% of the adducts were released during 1 hr at room temperature. Exposure to UV increased the release of adducts. Thereleased BP derivative showed a BPT-like spectrum.
For the subsequent studies, we used freshly extracted samples and kept the UV illumination of the samples at a minimum,e.g., by using small spectral bandwidths in the spectroscopicmeasurements.
Absorption Spectroscopic Measurements. The absorptionspectra of anf/'-BPDE-DNA and syn-BPDE-DNA are presented in
Chart 2a. The absorption spectrum of anf/-BPDE-DNA (full line)
is similar to the spectrum of BPT in water (16) except for a slightshift of 2 nm toward longer wavelengths and a spectral broadening by approximately a factor of 2 in apparent line width.
The syn-BPDE-DNA absorption spectrum in Chart 2a ( )
may be described as composed of 2 spectral components. Thespectral properties of these 2 components correspond approximately to the BPT spectra obtained from 2 types of physicalbinding Sites I and II in DNA (16). We have adopted the samenomenclature for the 2 syn-BPDE components. One component(I) is a BPT-like spectrum shifted 11 nm toward longer wavelengths, while the other component (II) is very much like the anti-
-0.1
-0 05
0.08
004
0002
-0.002
250 300 350Wavelength(nm)
Chart 2. Light absorption and LD spectra of native anf/-BPDE-DNA ( ) andnative syn-BPDE-DNA ( ). A, absorption spectra; oars, peaks of ComponentsI and II, described in the text. B, LD spectra. C, reduced LD, LD/A. When 2 ordinatescales are indicated, the left and right scales refer to the short- and long-wavelengthparts of the spectra, respectively. All spectra are normalized to 1 cm path length.The samples were prepared in 10 mM sodium cacodylate buffer, pH 7.0, withAJeSm= 3.5 and approximately 1 BPDE residue per 250 DNA bases.
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BPDE-DNA spectrum. Resolution and sensitivity do not permitdetection of possible minor components.
Visual inspection of the 2 spectra suggests that the anf;'-
BPDE-DNA spectrum consists of a fairly pure type II component,whereas the syn-BPDE-DNA spectrum is a mixture of the 2components, with type I as the major one. A quantitative estimation of the relative contributions of the 2 components in thesespectra was made in the following way. First, we observed thatthe shapes of the spectra of anti- and syn-BPDE-DNA on thelong-wavelength side are nearly identical. Hence, as a first approximation, the recorded anf/'-BPDE-DNA spectrum is taken as
representative of pure Component II, and the same spectrumshifted 9 nm toward longer wavelengths as representative ofComponent I. Using the absorbances of syn-BPDE-DNA at 345
and 354 nm, it is easy to calculate that this spectrum is composedof 64% type I and 36% type II components. As the next approximation, we assumed that the recorded anf/-BPDE-DNA spec
trum contained a 10% contribution of the type I spectrum andcorrected the 2 component spectra accordingly. With the newpair of type I and type II spectra, the composition of the syn-BPDE-DNA spectrum was again calculated and found to be thesame as given above. It was observed for the new correctedtype II spectrum that the slope on the long-wavelength side was
significantly altered, showing that it was not realistic to try withlarger contributions of Component I in the anf/'-BPDE-DNA spec
trum. The relative contributions of the 2 spectral componentsshould correspond to concentrations of components if theirextinction coefficients are identical.
Fluorescence Excitation Spectra. The fluorescence excitation spectra of the BPDE-DNA samples are shown in Chart 3.
The excitation spectrum of anf/'-BPDE-DNA resembles the
absorption spectrum closely. On the other hand, the excitationspectrum of syn-BPDE-DNA is significantly different from theabsorption spectrum. This is evidence of at least 2 species ofchromophores with different quantum yields.
To explain the properties of the absorption and excitationspectra of the syn-BPDE-DNA complex, we therefore will de
scribe them as the sum of 2 spectral components (correspondingto the components discussed in the absorption spectroscopysection above): spectrum type I (resembling the spectrum ofBPT in water but broadened and shifted 11 nm toward longerwavelengths) with peaks at 322,337, and 354 nm, and spectrumtype II of similar shape but with peaks at 316, 330, and 345.5
320 360Wavelength (nm)
Chart 3. Fluorescence excitation spectra of native anf/-BPDE-DNA ( ) andnative syn-BPDE-DNA ( ). The samples were prepared in 10 mm sodiumcacodylate buffer, pH 7.0, with AJSSm= 0.66 and approximately 1 BPDE residueper 250 DMA bases. The excitation spectra were recorded with a spectral bandwidth of 3 nm and with the emission monochromator set at 420 and 40 nm spectralbandwidth. Bars, peaks of Components I and II, described in the text.
nm. The peaks of the 2 components are indicated by oars abovethe syn-BPDE-DNA absorption spectrum in Chart 2A and the
excitation spectra in Chart 3.LD Measurements. The LD spectra, LD = A „- A±,of the
BPDE-DNA samples are shown in Chart 2ßtogether with the
reduced LD, LD/A, in Chart 2C.The positive sign of the LD at 345 nm of the anf/-BPDE-DNA
indicates that, for the majority of chromophores, the angle between the DNA helix axis and the transition dipole moment ofthe chromophore is less than 55°.With the simple first assump
tion that there is only one chromophoric species, it is possible toestimate the binding angle. S in Equation A is first obtained fromthe LD at 258 nm (S = 0.043). Together with the LD/A at 345
nm (corresponding to maximum optical absorption), this givesthe value a = 35°.This is an upper limit, since minor chromo
phoric components (see below), whether unoriented or with a >35°,will tend to give an apparently too large angle.
In the case of syn-BPDE-DNA, the negative LD indicates abinding angle which is larger than 55°.A calculation under thesame assumptions as for anf;'-BPDE-DNA but at 354 nm (also at
the maximum optical absorption) gives a transition dipole moment angle of 65°relative to the DNA helix axis as a lower limit.
In Chart 2C, it is seen that, for both anti- and syn-BPDE-DNA,
the reduced LD spectra are not constant over the entire absorption band assigned to a single transition. This observation deserves further attention. In reduced LD spectra, a single transition, which is either ordered uniquely or has several ais, shouldgive a constant value LD/A over the whole absorption spectralrange of the transition. In the case of components with differentabsorbances (A,)but with the same orientation angle (a), we alsoexpect a constant LD/A over the total spectral range. All othercases (e.g., 2 spectral components with different A, and differenta/) will give a nonconstant reduced LD (8). In our case, thepresence of an additional minor species with different LD properties would be expected to show up in the total reduced LDspectrum as peaks or troughs approximately at the peaks of itsabsorption spectrum.
The reduced LD spectrum of anf/-BPDE-DNA has troughs
overlaying its positive constant LD component at about 335 and360 nm, close to the absorption peaks of Component I. Thereduced LD spectrum of syn-BPDE-DNA has peaks overlaying
its negative constant LD component at about 314, 327, and 346nm, which coincide with the absorption peaks of Component II.
Taken together with the absorption and fluorescence spectraldata, the reduced LD spectra of both anti- and syn-BPDE-DNA
are consistent with the 2 types of Spectra I and II in differentproportions. The anf/-BPDE-DNA is dominated by Component II,which is nearly parallel with the helix axis («< 35°),whereas the
syn-BPDE-DNA has Component I as its major part, which hasits plane more perpendicular to the helix axis (a > 65°).
Denaturation of BPDE-DNA With Heat and Formaldehyde.In denatured DNA, the anf/-BPDE-DNA adduci was more stablethan was the syn-BPDE-DNA adduci.
The resulting absorbance and fluorescence excilalion speclrafrom ani/'-BPDE-DNA are shown in Chart 4. Bolh are similarly
shifted toward longer wavelenglhs and have peaks al 316, 334,and 351 nm (speclrum lype III). The inlensily of Ihe fluorescencespeclrum increased by a factor of 2.3 lo 2.6 compared lo Iheundenalured sample for bolh Ihe anf;'- and syn-BPDE-DNA com
plexes.
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Fluorescence and Linear Dichroism of BPDE-DNA
004 -
S 0.02 -
-_
320 360Wavelength ( nm )
Chart 4. Absorption and fluorescence excitation spectra of denatured anti-BPDE-DNA. The spectra were recorded as described in legends to Charts 2 and3, respectively.
Titrations With Ag*. Ag+ ions at [Ag+]:[DNA phosphate] ratios
below 0.5 bind preferentially to the DNA bases and have beenused as a probe (internal quencher of fluorescence) for chromo-
phores which form classical ¡ntercalativecomplexes with DNA(10). A conformational change of the DNA accompanies thebinding of Ag+ to DNA (3).
To monitor the conformational changes caused by Ag+ on
pure DNA, we measured the LD and CD of DNA with and withoutadded Ag+ to a concentration corresponding to 0.5 Ag+ per DNA
phosphate. The results are shown in Chart 5. The LD spectrumshows that the DNA is still oriented in the flow field; thus, theDNA does not aggregate upon addition of silver ions. The CDsignal completely changed its shape and increased about 10times upon addition of silver, indicating an internal conformationalchange (3).
We previously found a significant increase in the fluorescenceof anff-BPDE-DNA after addition of Ag* (42). We now recordedfluorescence excitation spectra at different Ag+ concentrationsfor anf/'-BPDE-DNA, syn-BPDE-DNA, and denatured anf/'-BPDE-
DNA. For the following evaluation, we have assumed that thespectra of both native complexes are composed of ComponentsI and II and have adopted a similar mode of analysis as describedfor the light absorption spectra.
Chart 6 depicts the calculated fluorescence intensities of themajor spectral components II of anf/-BPDE-DNA and I of syn-BPDE-DNA, respectively. The measurements on the denaturedanf/'-BPDE-DNA are also included in the chart. With increasing
concentrations of Ag+ added to the anf/-BPDE-DNA, the type II
spectrum increases significantly. Addition of silver to the syn-BPDE-DNA sample also results in increased intensity of the type
II spectrum (data not shown). The type I spectral component ofsyrc-BPDE-DNA is slightly decreased by Ag+ (Chart 6). The
spectrum of denatured anf/-BPDE-DNA is strongly quenched byAg+. The denatured syn-BPDE-DNA shows the same properties
(data not shown).The fluorescence of the DNA samples with added Ag+ re
mained unchanged after 2 extractions with ethyl acetate. Thesemeasurements show that the type II spectral component mustbe strongly quenched by native DNA at its binding site, since thechange of DNA conformation induced by Ag+ can cause the
o.e
O 0.4
o -25X
O
-50
250Wavelength
300nm )
Chart 5. Absorption, LD, and CD spectra of native DNA ( ) and native DNAwith AgNO3 added to a concentration of [Ag*):[DNA phosphate] = 0.5 ( ).
The samples were prepared in 10 HIMsodium cacodylate buffer (pH 7.0). All spectraare normalized to 1 cm path length.
0.1 0.2 0.3 0.4 0.5 0.6[fig*] / [DNfl-phosphate]
Charte. Effect of Ag* ions on the fluorescence of native an(/-BPDE-DNAComponent I (O). native syn-BPDE-DNA Component II (O), and denatured anfi-BPDE-DNA (A). The samples were prepared and the fluorescence was measuredas described in the text, with AifSâ„¢= 0.66 and approximately 1 BPDE residue per250 DNA bases. F/F0 is the ratio between the intensities with and without addedsilver ions. The excitation wavelength was 345 or 354 nm, and the emission wasobserved at 420 nm.
observed large increase in fluoresence intensity. The chromo-
phores giving the type III spectrum must be in close contact withthe single-stranded DNA bases, since here, Ag* acts as an
internal fluorescence quencher.Fluorescence Quenching By 02. The fluorescence quencher
O2 has been used as a probe for externally DNA-bound chro-
mophores (23). 02 does not penetrate into or bind to the DNAhelix; therefore, mainly chromophores bound on the outside ofDNA will be affected. The rate of quenching can be evaluatedusing the Stern-Volmer equation (32)
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O. Undeman et al.
(C)
where F and F0 are the measured fluorescence intensities withand without added quencher 0 and K is the Stern-Volmer
quenching constant.Chart 7 shows Stern-Volmer plots for O2 quenching of Com
ponents I and II of syn-BPDE-DNA and anf/'-BPDE-DNA, respec
tively, calculated as described above from measurements at theexcitation wavelengths 354 and 345 nm (see Ag+ titrations). Theplot for Component II of anf/'-BPDE-DNA is curved for the O2
quenching, indicating the existence of chromophores with different quenching constants K, although with the same spectra.Component I of syn-BPDE-DNA is similarly quenched by O2. Thefluorescence quenching of denatured anf/'-BPDE-DNA is strong
for 02. Again, the curvature indicates that several chromophoricspecies with different quenching constants are present.
It is not possible from the quenching results alone to draw anyconclusions about the accessibility of the quencher to the DMA-
bound chromophores. The fluorescence lifetimes of the chromophores have to be known, since the Stern-Volmer quenching
constant is proportional both to the encounter frequency between the quencher and the chromophore and to the lifetime ofexcited state of the chromophore.
Fluorescence Decay Measurements. With the aim to furthercharacterize different species of DNA-bound BPDE and evaluate
the O2 quenching experiments, we measured the fluorescencedecay of anf/'-BPDE-DNA and syn-BPDE-DNA. The decay data
were recorded with an apparatus using the single-photon count
ing technique. Chart 8 shows the results of a typical decaymeasurement and evaluation of anf/'-BPDE-DNA. The decay time
4 -
2 -
10 20 30 40 50 60 70 80Oxygen concentration (mM)
Chart 7. Stern-Volmerplots of oxygen quenching of the fluorescenceof BPDE-DNA samples. Native anf/-BPDE-DNA Component I (D); native syn-BPDE-DNAComponent II (O); denatured anf/-BPDE-DNA(A). The fully drawn curves in Chart7 represent theoretical Stern-Volmerquenching curves, calculated as described inthe text. The a3 (see text) of an(/-BPDE-DNAwas somewhat higher than given inTable 3 for the preparation used in the O2quenching measurements; therefore, a3= 0.12 was used in the calculation. The samples were prepared and the fluorescencewas measuredas described in the text, with AjgSyt.= 0.66 and approximately1 BPDE residue per 250 DNA bases. F0/F is the ratio between the intensitieswithout and with the quencher. The excitation wavelength was 345 or 354 nm,and the emission was observed at 420 nm.
and amplitude parameters are presented in Table 1.For syn-BPDE-DNA, the excitation was at 355 nm, which is
close to the absorption maximum of the type I spectrum. Twoexponential terms were sufficient to make a good fit betweenthe experimental decay and the model. The fast-decaying com
ponent with an estimated lifetime of 2.7 nsec is responsible forabout 80% of the absorption at the excitation wavelength 355nm, assuming there is no static quenching of the DNA-bound
chromophores. Since the lifetime of the free chromophore (BPT)in water solution is about 200 nsec, this component representschromophores which are strongly quenched by the DNA.
The fluorescence decay curve of anf/'-BPDE-DNA after exci
tation at 335 nm could not be fitted satisfactorily with 2 exponential terms (x2 = 3.04). Three terms were needed to obtain agood fit (x2 = 1.11). This indicates that there are at least 3different fluorescence species in the anf/'-BPDE-DNA. There are
2 major components with lifetimes of 1.6 and 7 nsec, respectively. The third component, which is less quenched by the DNA,represents only a minor fraction of the chromophore moleculesseen by fluorescence. Its relative amplitude varied from 3 to 12%in different preparations, whereas its contribution to the totalsteady-state fluorescence was in the range of 20 to 80%.
In the decay measurements on anf/'-BPDE-DNA with added
silver ions, the component with the shortest decay time disappears completely and the amplitude of the longest lifetime component increases about 10 times. Evidently, the chromophoresexperience different microenvironments at the binding sites onDNA upon addition of silver ions.
Although no lifetime measurements could as yet be made onsamples quenched with oxygen, we could make rough estimatesof the limiting quenching constants for the oxygen quenching bycombination of the results from the steady-state fluorescence
Co32
8
20 40 60 80 100
Time (nsec)Chart 8. Fluorescence decay curves after excitation with a short flash. Com
puted decay of anfi-BPDE-DNA in 10 mM cacocylate buffer, pH 7.0 ( ), withAJeSxn= 0.66 and approximately 1 BPDE residue per 250 DNA bases. Theparameters used in the computation are indicated in Table 1. The fluorescencedecay of 0.5 ^M POPOP in benzene (x). This decay curve was used to generatethe apparatus response function, as described in "Materials and Methods." Top
curve,weighted residuals.The excitation wavelengthwas 335 nm,and the emissionwas observed at 402 nm. The counts for the POPOPsamplehave been divided by10.
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Fluorescence and Linear Dichroism of BPDE-DNA
Table 1Typical fluorescence decay parameters lor BPDE-DNA samples
The samples were prepared and the decay parameters estimated as described in "Materials and Methods." Amplitudes (a,) are normalized to
unity. A confidence interval on the 95% significance level is given for those parameters that did not correlate strongly with each other (see also"Materials and Methods"). Repeated measurements on different preparations of the same system (anf/-BPDE-native DMA, 4 samples; anti-BPDE-native DMA plus Ag*, 3 samples; ant/-BPDE-denatured DNA, 3 samples; and syn-BPDE-native DNA, 1 sample) gave results with parameter values
typically within ±30%of one another within each group.
SystemBPT
inH2Oani/-BPDE-nativeDNAanf/-BPDE-native
DNA +Ag*6anff-BPDE-denatured
DNAsyn-BPDE-nativeDNAa,0.52
-i-0.040.49
±0.040.76±0.08^^
(nsec)1.6
-1-0.31.1
±0.22.7±0.4Ot0.42
+0.040.350.27
±0.020.24±0.04T2
(nsec)7.0
+0.6179.2
±131±28s1.00.060.650.24T3
(nsec)199±1a4211067x21.121.111.521.151.41
Mean ±S.D.At an (Ag*]:[DNA phosphate] ratio of 0.5.
experiments with oxygen quenching (Chart 7) and the time-
resolved experiments in the absence of quencher (Table 1). TheStern-Volmer relation may be written in the following form whenseveral fluorescing species are present:
2F«,
ZF,Fffl(1 + K,[0]}- (D)
Here, F,0and F, are the fluorescence intensities of the rth specieswithout and with quencher added, respectively. F/0is proportionalto a¡TÕO,and K, is the Stern-Volmer quenching constant of the ;'th
species. Equation D was fitted to the experimental F0/F versusQ points of Chart 7 by varying the K/s, using the 3 componentamplitudes a, and lifetimes TK,= T,for the BPDE-DNA complexes
in absence of quenchers given in Table 1. This was accomplishedon a computer using a program for nonlinear least-squares fitting
(33). The bimolecular quenching rate constant k¡is related to K,through k, = K//T, (32). The estimated quenching rate constantski are presented in Table 2 and the corresponding curves areplotted in Chart 7. The numerical values of k¡should be comparedto the "ideal" diffusion-controlled rate constant in water at 20°:frc = 6.5 x 109vrV1 (32).
DISCUSSION
The results presented in this study give clear evidence of atleast 2 structurally different DNA adducts formed when eitheranf/'-BPDE or syn-BPDE reacts covalently with native DNA.
The syn-BPDE-DNA displays a mixture of 2 spectral components both in the absorption and fluorescence excitation spectra.We have referred to these 2 components as Component I withpeaks at approximately 322, 337, and 354 nm and ComponentII with peaks at 316, 330, and 345 nm (Table 3).
Two similar spectral types were also observed by Ibanez efa/. (16) for BPT physically bound to DNA. Their spectrum type Iwas assigned to intercalated BPT molecules. BPT is a chromo-phoric analogue to the DNA-bound BPDE, since the aromaticstructure is the same in both cases. We expect that also for theBPDE adducts, the spectral properties should reflect the interaction of the chromophores with the components of DNA.
Lianos and Georghiou (25) have previously studied complexesbetween pyrene and the mononucleotides of DNA. In thesecomplexes, they observed a shift to longer wavelengths of thepyrene light absorption spectrum. The purine nucleotides causeda much stronger shift than did the pyrimidine nucleotides. Wetherefore suggest that the type I spectrum arises from thepyrene-like chromophoric moiety of the BPDE molecule intercalated into the DNA helix and interacting strongly with the bases.
Table 2Bimolecular quenching rate constants lor fluorescence quenching of BPDE-DNA
samples by oxygen
The samples were prepared and the quenching was measured as described in' Materials and Methods." The quenching rate constants (/(/) were calculated as
described in the text. The subscripts of k refer to the lifetime components given inTable 1. The quenching was measured for 2 different samples of each system, andthe maximal variation in the quenching was ±15%.
Systemanf/-BPDE-DNA,
Compo
nent lsyn-BPDE-DNA, Compo
nent IIDenatured anfi-BPDE-DNAÄ,0
1.00.03*,1.1 2.3
2.8ks
[M''S~']x
IO'99.3
3.8
Our LD measurement on syn-BPDE-DNA, which gives a negative type I spectrum and «> 65°, is further evidence of an
intercalated complex. Comparison of Charts 2A and 2B revealsfor syn-BPDE-DNA an apparent absence in the LD spectrum of
the type II spectral component which is present in the lightabsorption spectrum. It is calculated that about 35% of thebound chromophores contribute as spectral Component II to thelight absorption spectrum. If these syn-BPDE molecules arebound with a ^ 55°,the peaks at 327 and 346 nm in the LD
and reduced LD spectra of syn-BPDE-DNA are well explained.
Since the type II absorption spectrum also contributes at 354nm, it follows that the a value of 65°calculated for the type I
chromophore is an underestimate.The absorption spectrum of anf/'-BPDE-DNA is shifted and
broadened in comparison with a spectrum from BPT in aqueoussolution. This broadening is indicative of a heterogeneity in thebinding sites.
Also, in the case of anf/-BPDE-DNA, the reduced LD is notconstant over the absorption range of the A —»/.„'transition.
The significance of the troughs at 335 and 360 nm in the reducedLD is difficult to assess, but they could arise from a minor speciesof DNA-bound BPDE molecules which display a type I spectrumand are oriented nearly parallel to the DNA bases. The magnitudeof the reduced LD at 345 nm indicates, however, that the majorityof chromophores are bound with the long symmetry axis of thepyrene moiety at an angle of less than 35°to the DNA helix axis.
We may conclude that in the covalent reaction product(s) ofsyn-BPDE and DNA, the spectral Component I is dominating,with a significant contribution also of Component II, whereas theanf/'-BPDE-DNA complex is an almost pure Component II type,
with only a minor contribution of Component I. The spectralComponent I may be due to classic intercalation, with the molecular plane of the chromophore being close to perpendicular tothe DNA helix axis. Component II has a very different geometry,
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O. Undeman et al.
Table 3Experimental properties of the spectral types of BPDE-DNA complexes
Spectraltype of
BPDE-DNAcomplex1IIIIIPeaksof absorption or
fluorescenceexcitationspectrum(nm)322,337,354316,330,345316,332,351Majorcomponentinsyn-BPDE-DNA(-65%)ant/-BPDE-DNA
(>90%)Denatured
and-andsyn-BPDE-DNAChemical
stabilityLowHighNot
measuredAngle
betweentransition moment vector (in
molecular plane)and av.DNAaxis">65°<35°
8 Orientation axis in LD measurements.
with the transition moment vector of the chromophore formingonly a small angle with the DNA helix axis.
As has been pointed out by Hogan ef al. (15), the calculatedangles «need not be the angle between the DNA helix axis andthe transition moment vector of the pyrene moiety at the bindingsite, since the DNA may be bent at the binding site. The a valuemay rather represent an average of angles between straightDNA segments and the chromophoric transition moment vectors.
Heat denaturation of the DNA with bound chromophoreschanges the spectral properties and the fluorescence intensitiesconsiderably. The type III spectrum, which results from both theanti- and syn-BPDE-DNA, is consistent with a complex in whichthe chromophore is sandwiched between bases on a single DNAstrand. Such a structure was proposed by Frenkel ef al. (6) fora BPDE-dinucleotide monophosphate complex.
The spectral types of BPDE-DNA complexes identified by their
light absorption, fluorescence excitation, and LD spectra aregiven in Table 3.
Next, we consider the titrations with silver ions and the fluorescence quenching by oxygen ions together with the results oflifetime measurements. We have earlier reported that the additionof silver ions to anf/'-BPDE-DNA causes an increased fluores
cence intensity (42). The data presented here on the ComponentI spectrum of anf/'-BPDE-DNA is in full agreement with the
previous observations. The fluorescence increase was interpreted in terms of a DNA conformational change induced bysilver ions which forced the BPDE chromophoric moiety from abinding with strong quenching interaction with the DNA basesto a geometry with less such quenching.
That the fluorescent chromophores in the presence of silverions ([Ag+]:[DNA phosphate] ratio, 0.5) really are exposed on
the outside of the DNA was confirmed in a separate 02 quenchingexperiment (data not shown). The chromophores are easilyaccessible with kq ~ 5-109 NT1sec'1, which is only a factor of 2
below the value expected for free diffusion-controlled quenching.
The small change in light absorption of DNA itself caused byaddition of silver ions (Chart 5) is indicative of the persistence ofa double-stranded structure after complexing with Ag+. The
strong LD observed shows that good orientation occurs in theflow field and suggests that the structure must still be ratherextended and rigid. In presence of Ag+, the LD becomes positive
below 260 nm, and the CD spectrum shows a very pronouncedtrough at 270 nm. This is best explained as caused by a tilt ofthe bases induced by the binding of silver ions, as also suggestedby Dattagupta and Crothers (3). The tilt would orient some ofthe transition moment vectors of the bases at an angle less than
55°to the DNA helix axis, thereby giving a positive LD in the
spectral region of the transitions. The tilt would also facilitate acoupling of oscillators along the DNA helix, giving the increasedrotatory strength we observed for the DNA:silver ion complex.
The model proposed for the effect of Ag+ complexing to anti-
BPDE-DNA is further supported by the measurements of fluo
rescence lifetimes of the bound chromophores (Table 1). In thepresence of Ag+, the fastest decaying component is completely
abolished, and the lifetimes of the other 2 components areincreased considerably. The only spectral change observed inthe presence of Ag+ is a sharpening of the lines and an increase
in intensity of Component II. Also, in the case of syn-BPDE-DNA,the addition of Ag* leads to an increase of the type II component
in the fluorescence excitation spectrum, which most likely is alsodue to unquenching of chromophoric species when DNAchanges conformation. The decrease in intensity of the type Icomponent is not significant enough to permit safe conclusionsconcerning quenching by Ag+ ions in this case.
The Stern-Volmer plots showing the fluorescence quenchingof anf/-BPDE-DNA by oxygen are curved. This fact indicates the
existence of 2 or more chromophoric species which displaydifferent quenching constants.
The estimated bimolecular quenching constants from the oxygen quenching of the steady-state fluorescence (Table 2) revealthat both for anf/'-BPDE-DNA and syn-BPDE-DNA, there are
chromophoric species which are relatively inaccessible to oxygen. Since oxygen is a small uncharged quencher, these chromophoric species must be sterically protected from collisionswith the oxygen molecules, e.g., by insertion into the DNA helix.This is most pronounced for syn-BPDE, where about 80% of thechromophores show a quenching which is 7-fold lower than thediffusion-controlled limiting quenching (cf. Table 1). Only the longlifetime component of anf/'-BPDE-DNA seems to be freelyquenched by oxygen. The denatured anf/'-BPDE-DNA shows an
intermediate quenching of the 2 longer lifetime components. Itshould be noted that for the shortest lifetime components, theestimated kq values are uncertain due to the low fluorescenceyield from these components.
For the major type II component of anf/'-BPDE-DNA, fluores
cence decay measurements reveal at least 3 components. About50% of the chromophores are strongly quenched by the DNA.The interaction causing this strong quenching is probably different from the interaction shifting the absorption spectrum, sincethe quenched chromophores are of type II that has only a smallshift. The nature of this interaction is still obscure. One possibilitywould be that it is related to the strong electric field gradient
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Fluorescence and Linear Dichroism of BPDE-DNA
from the phosphate groups of DNA. Another alternative wouldbe that this fraction of DMA-bound chromophores has a wedge-
like geometry in a bent DNA helix, as suggested by Hogan ef al.(15). This would give an intermediate form of interaction betweenthe bases and the chromophore, which could explain the strongfluorescence quenching that the native DNA exerts on the chromophore. At the same time, this would also be in accord withthe positive LD spectrum of anf/'-BPDE-DNA.
For syn-BPDE-DNA, the major type I component is, in agree
ment with the proposed intercalation, much less accessible toquenching by O2 than is the type II component of anf/-BPDE-DNA. The reason for the small effect of Ag+ on the type I
component of syn-BPDE-DNA is, however, not clear.Upon heat denaturation, both ani/- and syn-BPDE-DNA give
rise to type III spectra. To obtain the long-wavelength shift
observed, the chromophores must probably be sandwiched between bases of the DNA. The strong fluorescence quenching byAg+ is in accord with such a structure. Because of its size, a
sandwiched chromophore is still exposed and rather well accessible to O2 quenching, which is consistent with the fairly largequenching constants (Table 2).
The present investigation demonstrates that it is possible andmeaningful to describe anti- and syn-BPDE-DNA complexes in
terms of 2 spectral types I and II and further subcategoriesaccording to fluorescence lifetime components and their response to various quenching agents. This classification is notclaimed to be rigorous, since each component is far from homogeneous. Since anf/'-BPDE is much more carcinogenic thansyn-BPDE (2), the properties of the anf/'-BPDE-DNA complexes
and their differences relative to the syn complexes or of thecorresponding single-stranded species, possibly mimicked bythe heat-denatured BPDE-DNA, might be most relevant, depend
ing on the level at which the interference with normal physiological activity takes place.
ACKNOWLEDGMENTS
We wish to thank Rolf Brandes for essential modifications in the computerprogram used in the fluorescence decay analysis. The Cancer Research Program,Division of Cancer Cause and Prevention, National Cancer Institute (Bethesda,Md.) is gratefully acknowledged for supplying the BP derivatives.
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1983;43:1851-1860. Cancer Res Ola Undeman, Per-Olof Lycksell, Astrid Gräslund, et al. and Linear Dichroism)pyrene 7,8-Dihydrodiol-9,10-epoxide Studied by Fluorescence
aCovalent Complexes of DNA and Two Stereoisomers of Benzo(
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