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Role of Fluorescence Line-Narrowing Spectroscopy and Related Luminescence-Based Techniques in the Elucidation of Mechanisms of Tumor Initiation by Polycyclic Aromatic Hydrocarbons and Estrogens† Ryszard Jankowiak,*,‡ Eleanor G. Rogan,§ and Ercole L. Cavalieri*,§ Ames Laboratory, U.S. Department of Energy and Department of Chemistry, Iowa State UniVersity, Ames, Iowa 50011, and Eppley Institute for Research in Cancer, UniVersity of Nebraska Medical Center, Omaha, Nebraska 68198 ReceiVed: April 8, 2004
Formation of DNA adducts by various carcinogens represents the first critical event in the mechanism of tumor initiation. The carcinogenic polycyclic aromatic hydrocarbons (PAHs) are biologically activated by two major mechanisms: one-electron oxidation to produce radical cations and monooxygenation to form bay-region diol epoxides. The PAH-DNA adducts formed by these mechanisms are stable adducts that remain in DNA unless removed by repair and depurinating adducts that are lost from DNA by cleavage of the glycosyl bond. Identification of PAH-DNA adducts has relied heavily on low-temperature, laser-based fluorescence spectroscopy under non-line-narrowing (NLN) and line-narrowing (FLN) conditions. These spectroscopies can be used for chemical identification, conformational analysis, and/or probing the microenvironment of DNA (or protein) adducts. Small and co-workers have pioneered the use of FLN spectroscopy in this research. For example, the structures of the depurinating adducts formed by the PAHs benzo[a]pyrene, 7,12dimethylbenz[a]anthracene, and dibenzo[a,l]pyrene have been elucidated. Understanding of the mechanism of tumor initiation by PAHs has relied on identifying and quantifying the DNA adducts formed. The insights gained from the study of PAH-DNA adducts enabled us to discover the estrogen metabolites that form depurinating DNA adducts and can be potential endogenous initiators of human cancer. Small and co-workers have also studied the estrogen-DNA adducts and estrogen-thioether conjugates by using FLNS and related luminescence-based techniques and have demonstrated that the level of the 4-hydroxyestrone-1-N3-adenine depurinating adduct in breast tissue from a woman with breast carcinoma was significantly higher than that in breast tissue from women without breast cancer. The fluorescence- and phosphorescence-based techniques they are developing will be applied to analyzing estrogen adducts and conjugates as biomarkers of susceptibility to breast and other types of human cancer.
1. Introduction In the 1960s James and Elizabeth Miller postulated that chemical carcinogens initiate cancer by covalent binding to the cellular macromolecules DNA, RNA, and protein.1,2 Most carcinogens need metabolic activation to form electrophilic species that react with nucleophilic groups of cellular macromolecules. This principle is important because it unifies the various structures of chemical carcinogens that directly or via metabolic activation generate reactive electrophilic species. On the basis of cellular function, it is logical to hypothesize that DNA is the key macromolecule in initiation of carcinogenesis. Formation of DNA adducts by various carcinogens represents the first critical event in the mechanism of tumor initiation. Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of chemical carcinogens. One of the great surprises concerning metabolic activation of PAHs has been the demonstration that more than one mechanism is involved in the covalent binding to DNA that leads to tumor initiation. PAHs are biologically activated by two major mechanisms: one†
Part of the special issue “Gerald Small Festschrift”. * Corresponding authors. E-mail:
[email protected] (R.J.);
[email protected] (E.L.C.). ‡ Ames Laboratory and Iowa State University. § University of Nebraska Medical Center.
electron oxidation to produce radical cations and monooxygenation to form bay-region diol epoxides (Figure 1).3,4 For example, the potent carcinogen benzo[a]pyrene (BP) forms three adducts by one-electron oxidation and three adducts via the bayregion diol epoxide (BPDE) intermediate (Figure 1). All of the adducts formed by one-electron oxidation and two of the diol epoxide adducts are depurinating adducts, which are lost from DNA by cleavage of the glycosyl bond, and one stable adduct, BPDE-10-N2dG, is formed by the diol epoxide and remains in DNA unless removed by repair. The depurinating adducts derived from covalent binding of BP to DNA can lead to the critical mutations that initiate cancer.3-5 Formation of stable and depurinating DNA adducts entails various processes with major differences in carcinogenic potential. The approach to understanding mechanisms of tumor initiation is based on several lines of investigation, which include metabolism studies, carcinogenicity experiments in various target organs, and identification and quantitation of PAH-DNA adducts.3,4 Evidence that depurinating PAH-DNA adducts play a major role in tumor initiation derives from a correlation of the levels of depurinating adducts and oncogenic Harvey (H)ras mutations in mouse skin papillomas (Table 1).4,5 For example, 7,12-dimethylbenz[a]anthracene (DMBA)5-9 and dibenzo[a,l]pyrene (DB[a,l]P)4,5,10,11 form predominantly depurinating
10.1021/jp0402838 CCC: $27.50 © 2004 American Chemical Society Published on Web 06/11/2004
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Figure 1. Metabolic activation of BP by one-electron oxidation with formation of radical cations and by monooxygenation with formation of diol epoxides. Five of the six adducts are depurinating adducts and one (BPDE-10-N2dG) is a stable adduct.
TABLE 1: Correlation of Depurinating Adducts with H-ras Mutations in Mouse Skin Papillomas H-ras mutations PAH DMBA
major DNA adducts N7Ade (79%)
DB[a,l]P N7Ade (32%) N3Ade (49%) BP
C8Gua + N7Gua (46%) N7Ade (25%)
no. of mutations/no. of mice codon
The objective of this article is to report the contributions of fluorescence line-narrowing spectroscopy (FLNS) and related luminescence-based techniques in the determination of mechanisms of tumor initiation by PAHs and estrogens.
4/4 CAA f CTA
61
2. Stable and Depurinating PAH-DNA Adducts
10/12 CAA f CTA
61
10/20 GGC f GTC 5/20 CAA f CTA
13 61
Carcinogens react with DNA to form two types of adducts: stable adducts and depurinating adducts. Investigators in chemical carcinogenesis have always dealt with stable adducts, which remain in DNA unless removed by repair. These adducts are usually detected by the 32P-postlabeling technique, although their identification has rarely been accomplished. In general, activated PAHs and estrogens (see below) predominantly form adducts at nucleophilic groups of adenine (Ade) and guanine (Gua), with destabilization of the glycosyl bond and subsequent depurination. As shown in Figure 2, which represents a Watson-Crick DNA, the backbone is constituted by deoxyribose and phosphate groups. The Ade is hydrogen bonded to thymine, and Gua to cytosine. The Ade has an exocyclic NH2 group that reacts with electrophiles to form stable adducts (Figure 2, hollow arrow), whereas binding to the N-3 or N-7 moieties of Ade gives rise to depurinating adducts (Figure 2, filled arrows). If reaction occurs at the exocyclic NH2 group of Gua, stable adducts are obtained (Figure 2, hollow arrow), whereas reaction at the N-7 (and sometimes C-8) of Gua produces depurinating adducts
Ade adducts and induce A f T transversions in codon 61. In contrast, BP forms approximately twice as many Gua depurinating adducts as Ade depurinating adducts and twice as many codon 13 G f T transversions as codon 61 A f T transversions.5,12-15 These results have provided the impetus for discovering the estrogen metabolites that form depurinating DNA adducts and can be potential endogenous initiators of human cancer.16-18 Experiments on estrogen metabolism,19-22 formation of DNA adducts,16-18 carcinogenicity,23-25 mutagenicity,26-28 and cell transformation29-32 have led to the hypothesis that reaction of certain estrogen metabolites, specifically catechol estrogen-3,4quinones, with DNA can generate the critical mutations initiating breast and other cancers.
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Figure 2. Formation of stable and depurinating DNA adducts and generation of apurinic sites.
(Figure 2, filled arrows). Following reaction at the N-7 of Gua, destabilization of the glycosyl bond is assisted by the antielimination of the hydrogen atom at the C-2′ of the deoxyribose moiety, with subsequent depurination. The resulting apurinic site in the DNA contains a C-2′-C-3′ double bond, which by addition of water forms an apurinic site containing deoxyribose.33 Evidence that depurinating PAH-DNA adducts play a major role in tumor initiation derives from a correlation of depurinating adducts and oncogenic H-ras mutations in mouse skin papillomas.4,5 Identification of the depurinating adducts has been accomplished by comparison to standard synthesized adducts by using HPLC [or capillary electrophoresis (CE)] and FLNS. 3. Fluorescence Line-Narrowing Spectroscopy (FLNS) for Identification and Characterization of Analytes Vigny and Dequesne34,35 and Geacintov et al.36 pioneered fluorescence studies of PAH-derived DNA adducts at room temperature (T). However, a room-T study of PAH-DNA adducts often does not provide sufficient resolution to distinguish spectra of PAH-DNA adducts from spectra of closely related PAH metabolites and/or PAH-DNA-derived decomposition products. To increase sensitivity and selectivity Geacintov and co-workers36 initiated 77 K studies of various PAH-DNA adducts. Although sensitivity was improved due to increase of the fluorescence quantum yield, the selectivity was not impressive. The spectra of PAHs, PAH metabolites, and PAH-DNA adducts obtained with broad-band excitation sources are very broad (even at 4.2 K) with bandwidths of several hundred reciprocal centimeters. As a result species with similar structures cannot be distinguished, particularly in intact DNA and/or complex mixtures. Although in general the low-T spectra provided more insight on various PAH activation processes, selectivity is not improved due to large inhomogeneous broadening. Subsequently, it was shown for parent PAHs that inhomogeneous broadening can be strongly reduced by freezing the sample to low temperatures in crystalline (usually n-alkane) matrixes; these findings led to the so-called Shpol’skii spectroscopy.37-39 However this approach, powerful as it is, is not suitable for more polar derivatives, such as polyhydroxylated metabolites or PAHs bound to biological macromolecules (e.g.,
DNA and proteins). The main disadvantage of Shpol’skii spectroscopy is its limited applicability range since the analytes have to be compatible with the n-alkane solvent.37-39 This led G. J. Small and co-workers to use fluorescence line-narrowed (FLN) spectroscopy (FLNS),40 which is a solid-state laser-based low-temperature fluorescence methodology that affords line widths of several cm-1 (see below). This approach requires only a solution that forms a clear glass upon freezing to cryogenic temperatures. Many such solvents are available. Because of this freedom in solvent choice, FLNS could be successfully applied to the study of PAHs adducted to DNA and proteins.39-47 In the last 20 years G. J. Small and co-workers,40,43-59 and several other groups,41,42,47,60-70 demonstrated that FLNS is very valuable in the study of various biomolecules, including those resulting from animal and human exposure to various chemical carcinogens.55,57,59,71-78 In particular, it has been demonstrated that FLNS can be used for chemical identification, conformational analysis, and/or probing the microenvironment of DNA (or protein) adducts.10,43,44,51,52,79-81 We hasten to add that FLNS emerged from the work of Denisov and Kizel,82 Szabo,83 and Personov et al.84 and is being successfully used for detection and characterization of many different “systems”. Due to the large gain in spectral resolution (several orders of magnitude), the field of FLNS applications in biophysical and biochemical sciences is growing rapidly. Examples include a large number of organic molecules,44,60,61 inorganic systems,62,63 and a wide variety of biomolecular systems including photosynthetic pigments,63-67,85 antenna protein-pigment complexes,68 and protein adducts.47 Comprehensive theoretical and experimental aspects of FLNS can be found in refs 39, 51, 60, and 86-90. Briefly, a molecule embedded in an amorphous solid such as glass or polymer typically exhibits at 4.2 K vibronic absorption bandwidths of about 300-400 cm-1. This is referred to as static inhomogeneous broadening and is a direct result of the host disorder.39,60,91-93 An inhomogeneously broadened absorption origin band [(0,0)-band] has no structure at low temperature (as shown in Figure 3A), but consists of many sharp spectral features due to individual sites [only two sites labeled as A and B are shown for simplicity], which correspond to the absorption zero-phonon lines (ZPLs) of the “guest” molecules occupying inequivalent sites A and B. A zero-phonon transition is one for
Study of Tumor Initiation Mechanisms by FLNS
Figure 3. Frame A: Principles of vibronic excitation in FLNS. Top of frame: Selective laser excitation (ωL1) within the (0,0)-band of subset of molecules B and selective laser excitation at ωL2 of two subsets of molecules (A and B) within the vibronic region. Bottom of frame: Schematic of the resulting fluorescence spectrum for ωL2 with two (0,0)A and (0,0)B transitions. ωR′ and ωβ′ are the vibrational frequencies in the excited state. Frame B: An example of the vibronically excited FLN spectrum is shown for the syn-DB[a,l]PDE-14-N7Gua adduct; λex ) 376.0 nm, T ) 4.2 K. The sharp ZPLs at 749 and 772 cm-1 correspond to excited-state vibrational frequencies.
which no net change in the number of phonons accompanies the electronic transition. Building to higher energy on each ZPL is a broad phonon (lattice vibrational) wing, referred to below as the phonon sideband (PSB). The latter band is due to phototransitions accompanied by the creation or annihilation of low-frequency phonons.60,91,93 Each single-site ZPL carries a homogeneous line width, Γhom, which is determined by the total dephasing time of the optical transition.51,60,88,93 Since the PSBs contribute to the absorption and/or fluorescence origin bands, the width of the (0,0)-absorption band shown in Figure 3A is approximately given by Γinh + Sωm,93,94 where ωm is the mean phonon frequency (for organic solvents typically about 20-30 cm-1) and S is the Huang-Rhys factor (see below). Classical broad-band excitation results in excitation of all sites and, as a result, a broad emission spectrum. However, if excitation occurs within the origin band (e.g., at ωL1, using a spectrally narrow laser), only those chromophores can be excited (isochromat) whose transition energies coincide with the frequency of the laser at ωL1. At temperatures near 4.2 K (to reduce thermal broadening) and at sufficiently low concentrations (to avoid intermolecular electronic energy transfer), dramatic narrowing of the fluorescence spectrum can result, and the resonant fluorescence provides a FLN spectrum. The originexcited FLN spectrum is composed of the ZPL [corresponding to the transition between the zero-point levels of the fluorescent (S1) and ground electronic (S0) states] labeled as (0,0)B and a series of vibronic ZPLs at lower energies [for simplicity only the (0,0)B band is shown in Figure 3A]. The vibronic ZPLs correspond to transitions terminating at vibrational levels of S0. Such FLN spectra allow the measurement of all ground-state vibrational frequencies.43,44,51,60 However, vibronically excited FLNS that probes the S1-state vibronic levels is more often used for identification of closely related analytes. Its principles are illustrated in Figure 3A. When the laser is tuned to frequency at ωL2, it excites two overlapping vibronic transitions (1R,0) and (1β,0), which are not resolved in the absorption spectrum. In this case only the two selected isochromats undergo fast vibrational relaxation to two different correlated points (A and B) in the zero-point site energy distribution of the S1 state. This is followed by fluorescence from the energetically distinct isochromats (A and B), resulting in an FLN spectrum that consists of two ZPLs [(0,0)A and (0,0)B], often referred to as a “multiplet origin structure.” The displacements between ωL2 and the doublet components of the
J. Phys. Chem. B, Vol. 108, No. 29, 2004 10269 origin transition yield the excited-state vibrational frequencies ωR′ and ωβ′. Also in this case the vibronic bands that build on (0,0)A and (0,0)B to lower energy are not shown for simplicity. In actual experiments typically several ZPLs are observed that contribute to the multiplet origin structure for each excitation frequency, providing many “fingerprints” of studied molecules. Interestingly, it has been established that the S1-state vibrational frequencies and intensities are most sensitive to subtle changes in the structure of the chromophore and its environment.44,45 Due to this superior sensitivity of vibronically excited FLNS, relative to origin-excited FLNS, the former has been used exclusively for identification and conformational analysis of closely related PAH-DNA adducts and PAH metabolites.44,45 This sensitivity has a firm theoretical understanding based on the Duschinsky effect (a mixing of the normal coordinates in the excited state and vibronically induced anharmonicity).95 An example of a vibronically excited FLN spectrum obtained at 4.2 K for the syn-dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE)14-N7Gua adduct, derived from the one of the most potent carcinogens, DB[a,l]P, is shown in frame B of Figure 3. In this case laser excitation at 378 nm selectively excites several modes, with the two strongest bands corresponding to 749 and 772 cm-1 excited-state vibrations. The prominence of the vibronic ZPLs indicates weak electron-phonon coupling that is briefly discussed below. By tuning the laser frequency across the S1 r S0 absorption spectrum one can determine all active excitedstate vibrations.41,45,60,88 As mentioned above, for each laser excitation a distinct fingerprint of the analyte can be obtained. We note that FLN is normally observed with excitation frequencies located within the S1 r S0 absorption spectrum. This is a consequence of the site excitation energies of different electronic states being largely uncorrelated. For example, S2 r S0 excitation followed by rapid S2 ' S1 internal conversion (prior to fluorescence) does not result in a line-narrowed S1 f S0 fluorescence spectrum since the site excitation energies of the S1 and S2 states are uncorrelated. 3.1. Electron-Phonon Coupling and Model Calculations of FLN Spectra. Before discussing the shape of FLN spectra, the coupling of the electronic excitation with intermolecular modes (phonons) is briefly addressed. In general, the coupling is strong for molecules that upon electronic excitation undergo large changes in geometry and/or large changes of the electronic charge distribution. The coupling strength can be expressed by linear and quadratic coupling coefficients.96-100 The linear coupling coefficients describe the changes in geometry of the host-guest system (along different normal phonon coordinates) that accompany the electronic transition. The diagonal quadratic coupling coefficients define the changes in phonon frequencies that accompany the transition. The off-diagonal quadratic electron-phonon coupling results in mixing of the ground-state normal phonon coordinates in the excited electronic statesa mechanism for pure dephasing (for details see refs 51, 88, and 96-100). The strength of the linear electron-phonon coupling is conveniently defined in terms of the dimensionless HuangRhys factor S. The total Huang-Rhys factor St ) ∑k Sk, with the sum over Franck-Condon active phonons. The FranckCondon factor for the ZPL is given by exp(-St).62,93 Weak coupling is defined by St < 1, since in the low-T limit the integrated intensity of the ZPL is greater than that of the PSB. This situation is reversed for St > 1 (strong coupling). The strength of the linear coupling is also often expressed in terms of the Debye-Waller factor R ) IZPL/(IZPL + IPSB).60,88,100 Here I designates integrated intensity. R, or equivalently St is a decreasing function of temperature60,100 (see below).
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The width of the ZPL is Γhom. The shape of the ZPL has the form of a Lorentzian with a width defined by lifetime broadening. However, due to the presence of electron-phonon coupling and interactions with the low-energy excitations (i.e., two-level systems, TLS), the width of the ZPL is temperature dependent.60,88,101,102 Typically, at very low T (up to ∼8 K), Γhom varies with temperature as ∼T1+µ with µ ∼ 0.3.93,96,103-109 The line width of Γhom is related to the T2 relaxation time by the following relation91,100,110
1 1 1 ) πcΓhom ) + T2 2T1 T2′
(1)
where T1 is the excited-state lifetime and T2′ is the pure dephasing time. Here it suffices to say that T2′ is due to the modulation of the single-site transition frequency, which results from the interaction of the excited state with bath phonons (and other low-energy excitations in glasses). This interaction does not lead to depopulation of the excited state but rather to a decay of the phase coherence of the superposition state initially created by the photon.93,110,111 Dephasing times are directly measured in photon echo experiments.112-114 In the low-T limit the single-site absorption (emission) spectra have been derived by Small and co-workers.94,101 The singlesite fluorescence profile (assuming a single-peaked one-phonon profile with peak frequency of ωm) is described by the following equation51,94
F(ω) ) e-Sl0(ω - Ω) +
∞
∑
R)1
SR
e-S R!
lR(ω - Ω + Rωm) (2)
where l0 (ω - Ω) is the Lorentzian line shape function of the ZPL with peak position Ω and homogeneous width Γhom, and lR (ω - Ω + Rωm) is the normalized line shape function of the R-phonon transition (R ) 1, 2, ...) peaking at Ω - Rωm. A Poisson weighting factor for every number R determines the intensity distribution of the PSB. Within the framework of the mean frequency approximation, the one-phonon profile l1 (ω - Ω + Rωm) represents the product g(ω)D(ω), with g(ω) and D(ω) being the phonon density of states and an electron-phonon coupling term, respectively. The profile lR is obtained by folding l1, R times, with itself. Various line shapes for l1 can be used;94,101 for example, if l1 is Gaussian with a full width at half-maximum (fwhm), σ, the profile lR is also Gaussian with fwhm ) xRσ.94 Finally, the spectrum for the whole ensemble of chromophores can be obtained by convolution with the ZSD function; the shape of the latter is typically taken as Gaussian with fwhm corresponding to Γinh. Of course at higher temperature, creation (and annihilation) of phonons takes place. Therefore, for T > 0 K a thermal population of phonon levels, according to Bose-Einstein statistics, has to be taken into account.100 The temperature-dependent expression for the singlesite absorption spectrum (again, assuming a single-peaked onephonon profile) has also been obtained by Small’s group, which in the case of fluorescence can be written as94,101
F(ω) ) e
-S(2nj+1)
∞
R
∑∑
R)0P)0
[S(nj + 1)]R-P[Snj]P
× (R - P)!P! lR,P[ω - Ω + (R -2P)ωm] (3)
nj is the phonon occupation number, ωm is the mean phonon frequency, while S(nj + 1) and Snj represent phonon creation and annihilation factors, respectively. As above, S is defined
Figure 4. Frame A: Single-site fluorescence profiles (solid lines) calculated according to eq 13 (with ωm ) 30 cm-1, Γ ) 40 cm-1, Γhom ) 2 cm-1, and Γinh ) 100 cm-1) for different values of S. For parts a, b, c, and d the values of S are 0.5, 1.5, 5.0, and 8.0. The dashed lines represent the corresponding total (convoluted) fluorescence spectra. Frame B: FLN spectra of BO-IMI-dAMP, λex ) 496.5 nm, T ) 4.2 K. Spectrum a is obtained with a laser excitation power density, I ) 30 mW/cm2 and an exposure time of ∼60 s. Spectrum b is obtained under experimental conditions as in (a), following a 300 s exposure to 50 mW/cm2 excitation power density. The lowest spectrum is the difference spectrum (a - b), obtained as discussed in the text.
so that Sωm (optical reorganization energy) ) ∑i Siωi. By analogy to eq 2, the value of R corresponds to the 0, 1, ..., R-phonon transition, while the second sum over P leads to a redistribution of intensity within the PSB according to the number of created or annihilated phonons. An increase of temperature not only leads to more intense PSBs at the expense of the ZPL but also gives rise to the anti-Stokes part of the PSB. Model calculations,51 performed at T ) 4.2 K with the abovedescribed theory, are shown in Figure 4A. Only the effect of S on the calculated single-site (solid lines) and convoluted fluorescence spectra (dashed lines) is illustrated. Calculations were performed for S ) 0.5, 1.5, 5.0, and 8.0 (for the origin band only), as shown in frames a, b, c, and d, respectively. The shape of the fluorescence spectra strongly depends on the value of S. This example demonstrates that an increase of S leads to an increase in PSB intensity compared to ZPL. Increasing the value of S also leads to a red shift of the total fluorescence peak position and, as a result, to larger Stokes shifts given by approximately 2Sωm.60,88,94 Typically, the value of the Γhom width is
