Chem. Res. Toxicol. 1991,4, 256-269
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Invited Review Fluorescence Line Narrowing: A High-Resolution Window on DNA and Protein Damage from Chemical Carcinogens Ryszard Jankowiak and Gerald J. Small* Department of Chemistry and Ames Laboratory-USDOE, Iowa State University, Ames, Iowa 5001 1 Received October 24, 1990
I . Introductlon Covalent binding of chemical carcinogens to DNA is believed to be the first critical step in the initiation of the tumor formation process (1-3). The polycyclic aromatic hydrocarbons (PAH) constitute a class of carcinogens for which metabolic activation to electrophilic intermediates is a necessary condition for DNA and other macromolecular damage. By virtue of the rich heterogeneous distribution of adducts they produce, the PAH provide a full spectrum of the complexity associated with understanding the initial phase of chemical carcinogenesis. There are several types of heterogeneity that one needs to consider. They stem from the existence of different metabolic pathways [for PAH the monooxygenation or diol epoxide ( 4 , 5 ) and one-electron oxidation or radical cation (6, 7) pathways have been quite extensively studied], reaction of metabolites with different bases, reaction of a metabolite with different nucleophilic centers of a given base, different DNA sites for a given chemical adduct, stereospecific reactions, and base sequence specificity. In addition to the problem of heterogeneity, one needs to be concerned with the persistence of macromolecular adducts, which depends on factors such as enzyme repair efficiency, chemical stability, and DNA replication rate. During the past several years we have been attempting to develop bioanalytical spectroscopic techniques that possess the selectivity and sensitivity required to address the problem of heterogeneity. Of particular interest are techniques that can be used for the characterization and determination of macromolecular adducts. The primary purpose of this paper is to review the progress that has been made with fluorescence line narrowing spectroscopy (FLNS).’ Abbreviations: ACR, acrylamide;ALA-BP, 7,8,9-trihydroxy-r-7,t8,t-9,~-10-tetrahydrobem[a]pyren-l0-y1 N-t-BOC-talaninata; BP, benzo[a]pyrene; BP-CEdG, one-electron oxidation adduct formed by covalent bonding between C-6 of BP and the N-7 position of deoxyguanosine; BP-N7-Gua, one-electron oxidation adduct formed by covalent bonding between C-6 of BP and the N-7 position of guanine; BPDE, benzo[o]py-rene diol epoxide(8);BPDE-DNA, covalent adduct of BPDE and DNA BPDE-N2-dG, monooxygenation adduct formed by covalent bonding between C-10 of the BP-diol epoxide and the 2-amino group of dC; BFT, dG, deoxybenzo[a]pyrene tetraol; (dG-dO2,poly(dG-dC).poly(dG-dC); guanosine; ELISA, enzyme-linked immunosorbent assay; FLNS, fluorescence line narrowing spectroscopy; FWHM, full width at halfmaximum; GMP, guanine monophosphate; HB, hole burning; HPLC, high-pressure liquid chromatography;NPHB, nonphotochemical hole buming; PAH, polycyclic aromatic hydrocarbon(s);PHB, photochemical hole buming; PSB, phonon sideband; PSBH, phonon sideband hole; S, Huang-Rhya factor; S, and SI,electronic ground and fvet excited singlet state, respectively,TLC, thin-layer chromatography; ZPL, zero-phonon homogeneous line width; rinh, siteline(s); l’, phonon bandwidth; rho,,,, inhomogeneous line broadening: X, burn wavelength; d, and a’ , excited-state vibronic frequencies; q o 0the , center frequency of t i e (0,O) absorption band; wL,frequency of laser; w, phonon frequency.
Fluorescence line narrowing is a low-temperature solid-state technique that can eliminate or greatly reduce the contribution of site-inhomogeneous line broadening ( r ~ to vibronic fluorescence bandwidths. For a molecule imbedded in an amorphous host, such as a glass or polymer or protein, this broadening is very large (- 100-300 cm-’) and reflects the intrinsic structural disorder of the host. In FLNS, narrow-line laser excitation into the inhomogeneously broadened vibronic absorption bands of the S1 So absorption system leads to site excitation energy selectivity. Fluorescence vibronic bandwidths of 1-5 cm-’ are readily achievable (low temperatures near 4 K are required to eliminate the thermal broadening contribution to the bandwidth). This represents an improvement in spectral resolution of about 2 orders of magnitude when measured relative to rinh of the vibronic absorption widths in amorphous hosts. Site-inhomogeneous line broadening of PAH in crystalline and amorphous hosts has been extensively studied. In crystalline hosts rinh is about 2 orders of magnitude smaller than in glasses. Thus,one might say that, in the application of FLNS to molecules imbedded in amorphous hosts, a narrow-line laser is used to “trick” an amorphous host into behaving like a well-ordered crystalline host, albeit at a loss of about 2 orders of magnitude in sensitivity. Fluorescence line narrowing emerged from the work of Szabo (8) and Personov et al. (9) on inorganic and organic doped solids, respectively. Since then it has been applied to a variety of spectroscopic and photophysical problems (10-12) very often associated with the lowest excited singlet (S,)state of planar aromatic molecules. From these studies it became evident that the (0,O)or origin absorption band of a chromophore imbedded in an amorphous host is generally site inhomogeneously broadened with rid 100-300 cm-’ and a contribution to the profile that is Gaussian. One could expect, therefore, that the inhomogeneous line width of a specific DNA-PAH adduct imbedded in a glass should lie in this range. More recently, FLNS has been applied to numerous biomolecules and biological systems: chlorophylls a and b (13-15) pheophytin a (16,17),protochlorophyll and protopheophytin (18), heme proteins (19), iron-free cytochrome c (201, etiolated leaves (21),and bacteriochlorophyll a and bacteriopheophytin a (22). The closely related line narrowing technique of spectral hole burning (10, 23-25) has now been applied to a wider variety of photosynthetic protein-pigment complexes (14, 22, 26-30). For such complexes, rinh also lies in the aforementioned range and it is clear that there is a significant contribution to ri,,,,from statistical fluctuations in protein-pigment structure.
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0893-228~/91/2104-0256%02.5~IQ0 1991 American Chemical Societv
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Chem. Res. Toxicol., Vol. 4, No. 3, 1991 257
Invited Review
The first analytical applications of FLNS with glasses emerged from this laboratory and were concerned with the distinction between and quantitation of PAH in complex mixtures (31). That FLN is operative for a macromolecular DNA-PAH adduct was first established in 1984. The adduct studied was the dominant adduct from the (+)-anti stereoisomer of the benzo[a]pyrene diol epoxide (32). Its structure is given later in this review. Shortly thereafter, a detection limit for this adduct (which has pyrene as the fluorescent chromophore) of about 1 damaged base pair in los bases for 100 pg of DNA was demonstrated at a spectral resolution of 10 cm-l (33). It was shown that structurally similar DNA adducts could be distinguished by FLNS (34). Comparable selectivity was demonstrated for polar PAH metabolites (35). It was also shown that FLNS can be applied to nucleoside adducts sorbed on TLC plates and that absolute quantitation is possible (36). In this review we will discuss only our most recent work that pertains to the versatility and selectivity of FLNS for adduct analysis. Attention will be focused on benzo[a]pyrene (BP), the most extensively studied PAH in chemical carcinogenesis. The physicochemical properties of DNA adducts derived from BP and its biological effects have been recently reviewed by Geacintov (37, 38) and Graslund and Jernstrom (39). The remainder of this review is organized as follows: In section I1 a nonmathemtical discussion of the principles of line narrowing spectroscopies is given that covers both FLN and "competitive" spectral hole burning. In section I11 the FLNS apparatus currently employed in our laboratory is described. In section IV the mechanistic aspects of the initial phase of chemical carcinogenesis from PAH are briefly reviewed. Section V covers five recent applications of FLNS to the analysis of carcinogen-macromolecular (DNA, globin), -polynucleotide, and -nucleoside adducts; included here is a description of a powerful methodology that combines FLNS with laser-excited Sz S,fluorescence spectroscopy at 77 K and fluorescence quenching with acrylamide. Section VI is devoted to some final remarks and a discussion of future prospects. It is in this section that the question of the relevance of DNA adduct structure types determined in a glass at 4.2 K to structure at biological temperatures is considered. +
I I . Princlples of Solid-state Line Narrowing Spectroscopies In condensed-phase molecular systems that are several mechanisms by which optical transitions and spectra are broadened. However, all broadening mechanisms can be categorized as homogeneous or inhomogeneous. Often homogeneous broadening and inhomogeneous broadening are defined and contrasted in terms of a single well-defined (i.e., all relevant quantum numbers for the initial and final states specified) transition. Homogeneous broadening is the broadening that is the same for each and every chemically identical molecule in the ensemble. In the gas phase one could consider, for example, absorption from the rotationless and vibrationless (zero-point)level of the ground electronic state to a particular rotational-vibrational level of an electronically excited state. At sufficiently low pressure this rovibronic trimition would be homogeneously broadened due to the finite lifetime (7') of the excited state with a full width half-maximum of FWHM (cm-') =
(2?rCTl)-'
(1)
where c is the speed of light. For 7, = 50 ns, which is typical for the lowest excited electronic state (S,)of polycyclic aromatic hydrocarbons, FWHM = O.OOO1 cm-l. At
U(0,O)
+
1 , ,,
,
, , I : l o I
,.
,, &< ,, , u . :i lJ.,vL ~v \r -( \ -, 7 - 3 - 6 . b . \. b f
4
0'
\ _ \ \
0-
Figure 1. Schematic representation of homogeneous (rh)and ) Profiles of the zero-phonon inhomogeneous ( r ~broadening. lines (ZPL) and their associated phonon sidebands (PSB) for specific sites at different frequencies have been enlarged compared to the inhomogeneous line to provide more detail. q is the laser frequency that selectively excites a narrow isochromat of an inhomogeneously broadened absorption band.
room temperature this lifetime broadening is negligible compared to the Doppler broadening, which is a manifestation of the distribution of molecular velocities which leads to a Guassian distribution of transition frequencies. This "heterogeneity" in frequencies is an example of inhomogeneous line broadening. However, gas-phase spectra of large molecules exhibit vibronic line widths that are most often determined by overlapping rotational transitions. This structure may also be viewed as a homogeneous broadening mechanism since the structure is common to each and every molecule. The solid-state analogue of rotational structure is phonon (lattice-vibrational) sideband structure; it is a manifestation of the change in the lattice equilibrium structure which accompanies the electronic excitation. The analogue of Doppler broadening is site-inhomogeneous broadening, which is the result of an analyte or probe molecule in a solid host adopting a very large number of energetically inequivalent sites. This leads to a Gaussian distribution of frequencies for any given vibronic transition. For amorphous hosts, such as glasses and polymers, the inhomogeneous broadening, Fin,,, is ~100-300cm-'. For crystalline hosts, rinhis reduced by about 2 orders of magnitude. Fluorescence line narrowing spectroscopy (FLNS) is a member of a class of laser-based spectroscopies that can eliminate or significantly reduce the contribution of rinh to the vibronic bandwidths. Spectral hole burning spectroscopy is another member of this class. It should be noted that spectral hole burning experiments have shown that for chlorophylls of light harvesting and reaction center ==100-200cm-'(M), essentially the same complexes rinh as for chlorophyll embedded in organic glass hosts (14). This is also the case for PAH metabolites covalently bound to DNA. A. Principles of Fluorescence Line Narrowing. Fluorescence line narrowing spectroscopy has been the subject of several reviews (10-12). Below, a nonmathematical discussion is presented (for a theoretical discussion see ref 10). Figure 1 is a schematic representation of an inhomogeneously broadened electronic absorption origin band, (O,O), a t low temperature. The relatively sharp dashed bands depict the (0,O)transitions of the "guest" molecule occupying inequivalent sites. It is customary to refer to the dashed band as the zero-phonon line (ZPL).
Jankowiak and mall
258 Chem. Res. Tonicol.,Vol. 4, No. 3, 1991
A zero-phonon transition is one for which no net change in the number of phonons accompanies the electronic transition. Building to higher energy on each ZPL in Figure 1 is a broader phonon wing. This is the phonon sideband (PSB). We return to a discussion of the PSB later. As mentioned earlier, rinh =1&300 cm-' for glassy hosts. Even for a picosecond excited-state lifetime ( T I ) , the pure lifetime broadening is well over an order of magnitude less than r b h . Each single site ZPL carries a homogeneous line width, rhom, which is determined by the total dephasing time 72 of the optical transition: 1 -1= - +1(2) 72
271
A
T
l-
72'
71 is defined above, and 72) is the pure dephasing time. This equation would be familiar to practitioners of magnetic resonance spectroscopies. The time 72) is a fundamentally important and interesting quantity, best understood by means of the density matrix formulation of spectroscopic transitions (45, 46). For our purposes it suffices to say that 72) is due to a modulation of the single-state transition frequency that results from the interaction of the excited state with the bath phonons [and other low-energy excitations in glasses (23)]. This interaction does not lead to electronic relaxation of the excited state but rather to a decay of the phase coherence of the superpositionstate initially created by the photon. Loosely speaking, one can say that 72' leads to an uncertainty broadening associated with the time required for the system to "forget" how it was excited. In units of cm-I rhom = (7r72C)-l (3) where c is the speed of light in cm s-l. Obviously rhom determines the ultimate spectral resolution (selectivity)attainable by line narrowing techniques and since, for moderate to good fluorescers, the broadening from 71 is very small, it is 7; that is of concern. The key point for analytical fluorescence spectroscopy is that 7; is strongly temperature dependent. Pure dephasing theories are now well developed (23,47, &), and photon echo (49-51,53) and spectral hole burning (23-25,5245) have been used to study the temperature dependence of 7; in a wide variety of molecular systems. At room temperature, rhom from 7 4 (determined by the interaction with both phonons) is =kT,i.e., -200 cm-', which is comparable to rinh for glasses. Line narrowing spectroscopies cannot eliminate rhom, which means that low temperatures are required to minimize the number of thermally populated low-frequency phonons responsible for rhom. For glass hosts it is now firmly established that rhom from pure dephasing is 50.1 cm-' a t 4.2 K (23, 24, 53-55). The basic principles of FLN can be understood from Figure 1. First, we note that if a broad-band classical excitation source is used to excite the (0,O)band, all sites (ZPL) will be excited and all sites will fluoresce, resulting in a broad fluorescence spectrum characterized by vibronic bandwidths equal to rinh.When a laser of frequency wL and line width AWL
