Fluorescence Line Narrowing-Nonphotochemical Hole Burning ...wxjs.chinayyhg.com/upload/Files/Chemical_Research... · fluorescence line widths, selective excitation (from the laser),

60 Chem. Res. Toxicol. 1988,1, 60-68

Fluorescence Line Narrowing-Nonphotochemical Hole Burning Spectrometry: Femtomole Detection and High Selectivity for

Intact DNA-PAH Adducts

R. Jankowiak, R. S. Cooper, D. Zamzow, and G. J. Small* Ames Laboratory-USDOE and Department of Chemistry, Iowa State University,

Ames, Iowa 5001 1

G. Doskocil and A. M. Jeffrey Cancer Centerllnstitute for Cancer Research, Columbia University,

New York, New York 10032

Received October 2, 1987

A new fluorescence line narrowing (FLN) apparatus is described and evaluated through experiments on intact DNA-PAH (polycyclic aromatic hydrocarbon) and globin-PAH adducts, as well as polar PAH metabolites. A detection limit of -3 modified bases in lo8 for a DNA adduct formed with a diol-epoxide of benzo[a]pyrene (BPDE-DNA) is reported for 20 pg of DNA a t a spectral resolution of -8 cm-l. The methodology employed avoids or minimizes spectral degradation and loss of sensitivity due to photooxidation and nonphotochemical hole burning (NPHB). A new double selection technique that employs both FLN and NPHB is described and found to lead to a significant improvement in selectivity over that obtained with conventional FLN.

1. Introduction Formation of an adduct between the genetic material

DNA and a chemical agent is thought to be a crucial step in the initiation of carcinogenesis and tumorigenesis (1-3). As typical DNA damage levels are low (;51:108 base pairs), there has recently been considerable interest in the development of highly sensitive bioanalytical techniques for damage analysis. As important as high sensitivity is the requirement of high selectivity of the type which allows for the distinction between structurally very similar DNA adducts. For example, the distinction between adducts formed from metabolites of a polycyclic aromatic hydrocarbon (PAH) and an alkylated derivative thereof is de- sirable. Advantages also exist for a technique which is applicable to the intact DNA adduct (rather than only to the isolated damaged nucleotide), is rapid and nonde- structive.

Most genotoxic agents require metabolic activation to intermediates that react with cellular nucleophiles. In this regard the PAH have been most extensively studied and appear to undergo activation by two main routes: one- electron oxidation to yield radical cations ( 4 ) and mono- oxygenation to produce bay-region diol-epoxides (5-7). Detailed studies of competing metabolic activation pathways and subsequent reactions with DNA bases have been hampered by the unavailability of rapid methodologies for determination and characterization of reactive intermediates and adducts. The availability of such would also be valuable for the study of DNA repair and synergistic and antagonistic effects associated with the components of mixtures of carcinogens and mutagens.

Recent attempts at identifying DNA-PAH adducts have led to the development of several techniques, such as enzyme-linked immunosorbent assay (ELISA) and Rander- ath procedure (8-10). A fluorescence-based approach which affords high selectivity and one that has been applied to intact DNA adducts is fluorescence line narrowing (FLN) spectrometry (11-20). In this methodology the

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analytes are dissolved in a glass-forming solvent which is cooled to cryogenic temperatures (typically 4-20 K). Ap- propriate excitation with a pulsed tunable dye laser into the lowest singlet (SI) absorption system of the analyte can result in vibronic fluorescence bands whose widths are as narrow as a few cm-l, i.e., about 2 orders of magnitude sharper than the SI vibronic absorption bands of the analyte in the glass at low temperatures. The selectivity of FLN for mixtures of analytes derives from the narrow fluorescence line widths, selective excitation (from the laser), and gated fluorescence detection. FLN, as an analytical methodology, was first developed for polycyclic aromatic hydrocarbons (PAH) (15) and their derivatives (12,18,20) and has been shown to be applicable to PAH in real samples such as solvent refined coal (19). I t has also been shown to be quantitative (20). More recently FLN has been used to directly distinguish between five intact DNA adducts formed from benzo[a]pyrene, 5- methylchrysene, chrysene, and benz[a]anthracene (13), three of these adducts possessing phenanthrene as the parent fluorescent chromophore. Furthermore, each intact adduct could also be distinguished from its corresponding unbound tetrol (13). The distinction between these metabolites in a mixture has been demonstrated earlier (12). Utilization of an ordinary prototype FLN system for the DNA adduct formed from mouse fibroblast 10T1/2 cells exposed to [3H]benzo[a]pyrene led to characterization at a spectral resolution of -10 cm-l for a damage level of -5:106 base pairs (-30 r g of DNA) (12). Improvements in instrumentation which might lead to a 3 order of magnitude improvement in damage level detectability were discussed (13).

In this paper a new FLN system that incorporates the design criteria discussed in ref 13 is described. Its application to the most frequently studied PAH adduct, BPDE-DNA, is discussed. Data are presented which establish a detection limit of -1 fmol for the bound metabolite. Obstacles, such as photooxidation and nonpho-

0 1988 American Chemical Society

FLN-NPHB Spectrometry Chem. Res. Toxicol., Vol. 1, No. 1, 1988 61

frequency

PHB (b) After burning

Figure 1. (a) Inhomogeneous line broadening and (b) photochemical and nonphotochemical hole burning. l?I - few hundred cm-l for glasses; rH (homogeneous line width) 5 0.1 cm-’ at T - 1.5 K for organic molecules in glasses. In b (the bottom schematic) the L(productn corresponds to new impurity-glass site configurations produced by light-induced TLS,,, relaxation.

tochemical hole burning, which had to be overcome in order to attain this limit, are discussed. A new double spectral selection method which incorporates hole burning and FLN is described. This method provides the FLN spectrum of sites which have been spectrally bleached (burnt) and allows for optimum utilization of the hole burning effect. In addition, spectra are presented which establish that FLN is applicable to polar metabolites in urine and analysis of globin damage from one and the same metabolites which damage DNA.

I I. Principles of Fluorescence Line Narrowing and Hole Burning

Fluorescence line narrowing (1 1-20) and spectral hole burning (21-23) are site excitation energy (isochromat) selective spectroscopies that can eliminate or significantly reduce the contribution of site inhomogeneous line broadening, rI, to spectral profiles. A site inhomogene- ously broadened absorption profile is depicted in Figure l a and is the convolution of a very large number of individual site absorptions possessing a homogeneous line width I?. At liquid helium temperatures r 5 0.1 cm-l, while for amorphous molecular hosts such as glasses or polymers, I’I - 300-500 cm-l. Classical broad-band excitation of all the energetically inequivalent sites which contribute to the profile of Figure l a results in an equally broad fluorescence

I

Figure 2. Schematic representation of laser site excitation energy selection in FLNS (see explanation in text).

spectrum and, as a consequence, poor selectivity. However, selection of a narrow isochromat of the absorption profile with a laser of width AoL << rI can result in dramatic line narrowing provided certain conditions are met (20). For example, the fluorescence observed must originate from the electronic state being excited-a consequence of the fact that site energy distribution functions for different electronic states are generally not correlated.

For practical applications of FLN on molecular systems the origin or lower lying vibronic transitions of the lowest excited singlet (SI) state are generally excited. For origin excitation, the resonance fluorescence (origin) transition occurs at the laser frequency and is, therefore, not a reliable analytical line. Furthermore, the vibronic transitions provide only the ground-state vibrational frequencies. Utilization of vibronic excitation is significantly better from the standpoints of characterization and selectivity. Figure 2a depicts an absorption spectrum in which there are two overlapping vibronic transitions to fundamental excited state vibrations 1, and 1,. To the left is an energy level diagram in which the slanted parallel lines indicate the site inhomogeneous broadening for the ( l,,O) and lD,O) transitions. The laser excitation frequency (aL) is chosen to excite different isochromats belonging to the v,‘ = 1 and v i = 1 levels. For example, wL selects an isochromat associated with v i = 1 that lies near the bottom of its distribution while that associated with v,’ = 1 lies near the top of its distribution. As vibrational relaxation occurs on the picosecond time scale, it precedes the fluorescence which originates from the zero-point vibrational level. Vibrational relaxation is indicated by the downward wiggly arrows which show that fluorescence originates from two different isochromats that are linked to the two vibrations initially excited. The resulting fluorescence spectrum has the appearance of Figure 2b in which the laser line (AL) lies to higher energy of all fluorescence bands. Fluores- cence from the two isochromats results in a doubling of

62 Chem. Res. Toxicol., Vol. 1, No. 1, 1988 Jankowiak et al.

F} - - - - - - - - - 0" !

Figure 3. Block diagram of FLNS instrumentation showing excimer laser (Exc. L), dye laser (Dye L), cryostat (C), sample (S), photodiode (PD), zero drift control (ZDC), delay generator (DG), high-voltage switch (HV), photodiode array (PDA), monochromator (M), optical multichannel analzyer (OMA), and printer (P).

the origin or (0,O) transition as well as the (0,l) vibronic transitions. An important point is that the displacements between XL and the (0,O) bands provides the excited state vibrational frequencies (u: and w i ) , while the displacements between the (0,O) and (0,l) bands yield ground-state vibrational frequencies.

For the distinction between the DNA-PAH adducts referred to earlier, the utilization of vibronic rather than origin excitation proved to be essential (13). The reason is that (0,O) or origin excited fluorescence spectra from a given parent PAH are not significantly perturbed by substituent groups.' On the other hand, the vibrational structure and dynamics in the S1 state are often sensitive to ring substitution (24). For the DNA-PAH adducts and unbound metabolites, vibronic excitation a t - 500 cm-l above the (0,O) transition revealed a rich and very diag- nostic multiplet origin structure in fluorescence consisting of several bands (analogous to the doublet shown in Figure 2b) (13).

With an understanding of FLN, photochemical hole burning (PHB) spectroscopy followed in a natural way. It is apparent that if the species giving rise to the absorption is photoreactive, isochromat selective photobleaching can occur. Hole burning spectroscopy is based on the ability of a matrix-isolated optical absorber to undergo a reaction while in an excited state (17, 22, 23). This reaction can be a chemical modification, e.g., irreversible molecular fragmentation, reversible tautomerization, or photoioni- zation (in the case of trapped electrons in organic glasses) (17,22). In the case of nonphotochemical hole burning (NPHB), the mechanism involves an absorber-glass cage structural relaxation that changes the physical state of the absorber. This leads to a shift of the optical transition energy and, consequently, the production of a spectral hole (at the laser frequency) whose width is determined by dephasing processes and spectral diffusion (25, 26). Zero-phonon hole widths are usually very narrow (50.2 cm-l) for T 5 4 K, when a single-frequency burn laser and low-burn intensities are used (21-23).

I I I. Experimental Procedures Instrumentation. A block diagram of the instrumentation

is shown in Figure 3. The pulsed excitation system employed is an excimer (Lambda Physik EMG 102 MSC) pumped dye laser (Lambda Physik FL-2002), which provides a spectral line width of -0.2 em-' and a temporal pulse width of -10 ns. XeCl is the excimer gas mixture, providing a pump beam of 308 nm, and various laser dyes can be used to provide wavelength tunability. The system was operated at 30 Hz, and average power densities at the sample ranging from 5 to 600 mW/cm2 were employed. Two lenses are used to form the dye lase output beam into a 10 mm X 3 mm beam at the sample, and two quartz lenses collect and focus the fluorescence into the monochromator. The 1-m focal length McPherson 2061 monochromator (F 7.0) provides a re- ciprocal linear dispersion of 0.416 nm/mm (via a 2400 grooves/" grating) and was operated at a band-pass of 0.11 nm or -8 cm-l

Figure 4. BPDE-DNA adduct with binding of the B[a]P derivative occurring at the C-10 position of the diol epoxide to the exocyclic nitrogen of guanine. for the spectra presented here. The widths of the zero-phonon lines in the spectra are therefore monochromator-limited.

Fluorescence is detected by a Tracor Northern TN-6134 in- tensified blue-enhanced gateable diode array consisting of 1024 diodes spanning a spectral window of -6 nm. Gated detection is accomplished by using a Lambda Physik EMG-97 zero drift control (to synchronize the sync out pulse to within a few na- noseconds of the laser light pulse), a Berkely Nucleonics Cor- poration Model 8010 pulse generator (used as a delay generator to set the delay, 0-1 s, between the excitation pulse and the detector's temporal observation window), and an Avtech model AVL-TN-1 high-voltage pulse generator (to set the width of the observation window, 5-120 ns). Gated operation allows for time discrimination to eliminate scattered laser light from the fluorescence and/or to temporally resolve the fluorescence of components having differing fluorescent lifetimes. All spectra presented were obtained by using gated detection with the ex- ception of the 6-MeB[a]P spectra, which were obtained in the continuous detection mode. Background subtraction and data handling and storage are performed by a Tracor Northern TN-6500 Optical Multichannel Analyzer (OMA).

Compounds. The structure of BPDE-DNA is shown in Figure 4. High-modification BPDE-DNA samples (1:183 DNA base pairs modified) were prepared by reacting anti-7,8-dihydroxy- 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with calf thymus DNA, as described previously (7). The low modification samples (5:106 and 8:107 base pairs modified) were obtained from mouse fibroblast 10T1/2 cell cultures treated with [3H]benzo[a]pyrene ([3H]B[a]P) for 24 h. Following the reaction period, the DNA was extracted and purified (27); adduction levels were determined by associated 3H label and the Randerath procedure (28). Calf thymus DNA was purchased from Sigma, St. Louis, MO, and [3H]B[a]P from Amersham Searle, Arlington Heights, IL. anti-BPDE was obtained through the NCI Chemical Carcinogen Reference Standard Repository from Midwest Re- search Institute, Kansas City, MO. s. Hecht, Naylor Dana In- stitute for Disease Prevention, American Health Foundation, Valhalla, NY, provided 1,2,3,4-tetrahydro-l,2,3,4-tetrahydroxy- 5-methylchrysene (5-MeC-tetrol), along with the diol epoxide used to prepare the corresponding DNA adduct (5-MeCDE-DNA). The adduct was prepared in the kame manner as the high modification level BPDE-DNA adduct and had a similar modification level, as determined by UV absorption (13). The benzo[a]pyrene derivatives studied, along with their acronyms to be used hereafter, are as follows: 7,8,9,10-tetrahydro-7,8,9,lO-tetrahydroxybenzo- [a] pyrene (B [a] P-tetrol), 6-methylbenzo[a]pyrene (6-MeB [ alp), . 7,8,9,10-tetrahydro-7-hydroxybenzo [ a] pyrene (7-H-THB [a] P), 7,8,9,10-tetrahydro- 10-hydroxybenzo [ a] pyrene (10-H-THB [a] P), 4,5-dihydro-9-methoxybenzo[a]pyrene-4,5-diol (B[a]P-diol), and 7,8,9,10-tetrahydrobenzo[a]pyrene (THB[a]P). The structures of each of these metabolites can easily be derived from the

FLN-NPHB Spectrometry Chem. Res. Toxicol., Vol. I , No. 1, 1988 63

5-MeC tetra1 L

I

I I 1 1 1 / I

345 350 355 360 A (nm)

Figure 5. FLN spectra of a mixture of a DNA adduct and corresponding tetrol. Top spectrum (pure 5-MeC-tetrol) and bottom spectrum (pure 5-MeCDE-DNA) show lines labeled in cm-' used for identification. All were obtained with (1,O) excitation, A,, = 343.1 nm, T = 4.2 K.

numbered structure shown in Figure 4. The 6-MeB[a]P was obtained from E. Cavalieri and E. Rogan, Eppley Institute for Cancer Research, University of Nebraska Medical Center, and B[a]P-tetrol was obtained through the NCI Chemical Carcinogen Reference Standard Repository from Midwest Research Institute, Kansas City, MO. The remaining metabolites were kindly sup- plied by R. G. Harvey, Ben May Laboratory for Cancer Research, University of Chicago. BPDE modified globin was obtained from R. M. Santella and H. Wallin, Institute for Cancer Research, Columbia University.

Glasses. The same glass-forming solvent, a mixture consisting of 50% glycerol, 40% water, and 10% ethanol by volume, was used for all samples (except where specified). Quartz tubing (3 mm 0.d. X 2 mm i.d. x 2 cm) sealed at one end was used to contain the samples (-3O-gL total volume). For the degassed samples the tube was filled with sample, subjected to several freeze/ pump/thaw cycles and then sealed off. Samples held vertically by an aluminum sample holder designed to occlude laser scatter from the sample tube edges were lowered into the Dewar ( N 2-min total immersion time). A slow cool down procedure, as used previously (15), is not necessary due to the small sample volumes used. The bulk of the experiments discussed in this paper were performed by using a double-nested 3-L glass liquid helium Dewar (Pope Scientific) having quartz windows and designed to have no liquid nitrogen in the optical pathways. For studies that required temperatures other than 4.2 K, a Janis Research Model 8-DT Super Vari-Temp liquid helium cryostat equipped with an optical access tail section was used. Sample temperatures were measured by using a silicon diode thermometer (Lake Shore Cryogenics Model DT-500 K).

IV . Results and Discussion A. Detection Limits and Selectivity. Although this

paper is primarily concerned with detection limits for FLN, we begin by presenting some data which illustrate the selectivity of the technique which were not presented in ref 12 and 13. These works dealt with the characterization of an distinction between DNA-PAH adducts and the unbound metabolites in laboratory prepared mixtures. It was noted (13) that the energy of the SI state of the unbound tetrols relative to that of their respective DNA adduct is higher by - 100-200 cm-'. This means that the distinction by FLN between a tetrol and its associated adduct is straightforward. Figure 5 illustrates this for 5-MeC-tetrol and 5-MeCDE-DNA, as defined in section 111, ref 13. The excitation wavelength chosen (Aex = 343.1

Table I. Ground- and Excited-State Vibrational Frequencies (cm-') of B[a ]P-tetrol and BPDE-DNA in

Glass at T = 4.2 K ground-state excited- ground-state excited-

(Aex = 377.1 nm) statea (AoT = 377.1 nm) statea

354 w 428 w 464 s 477 s 510 m 559 8

594 s 745 m 786 s 848 s

354 w 428 w 468 w 478 w 512 w 562 s 596 s 751 m 787 m 855 s

B [alp-tetrol 454 1118 s 47 1 1179 m

1249 s 1290 m

532 1414 s 546 1556 s 578 1592 m 757 1631 m 827 956

BPDE-DNA 452 1118 s 465 1179 m 476 1249 s 504 1289 m

1413 s 541 1560 s 579 1592 m 761 1637 m 830 957

1049 1111 1158 1170 1330 1379 1441 1517 1562 1615

1045 1109

1379 1435

1617

Excited-state vibrational frequencies for BPDE-DNA were measured from four spectra obtained with four A,, values ranging from 371.5 to 353.5 nm. Those for B[a]P-tetrol were measured from six spectra obtained with six A,, values ranging from 368.3 to 355.4 nm. The symbols s, m, and w denote intensities (strong, medium, and weak). Intensities are not given for the excited state vibrations since they depend on the A,, value, see section 11.

nm) provides for vibronic pumping at -865 and 1056 cm-' above the zero-point level for the tetrol and adduct, respectively. The cm-' values of the labeled bands are S1 excited-state vibrational frequencies. The FLN spectrum for the binary mixture is a combination of the individual component spectra given in the figure. The distinction between BPDE-DNA and B[a]P-tetrol in the presence of six other compounds is shown in ref 13. A A,, value of 366.1 nm provides for a clear distinction between BPDE- DNA and B[a]P-tetrol in a binary mixture. In this case, also, A,, provides for vibronic excitation of both species.

Continuing with the topic of selectivity we present in Table I the ground- and excited-state (S,) vibrational frequencies and intensities for B[a]P-tetrol and BPDE- DNA. The ground- and excited-state frequencies were obtained by origin and vibronic excitations, respectively. These data serve to illustrate an important point made earlier. It is that the ground-state vibrational frequencies (intensities) of the parent PAH chromophore (pyrene in this case) are quite insensitive to substituent effects while those of the S1 state are sensitive. Thus, for example, the ground-state vibrational frequencies (intensities) for B- [alp-tetrol and BPDE-DNA are very similar. From section I1 it can be understood that when this is the case and when rI (site inhomogeneous line broadening) is greater than the energy gap between the S1 states, distinction between the two species would be very difficult. However, strong substituent perturbational effects on the S1 vibrational modes coupled with the vibronic excitation allows for distinction even when the above inequality involving rI holds.

Turning now to the question of detection limits we note that with a conventional prototype FLN system, which employed a low power (-2-mW average power) N2- pumped dye laser, a detection limit for damage of -5:106 base pairs for -30 pg of DNA has been reported for the BPDE-DNA adduct a t an FLN resolution of -10 cm-'


- u L*/ L

374 375 376 377 378 379 Xlnm)

Figure 6. FLN spectrum of B[a]P-tetrol at T = 4.2 K, h, = 355.4 nm, (1,O) excitation. Upper curve is taken with low average laser excitation power density, I = 5 mW/cm2. Lower curve is taken after scanning the laser through the very broad absorption region, (0,O) band, at high laser power ( I = 600 mW/cm2).

(13). This corresponds to the detection of -1 pmol of the bound metabolite, which is roughly 3 orders of magnitude too high for practical biological applications. Our calculations indicated that the FLN system described in section I11 should provide a detection limit of -1 fmol. I t should be noted that the SI +- So absorption transition of pyrene is weak (emsll -250 a t room temperature in ethanol solution) and that its fluorescence quantum yield is -0.65 (29). For more strongly absorbing and fluorescent chromophores such as the aflatoxins one would anticipate lower detection limits.

Prior to the studies on PBDE-DNA and B[a]P-tetrol, the FLN system of Figure 3 was used to determine the detection limits for the moderately strong fluorescers 1- methylpyrene and anthracene. For both compounds a FLN detection limit of 1 fmol or -0.2 pg was achieved at a spectral resolution of -10 cm-l. This value would correspond to the detection and characterization of an equivalent 30-pg DNA sample having a damage level of l:lOs bases. Thus, the FLN system appeared to have the capability to detect and characterize the low DNA damage levels.

To detemine whether this is the case, B[a]P-tetrol and BPDE-DNA were selected for study. Using nondegassed samples, excitation of the pyrene chromophore with suf- ficiently high laser intensity led to photodegradation and, as a consequence, spectral degradation. Figure 6 shows the effect of photodegradation on the FLN spectrum of a nondegassed B[a]P-tetrol sample; the upper curve in Figure 6 is taken with low laser flux (5 mW/cm2), and the lower curve after scanning the laser through the very broad absorption region ((0-0) band) a t high laser flux (-600 mW/cm2). Significant changes are observed-the zero- phonon lines (ZPLs) have disappeared, leaving only a broad fluorescence background. Warming the sample to room temperature and then cooling to low temperature (4.2 K) did not restore the ZPLs-the photodegradation was not thermally reversible (similar results have been obtained for nondegassed BPDE-DNA samples).

For degassed samples the spectral degradation is greatly reduced. The results indicate that a significant photodegradation route in nondegassed samples is due to photooxidation. Figure 7 shows the FLN spectrum of a degassed BPDE-DNA sample having a damage level of 810' bases (curve 1) and a higher modification BPDE-DNA sample (curve 2). The signal/noise ratio of the strongest peak in curve 1 is greater than 201, resulting in a detection limit of - 1: lo7 bases for BPDEDNA. For a DNA sample of 30 pg, this corresponds to - 10 fmol of adduct detected.

2000

3 , I LL 380 381 376 377 370 379

Unm) Figure 7. FLN spectra of BPDE-DNA (degassed) with a modification level of 8 adducts in lo7 DNA bases (curve 1) and a modification level of 1 adduct in 183 DNA bases (curve 2), A,, = 369.6 nm, (1,O) excitation, T = 4.2 K.

( a ) I 1579

u 376 381

Ainmi

Figure 8. (a) FLN spectrum of BPDE-DNA (degassed). Ex- citation laser power density I = 85 mW/cm2, A,, = 369.6 nm, T = 4.2 K. The numbers correspond to excited state vibrations (in cm-'). (b) Decay of the integrated fluorescence intensity of the 579 cm-l peak (Figure 8a) for a laser excitation power density of 400 mW/cm2. (c) Resulting FLN spectrum after burning (at 400 mW/cm2) for 30 min. The experimental conditions are as in Figure 8a.

This is still an order of magnitude greater than the 1-fmol detection limit mentioned earlier, and, indeed, while the photodegradation due to photooxidation has been eliminated in the degassed samples, an efficient nonphotochemical hole burning (NPHB) process is still present. The NPHB is responsible for the higher than expected limit of detection.

Figure 8 illustrates the effect of NPHB on the FLN spectrum of a degassed BPDE-DNA sample. Figure 8a shows the FLN spectrum a t s h r t irradiation time and low laser power density (85 mW/cm2). Spectral hole burning was then carried out with high laser power density (400 mW/cm2), and Figure 8b shows the decrease of the intensity of the strongest ZPL ((0-0) site at o = 579 cm-') as a function of irradiation time. The fluorescence spectrum after hole burning is shown in Figure 8c, where one can readily observe that the ZPLs have been eliminated.

Nonphotochemical hole burning is a thermally reversible process; that is, by simply warming the sample to room

FLN-NPHB Spectrometry Chem. Res. Toricol., Vol. 1, No. 1, 1988 65

300 -

200 - I

’ooY50 500 550 600 650 760 750 800

WAVEN UM BERS ( c m-’)

Figure 9. FLN spectrum of BPDE-DNA (degassed) with a modification level of &lo7 synchronously scanning A,, and hobs over 40 cm-’ and using white light restoration pulses (see text) at T = 20 K, Z = 450 mW/cm2. Signal/noise ratio of the most intense peak is greater than 80. Note that the bottom axis is in cm-’, which represents the frequency separation between the laser and the observed fluorescence.

temperature the holes in the absorption profile are “refilled” and the absorption spectrum returns to its ori- ginal form. However, during the actual experiment, NPHB reduces both the selectivity and sensitivity of FLNS. The intensity of the ZPLs is diminished by photobleaching of the sites that have their zero-phonon transition at the laser frequency. As a result, the characteristic FLN fingerprint of the molecule under study can be eventually destroyed. The NPHB is particularly troublesome for the measure- ment of very weak fluorescence signals from low concen- tration (-1 fmol) samples, since the high laser power densities required for detection also accelerate the NPHB process. Thus, in order to improve the limits of detection for BPDE-DNA (and other samples in which NPHB occurs), methods for circumventing NPHB are required.

The underlying principles of NPHB (see section 11) suggest that the deleterious effect of NPHB on FLN can be reduced by the simultaneous and synchronous scanning of the laser excitation and monochromator wavelengths, provided the scan interval is less than rI (Figure 1). Scanning of the laser limits the time of exposure for each isochromat. It was found that simultaneous scanning does not significantly alter the FLN structure for scans of -50 cm-’ or less and that the ZPL intensities are increased. Zero-phonon hole burning can also be decreased by sub- jecting the sample to white light restoration pulses (30). Furthermore, the NPHB quantum efficiency can be reduced by operating at elevated temperatures (- 10-20 K) (23). Utilization of temperatures much greater than 20 K leads to undesirable thermal broadening of the ZPL.

Figure 9 shows the result of an experiment on BPDE- DNA in which synchronous scanning a t T = 20 K and white light pulses (1-ms duration delayed 1 ms after the laser pulse) were employed. The signal/noise ratio of the strongest peak in Figure 9 is greater than 80, so a limit of detection of -3 adducts:lOs bases, for a 20-pg DNA sample, is obtained. This corresponds to -2 fmol of adduct detected and illustrates that FLNS does have the requisite sensitivity (comparable to that of ELISA (8, 10)) to characterize DNA damage at the low levels found in biological samples.

To conclude this subsection we discuss and present data for a new analytical method that combines FLN and NPHB. It is an extension of the procedure utilized by Bogner and Schwarz (31) and Fiinfschilling and co-workers (32) to the case of vibronic excitation. The method is

I C )

BPDE-DNA

5,

381 Xhm)

376

Figure 10. FLN spectra of BPDE-DNA, A,, = 369.6 nm, T = 4.2 K. (a) Spectrum obtained with a laser excitation power density, Z = 85 mW/cm2 and an exposure time of -100 s. (b) Spectrum obtained under experimental conditions as in a, following a 100-s exposure to 400-mW/cm2 excitation power density. (c) The “difference” spectrum (a - b) obtained, as discussed in detail in the text. The numbers correspond to excited state vibrations in cm-’ (see Table I).

designed to eliminate the broad fluorescence contribution, which arises from excitation of sites via phonon sideband absorption, from the spectrum. Such elimination would result in a spectrum originating only from sites excited by zero-phonon absorption and, as a consequence, improved selectivity. The method involves recording the FLN spectrum at different stages of the NPHB process. A fixed A,, can be utilized. As the NPHB a t A,, proceeds, the fluorescence spectrum degrades due to the diminution of fluorescence originating from sites excited by their zero- phonon transitions. Consider that spectrum “a” is obtained a t an earlier irradiation time than spectrum “b”. The difference spectrum, “a” - “b”, will then represent the FLN spectrum of the molecules that have been burned out by irradiation during the time interval between the recording of the two spectra. The results of such an experiment are presented in Figure loa-c for BPDE-DNA. Spectrum c is the difference spectrum, which is considerably sharper than spectrum b and even spectrum a. Spectrum c can be compared directly with spectrum a of Figure 8; the former is better resolved than the latter since the latter is contributed to by fluorescence from sites excited by zero-phonon and phonon sideband transitions. The latter type of transition does not contribute to spectrum c of Figure 10. The results of Figure 10 are important also because higher laser intensities can be utilized while pre- serving the structure necessary for characterization. The difference spectra methodology can also be applied to short-lived fluorescing species for which gated fluorescence detection is not possible and scattered laser light poses a problem. The FLN and difference spectra for 6-MeB[a]P are presented in Figure 11. The spectra were obtained with vibronic excitation and were recorded in the continuous mode (not gated) of the OMA system; the high scattered laser background apparent in spectra a and b is virtually eliminated in the difference spectrum.


408 412

X(nm)

Figure 11. FLN spectra of 6-MeB[a]P, A,, = 388 nm, (1,O) excitation, T = 4.2 K. (a) Laser excitation power density, I = 80 mW/cm2. (b) Identical conditions with a after several minutes of continuous irradiation with the same laser power density, I = 80 mW/cm2. (c) The ”difference” spectrum showing the useful analytical peaks labeled in cm-’.

> m z w F-

k

z W 0 Z W 0 W LL 0 3 L L

Figure 12. FLN spectra of BPDE-globin (13.4 nmol of BPDE- adduct/mL of globin) using (1,O) excitation: (a) A,, = 369.6 nm and (B) A, = 368.6 nm. The labeled peaks correspond to excited-state vibrational frequencies, in cm-l (see Table 11).

B. New Applications of FLN. (1 ) BPDE-Globin Adducts. Given that FLN is applicable to DNA-PAH adducts, there are no apparent reasons why it should not be applicable to globin-PAH adducts. The question of whether or not it is is significant because there appears to be some correlation between DNA damage and globin damage (33). As proof of principle, Figure 12 shows the FLN spectra for BPDE-globin obtained by vibronic excitation a t A,, = 369.6 nm and A,, = 368.6 nm.

The modification level for the adduct is 13.4 nmol of BPDE-adduct/mL of globin. All fluorescence peaks in Figure 12 correspond to the multiplet origin structure so that the frequency labels correspond to excited state vibrational frequencies. The upper dashed spectrum (A) in Figure 12 was obtained with the same A,, that was used to obtain spectrum a of Figure 8 for BPDE-DNA. The

Table 11. The Ground- and Excited-State Vibrational Frequencies (cm-’) of BPDE-Globin in Glass at T = 4.2 K

ground-state excited- ground-state excited- (Aex = 377.0 nm) state (Aex = 377.0 nm) state

341 w 347 1411 s 675 360 w 381 1558 m 720 425 w 409 1593 w 745 464 m 457 1633 w 764 688 w 473 833 760 w 485 894 786 s 502 960 821 w 542 1021 845 s 552 1028

1113 m 583 1049 1176 w 622 1114 1246 m 653

Excited-state vibrational frequencies greater than - 1200 cm-’ were not measured. The excited-state vibrational frequencies were obtained from seven FLN spectra obtained with seven different A, values ranging between 371.5 and 362.9 nm. See caption to Table I.

583- and 473-cm-l bands of the former spectrum correlate with the 579- and 476-cm-’ bands of the latter. The intensity distribution differences in the two spectra are the result, in part, of the fact that the S1 state of BPDE-globin is red-shifted by -70 cm-l relative to the S1 state of BPDE-DNA. The lower spectrum of Figure 12 bears a resemblance to spectrum a of Figure 8 because it was obtained with a A,, value that corresponds to an excitation energy that is 73 cm-l higher than that associated with A,, = 369.6 nm. By decreasing and increasing A,, relative to A,, = 369.6 nm, the excited vibrational frequencies for BPDE-globin given in the second column of Table I1 were obtained from a series of spectra. The ground-state vibrational frequencies given in Table I1 were obtained by origin excitation a t A,, = 377.0 nm. Comparison of these vibrational frequencies with those presented in Table I, taking into account that frequencies between 464 and 688 cm-’ for BPDE-globin were not measured, shows a strong similarity in the ground-state vibrational frequencies between these two adducts.

No attempt was made to determine the detection limit for BPDE-globin as was done for BPDE-DNA. However, there is no apparent reason why its detection limit should not be comparable to that for BPDE-DNA.

(2) Metabolites in Urine. As a further demonstration of applicability, FLNS has been applied to the direct analysis of several polar metabolites of B[a]P in urine: B[a]P-tetrol, 7-H-THB[a]P, lO-H-THB[a]P, THB[a]P, and B[a]P-diol (see section 111). The importance of de- tecting polar metabolites of PAH in urine arises from the fact that a significant percentage of PAH absorbed by the body can eventually show up in urine as polar metabolites. A technique which can detect PAH metabolites in urine can therefore be used to monitor human exposure to PAH.

Figure 13 shows the FLN spectra of B[a]P-tetrol in a 1:l urine/pure glass mixture, “urine glass”. The spectrum in Figure 13a was obtained by using vibronic excitation, and, therefore, the labeled peaks (in cm-‘) correspond to excited-state vibrations. The spectrum in Figure 13b was acquired with origin excitation and, therefore, its peaks correspond to ground-state vibrations. Comparison of the vibrational frequencies found in these two spectra with those listed in Table I for B[u]P-tetrol in pure glass shows that there is a good agreement.

For B[a]P-tetrol in urine, a -1-pmol limit of detection has been obtained; further lowering of this limit is hampered by the presence of many other fluorescing compounds present in urine, which yield a relatively strong and

FLN-NPHB Spectrometry

r I t

v) z w I-

w 0

z w

t

2

A ) * 4 J I 8 (a ) P tet rol

P- IC t P 1 lB

t

v) z w

t

i W 0 z w u v)

w K 0 3 Y

8 )

393 394 395 396 587 398 399 h(nm)

Figure 13. FLN spectra of B[a]P-tetrol in “urine glass”. (a) Spectrum obtained with (1,O) excitation (Aex = 369.6 nm). The labeled peaks correspond to excited state vibrational frequencies, in cm-’. (b) Spectrum obtained with (0,O) excitation (Aex = 377.1 nm). The labeled peaks correspond to ground-state vibrational frequencies, in cm-’.

broad background. A simple sample cleanup procedure could be used to remove these interfering compounds, resulting in a lower detection limit. Such a procedure would probably involve either an extraction with a non- polar solvent or preconcentration on a disposable chro- matographic column.

Similar results were obtained for the other metabolites studied. These results indicate that FLNS could provide a convenient method to monitor human exposure to carcinogens.

V. Concluding Remarks The principal objective of this work was to determine

the detection limit for intact DNA-carcinogen adducts achievable with FLN spectrometry. With the often studied BPDE-DNA adduct as a model adduct, a detection limit of -3 modified bases in lo8 (20 pg of DNA) was determined. This value is comparable to the best limits of detection for intact adducts that have been achieved with other techniques, e.g., ELISA. It should be noted, however, that FLNS is readily applicable to DNA-PAH adducts, whereas ELISA requires that a specific antibody to made for each adduct of interest, a difficult and time-consuming process. In obtaining the approximate femtomole FLNS detection limit for the bound metabolite a t a spectral resolution of -8 cm-l, it was necessary to eliminate photooxidation and the deleterious effects of nonphotochemical hole burning (NPHB). The former task was trivial, while the latter necessitated synchronous scanning of the excitation laser and monochromator together with two straightforward approaches that led to spectral diffusion/hole filling. In so doing a novel double selection methodology, which employs FLN and NPHB, was developed that enhances the selectivity of standard FLN spectrometry. In essence, this methodology minimizes the intensity of the broad phonon sidebands that can often lead to a degradation in selectivity.

Chem. Res. Toxicol., Vol. 1 , No. 1, 1988 67

In addition to being highly selective and sensitive, FLN spectrometry is a practical technique. For a laser-based method the technology is simple (components available commercially) and the procedure for rapid sample cool- down (-2 min) ensures a sample turnover rate that is high. Each spectrum presented here was acquired on average in about 5 min. Thus, when the optimum excitation wavelengths for species of interest are known, dozens of samples can be analyzed during the course of a day. At the same time, spectral features due to unknown components in a mixture would be identified.

With the attributes of high sensitivity and selectivity and rapid sample throughput, along with the results presented here and in ref 1 2 and 13, one can conclude that FLNS is ideally suited for the analysis of mixtures of similar but distinct intact DNA-PAH adducts. Its applicability to globin-PAH adducts and polar metabolites in urine has also been established. Moreover, FLN is operative for heme proteins (34) and photosynthetic an- tenna protein complexes (35). Recently FLNS has been applied to the investigation of the metabolic processes involved in the formation of DNA adducts in mice exposed to B[a]P (36). The important conclusion that should be drawn from all of these applications is that FLNS is applicable to a wide range of biomolecules and biomolecular problems.

Acknowledgment. This research was supported by the Office of Health and Environmental Research, Office of Energy Research, and by NCI Grant CA02111. We are indebted to Dr. H. Wallin and Dr. R. Santella for providing us with the BPDE-globin adduct and thank Dr. J. M. Hayes for many useful discussions.

References (1) Maugh, T. H., IL (1984) “?‘racking exposure to toxic substances“.

Science (Washington, D.C.) 226, 1183-1184. (2) Miller, E. C. (1978) “Some current perspectives on chemical

carcinogenesis in humans and experimental animals: presidential address”. Cancer Res. 38, 1479-1496.

(3) Nicolni, C., Ed. (1982) Chemical Carcinogenesis, Plenum, New York.

(4) Cavalieri, E., and Rogan, E. (1985) “Role of radical cations in aromatic hydrocarbon carcinogenesis”. EHP, Enuiron. Health Perspect. 64, 69-84.

( 5 ) Sims, P., Grover, P. L., Swaisland, A., Pal, K., and Hewer, A. (1974) “Metabolic activation of benzo[a]pyrene proceeds by a diol-epoxide”. Nature (London) 252, 326-328.

(6) Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H., and Naka- nishi, K. (1976) “Benzo[a]pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo”. Science (Washing- ton, D.C.) 193, 592-595.

(7) Jeffrey, A. M., Weinstein, I. B., Jennette, K. W., Grzeskowiak, K., Nakanishi, K., Harvey, R. G., Autrup, H., and Harris, C. (1977) “Structures of benzo[a]pyrene-nucleic acid adducts formed in human and bovine bronchial explants”. Nature (London) 269,

(8) Jeffrey, A. M. (1985) “Polycyclic aromatic hydrocarbon-DNA adducts: formation, detection and characterization”. In Poly- cyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) pp 187-208, American Chemical Society, Washington, DC.

(9) Randerath, K., Randerath, E., Agrawal, H. P., Gupta, R. C., Schurdak, M. E., and Reddy, M. V. (1985) “Postlabeling methods for carcinogen-DNA adduct analysis”. EHP, Enuiron. Health Perspect. 62, 57-65.

(10) Harris, C. C., Yolken, R. H., and Hsu, I. C. (1982) “Enzyme immunoassays: applications in cancer research”. In Methods in Cancer Research (Busch, H., and Yoeman, L. C., Eds.) pp 213-243, Academic, New York.

(11) Heisig, V., Jeffrey, A. M., McGlade, M. J., and Small, G. J. (1984) “Fluorescence-line-narrowed spectra of polycyclic aromatic carcinogen-DNA adducts”. Science (Washington, D.C.) 223,

(12) Sanders, M. J., Cooper, R. S., Small, G. J., Heisig, V., and

348-350.

289-291.

68 Chem. Res. Toxicol., Vol. 1, No. 1, 1988

Jeffrey, A. M. (1985) “Identification of polycyclic aromatic hydrocarbon metabolites in mixtures using fluorescence line narrowing spectrometry”. Anal. Chem. 57, 1148-1152.

(13) Sanders, M. J., Cooper, R. S., Jankowiak, R., Small, G. J., Heisig, V., and Jeffrey, A. M. (1986) “Identification of polycyclic aromatic hydrocarbon metabolites and DNA adducts in mixtures using fluorescence line narrowina sDectrometrv”. Anal. Chem. 58,

(14) Personov, R. I. (1983) “Site selection spectroscopy of complex molecules in solutions and ita applications”. In Spectroscopy and Excitation Dynamics of Condensed Molecular Systems (Agra- novich, V. M., and Hochstrasser, R. M., Eds.) pp 555-619, North-Holland, New York.

Brown, J. C., Edelson, M. C., and Small, G. J. (1978) “Fluorescence line narrowing spectrometry in organic glasses containing park-per-billion levels of polycyclic aromatic hydrocarbons”. Anal. Chem. 50, 1394-1397.

(16) Personov, R. I., and Kharlamov, B. M. (1973) “Extreme narrowing of bands in the fluorescence excitation spectra of organic molecules in solid solutions”. Opt. Commun. 7, 417-419.

(17) Weber, M. J., Ed. (1987) “Optical linewidths in glasses” J . Lumin. 36 (4, 5).

(18) Chiang, I., Hayes, J. M., and Small, G. J. (1982) “Fluorescence line narrowing spectrometry of amino polycyclic aromatic hydrocarbons in an acidified organic glass”. Anal. Chem. 54,315-318.

(19) Brown, J. C., Duncanson, J. A., Jr., and Small, G. J. (1980) “Fluorescence line narrowing spectrometry in glasses for direct determination of polycyclic aromatic hydrocarbons in solvent-refined coal”. Anal. Chem. 52, 1711-1715.

(20) Brown, J. C., Hayes, J. M., Warren, J. A., and Small, G. J. (1981) “New laser-based methodologies for the determination of organic pollutants via fluorescence”. In Lasers in Chemical Analyeis (Hieftje, G. M., Travis, J. C., and Lytle, F. E., Eds.) Chapter 12, Humana, Clifton, NJ.

(21) Personov, R. I., Al’shita, E. I., and Bykovskaya, L. A. (1972) “The effect of fine structure appearance in laser-excited fluorescence spectra of organic compounds in solid solutions”. Opt. Commun. 6,169-173.

(22) Hayes, J. M., Jankowiak, R., and Small, G. J. (1987) “Two- level-system relaxation in amorhous solids as probed by non photochemical hole-burning in electronic transitions”. In Topics in Current Physics. Persistent Spectral Hole Burning: Science and Applications (Moerner, W. E., Ed.) Chapter 5 and other chapters therein, Springer-Verlag, New York.

(23) Jankowiak, R., and Small, G. J. (1987) “Hole-burning spectroscopy and relaxation dynamics of amorphous solids a t low temperatures”. Science (Washington, D.C.) 237, 618-625.

816-820.

(15)

Jankowiak et al.

(24) Warren, J. A,, Hayes, J. M., and Small, G. J. (1986) ‘Vibronic mode mixing in the SI state of P-methylnaphthalene”. Chem.

(25) Jankowiak, R., Shu, L., Kenney, M. J., and Small, G. J. (1987) “Dispersive kinetic processes, optical linewidths and dephasing in amorphous solids”. J . Lumin. 36, 293-305.

(26) Walsh, C. A., Berg, M., Narasimhan, L. R., and Fayer, M. D. (1986) “Optical dephasing of chromophores in an organic glass: picosecond photon echo and hole burning experiments”. Chem. Phys. Lett. 130, 6-11; (1987) “Dynamics in low temperature glasses: theory and experiments on optical dephasing, spectral diffusion, and hydrogen tunneling”. J . Chem. Phys. 86, 77-87.

Brown, H. S., Jeffrey, A. M., and Weinstein, I. B. (1979) “Formation of DNA adducts in 10T1/2 mouse embryo fibroblasts incubated with benzo[a]pyrene or dihydrodiol oxide derivatives”. Cancer Res. 39, 1673-1677.

(28) Reddy, M. V., Gupta, R. C. , Randerath, E., and Randerath, K. (1984) “32P-postlabeling test for covalent DNA binding of chem- icals in vivo: application to a variety of aromatic carcinogens and methylating agents”. Carcinogenesis 5, 231-243.

(29) Birks, J. B. (1970) Photophysics of Aromatic Molecules, pp 71, 128, Wiley-Interscience, New York.

(30) Kharlamov, B. M., Personov, R. I., and Bykovskaya, L. A. (1974) “Stable ‘gap’ in absorption spectra of solid solutions of organic molecules by laser irradiation”. Opt . Commun. 12,

(31) Bogner, U., and Schwarz, R. (1981) “Laser-induced changes in the sideband shape of selectively excited dyes in noncrystalline organic solids a t 1.3 K”. Phys. Reu., B: Condens. Matter B24,

(32) Funfschilling, J., Glatz, D., and Zschokke-Granacher, I. (1986) “Hole-burning spectroscopy as a tool to eliminate inhomogeneous broadening”. J . Lumin. 36, 85-92.

(33) Osterman-Golkar, S., Ehrenberg, L., Segerback, D., and Hal- strom, I. (1976) “Evaluation of genetic risks of alkylating agents. 11.

(34) Koloczek, H., Fidy, J., and VanderKooi, J. M. “Fluorescence line-narrowing spectra of Zn-cytochrome c. Temperature dependence” J. Chem. Phys. (in press).

(35) Avarmaa, R., Renge, I., and Mauring, K. (1984) “Sharp-line structure in the fluorescence and excitation specra of greening etiolated leaves”. FEBS Lett. 167, 186-190.

(36) Zamzow, D., Jankowiak, R., Cooper, R. S., Small, G. J., Rogan, E. G., and Cavalieri, E. L. “Fluorescence line narrowing investigation of DNA adducts formed by one-electron oxidation from benzo[a]pyrene”. J . Am. Chem. SOC. (to be submitted for publi- cation).

Php . 102, 313-323.

(27)

191-193.

2846-2849.

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