Chem. Res. Toxicol. 1988,1, 60-68
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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 50011
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 P A H 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. T h e methodology employed avoids or minimizes spectral degradation a n d loss of sensitivity due t o photooxidation and nonphotochemical hole burning (NPHB). A new double selection technique t h a t employs both FLN and N P H B is described and found to lead t o a significant improvement in selectivity over t h a t 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 desirable. 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 nondestructive. 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: oneelectron oxidation to yield radical cations ( 4 ) and monooxygenation 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 Randerath 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
analytes are dissolved in a glass-forming solvent which is cooled to cryogenic temperatures (typically 4-20 K). Appropriate 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). It 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, 5methylchrysene, 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]pyreneled 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-
0893-228~/88/2701-0060$01.50/0 0 1988 American Chemical Society
Chem. Res. Toxicol., Vol. 1, No. 1, 1988 61
FLN-NPHB Spectrometry
frequency
PHB
(b) After burning
I
Figure 2. Schematicrepresentation of laser site excitationenergy selection in FLNS (see explanation in text).
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Figure 1. (a) Inhomogeneous line broadening and (b) photochemical and nonphotochemicalhole 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(productncorresponds to new impurity-glass site configurations produced by light-induced TLS,,, relaxation.
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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 (11-20) 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 inhomogeneously 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
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spectrum and, as a consequence, poor selectivity. However, selection of a narrow isochromat of the absorption profile with a laser of width AoL k m
z
w F-
z W
0 Z W
0 W LL
0
3
L L
Figure 12. FLN spectra of BPDE-globin (13.4 nmol of BPDEadduct/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
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 S1state 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 detecting 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
Chem. Res. Toxicol., Vol. 1, No. 1, 1988 67
FLN-NPHB Spectrometry r t
t v)
4 J
z
w
I
I-
2
IC
t
1
w z
t
8 ( a ) P tet rol
P-
0 w
t
I
*
A)
P lB
8)
z v)
w
i
W
0
z w u v) w K
0
3 Y
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 nonpolar solvent or preconcentration on a disposable chromatographic 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.
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 cooldown (-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 antenna 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 Nakanishi, K. (1976) “Benzo[a]pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo”. Science (Washington, 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, 348-350. (8) Jeffrey, A. M. (1985) “Polycyclic aromatic hydrocarbon-DNA adducts: formation, detection and characterization”. In Polycyclic 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-narrowedspectra of polycyclic aromatic carcinogen-DNA adducts”. Science (Washington, D.C.) 223, 289-291. (12) Sanders, M. J., Cooper, R. S., Small, G. J., Heisig, V., and
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, 816-820. (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 (Agranovich, V. M., and Hochstrasser, R. M., Eds.) pp 555-619, North-Holland, New York. (15) 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) “Theeffect 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) “Twolevel-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 at low temperatures”. Science (Washington, D.C.) 237, 618-625.
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