2692
Anal. Chem. 1988, 60, 2692-2694
without recompressing the entire library. A plethora of applications exist that require calculating eigenvectors. Robust eigenvectors may prove useful for other applications.
(2) (3) (4) (5) (6)
ACKNOWLEDGMENT The authors thank John Marshall and David Booth for their valuable comments and criticisms. The previous workers at the University of North Carolina, Chapel Hill, NC, are thanked for supplying and assisting in the collection of the GC/FT-IR data. LITERATURE CITED (1) Hangac, G.; Wielboldt, R. C.; Lam, R. B.;Isenhour, T. L. Appl. Specfrosc. 1982, 36, 40.
(7) (8) (9)
Harrlngton, P. 8.; Isenhour, T. L. A w l . S p e c m c . 1987, 4 1 , 449. Harrlngton, P. B.; Isenhour, T. L. Anal. Chim. Acta 1987, 797, 105. Giilette, P. G.; Koenig, J. L. Appl. Spectrosc. 1982, 36, 535. Williams, S. S.: Lam, R. B.; Isenhour. T. L. Anal. Chem. 1983, 55, 1117. Maiinowski, E. R.; Howery, D. G. Factor Analysis In Chemisfry; WileyInterscience: New York, 1960. Phillips, G. R.; Eyring. E. M. Anal. Chem. 1983. 55, 1134. Campbell, N. A. Appl. Stafkf. 1980, 29, 231. Harrington, P. B.: Isenhour, T. L., Chapel Hill, NC, unpublished work, 1987.
RECEIVED for review August 18, 1987. Resubmitted August 26,1988. Accepted September 15,1988. This research was by the National Science Foundation under Grant CHE-8415670.
Fluorescence Line Narrowing Spectrometry of Nucleoside-Polycyclic Aromatic Hydrocarbon Adducts on Thin-Layer Chromatographic Plates R. S. Cooper, Ryszard Jankowiak, J. M. Hayes, L u Pei-qi, and G. J. Small*
Ames Laboratory-USDOE
and Department of Chemistry, Iowa State University, Ames, Iowa 50011
Fluorescence llne narrowlng spectrometry (FLNS) is applied to a polycycllc aromatic hydrocarbon-nucleoside adduct sorbed on a thin-layer chromatography (TLC) plate. A caiibration curve over more than 5 decades of concentration Is presented and yleids a detectlon ilmlt of -10 fmol. The effects of nonphotochemkal hole burning (NPHB) are investigated and dlscussed. Results Indicate that FLN spectrometry can be Interfaced wlth the s2P-postlabeflngprocedure to provide for high sensltlvlty and selectivity analysis of TLC spots assoclated with fluorescing nucleotide adducts.
I. INTRODUCTION Fluorescence line narrowing (FLN) spectrometry has recently been shown to possess the attributes required for the analysis of cellular macromolecular (e.g., DNA, globin) damage resulting from covalent binding between the macromolecule and carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs) (I). The formation of a DNA adduct is generally believed to be a crucial step in the pathway that leads to carcinogenesis and tumorigenesis (2-4). Because of their environmental prevalence (5) and broad spectrum of activity in animal tissues (6))PAHs have received considerable attention in connection with chemically initiated carcinogenesis. Since many PAHs and their polar metabolites (unbound or bound to a cellular macromolecule) exhibit high fluorescence quantum yields, fluorescence-based bioanalytical techniques are well-suited for addressing a variety of analysis problems associated with PAH induced carcinogenesis. In ref 1 a detection limit of -3 modified bases in los for a DNA adduct (7) formed with a diol-epoxide of benzo[a]pyrene (BP) was reported for 20 rg of DNA at a spectral resolution of 8 cm-I. This limit corresponds to the detection of about 1fmol of the bound metabolite. Such a level of detectability is required for practical analysis of DNA damage on samples 0003-2700/88/0360-2692$01 SO10
from animals or humans (8). Selectivity is also an important consideration. Studies, which preceded the work reported in ref 1, had established that FLN spectrometry possesses the selectivity to directly distinguish between five DNA adducts formed from BP, 5-methylchrysene, chrysene, and benz[a]anthracene via the monooxygenation diol-epoxide route (9). Comparable selectivity has also been demonstrated for rather complex mixtures of structurally similar PAH metabolites (IO). Only enzyme-linked immunosorbent assay (ELISA) possesses a selectivity and sensitivity for DNA adduct analysis that is comparable to that of FLN spectrometry (11). Although the 32P-postlabeling (Randerath) procedure (12-15) is limited to the analysis of nucleotide-metabolite adducts, it is noteworthy since its detectability exceeds that of ELISA and FLN by 2-3 orders of magnitude (12,14).In the Randerath procedure adducted and nonadducted (normal) labeled nucleotides are applied as a spot to a thin-layer chromatographic (TLC) plate which is then developed in four dimensions, two of which serve to resolve different adducts. Detection of the adducts is performed autoradiographically. However, when suitable standards for w e with TLC are not available, the characterization of the unknown spots is very difficult. This is particularly so for analysis of DNA adducts formed in vivo (16,17).Even when a match between a TLC spot of a sample component with that of a standard is achieved, it would be highly advantageous to be able to spectroscopically confirm that the suspected adduct is the actual adduct. An additional complication arises in the Randerath procedure when the developed TLC plate exhibits diffuse multicomponent zones. A spectroscopic technique that allows for resolution of such a zone into constituent components would be particularly valuable. With this and the versatility of FLN spectrometry in mind, we have initiated studies designed to explore the feasibility of coupling FLN with the 32P-postlabeling methodology. Fluorescence line narrowing for PAH sorbed on TLC plates @ 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 2603
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had previously been reported by Hofstraat et al. (18,19). In this paper we report FLN spectra and a detection limit of 10 fmol for a deoxyguanosine adduct, formed from BP, sorbed on a TLC plate. A calibration curve for this adduct over 5 decades is also presented. The complications for analysis that arise from spectral hole burning (1,20) are considered and compared with the same for glassy matrices. A detection limit of -1 fmol is projected for the above adduct based on the recent advances in FLN spectrometry presented in ref 1.
11. EXPERIMENTAL SECTION An excimer (Lambda Physik EMG 102 MSC) pumped dye laser (Lambda Physik FL-2002), having a spectral line width of -0.2 cm-* and a pulse width of -10 ns, and operating at 30 Hz was the excitation e~urceemployed. XeCl was the excimer gas mixture utilized, providing a pump beam of 308 nm, and PBBO (Exciton) was used to obtain wavelength tunability between 386 and 420 nm. Tunability over this wavelength range is sufficient for excitation of the origin and low-lying vibronic transitions of BP and the Model BP adduct analyzed, 8-[benzo[a]pyren-6-yl]deoxyguanosine (C8dG-BP). The output from the dye laser was shaped into a 5 mm X 5 mm beam at the sample, with excitation power densities ranging from 5 to 250 mW/cm2. Fluorescence was collected at a right angle to the excitation and focused into a 1-m McPherson 2061 monochromator (F 7.0) having a reciprocal linear dispersion of 0.416 nm/mm. Fluorescence was detected by using a Tracor-Northern TN-6134 intensified blue-enhanced gateable photodiode array (PDA) of 1024 diodes spanning a spectral window of -6 nm. Gated detection of the fluorescence was accomplished with a Lambda Physik EMG-97 zero drift control (ZDC) to synchronize the sync out to within a few nanoseconds of the excimer light pulse, a Berkeley Nucleonics Corp. Model 8010 pulse generator used as a delay generator (DG) to set the delay (0-1 s) between the laser and the detector's temporal observation window, and an Avtech Model AVL-TN-1 high-voltage pulse generator (HV) to set the width of the observation window (5-120 ns). All data handling, including background subtraction and data storage, were performed with a Tracor-Northern TN-6500 optical multichannel analyzer (OMA). The synthesis of C8dG-BP by electrochemical oxidation has been described in ref 21. Ita structure is shown in Figure 1from which it is apparent that BP is the parent fluorescent chromophore. Samples of the C8dG-BP adduct were dissolved in water and were diluted to different concentrations (104-104 M). A 5-rL portion of each prepared concentration was spotted onto a TLC Whatman, Clifton, NJ) using plate (KC18 reversed phase, 200 e, a 50-pL glass syringe. Prior to spotting, the TLC plates were purified by developing them overnight in methanol (Altech, Deerfield, IL) followed by drying. Subsequently, the plates were cut into 1cm X 3 cm rectangles and the majority of the stationary phase was removed by scraping with a razor blade. A square (-5 mm X 5 mm) was left on the plate and to this area the sample was spotted. This procedure was used to ensure that the sample was deposited into a precisely defined area of the plate. Following the application of the sample, the plates were allowed to dry. The plates were attached to a sample holder and cooled to 4.2 K by
Flgure 2. FLN spectrum of the C8dGBP nucleoside adduct sorbed onto a TLC plate. Excitation laser flux = 30 mW/cm*, A, = 395.7 nm (vibronic excitation), T = 4.2 K. The numbers above the peaks correspond to excited state vibrations (in cm-I).
immersion in boiling helium contained in a glass liquid helium cryostat (Pope Scientific). The laser excitation beam was directed at an angle of -30° to the normal of the TLC plate in order to reduce specular reflection of the beam onto the entrance slit of the monochromator. Gated detection was used to eliminate scattered laser light from the fluorescence signal.
111. RESULTS AND DISCUSSION The FLN spectra of five BP-nucleoside adducts (including C8dG-BP) imbedded in a glycero1:water based glass have recently been reported (22). All adducts were synthesized by electrochemical oxidation of BP in the presence of guanosine, deoxyguanosine, and deoxyadenosine (21). Figure 2 shows the FLN origin multiplet structure of C8dG-BP sorbed on a TLC plate. The spectrum was obtained with vibronic excitation a t A,,(excitation wavelength) = 395.7 nm, and a 20-118 delay time. These values were determined to provide maximum intensity of the origin band multiplet. The origin components are labeled with cm-' values corresponding to their displacements from the laser frequency. As discussed at length in ref 1,these displacements yield the excited-state frequencies of the fundamental vibrations pumped by the laser. The structure in Figure 2 is very similar to that observed for C8dG-BP in the glass (22). The spectrum in Figure 2 was obtained with a relatively low excitation flux (-30 mW/cm2) and short acquisition time (-9 s) in order to minimize the deleterious effects of nonphotochemical hole burning (NPHB) (1,22). The NPHB produces a persistent spectral hole at AB whose growth is governed by dispersive kinetics stemming from the inherent structural disorder of the host medium (20,23,24). As discussed in ref 1,the production of a spectral hole can lead to a diminuation in the intensities of the zero-phonon lines (ZPL) in the FLN spectrum. In the extreme, the ZPL structure is lost and replaced by broad fluorescence which originates from analyte sites excited via their phonon sidebands. Figure 3 illustrates the effect of laser intensity on the rate at which the ZPL intensity decreases due to NPHB. The ZPL whose intensity was monitored corresponds to the 523-cm-' band of Figure 2. For region "A" a low excitation flux of -30 mW/cm2 was used and it can be seen that ZPL signal, although noisy, is nearly constant during the irradiation time. The noise in region "A" is due to the adduct concentration being near the detection limit. For region "B", obtained with an excitation flux of -170 mW/cm2, a significant decay in the ZPL intensity is observed after the initial increase in the intensity produced by the onset of the higher flux. We have analyzed the decay following the procedure of ref 20 and find that the kinetics are dispersive as expected for NPHB. Comparison of the ZPL decays for C8dG-BP in the glass (22) and on the TLC plate indicates that NPHB for the latter is about 10 times less
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
IV. CONCLUSIONS The improvements proposed above for achieving 1fmol detection limits are those that were used in ref 1 to achieve similar detection limits in glasses. Although femtomole (or 1 adduct in lo8 bases) sensitivity is not compatible with the most sensitive variants of the Randerath procedure, several recent studies of DNA adducts by the postlabeling procedure (16,25-27)have indicated adduction levels in the 1in 108 or higher range. Additionally, the adducts detected in those studies had mobilities distinct from the adduct formed by reaction of 7,8-diolg,lO-oxide of benzo[a]pyrene or from the adducts formed with the principal polycyclic aromatic compounds that have been characterized. Thus, the problem of identifying the adducts detected by the Randerath procedure presents a significant limitation to epidemiological studies. Fluorescence l i e narrowing spectrometry of the adduct spots on TLC plates provides a means of obtaining important spectroscopic information to aid in the identification.
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Flgure 4. Plot of moles of C8dQ-BP sorbed on a TLC plate versus fluorescence intensity (523-cm-‘ peak, Figure 2), kx= 395.7 nm, T = 4.2 K. Intensities were normalized to a 15-s detector integration time.
efficient than in the glass. A similar observation was made for tetracene in ref 19. The deleterious effects of NPHB on FLN can be significantly minimized by synchronous scanning of the excitation laser and monochromator (so that a fresh isochromat is continuously excited) and by increasing the sample temperature to -15-20 K (1). At higher temperatures spontaneous hole filling is more efficient than at 4.2 K. This filling serves to restore the ZPL intensities. However, synchronous scanning and a higher sample temperature were not used to obtain the data that are presented next. Figure 4 is a plot of the 523-cm-l ZPL line intensity for C8dG-BP versus concentration on the TLC plate. The data yield a detection limit of -10 fmol for a signal-to-noise (SIN) ratio of 3. It is important to note that the TLC plates were spotted in such a manner that the sample covered an area 5 mm X 5 mm in order to mimic the spot dimensions encountered with 32P-postlabelingand that the monochromator aperature used only sampled 6% (0.3 mm X 5 mm) of the spot area. Taking this into account yields a limit of detection of 0.6 fmol/l.5 mmz (sampled spot area). Additionally, the data for Figure 4 were obtained by using a laser excitation flux of only 30 mW/cm2 in order to avoid NPHB. Utilization of a flux of -300 mW/cm2 together with synchronous scanning and a sample temperature of 15-20 K can be expected to decrease the detection limit for C8dG-BP to 1fmol (or 60 amol/l.5 mm2) based on the results of ref 1. Use of a more suitable collection aperature can be expected to yield an even lower limit of detection.
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LITERATURE CITED (1) Jankowiak, R.; Cooper, R. S.;Zamzow, D.; Small, G. J.; Doskocll, G.; Jeffrey, A. M. Chem. Res. Toxlwl. 1988, 1 , 60-68. (2) Mlller, E. C. Cancer Res. 1978, 38, 1479-1496. (3) Maugh, T. H. Science (Washington, D.C.) 1984, 226, 1183-1184. (4) ChemlcelCarclnogenesls; Nlcollnl, C., Ed.; Plenum: New York, 1982. (5) World Health Organlzatlon Monograph on the Evaluation of the Carclnogenic Risks of Chemicals to Man: Polycyclic Ammatlc Compounds, Int. Agency Res. Cancer, W.H.O.: Lyon, France, 1983; Part I, Vol. 32. (6) Hlgglns, C. B. ExperimentalLeukemk and Mammary Cancer; Unlversky of Chlcago Press: Chicago, IL, 1979. (7) Brown, H. S.;Jeffrey, A. M.; Welnsteln, I. B. Cancer Res. 1979, 39, 1673-1677. (8) Santella, R. M. Mufat. Res. 1988, 205, 271-282. (9) Sanders, M. J.; Cooper, R. S.; Jankowiak, R.; Small, G. J.; Helsig, V.; Jeffrey, A. M. Anal. Chem. 19888 56, 816-820. (10) Sanders. M. J.; Cooper, R. S.; Small, G. J.; Helslg, V.; Jeffrey, A. M. Anal. Chem. 1985, 5 7 , 1148-1152. (11) Harris, C. C.; Yoiken, R. H.; Hsu, I. C. I n Methods In Cancer Research;Busch, H.,Yoeman, L. C., Eds.; Acedemlc: New York, 1982. (12) Reddy, M. V.; Gupta, R. C.; Randerath, E.; Randerath, K. Carclmgenesis 1984, 5 , 231-243. (13) Gupta, R. C.; Reddy, M. V.; Randerath, K. Carclnogenesls 1982, 3. 1081- 1092. (14) Randerath, K.; Randerath, E.; Agrawal, H. P.; Gupta, R. C.; Schurdak, M. E.; Reddy, M. V. EHP, Environ. HeaM Perspect. 1985, 6 2 , 57-65. (15) Randerath, K.; Reddy, M. V.; Gupta, R. C. Roc. Nan. Acad. Sci. U . S.A. 1981, 78, 6126-6129. (16) Dunn, P.; Black, J. J.; Maccubln, A. Cancer Res. 1987, 47, 8543-6548. (17) Everson, R. 6.; Randerath, E.; Santella. R. M.; Cefab, R. C.; Avlltis, T. A.; Randerath, K. Sc/ence (Washhgton. D.C.) 1988, 231. 54-57. (18) Hofstraat, J. W.; Engelsma, M.; Ceflno, W. P.; Hoornweg, G. P.; Gooljer, C.; Velthorst, N. H. Anal. Chim. Acta 1984, 159, 359-363. (19) Hofstraat, J. W.; Engelsma, M.; Van de Nesse, R. J.; Brinkman, N. A. Th.; Gooijer, C.; Velthorst, N. H. Anal. Chlm. Acta 1987, 193, 193-207. (20) Jankowiak, R.; Small, G. J. Science (Washlngton, D.C.) 1987, 237, 618-625. (21) Rogan, E. G.; Cavalierl, E. L.; Tlbbels, S. R.; Cremonosi, P.; Wamer, C. D.; Nagel, D. L.; Tomer, K. 6.; Cerny, R. L.; Gross, M. L. J. Am. Chem. SOC., in press. (22) Zamzow, D.; Jankowiak, R.; Cooper, R. S.; Small, G. J.; Tibbels, S.; Cremonosl, P.; Rogan, E. G.; Cavallerl, E. L., submitted for publicatlon In Chem Res. Toxlcol. (23) Jankowiak, R.; Richert, R.; Bassler, H. J. Phys. Chem. 1985, 8 9 , 3896-4574. (24) Jankowlak, R.; Shu, L.; Kenney, M. J.; Small. G. J. J. Lumin. 1987, 36,293-305. (25) Everson, R. 6.; Randerath, E.; Santella, R. M.; Cefalo, R. C.; Avllts, 1. A.; Randerath, K. Science (Washington. D.C.) 1988, 231. 54-57. (26) Phillips, D. H.; Hewer, A.; Grover, P. L. Carcincgenesls 1988. 7 , 207 1-2075. (27) Phillips, D. H.; Hemminkl. K.; Alhonen, A,; Hewer, A,; Grover, P. L. Mutat. Res. 1988, 204, 531-541.
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RECEIVED for review June 28, 1988. Accepted September 21, 1988. Ames Laboratory is operated for the US. Department of Energy by Iowa State University under Contract No. W-7405-Eng-82. This work was supported by the Office of Health and Environmental Research, Office of Energy Research. We are indebted to E. L. Cavalieri and E. G. Rogan for providing us with the C8dG-BP nucleoside adduct.
