lawing in the Studv of Chemicd Carcinogenesis Ryszard Jankowlak and Gerald J. Small Ames Laboratory-USWE of Chemisby Iowa State University A m . IA 50011
and Department
Fluorescence line-narrowing spectroscopy (FLNS) is useful for obtaining high-resolution optical spectra of molecules or atomic ions imbedded in amorphous solids for which conventional low-temperature absorption spectra yield broad vibronic bands. The potential of FLNS as a direct analysis methodology was first explored in our laboratory for determination of polycyclic aromatic hydrocarbons (PAHs) in complex samples such as solvent-re-
REPORT fined coal (I). This work yielded encouraging resulta, but we were also interested in biomolecular problems, which FLNS cnn solve more readily than other high-resolution techniques such as GC/MS. FLNS has been applied to a wide variety of biomolecules and biomolecular systems including photosynthetic pigments (2, 3), antenna protein-pigment complexes (4), and proteins (5). In this REPORT, we will discuss the principles and instrumentation involved in fluorescence linenarrowing spectroscopy and the application of FLNS to the study of cellular macromolecular damage and chemical carcinogenesis. 0003-2700/89/A361-1023/$0 1.50/0 @ 1989 American Chemlcal Soclsty
PrlnCiPles of FINS
In conventional low-temperature solid-state absorption spectra, the linewidth attributable to site heterogeneity, rfi, is commonly referred to as inhomogeneous line broadening, whereas thermal broadening of a single vibronic transition is considered a homogeneous broadening mechanism. Because the homogeneous broadening contribution to the linewidth is approximately equal to kT, it can be effectively eliminated by a sufficient reduction in sample temperature. For example, the pure depbasing contribution from analfie-host interactions to is tvoicallv < 0.1 cm-1 at 4.2 K’:”Even a i iiquid helium temperatures, however, the band-broadening is still -300 cm-1. contribution of rrnh
(For pure vibrational transitions, rinh is reduced from this value by about 1 order of magnitude.) Figure 1 depicts the contributions from both homogeneous and inhomogeneous broadening. By definition, line-narrowing spectroscopies can “get under the skin” of the inhomogeneously broadened profile and, in the process, significantly reduce the inhomogeneous line-broadening contribution to the linewidth. The extent to which broadening from rinh can be “narrowed out” depends on the type of transition being probed and the state of the analfie initially prepared by the light field. If the inhomogeneous contributions to band broadeGng are much less than the homogeneous contributions, however, the inhomogeneous contributions are ef-
ANALYTICAL CHEMISTRY. VOL. 61, NO. 18. SEPTEMBER 15. 1989
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fectively eliminated. FLNS is a relatively simple technique (6). In Figure 1, a nmow-frequency laser whose width, WL, is much less than rhh excites only a narrow
isochromat (the signal from a smallVOhme element of the sample) of an inhomogeneously broadened absorption band associated with the fluorescent state (usually SI). The sharp un-
derlying absorption profdes with width indicate the zero-phonon lines (ZPLs) of different analyte sites in the host. If the absorption corresponds to the origin, or (0,O)band, rhom is < 0.1 em-’ at 4 K for compounds that have moderately strong fluorescence. For analytical applications, AWL need not be less than 1cm-1. Initially the laser onlv excites the ZPIB of sites within the isochromat defmed by the laser. At sufficiently low analyte con. centrations (< 10-3 M),intermolecular energy transfer-which can lead to site randomization-does not compete with fluorescence 80 that the fluorescence spectrum is a structured, he-narrowed array of ZPLs associated with the eacited isochromat. For (0,O)band excitation the ZPLs correspond to transitions originating from the zero-point vibrational SI level and terminating at the zero-point and vibrational sublevels of So. The former transition is of the resonant type degenerate with q. Although one could try to excite electronic states lying higher in energy than SI (e.g., S t ) to generate FLN specSO transition, this tra for the SI strategy would not be successful because the isochromat selected by the laser for the Sz SOtransition generally maps onto a broad “polychromat” of SI following internal conversion. That is, the site excitation energy distribuSo transitions are not tions for S, correlated. As shown in Figure 1, the FLN spectrum will shift as W.I is tuned across the inhomogeneous profile. Fortunately, thesite excitationenergy distributions of different vibrational levels of the SIstate generally are high. ly correlated. This means that excitation of a vibronic band, (LOJ, of SI can still yield an FLN spectrum that originates from the zero-point level of SI. The use of vibronic excitation offers several advantages over origin band excitation, including improved selectivity and the ability to determine both the ground- and excited-state vibrational frequencies of the chromophore. The improved selectivity is attributable to the vibronic features in the SI SO absorption spectrum, which are often more sensitive to structural perturbations than the SI SO fluorescence spectrum (7). Figure 2 shows a vibronic excitation scheme in which W L excites two overlapping one-quantum vibronic transiand (la,o), which will not be tions (lo,”) resolved in the absorption spectrum. The laser excites two different isochromats, one for a and one for 8, and because of the correlation, the two isocbromats undergo vibrational relaxation (wiggly mows) to two different points in the zero-point distribution of rhom
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Flgure 1. Schematic of an inhomogeneously broadened absorption profile of width Plnhin solid matrices.
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Figure 2. Schematic of laser site selection in FLNS, me slope of exciI&tele iweis mpr-nls he variation d heir energies as a functionof he site. rhh &no108 ms inhorogeneaus brm&nlng of he (0.0) transition. Using he laser excite~lon.w. two Sub8816 01 molecules wiihin rbh are selectively excitd. 1024A
ANALYTICAL CHEMISTRY, VOL. 61, NO. 18. SEPTEMBER 15. 1989
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 18. SEPTEMBER 15. I989
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REPORT the SIstate. Following population of the zero-point level, fluorescence occurs from these two energetically distinct isochromats and produces a “doubling” of every line in the FLN spectrum. The displacements between OL and the doublet components of the origin transition yield the excited-state and vibrational frequencies w.I and q’, the displacements between the origin doublet and lower energy doublets in the FLN spectrum yield the groundstate frequencies w. and up By measuring the FLN spectrum as a function of a located within the SI SOahsorption spectrum one can, in principle, determine the frequencies of all modes active in the absorption spectrum. Each a value yields a distinct fmgerprint for the analyte, and the OL variable can he used to unravel spectral interferences for mixtures.
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I M i O n The FLNS system employed in our laboratory (see Figure 3) waa designed to provide a spectral resolution of -3 cm-1 at 400nm with photon-counting sensitivity. Because PAH metabolites and DNA adducte absorb in the 34WO-nm region, an excimer (XeC1 gas) pumped dye laser with a 20-ns pulse width provides a convenient excitation source. Average power densities typically range from 5 to 100 mW/cm2 at a pulse repetition rate of -30 Hz. A 1-m focal length McPherson 2061 monochromator (f7.0)with a reciprocal linear diapersion of 0.42 n d m m (2400 groovelmm grating) is used to disperse the fluorescence. Optimum resolution is determined by an intensified blueenhanced gateable photodiode array (PDA). The PDA and monochromator provide a -7.0-nm segment of the fluorescence spectrum for a given monochromator setting. (For survey studies of uncharacterized samples, it is beat to use a monochromator with higher rec i p r d linear dispersion, which gives lower resolution. Fruitful studies could even be conducted at 77 K, followed by higher resolution studies at 4.2 K once a qualitative assessment of the types of fluorescent chromophores present has been made.) A double-nested glass liquid helium Dewar (with fused-quartz optical windows) was designed to eliminate liquid nitrogen from the optical pathways. A gks-forming solvent of 50% glyceml, 40% water, and 10% ethanol by volume is most often employed for macromolecular DNA and globin adducts, nucleoside adducts, and the PAH metabolites themselves. Quartz tubing (3 mm 0.d. X 2 mm i.d. X 1cm) is used to contain the solvent (-30 FL total volume). For metabolites and ad1026A
Flguri
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Flgure 4. One-electron oxidation and monooxygenation in the metabolic activd Of BP. Two dinerem types of Bdducts am tonned wim m e DNA base guanine acting as me nucleophile.
duds prone to facile photooxidation, the sample is subjected to several freeze-pumpthaw cycles and sealed under vacuum. Samples are held vertically by an aluminum sample holder designed to occlude laser light scatter from the sample tube edges. They can be cooled from 300 K to 4.2 K in about 2 min by direct immersion in boiling helium. Mechaniiofcarchogenesis Most carcinogens requiremetabolicactivation; covalent binding of metabolites to DNA, RNA, and protein is generally believed to be the first critical step in the multistage process that leads to tumor formation (7,8).PAHs
ANALYTICAL CHEMISTRY, VOL. 61, NO. 18, SEPTEMBER 15. 1989
rank second to mycotoxin mold metabolites in carcinogenic potency (8).The principal events in chemical carcinogenesis are strongly influenced and often determined by host-dependent factors that may vary according to cell type, tiasue, individual, strain, and species. The current view for PAHs is that metabolic activation to electrophilic intermediates can occur by two pathways: monooxygenation to yield diol epoxides (9, 10) and one-electron oxidation to produce radical cations (11). Figure 4 shows these two pathways for henzo[a]pyrene (BP). The top of the figure shows a radical cation-type ad. duct in which BP is bound at its C-6
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position to the C-8 of guanine. Oneelectron oxidation can occur through cellular peroxidases, prostaglandin H synthase, or cytochrome P-450 catalysis of the one-electron oxidation. The microsomal enzyme systems present in cells (including cytochrome P-450 and aryl hydrocarbon hydroxylase) catalyze the monooxygenation of PAH to more water-soluble oxygenated derivatives. Arene oxides (epoxides) are the likely precursors of numerous PAH metabolites, including phenols, dihydrodiols, diol epoxides, tetrols, and conjugation products. Considerable support for this mechanism has come from the determination of the structure of a major adduct formed between BP and DNA in numerous in vitro and in vivo experiments. The structure of the adduct at the bottom of Figure 4 involves covalent binding of the (+)enantiomer of the metabolite trum-7,8-dihydroxy-anti-9,lO-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] through its 1 0 - m i tion to the exocyclic nitrogen (N-2) of guanine. The diol epoxide and radical
,
cation pathways for PAH lead to parent fluorescent chromophores that are distinctly different, such as the pyrene and BP derivatives in Figure 4. Our understanding of the initial phases of chemical carcinogenesis has been hampered by the unavailability of practical high-resolution and sensitive bioanalytid techniques for the determination and characterization of both cellular macromolecular (i.e., intact DNA) adducts and nucleotide adduds. The problem is difficult; one needs to detect a DNA damage level of -1 base pair in los in -100 pg of DNA for in vivo studies at a sufficiently good remlution to distinguish between structurally similar adducts. Distinction between stereoisomersof a given metabolite bound to a particular nucleic acid base is a good example of the necessary selectivity. lnvibo FLNSot DNAandnucleoside adduds FLNS can be applied to macromolecular DNA, globin, and nucleoside adducts (6,12,13)and polar metabolites
dPDE-DNA
I .gwe 5. Schematic of the nucleotide adduct within the DNA macromolecule. Wectiw l a w excitation leads to an FLN specbun that repesmts a s w a l “fingerpint” 01 ht q e t molecule.
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 18. SEPTEMBER 15, 1989
(14). Figure 5 shows one of the first FLN spectra obtained for a macromolecular DNA-carginogen adduct formed from a highly reactive diol epoxide of BP. The vibronic bands in the structured spectrum are limited to a width of 10 cm-1 by the monochromator. Prior to this work (14) it was not clear whether interactions between DNA and the fluorescent chromophore would render FLN inoperative, that is, produce a large homogeneous broadening that no amount of magic could circumvent. The stereoisomers of BPDE exhibit remarkably different tumorigenic and mutagenic activities (15). Adduct formation with DNA appears to occur primarily at guanine, and the relative yields from the anti-BPDE and synBPDE isomers are dependent on the species under study (16, 17). In the anti-diastereomer (anti-BPDE) the benzylic hydroxyl group and the epoxide oxygen atom are on opposite faces of the molecule, whereas in the synisomer (syn-BPDE) these groups are on the same face. Because each diastereomer may exist as a pair of enantiomers, four stereoisomers of BPDE are possible (in Figure 4, the structure of (+)-anti-BPDE is shown). FLN spectra for native DNA adducts derived from (+) and (-)-anti-BPDE and syn-BPDE (18) along with a spectrum of the tetraol of BPDE (BPT)are shown in Figure 6. (BPDE-DNA adduets are known to be unstable and dissocisteto the tetraol, particularly in the presence of light.) T h e spectra were obtained using an excitation wavelength of 371.6 nm (vibronic excitation), and the bands in each spectrum represent the multiplet origin structure. Not only is the tetraol distinguished from the adducts; the adducts themselves can be distinguished primarily by vibronic intensity distributions. Interpretation of the vibronidly excited FLN spectra indicates that the SI state energy increases in going from the syn-BPDE adduct to the (-)-antiBPDE to the (+)-anti-BPDE adduct to the tetraol. The ability of FLNS to resolve different stereoisomeric adducts appears to result from DNA-metabolite intermolecular interactions. Recent work in our laboratory (19) indicates that the spectra of (+)-antiBPDE-DNA, (-)-anti-BPD&DNA, and syn-BPDE-DNA correspond to the binding of the BPDE isomers to the N-2 of guanine and that a given BPDE stereoisomer can assume different binding configurations (20, 21). Thus “DNA host-engineeredselectivity” has imparted a degree of resolution to FLNS that was not anticipated at the outset of our studies.
FLNS has also been used to determine five BP-nucleoside adducts synthesized by one-electron oxidation of BP in the presence of guanosine, deoxyguanosine, and deoxyadenosine (13). The resulta showed that a major depurination adduct from the hinding of BP to DNA in rat liver nuclei is 7(benzo[a]pyren-6-yl)guanine(N7Gua). Only 20 pg of the adduct was required, an amount that can he obtained from one rat, whereas analysis by the common method of collisionally activated decomposition MS would require sacrificing many rats.
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FINS analysis d In vivo DNA akbcb FLNS also can be used for in vivo studies. FLN spectra of (+)-anti-BPDEDNA and syn-BPDE-DNA (Figure 7) have been used to identify the major diol epoxide adduct of fish liver DNA from English sole exposed to BP in laboratory-controlled experiments (22, 23).Of particular interest in this study
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rigure 7. Comparison of the FLN spectrum of fish liver DNA (a)extracted from fish exposed to BP. (b)with standard FLN spectra of symBWE-DNA, and (c) (+kantCBWE-ONA.
me mdificetim levels me -1 adduct in 10' bares. -I adduct in 10' baaes (determined ,adlD meblcally). and -1 adxlct in -200 baaes. r e SpeCtlV.3W.
I
,.dum 8. Comparison of the vibrh.,, ly excited FLN spectra for three diH
enl DNA adducts.
All spectra w e obtained In me standard giy/H& glass at 4.2 K wl(h an exciunim wavelengh of 371.8 nm. (a) M i w e of BPT and DNA at a conwnlraticm of M. (b) (+kmtBFDE-DNA wilh 0.5% basas mdiRed. (c)(-tantCBPDE-DNA vim 1.5% bares moditled. snd (qSyrrBpDEDNA at a c a ~ x l ~ l l of o n-1 adduct in 10' b a s a s . h peaksarelebelad winmeir co~r* spmding excitBdbtale vibrational fmquencim (in m-').
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was whether the expected N-24eoxyguanosine (N-2-33 adduct from (+)anti-BPDE is formed. Despite the very low damage level of the DNA (-1 adduct in 10' bases as determined by an independent method), the FLN spectrum exhibits a good signal-to-noise ratio. As expected, a major adduct is derived from BPDE, establishing the importance of the monooxygenation mechanism. However, comparison of the fish DNA spectrum with the FLN spectra of syn-BPDE and (+)-antiBPDE-DNA shows that the adduct is not derived from (+)-anti-BPDE hut from syn-BPDE. Although the major adduct is derived from syn-BPDE, weaker contributions from (+)-anti- and (-)-antiBPDE-DNA adducts cannot be excluded. Both anti- and syn-BPDE are strongly mutagenic in bacterial and mammalian cells, but anti-BPDE generally shows greater activity than synBPDE in most tests (24).Thus the hieh proportion of syn-BPDE-DNA 2ducts in English sole exposed to the high dosage of BP used in these experi-
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REPOR7 ments presents an interesting problem. Similar results were obtained recently by Varanasi et al. (22)using32P postlabeling. They showed that whereas fwh exposed to 2-15 mg B P k g b.w. yielded mainly the anti-BPDE-DNA adduct, fish exposed to 100 mg B P k g b.w. exhibited a dominant contribution from syn-BPDE-DNA adducts. Moreover, in rat dermal fibroblasts (16) the relative quantities of (+)-anti-BPD& dG (-7%) have been much smaller than those determined in rabbit dermal fibroblasts (-90%). In rat dermal fibroblasts, syn-BPDWG represented more than 40% of the adducts, whereas in rabbit cells it accounted for only -5%. Large quantities of synBPDE-DNA adducts were also observed in hamster epidermal cells (2.5). Given that the level of adduct formation and the relative amounts of different adducts appear to depend on the BP dosage level, FLNS should prove useful for a detailed study of this effect. Future directions Because it can provide high-resolution analyses of macromolecular adducts at the low concentration levels produced by in vivo exposureto genotoxic agents, FLNS should play an important role in future studies of the initial phases of chemical carcinogenesis as well as DNA repair mechanisms. The versatility of FLNS is underscored by its application to nucleoside adducts both in glasses and sorbed on TLC plates (12).Because it is difficult to identify TLC spots from complex samples, and because the spots can diffuse and are likely to be heterogeneous, interfacing FLNS with the sensitive 3T-pcatlabeling procedure (26) for detection of nucleotide adducta is potentially useful. It is important to analyze for both stable macromolecular adducts and depurinated adducts; one cannot assume that the DNA “nick” prcduced by depurination cannot produce misccding or error-prone DNA repair. Another future avenue for FLNS is suggested by recent studies of globincarcinogen adducts and by the apparent correlation of globin adduct levels with DNA adduct levels in target tissues (27). Because globin adducts are not subject to repair, and because greater amounts of globin than DNA are available from blood. FLNS of globin might be the basis for a practical and reliable body burden assessment methodology (28). Recent 77 K selective laser-excited fluorescence studies of (+) and (-)anti-BPDE-DNA and syn-BPDEDNA have demonstrated that even non-line-narrowedspectra can be used
to distinguish between these adducts and to establish that a given stereoisomer can assume more than one DNA binding site (19).The use of fluorescence quenchers such 88 acrylamide allows classification of adduct sites ex. terior or interior (quasi-intercalated) types. The combination of selectively laser-excited 77 K fluorescence spectroscopyand FLNS with modeling and energy minimization calculations should enhance understand@
ing of the structure of DNA adduct sites as well as structure-DNA repair relationships. Am- Laboratory is operated for the U.S. Department of Energy by Iowa State Univemih, under contract no. W-1405-Eng-82. This work w a supported bythe omiee ofHd*mdfi~ronmmd Research. Office of E n e m Research. wean, indebted to OUT mllabmatom A. M. Jemey (who sparked OUT interest in chemical carcinogenesb). U. Varanasi, N.E Geacintov, E.L. Cavalieri, and E. G.R , , ~ ~without , whom work desaibed herein would not have been possible. n e develop-
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nent of FLNS &s an analytical twl owes much to m v i o u s members of the group: J. C. B r m .
M.J. h d e r s , U MffiMe. I. C h i . R S.Cwwr,and
tng DNA Damaging Agenla in Humom. Applreation in Comer Eprdemiology and Reuentron: Bartseh. H.: Hemminki. K..
D. Zammw.
Reterences :1) Brown, J. C.; Duncanson, J. A., Jr.; Small. G. J. Anal. Chem. 1980,52,1711-
15. 12) Renge, I.; Mauring. K.; Sarv, P.;Avarmas, R. J.Phys. Chem. 1986,90,6611-16. 13) Fiinfdilling, J.; Walz, D. Photochem. Photobiol. 1983.38,38%93. (4) A v m a a , R.; Renge, I.; Mauring, K. FEES Lett. 1984,167,18&90. (5) Kolanek, H.;Fidy, J.;Vanderkooi, J. R. J. Chem. Phys. 1981,87,438&94. (6) Jankowiak, R.; Cooper, R. S.;Zamzaw,
France, 1988, pp. 3251. (28) Weston, k: Willey, J. C.; Manchester, D. K.; Wilson, V. L.;Brooks, B. R.; Choi, J-S.: Poiner. M.C.: Tnvers. G. E.: Newm' M. ,J.; h n , D. L.;H&i, 6. C. In Methods for Detecting DNA. Da.m ing Agents in Humans: Applwatron in tancer Epidemiology and Preuention; Bartach, H.; Hemminki, K., E&, IARC Scientific Publications: Lyon. France, 1988; pp. 181-89.
D.; Small, G. J.; Doskocll, G.; Jeffrey, A. M. Chem. Res. Toricol. 1988,1, W68. ( I ) Miller, E. C.; Miller, J. A. Cancer 1981, 47,23274. (8) World Health Organization Monograph on the Eualuation o the Carerno
genicRisk8of the ChemicaftoMarxPoly: nuclear Aromatic Compounds; International Agency for Research, in Cancer. World Health Organization: Lyon, France, 1983, Vol. 32. (9) Harvey, R.G.; Geacintov, N. E. Ace. Chem. Res. 1988,21,6611. (10) J e f f 2 A. M. In Polycyclic Hydrocarbons a Carcinogenesis; Harvey, R G., Ed.; American Chemical Society: Washington, DC, 1985, Chapter 8. (11) Cavalieri, E.; Rogan, E. Enuiron. Health Perspect. 1985,64,69-84. (12) Cooper, R. S.; Jankowiak, R.; Hayes. J. M.; Lu, P.; Small, G. J. Anal. Chem. 1988,60,269294. (13) Zamzaw, D.; Jankowiak, R.; Coo I,
R.S.; Small, G. J.; Tihhels, S.R.; E e monosi, P.; Devanesan, P.; Rogan, E. G.; Cavalieri, E. L. Chem. Res. Toxicol. 1989,
2,2934. (14) Sanders,
M. J.; Cooper, R. 5.; Jankowiak. R.; Small. G. J.; Heisig, V.; Jeffrey, A.M. Anal. Chem. 1986,58,816
20. (15) Slaga, T.J.; Bracken,
W.J.; Gleason, G.; Levin, W.;Yagi, H.; Jerina, D.M.; Conney, A. H. Cancer Res. 1919,38, 6771
(16j'ALerandrov. K.; Sala, M.; RBjas, M. Cancer Res. 1988,48,7132-39. (17) Moore, C. J.; Pruess-Schwartz, D.; Mauthe, R. J.; Gould, M. N.; Baird, W.M. Cancer Res. 1987,47,44024& (18) Zin er, D.; Geacintov. N E, Harvey, R. G. %ioohvs. Chem. 1987.27..;31-38. (19) Jank6&, R.; Lu, P.; Small, G. J.; Geacintov, N. E., submitted for public+ tion in Chem. Res. Toricol. (20) Kim. S.K.; Brenner, H. C.; Soh. B: J.; Ceacmtov. N.E. Photochem. Pholobrol.. in press. (21) Kim, 5. K.; Geacintov, N. E.; Brenner. H. C.; Harvey, R. G. Carcinogenesis, in PIeSS. (22) Varanasi, U.; Reichert, W.L.; Le Eherhart, B-T.; Stein, J. E. Chem. Bioi. Interact., in press. (23) Jank?wiak, R.; Lu, P.; Nanhimoto, M.; Varanaai.. U.:. Small. G. J.,. unpublished . work. (24) Newbold, R F . ; Brooks, P. Nature (London) 19'16,261,5365. (25) DiGiovanni, J.; Sina, J.F.; Aahurst, 5. W.;S i e r , J. M.; Diamond, L. Cancer Res. 1983,43,163-70. (26)Randerath, K.; Fhderath, E.; Agraval, H. D.; Gupta, R. C.; Schurdak, M. E.; Fleddy, M. V. Enurron. Health Perspect. 1985.62.574. (27) Wogan, G. N. In Methods for Detect-
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Gerald J. Small is professor of chemistry at Iowa State University and senior chemist at Ames LaboratoryUSDOE. He receiued his PhD. in physical chemistry from the Uniuersit y of Pennsyluania in 1967. Following a two-year appointment as a research fellow at the Australianh'atioml Uniuersity, he joined the faculty of ISU in 1969. His research interests include molecular electronic spectroscopy, linear and nonlinear laser spectroscopies, energy and electron transfer in photosynthesis, carcinogen metabolism and cellular macromolecular damage, and struceural disorder and tunneling in amorphous solids.
Ryszard Jankowiak is an associatescientist at Ames Laboratory-USDOE. He receiued his PhB. in physics from the Technical University in Gdahsk (Poland) in 1981 and was a research associate and uisiting scientist at the Philipps Uniuersity in Marburg (West Germany) from 1981 to 1985. His research interests include molecular electronic spectroscopy, laser bioanalytical spectroscopy, chemical carcinogenesis, photosynthesis, and structural disorder and tunneling in amorphous solids at uery low temperatures.
