Comparative laser spectroscopic study of DNA and polynucleotide

Guohui Jiang, Ryszard Jankowiak, Nenad Grubor, Marzena Banasiewicz, Gerald J. Small, Milan Skorvaga, Bennett Van Houten, and J. Christopher States...
0 downloads 0 Views 1MB Size
Download PDF
Chem. Res. Toxicol. 1991, 4 , 58-69

58

Comparative Laser Spectroscopic Study of DNA and Polynucleotide Adducts from the ( +)-anti-Diol Epoxide of Benzo[ a Ipyrene Peiqi Lu, H y u k Jeong, Ryszard Jankowiak,* and G. J. Small* Ames Laboratory-USDOE

and Department of Chemistry, Iowa State University, Ames, Iowa 50011

S. K. Kim, Monique Cosman, and N. E. Geacintov Department of Chemistry, New York University, New York, New York 10003 Received July 24, 1990

A recently developed methodology [Jankowiak, R., Lu, P., Small, G. J., and Geacintov, N. E. (1990) Chem. Res. Toxicol. 3,3%-461, which combines fluorescence line narrowing spectroscopy So laser excitation) fluorescence spectroscopy at 77 K a t 4.2 K with non-line-narrowed (S,

-

and fluorescence quenching, is used to characterize adducts formed from (+)-anti-BPDE and the alternating copolymers poly(dG-dC).poly(dG-dC) and poly(dA-dT)*poly(dA-dT),the nonalternating poly(dG).poly(dC), single-strand poly(dG), and the oligonucleotide d(ATATGTATA). Detailed comparisons of the fluorescence spectra and quenching (with acrylamide) of the properties of the adducts with those of (+)-anti-BPDE-DNA adducts are made. Fluorescence spectra of the trans and cis isomers of the adduct formed from guanosine monophosphate and the adducts of d(ATATGTATA) are used t o assign the stereochemistry of the two major DNA adducts as trans-N2-dG moieties which occupy two different DNA sites. Evidence for the existence of minor cis-type guanine adducts is provided. Finally, a fourth type of DNA adduct (minor) is identified and assigned as trans-N6-dA.

the study of carcinogenic mechanisms a t the molecular level is the availability of sensitive and selective methods The mutagenic and tumorigenic properties of polycyclic for the analysis of adducted macromolecules. aromatic hydrocarbons (PAH) have been the subjects of At present, the highest resolution frequency domain numerous investigations (1-3). Benzo[a]pyrene (BP) is technique available for the analysis of DNA-PAH adducts one of the most widely studied compounds of this class of is laser-excited fluorescence line narrowing spectroscopy carcinogens. The physicochemical properties of DNA (FLNS) (13-21). FLNS is also applicable to nucleotide adducts derived from BP and its biological effects have and nucleoside adducts (7,21). FLNS provides the ultibeen recently reviewed by Geacintov ( 4 , 5 ) and Graslund mate spectral resolution for fluorescence-based characand Jernstrom (6). The metabolic pathways [diol epoxide terization of biomolecular samples. Vibronic line widths and one-electron oxidation or radical cation (7, S)] of BP of only a few cm-' are attainable, meaning that detailed and base-nucleophilic center specificity of its electrophilic analysis of both the ground- and excited-state vibrational metabolites are rather well understood. However, the modes of the fluorescent chromophore is possible. The relationships between basemetabolite chemical structure, principles of FLNS for analysis of DNA- and globin-PAH site structure and genetic mutation (e.g., of protooncogadducts have been recently reviewed (13). Briefly, the enes), and error-free and error-prone DNA repair are not phenomenon of FLN is generally observable for narrow (4449-12). Thus, the problem of base sequence specificity line laser excitation of the inhomogeneously broadened of the binding of metabolites and properties of adducts vibronic absorption bands of the SI So absorption sysshould receive greater attention. tem. A reduction in the inhomogeneous broadening conThe solution of such problems will depend, in part, on tribution to the vibronic fluorescence bands of up to 2 the availability of highly selective and sensitive bioanaorders of magnitude can be achieved. Either origin or lytical methodologies for the characterization of both stable vibronic (1,O) excitation may be employed. The former and labile covalent DNA-PAH adducts. The required yields a spectrum that contains only information pertaining selectivity is well illustrated by noting that the primary to the ground-state vibrational modes. In contrast, vidiol epoxides of BP which react with DNA are (+)trans-7,8-dihydroxy-anti-9,lO-epoxy-7,8,9,lO-tetrahydro-bronic excitation at different frequencies yields a series of spectra which provide the excited-state mode frequenBP [ (+)-anti-BPDE], (-)-anti-BPDE (see Figure l),and cies and their relative absorption cross sections. Because their syn diastereomers. Moreover, each stereoisomer the excited-state modes are more sensitive than groundyields a distribution of adducts; vide infra. For studies of state modes to substituent and environmental perturbaDNA from mammalian cells exposed to ambient concentions, vibronically excited FLNS is the more selective trations of PAH the required detection limit for adducts approach. The reader who is not familiar with vibronically is about 1 fmol/ 100 pg of DNA. A key requirement in

Introduction

-

-

0 1991 American Chemical Society 0893-228~/9~/2704-0058$02.50/0

Chem. Res. Toxicol., Vol. 4, No. 1, 1991 59

Laser Spectroscopy of BPDE-DNA Adducts

-

trans BPT

f

'

-

-

-

Cis BPT

anti 7, 8 dihydrodiol 9,lO epoxide

-

OH

OH

-

\

(-)- anti 7, 8 dihydrodiol 9,lO- epoxide

-

cis BPT

*OH OH

Figure 1. Structures of BPDE stereoisomers and associated cisand trans-tetraols.

excited FLNS may find the following discussion helpful. Let w ( ~ ,and ~ ) r I N H be the center frequency and inhomogeneous width of the S1 So absorption origin. To generate a spectrum characterized by excited-state vibrational modes in the frequency range =Q f rINH/2 requires excitation a t we, = w ( ~ , + ~ ) Q . This frequency will generally excite several inhomogeneously broadened and overlapping vibronic absorption bands, a,0,.... The "isochromats" of bands a,P, selected by we, undergo rapid vibrational relaxation (= 1 ps) to their respective and energetically distinct positions in the overall distribution (inhomogeneous) of the zero-point vibrational level of the S1state. Subsequently, (0,O) fluorescence of the a,0,... isochromats occurs to produce a multiplet origin structure in the Z U ( ~ , ~ ) f rINH/2 spectral region, with as many (0,O) components as vibrations excited by wer The differences between the frequencies of these components and wex yield the excited-state mode frequencies (13, 35, 36). For example, to generate excited-state modes in the vicinity of 600 cm-l for the (+)-1-DNA adduct (cf. Table 11), one would employ 0 An important a laser excitation wavelength of ~ 3 7 nm. point is that the multiplet origin structure is located in the region of the inhomogeneously broadened fluorescence origin produced under non-line-narrowing conditions (i.e., S2 So excitation). Most recently, this technique has been used for analysis of macromolecular DNA and globin damage from in vivo exposure to PAH (20,22). For example, it was shown that the major stable DNA adduct from the liver of fish exposed to high dosage levels of BP is derived from syn-BPDE (20), in agreement with previous work (23). At low dosage levels, the major adduct was found to originate from (+)-antiBPDE (23, 24). FLNS was used to prove (22) that the major human globin adduct from BPDE is a carboxylic ester and that this adduct is of the interior type (i.e., undetectable in the intact protein by monoclonal antibody approaches). Analysis by FLNS of the urine and feces of rats treated with B P has proven, for the first time, that the cytochrome P-450 catalyzed one-electron oxidation of B P is a major pathway for DNA damage in mammalian

-

...

-

1

-

trans BPT

-

Table I. Biomolecules Used in Adduction Experiments with (+)-anti-BPDE adduct concentrabiomolecule abbreviation tion: M poly(dG-dC).poly(dG-dC) (dG-dC)Z poly(dA-dT).poly(dA-dT) (dA-dT)z 10" poly(dG)*poly(dC) (dG)*(dC) poly(dG) (dG) guanosine monophosphate GMP trans -10" cis -io-' d(ATATGTATA) oligonucleotide d(-TGT-) trans -10" cis -2 x calf thymus DNA DNA 10" At 20 mM sodium cacodylate buffer and 20 mM NaCl, pH 7.0. For the BPDE-d(ATATGTATA) adducts the buffer was 20 mM Na2HP04,pH = 7.0.

cells (25). The nucleotide adduct identified was BP bound at its C6 position to N7 of guanine. Distinction by FLNS between this adduct and that formed at C8 of guanine had been demonstrated earlier (17). However, we recently reported that FLNS in combination with fluorescence quenching and 77 K selectively excited (S, So) fluorescence spectroscopy is a more powerful methodology for DNA adduct analysis (19). In that work five adducts from the reaction of (-)-anti-BPDE with purified DNA were identified and classified as type I (interior or quasi-intercalated) or type I1 (exterior, solvent accessible). The adducts were denoted as (-)-j, j = 1-5. The (+)-anti-BPDE enantiomer yielded three adducts, with (+)-lbeing dominant at -85% (19). That the (-)-enantiomer yielded the more heterogeneous distribution of adducts confirmed the results of earlier work (26, 27). It is important for the present paper to note also that type I1 adducts are characterized by a higher value of the ratio ( R )of the intensity of the fluorescence origin to that of the prominent 1400-cm-l vibronic band whose wavelength is 20 nm greater than that of the origin. The more hydrophobic the environment of the pyrenyl chromophore, the lower is the R value. With increasing hydrophobicity the linear electron-phonon coupling increases; for example, line narrowing in fluorescence or fluorescence excitation was not observed for the intercalated and solvent-inaccessible (+)-3- and (-)-3-DNA adducts (19). The strong coupling is presumably the result of a strong interaction between the pyrenyl chromophore and the bases, which imparts significant charge-transfer character to the S1 state. On the basis of FLN spectra both (+)-l-and (-)-l-DNA were assigned as N2-dG-BPDE adducts. The (-)-4- and (-)-5-DNA species were attributed to adducts of BPDE with a base(s) other than guanine, e.g., adenine. Unavailability of appropriate standard adducts prevented definitive assignments. The results of ref 19 suggest that the above methodology may prove useful for the study of DNA repair and base sequence specificity of covalent binding in vivo. It is this latter potential application that this work is concerned with. Specifically, we report the results of studies on adducts formed from (+)-anti-BPDE with the alternating copolymers poly(dG-dC)-poly(dG-dC)and poly(dA-dT)poly(dA-dT) and the nonalternating poly(dG).poly(dC), as well as single-strand poly(dG) and the oligonucleotide d(ATATGTATA). Table I lists the polynucleotides and oligonucleotides (and abbreviations used) whose adducts from (+)-anti-BPDE have been studied. Detailed comparisons of their fluorescence spectra, fluorescence quenching, and other properties with those of (+)-antiBPDE-DNA adducts are given in order to gain some insight about base sequence specificity. In addition, the

-

- -

60 Chem. Res. Toxicol., Vol. 4, No. 1, 1991

-

(+) - anti - BPDE (dGdC),

09

E

A ex=346nm

B

ex=355nm

Lu et al. purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository. The polynucleotides poly(dG-dC)-poly(dG-dC),poly(dA-dT).poly(dA-T),and poly(dG)-poly(dG)were purchased from Pharmacia Inc. (Piscataway, NY). The synthesis and purification of the single-strand oligonucleotide d(ATATGTATA)is described elsewhere (32). For all adducts studied (includingDNA) the percent modification levels fell in the range 0.2-1.5, except for poly(dG) for which the level was 0.03. In the case of d(ATATGTATA)each strand contained one covalently bound BPDE residue. The synthesisof the addud from (+)-anti-BPDE and guanosine monophosphate and the isolation procedure for the trans and cis isomers were similar to those used by Cheng et al. (33). For all reaction products extreme care was taken to remove physically bound tetraols (34,35). The adducts were diluted in the glass-formingsolvent (see above) to a pyrenyl chromophore concentration of M. For formation of noncovalent complexes, BPT (50 pL, 4 X M) was mixed with DNA or poly(dG) (50 pL, 1 mg/mL). The mixture was sonicated for 5 min at 20 “C. Then, 150 pL of glycerol-ethanol solvent (5:l v/v) was added to the mixture, followed by sonication for another 5 min. For the fluorescence quenching studies, acrylamide (ACR) was employed at a concentration of 1 M, an optimal concentration for such studies.

-

360

390

460

4io

WAVELENGTH (nm)

Figure 2. Laser-excited fluorescence spectra of (+)-antiBPDE-(dG-dC)* for A,, = 346 nm (A) and A,, = 355 nm (B),

respectively. The band at 381.1 nm corresponds to quasi-intercalated tetraol. Spectrum C is mainly due to the (+)-l-(dG-dC), N2-dG adduct.

fluorescence spectra of the trans and cis isomers of the adduct formed fram guanosine monophosphate and d(ATATGTATA) oligomer with (+)-anti-BPDE are used to assign the stereosymmetry of the (+)-1- and (+)-%-DNA adducts. A fourth minor DNA adduct from (+)-antiBPDE, (+)-4-DNA, is identified and assigned as N6-dA (type 111).

Experimental Section Instrumentation. The underlying principles of the low-tem-

perature FLNS and the associated instrumentation have been described in detail (13, 17,18,21). The excitation source was a Lambda Physik FL-2002 pulsed dye laser pumped by a Lambda Physik EMG 102 MSC excimer laser. Typical intensities used for the FLNS (4.2 K) and S2 So (77 K) laser excited experiments were -3 and -30 mW/cm2 (at a pulse repetition rate of 30 Hz), respectively. Fluorescence was detected with a Princeton Instruments IRY-l024/G/R/B intensified blue-enhanced gateable photodiode array. Gated detection was accomplished by using a Lambda Physik EMG-97 zero-drift controller to trigger a FG-100 high-voltage gate pulse generator which provided adjustable delay and width of the detector’s temporal observation window. Laser-excited fluorescence spectra at 77 K, obtained under non-line-narrowing conditions (S, Soexcitation),were measured by using an SRS Model SR280 boxcar averager (Stanford Research) equipped with two Model SR250 processor modules for channels A and B. Channel A was used to monitor the fluorescence signal, while channel B was used to monitor the pulse-topulse intensity jitter of the excimer pumped dye laser. All spectra reported were normalized for pulse jitter. The boxcar averager was interfaced through a SR245 computer interface module with a PC-compatible computer for data acquisition and analysis. The delay time for fluorescencemeasurements was 25 ns, and the width of the observation window was 60 ns. Samples were dissolved in 30 p L of 5:4:1 glycerol-H20-EtOH in quartz tubes, taken through several freeze-pump-thaw degassing cycles, sealed, and cooled to either 77 or 4.2 K. Materials. Calf thymus DNA was purchased from Worthington Chemicals (Freehold, NJ). The DNA was prepared and reacted with (+)-anti-BPDE or (-)-anti-BPDE (cf. Figure 2) as described previously (30, 31). The BPDE enantiomers were

-

-

Results and Discussion Spectrofluorimetric analysis of native DNA and polynucleotide samples for covalent adducts formed with BPDE is complicated by the presence of 7,8,9,10-tetrahydrotetrahydroxybenzo[a]pyrene (BPT) which is not covalently linked with the nucleic acids and which possesses pyrene as the parent fluorescent chromophore. In this work, BPT is of concern since it can be produced either by hydrolysis or photodecomposition of the adducts. To identify interferences from BPT, it is not sufficient to have only determined the fluorescence spectra of BPT in the solvent (glass) of interest. Although a considerable fraction of the BPT in a solution containing DNA is solvated by a solvent cage (’’free- BPT), BPT also forms physical complexes with DNA (29). The differences in the intermolecular interactions between BPT and its environment in these two situations are sufficient to produce spectral shifts of several nanometers and differences in vibronic intensity distributions. Thus, it is essential to generate standard BPT fluorescence spectra from samples for which BPT has been physically mixed with DNA (or polynucleotide) in the solvent. We have performed such experiments for tetraols derived from the hydrolysis of anti-BPDE mixed with DNA So) and 4.2 and poly(dG). Both 77 K laser-excited (S, K FLN spectra for several excitation wavelengths were obtained and analyzed. In addition, fluorescence quenching studies with acrylamide (ACR) were conducted in order to determine the degree of solvent accessibility of BPT in different environments. The results, although interesting from physicochemical and spectroscopic points of view, are not central to the present paper and, therefore, are given in the Appendix. That is, the spectral features of B P T species shall be viewed here as a potential interfering factor in the spectra of covalent adducts. Thus, in what follows, we need only to summarize the essential findings: there are three BPT species with fluorescence origin bands at 376,377, and 381 nm; the first two bands are assigned to trans- or cis-BPT which are “free” or externally bound to a DNA base, while the latter is due, at least in part, to quasi-intercalated BPT. All three types of BPT are subject to quenching by ACR the degrees of quenching are discussed in the Appendix. The BPT species emitting a t 376 and 377 nm are distinguishable from covalent BPDE adducts because their origins are significantly blue-shifted relative to those of the latter. In

-

-

Laser Spectroscopy of BPDE-DNA Adducts

Chem. Res. Toxicol., Vol. 4, No. 1, 1991 61

+

adducts

Table 11. Spectral Characteristics of Adducts of ( )-anti-BPDECovalent Adducts ~tlco,o,, nm fwhm (O,O),cm-' R site type

(+)-l-DNA, major (+)-$-DNA (+)-3-DNA (+)-I-DNA (+)-l-(dG-dC),, major (+)-t(dG-dC)Z (+)-J-(dG-dC),' (+)- l-(dG)*(dC) (+)-%-(dG).(dC),major (+)-3-(dG)*(dC) (+)-I-(dA-dT) (+)- l-poly(dG) (+)-$-poly(dG), major (+)-3-p0ly(dG)

378.0 379.3 380.6 382.7 377.6 379.4 -380.5 377.9 (with ACR) 378.3 (no ACR) 380.5 -382.5 378.5 379.3 380.5

-

190 180 340 200 180 200

210 230 -250 200 270 300 340

-

I1 I" I I11 I1 I" I I In I IIId I I" I

1.75 1.25 1.15 1.55 1.25 1.2 1.35 1.1

1.15 1.0 1.1

quenchable by ACR Yes yesb no Yes yes yesb slightly yes, less than (+)-2 yes, more than (+)-l slightly Yes Yes Yes slightly

" We assign (+I-$ as site I type adducts with a 'degree" of interior binding (and/or stacking interactions between the bases and the pyrenyl residues) that is less than that for (+)-3. bBut less than (+)-l.CExistenceof this minor adduct is based on FLN data. fwhm and R values were not measured. *We call it site I11 since its properties are different from those of sites I and sites 11. addition, these BPT species exhibit relatively narrow fluorescence bands in the 77 K spectra. This is not the case for the 381-nm emitting species, which is referred to as BPT-381 in what follows. BPT-381 represents a potentially serious interference for adducts since it can be easily misassigned to an intercalated covalent DNA adduct from (+)-anti-BPDE, which exhibits an origin at 381 nm (19). This covalent adduct, however, can be differentiated from BPT-381 since its fluorescence is not significantly quenched by ACR. (A) Synthetic Polynucleotide Adducts from (+)anti-BPDE. The synthetic polynucleotides studied (Table I) are alternating or nonalternating purine-pyrimidine copolymers with backbone conformational parameters which place them in the B-DNA class. Since we have found that the alternating (dG-dC)z duplex adducts are most similar to those of DNA, we focus on their 77 and 4.2 K FLN spectra. Although it has been established that the excitation and emission spectra of (A)-anti-BPDE( d G - d Q are heterogeneous in nature (30, 33), the improved resolution of the spectra reported here are required for definitive distinction between different adduct types. (1) Laser-Excited (S, So) Fluorescence Spectra at 77 K. Figure 2 shows the 77 K fluorescence spectra for (+)-anti-BPDE-(dG-dC), obtained with A,, = 346 nm (A) and 355 nm (B). The "free" BPT bands at 376.1 and 376.8 nm are absent in spectrum A (cf. Appendix), which exhibits a dominant band a t -377.8 nm (solid arrow) and a shoulder a t 381 nm (dashed arrow). The shoulder is assigned first. The lower energy excitation a t 355 nm results in a spectrum (B) for which the shoulder is resolved into the dominant origin, i.e., A,, = 355 nm discriminates against the 377.6 nm emitting species. Spectrum B is similar to that of BPT-381 (cf. Appendix), e.g., the R ratios are 1.35 and 1.4. Moreover, the intensity of the band a t -381 nm increases in aged and/or damaged (by white light illumination) samples. Furthermore, the 381 nm emitting species of spectrum B (Figure 2) and BPT-381 are both subject to significant and comparable quenching by ACR. There are other similarities; cf. Appendix. Thus, we conclude that the 381 nm emitting species of (+)anti-BPDE-(dG-dC), is mostly BPT-381 and not the (+)-3 adduct formed with DNA, vide supra. The latter is not subject to significant quenching and has an R ratio of 1.15 (19) (Table 11). Furthermore, the (+)-3adduct, unlike BPT-381, does not exhibit fluorescence line narrowing due to strong electron-phonon coupling produced by intercalation (19). However, a small contribution from the (+)-3-(dG-dC), adduct with a maximum a t -380.5 nm and properties similar to (+)-3-DNA cannot be ruled out

-

A ex = 346 nm, with ACR B ex = 355 nm, with ACR C (+)1 - (dG-dC),

375

380

385

WAVELENGTH (nm)

Figure 3. Origin bands of the 77 K laser-excited fluorescence spectra of (+)-anti-BPDE-(dG-dC)2 in the presence of 1 M ACR, obtained with X,= 346 nm (A) and X, = 355 nm (B),respectively. Spectrum C represents the incompletely quenched contribution from (+)-l-(dG-dC), (see curve C in Figure 2).

on the basis of these broad fluorescence spectra (see also section C). The contribution of BPT-381 to spectrum A of Figure 2 is readily eliminated by subtracting spectrum B mostly due to BPT-381 (appropriately scaled after spectral deconvolution) from spectrum A. The resulting A - B difference spectrum is shown as C in Figure 2. The difference spectrum is due primarily to the covalent adduct (+)1-(dG-dC),; cf. Table 11. Excitation a t 350 nm revealed nearly comparable contributions from 377.6 nm and from BPT-381 bands since this A,, is not very selective for either of these species and confirmed that the above deconvolution by subtraction is valid. There is also a weak contribution to spectrum C from a second adduct, (+)-2-(dGdC),. Evidence for this is provided by Figure 3, which shows the origin region for (+)-anti-BPDE-(dG-dC)z in the presence of ACR obtained with A,, = 346 nm (A) and 355 nm (B). Comparison of spectrum A with spectrum A

Lu et al.

62 Chem. Res. Toxicol., Vol. 4,No. I , 1991

n

ex 369.6nm

1

b

!

i

2 1

ii,\.,j r.\ . j v /"\, \,A E

376

378

380

3

..f,,,r, \.P.+.

WAVELENGTH (nm)

I

376

.\.,.' .i 377

378

/"'

379

Figure 4. Comparison of FLN spectra of (+)-anti-BPDE-(dGdC), for experimental conditions which provide, as the major adduct, (+)-l(B) and (+)-2 (A), respectively (see also Figure 2). A,, = 369.6 nm, T = 4.2 K. Zero-phonon lines (ZPL) are labeled with excited-state vibrational frequencies. ZPLs at 763 and 830 cm-' correspond to BPT-381 (see Figure 12).

WAVELENGTH ( n m ) Figure 5. Comparison of FLN spectra: (+)-anti-BPDE-DNA, (+)-1 adduct (A); (+)-anti-BPDE-(dG-dC)2(B);trans isomer of (+)-anti-BPDE-NS-GMP (C); (+)-anti-BPDE-(dG).(dC) (D); (+)-anti-BPDE-poly(dG) (E). A,, = 369.6 nm, and T = 4.2 K.

of Figure 2 (no quencher) shows that ACR significantly quenches fluorescence from (+)- l-(dG-dC),, resulting in the dominance of the former by a band a t ~ 3 7 nm 9 [due mainly to (+)-b(dG-dC),]. Nevertheless, (+)-1-(dG-dC), is not completely quenched and is responsible for the shoulder at ~ 3 7 7 . 8nm (solid arrow). Utilization of A, = 355 nm leads to essentially complete suppression of (+)-1-(dG-de), emission and a spectrum (B) due to the (+)-2-(dG-dC), adduct (the fraction of BPT-381 which is not quenched makes, at most, a weak contribution). Spectrum C in Figure 3 (obtained by scaling the origin band of spectrum C from Figure 2) is that which, when added to B, yields A. Thus, the 377.8-nm shoulder of spectrum A in Figure 3 is due to incompletely quenched (+)-1-(dG-dC),. The S1 origins for (+)-1- and (+)-2(dG-dQp are at 377.6 and 379.4 nm. These wavelengths are very similar to those for the (+)-l-and (+)-2-DNA adducts; cf. Table 11. Furthermore, the (+)-2-(dG-dC), adduct was also found to be significantly less quenchable by ACR than the (+)-1-(dG-dC), adduct, which was also observed for (+)-land (+)-2-DNA adducts. (2) Laser-Excited FLN Spectra. The 4.2 K FLN spectra provide additional support for the above assignments of the (+)-1- and (+)-2-(dG-dC), adducts. Figure 4 presents spectra obtained with A,, = 369.6 nm for (+)-anti-BPDE-(dG-dC), in the presence (A) and absence (B) of ACR. This wavelength is that which was used to obtain multiplet origin structure for (+)-1-DNA in the -600-cm-' region (19). Spectrum B is dominated by the vibronic line a t 579 cm-'. This is also the case for (+)-1DNA (19). However, the higher energy vibrations at 763 and 828 cm-' are more prominent in spectrum B of Figure 4. The reason is that the (+)-2-(dG-dC), adduct makes a greater contribution to the FLN spectrum. This follows from spectrum A of Figure 4, which shows that quenching by ACR of the contribution from (+)-1-(dG-dC), reveals modes at 648, 757, and 830 cm-'. Therefore, their relative intensity to the mode near 579 cm-' (now 581 cm-') in-

creases. Because the (+)-1-(dG-dC), adduct is more efficiently quenched than (+)-2-(dG-dC),, and their S1origins are located a t 377.6 and 379.4 nm, respectively, we conclude that spectrum A is due predominantly to the (+)-2-(dG-dC), adduct. In the Appendix, it is shown that BPT-381 cannot be contributing appreciably to the 757and 828-cm-' bands of spectrum A (with ACR) but does contribute to the 763- and 830-cm-' bands of spectrum B. It is noteworthy that the modes for (+)-l-and (+)-2(dG-dC), are identical with those measured for (+)-1- and (+)-2-DNA (19). The possibility that the (+)-1-(dG-dC), adduct contributes to the 581-cm-' band of spectrum A in Figure 4 cannot be excluded since ACR does not completely quench its fluorescence. (B) Comparison of (+)-anti-BPDEAdducts of DNA and Polynucleotides. In this subsection Table I1 is used for a discussion of the similarities and differences between the fluorescence characteristics of the (+)-1-, (+)-2-, and (+)-3-DNA adducts and those of adducts formed with (dG-dC),, (dG).(dC), and (dG). For the sake of brevity, spectra for the latter two polynucleotides have not been presented and only a portion of the FLN spectra obtained for (dG-dC), are shown. The first column of Table I1 lists the adducts; when more than one adduct from the precursor is observed, the major adduct is so labeled. Thus, for example, (+)-1-DNA and (+)-1-(dG-dC), are major adducts. The second and third columns provide the wavelength [Xfl(O,O,] and width (fwhm) of the inhomogeneously broadened fluorescence origin, while the fourth column gives the R value. These parameter values, together with the degree of quenchability by ACR, are most important for the above comparison. From Table I1 one observes that all polynucleotides, except (dA-dT),, yield an adduct with a fluorescence origin at -378 nm, a (+)-1 adduct. We emphasize again, however, that the adducts for each polynucleotide are labeled as (+)-j,with increasing j corresponding to increasing Afl(o,o). That is, adducts from different polynucleotides with the

Laser Spectroscopy of BPDE-DNA Adducts

Chem. Res. Toxicol., Vol. 4, No. 1, 1991 63

-

3

In

1 (+)- cis - BPDE-GMP

-

A (+) anti - BPDE DNA + ACR B (+) - anti - BPDE - (dGdC),+ ACR

ex 356.9 nm

ex = 369.6 nm

2

i I 5

376

377

378

379

380

3?6

378

380

382

WAVELENGTH (nm) Figure 6. FLN spectrum of (+)-anti-cis-BPDE-GMPobtained at T = 4.2 K for &I = 369.6 nm. FLN peaks are labeled with their

excited-state vibrational frequencies (cm-l).

WAVELENGTH (nm) Figure 7. FLN spectra of (+)-anti-BPDE-DNA (A) and (+)anti-BPDE-(dG-dC), (B) in the presence of 1M ACR. &I = 356.9 nm, and T = 4.2 K.

same j value need not necessarily be the same (chemically and/or structurally). Figure 5 gives the FLN spectra for the standard trans isomer of the (+)-anti-BPDE-N2-GMP adduct (spectrum C), (+)-1-DNA (spectrum A), (+)anti-BPDE-(dG-dC), (spectrum B), (+)-anti-BPDE(dG).(dC) (spectrum D), and (+)-anti-BPDE-(dG) (spectrum E). All spectra were obtained with A,, = 369.6 nm, which is quite highly selective for (+)-ladducts whose S, state is located a t -378 nm. That the spectrum for (+)-l-DNA is dominated by the 579-cm-' mode was established earlier (19). The close similarity of the spectra in Figure 5 suggests that all adducts are of the N2-dG type. Furthermore, the standard spectrum for A,, = 369.6 nm of the cis isomer of (+)-anti-BPDE-N2-GMP shown in Figure 6 is distinctly different from that of the trans isomer shown in Figure 5 (spectrum C). Thus, we may conclude that the (+)-1 adducts are trans isomers, a conclusion consistent with that of earlier work (41). We note that a (+)-2 adduct from (dG).(dC) probably contributes to spectrum D since its S1 state is almost degenerate with that of the (+)-1 adduct. The (+)-1-DNA adduct and the trans isomer of (+)anti-BPDE-GMP exhibit the same R value of 1.75 (Tables I1 and 111). This result, together with spectra A and C of Figure 5, supports our earlier assignment (19) for (+)-1DNA to a type I1 (external) adduct with high quenching efficiency. Inspection of Table I1 reveals that it is the (+)-1 adduct of (dG-dC)* whose R value (1.55) and other fluorescence characteristics are most similar to those of (+)-1-DNA. For example, the R values for the (+)-1 adducts of (dG).(dC) and (dG) are considerably smaller (1.20 and 1.15), indicating that they may correspond to a type I (interior) site (19). Consideration of the values given in Table I1 for the widths of the fluorescence origin bands due to inhomogeneous broadening [the linear electronphonon coupling is weak for all (+)-1 adducts, as judged by the FLN spectra, and makes only a negligible contribution to the bandwidth] reveals that the structure of (dG)

is highly irregular, thereby providing a relatively less ordered environment for the trans-(+)-l-N2-dG adduct. Coiling of (dG) probably contributes to the heterogeneity. It should also be noted that the adducts from (dG).(dC) are distinct from those of DNA and (dG-dC), in that the (dG).(dC) (+)-1 adduct is less quenchable by ACR than the (+)-2 adduct. Table I1 also reveals that it is the (+)-2 adduct of (dG-dC), which is most similar to (+)-%-DNA. Thus, the alternating (dG-dC), copolymer affords a structure conducive to the formation of two adducts with spectral and other properties which are very similar to the (+)-l(type 11) and (+)-2 (type I) adducts identified in previous work in native DNA (19). However, this copolymer does not yield an adduct of the intercalated and nonquenchable (+)-3-DNA type with a fluorescence origin at -381 nm. This latter adduct does not exhibit line narrowing in its fluorescence or excitation spectra due to strong electron-phonon coupling (19). In the Appendix it is shown that BPT-381, which can be easily mistaken for a covalent adduct of the (+)-3type, does exhibit line narrowing and is quenchable by ACR. Figure 7 compares the 4.2 K fluorescence spectra of (+)-anti-BPDE-DNA and -(dG-dC), obtained with A,, = 356.9 nm [SI So vibronic excitation, which also reveals the contribution from (+)-3-type adducts] and in the presence of 1 M ACR. Although fluorescence from the (+)-1adduct is significantly quenched, (+)-land (+)-2 still contribute to the sharp vibronic features a t 1442,1519,1560,and 1606 cm-I [(+)-2 is a minor adduct for DNA (19) and (dG-dC),; its relative contribution is revealed by using ACR quencher]. The close similarity between these features for DNA and (dG-dC), further confirms that (dG-dC), yields adducts of the (+)-1- and (+)-2-DNA type. It is, however, the broad feature at -381 nm for (+)-anti-BPDE-DNA that is of primary interest (spectrum A). It is the fluorescence origin of (+)-3-DNA (19). Spectra obtained without ACR (not shown) show that this fluorescence is not quenchable. In contrast, BPDE-(dG-dC), emission at 381 nm is

-

64

Chem. Res. Toxicol., Vol. 4 , No. I , 1991

Table 111. Spectral Characteristics of (+)-anti-BPDE-GMP and ( )-anti-BPDE-d(ATATGTATA) Adducts fluorescence band fwhmb adducts of max (nm) (O,O), (+)-anti-BPDE f 0.1 nm cm-' R (f0.05) site type NZdG, trans-GMP 377.3a 130 1.75 N2-dG, cis-GMP 377.4" 150 1.75 N2-dG, 379.5 250 1.3 I' trans-d(-TGT-) N2-dG, 380.5 190 1.25 I' cis-d(-TGT-)

+

-

"Band maxima and width of (0,O) fluorescence bands were obtained after deconvolution. bfwhm was calculated as a twice the half-width on the high-energy side. 'This adduct shows basestacking interactions.

quenchable. (The degree of quenching for 1 M ACR is higher than for 0.5 M ACR; unpublished data). Figure 7 and other results establish that ( d G - d Q does not yield an appreciable amount of an intercalated covalent adduct of the (+)-3-DNA type. This is consistent with the finding that the intercalation association constant for this duplex is small relative to that of DNA (37, 38, 40). The explanation may lie in the fact that DNA has many intercalation binding sites, 10 when strand polarity is taken into account. In addition, the reactivities of guanines with (+)-antiBPDE are distinctly nonstatistical with respect to sequence (11, 1 2 , 4 1 , 4 3 ) . However, both spectra in Figure 7 were obtained for (+)-anti-BPDE. Therefore, not only trans type but also a small contribution from cis-type adducts is expected. This will be discussed further in section C. In summary, all polynucleotides studied yield a (+)-l adduct of the trans-NZdG type whose special characteristics are very similar to those of (+)-1-DNA. However, it is the alternating (dG-dCIzthat provides the best model for the (+)-l-and (+)-2-DNA adducts. The FLN spectra of the ( + ) - l ,when compared with those of the standard trans and cis isomer of (+)-anti-BPDE-GMP, reveal that (+)-1 is the trans isomer of the N2-dG covalent adduct. Furthermore, HPLC analysis of the nucleotide adducts from (+)-anti-BPDE-DNA ( 2 7 , 3 3 , 4 1 )have shown that trans adducts dominate. Of course, with HPLC analysis all information pertaining to different DNA site conformations for the same chemical adduct is lost. It is argued in section C that both the (+)-1 and (+)-2 are trans-N2-dG adducts in two different site conformations. The present and earlier work (19) establishes that the (+)-ladduct for DNA and (dG-dC)pis of type I1 or exterior (solvent accessible) with a relatively weak interaction between the pyrene chromophore and bases. The (+)-2 adduct is characterized by a stronger interaction (with concomitant red shift of the SI state) and has a more interior (hydrophobic) configuration, albeit not an intercalated configuration (quasi-intercalated) as is the case for (+)-3-DNA. (C) anti-BPDE Adducts of an Oligodeoxynucleotide with Specific Base Composition. According to the above analysis, the (+)-1 and (+)-2 adducts for DNA and (dC-dOzare not of the intercalated type. Thus, one might expect to observe similar adducts for single-stranded oligodeoxynucleotides such as d(ATATGTATA1. On the other hand, an intercalated adduct of the (+)-3-DNA type should not be observed. The single strand d(-TGT.-) provides for high yields of N2-dG adducts ( 3 2 ) . For (+)-anti-BPDE and (-)-anti-BPDE the trans:cis ratios are 7:l and 2:1, respectively, as determined by HPLC analysis following enzymatic digestion (32). Table 111summarizes the spectral characteristics (spectra not shown) of the

Lu et al. trans- and cis-BPDE standard isomers of the N2-dG adduct from GMP and the trans- and cis-BPDE isomers from d(-TGT-). For example, the origin bands of (+)-anti-BPDE-d(.. TGT-) and (+)-anti-BPDE-GMP were observed at 379.5 and -377.3 nm, respectively. The band positions were observed to be independent of excitation wavelength within the Sz So absorption system, indicating the existence of only one dominant emitting species for each sample. Both samples contained only the trans isomer of the NZdG adduct (as determined by HPLC analysis). The substantial -2.2-nm red shift of the origin for the d(TGT-) trans-N2-dG adduct suggests that it is characterized by a BPDE-base stacking interaction. This adduct is quite stable; prolonged laser irradiation at 355 nm or heating to 80 OC had no significant effect on the low-temperature fluorescence spectra. However, the Sz So absorption spectrum measured at 80 "C is substantially blue-shifted relative to the room temperature spectrum (32). Upon subsequent cooling to room temperature, the original absorption spectrum was recovered. This is consistent with BPDE-base stacking since such stacking is disrupted at higher temperatures. From Table I1 and I11 it is apparent that the S1 state energy, fwhm, and R value for this adduct are similar to those for the (+)-2 adducts of DNA and (dG-d(&. This confirms that the latter two adducts are trans-N2-dG. On the other hand, the spectral characteristics of the d(-TGT-) adduct are very different from those of the (+)-ladducts of DNA and (dG-dC),; cf. Tables I1 and 111. It appears, therefore, that the d(-TGT-) oligonucleotide structure does not lead to appreciable formation of a trans-N2-dG adduct of the (+)-1 conformation (exterior, type 11). With reference to the band position of (+)-trans-BPDE-GMP, we note that the origin of the trans-N2-dG adduct of GMP is only slightly blueshifted (by 0.3 nm) relative to that of the (+)-ladduct of (dG-dC), and that both adducts are characterized by a high R value; cf. Tables I1 and 111. Our results show that the (+)-3 adduct is not formed with d(-TGT-), as anticipated since this is an intercalated adduct. Figure 8 presents FLN spectra obtained with A,, = 356.9 nm for (+)-trans-BPDE-d(-TGT-) (A) and (+)-cisBPDE-d(-TGT-) (B). Spectrum A should be compared with those of Figure 7, from which it may be concluded that spectrum A of Figure 8 exhibits narrow vibronic features (1388-1603 cm-') which are essentially identical with those of (+)-anti-BPDE-DNA + ACR and (+)anti-BPDE-(dG-dC)2 ACR due primarily to the dominant (+)-2 adduct. A striking difference between spectra A and B of Figure 7 is the absence of the broad -381-nm emission in spectrum B. Thus the (+)-3 adduct does not contribute significantly for the (dG-dC)z deoxypolynucleotide. However, careful comparison of spectrum A in Figure 8 for (+)-trans-type adduct with spectrum B in Figure 7 for (+)-anti-BPDE-(dG-dC), + ACR (in which a mixture of trans and cis adducts is anticipated) shows differences in the long-wavelength region, e.g., -379-382 nm. This broad emission with a maximum at -381 nm (at 4.2 K) contributes even more in (+)-anti-BPDE(dG).(dC) and (+)-anti-BPDE-poly(dG) (spectra not shown). One possible explanation is that this contribution is associated with (+)-cis-type adducts. This is confirmed by the spectrum of pure (+)-cis-BPDE-type adduct separated by HPLC from (+)-anti-BPDE-d(-TGT-), whose spectrum is shown in Figure 8, curve B. These and other unpublished data show that cis adducts can adopt a site I configuration (Geacintov et al., to be submitted for publication). Although we are still investigating the

-

-

+

Chem. Res. Toxicol., Vol. 4, No. 1, 1991 65

Laser Spectroscopy of BPDE-DNA Adducts A: (+)-@ans-BPDE-d(ATATGTATA) B: (+)-cis-BPDE-d(ATATGTATA)

A (+) - anti - BPDE - DNA B (+) - anti - BPDE - (dA-dT),

3 2lm

t z

CJ

d

m m

A

c

m

v,

z z -

W -I

W

0

z W

0 v,

A

W

il

LT

0

3 LL

376

378

380

382

WAVELENGTH (nm)

380

381

382

383

384

WAVELENGTH (nm)

Figure 8. Comparison of FLN spectra of (+)-trans-and (+)cis-type adducts obtained from (+)-anti-BPDEwith d(ATATGTATA) oligomer (spectra A and B, respectively). A,, = 356.9 nm, and T = 4.2 K.

Figure 9. Comparison of FLN spectra: (+)-anti-BPDE-DNA (A) and (+)-anti-BPDE-(dA-dT)2.A,, = 374.2 nm, and T = 4.2 K.

spectroscopic properties of cis-type adducts, we tentatively assign the (+)-3-(dG-dC),, (+)-3-(dG)-(dC), and (+)-3poly(dG) from (+)-anti-BPDE as (+)-cis isomers with properties similar to those of (+)-3-DNA. (D) Assignment of a (+)-aoti-BPDE-Deoxyadenosine Adduct of DNA. As discussed in the introduction, (-)-anti-BPDE yields a more heterogeneous distribution of DNA adducts than (+)-anti-BPDE, which consists of five covalent adducts, (-)-j (j = 1-5). Although the relative levels of (-)-j (j = 1-3) differ significantly from those of (+)-j (j = 1-31, the spectral and quenching characteristics of the adducts with the same j value are identical. It was suggested (19) that the (-)-4 adduct could be associated with a base other than guanine, e.g., adenine, since its A,(,, of 382.7 nm is higher than those of (-)-j (j = 1-3) and, yet, the adduct is of type 11. Brookes and Osborne (26,27) have reported that (+)- and (-)-BPDEDNA consists of 2% and 18% of N6-dA adducts, respectively. Also, recent data obtained by Cheng et al. (33) showed that the percentage of total adducts for deoxyadenosine from reaction of (+)- and (-)-BPDE with DNA is 5% and 15%, respectively. These data explain why the (+)-4 adduct was not detected in our earlier work (19). In an attempt to identify the minor (+)-4 adduct, further studies of (+)-anti-BPDE-(dA-dT), and (+)-anti-BPDEDNA were undertaken. First, the results from 77 K fluorescence studies of (dA-dT)*summarized in Table I1 show, as expected, that the (+)-l and (+)-2 adducts from dG are absent. Only one adduct with a fluorescence origin at -3382.5 nm was identified. A small contribution from BPT was also observed [BPDE adducts of poly(dA-dT)* are less stable than those of (dG-dC), (28)]. The spectral characteristics of (+)-4-DNA are identical with those of (-)-4-DNA (Table 11). Figure 9 compares the FLN spectra of (+)-anti-BPDEDNA (A) and (+)-anti-BPDE-(dA-dT), (B) obtained with A,, = 374.2 nm. This wavelength was chosen to reveal

mode structure in the 600-cm-' region for adducts with a fluorescence origin near 382 nm. The spectra exhibit the same modes, e.g., 472 and 585 cm-l. The latter is the counterpart of the 579-cm-' mode of the (+)-l and -2 adducts. The frequency shift of 6 cm-' is large and, by itself, could be used to distinguish between N2-dG and N6-dA adducts of BPDE. However, the vibronic intensity distributions of spectra A and B in Figure 9 are different. Two possible explanations for this are that (+)-2-DNA makes a significant contribution to the 472-cm-' band of spectrum A and/or that the trans to cis distributions for DNA and (dA-dT), are different. The latter is supported by recent results obtained by Cheng et al. (33),who showed that the relative percentage of adducts for cis- and trans-deoxyadenosine from reaction of (+)-anti-BPDE with DNA and with deoxyadenylic acid is 1:4 and 1:1, respectively. The FLN spectrum for (-)-I-DNA is similar to spectrum A of Figure 9. The FLN data indicate that the (+)-I-DNA and (-)-4-DNA adducts possess the same chemical structure and, furthermore, that they are not N2-dG adducts. Consistent with the results of Brookes and Osborne (26,27) and Cheng et al. (33), we have observed a higher relative signal strength from (-)-I-DNA than from (+)-4-DNA. On the basis of their work it is not unreasonable to assign both (+)- and (-)-I-DNA adducts as trans-N6-dA. The structures of the trans isomer of this adduct and trans-N2-dG are shown in Figure 10. Adenine adducts were suggested to be predominantly intercalative in nature (28). However, linear dichroism experiments of (+)-anti-BPDE-(dA-dT), yield no signal from the BPDE chromophore absorption region, indicating significant disorder at the binding site (C. Roche and N. E. Geacintov, unpublished results). Although the pyrenyl residue of dA adduct in the vicinity of the negatively charged phosphodiester groups could account for the high R value, in general, the properties of (+)- and (-)-4 adducts are different from those of sites I and sites 11. Therefore, in Table 11, we designate the ( f ) - 4 adducts as site 111.

Lu et al.

66 Chem. Res. Toxicol., Vol. 4 , No. I, 1991 0

OH

A)

N2-dG

B)

N6-dA

Figure 10. Structures of the NP-dG (A) and N6-dA (B) adducts formed from (+)-anti-t~an.~-BPDE.

Conclusions This work shows that FLNS in combination with So fluorescence quenching and laser-excited S2 fluorescence spectroscopy can distinguish between chemically and/or structurally (site) different adducts of BPDE in the distribution of adducts from a polynucleotide. Furthermore, differences in the type and heterogeneity of distributions from different polynucleotides and singlestrand oligonucleotides can be detected. By themselves the results establish that the major DNA adduct from the stereoselective reaction with (+)-anti-BPDE is the trans isomer of N2-dG, which is in accord with previous work (33,411. The (+)-2-DNA is also an N2-dG adduct which, on the basis of the results for (+)-anti-BPDE-d(-TGT-), is suggested to be the same stereoisomer in a different site configuration. The (+)-3-(dG-dC),, (+)-3-(dG).(dC), and (+)-poly(dG) adducts, which also involve binding to dG, are assigned as cis adducts (site I) with properties similar to those of (+)-3-DNA. In the future it will be important to establish the structure of (+)-3-DNA-type adducts, especially that at low damage level (expected in vivo samples) their relative contribution significantly increases (unpublished data). Our recent unpublished data with DNA and different polynucleotides indicate that the base-stacked (+)-%-type adduct exists also at room temperature and is responsible for room temperature excimer formation (fluorescence maximum a t -475 nm). Favored covalent binding to bases close to already modified guanines [even at small (0.008) BPDE/nucleotide ratios] has been observed (43). However, the biological significance of closely spaced adducts has yet to be determined. In addition, a question that often arises is whether or not the identification of different DNA configurations (sites) for a base-metabolite adduct of specified chemical structure at low temperatures in glasses is relevant to structures a t biological temperatures. We have in mind our results that show that there are two configurations for the trans-N2-dG isomer from (+)-anti-BPDE. The answer to this question emerges when one recognizes that a DNA

-

adduct configuration can only be trapped at low temperatures provided it is thermally accessible a t high temperatures. The only relevant question is whether what one observes in a solid at low temperatures reflects all of the configurations accessible at room temperature. However, it is interesting to note that our findings concerning the heterogeneity of DNA adducts from (+)-anti-BPDE and (-)-anti-BPDE are in qualitative accord with those based on HPLC analysis of nucleotide adducts (26, 27). A fourth, very minor adduct [ (+)-4-DNA] was observed and assigned as trans-N6-dA. It has been shown, for example, that 36% of the activation of the ras protooncogene in vitro by (+)-anti-BPDE occurs at the adenine residue (45),despite the latter’s low reactivity. Therefore, the characterization and determination of minor stable adducts and labile adducts which undergo elimination [e.g., the alkali labile N7-dG adduct ( l I , 1 2 , 4 4 ) ]is very important (26, 27, 45, 46). In this work, only three polynucleotide duplexes [(dGdC)2,(dG).(dC), (dA-dT),] were studied; of these, it is the alternating ( d G - d Q that yields (+)-land -2 adducts which are most similar to the trans-N2-dG adducts of DNA. However, the R ratio for (+)-l-(dG-dC), of 1.55 is somewhat lower than the value of 1.75 for (+)-1-DNA. Since (dG).(dC) yields an N2-dG adduct with spectral properties similar to those of (+)-1-DNA and dG tracts have been found to be particularly conducive to reactions of (+)anti-BPDE with dG ( 1 1 , 12,32), it is most likely that in native DNA more than one type of base triplet involving dG contributes to the (+)-1-DNA fluorescence. This is probably also the case for (+)-2- and (+)-3-DNA. Although our results for the adducts from (-)-antiBPDE with the above polynucleotides have not been presented, they support our finding that the laser-excited S2 Sofluorescence spectra, FLN spectra, and quenching properties of the (+)-j-DNA and (-)-j-DNA adducts with the same j value (j = 1-4) are exceedingly similar (only the distributions differ). Since we have shown that the trans and cis isomers of the N2-dG adduct with GMP are readily distinguishable by FLNS, the above close similarity suggests that (+)-)-DNA and (-)-j-DNA are enantiomeric pairs. Distinction between the two enantiomers would depend, in part, on interactions with chiral centers of neighboring bases which, if weak, would not allow for distinction by FLNS. Although (+)-anti-BPDE and (-)-anti-BPDE form stable adducts, the former is significantly more mutagenic for the same total adduction (stable) level in repair-competent cells ( I , 27). Both are comparably mutagenic on the repair-deficient Salmonella typhimurium strain (46). Our results provide a more detailed picture of the differences in the adduct distributions from (+)- and (-)-antiBPDE-DNA, but the different mutagenic activities of the two enantiomeric forms cannot be understood in terms of the adduct (stable) distributions without invoking unique site-structural properties of the (+)-j and (-1-j adducts ( 4 , 6, 44, 45). However, if one is to hold to the assumption that it is the diol epoxide metabolic pathways of BP which, in vivo, lead to mutagenesis and tumorigenesis, then the roles of labile adducts and (+)- and (-)-syn-BPDE must be more carefully assessed. In addition, the possibility that the radical cation (one-electron oxidation) metabolic pathway may be important for mutagenesis and carcinogenesis is now distinct (8, 25).

-

Appendix Physical Mixtures of anti-BPT with Native DNA and (dG). Figure 11 shows 77 K laser-excited (S2 So)

-

Laser Spectroscopy of BPDE-DNA Adducts

Chem. Res. Toxicol., Vol. 4, No. 1, 1991 67

~~

i

anti-BPT + DNA ex 369.6 nm A without ACR B with ACR

A (BPT+DNA) B (BFT+DNA+ACR) C A - B

T

m t-

380

390

460

4io

WAVELENGTH ( n m ) Figure 11. Laser-excited fluorescence spectra: racemic BPTDNA (A) and racemic BPT-DNA + 1 M ACR (B). A,, = 355 nm, and T = 77 K. Spectrum C is the difference between A and B, which identifies the ACR-quenched bands.

spectra of racemic anti-BPT (6 X lo4 M) which has been M). Spectra A and B physically mixed with DNA were obtained in the absence and presence of the fluorescence quencher (ACR, 1 M) with A,, = 355 nm. The former is characterized by three origins at -376.1, 376.8, and 381.1 nm. Utilization of A,, = 346 nm yielded a spectrum (not shown) that is dominated by the BPT responsible for the 376.1- and 376.8-nm bands. These two bands are assigned to “free” and/or weakly physically bound (to DNA) BPT. Spectrum C (dashed) in Figure 11 is the A - B difference spectrum which illustrates (as does a comparison of spectra A and B) that with 1 M ACR the 381.1-nm fluorescence is almost entirely quenched (considerably more so that the “free” BPT bands; the 376.1-nm band is more efficiently quenched than the 376.8-nm band). Since these are low-temperature glass spectra, it is clear that the observed quenchings are of the static type dictated by frozen-in configurations of ACR-BPT-DNA which exist a t room temperature. The 381.1-nm band is referred to as BPT-381. BPT-381 and “free” BPT was also observed for a physical mixture of BPT with poly(dG) and/or DNA. The significant red shift of the S,-state energy of BPT for the BPT-381 entity indicates that in the latter the pyrenyl chromophore is strongly interacting with nucleic acid bases, e.g., base-base stacking interaction. Yet the structure of the physically bound BPT-381 is sufficiently open to permit solvent accessibility, i.e., ACR quenching. This should be contrasted with room temperature spectra of aqueous BPT-DNA solutions, in which the red-shifted emission peak at 382 nm, presumably, due to intercalated BPT, is quenched less than the emission of “free” BPT whose emission maximum is at 379 nm (29). A t sufficiently high laser intensity (-30 mW/cm2) it was observed that the fluorescence intensity of BPT-381 relative to that of the “free” BPT bands increased, suggesting the possibility that BPT-381 could be contributed to by a photoproduct or that interconversion in the excited state

3?6

378

380

382

WAVELENGTH (nm)

Figure 12. FLN spectra of (+)-anti-BPT + DNA obtained for vibronic excitation: without (A) and with quencher (1M ACR) (B). A,, = 369.6 nm, and T = 4.2 K. between the “free” BPT and BPT-381 is occurring. FLN spectra of BPT-DNA and BPT-(dG) were also obtained. As an example, Figure 12 shows spectra for the BPT-DNA mixture obtained with A,, = 369.6 nm in the absence (A) and presence (B) of ACR. The sharp zerophonon lines (ZPL) are labeled with the &-state vibrational frequencies since the ZPLs for “free” B P T and BPT-381 should be centered near 376-377 and 381 nm, respectively. Comparison of FLN spectra A and B and the results of Figure 11 establish that the 763- and 830-cm-’ ZPL are due to BPT-381 while the lower frequency modes, e.g., 469 and 579 cm-’, are due to “free” BPT. As discussed in the main body of the text, BPT-381 can be easily misassigned to the (+)-3-DNA type I adduct. However, the latter is distinct in that its strong linear electron-phonon coupling precludes the observation of line narrowing (of the type observed for BPT-381) and its fluorescence is not significantly quenched by ACR. Registry No. (+)-anti-BPDE, 63323-31-9; trans-N2-dGBPT, 78779-87-0; cis-N2-dG-BPT, 130694-28-9; trans-N6-dA-BPT, 130694-27-8;GMP, 85-32-5;poly(dG-dC).poly(dG-dC),62081-33-8; poly(dA-dT)*poly(dA-dT),26966-61-0; poly(dG)*poly(dC), 25512-84-9; poly(dG), 25656-92-2; d(ATATGTATA), 130574-83-3.

References (1) Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. (1985) Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens. In Bioactiuation of Foreign Compounds (Anders, M. W., Ed.) pp 177-242, Academic Press, New York. (2) Stevens, C. W., Bouck, N., Burgess, J. A., and Fahl, W. E. (1985) Benzo(a)pyrene diol epoxides: different mutagenic efficiency in human and bacterial cells. Mutat. Res. 152, 5-14. (3) Pelling, J. C., Neades, R., and Strawhecker, J. (1988) Epidermal papillomas and carcinomas induced in uninitiated mouse skin by tumor promoters along contain a point mutation in 61st codon of the Ha-ras oncogene. Carcinogenesis 9, 665-667. (4) Geacintov, N. E. (1988) Mechanisms of reaction of polycyclic aromatic epoxide derivatives with nucleic acids. In Polycyclic

68

Chem. Res. Toxicol., Vol. 4, No. 1, 1991

Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relationships (Yang, s. K., and Silverman, B. D., Eds.) Vol. 2,pp 181-206,CRC Press, Boca Raton, FL. (5) Geacintov, N. E. (1985)Mechanisms of interaction of polycyclic aromatic diol epoxides with DNA and structures of the adducts. In Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) pp 107-124,American Chemical Society, Washington, DC. (6) Graslund, A,, and Jernstrom, B. (1989)DNA-carcinogen interaction: covalent DNA-adducts of benzo(a)pyrene 7,8-dihydrodiol 9,10-epoxides studied by biochemical and biophysical techniques. Q.Rev. Biophys. 22, 1-37. (7) Lehr, R. E., Kumar, S., Levin, W., Wood, A. W., Chang, R. L., Conney, A. H., Yagi, H., Sayer, J. M., and Jerina, D. M. (1985) The bay region theory of polycyclic aromatic hydrocarbon carcinogenesis. In Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) pp 63-84, American Chemical Society, Washington, DC. (8) Cavalieri, E., and Rogan, E. (1985)Role of radical cations in aromatic hydrocarbon carcinogenesis. Environ. Health Perspect. 64,69-84. (9) Glatt, H., and Oesch, F. (1986)Structural and metabolic parameters governing the mutagenicity of polycyclic aromatic hydrocarbons. In Chemical Mutagens (de Serees, F. J., Ed.) Vol. 10, pp 73-127, Plenum Publishing Corp. (10) Stevens, C. W.,Manoharan, T. H., and Fahl, W. E. (1988) Characterization of mutagen-activated cellular oncogenes that coner anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: sequence analysis of an enzymatically amplified mutant HRAS allele. Roc. Natl. Acad. Sci. U.S.A. 85, 3875-3879. (11) Boles, T.C., and Hogan, M. E. (1986)High-resolution mapping of carcinogen binding sites on DNA. Biochemistry 25,3039-3043. (12) Lobanenkov, V. V.,Plumb, M., Goodwin, G. M., and Grover, P. L. (1986)The effect of neighboring bases on G-specific DNA cleavage mediated by treatment with the anti-diol epoxide of benzo(a)pyrene in vivo. Carcinogenesis 7, 1689-1695. (13) Jankowiak, R., and Small, G. J. (1989)Fluorescence line-narrowing spectrometry in the study of chemical carcinogenesis. Anal. Chem. 61,1023-1032. (14) Heisig, V.,Jeffrey, A. M., McGlade, M. J., and Small, G. J. (1984)Fluorescence-line-narrowed spectra of polycyclic aromatic carcinogen-DNA addcts. Science 223, 289-291. (15) Sanders, M. J., Cooper, R. S., Small, G. J., Heisig, V., and Jeffrey, A. M. (1985)Identification of polycyclic aromatic hydrocarbon metabolites in mixtures using fluorescence line narrowing spectrometry. Anal. Chem. 57, 1148-1152. (16) 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 narrowing spectrometry. Anal. Chem. 58, 816-820. (17) Zamzow, D., Jankowiak, R., Cooper, R. S., and Small, G. J. (1989) Fluorescence line narrowing spectrometric analysis of benzo[a]pyrene-DNA adducts formed by one-electron oxidation. Chem. Res. Toxicol. 2, 29-34. (18) Jankowiak, R., Cooper, R. S., Zamzow, D., Small, G. J., Doscocil, G., and Jeffrey, A. M. (1988)Fluorescence line narrowing-nonphotochemical hole burning spectrometry: femtomole detection and high selectivity for intact DNA-PAH adducts. Chem. Res. Toxicol. 1, 60-68. (19) Jankowiak, R., Lu, P., Small, G. J., and Geacintov, N. E. (1990) Laser spectroscopic studies of DNA adduct structure types from enantiomeric diol epoxides of benzo[a]pyrene. Chem. Res. Toxicol. 3, 39-46. (20) Jankowiak, R.,Lu, P., & Small, G . J. (1990)Fluorescence line narrowing spectrometry: A versatile tool for the study of chemically initiated carcinogenesis. J. Pharm. Biomed. Anal. 8, 113-121. (21) Cooper, R. S.,Jankowiak, R., Hayes, J. M., Lu, P., and Small, G. J. (1988)Fluorescence line narrowing spectrometry of nucleoside-polycyclic aromatic hydrocarbon adducts on thin-layer chromatographic plates. Anal. Chem. 60,2692-2694. (22) Jankowiak, R.,Day, B. W., Lu, P., Doxtader, M. M., Skipper, P. L., Tannenbaum, S. R., and Small, G. J. (1990)Fluorescence line-narrowing spectral analysis of in vivo human hemoglobinbenzo[ajpyrene adducts: Comparison to synthetic analogues. J . Am. Chem. SOC. 112,5866-5869. (23) Varanasi, U., Reichert, W. L., Eberhart, B. T. L., and Stein, J. E. (1989)Formation and persistence of benzo[a]pyrene-diolepoxide-DNA adducts in liver of English sole (Parophrys uetulus).

Lu et al. Chem.-Biol. Interact. 69,203-216. (24) Lu, P. (1990)Laser excited fluorescence studies of cellular macromolecular damage from chemical carcinogens. Ph.D. Dissertation, Iowa State University, Ames, IA. (25) Rogan, E. G., Ramakrishna, N. V. S., Higginbothan, S., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1990) Identification and quantitation of 7-(benzo[a]pyrene-6-yl)guanine in the urine and feces of rats treated with benzo[a]pyrene. Chem. Res. Toxcol. 3,441-444. (26) Osborne, M. E., Jacobs, S., Harvey, R. G., and Brooks, P. (1980) Minor products from the reaction of (+) and (-) benzo[a]pyrene-anti-diol-epoxide with DNA. Carcinogenesis 2, 553-558. (27) Brookes, P., and Osborne, M. R. (1982)Mutation in mammalian cells by stereoisomers of anti-benzo[a]pyrene-diolepoxidein relation to the extent and nature of the DNA reaction products. Carcinogenesis 3, 1223-1226. (28) Kim, S.K.,Geacintov, N. E., Zinger, D., and Sutherland, J. C. (1989)Fluorescence spectral characteristics and fluorescence decay profiles of covalent polycyclic aromatic carcinogen-DNA adducts. In Synchrotron Radiation in Structural Biology (Sweet, R. M., and A. D., Woodhead, Eds.) Basic Life Sciences 51, pp 187-205, Plenum Press, New York. (29) Kim, S. K.,Brenner, H. C., Soh, B. J., and Geacintov, N. E. (1989) Fluorescence spectroscopy of benzo[a]pyrene diol epoxide-DNA adducts. Conformation specific emission spectra. Photochem. Photobiol. 3, 327-337. (30) Geacintov, N. E., Ibanez, V., Gagliano, A. G., Jacobs, S. A., and Harvey, R. G. (1984)Stereoselective covalent binding of antibenzo[a]pyrene diol epoxide to DNA. Conformation of enantiomer adducts. J. Biomol. Struct. Dyn. 1, 1473. (31) Geacintov, N. E., Yoshida, H., Ibanez, V., Jacobs, S. A,, and Harvey, R. G. (1984)Conformations of adducts and kinetics of binding of the optically pure enantiomers of anti-benzo[a]pyrene diol epoxide. Biochem. Biophys. Res. Commun. 122,33. (32) Cosman, M., Ibanez, V., Geacintov, N. E., and Harvey, R. G. (1990)Preparation and isolation of adducts in high yield derived from the binding of two benzo[a]pyrene-7,8-dihydroxy-9,lO-oxide stereoisomers to the oligonucleotide d(ATATGTATA). Carcinogenesis 11, 1667-1672. (33) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1988) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, 334-339. (34) Chen, F. M. (1986)Binding of enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene to polynucleotides. J. Biomol. Struct. Dyn. 4,401-418. (35) Zinger, D., Geacintov, N. E., and Harvey, R. G. (1987)Conformations and selective photodissociation of heterogeneous benzo[a]pyrene diol epoxide enantiomer-DNA adducts. Biophys. Biochem. 27, 131-138. (36) Geacintov, N. E. (1986)Is intercalation a critical factor in the covalent binding of mutagenic and tumorigenic polycyclic aromatic diol epoxides to DNA? Carcinogenesis 7, 759-766. (37) Brown, J. C., Hayes, J. M., Warren, J. A., and Small, G. J. (1981)in Lasers in Chemical Analysis (Hiefje, G. M., Travis, J. C., and Lyfle, F. E., Eds.) Chapter 12,Humana Press, Clifton, NJ. (38) Personov, R. I. (1983)Site selection molecular spectroscopy. In Spectroscopy and Excitation of Condensed Molecular Systems (Agranovich, V. M., and Hochstrasser, R. M.,Eds.) pp 555-619, North-Holland Press, New York. (39) Geacintov, N. E., Shahbez, M., Ibanez, V., Moussaoui, K., and Harvey, R. G. (1988)Base-sequence dependence of noncovalent complex formation and reactivity of benzo[a]pyrene diol epoxide with polynucleotides. Biochemistry 27, 8380-8386. (40) Chen, F. M.(1984)Sequence specific binding of tetraols of benzo[a]pyrene diol-epoxide to DNA in neutral and acidic solution. Carcinogenesis 5, 753-758. (41) Meehan, T., and Straub, K. (1979)The covalent binding of enantiomeric BaF’DEs to double stranded DNA is stereoselective. Nature 227,410-412. (42) Yang, N.-C. C., Hrinyo, T. P., Petrich, J. W., and Yang, D.-D. H. (1983)Base sequence selectivity in binding of aromatic hydrocarbons with synthetic polynucleotides. Biochem. Biophys. Res. Commun. 114,8-13. (43) Erikson, M., Erikson, S., Norden, B., Jernstrom, B., and Graslund, A. (1990)Excimer fluorescence of (+)-anti-benzo(a)pyrene diol epoxide covalently bound to poly(dG-dC): structural implications. Biopolymers 29, 1249-1260. (44) Rill, R. L., and Marsh, G. A. (1990)The sequence preference of covalent DNA binding by anti-(+)- and anti-(-)-benzo[a]pyrene

Laser Spectroscopy of BPDE-DNA Adducts diol epoxide. Biochemistry 29, 6050-6058. (45) Di Giovanni, J., Sawyer, T. W., and Fisher, E. P. (1986) Correlation between formation of a specific hydrocarbon-deoxyribonucleoside adduct and tumor initiating activity of 7,12-dimethylbenz[a]anthracene and its 9- and IO-monofluoro derivatives in mice. Cancer Res. 46, 4336-4341. (46) Vousden, K. H., Bos, J. L., Marshall, C. J., and Phillips, P. H.

Chem. Res. Toxicol., Vol. 4, No. 1, 1991 69 (1986) Mutations activating human C-Haras 1 proto-oncogene (HRAS,) induced by chemical carcinogens and epurination. Roc. Natl. Acad. Sci. U.S.A. 83, 1222-1226. (47) Burgess, J. A., Stevens, C. W., and Fahl, W. E. (1985) Mutation a t separate gene loci in Salmonella typhimurium TA,, related to DNA nucleotide modification by stereoisomeric benzo[a]pyrene 7,8-diol 9,lO-epoxides. Cancer Res. 45, 4257-4262.