Diethylstilbestrol-DNA Interaction Studied by Fourier Transform Infrared and Raman Spectroscopy*

The interaction of diethylstilbestrol (DES) with calf thymus DNA was investigated at physiological pH with drug/DNA (phosphate) molar ratios ( r ) of 1:40, 1:20, 1:10, 1:4, 1:2, and 1. Fourier transform infrared and laser Raman difference spectroscopy were used to establish cor- relations between spectral changes and drug binding mode, sequence selectivity, DNA conformation, and structural properties of DES (cid:122) DNA complexes in aqueous solution.Spectroscopic results indicated that DES is a weak intercalator with affinity for A-T-rich regions. It is also a groove binder with a major interaction with the thymine O-2 atom. At low drug concentration ( r (cid:53) 1:40), the A-T-rich region is the main target of drug intercalation, while at a higher drug content ( r > 1:5), external binding to the G-C bases also occurs with a partial helix desta- bilization. Evidence for this comes from the spectral alterations of the A-T vibrational frequencies at 1661 cm (cid:50) 1 (Raman) and 1663 and 1609 cm (cid:50) 1 (IR) and of the G-C vibrations at 1581 and 1491 cm (cid:50) 1 (Raman) and 1717 and 1492 cm (cid:50) 1 (IR). Drug intercalation leads to a major reduction of B-DNA structure in favor of A-DNA. standard. This band related to deoxyribose C–C stretching vibrations, exhibits no major alterations (intensity or shift- ing) on DES (cid:122) DNA complexation, and is canceled upon spectral subtraction.LaserRaman spectra were recorded on a DILOR Omars-89 Raman spectrometer using a 514.4 nm line of an argon laser (Spectra-Physics Model 2020-03). The laser power was 300 milliwatts at the sample. The spectra were typically recorded at a 5 cm (cid:50) 1 slit width with a 2-s integration time at a 2 cm (cid:50) 1 frequency increment. The spectra were routinely background-corrected by subtracting an appropriate third

Diethylstilbestrol (DES) 1 (Structure 1), a synthetic estrogen, is known to be a carcinogen in humans and in animals (1,2). Radioactively labeled DES was found to bind to DNA in vivo (3)(4)(5) and in vitro (6), but the nature of complexation could not be clarified. The major difficulties in defining the nature of DES-DNA interaction were partially related to the instability of DES⅐DNA and DESQ⅐DNA complexes (DESQ is a metabolic intermediate product derived from DES oxidation) formed in vivo and in vitro (6,7). Similarly, the possibility of intercalate formation of both DES and DESQ with DNA was excluded (7,8). It has also been suggested that DES-DNA adduct formation may occur under oxidative stress (9,10). However, a number of biological and biochemical effects of DES were noted that depended on metabolic activation of the stilbene and that are commonly associated with genotoxic activity (11). Since DESinduced carcinogenesis can be related to its complexation with DNA (2), the structural analysis of DES⅐DNA complexes has major biological importance, and thus, this investigation was undertaken. To our knowledge, there has also been no report on the interaction of DES with DNA using infrared or Raman spectroscopic techniques. Vibrational spectroscopy has been widely used as a major tool to characterize the nature of drug-DNA complexation and the effect of such interaction on the secondary structure of nucleic acids (13,14). For example, in recent years, Raman and infrared spectroscopic techniques were applied to analyze the interactions between DNA and Adriamycin, 11-deoxycarminomycin (15), aclacinomycin, saintopin (16,17), anthracyclines (18), intoplicine (13), doxorubicin (19), and platinum (20). Recently, we used FTIR and laser Raman spectroscopy to characterize the nature of DNA complexation with vitamin C (21) and aspirin 2 in order to evaluate the effects of drug interaction on the DNA conformation.
In this work, we applied FTIR and laser Raman difference spectroscopy to analyze the structural properties of DES⅐DNA complexes formed at physiological pH with drug/DNA (phosphate) molar ratios (r) of 1:40 to 1. The FTIR spectra of DES⅐DNA complexes were compared with those of strong intercalating agents such as acridine orange, methylene blue (MB), and ethidium bromide (EB), and the results are reported. Furthermore, the effects of DES-DNA interaction on biopolymer secondary structure and helix stability are evaluated.

EXPERIMENTAL PROCEDURES
Materials-Highly polymerized calf thymus DNA sodium salt (7% sodium content) was from Pharmacia Biotech Inc. and was used as supplied. DES was purchased from Sigma and was used without further purification. All other chemicals were reagent-grade.
Preparation of Stock Solutions-Sodium DNA was dissolved to 4% (w/w; 0.1 M phosphate) in 0.1 M NaCl solution at 5°C for 24 h with occasional stirring to ensure formation of a homogeneous solution. A solution of 1-50 mM DES was also prepared in water/ethanol solution (50:50, v/v). Mixtures of drug and DNA were prepared by adding DES solution dropwise to DNA solution with constant stirring to give the desired drug/DNA molar ratios of 1:40, 1:20, 1:10, 1:4, 1:2, and 1 at a final DNA concentration of 2% (w/w) or 0.05 M DNA (phosphate). Solution pH was kept near 7.5 to 6.5 with NaOH solution (0. 1 M). The IR and Raman spectra were recorded 4 h after initial mixing of drug and DNA solutions.
Infrared spectra were recorded on a Bomem DA3-0.02 Fourier transform infrared spectrometer with a nitrogen-cooled HgCdTe detector and a KBr beam splitter. Solution spectra were taken using AgBr windows with a resolution of 2 cm Ϫ1 and 100 -500 scans. Water subtraction was carried out as reported (23). A good water subtraction was achieved as shown by a flat base line around 2200 cm Ϫ1 , where the water combination mode is located. The FTIR difference spectra ((DNA solution ϩ DES solution) Ϫ DNA solution) were produced using the band at 968 cm Ϫ1 as internal standard. This band related to deoxyribose C-C stretching vibrations, exhibits no major alterations (intensity or shifting) on DES⅐DNA complexation, and is canceled upon spectral subtraction.
Laser Raman spectra were recorded on a DILOR Omars-89 Raman spectrometer using a 514.4 nm line of an argon laser (Spectra-Physics Model 2020-03). The laser power was 300 milliwatts at the sample. The spectra were typically recorded at a 5 cm Ϫ1 slit width with a 2-s integration time at a 2 cm Ϫ1 frequency increment. The spectra were routinely background-corrected by subtracting an appropriate third * This work was supported by the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 819-376-5077 (ext. 3321); Fax: 819-376-5057. 1 The abbreviations used are: DES, diethylstilbestrol; DESQ, diethylstilbestrol-4Ј,4Љ-quinone; FTIR, Fourier transform infrared; r, drug/ DNA (phosphate) molar ratio. degree polynominal function from the original curve. The spectra presented here are not smoothed. The difference spectra ((DNA solution ϩ DES solution) Ϫ DNA solution) were obtained according to our previous report (24) using the intense Raman line at 787 cm Ϫ1 as internal reference. This line, due to the coupling of cytosine and phosphodiester modes, exhibits no major spectral changes (intensity or shifting) on drug-DNA interaction and is canceled upon spectral subtraction.
The intensity ratios of several DNA in-plane vibrations (against the band at 968 cm Ϫ1 ) related to the A-T and G-C base pairs were measured as a function of DES concentration with error of Ϯ5%. The detailed infrared spectral manipulations and intensity ratio calculation are presented in our recent report (25).

RESULTS AND DISCUSSION
DES⅐DNA Complexes-At low DES concentration (r ϭ 1:40), a minor increase in the intensity (20%) of several infrared bands was observed at 1717 cm Ϫ1 (G and T), 1663 cm Ϫ1 (T, A, G, and C), 1609 cm Ϫ1 (A), and 1492 cm Ϫ1 (C and G) related to both A-T and G-C in-plane vibrational frequencies (23)(24)(25)(26)(27)(28)(29)(30). These intensity variations were also associated with a minor shift of the band at 1717 cm Ϫ1 toward a lower frequency at 1715 cm Ϫ1 (Figs. 1 and 2). On the other hand, the Raman spectra of DES⅐DNA complexes formed at a low drug content (r ϭ 1:40) exhibited a major decrease (50%) in the intensity of the thymine line at 1661 cm Ϫ1 (30) with the shift of this band toward a higher frequency at 1669 cm Ϫ1 (Fig. 3). The presence of a negative derivative feature at 1669 cm Ϫ1 in the Raman difference spectrum of the DES⅐DNA complex is due to a major loss of intensity and shifting of the thymine line at 1661 cm Ϫ1 (Fig. 3, r ϭ 1:40). Since the major spectral changes observed are for the thymine bands at 1663 cm Ϫ1 (IR) and 1661 cm Ϫ1 (Raman), drug interaction is mainly with the thymine O-2 atom with minor intercalation along the A-T-rich region. A similar increase in the intensity of several A-T Raman lines was observed upon metalloporphyrin intercalation with poly(dA-dT) 2 biopolymer (33,34).
Theoretical calculations on DNA fragments have suggested (31, 32) that the minor groove frequently carries a strong negative surface potential, and therefore, strong electrostatic interactions between the positive center (on the intercalating agent) and the negative charges found in the minor groove may facilitate the external binding mode. External binding to the A-T-rich region was also found in a series of DNA intercalators (34 -36).
Evidence for no major drug interaction with the G-C region comes from only slight alteration of the guanine and cytosine lines at 1580 cm Ϫ1 (G), 1490 cm Ϫ1 (G), and 1257 cm Ϫ1 (C) in the Raman spectrum of the DES⅐DNA complex (Fig. 3, r ϭ 1:40). However, it should be noted that drug intercalation with the A-T base pairs is weak since the overall spectral changes (intensity variations) of the A-T bands are not so large. The addition of more drug (r Ͼ 1:40) leads to DES⅐DNA complexation via the A-T base pairs in a similar fashion with no major alteration of the helix structure (Fig. 2).
At r Ͼ 1:10, a minor reduction of intensity (20%) was observed for the A-T infrared bands at 1663 and 1609 cm Ϫ1 upon drug interaction (Fig. 2). The reduction in the intensity of the A-T vibrations was associated with the shift of the PO 2 antisymmetric vibration at 1222 cm Ϫ1 to 1232 cm Ϫ1 (Fig. 1). These infrared spectral changes were accompanied by the shifting of the Raman marker line (B-DNA) at 838 cm Ϫ1 (phosphodiester mode) toward a lower frequency at 832 cm Ϫ1 (Fig. 3). These spectral changes are due to a partial alteration of the A-T backbone B-structure toward A-DNA conformation upon drug interaction.
At high DES concentration (r Ͼ 1:5), a major intensity increase (60%) was observed for the G-C bands at 1717 and 1492 cm Ϫ1 as well as for the A-T vibrations at 1663 and 1609 cm Ϫ1 ( Fig. 2). These intensity variations were associated with major shifts of the G-C and A-T infrared bands at 1717 cm Ϫ1 to 1710 cm Ϫ1 , 1492 cm Ϫ1 to 1490 cm Ϫ1 , 1663 cm Ϫ1 to 1658 cm Ϫ1 , and 1609 cm Ϫ1 to 1607 cm Ϫ1 (Fig. 1). Similar intensity variations were observed for the guanine Raman lines at 1580 and 1490 cm Ϫ1 (Fig. 3). The positive derivative features observed at 1580 and 1490 cm Ϫ1 in the Raman difference spectra of DES⅐DNA complexes formed at high drug concentration (r ϭ 1) are consistent with a partial helix destabilization and major drug interaction (externally) with the A-T and G-C base pairs. The negative feature at 1670 cm Ϫ1 is due to the loss of intensity of the thymine line (1661 cm Ϫ1 ). Similarly, the positive features observed at 1380 cm Ϫ1 (thymine) and 1340 cm Ϫ1 (adenine) in the Raman difference spectra are also caused by a major drug interaction with the A-T region (Fig. 3, r ϭ 1). A partial unwinding of double helix structure was also observed upon other drug intercalation, which resulted in an increase in the intensity of several DNA in-plane vibrations (33,34).
It should be noted that other positive features observed at 1286, 1083, and 1046 cm Ϫ1 in the Raman difference spectra of DES⅐DNA complexes at high DES concentration are due to drug vibrational frequencies and are not related to DNA vibrations (Fig. 3, r ϭ 1). Similarly, the positive features observed at 1590, 1513, 1245, 1101, and 885 cm Ϫ1 in the IR difference spectra of drug-DNA complexes formed at high drug concentrations are due to DES vibrations and not to the DNA molecule (Fig. 1, r ϭ 1).
However, at high DES concentration (r ϭ 1), a major alter-ation of the biopolymer secondary structure also occurs, which is characterized by the reduction in the intensity of several DNA in-plane vibrations at 1717, 1663, and 1609 cm Ϫ1 in the infrared spectra of DES⅐DNA complexes ( Figs. 1 and 2, r ϭ 1). This is also consistent with the shift of B-DNA marker bands at 1717 cm Ϫ1 (G and T) to 1710 cm Ϫ1 and of the PO 2 band at 1222 cm Ϫ1 to 1232 cm Ϫ1 as the results of a partial B-DNA conversion to A-DNA (30). It is worth mentioning that the marker infrared band at 841 cm Ϫ1 (phosphodiester mode of B-DNA) was overlapped by the strong DES vibration (838 cm Ϫ1 ) at high drug concentration (Fig. 1, r ϭ 1). However, the reduction in the intensity and shifting of the Raman marker line at 838 cm Ϫ1 (B-DNA) toward a lower frequency at 833 cm Ϫ1 together with the appearance of a shoulder line at 820 cm Ϫ1 (A-DNA) are also evidence for a major reduction of B-DNA structure in favor of A-DNA upon drug interaction (Fig. 3, r ϭ 1). It should be noted that, although the PO 2 band at 1222 cm Ϫ1 shifted toward a higher frequency at 1232 cm Ϫ1 (IR spectra) and the Raman line at 833 cm Ϫ1 (phosphodiester mode) moved toward a lower frequency at 820 cm Ϫ1 on drug complexation, no major drug-PO 2 interaction occurred in these DES⅐DNA complexes. Evidence for this comes from no considerable intensity variations of the backbone PO 2 band at 1222 cm Ϫ1 upon DES intercalation (Fig. 2). The major spectral shiftings observed for the phosphate vibrations are due to the alterations of the DNA secondary structure during drug complexation. Additional evidence for drug-DNA complexation comes also from the spectral changes of some of the DES in-plane vibrational frequencies. The shifts of the infrared bands of free DES at 1513 cm Ϫ1 to 1510 cm Ϫ1 , 1370 cm Ϫ1 to 1375 cm Ϫ1 , 1257 cm Ϫ1 to 1260 cm Ϫ1 , 1045 cm Ϫ1 to 1037 cm Ϫ1 , 884 cm Ϫ1 to 871 cm Ϫ1 , and 836 cm Ϫ1 to 838 cm Ϫ1 upon DNA interaction are characteristic of drug-DNA complexation (Fig. 1). Similarly, the shifts of the free DES Raman lines at 1457 cm Ϫ1 to 1454 cm Ϫ1 and at 1048 cm Ϫ1 to 1050 cm Ϫ1 are related to drug-DNA complexation (Fig. 3). A strong line at 881 cm Ϫ1 in the Raman spectra of free DES, which was observed at 879 cm Ϫ1 in the spectra of drug-DNA complexes, is due to ethanol vibration, and it is eliminated from difference spectra for clarity (Fig. 3).
Comparison with Other DNA Intercalators-The infrared spectra of DES⅐DNA complexes were compared with those of strong DNA intercalators (12,22,37,38) such as ethidium bromide, acridine orange, and methylene blue, recorded in our laboratory, and the results are compared here accordingly.
A strong dye-PO 2 interaction was observed for ethidium bromide, acridine orange, and methylene blue upon intercalation (external binding with the backbone phosphate groups) due to major intensity variations of the phosphate vibration at 1222 cm Ϫ1 . The DES-PO 2 interaction was not observed in these drug-DNA complexes. Major DES interaction with the thymine oxygen atom occurred (groove binding) upon drug intercalation, while such interaction was not observed in the case of dye-DNA intercalation (12,22,38). Although the dye-DNA intercalation resulted in a minor local alteration of the backbone phosphate geometry, no major departure from B-DNA structure was observed upon dye complexation. However, the DES-DNA binding resulted in a major reduction of B-DNA structure in favor of A-DNA. The dye-DNA complexation was not mainly sequencespecific (38), while DES interaction was with the A-T bases at low drug concentration and progressed toward G-C base pairs (external binding) at a high drug content.
In conclusion, the vibrational spectroscopic results presented here, for the first time, clearly show that DES intercalates along the A-T-rich region in a way that the aromatic parts are inserted into the A-T base pairs, while the OH groups stretch externally toward the thymine oxygen atom (groove binding).