The solid-matrix phosphorescence of (±)-anti-dibenzo[a,l]pyrene diol epoxide-DNA adducts and benzo[e]pyrene

Analytica Chimica Acta 560 (2006) 134–142

The solid-matrix phosphorescence of (±)-anti-dibenzo[a,l]pyrene diol epoxide-DNA adducts and benzo[e]pyrene Allison L. Thompson, Robert J. Hurtubise ∗ Department of Chemistry, University of Wyoming, Laramie, WY 82071-3838, United States Received 20 October 2005; received in revised form 16 December 2005; accepted 21 December 2005

Abstract New solid-matrix phosphorescence (SMP) methods for (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P were developed. The methods can be used to detect and characterize (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P by employing SMP spectra, intensities, and lifetimes acquired with the heavy-atom salt, TlNO3 , on Whatman 1PS paper. With the SMP data, a number of photophysical parameters were calculated such as biexponential SMP decay curves, pre-exponential factors, and fractional contribution to SMP decay curves. The SMP results were compared with earlier SMP data for (±)-anti-BPDE-DNA adducts and tetrol I-1. The SMP results show that small molecular-weight compounds like B[e]P can be readily detected and characterized by SMP. For example, the limit of detection for B[e]P was 0.60 pmol. Comparison of the SMP properties of the (±)-anti-DB[a,l]PDE-DNA adducts with earlier SMP data for the (±)-anti-BPDE-DNA adducts showed major differences in the SMP spectra, intensities, and lifetimes. The methods developed are important for the comparison of the SMP properties of different diol epoxides of PAH bonded to DNA. © 2006 Elsevier B.V. All rights reserved. Keywords: Solid-matrix phosphorescence; Benzo[e]pyrene; Dibenzo[a,l]pyrene diol epoxide-DNA; Heavy-atom effect

1. Introduction Polycyclic aromatic hydrocarbons (PAH) are an important class of compounds known for their carcinogenic and mutagenic properties. Dibenzo[a,l]pyrene (DB[a,l]P) is the most carcinogenic of the PAH [1,2]. DB[a,l]P is metabolically activated to its diol epoxide, dibenzo[a,l]pyrene-11,12-diol13,14-epoxide (DB[a,l]PDE), by cytochrome P450 enzymes [3]. After formation of the diol epoxide, DB[a,l]PDE binds to the DNA to form DNA adducts [4]. The DP[a,l]PDEDNA adducts are found in several different forms, namely, external, external with base stacking, and intercalated. It has been shown that DP[a,l]PDE-DNA adducts are primarily intercalated [5]. Solid-matrix phosphorescence (SMP) has proven to be useful in a variety of trace organic analyses [6–9]. Various types of solid matrices such as filter paper [10–12], cyclodextrin-salt mixtures [13–15], and sugar glasses [16] have been used to acquire solid-

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0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.12.053

matrix fluorescence (SMF) and SMP. The most utilized solid matrix is filter paper due to its effectiveness in permitting SMP and its low cost [8–10,17]. In this study, DB[a,l]PDE-DNA adducts and benzo[e]pyrene (B[e]P) were examined using SMP to study their phosphorescence spectral features and lifetimes in the presence of TlNO3 . Several reports have appeared on the (±)-antibenzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide ((±)-antiBP[a]DE)-DNA adducts using solid-matrix luminescence (SML). Tjioe and Hurtubise [17] used SMF and SMP to detect and characterize (±)-anti-B[a]PDE-DNA adducts. They showed with the (±)-anti-B[a]PDE-DNA adducts at high levels of modification (0.50–0.98%) that different forms of the B[a]PDE-DNA adducts could be detected using Whatman No. 1 and Whatman 1PS paper [17]. Li et al. [18] reported that linear relationships could be obtained between SMP and the percent modification of DNA with (±)-anti-B[a]PDE-DNA adduct. Also, they showed that the limit of detection by SMP was close to one adduct in 107 nucleotides. More recently, Smith and Hurtubise [19,20] used Whatman 1PS paper to study the fluorescence quenching [19] and the phosphorescent enhancement [20] with heavy-atom

A.L. Thompson, R.J. Hurtubise / Analytica Chimica Acta 560 (2006) 134–142

salts to further characterize the (±)-anti-B[a]PDE-DNA adducts. To date, DB[a,l]PDE-DNA adducts have been investigated using various methodologies. 32 P-post-labeling has been employed extensively to characterize the DB[a,l]PDE-DNA adducts [5,21–23]. The technique of 35 S-phosphorothioate postlabeling has also been used to study the DB[a,l]DE-DNA adducts [24]. Some non-radioactive methods have also been utilized to investigate PAH-DNA adducts. Fluorescence linenarrowing spectroscopy along with low-temperature fluorescence spectroscopy have been used with enzyme digestion and high-performance liquid chromatography to isolate and identify the stable DB[a,l]PDE-DNA adducts in mononucleotides [5,23,25,26]. In this research, the SMP spectral features and lifetimes of (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P were obtained in the presence of the heavy-atom salt, TlNO3 . The results were compared with earlier SMP results for the (±)-antiB[a]PDE-DNA adducts [20]. Benzo[e]pyrene was used as a model compound because its aromatic ring system is the same as DB[a,l]PDE as shown in Fig. 1. Thus, the SMP enhancement properties and the SMP lifetimes for the two aromatic systems should be similar. This is the first report of the SMP properties of the (±)-anti-DB[a,l]PDE-DNA adducts.

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2. Experimental 2.1. Apparatus and software The SMP data were obtained using a Perkin-Elmer LS-50B spectrometer (Norwalk, CT). This system is designed with a pulsed source and gated detector and has a spectral resolution of ±1 nm. The pulsed source is a xenon flash lamp which is pulsed at line frequency. The pulse width at half peak height is less then 10 ␮s and has a power equivalent to 20 kW for 8 ␮s duration. The instrumental parameters were controlled by the Fluorescence Data Manager Software (FLDM). The data were analyzed using GraphPad Prism Version 3.00 for Windows, GraphPad Software (San Diego, CA). 2.2. Materials and reagents The benzo[e]pyrene (B[e]P) used was purchased from Aldrich Chemical Company Inc. (Milwaukee, WI) and was 99% pure. The (±)-anti-r-11,t-12-dihydroxy-t-13,14epoxy-11,12,13,14-tetrahydodibenzo[a,l]pyrene [(±)-anti-DB [a,l]PDE] was purchased from Midwest Research Institute (Kansas City, MO). Calf thymus DNA (sodium salt Type I “Highly Polymerized”) was purchased from Sigma Chemical Company (St. Louis, MO) and was purified as described earlier [27]. The methanol and water were HPLC grade and were purchased from EMD Chemicals Inc. (Gibbstown, NJ). The thallium nitrate was 99.999% pure and was purchased from Aldrich Chemical Company Inc. (Milwaukee). Thallium nitrate is a highly toxic material. It is necessary to wear safety glasses, and a laboratory coat when using thallium nitrate. Also, thallium nitrate has to be disposed of with appropriate safety guidelines. However, it is a salt, and it is not volatile. If treated properly, thallium nitrate is easily handled. The filter paper used was Whatman 1PS. It was developed with distilled ethanol four times to move impurities to the top of the paper. All work was done in limited lighting, and solutions were wrapped in foil and stored in a refrigerator when not in use. 2.3. Preparation of (±)-anti-DB[a,l]PDE-DNA adducts The (±)-anti-DB[a,l]PDE-DNA adducts were prepared using the methodology and procedures for benzo[a]pyrenetrans-7,8-dihydrodiol-9,10-epoxide-DNA adducts discussed in earlier work [27–29]. The percent modification of (±)-antiDB[a,l]PDE-DNA adducts was determined by using UV absorbance as described in the literature [28–30]. It was determined that the DNA was modified at 0.07% with (±)-antiDB[a,l]PDE, which is equivalent to one adduct in 1430 bases. 2.4. Solid-matrix phosphorescence

Fig. 1. Molecular structures for B[e]P and (±)-anti-DB[a,l]PDE-N2 dG.

The procedure to prepare the SMP samples was described earlier [29]. B[e]P was dissolved in MeOH:H2 O (50:50) and the (±)-anti-DB[a,l]PDE-DNA adducts solutions were prepared in MeOH:H2 O (30:70). SMP spectra and lifetimes were obtained for B[e]P, (±)-anti-DB[a,l]PDE-DNA adducts, and purified

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calf thymus DNA. Blank SMP spectra and lifetimes of either MeOH:H2 O (50:50) or calf thymus DNA in MeOH:H2 O (30:70) solutions were taken in the same manner as B[e]P and (±)-antiDB[a,l]PDE-DNA adducts, respectively, and subtracted from the appropriate SMP sample data. The instrumental settings used to collect the SMP intensity were set to maximize the SMP intensity. The delay time was set at 0.1 ms with a gate time of 9.9 ms. The excitation and emission slits were set at 4 nm. The excitation and emission wavelengths used to obtain the spectra and lifetimes were 334 and 542 nm, respectively, for B[e]P, and 347 and 554 nm for (±)-anti-DB[a,l]PDE-DNA adducts. 3. Results and discussion 3.1. Phosphorescence spectra of B[e]P and DB[a,l]PDE-DNA adducts Considering that B[e]P and DB[a,l]PDE-DNA adducts have the same aromatic ring structure, one can assume that the SMP spectra would be similar. The SMP excitation spectrum of B[e]P shows two major peaks, one at 290 and 335 nm (Fig. 2). Also, there are peaks at 232, 280, and 321 nm. The emission spectrum shows a major band at 542 nm and smaller peak at 589 nm. The excitation spectrum for (±)-anti-DB[a,l]PDE-DNA adducts showed a major peak at 347 nm and a peak near 300 nm (Fig. 2). The SMP emission spectrum gave a major peak at 557 nm with a shoulder at 600 nm. When comparing the emission spectrum for B[e]P to the emission spectrum for (±)-anti-DB[a,l]PDEDNA adducts, one can see the two spectra are very different with a 15 nm shift to the red for the peak maximum for the (±)-anti-DB[a,l]PDE-DNA adducts. Because significant broadening occurred for the SMP emission spectrum of the (±)-antiDB[a,l]PDE-DNA adducts, its shape is quite different than the spectrum for B[e]P. The same is true in comparing excitation spectra of B[e]P and (±)-anti-DB[a,l]PDE-DNA adducts. The peaks below 300 nm that appeared for B[e]P did not emerge in

the spectrum for (±)-anti-DB[a,l]PDE-DNA adducts because DNA absorbs ultraviolet light very strongly in this region. Fig. 1 shows a representative structure that results when DB[a,l]PDE reacts with deoxyguanine. The B[e]P moiety is attached to a non-aromatic ring. Thus, the SMP from the B[e]P ring system would not be influenced much by the substitution on the aromatic ring system of the B[e]P component. The spectral broadening and the red shift in the SMP spectra of the (±)anti-DB[a,l]PDE-DNA adducts compared to the SMP spectra of B[e]P were also reported for the SMF spectra of B[e]P and (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P [29]. The broadening and the red shift occur because the conformations of the (±)-anti-DB[a,l]PDE-DNA adducts allows for strong interactions of the adducts with the DNA bases [5,25]. It has been shown previously by Jankowiak et al. [5], with fluorescence linenarrowing spectroscopy and non-line-narrowing spectroscopy, that a red shift occurs for the DB[a,l]PDE-DNA adducts. They concluded that the red shift was because of ␲–␲ interactions of the adducts and the DNA bases and conformational changes in the cyclohexenyl ring of the DB[a,l]PDE-DNA adducts. In addition, inhomogeneous broadening would also cause broadening of the emission spectrum. Inhomogeneous broadening results from the statistical average of fluorescence emission that is not the same for all the emitting species in the sample. The spectral bands that are dominated by inhomogeneous broadening can be described by a Gaussian function [31]. Apparently, ␲–␲ interactions and inhomogeneous broadening cause the spectral shift and spectral broadening of the SMP spectra. In earlier work, Li et al. [18] showed that the SMP maximum excitation and emission wavelength for (±)-anti-B[a]PDEDNA adducts on 1PS paper with TlNO3 were 350 and 609 nm, respectively. As Fig. 2 shows the maximum emission wavelength for (±)-anti-DB[a,l]PDE-DNA adducts is 557 nm. This wavelength is shifted 52 nm to the red. Present research involves modifying a single sample of DNA with both (±)-anti-B[a]PDE and (±)-anti-DB[a,l]PDE and obtaining the SMP properties of the adducted DNA sample. 3.2. SMP enhancement

Fig. 2. SMP spectra of B[e]P (3 ng) and of (±)-anti-DB[a,l]PDE-DNA adducts (18 ␮g) adsorbed on 1PS paper with 25 ␮g of TlNO3 .

The use of external heavy-atom salts such as TlNO3 and NaI to enhance phosphorescence emission is especially beneficial for SMP. In a solid matrix, the probability of close contact of the heavy-atom salt increases since the solid matrix, in essence, traps the heavy-atom salt and the phosphor in a relatively small sample area. With a solid matrix such as Whatman 1PS paper, there are a limited number of sites for both the heavy-atom salt and the phosphor in the paper after the sample has been dried making it even easier to obtain close proximity between the two [20]. The rigidity of the environment that contains the sample also contributes to the SMP enhancement. Upon examination of the SMP data for B[e]P, plotted as intensity versus the amount of TlNO3 (Fig. 3), it can be seen that the phosphorescence intensity (Ip ) was enhanced as the amount of TlNO3 increases up to 50 ␮g. However, the SMP intensity decreased when the quantity of TlNO3 was at 75 ␮g. The SMP heavy-atom salt enhancement data for (±)-anti-DB[a,l]PDE-

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Fig. 3. Plot of Ip and IF vs. TlNO3 (␮g) for B[e]P (3 ng) adsorbed on 1PS paper.

DNA adducts gave a plot somewhat similar to B[e]P as can be seen in Fig. 4A. An approximate plateau was reached around 20 ␮g of TlNO3 , but there was a significant increase in the scatter of the data as the amount of TlNO3 increased (Fig. 4A). In this study, since TlNO3 was more effective at enhancing the SMP, emphasis was placed on this salt rather than on NaI. In comparing our results with the results obtained by Smith and Hurtubise [20] for tetrol I-1 and (±)-anti-B[a]PDE-DNA adducts, generally, the SMP enhancement for both tetrol I1 and (±)-anti-B[a]PDE-DNA adducts was greater compared to the SMP enhancement of B[e]P and (±)-anti-DB[a,l]PDEDNA adducts with TlNO3 . They showed, that for tetrol I-1 with 25 ␮g of TlNO3 , the SMP increased 119 times when compared to the SMP with no TlNO3 [20]. For the (±)-anti-B[a]PDEDNA adducts, with 25 ␮g of TlNO3 , the SMP increased by 151 times compared to the SMP intensity with no TlNO3 present. In this work, the SMP of B[e]P with 50 ␮g of TlNO3 improved by a factor of 9 compared to the SMP with 0.1 ␮g of TlNO3 . For the (±)-anti-DB[a,l]PDE-DNA adducts, the SMP intensity with 37.5 ␮g of TlNO3 was 26 times higher than the SMP intensity with no heavy-atom salt present. It is important to note that it is not possible quantitatively compare the SMP data obtained by Smith and Hurtubise and the data from this study since different Perkin-Elmer LS-50B instruments and different instrument setting were used to obtain their data and our data. NaI was also investigated as a heavy-atom salt. A plot of SMP versus amount of NaI reached an approximate plateau at 20 ␮g of NaI (the graph is not shown). The SMP enhancement of (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P was much greater with TlNO3 than with NaI. There was little SMP enhancement with NaI. With 80 ␮g NaI, the SMP enhancement was small for the (±)-anti-DB[a,l]PDE-DNA adducts with only an increase of five times in intensity compared to the intensity with no heavy-atom salt present. Ramamurthy and Turro

Fig. 4. Plots of Ip and IF vs. TlNO3 (␮g) for (A) (±)-anti-DB[a,l]PDE-DNA adducts modified at 0.05% (18 ␮g), and (B) (±)-anti-B[a]PDE-DNA adducts modified at 0.01% (18 ␮g) adsorbed on 1PS paper; (B) taken from Ref. [20].

[32] have shown that PAH included in Tl+ exchange zeolites show enhanced phosphorescence because the ␲-cloud of the PAH interacts with the Tl sites. Thus, the close contact of the Tl+ in the Whatman 1PS paper with the aromatic ring system in the (±)-anti-DB[a,l]PDE-DNA adducts would strongly favor enhancement of SMP by the heavy-atom effect. With I− , most likely the I− would not interact as well with the ␲-cloud of the aromatic ring system because of the negative charge on the I− . This would result in a less effective heavy-atom effect with I− .

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3.3. Comparison of SMF and SMP The SMF quenching of both B[e]P and (±)-antiDB[a,l]PDE-DNA adducts has been discussed previously, and those SMF quenching results will not be considered in detail in this manuscript [29]. There are significant differences in the pattern of SMF intensity (IF ) as a function of the amount of TlNO3 compared to the pattern for the SMP intensity for B[e]P shown in Fig. 3. The SMP enhancement for B[e]P with TlNO3 showed a gradual increase in the SMP intensity as the amount of TlNO3 increased. The SMF decreased very rapidly up to 1 ␮g of TlNO3 , then as the amount of TlNO3 increased, the magnitude of the decrease in SMF became less significant. Smith and Hurtubise [20] compared the SMF intensities with the SMP intensities of tetrol I-1, and they reported that the SMF and SMP intensities were almost mirror images of one another, and at 25 ␮g of TlNO3 the minimum SMF and maximum SMP intensities were obtained. They indicated that at greater than 25 ␮g of TlNO3 the rate constant for intersystem crossing from excited singlet state to excited triplet state, the rate constant for intersystem crossing for excited triplet state to ground state, and rate constant for SMP become independent of TlNO3 because the SMF and SMP signals remained constant. This was not the case with B[e]P (Fig. 3). The results in Fig. 3 indicate there is not a mirror image relationship between SMF and SMP, and the SMP does not become constant even at 50 ␮g of TlNO3 . The data in Fig. 3 show that the interactions between TlNO3 , the solid matrix, and B[e]P are complex. For B[e]P and the (±)-anti-DB[a,l]PDE-DNA adducts, the main difference with TlNO3 present is how the SMF changes compared to the SMP. Fig. 3 shows that for B[e]P the SMF was detected out to 50 ␮g, whereas the (±)-anti-DB[a,l]PDE-DNA adducts showed no SMF with TlNO3 greater than 1 ␮g (Fig. 4A). Even though the SMF intensity of the (±)-anti-DB[a,l]PDEDNA adducts was completely quenched with 1 ␮g of TlNO3 , the SMP intensity continued to increase as the amount of TlNO3 increased up to about 40 ␮g (Fig. 4A). Comparing the SMF quenching and SMP enhancement as function of TlNO3 for (±)-anti-DB[a,l]PDE-DNA adducts (Fig. 4A) and the results reported by Smith and Hurtubise [20] with (±)-anti-B[a]PDE-DNA adducts (Fig. 4B), it can be seen that TlNO3 interacted differently with the two types of DNA adducts. Fig. 4B shows that with the (±)-anti-B[a]PDE-DNA adducts as the amount of TlNO3 increased the SMF intensity leveled off to a constant intensity by 15 ␮g. However, the SMP continued to increase up to 80 ␮g of TlNO3 [20]. In comparing the SMF plots in Fig. 4A and B, the main difference is that (±)-anti-B[a]PDE-DNA adducts still had detectable SMF at the higher levels of TlNO3 , but the (±)-anti-DB[a,l]PDE-DNA adducts had no detectable fluorescence. As discussed earlier, the SMP was enhanced for both types of adducts as the TlNO3 increased (Fig. 4A and B). The complex structures and the conformations of the (±)anti-DB[a,l]PDE-DNA adducts and (±)-anti-B[a]PDE-DNA adducts are responsible for the overall shapes of the SMP plots in Fig. 4A and B and how the TlNO3 interacts with the paper and DNA. It has been shown that Tl+ ions bonds to the N-donor atoms

of the nucleobases in DNA [33,34]. Smith and Hurtubise [20] postulated that because the externally bound (±)-anti-B[a]PDEDNA adducts are predominate [35,36] in the DNA that the Tl+ ions would mainly interact with these adducts. The quasiintercalated adducts in (±)-anti-B[a]PDE-DNA adducts, which are present at a smaller percentage, would have to be very close to the Tl+ ions bonded in the DNA to allow for SMP enhancement. It is important to mention that the Tl+ ions in solution would have interacted with the DNA on the solid matrix before the solution of TlNO3 was completely dried and salt formation occurred. Thus, Tl+ ions in solution can initially interact with DNA adducts in the solid matrix and after drying the solid matrix, solid TlNO3 salt would be present in the matrix. With the (±)-anti-DB[a,l]PDE-DNA adducts, approximately 75% of the adducts are intercalated [5]. With these DNA adducts modified at a fairly high level of 0.01% (one adduct in 1000 bases), the probability that the intercalated form of (±)-antiDB[a,l]PDE-DNA adducts would interact with the Tl+ bonded to the N-donor nucleobases is high. We have already discussed that the binding of the Tl+ ions to the DNA bases permits the efficient quenching of the intercalated adducts in (±)-antiDB[a,l]PDE-DNA adducts [29]. The SMF quenching of the (±)anti-DB[a,l]PDE-DNA adducts with TlNO3 is much more efficient than for the (±)-anti-B[a]PDE-DNA adducts because of the close contact of the Tl+ ions with the (±)-anti-DB[a,l]PDEDNA adducts. The previous SMF quenching data for the (±)anti-DB[a,l]PDE-DNA adducts indicate that the close contact of the Tl+ ions bonded in the DNA with the adducts allows for the enhancement of the adducts’ SMP. 3.4. SMP lifetimes and pre-exponential factors SMP lifetime decay curves were obtained for B[e]P and (±)anti-DB[a,l]PDE-DNA adducts as a function of the amount of TlNO3 (0–80 ␮g of TlNO3 ) adsorbed on 1PS paper. With nonlinear regression analysis, the decay data were fit with either a monoexponential equation or biexponential equation, although essentially all of the SMP lifetime data fit a biexponential equation. The lifetime components were calculated using the equations that follow: Ip = α e−t/τ

(1)

Ip = α1 e−t/τ1 + α2 e−t/τ2

(2)

where Ip is the SMP intensity at time, t, α1 and α2 the preexponential factors for the short and long decaying components, and τ 1 and τ 2 are the lifetimes. The correlation coefficients ranged from 0.975 to 0.999. Selected values of α1 , α2 , τ 1 , and τ 2 for B[e]P and (±)-anti-DB[a,l]PDE-DNA adducts are shown in Tables 1 and 2, respectively. The αi values for the data that fit the
biexponential decay have been normalized using the function αi / αi [37]. With no heavy-atom salt present, the SMP intensities were moderately strong so SMP decay curves for B[e]P (2 ng) and (±)-anti-DB[a,l]PDE-DNA adducts modified at 0.07% could be obtained. For both B[e]P and the (±)-anti-DB[a,l]PDE-DNA adducts, with no heavy-atom salts, the SMP lifetimes were much

A.L. Thompson, R.J. Hurtubise / Analytica Chimica Acta 560 (2006) 134–142 Table 1 Pre-exponential factors and SMP lifetimes for biexponential fits of B[e]P (2 ng) adsorbed on 1PS paper with various amounts of TlNO3 TlNO3 (␮g)

α1

τ 1 (ms)

α2

τ 2 (ms)

0 0.3 1 5 10 25 50

– 0.72 0.80 0.62 0.75 0.78 0.81

– 8.20 7.65 5.00 6.78 6.44 5.96

47.9 0.28 0.20 0.39 0.25 0.22 0.19

970 297 152 72.1 88.7 71.9 71.7

Pooled standard deviation values for the lifetimes and pre-exponential factors are as follows: α1 = ±0.031, α2 = ±0.028, τ 1 = ±0.65 ms, and τ 2 = ±16.4 ms.

longer than the lifetimes with TlNO3 present. For B[e]P with no TlNO3 , the lifetime was monoexponential and calculated to be 970 ms (Table 1). The lifetime decay curve for (±)-antiDB[a,l]PDE-DNA adducts was also monoexponential, and the SMP lifetime was 1080 ms (Table 2). When TlNO3 was added, the lifetimes for both B[e]P and (±)-anti-DB[a,l]PDE-DNA adducts dropped significantly and the decay curves became biexponential. Smith and Hurtubise [20] found similar biexponential decay curves with tetrol I-1 and the (±)-anti-B[a]PDE-DNA adducts in the presence of TlNO3 . With a small amount of TlNO3 added (0.3 ␮g), the longer component of the two component lifetime for B[e]P was calculated as 297 ms, and the shorter component was 8.20 ms. As the amount of TlNO3 was increased, the longer component continued to decrease, and then became approximately constant near 5 ␮g of TlNO3 (Table 1). The short component became fairly constant beginning at 5 ␮g of TlNO3 . With the (±)-anti-DB[a,l]PDE-DNA adducts, with a small amount of TlNO3 (0.30 ␮g), the longer component was calculated to be 33.4 ms with the shorter component at 4.79 ms (Table 2). Both the long and short components remained approximately constant as the amount of TlNO3 was increased (see Tables 1 and 2). The decrease in the phosphorescence lifetime results from the heavy-atom effect [8,9,20]. The values for τ 1 are smaller than the τ 2 values for both B[e]P and (±)-antiDB[a,l]PDE-DNA adducts because the phosphors contributing to the τ 1 are in closer contact to Tl+ compare to the component contributing to τ 2 . The pre-exponential factors, α1 and α2 , for B[e]P essentially did not change as the amount of TlNO3 increased (Table 1). Table 2 Pre-exponential factors and SMP lifetimes for the biexponential fit of (±)-antiDB[a,l]PDE-DNA adducts (18 ␮g of a sample modified at 0.076%) adsorbed on 1PS with various amounts of TlNO3 TlNO3 (␮g)

α1

τ 1 (ms)

α2

τ 2 (ms)

0 0.3 1 5 10 25 50

– 0.47 0.53 0.68 0.66 0.87 0.77

– 4.79 3.18 3.21 2.34 3.14 2.80

122 0.53 0.47 0.32 0.34 0.13 0.23

1080 33.4 30.8 28.0 21.4 34.5 28.3

Pooled standard deviation values for the lifetimes and pre-exponential factors are as follows: α1 = ±0.038, α2 = ±0.038, τ 1 = ±0.84 ms, and τ 2 = ±3.6 ms.

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Table 3 Fractional contributions of the short and long lived SMP components of B[e]P with various amounts of TlNO3 TlNO3 (␮g) 0.3 1 5 10 25 50

P1 0.066 0.17 0.10 0.19 0.26 0.26

P2 ± ± ± ± ± ±

0.058 0.02 0.22 0.01 0.20 0.02

0.93 0.83 0.90 0.81 0.76 0.74

± ± ± ± ± ±

0.14 0.13 0.29 0.09 0.13 0.06

Because the αi terms represent the fraction of the two major populations of phosphors, the α values show that there is no significant change in the populations as the amount of TlNO3 increases. Smith and Hurtubise [20] showed similar results for tetrol I-1. They reported that the consistency in the α values as the amount of TlNO3 increased indicated that the phosphor was adsorbed strongly to the solid matrix. Thus, with the α1 and α2 values for B[e]P showing the same trend, B[e]P is also adsorbed strongly to the solid matrix. The α1 values for (±)-anti-DB[a,l]PDE-DNA adducts increased as TlNO3 increased as shown in Table 2. However, the α2 values decreased with an increase in TlNO3 . This is in contrast to the α1 and α2 values for the (±)-anti-B[a]PDE-DNA adducts reported by Smith and Hurtubise [20]. They found that both the α1 and α2 values remained constant as the amount of TlNO3 increased. The changes in α values with the increase in TlNO3 for the (±)-anti-DB[a,l]PDE-DNA adducts is most likely due the interaction of the Tl+ ions with the DNA and the large percent of intercalated DNA adducts. The population of the component represented by α1 increases, the population of the component represented by α2 decreases. Because the (±)anti-B[a]PDE-DNA adducts are predominately external, they would interact differently with the Tl+ ions and allow the α1 and α2 values to remain constant. 3.5. Fractional contributions to SMP intensities The fraction contributions to the SMP intensities for the two populations of phosphors for B[e]P and DB[a,l]PDE-DNA adducts were calculated using the following equation: αi τi Pi =
(3) (αi τi ) where Pi is the fractional contribution of each decay time to the steady-state SMP intensity [20,37]. The fractional contributions calculated (P1 and P2 ) are shown in Tables 3 and 4 for some of the samples with different amounts of TlNO3 . P1 represents the fractional contribution for the short lifetime component (τ 1 ) and P2 represents the fractional contribution for the long lifetime component (τ 2 ). When the α1 and α2 values of B[e]P and (±)-anti-DB[a,l]PDE-DNA adducts in Tables 1 and 2 are compared to the P1 and P2 values, it can be seen that whereas α1 values are greater than α2 , the P1 values are smaller than the P2 values. This is expected since the equation to calculate P1 and P2 values include τ 1 and τ 2 , and the τ 2 values are considerably larger than the τ 1 values. The same trend was observed by Smith

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Table 4 Fraction contribution of the short and long lived SMP components of (±)-antiDB[a,l]PDE-DNA adducts with various amounts of TlNO3 TlNO3 (␮g)

P1

0.3 1 5 10 25 50

0.11 0.10 0.19 0.18 0.38 0.25

P2 ± ± ± ± ± ±

0.55 0.10 0.17 0.16 0.17 0.04

0.89 0.90 0.81 0.82 0.62 0.75

± ± ± ± ± ±

0.09 0.07 0.22 0.20 0.42 0.04

and Hurtubise [20] with tetrol I-1 and (±)-anti-B[a]PDE-DNA adducts. The values for P1 and P2 are essentially the same for the corresponding amount of TlNO3 for both B[e]P and (±)anti-DB[a,l]PDE-DNA adducts. 3.6. ln Ip versus time plots for SMP Plots of ln Ip versus time for (±)-anti-DB[a,l]PDE-DNA adducts obtained with different amounts of TNO3 were compared to previous results for (±)-anti-B[a]PDE-DNA adducts considered by Smith and Hurtubise [20]. They showed that for (±)-anti-B[a]PDE-DNA adducts there was an upward global shift in the ln Ip versus time plots as the amount of TlNO3 increased. The plots of ln Ip versus time (±)-anti-DB[a,l]PDEDNA adducts did not show a global shift as was observed for (±)anti-B[a]PDE-DNA adducts (compare Fig. 5A and B). Smith and Hurtubise [20] reported that the global shift for the (±)anti-B[a]PDE-DNA adducts was caused by how the DNA and the DNA adducts interact with the solid matrix. The DNA would form hydrogen bonds with the filter paper since the phosphate groups in the DNA would interact strongly with the hydroxyl groups in the filter paper. With the DNA held rigidly by the solid matrix, the (±)-anti-B[a]PDE-DNA adducts would be held in fix positions. Since there is an increase in the rate constant of phosphorescence, kp , and the rate constant for non-radiative transition from the excited triplet state, km , as the amount of TlNO3 increases, the global shift for (±)-anti-B[a]PDE-DNA adducts indicates that both kp and km increased in a similar manner [20]. However, with the (±)-anti-DB[a,l]PDE-DNA adducts, there was no global shift for the ln Ip versus time plots. Therefore, both kp and km did not change in a similar manner. Also, the lack of a global shift indicates that the adducts interact differently with the filter paper and TlNO3 . The decay plots in Fig. 5A and B show that the SMP for the (±)-anti-B[a]PDE-DNA adducts decays more rapidly than the (±)-anti-DB[a,l]PDE-DNA adducts. This indicates that the (±)-anti-B[a]PDE-DNA adducts are in closer proximity to TlNO3 compared to the (±)-anti-DB[a,l]PDEDNA adducts. For example, with the (±)-anti-B[a]PDE-DNA adducts, the DNA is strongly absorbed to the solid matrix and the adducts are mainly externally bound to the DNA [20,35,36]. These conditions permit the adducts to remain in close contact with the TlNO3 in the 1PS paper as the amount of TlNO3 is increased [20]. However, with the (±)-anti-DB[a,l]PDE-DNA adducts, the adducts are predominately intercalated, and the ability of the adducts to interact with Tl+ ions depends on how close

Fig. 5. Plots of ln Ip vs. time (ms) for (±)-anti-B[a]PDE-DNA adducts modified at 0.0001% (18 ␮g) adsorbed on Whatman 1PS with increasing amounts of TlNO3 (A). The data for (A) were taken from Ref. [20]. Plots of ln Ip vs. time (ms) for (±)-anti-DB[a,l]PDE-DNA adducts modified at 0.05% (1 ␮g) adsorbed on Whatman 1PS with increasing amounts of TlNO3 (B).

the DNA adducts are to the Tl+ ion that are bonded to the Ndonor bases [5]. The ln Ip versus time plots for B[e]P and (±)-antiDB[a,l]PDE-DNA adducts, and tetrol I-1 and (±)-antiB[a]PDE-DNA adducts [20] were linear after a certain period of SMP decay within a large range of the microgram amounts of TlNO3 on the 1PS paper. The ln Ip plots for (±)-antiDB[a,l]PDE-DNA adducts were linear from 45 to 160 ms (Fig. 6A). Typical linear correlation coefficients for selected amounts of TlNO3 for the linear range were calculated. For example, a linear correlation coefficient of 0.985 for 10 ␮g of TlNO3 was obtained. The ln Ip plots for (±)-anti-B[a]PDEDNA adducts were linear in the range of 24–38 ms as shown in Fig. 6B [20]. A typical linear correlation coefficient was cal-

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systems should be investigated via SMP lifetimes to determine if similar plots are acquired. 4. Conclusions

Fig. 6. Plots of ln Ip vs. time (ms) for (±)-anti-DB[a,l]PDE-DNA adducts modified at 0.05% (18 ␮g) adsorbed on Whatman 1PS with various amounts of TlNO3 . The time range is 45–155 ms (A). Plots of ln Ip vs. time (ms) for (±)-antiB[a]PDE-DNA adducts modified at 0.0001% (18 ␮g) adsorbed on Whatman 1PS with various amounts of TlNO3 . The time range is 24–38 ms (B). The data in (B) were taken from Ref. [20].

culated to be 0.998 for 80 ␮g TlNO3 . The linear range for tetrol I-1 was from 24 to 38 ms with a representative linear correlation coefficient of 0.995 for 5 ␮g TlNO3 [20]. For B[e]P, the linear range was from 50 to 200 ms. A typical correlation coefficient was 0.925 with 3.0 ␮g TlNO3 . However, for tetrol I-1, B[e]P, and (±)-anti-DB[a,l]PDE-DNA adducts, the linear ranges did not give global shifts and parallel lines like those in Fig. 6B for (±)-anti-B[a]PDE-DNA adducts. The lines for tetrol I-1, B[e]P, and (±)-anti-DB[a,l]PDE-DNA adducts were linear, but they intersected one another. The linearity of the SMP time plots shows that only the long lifetime component was observed and the decay curves for these time frames were monoexponential. The plots in Fig. 6B are unique and other PAH-DNA adduct

Comparison of the SMP excitation and emission spectra of B[e]P and the (±)-anti-DB[a,l]PDE-DNA adducts clearly showed major differences which indicated that the (±)-antiDB[a,l]PDE-DNA adducts were intercalated in the DNA. Also, the SMP emission maximum for the (±)-anti-DB[a,l]PDE-DNA adducts were shifted 52 nm to the red compared the SMP emission maximum for the (±)-anti-B[a]PDE-DNA adducts. This indicates that with a single sample of DNA that would be modified with both the diol epoxides could be characterized for the two different adducts by SMP. These experiments are presently being pursued. Because the (±)-anti-B[a]PDE-DNA adducts are mainly in the external form and the (±)-anti-DB[a,l]PDE-DNA adducts are predominately intercalated, some important differences appeared in the SMF and SMP data for the different DNA samples. Also, TlNO3 was shown to be a much better heavy-atom salt in enhancing the SMP of both the (±)-anti-DB[a,l]PDEDNA adducts and B[e]P than NaI. In comparing the patterns of the changes in the SMF and SMP intensities as the amount of TlNO3 increased for the (±)-anti-DB[a,l]PDE-DNA adducts with the corresponding patterns of the changes in the SMF and SMP intensities of the (±)-anti-BPDE-DNA adducts significant differences were observed. These differences showed that at least for these two DNA adduct systems that the predominately external adducts for the (±)-anti-BPDE-DNA behaved quit differently than the predominately intercalated adducts of the (±)-anti-DB[a,l]PDE-DNA. These results strongly suggest that the comparison of the SMF and SMP intensity patterns of other DNA adducts systems as a function of the heavy-atom salt content in the solid matrix could be very useful in characterizing external and intercalated adducts. In addition, a dramatic difference was shown in the SMP lifetime patterns for the two different adduct systems as the amount of TlNO3 increased. The entire set of SMP decay curves for the (±)-anti-BPDE-DNA adducts showed a global shift in intensity as the amount TlNO3 increased. However, the entire set of SMP decay curves for the (±)-anti-DB[a,l]PDE-DNA adducts did not show a global shift in the decay curves as the TlNO3 increased, but the decay curved crossed one another. Also, the long decaying components for the DNA adduct systems were mono-exponential. In the future, it should be possible to use time-resolved SMP to further characterize both the short- and long-decaying components of (±)-anti-BPDE-DNA adducts and (±)-anti-DB[a,l]PDE-DNA adducts. The methods developed are useful for DNA samples modified at high levels, and addition work is needed to determine if the approaches developed can be applied to human DNA samples. Acknowledgements This work was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sci-

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ences, Office of Science, U.S. Department of Energy (DE-FG0204ER15545), and the National Institutes of Health, National Cancer Institute (R03CA97723).

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