765
Chem. Res. Toxicol. 1992,5, 765-772
Evidence of Involvement of Multiple Sites of Metabolism in the in Vivo Covalent Binding of Dibenzo[a,h]pyrene to DNA G. A. Marsch, R. Jankowiak, and G. J. Small' Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
N. C. Hughes and D. H. Phillips Haddow Laboratories, Institute of Cancer Research, Cotswold Road, Sutton, Surrey, SM2
5NG U.K.
Received April 27, 1992
The in vivo formation of dibenzo[a,hlpyrene-DNA adducts in mouse skin was assessed by laser-excited fluorescence spectroscopy at 77 and 4.2 K. Two adducts were identified with fluorescence origin bands at -383.5 and 407.2 nm, and these were shown to possess pyrene and benzo[a] pyrene (B[alP) chromophores, respectively. Both DNA-bound chromophores displayed considerable electron-phonon coupling and likely assume a highly base-stacked or quasiintercalated configuration within DNA duplexes. The presence of B[a]P and pyrene aromatic systems indicates that two-electron or monooxygenation metabolism occurred on either the a or h benzo moieties (which are equivalent) in the former case, and on both these rings in the latter case. The presence of two adduct species agrees with 32P-postlabeling analysis of the DNA, which showed the presence of two major adducts in both thin-layer and high-performance liquid chromatographic separations. Introduction Polycyclicaromatic hydrocarbons(PAHs)lare products of the incomplete combustion of carbonaceous material ( I ) . It is now generally accepted that PAHs exert their biological effects of mutagenicity and carcinogenicity via metabolic activation by cytochrome P-450 and epoxide hydrolase to electrophilic speciesthat react covalentlywith nucleophilicsites in cells to form adducts (2,3). A wealth of evidence supports the view that DNA is the critical cellular target nucleophile (4,5). The hexacyclic aromatic hydrocarbon dibenzo[a,hlpyrene (DB[a,h]P) is an environmentalpollutant (1)which has high mutagenic and tumorigenic activities in many bacterial and mammalian cell lines (6-11). Since DB[a,h]P is a symmetrical molecule with two equivalent bay regions (at rings a and h, see Figure 4), metabolism is anticipated not only at the a or h ring, but also at both rings simultaneously. Quantum mechanical calculations showing AE1JJB of 0.845 for DB[a,hlP (9, IO), one of the highest for any hydrocarbon whose diol epoxide has been studied (12),predict high chemical reactivity of the bayregion diol epoxides. The nature of the DNA reactive species, however, remains to be verified. Therefore, it is of interest to establish the structure of stable DB[a,hlPDNA adducts formed by in vivo systems. The 32P-postlabelingassay,a highly sensitivetechnique that enables the detection of DNA adducts in only microgram quantities of DNA ( 1 3 , 1 4 ) ,was used recently to examine the covalent binding of DB[a,hlP to DNA in 1 Abbreviations: B[alP, benzo[alpyrene;BPDE, anti-benzo[alpyrene 7,B-dihydrodiol 9,lO-epoxide;DB[a,hlP, dibenzo[a,hlpyrene;DDBP, benzo[a] pyrene tram-7,8-dihydrodio1;FLN,fluorescenceline narrowing; FWHM, full width at half-maximum;HPLC, high-performanceliquid chromatography; PAH, polycyclic aromatic hydrocarbon; PEI, poly(ethylenimine);THF,tetrahydrofuran;ZPL,zero-phononline:TLC,thinlayer chromatography;SDS, sodium dodecylsulfate;FLNS, fluorescence line narrowingspectroscopy;ESVF,excited-statevibrationalfrequencies.
mouse tissues (15). Two adducts were detected in the skin of treated animals, although the nature of these two adducts remains to be characterized. In a series of papers (16-22) we have demonstrated that fluorescence line-narrowed (FLN) spectroscopy can be used for the analysis of DNA damage by PAHs in vivo (19, 20,22),since the present limit of detection is ca. 1adducted base pair per lo8 base pairs in 100 pg of DNA (22). For example, a FLN spectral analysis of in vivo human hemoglobin was used to determine the structure of the major human globin adduct from benzo[alpyrene (B[a]P) (20). FLN spectroscopy is also capable of assisting in the assignment of adduct stereochemistries (21,22). In this paper we provide information on the route of metabolic activation of DB [a,hlP by using fluorescence line narrowing spectroscopy to identify the presence of two distinctly different chromophores in DB[a,hlPmodified DNA from mouse skin, analogous to the two distinct DB [a,hlP-DNA adducts detected by 32P-postlabeling. Experimental Procedures The carcinogen (DB[a,h]Pandradiolabel (J2P)used in these experiments were in liquid form. For both, contamination must be eliminated by wearing impermeable laboratory gloves, laboratory coats,and splash-proofgoggles. Hazardous materials should besequestered from the rest of the laboratory. Radiolabel exposure is kept ALARA ("aslow as reasonably achievable") by working with plexiglass shielding and the proper dosimeters (Geiger counters and body dosimeters). Wastes must be placed only in approved containers and disposed of by the proper Environmental Safety and Health personnel. Instrumentation. The fluorescence line-narrowing spectroscopy system employed in these studies has been described in detail (22). The excitation source was a Lambda Physik FL2002 dye laser pumped by a Lambda Physik EMG 102 MSC excimer laser. Fluorescence was detected with a Princeton Instruments IRY-l024/G/R/B intensified blue-enhanced pho-
0893-228~/92/2705-0765$03.00/0 0 1992 American Chemical Society
766 Chem. Res. Toxicol., Vol. 5, No. 6,1992 todiode array. Gated detection was accomplished using a Lambda Physik EMG-97 zero drift controller to trigger a FG-100 highvoltage gate pulse generator, allowing the delay time and the width of the detector's temporal observation window to be adjusted. Samples were dissolvedin 30 pL of either 5:4:1 glycerolH20-EtOH or HzO in quartz tubes, taken through several freezepump-thaw degassing cycles, sealed, cooled to 4.2 K, and probed with the laser in the (0,l) excitation regions. Laser-excited fluorescence spectra at 4.2 and 77 K, obtained under non-line-narrowing conditions (Sz SOexcitation), were measured by using an SRS Model SR280 boxcar averager (Stanford Research, Palo Alto, CA) 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-to-pulse intensity jitter of the excimer pumped dye laser. All spectrareported were normalized for pulse jitter. The boxcar averager was interfaced through a SR425 computer interface module with a PC-compatible computer for data acquisition and analysis. Chemicals. DB[a,h]P (purity99.52 f 0.045 %)waspurchased from the Community Bureau of Reference (Brussels, Belgium). Poly(ethy1enimine)-cellulose (PEI-cellulose) TLC sheets were manufactured by Macherey-Nagel and supplied by Camlab (Cambridge, U.K.). All other chemicals and biochemicals were obtained from previously mentioned sources (23). Topical Treatment of Mice and Isolation of DNA. Male Parkes mice (4-6 weeks) were purchased from the MRC National Institute for Medical Research (Mill Hill, London). DB[a,h]P was applied to the shaved dorsal skin of mice 11.0 pmol/mouse in 200 pL of tetrahydrofuran (THF)]; control mice received T H F only. Groups of 4 animals were killed by cervical dislocation 2 days after treatment, and the treated areas of skin removed. The dermalsurface of the frozen skin was then scraped with a scalpel blade, and the remaining frozen epidermal layer was powdered in liquid nitrogen (24).The powdered skin samples were then thawed in 10 mM EDTA, and after homogenization, a 10% solution of SDS (0.1 vol) was added and the DNA isolated and purified using a previously published phenol extraction method (25,26). 32P-PostlabelingAnalysis. The nuclease P1 version of the 32P-postlabeling assay was used (14). Samples of DNA (4 pg) were digested to deoxyribonucleoside 3'-monophosphates with micrococcal nuclease (0.19 unit) and spleen phosphodiesterase (1.6 pg) for 18 h a t 37 "C in 4.8 pL of 17 mM sodium succinate and 8 mM CaCl2 (pH 6.0). A solution, containing 0.96 pL of nuclease Pl(1.25 pg/pL), 2.4 pL of 0.5 M sodium acetate (pH L O ) , and 1.44 p L of 0.3 M ZnClz, was added to each DNA digest, to selectively dephosphorylate nonadducted nucleotides (14). After incubation at 37 OC for 1 h, 1.9 pL of 0.5 M Tris base was added. The DNA digests were then 32P-postlabeled by incubation with a buffer solution [prepared by mixing 1.0 pL of 0.2 M bicine, 0.1 M MgC12,O.l M DTT, and 10 mM spermidine (pH 9.0), 0.5 pL of laboratory(25 pCi), and 2 pL of synthesized (27) carrier-free [T-~~PIATP T4 polynucleotide kinase (3 units/pL)]. After incubation at 37 "C for 30 min, any residual [rJ2P]ATP was destroyed by the addition of 2 pL of potato apyrase (20 milliunitslpl), followed by incubation at 37 "C for a further 30 min. Thin-LayerChromatography. To remove residual labeled normal nucleotides, unused [ T - ~ ~ P I A TPi, P ,and other radioactive contaminants and to resolve 32P-labeledadducts, multidirectional anion-exchange TLC was used as described previously (15,281, with the following solvent system: D1,l.O M sodium phosphate (pH 6.0); D2,3.5 M lithium formate and 8.5 M urea (pH 3.5); D3, 0.8 M lithium chloride, 0.5 M Tris-HC1, and 8.5 M urea (pH 8.0); D4, 1.7 M sodium phosphate (pH 6.0). Adduct spots on the chromatograms were visualized by autoradiography a t -75 "C using intensifying screens. Quantitation of DNA Adducts. The specific activity of the ATP P ] was determined by measuring laboratory-synthesized[ T - ~ ~ the T4 polynucleotide kinase-catalyzed incorporation of radioactivity into a known amount of dAp (14). The areas of the
-
Marsch et al. chromatograms containing the 32P-labeledadducts were excised and the levels of radioactivity determined by Cerenkov counting. Background levels were obtained by counting areas of the chromatograms that did not contain adducts, and were subtracted. The levels of adducts present in a DNA sample are expressed as fmol of adducts/pg of DNA (1fmol/pg of DNA is equivalent to 33 adducts/l@ nucleotides) and were calculated from the formula: fmol of adducts/pg of DNA = adduct radioactivity (dpm/pg of DNA) specific activity of [y3'P1ATP (dpm/fmol)
High-Performance Liquid Chromatography. HPLC analyses were carried out with the apparatus described by Pfau and Phillips (29),consistingof two Waters 501HPLC pumps, a Waters WISP autosampler, a Waters 440 absorbance detector a t 280 nm, and Berthold LB 507 HPLC radioactivitymonitor. Gradient control and other data processing were acheived with Waters Baseline-810 software. Radioactive spots of DB[a,h]P-DNA adducta were eluted from PEI-cellulose by overnight extraction with 400 pL of 4.0 M pyridinium formate (pH 4.5). Eluates were filtered and then reduced to dryness in a Savant Speed Vac concentrator. Recovery was 70% or greater. Adduct residues were redissolved in water and separated to HPLC separation on a Zorbax phenyl-modified reversed-phase column (particle size 5 pm, 250 X 4.6-mm i.d.), with the following linear gradients at a flow rate of 1 mL/min: time (min)
%A
%B
0
90
15
54 52 20
10 46 48 80
90
10
60 80 90
Preparation of Samples of Fluorescence Analysis. DNA isolated from mouse skin treated with DB[a,h]P was precipitated with 0.1 volume of 5 M NaCl and 2 volumes of ethanol precooled to -20 OC. The DNA precipitate, containing 1pmol of adducts, was taken up in 30-40 p L of the glass-forming solvent (glycerol/ water/ethanol, 5 4 1 ) in quartz glass tubes (3-mm 0.d. X 2-mm i.d. X 1cm) and was analyzed spectroscopically a t 77 and 4.2 K. Results In this section, data are presented which establish that two DNA adducts were formed in vivo when DB[a,hlP is applied topicallyto mouse skin. The adducts were assessed by laser-excited fluorescence spectroscopy, performed under line-narrowed (at 4.2 K) and non-line-narrowed conditions (S2 SOlaser excitation at T = 77 K),and by 32P-postlabeling(15). It is shown below that the origin or (0,O)transition of fluorescing adduct chromophore and its fluorescence line-narrowed (FLNS) spectra provide useful information for structural adduct assignment. (A) Laser-Excited Fluorescence Spectra at 77 K. Figure 1 shows the fluorescence spectra a t T = 77 K for DB[a,h]P-DNA isolated from mouse skin (15) obtained for three representative excitation wavelengths, revealing different fluorescing chromophores. Frame A presents two spectra obtained with Lx= 308 nm (dashed line) and A,, = 355 nm (solid line), whereas frame B shows a fluorescence spectrum (solid line) selectively excited a t A,, = 370 nm. Comparison of the latter with the spectra of Figure 1A (and other data for different excitation wavelengths, spectra not shown) revealed that the DNA had two different adducts with origin bands at -383.5 and -407.2 nm. These adducts will be designated as
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In Viuo Binding of DB[a,h]P to DNA
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 767
380 I-
/
380
400
420
382
384
386
Wavelength (nm)
\
440
Figure 2. FLN spectra obtained by laser excitation at hX=
Wavelength (nm) Figure 1. Selectively excited fluorescence spectra of DNA from mouse skin after topical treatment with 1.0 bmol of DB[a,h]P in 200 p L of THF. Spectra obtained for laser excitation wavelengths of 355 nm (solid line), 308nm (dashed line), and 370 nm are shown in frames A and B,respectively. Vertical arrows identify the origin bands of different adducts. The fluorescence spectrum for adduct I (curve d), obtained as the difference, spectrum a - spectrum b, is shown in frame B,see text. T = 77
K.
adducts I and 11, respectively. There is a 2-fold support for these assignments. First, spectra c and d can be generated from the deconvolution of spectrum a. Also, the band at 383.5 nm is clearly more pronounced at bX= 355 nm (spectrum a) than at bx= 308 nm (spectrum b). The subtraction of spectrum b from spectrum a yielded the same spectrum d as generated by the deconvolution of spectra a, exhibiting an origin band at 383.5 nm and a major vibronic progression at 404.5 nm. These data demonstrate that adducts I and I1have pyrene- and B [alplike fluorescence, respectively. Thus, saturation of the a and/or h benzo rings of DB[a,hlP appears to be implicated in the in vivo reaction of DB[a,h]P with DNA. The absenceof benzo[blchrysene-likefluorescence(originband at -397 nm) indicates little or no K-region oxidation (see Figure 4). The DB[a,hlP fluorescence (origin at -393 nm) is readily distinguishable from adduct I and adduct I1 fluorescence (data not shown). No contamination of the adduct spectra either by “pure”(nonreacted)DB[a,hlP or one-electron oxidation adduct (with DB [a,hlP chromophore)could be discerned for a wide range of excitation wavelengths, Le., 308-370 nm. (B) Adduct I with Pyrene-like Fluorescence. In this subsection we first explore the nature of adduct I. Although 77 K broad fluorescence spectra are particularly useful in revealing the (0,O)transitions of the SI SO fluorescence spectra, high-resolution fluorescence linenarrowed (FLN) spectra provide more information and have been often used for structure elucidation (16-22). Figure 2 presents two FLN spectra at the origin band of adduct I obtained with laser excitation at 359.8nm (dashed line) and 363.4 nm (solid line), respectively. The zerophonon lines (ZPLs) are labeled in wavenumbers correspondingto excited-statevibrationalfrequenciesof adduct I. These spectra are characterized by weak ZPLs indicating strong electron-phonon coupling (22). An analysis of the excited-state vibronic frequencies also indicates that the frequenciesare characteristic for the pyrene chromophore
-
359.8nm (upper spectrum) and hX= 363.4nm (lower spectrum) in the region of the (0,O)band of adduct I. Bands are labeled in wavenumbers from the laser line and correspond to the excitedstate vibrational frequencies. Note strong electron-phonon coupling. T = 4.2 K.
(Table I). The excited-state vibronic frequencies of AlaB[alP ester, BPDE-deoxyguanosine adduct, and two oligomerscontainingcovalentlybound BPDE are provided in Table I and compared to the frequencies of adduct I. All these adducts have pyrene as the fluorescing chromophore. The frequency range covers 1100-1600 cm-’, the region of greatest activity. The similarity of the excited-state vibrational frequencies of adduct I with various BPDE-type adducts supports the above assignment. Additional support for this assignment is provided by the observation that the SI SOtransition of adduct I is weakly allowed, typical for the pyrene chromophore (1 7, 22). Therefore, we conclude that metabolism (by twoelectron oxidation) occurred at both the a and h rings of DB[a,h]P, leavingapyrene chromophore. This is the first direct experimental evidence of multiple sites of metabolism in the activation in vivo of DB[a,hlP. Our results are in agreement with earlier tumorigenicity studies of DB[a,hlP and its derivatives (8-10). DB[a,h]PlI2-dihydrodiol, the immediate metabolic precursors of the bay-region diol epoxides, had tumor-initiating activity on mouse skin about equal to its parent hydrocarbon (IO). However, DB[a,hlP-H1-1,2-diol also showed appreciable tumor-initiating activity on mouse skin (10). The double bond adjacent to the two hydroxyls is now saturated, so further activating metabolism on that ring is impossible. Therefore,further metabolicreactionsmust have activated the molecule elsewhere,most likely at the other bay-region benzene ring. From these studies, the metabolism of DB[a,hlP can best be explained by the addition of hydroxyls to the 1, 2,8, and 9 positions followed by epoxidation at the 3, 4 and 10, 11 positions. Therefore, the high tumorigenic activity of DB[a,hlP can be explained by multiple sites of metabolism from monooxygenation pathway reactions without invoking other non-bay-region metabolic pathway@). This is also supported by the lack of activity of 2,10-difluoro-DB[a,hlPas a tumor initiator on mouse skin and in causing pulmonary and liver tumors in newborn mice (8). (C) Adduct I1 with Benzo[a]pyrene-like Fluorescence. In this subsection we discuss FLN data, obtained
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Marsch et al.
768 Chem. Res. Toxicol., Vol. 5, No. 6, 1992
Table I. Excited-State (SI) Vibrational Frequencies (cm-*)of Adduct 1 Compared to Vibrational Frequencies of Carboxylic Ester of Anti-BPDE (Ala-B[a]P), Benzo[a]pyrene Diol Epoxide-DNA Adduct [BPDE-DNA; (+)-1 Type Adduct (ZI)], and (+)-tram-and (+)-cis-BPDE-d(CACATGTACAC)2 Double-Stranded Oligomer Adducts3 adduct I Ala-B[alp BPDE-DNA (+)-l (+)-tram-BPDE(+)-cis-BPDE(cm-l) esteP (cm-l) adductb(cm-l) d(CACATGTACAC)Z(cm-l) d(CACATGTACAC)z(cm-l) 1112 1111 1109 1108 1109 1329 1331 1332 1330 1333 1369 1387 1379 1385 1387 1434 1443 1435 1442 1442 1503 1503 1503 1503 1513 1518 1518 1518 1558 1543 1559 1559 1552 1596 1607 1617 1603 1600 a 7,8,9Trihydroxy-r-7,t-8,t-9,c-l0-tetrahydo[a]pyren-10-yl-N-t-BOC-~-alanate (Ala-B[alP);see ref 20. Note that although the excitedstate vibrational frequencies of these adducts are very similar, their &-state energies are different from those in ref 21.
Table 11. Excited-State (SI) Vibrational Frequencies of Adduct I1 from Two-Electron Oxidation Compared to Vibrational Frequencies of B[a]P, DB[a,B]P, and BP-CG-N7-Gua Adduct. adduct I1 B[alP DB[a,hlP BP-C6-N7-Gua (cm-1) (cm-1)b (cm-1) (cm-l)* 453 449 451 465 475 472 511 498 508 513 546 538 -57lC 549 569 569 580 614 588 626 624 659 633 Fromone-electronoxidation (26');see text for explanation. From ref 26. Unresolved band of closely spaced vibrations.
2
403 404 405
406
Wavelength (nm) Figure 3. Comparison of the vibronically excited FLN spectra (bX = 396.7 nm, T = 4.2 K) from benzo[a]pyrene standard and fromintactDB[a,h]P-DNA (dashedline) frommouseskin. Peaks are labeled with their excited-state vibrational frequencies in cm-l.
within the (0,O)band of adduct 11, that demonstrate the in vivo activation at either the a or h ring by the monooxygenation pathway. An FLN spectrum of adduct I1 is shown as the upper spectrum in Figure 3. Excitation at 395.7 nm induces fluorescencefrom the SIstate of the B[alP chromophore, and severalprominent excited-state vibronicbands at 453, 475, 508, -571, and 626 cm-1 are observed. The lower spectrum (with similar vibrations) is obtained for B[alP in standard glass; both spectra were obtained at T = 4.2 K, for the same excitation wavelength. As expected, the intensity distribution of the zero-phonon lines of adduct I1 differs from that of B[a]P, since the S1 state of adduct I1 (see section A) is red shifted from that of B[alP by 250 cm-1. The red shift of the fluorescenceis typicallyobserved when PAHs bind covalently to DNA. Table I1 shows comparison of the excited-state(SI)vibrationalfrequencies of adduct I1 with vibrational frequencies of B[alP, DB[a,hlP, and the B[a]P-C6-N7-Gua adduct from oneelectronoxidation (26). Similaritiesof B[alP's and adduct 11's excited-state vibrational frequencies are apparent (Table 11), even though metabolism at the a or h ring of DB[a,hlP and subsequent addition to DNA slightly perturb the frequencies. This indicates that covalent binding of DB[a,h]P to DNA does not dramaticallychange the frequencies of normal vibrations, implying that the vibrational frequencies arise from B[alP chromophore attached to DNA through at least two intervening bonds.
On the contrary, for the BP-CG-N7-Gua one-electron oxidation adduct, an atom which is part of the B[alP conjugated system is bound directly to the DNA, so the vibrational frequencies are expected to be significantly perturbed, as observed. This is demonstrated in Table I1 where the excited-state vibrational frequencies (ESVF) of adduct I1 are compared with the vibrational frequencies of B[alP and BP-CG-N7-Gua adduct. Therefore, on the basis of the data presented, we conclude that adduct I1 is a product of metabolism by monooxygenation at one ring (a or h, which are equivalent) of DB[a,hlP. The spectral characteristicsand assignments of adducts I and I1 formed in mouse skin and their relative contribution (seesections D and E) to the total level of binding are summarized in Table 111. The broader (0,O)bands [I'(o,o)I of adduct I [I'(o,O) 400 cm-'3 and adduct I1 [I'(O,o) 360 cm-'] versus I'(O,o) of pyrene (-220 cm-l) and B[alP (-290 cm-l), and especially the 4-5-nm red shift, reflect the role of a strong covalent PAH chromophore-DNA interaction. A red shift for a PAH bound to DNA is usually observed (21,22)and agrees with the analogy of simplemolecular systems,where the magnitude of the red shift reflects the binding energy of the complex. Possible pathways of metabolic activation culminating in stable DB[a,h]P-DNA adduds are schematically shown in Figure 4. In our studies (vide supra), adduct I [hflco,~) = 383.5 nml and adduct I1 [Xfl(o,o) = 407.2 nml were described as possessing pyrene- and B[alP-like chromophores, respectively. Theoretically, a bis-diol epoxide derivativeof DB[a,h]P, for example, compound C in Figure 4, could give rise to a DNA adduct with a pyrene-like chromophore. From studies of the related symmetrical PAH dibenz[a,h]anthracene, there is evidence for the involvement of a bis-diol epoxide in DNA binding.2 An
-
* Phillips et al., unpublished data.
-
3 Jankowiak et al., unpublished results. Preparation and characterization of these adducts are described in ref 30.
I n Vivo Binding of DB[a,hlP to DNA
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 769
Table 111. Characteristics of DB[a,b]P-DNA Adducts Formed in Mouse Skin and Their Relative Contribution to the Total Level of Modification
adducts of DB[a,h]P-DNA
I
I1
fluorescence band max fwhmO(cm-l) fluorescing (nm) (h0.2 nm) (A10cm-l) chromophore 383.5 400 pyrene
407.2
360
electronphonon coupling moderate
benzo[alpyrene strong
% of total binding of adducts I and 11, estimated spectrofrom 3*Pscopically* postlabeling' assignment -60-80 75.8 multiple sites of metabolism by monooxygenation pathway ( M P ) at a and h rings -20-40 24.2 metabolism by MP at a or h ring
*
fwhm was calculated aa twice the half-width on the high-energy side. Estimation baaed on the ratio of extinction coefficientsof fluorescing chromophores, assuming similar fluorescence quantum yield. e From ref 15;measured at time point of maximum binding.
Bay region Bay region
''K - recrion Dibenzo[a,h]pyr&e
1%
metabolism of
"* 1
HO
HO
.c
ad%$!
H
I
-:*: oHiaddi&;
addition o', on; ring to DNA ; hydro1 sis of other e oxi e
,
00
OH
adduct I
DNA
L
to
-;
- -addict21 -
to
,
I
L - - - - - J
Figure 4. Some possible pathways of metabolic activation of DB[a,hlP in mouse skin, which could lead to the formation of DNA adducts with pyrene or benzo[a] pyrene fluorescent chromophores. The existence of additional pathways is not excluded, and evidence for the formation of many of the postulated intermediates has yet to be obtained. Percentages of adducts I and I1 formed in vivo in mouse skin DNA are shown in Table 111. MFO = mixed-function oxidases; P-450 = cytochrome P450.
adduct formed by compound C would be distinguishable spectroscopically from adducts formed by AI, Az, or B (Figure 4) due to its possession of an olefinic bond. Although an adduct standard derived from compound C is not available, a comparison of the benzo[alpyrene trans-'l,&dihydrodiol (DDBP) fluorescenceemission with that from adduct I [Xn(o,~= 383.5 nml is appropriatesince the fluorophores would be similar if the adduct with the (0,O)origin band at -383.5 nm is formed by C (Figure 4). The fluorescence (0,O)band of the olefinic DB[a,hlP precursor to such an adduct would be red-shifted several nanometersfrom the fluorescence (0,O)origin band of free DDBP, because the addition of the saturated benzo moiety in the olefinic DB[a,hlP metabolite red-shifts the fluorescence emission. In addition, the formation of DNA adducts further red-shifts Xfl(o.0) by another few nanometers. The overall red shift of an adduct formed by compound C with respect to free DDBP should be less
than 10nm, but the adduct fluorescence will certainly not be blue-shifted with respect to free DDBP. However, at 77 K with hx= 365 nm, hn(o,o)for free DDBP is at 395.7 nm (Figure 5), a red shift of =12 nm from adduct I. In addition, the first vibronic progression of DDBP can be seen as a prominent fluorescence shoulder at 405 nm, corresponding to a 620-cm-l ground-state vibrational frequency (Figure 5). Pyrene and B[alP chromophores exhibit a less prominent transition of =750 cm-l from the (0,O)origin band. Line-narrowed spectra (datanot shown) also demonstrate that the vibronic frequencies of DDBP significantly differ from the vibrational frequencies of B b I P tetrols and BPDE-DNA adducts. We conclude that adduct I is formed by the oxidation of all eight sites of the a and h rings of DB[a,hlP prior to DNA attack. Thus, an adduct derived from compound C is not observed, but adducts I and 11, which contain pyrene and B[alP chromophores,respectively, could &e from intermediates
770 Chem. Res. Toxicol., Vol. 5, No.6, 1992
Marsch et al. A
BP-7,8-diol T=77K
he,= 365 nm
Q
'.
I
.-:
Q
B
Ii\/
U
I
380 400
410
420
430 440
Wavelength (nm) Figure 5. Fluorescence spectrum of benzo[a] pyrene trans-7,8
!I
ii
c
dihydrodiol (DDBP) obtained under non-line-narrowing conditions a t T = 77 K for hx= 365 nm. Fluorescence origin band (0,O)of DDBP is located a t 395.7 nm. Retention time (minutes)
Figure 7. HPLC of 32P-labeledDNA a d d u d s formed in mouse skin by DB[a,h]P. Profiles shown are of (A) DB[a,h]P-DNA adduct 1and (B) DB[a,hlP-DNA adduct 2. DNA adducts were 32P-labeledand chromatographed on PEI-cellulose TLC plates; the major adducts (Figure 5)were extracted from the TLC plates and further analyzed by HPLC using a phenyl-modified reversedphase column.
Figure 6. Autoradiographsof PEI-cellulose TLC maps of 32Plabeled digests of DNA from mouse skin treated with DB[a,h]P. Maps shown are of DNA from mouse skin treated with (A) T H F (control) and (B) DB[a,h]P (1.0 pmol/mouse). DNA was 32Ppostlabeled and chromatographedon 10- X 10-cm PEI-cellulose TLC plates as described in the text. The origin is located a t the bottom left-hand corner of each chromatogram and was excised before autoradiography, which was for 24 h at -75 "C.
AI, A2, and B. However, we cannot exclude that a crosslinked product (with pyrene chromophore), also arising from metabolite A2, may be formed. (D) Analysis of DB[a,h]P-DNA Adducts by 32pPostlabeling. T w o 32P-labeledDB [a,h]P-DNA adducts were detected in mouse skin that had been treated with the hydrocarbon (Figure 6). This profile of adducts is essentiallythe same as that described previously (15).The major adduct,DB[a,h]P-DNA adduct 1, represented more than 75% of the total binding of 3.0 fmol/pg of DNA and was more mobile than adduct 2 when chromatographed on PEI-cellulose TLC plates (Figure 6). It has been suggested that chromatographic separation on reversed-phase columns is more predictive of the polarity/lipophilicityof molecules than anion-exchange chromatography (29). DB[a,h]P-DNAadduct spots 1 and 2 were thereforeextracted from PEI-celluloseTLC sheets and analyzed by HPLC. This analysis yielded a major radioactivepeak for each adduct spot (Figure7), indicating that each adduct spot consists principally of a singleadduct species. By making comparisonsof the retention times of the adducts eluting from the column, DB[a,h]P-DNA adduct 1 would be predicted to be more polar than DB[a,h]P-DNA adduct 2, having retention times of 20.3 and 22.3 min, respectively (Figure 7). (E) SpectroscopicEstimation of the Relative Abundance of Adducts I and 11. We estimated from our fluorescencemeasurements and absorption spectra of the pyrene and B[a]P chromophoresthat the adduct resulting from metabolismat both a and h benzo rings predominates.
The extinction coefficient of B[a]P chromophore at 355 nm is -2-4 times stronger than for pyrene chromophore at the same wavelength, assuming a standard red shift of approximately 5 nm upon covalent attachment to DNA, yet the contributions of both chromophores to the DB[a,h]Padductspectrumat355aresimilar. Wealsoassume that both adducts have the same fluorescence quantum yield, a good assumption since pyrene and B[a]P have similar quantum yields, and the chromophores originate from the same parent DB[a,hlP molecule. Although quantitationsby fluorescencespectroscopyare uncertain, we estimated that adduct I comprises 60-80% of the total adduct population. In a preliminary investigationwe found that fluorescence spectroscopic analysis of 32P-labeled DB[a,h]P-DNA adducts extractedfromPEI-celluloseyielded nothingthat could be attributable to a PAH chromophore, because of an excessivebackground fluorescenceassociated with the PEI-cellulose (data not included). Fluorescenceanalyses were therefore made using intact DNA modified by DB[a,h]P in vivo. Thus it has not yet been possible to make direct comparisons between the two adducts detected by 32P-postlabeling(Figure 6B) and the two chromophores found by fluorescence spectroscopicanalysis of the intact DNA (Figures 1-3). However, from the relative mobilities of the two adducts when chromatographed using TLC and HPLC and their relative abundancies, we postulate that the major adduct detected by 32P-postlabeling(adduct 1) contains a hydrocarbon moiety that is more extensively metabolized (i.e., possesses more polar substituents) than the moiety in adduct 2. Therefore, we postulate that 32Ppostlabeled adduct 1 is adduct I characterized by FLNS and that adduct 2 (32P-postlabeled)is adduct I1 detected by fluorescence spectroscopy under non-line-narrowing and line-narrowingconditions. This assignment is also in agreement with the percent of total binding of adducts I and I1 estimated spectroscopically (see Table 111).
In Vivo Binding of DB[a,h]P to DNA
Discussion As expected, we found that DB[u,hlP is metabolized in vivo by two-electron transfer double addition dearomatization. Indeed, this is not surprising since DB[u,hlP, with high symmetry and two identical mesoanthracenic positions, is a perfect substrate for the two-ring metabolism. It is shown that selective laser excitations can provide direct analysis of multiple DNA adducts within a single DNA sample without degradation of the DNA. This procedure,as demonstratedhere and in the literature (16-22,261, offers the possibility of obtaining structural information about adducts at very low concentrations. Direct evidence obtained by fluorescence spectroscopy showed that the major adduct observed in vivo in mouse skin DNA is formed after metabolism in both benzo rings prior to reaction with DNA. The relative abundances of adduct 1and 2, and determined by 32P-postlabeling,are in good agreement with those obtaind from the spectral analysis of adduct I (metabolism in two rings, major adduct) and adduct I1 (metabolism in one ring, minor adduct). Completion of these analyses would include the identificationof the adducted DNA base and possible basesequence specificity. At this point, since the standard DB [a,hlP-DNA adducts are not available for analysis by FLNS, it is not possible to identify which DNA base has been modified: Nevertheless, on the basis of the spectra presented we can conclude that adducts I and I1 show moderate and strong electron-phonon coupling,suggesting that adducts I and I1 most likely assume somewhat basestacked and highly base-stacked, or quasi-intercalative, configurations, respectively. Acknowledgment. Ames Laboratory is operated for USDOE by Iowa State Universityunder Contract W-7405Eng-82. This work was supported by the Office of Health and Environmental Research, Office of Energy Research. G.A.M. was supported by an appointmentto the Alexander Hollaender Distinguished Postdoctoral Fellowship Program sponsored by the U.S. Department of Energy, Office of Health and Environmental Research,and administered by Oak Ridge Associated Universities. This work was also supported, in part, by grants from the Cancer Research Campaign and the Medical Research Council,and in part by Grant CA 21959, awarded by the US. National Cancer Institute, DHHS. One of us (N.C.H.) gratefully acknowledges receipt of a studentship from the Institute of Cancer Research, and the award of an ICRETT Fellowshipfrom the International Union Against Cancer.
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