Derivative fluorescence spectral analysis of polycyclic aromatic

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Chem. Res. Toxicol. 1989, 2, 104-108

Derivative Fluorescence Spectral Analysis of Polycyclic Aromatic Hydrocarbon-DNA Adducts in Human Placenta A. Weston,*pt D. K. Manchester,$ M. C. Poirier,s J.4. Choi,t G. E. Trivers,t D. L. Mann,t and C. C. Harris? Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, Departments of Pediatrics and Pharmacology, Division of Genetics, University of Colorado School of Medicine, The Children's Hospital, Denver, Colorado 80218, and Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received January 4, 1989 Metabolic activation in humans of chemical carcinogens found in the environment results in the formation of carcinogen-DNA adducts in vivo. Some polycyclic aromatic hydrocarbon-DNA adducts in human DNA can be hydrolyzed under mildly acidic conditions to yield tetrahydrotetrol derivatives which may then be detected by synchronous fluorescence spectroscopy. In an analysis of human placental DNA, second derivative spectroscopy alone was unable to resolve the synchronous fluorescent signature for r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene from a crude extract, because a complex array of other fluorescent materials was also present. Purification of the sample by a combination of chromatographic procedures including immunoaffinity chromatography and HPLC has now been shown to yield r-7,t-8,t-g,c-lO-tetrahydroxy-7,8,9,1O-tetrahydrobenzo[a]pyreneresidues from human DNA that are spectroscopically pure at the second derivative level. Immunoaffinity columns were prepared with rabbit antiserum raised against DNA that had been modified with (f)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene. This antiserum has now been shown to recognize DNA samples that have been modified with six different polycyclic aromatic hydrocarbon diol epoxides and is probably only specific for a broad spectrum of polycyclic aromatic hydrocarbon-DNA adducts. Adducts were eluted from the immunoaffinity columns, hydrolyzed with acid, and extracted into isoamyl alcohol, before being subjected to high-performance liquid chromatography. These experiments reveal important limitations of second derivative fluorescence spectroscopy as a tool in the analysis of complex environmental mixtures. Furthermore, they extensively define the ability of anti-benzo[a]pyrenediol epoxide-DNA antibodies to recognize different types of polycyclic aromatic hydrocarbon-DNA adducts.

Introduction Environmental exposure to chemical carcinogens was recognized as hazardous more than two centuries ago when soot was identified as an etiological agent in scrotal cancer among chimney sweeps (1). Polycyclic aromatic hydrocarbons (PAHs)' are a major class of carcinogens that have been isolated from coal tar pitch, soot, and mineral oils (2-4). It has been shown that PAHs, which are chemically inert, require metabolic activation in order to exert their biological effects (5-7). Thus, benzo[a]pyrene (BP) is metabolized in biological systems by the action of mixedfunction oxidases (P-450) and epoxide hydrolase to (&)r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (BPDE) or (&) -r-7 ,t-8-dihydroxy-c-9,lO-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, which can bind covalently to DNA to form adducts primarily at the exocyclic amino group of deoxyguanosine residues (8, 9). Radiolabeled PAHs have been used to elucidate mechanisms of carcinogen metabolism in laboratory studies. In addition, methods are currently being developed to detect PAH-DNA adducts formed in humans following envi-

ronmental exposures to PAHs (10). Two major obstacles to the development of these methods are the presence in human samples of complex mixtures of materials that confound simple assay systems and low levels of carcinogen-DNA adducts that challenge the detection limits of conventional techniques. The formation in humans of many types of carcinogen-DNA adducts of environmental origin is possible, and highly likely, e.g., aromatic amines (4-aminobiphenyl), N-nitrosamines (N-nitrosonornicotine),and nitrated PAHs (1-nitropyrene) as well as a multitude of PAHs other than BP (11,12). Hitherto, derivative spectroscopy has not been applied to the analysis of carcinogen-DNA adducts arising from environmental exposure of humans in vivo. Synchronous fluorescence spectroscopy (SFS)has previously been used to examine human DNA samples for the presence of carcinogen-DNA adducts, but the complex spectra that are generated are difficult to interpret (13). Theoretical studies have suggested that derivative spectroscopy may be able to resolve specific fluorescence signals (14);however, current methodology has previously been limited to relatively simple mixtures (15-18). Furthermore,

* To whom correspondence should be addressed at Building 37, Room 2C09, NCI, Bethesda, MD 20892. 'Laboratory of Human Carcinogenesis,National Cancer Institute. *The Children's Hospital. 1Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute.

Abbreviations: BA, benz[a]anthracene;B[k]F, benzo[k]fluoranthene; DB[a,c]A, dibenz[a,c]anthracene;BP, benzo(a1pyrene;ELISA, enzymelinked immunosorbent assay; PAH, polycyclic aromatic hydrocarbon; SFS, synchronous fluorescence spectroscopy; BPDE, (f)-r-7,t-8-di7 hydroxy-t-9,10-epoxy-7,8,9,lO-tetrahydrobenzo[a]p~ene;AA, wavelength difference.

0893-228~/89/2702-0104$01.50/0 0 1989 American Chemical Society

Fluorescence Analysis of PAH-DNA Adducts in Humans

preparative strategies that include immunoaffinity chromatography have already been combined to provide a practical approach to resolve specific DNA adducts present in complex human biological samples. This report describes the results of applying second derivative SFS to complex environmentally derived samples and shows that this technique alone is unable to resolve individual components of the mixtures. In addition, the extensive cross-reactivity profiles for the antibodies used in these studies are reported, and these data define a range of PAH-DNA adducts that may be purified from human DNA samples.

Chem. Res. Toxicol., Vol. 2, No. 2, 1989 105 7300). Levels of DNA modification for the unlabeled PAH

diol epoxides were leas than that for BPDE and ranged from 0.1% to 0.5%. These modification levels were corroborated by the

32P-postlabelingmethod of PAH-DNA adduct detection (Dr. R. C. Gupta, personal communication) (25). Enzyme-Linked Immunosorbent Assay. Competitive enzyme-linked immunosorbent assays (ELISAs) were performed as previously described (26,27). Briefly, polyvinylchloride microtiter plate wells were coated with 1.5 ng of either unmodified control DNA or DNA that had been modified with BPDE to a level of 28 pmol/pg of DNA. The polyclonal rabbit anti-BPDE-DNA antisera were diluted (3.2 X lo4) prior to use, as was alkaline phosphatase conjugated goat anti-rabbit IgG (5 X lo-?. The substrate used was p-nitrophenyl phosphate. Samples of PAH diol epoxide modified DNA constituted fluid-phase competitors Materials and Methods and were compared to DNA modified with BPDE that was used Chemicals. Racemic r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10- to construct the standard competition curve. All DNA samples tetrahydro[ 1,3-3H]benzo[a]pyrene and nonradiolabeled authentic were denatured (100 "C for 3 min) and cooled on ice (10 min), benzo[a]pyrene tetrahydrotetrols (BP-7,10/8,9-tetrol, BP-7/ the salt concentration was adjusted to 0.02 M with NaCl, and 8,9,10-tetrol, and BP-7,9,10/8-tetr01)~were obtained from the denatured carrier calf thymus DNA was added to give a final National Cancer Institute Chemical Carcinogen Reference concentration of 1 pg of DNA per microtiter plate well. ComStandard Repository (Bethesda, MD 20892). The unlabeled diol petition curves were obtained with samples adjusted to give values epoxides of benz[a]anthracene (BA), chrysene, benzo[k]fluoranin a readable range for five or six different concentrations of each. thene (B[k]F), and dibenz[a,c]anthracene (DB[a,c]A) were also Immunoaffinity Chromatography. Placental DNA samples obtained from this source. Calf thymus DNA was obtained from (15 mg) either before or after immunoaffinity chromatography Sigma Chemical Co. (St. Louis, MO 63178) and was repurified were acidified by addition of HC1 to a final concentration of before use by phenol extraction and ethanol precipitation. Reapproximately 100 mM (pH 1.5) and heated at 90 "C for 3 h. Each distilled phenol was purchased from Bethesda Research Labohydrolyzed sample was then extracted three times with an equal ratories (Gaithersberg, MD 20877), and RNase and DNase I were volume of isoamyl alcohol. The organic phases were then washed purchased from Sigma Chemical Co. Antibody isolation columns with an equal volume of water and evaporated to dryness under (IgM/IgG "Quick-Sep") were obtained from Isolab, Inc. (Pisreduced pressure, and the residues were redissolved in 600 pL cataway, NY 08854). Isoamyl alcohol (AR grade) was procured of water. Reverse-phase HPLC was conducted a t room temfrom Mallinckrodt Inc. (Paris, KY 40361). Tetrahydrofuran was perature by using a Vydac C-18 ODS column (25 cm X 4.6 mm). obtained from Aldrich Chemical Co. (Milwaukee, WI 53233). Isoamyl alcohol extracts were eluted with linear methanol/water Water and methanol (both HPLC grade) were obtained from gradients (30%-60% over 10 min, followed by 60%-100% over Baker Scientific (Pillipsburg, NJ 08865). 5 min) at a flow rate of 1 mL/min. Eluates were collected in Biological Samples. Placentas from term, uncomplicated 0.5-mL fractions, and the fractions were evaporated to dryness pregnancies were collected a t delivery at University Hospital, under reduced pressure and redissolved in water (600 pL) prior Denver, according to a protocol approved by the University of to analysis by second derivative SFS. Colorado Health Sciences Center Human Subjects Committee. Second Derivative Synchronous Fluorescence SpecFor each placenta studied, nuclear fractions were prepared from troscopy. Synchronous fluorescence spectroscopy is described approximately 40 g of freshly obtained placental villous tissue in detail elsewhere (29) and has previously been used as an assay (syncytiotrophoblast tissue) (21). From these fractions DNA was for BPDEDNA adducts in cells and tissues from humans exposed purified to optical density absorbance ratios (OD 260 nm/280 nm) to PAH (13,30). More detailed analysis of the specific fluorescence of 1.8 or greater by proteinase digestion, phenol extraction, ethanol spectral characteristics of PAH residues that were isolated from precipitation, and RNase digestion according to a protocol dehuman DNA was obtained upon generation of second derivative veloped to extract DNA from peripheral blood cells (19). SFS. The Savitsky-Golay algorithm (31) was used to convert Polycyclic Aromatic Hydrocarbon Diol Epoxide Modified spectral data acquired by driving the excitation and emission DNA. The preparation of PAH diol epoxide modified DNA monochromators of a fluorescence spectrophotometer (Perkinsamples was essentially the same as that previously described for Elmer Corp., Rockville, MD 20850) simultaneously with a fixBPDE-DNA (22, 23). Radiolabeled BPDE or unlabeled diol ed-wavelength difference of 34 nm (Ax 34 nm). epoxide derivatives of other PAHs (chrysene, BA, DB[a,c]A, and B[k]F) were dissolved in tetrahydrofuran (1mg/mL), and each Results and Discussion solution was made up to 5 mL with ethanol. The standard diol epoxide solutions were mixed with DNA solutions (10 mL of 1 When authentic BP-7,10/8,9-tetrol was analyzed by mg/mL solutions in Tris buffer, 100 mM, pH 7.4). The mixtures second derivative SFS (AA 34 nm), a single, sharp signal were incubated at room temperature (24 "C) for 12-16 h and then was observed at 379 nm (data not shown). The magnitude extracted with equal volumes of water-saturated diethyl ether of the signal was found to correspond to 230 pg of BP(an extraction procedure that was repeated eight times) and 7,10/8,9-tetrol (per 600 pL), and when a serial dilution of water-saturated isoamyl alcohol (repeated four times) to remove BP-7,10/8,9-tetrol in water was subjected to second deunreacted hydrocarbon residues. The DNA samples were prerivative SFS, signal linearity was observed in the range cipitated by the addition of sodium chloride (100 mM final concentration) and ethanol (21/2 volumes). The DNA precipitates 20-2000 pg/mL and a calibration curve was constructed were then washed in ethanol (70%) and redissolved in water. (data not shown). When human placental DNA (15 mg) DNA modification with BPDE was determined to be 1.3% (36 was subjected to mild acid hydrolysis, organic solvent expmol of BPDE/pg of DNA) by UV absorption spectroscopy (eu7= traction, and second derivative SFS, complex spectra were 29 OOO) and liquid scintillation counting. Modification levels for observed (Figure 1A). These data are indicative of the DNA samples that had been adducted with nonradiolabeled PAH presence in this sample of multiple fluorescent compodiol epoxide derivatives were estimated by UV absorption nents. Comparison with the second derivative SFS that spectroscopy from c values that were generated for the intact was generated for a 384 pg/mL concentration of an auaromatic nuclei ( 2 4 ) ,which were phenanthrene (egg5= 316), anthentic sample of BP-7,10/8,9-tetrol showed that this thracene 2880), triphenalene (emm 2760), and fluoranthene

* Nomenclature for the tetrahydrotetrols of BP is consistent with Yang et al. (1978), reference 43 (BP-7,10/8,9-tetrol, BP-7/8,9,10-tetrol, and BP-7,9,10/8-tetrol).

technique alone is not capable of resolving the components of this mixture. Clearly, second derivative SFS analysis of this sample (AX 34 nm) did not have sufficient power to resolve a recognizable signature peak (emission 379 nm

106 Chem. Res. Toxicol., Vol. 2, No. 2, 1989

Weston et al.

3 3 4 3 8 0 4 3 0 4 8 0 5 3 0 5 8 4

3 3 4 3 8 0 4 3 0 4 8 0 5 3 0 5 8 4

Emission Wavelength (nm) Figure 1. Second derivative synchronous fluorescence spectra (AA 34 nm) generated for (A) a crude isoamyl alcohol extract of acid-hydrolyzed (0.1 N HCl, 90 OC, 3 h) human placental DNA and (B) an HPLC-fractionated isoamyl alcohol extract of acid-hydrolyzed (0.1 N HCl, 90 "C, 3 h) human placental DNA. Synchronous spectra are shown by using an emission coordinate.

other components of the mixture (Figure 1B) since a sharp emission band is present at 379 nm. These data support previous HPLC/SFS analyses (20)of DNA isolated from the peripheral blood lymphocytes of coke oven workers where only zeroth-order SFS were considered. However, the presence of other large fluorescent signals at longer wavelengths may have implications for quantitative analysis (32). In an attempt to concentrate BPDE-DNA adducts prior to hydrolysis to tetrols and HPLC/SFS, antibodies that had been raised in rabbits against BPDE-modified DNA were used to prepare immunoaffinity chromatography columns (19). The cross-reactivities of these antibodies with BP-related PAH-DNA adducts have now been examined. Calf thymus DNA samples that had been modified with diol epoxide derivatives of PAHs structurally related to BP (BA, chrysene, B[k]F, and DB[a,c]A) were assayed by competitive ELISAs with the anti-BPDE-DNA antibody. The inhibition curves that were established (Figure 2) indicate the presence, in these differently modified DNA samples, of common immunological epitopes that are recognized by the antibodies. Thus, when human samples are used, materials eluting from immunoaffinity columns prepared with these antibodies are likely to contain PAH-DNA adducts other than those formed from the activation of BP. In other immunoreactivity studies, these antibodies were found not to recognize either aflatoxin-DNA adducts (13) or a series of aromatic amine-DNA adducts (unpublished observations). The analysis of cross-reactivities for the antiBPDE-DNA serum that is presented here is more extensive but consistent with previously published reports (13, 33, 34). When partial digests of human placental DNA (15 mg), which had been applied to immunoaffinity columns bearing anti-BPDE-DNA antibodies and eluted with NaOH (50 mM), were acidified (0.1 N HCl), hydrolyzed (90 "C, 3 h), extracted with isoamyl alcohol, and subjected

fmol ADDUCT IN MODIFIED DNA

Figure 2. Examination of the serological cross-reactivity profile for the rabbit anti-BPDE-DNA serum. These ELISA inhibition curves show that this antiserum recognizes DNA samples modified with benz[a]anthracene diol epoxides [bay region (0) and non bay region (D)] and chrysene diol epoxide (o),as well as the diol epoxides of benzo[a]ppene (a),benzo[k]fluoranthene (A),and dibenz[a,c]anthracene (A).

for BP-7,10/8,9-tetrol). A fluorescence signal at 374 nm exists only 5 nm from the signature signal (379 nm) at shorter wavelengths, but it is impossible without further experimentation to determine the identity of this signal AA 34 nm). Even if it were possible to assign (EmA374nm, the origin of this signal to BP-7,10/8,9-tetrol, its position in the spectrum and relative magnitude would probably vary as a function of the composition of the complex mixture under study (32). When SFS was performed on this material following HPLC, the spectrum was simplified (Figure 1B). Although the HPLC conditions are sufficiently rigorous to separate the four possible stereoisomers of BP-7,8,9,10-tetrahydrotetrol (data not shown), they may not completely separate the diverse derivatives of PAHs that are assumed to be components of the mixture. Consequently, the spectrum generated for material that had the correct HPLC retention time for BP-7,10/8,9-tetrol (Figure 1B) remains complex; however, second derivative SFS appears to resolve the BP-7,10/8,9-tetrol signature signal from

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Figure 3. Second derivative synchronous fluorescence spectra (AA 34 nm) generated for (A) an acid-hydrolyzed (0.1 N HCl! 90 "C, 3 h) immunoaffinity column eluate (50 mM NaOH) of DNase I partially digested human placental DNA and (B) an HPLC-fractionated acid hydrolysate (0.1 N HC1, 90 "C, 3 h) of an immunoaffinity column eluate of DNase I partially digested human placental DNA. Synchronous spectra are shown by using an emission coordinate.

Fluorescence Analysis of PAH-DNA Adducts in Humans

to SFS, a relatively simple second derivative spectrum was obtained (Figure 3A). Only one major fluorescence signal was found to be present, and this was coincident with that observed at 379 nm for the BP-7,10/8,9-tetrol. Quantitation of the fluorescence signal from the calibration curve suggested that the immunoaffinity column eluate contained 760 pg of BP-7,10/8,9-tetrol. However, the presence of other weak fluorescence emissions (Figure 3A; 340,370, 385, and 425 nm) in the immunoaffinity column eluates (NaOH, 50 mM), suggests the presence of other PAHDNA adducts. This result is expected from the antibody cross-reactivity data (Figure 2). Since fluorescence spectroscopy is not a destructive process, the isoamyl alcohol extracts of immunoaffinity column concentrated material were subjected to HPLC for further purification. The second derivative SFS for a fraction of this material that is chromatographically indistinguishable from BP-7,10/8,9-tetrol is shown in Figure 3B. This second derivative spectrum has only one synchronous signal at 379 nm, which was found to be superimposable upon that of the authentic BP-7,10/8,9-tetrol standard. Quantitative analysis of this specific signal by comparison with the calibration curve revealed the presence in this HPLC fraction of 630 pg of BP-7,10/8,9-tetrol, in the absence of fluorescence emissions of unknown origin. These analyses attempt to quantitate the level of BPDEDNA adducts in human placenta by second derivative SFS. Although it has not yet been possible to determine the efficiency of BP-7,10/8,9-tetrol recovery as percentage yield, a minimum adduct level can be calculated from the observed spectrum (Figure 3B). This level is 630 pg of BP-7,10/8,9-tetrolper 15 mg of DNA (0.13 fmol/pg), which is 1 base modification by BP in 2 X lo’ nucleotides. The presence of BP-7,10/8,9-tetrol in HPLC eluates of the human DNA extracts that were studied here was subsequently confirmed by GC-MS (19, 35). A variety of approaches to the fluorescence spectral analysis of biological and environmental samples have been used. The object of these analyses has been to identify and in some instances quantitate individual components of the mixture being studied. The application of second derivative synchronous fluorescence spectroscopy to mixture analysis has generally been limited to binary, tertiary, and quaternary mixtures (15-17). However, one case does exist in which second derivative SFS was applied to the resolution of a mixture containing seven components ( 17 ) . Limitations of SFS when applied to even relatively simple mixtures are known but have not yet been systematically explored (32). Furthermore, the usefulness of other novel analytical fluorescence techniques, e.g., fluorescence line narrowing and nonphotochemical hole burning spectroscopy, to complex environmental mixtures remains to be established (36, 37). For the precise quantitation of carcinogen-DNA adducts, the need for identification of a specific adduct is axiomatic. In this context, since antibodies may exhibit cross-reactivity patterns, they are most usefully applied either with physical methods (chromatography)to increase specificity (38, 39) or as a preparative tool where physicochemical analysis is the end point (40). Exceptions to this are the measurement of either cis-platinum- or psoralen-DNA adducts formed in patients following chemotherapy ( 4 1 , 42). Second derivative SFS is highly specific with a detection limit in the low picogram range (10-20 pg) and has been used to determine recovery of BP-7,10/8,9-tetrol from placental DNA samples that were irolatrd by immunoeffinity chromatography. The data indicate that there is no

Chem. Res. Toxicol., Vol. 2, No. 2, 1989 107 simple theoretical substitute for preparative chromatographic techniques in any analysis of complex environmentally derived mixtures of carcinogen-DNA adducts. Therefore, the goals of future studies will be the development of combined immunoaffinity column chromatography and HPLC for carcinogen-DNA adduct isolation and the identification of unknown fluorescent Components that were detected during the course of these studies. Acknowledgment. We thank Mr. Bob Julia for his excellent editorial assistance in the preparation of this report.

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