Chem. Res. Toricol. 1992,5, 685-690
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Identification of Individual Benzo[ clphenanthrene Dihydrodiol Epoxide-DNA Adducts by the 32P-Postlabeling Assay Karen A. Canella,*’?Kimmo Peltonen,tJ Haruhiko Yagi,s Donald M. Jerina,s and Anthony Dipplet Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received May 18, 1992 Purine deoxyribonucleoside3’-phosphates were reacted separately with the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol1,2-epoxide. Products resulting from the cis and trans opening of the epoxide ring by the exocyclic amino groups of deoxyadenosine and deoxyguanosine 3’-phosphates were separated by high-pressure liquid chromatography and identified by comparison of the observed circular dichroism spectra with the known spectra for the corresponding nucleoside adducts. The 16 structurally identified benzo[c] phenanthrenepurine deoxyribonucleoside 3‘-phosphate adducts were then separately postlabeled according to the Randerath method, and the positions of the individual bisphosphates were mapped by thin-layer chromatography. Chromatographicconditions were developed that allowed separation of the four adducts for 3 of the 4 dihydrodiol epoxide isomers.
Introduction Polycyclic aromatic hydrocarbons are widespread environmental pollutants that can express carcinogenic properties through metabolic activation to bay-region dihydrodiol epoxides (reviewed in refs 1-3). Reaction of these electrophilic dihydrodiol epoxide metabolites with DNA is the mutagenic event presumed to be responsible for initiation of the carcinogenic process (4-6). As illustrated for benzo[clphenanthrene (B[cIPh)l (Figure l),dihydrodiol epoxides can exist as four configurational isomers, i.e., two enantiomeric pairs of diastereomers with each pair differing only in the spatial relationship of the epoxide oxygen and the benzylic hydroxyl group. Thus, the epoxide oxygen and the benzylic hydroxyl are on the same face of the ring in the diastereomer referred to here as benzo[clphenanthrene dihydrodiol epoxide-1 (B[clPhDE-1)and on opposite sides in the diastereomer referred to as benzo[clphenanthrene dihydrodiol epoxide-2 (B[clPhDE-2). All four isomers are susceptible to reaction of the benzylic C-1 position with the exocyclic amino groups of deoxyadenosineor deoxyguanosine from above or below the plane of the hydrocarbonring, leading to both cis- and trans-opened epoxide adducts (Figure 1) (7, 8). When reacted separately with calf thymus DNA, each B[clPhDE isomer generated a unique distribution of adducts wherein the epoxide ring was opened either cis or
9%
OH
@&@OH
@%
%
(-)-DIHYDRODIOL EPOXIDE.1
(+)-DIHYDRODIOL EPOXIDE.1
(-)-DIHYDRODIOL EPOXIDE-?
(+)-DIHYDRODIOL EPOXIDE.?
+ NCI-Frederick
Cancer Research and Development Center. f Present address: Institute of Occupational Health, Topeluiksenkatu 41 aA,SF-00250 Helsinki, Finland. t National Institutes of Health. Abbreviations: B[clPh, benzo[clphenanthrene; B[clPhDE-1, the benzo[clphenanthrene 3,4-dihydrodioll,2-epoxide diastereomer in which the epoxide oxygen and benzylic hydroxyl are cis to one another; B[clPhDE-2, the benzotclphenanthrene 3,4-dihydrodiol 1,2-epoxide diastereomer in which the epoxide oxygen and benzylic hydroxyl group are trans;PEI-cellulose, poly(ethy1enimine)-cellulose;HPLC, high-pressure liquid chromatography; CD, circular dichroism; TLC, thin-layer chromatography; dG, deoxyguanosine 3’-phosphate; dA, deoxyadenosine 3’phosphate.
Figure 1. Structures of adducts formed in the reaction of each configurational isomer of B[c]PhDE with the exocyclic amino groups of either deoxyguanosine (dG) or deoxyadenosine (dA) 3’-phosphate. Each reaction resulted in both cis and trans opening of the epoxide ring.
trans by purine deoxyribonucleosides (7, 81, yet similar fractions, between 60 and 7576, of each of the dihydrodiol epoxides were bound to DNA in vitro. Despite this similarity, rodent bioassays showed different tumorigenic 0 1992 American Chemical
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686 Chem. Res. Toxicol., Vol. 5, No. 5, 1992 (+) or (-)-B[c]PhDE-1 or 2
+
purine 3'-phosphate
1-
Sip-pak unmodified Np 2 MeOH
t B[ C]P h-N p HPLC, CD
,/
cis adduct
A
\
trans adduct
32P-Postlabeling 2D TLC
Autoradiography
1
/
Figure 2. Strategy for characterizing eachpair of adducts shown in Figure 1.
activities for different isomers,with (-)-B[clPhDE-2 being the most active tumorigen and (-)-B[c]PhDE-l being the least active (9). Thus, factors such as chemical stability and stereochemical interactions with cellular components may influence the amount of compound that persists and that is therefore available for binding to cellular DNA. In addition, different individual adducts may have different biological potentials. Of the numerous methods available for the detection and quantitation of DNA adducts in biological systems, the Randerath 32P-postlabelingassay (10,11) is perhaps the most widely utilized because of its small sample requirements and its high sensitivity. One drawback to this method is that it is not inherently chemospecific in that it does not reveal structural information about the modified nucleotides that are visualized after autoradiography. To overcome this difficulty, we have previously developed an approach to the generation of markers for hydrocarbon-DNA adducts (12). The feasibility of t h e approach was demonstrated by generating markers for each of the eight postlabeled adducts which arise from the reaction of both enantiomers of benzo[alpyrene dihydrodiol epoxide-:! with the purine nucleotides of DNA (12). In the present paper, this approach was extended to the generation and identification of the 16 postlabeled adducts which result from the reaction of the four isomers of B[clPhDE with purine nucleotides in DNA ( 7 , 8 ) .
Materials and Methods Materials. The four configurational isomers of B[c]PhDE were synthesized as previously described (13). Caution: [Three
Canella et al. of these isomers exhibit substantial tumorigenic activity i n animals (9) and should be handled appropriately.] C-18 SepPaks and Sep-Pak Lights were purchased from Waters (Milford, MA). The PEI-cellulose plates were produced by Machery-Nagel (Westbury, NY). Nucleoside 3'-phosphates, calf thymusDNA, and enzymes were all obtained commercially. Instrumentation. HPLC was run on a Shimadzu Model 6-A equipped with an RF-535 fluorescence HPLC monitor. Ultraviolet absorption spectra were recorded on a Milton-Roy Spectronic 3000 diode array spectrophotometer. Circular dichroism spectra were recorded on a JASCO J-500A spectropolarimeter. Radioactive decay was quantitated by a Packard 1500 Tri-Carb liquid scintillation analyzer. Preparation of Modified Nucleotides. Aliquots (1mL) of either deoxyadenosine or deoxyguanosine 3'-phosphate (10 mg/ mL) in 0.1 M Tris-HC1 (pH 7) were separately mixed with 100 pL of an acetone solution of each of the four isomeric B[c]PhDES (1 mg/mL). After initial mixing, the reactions were kept in the dark a t 37 "C for 5 h. To remove dihydrodiol epoxide hydrolysis products, the reaction mixtures were extracted three times with water-saturated ethyl acetate (1mL), followed by two extractions with diethyl ether (1 mL). The aqueous solutions were then loaded onto C-18 Sep-Pak cartridges, and most of the unreacted nucleotide was removed by washing each cartridge with water (20 mL). The more lipophilic B[c]PhDE-nucleotide adducts were subsequently eluted with methanol (3 mL). The resulting eight methanol fractions were separately evaporated to dryness and redissolved in 0.05 M potassium phosphate buffer (pH 7.1; 0.5 mL). Analysis of Nucleotide Adducts. The phosphate buffer solutions containing the crude nucleotide adduct preparations were subjected to HPLC. Aliquots (20 pL) were injected onto a 4.6- X 250-mm, 5-pm Beckman ODS column. The peaks of interest were collected over a 60-min linear gradient from 2 to 30% acetonitrile in 0.05 M potassium phosphate (pH 7.1). UV absorbance was monitored at 260 nm, and fluorescence was monitored using an excitation wavelength of 250 nm and an emission wavelength of 360 nm. After multiple chromatographic runs, peaks were pooled, concentrated to dryness, taken up in water, and loaded onto C-18 Sep-Pak cartridges which were subsequently washed with water (40 mL) to ensure removal of all of the phosphate buffer. Again, the lipophilic component, this time consisting of a single HPLC peak, was eluted from the Sep-Pak with methanol (3 mL). The methanol fractions were concentrated to 0.85 mL for UV and CD spectroscopy. The CD spectra were normalized by dividing each spectrum by the UV absorbance a t Am=. 32P-Postlabelingand TLC Separation. After the UV and CD spectra were obtained and the purified adducts were identified, 1 pL of each of the 850-pL solutions was taken for postlabeling. The l-pL samples were concentrated to dryness and then redissolved in 10 pL of sodium succinate buffer and 2.0 pL of kinase buffer before the addition of 0.5 pL of polynucleotide kinase (10 units/pL) and 3.0 pL of [ T - ~ ~ P I A (Amersham TP Corp., Arlington Heights, IL) (10 pCi/pL) (10). After 40 min of incubation a t 37 "C, 1 pL of apyrase (40 units/pL) was added, and the reaction was left a t 37 "C for an additional 30 min. Each labeled mixture was then diluted with 0.1 M sodium phosphate (pH 7; 0.5 mL). In order to remove the [32P]inorganic phosphate, one of two different procedures was utilized, Le., a Sep-Pak procedure or a D1 procedure. ( A ) Sep-Pak Procedure. To remove the [32Plinorganic phosphate from the labeled mixture, above, before application to the thin-layer PEI-cellulose plates, the diluted samples were loaded onto preconditioned C-18 Sep-Pak Light cartridges and washed with 0.05 M sodium phosphate (pH 7.0; 10 mL). The radiolabeled bisphosphates were subsequently recovered from the cartridges with a solution of 50% acetonitrile in0.05 M sodium phosphate (pH 7.0; 0.5 mL). (B) D1Procedure. To remove the [32P]inorganicphosphate from the labeled mixture after application of the sample to the
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 687
Benzo[c]phenanthrene-DNA Adducts
0
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Figure 3. HPLC elution profiles of the reaction of each configurational isomer of B[clPhDE with either dG or dA 3'-phosphates. The reactions of (+) enantiomers of B[cIPhDE-l and -2 are shown in the left column (panels A-D) with the corresponding reaction of the opposite enantiomer in the right column (panels E-H). PEI-cellulose plates, wicks were attached (14) and the plates were developed for 14 h in 0.1 M sodium phosphate (pH 6.8) (Di). TLC Separation. Separation of the adducts was achieved by a modification of previously used buffer conditions (12,14).The solvent for development in the D3 direction consisted of 1.8 M lithium formate and 3.7 M urea (pH 3.4) run for 5 h. Development in the D4 direction (90" from D3) was with 0.4 M sodium phosphate, 0.25 M Tris, and 3.7 M urea (pH 8.2) for 4 h. Preparation and Postlabeling of B[c]Ph-Modified DNA. To a solution of calf thymus DNA (40 pg) in 0.01 M Tris-HC1 (pH 7.0; 40 pL) was added 1pL of a 1.03 pg/pL acetone solution of (+)-B[c]PhDE-2. The reaction was mixed by pipetting and left to incubate at 37 "C for 5 h. To remove the hydrolysis products of the dihydrodiol epoxide, the reaction mixture was extracted twice with water-saturated 1-butanol (40 pL) followed by an extraction with water-saturated ethyl acetate (40 pL) and an extraction with water-saturated diethyl ether (40 pL). An aliquot (10 pL) was removed and added to a vial containing 0.3 M sodium succinate and 0.1 M calcium chloride (pH 5.8; 1pL). Calf spleen phosphodiesterase and micrococcal nuclease (5 pg and 0.4 unit, respectively) were added according to the method of Randerath (IO),and the mixture was left for 3.5 h at 37 "C. After digestion
to nucleoside 3'-phosphates, half of the mixture was treated with 7.5 pg of nuclease P-1 in nuclease P-1 buffer (5 pL) (11). The remaining half was diluted with 5 pL of distilled deionized water. After 40 min at 37 "C, 0.5 M Tris base (3 pL) was added to both reactions, followed by 2.0 pL of kinase buffer, 0.5 pL of polynucleotide kinase (10 units/pL), and 3.0 p L of [-yJ2P]ATP (10 pCi/pL) (11). After 40 min of incubation a t 37 "C, 1pL of apyrase (40 units/pL) was added, and the reaction was left at 37 "C for an additional 30 min.
Results and Discussion The strategy for these studies (Figure 2) was to separately react each configurational isomer of B [cl PhDE with a purine nucleoside 3'-phosphate to yield two products resulting from both cis and trans opening of the epoxide ring at the benzylic C-1 position by the exocyclic amino group of the purine. Conditions would then be developed to separate these by HPLC. Since the 16 adducts shown in Figure 1have been rigorously characterized at the nucleoside level (8)and each was found to have a unique CD spectrum, comparisons of the CD spectra obtained for the
688 Chem. Res. Toxicol., Vol. 5, No.5, 1992 4Ay/(+)l
Canella et al. _-
dG trans
I
5A (-)-bihyd~diOi Epoxide-1
dG trans
5
mdeg 0
I \(+)2
dG trans
v 1
.
dQ trans
dG cis
2 )-(:'
L. 1.
a .
L A
l4:9- (
1
2
.2 a
I . . . . . . -
12 dA trans
5D (+)-DihydrodiOl Epoxide-2
14;
( + ) 2 dA cis h
dA trans +cis
I 220
nm
500 220
\(-)2
dA cis nm
500
Figure 4. CD spectra of all 16 isolated B[c]Ph dG and dA 3'phosphate adducts. The structural assignment was based on the data in ref 13.
nucleoside3'-phosphate adducts prepared herein with the CD spectra previously obtained for the correspondingnucleosides would facilitate identification of each of the purified products. The preparation and HPLC separation of all eight pairs of cis/trans isomers were carried out as described under Materials and Methods. By injection of a small aliquot of the ethyl acetate extract from the reaction it was determinedthat the major tetraol hydrolysis products had retention times of about 50 min under these chromatographic conditions. Each reaction mixture, despite the prepurification on a Sep-Pak, revealed a number of UVabsorbing peaks (Figure 3). However, only those peaks appearing after 30 min exhibited a fluorescence signal for the phenanthrene moiety, and these were pooled from multiplechromatographicruns. The CD spectrumof each peak, collectedbetween 30 and 50 min, was then examined to allow identification of the required B [c]PhDE-deoxyribonucleoside 3'-phosphate adducts. The CD spectra are illustrated in Figure 4 and the assignments of structure could be unambiguously made from these by comparison with the known spectra for the corresponding nucleosides (8). As found for the nucleosides, trans-opened nucleotide adducts arising from the B [c]PhDE-l diastereomer elute before the cis adducts, and cis adducts elute before trans adducts when derived from the B[c]PhDE-2 diastereomer (Figure 3). The nucleoside adducts described earlier (7, 8) were obtained from the nucleoside 5'-phosphates, and although product yields were higher with the 5'-phosphates, there are some similarities in the reactions of the 5'- and 3'phosphates. For example, substantial yields of both cisand trans-opened epoxide adducts arose from the B[c]PhDE-1enantiomerswith either 3'- or 5'-phosphates, with the ratio of cis/trans (determined from integration of the HPLC signal) being in the range 1-2.5 for the former and
dQ trans dQ cis
Figure 5. Autoradiograph resulmg from cochromatography o all four possible purine nucleoside bisphosphate products resulting from reaction of each configurationalisomer of B[c]PhDE. Though the origins are not all visible, they are all in the bottom left-hand corner, as seen in panel B.
1-5.5 for the latter. Adducts from B[c]PhDE2 arose predominantly from trans opening of the epoxide ring, such that cis/trans ratios fall in the 0.05-0.2 range for both 3'- and 5'-phosphates with the sole exception of the reaction of (+)-B[c]PhDE-2 with deoxyguanosine 3'phosphate, where this ratio was 2.7. Since the trans adduct from this latter reaction yielded two spots on postlabeling (see later), this may be an underestimate of the cis/trans ratio. After identificationof the nucleotide adducts, aliquots of each were separately postlabeled as described in Materials and Methods. Initial experimentsshowed that the chromatographicsystemsused in previousstudieswith 7,12-dimethylbenz[a]anthracene-DNA adducts (12, 14, 15)were not applicable to the benzo[c]phenanthrene adducts. For example, using the concentrated buffer used earlier for development in the D1 direction, two of the adducts, i.e., (-)-2 dA cis and (-)-2 dG trans, were lost. Also, using the solvents used earlier for D3 and D1 development, spots were diffuse. However, the problems experienced with the old D1 conditions were eliminated by using either a Sep-Pakprocedure or a much more dilute buffer for D1development,and the diffusenessof the spots was remedied by reducing the buffer concentrationsused for D3 and Dq development by 50%, all as described in the Materials and Methods section. Thus, when the Sep-Pak
Benzo[cJphenunthrene-DNA Adducts
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 689
dA clr
+ tnm
Figure6. Autoradiographsof the postrlabeledproducts resultingfrom (+)-B[cIPhDE2. PanelsA, B, D, and E represent the compounds prepared from the purine nucleoside 3'-phosphates purified by HPLC. Panels C and F were the result of cochromatography of the with ctDNA and can be contrasted with Figure 5D. The indicated materials. Panel G illustrates the reaction of (+)-B[c]PhDE2 . . origins are in the bottom left-hand corner.
procedure was used to remove the [32P]inorganic phosphate from the postlabeled preparations, and the volumes of the 16 preparations were adjusted so that all had the same radioactivity per unit volume, aliquots (1pL) of each HPLC-purified material ran as a single spot with the exception of the (+)-2 dG trans adduct. The latter exhibited two widely separated spots under the TLC conditions used (Figure 6D). To establish TLC maps of all four purine products that could arise from a given isomer of B[c]PhDE, the cis- and trans-opened adducts derived from deoxyguanosine and deoxyadenosine 3'-phosphate were combined and run together through the TLC procedures (Figure 5). To facilitate identifications, the cis- and trans-opened products for each purine nucleotide were also run together (not shown). The separations and the assignments ar-
rising from the studies are illustrated in Figure 5. In autoradiographs made with shorter exposure times, it was seen that (+)-1 dA cis and (+)-1 dG cis (Figure 5B) are two discrete spots in Figure 5B, but this was not the case with (+)-2 dA trans and (+)-2 dA cis (Figure 5D), which were inseparable under these solvent conditions. Figure 6 illustrates how the individual adducts were identified in Figure 5 through the analysis of chromatograms loaded with individual adducts (Figure 6A,B,D,E) and through analysis of chromatogramswhere cis and trans adducts were loaded together (Figure6C,F). The data for the (+)-B[c]PhDE-2 are illustrated in Figure 6 because this shows that the trans and cis deoxyadenosine bisphosphate adducts did not separate under these chromatographic conditions. These data also show that the small spot found on the right of the main spot for the trans
690 Chem. Res. Toxicol., Vol. 5, No. 5, 1992
adduct of deoxyguanosine bisphosphate was presumably an impurity since this was not present when DNA was reacted with the (+)-B[clPhDE-2. The latter (Figure 6G) showed, as expected, only three spots, corresponding to dG cis, dG trans, and the combined dA trans and dA cis. Thus, the small spot on the far right in Figures 5D and 6D was found to be a contaminant in the (+)-2 dG trans marker. It is of interest to note that no adducts were detected if the DNA preparation was incubated with nuclease P-l, indicatingthe P-l dephosphorylatesthe benzo[clphenanthrene-deoxyribonucleoside 3’-phosphate adducts.
Conclusions This work has demonstrated that the adducts formed by cis and trans opening of the epoxide ring of individual B[clPhDE configurational isomers by the amino groups of deoxyadenosine or deoxyguanosine 3’-phosphates can be separated by HPLC and structurally identified on the basis of their unique CD spectra. After postlabeling these characterized compounds by the Randerath 32P-postlabeling procedures, it was determined that fairly low buffer concentrations for development in the D1, D3, and Dq directions on TLC were necessary to visualize all of these adducts as discrete spots by autoradiography. By subjecting all four possible purine products of each single configurational isomer of B [clPhDE to cochromatography, four TLC maps linking the positions of the spots on the TLC plate with known structures were generated. The utility of this method was demonstrated by carrying out the 32P-postlabelingprocedure on a B[clPhDE-modified DNA sample and then identifying the adducts after twodimensional chromatography using the modified DI, D3, and Dq buffers (Figure 6). In addition, a caution was raised against the use of nuclease P-1 enrichment for adducts arising from B[clPhDE.
Acknowledgment. Research sponsored in part by the National Cancer Institute, DHHS, under Contract N01CO-74101with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services,nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S.Government. References (1) Dipple, A., Moschel, R. C., and Bigger, C. A. H. (1984) Polynuclear aromatic carcinogens. In Chemical Carcinogens (Searle, C. E., Ed.)
Canella et al. 2nd ed., pp 41-163, American Chemical Society, Washington, DC. (2) Harvey, R. G. (1986) The molecular mechanism of carcinogenesis of polycyclic hydrocarbons. In Molecular M e c h a n i s m of Carcinogenic and Antitumor Actiuity (Chagas, C., and Pullman, B., Eds.) pp 95-129, Pontificia Academia Scientiarvm, Vatican City. (3) Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. (1985) Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens. Bioact. Foreign Compd. 7, 177-242. (4) Brookes, P., and Lawley, P. D. (1964) Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acids of mouse skin. Nature 202, 781-784. (5) Jerina, D. M., and Daly, J. W. (1976) Oxidation a t carbon. In Drug Metabolism (Parke, D. V., and Smith, R. L., Eds.) pp 13-32, Taylor and Francis, London. (6) Levin, W., Wood, A. W., Wislocki, P. G., Chang, R. L., Kapitulnik, J., Mah, H. D., Yagi, H., Jerina, D. M., and Conney, A. H. (1978) Mutagenicity and carcinogenicity of benzo[alpyrene and benzo[elpyrene derivatives In Polycyclic Hydrocarbons and Cancer (Gelboin, H. V., and Ts’o, P. 0. P., Eds.) Vol. I, pp 189-202, Academic Press, New York. (7) Dipple, A., Pigott, M. A., Agarwal, S. K., Yagi, H., Sayer, J. M., and Jerina, D. M. (1987) Optically active benzo[clphenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327, 535536. (8) Amrwal, S. K.. Saver. J. M.. Yeh. H. J. C.. Pannell. L. K.. Hilton. BTD., Pigott, M. A,,Dipple,’A., Yagi, H., A d Jerina, D. M. (1987) Chemical characterization of DNA adducts derived from the configurationally isomeric benzo[clphenanthrene-3,4-dio1-1,2-epoxides. J . A m . Chem. SOC.109, 2497-2504. Levin, W., Chang, R. L., Wood, A., Thakker, D. R., Yagi, H., Jerina, D. M., and Conney, A. H. (1986) Tumorigenicity of optical isomers of the diastereomeric bay region 3,4-diol-1,2-epoxidesof benzo[c]phenanthrene in murine tumor models. Cancer Res. 46,2257-2261. Gupta, R. C., Reddy, M. V., and Randerath, K. (1982) 32P-Postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis 3, 1081-1092. Reddy, M. V., and Randerath, K. (1986) Nuclease P-1-mediated enhancement of sensitivity of 32P-postlabelingtest for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551. Canella, K. A., Peltonen, K., and Dipple, A. (1991) Identification of (+) and (-1 anti benzo[alpyrene dihydrodiol epoxide-nucleic acid adducts by the 32P-postlabeling assay. Carcinogenesis 12, 11091114. Yagi, H., Thakker, D. R., Ittah, Y., Croisy-Delcey, M., and Jerina, D. M. (1983) Synthesis and assignment of absolute configuration to the trans 3,4-dihydrodiol and 3,4-diol-1,2-epoxides of benzo[clphenanthrene. Tetrahedron Lett. 24, 1349-1352. Vericat, J. A., Cheng, S. C., and Dipple, A. (1989) Absolute stereochemistry of the major 7,12-dimethylbenz[alanthracene-DNAadducts formed in mouse cells. Carcinogenesis 10, 567-570. Schmeiser, H., Dipple, A., Schurdak, M. E., Randerath, E., and Randerath, K. (1988) Comparison of 32P-postlabeling and high pressure liquid chromatographic analysis for 7 , 1 2 - d i m e t h y l b e n z [ a l a n ~ a ~ n ~ DNA adducts. Carcinogenesis 9, 633-638.
