26
Chem. Res. Toricol. 1992,5, 26-33
32P-PostlabelingAnalysis of DNA Adduction in Mouse Skin following Topical Administration of (+)-7,8-Dihydroxy-7,8-dihydrobenzo[ a Ipyrene Ashok P. Reddy,t Donna Pruess-Schwartqt Chuan Ji,’ Peter Gorycki,’ and Lawrence J. Marnett*itJ Department of Chemistry, Wayne State University, Detroit, Michigan 48202, and The A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Departments of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received June 13, 1991
32P-Postlabelingwas employed for analysis of DNA adducts produced in mouse skin following topical administration of enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Deoxynucleoside 3’-monophosphates were isolated by digestion of epidermal DNA with micrococcal endonuclease and spleen phosphodiesterase and phosphorylated with [ Y - ~ ~ P I A T P . 32P-Labeleddeoxynucleoside 3’,5’-bisphosphate adducts to diastereomeric benzo[a]pyrene dihydrodiol epoxides (BPDE) were separated by four-directional thin-layer chromatography on poly(ethy1enimine)-cellulose plates using a recently described solvent system [Reddy, A. P., Pruess-Schwartz, D., and Marnett, L. J. (1992) Chem. Res. Toxicol. (preceding paper in this issue)]. When (+)-BP-7,8-diol was topically administered, a major adduct spot was detected that cochromatographed with a standard produced by reaction of 7(S),8(R)-dihydroxy-9(S),1O(R)-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene[ (+)-syn-BPDE] with DNA. The level of this adduct increased in a dose- and time-dependent fashion and was elevated in animals pretreated with 0-naphthoflavone. Relatively small amounts of radioactivity cochromatographed with standards of deoxynucleoside 3’,5’-bisphosphate adducts derived from 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,1O-tetrahydrobenzo [a]pyrene [ (-)-anti-BPDE]. Following topical administration of (-)-BP-7,8-diol, a single adduct spot was detected that cochromatographed with a standard of the major deoxyguanosine adduct derived from 7(R),8(S)-dihydroxy-9(S),1O(R)-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene[(+)-anti-BPDE]. The stereochemistry of epoxidation of the enantiomers of BP-7,8-diol indicates that cytochrome P-450 catalyzes the terminal activation step of benzo[a]pyrene activation to an ultimate carcinogen in mouse skin, a target organ for its carcinogenic activity.
Introductlon The ability of polycyclic aromatic hydrocarbons to induce tumors in skin is important from historical, practical, and theoretical viewpoints. Pott first proposed that the high incidence of scrotal cancer in British chimney sweeps was due to their chronic exposure to agents in soot ( I ) . Investigation of the carcinogenic activity of coal tar components on rodent skin led to the identification of benzo[a]pyrene as an important polycyclic hydrocarbon carcinogen (2, 3 ) . Mouse skin is routinely used to assay chemicals for carcinogenic activity, and investigations of the mechanism of skin tumor induction has led to many advances in our understanding of the individual steps in multiple-stage carcinogenesis. Among these was the discovery that metabolism is crucial for conversion of polycyclic hydrocarbons to reactive carcinogenic derivatives (4-6). Cytochrome P-450 enzymes play a major role in the oxidative metabolism of polycyclic hydrocarbons in vitro and in vivo (7). Different orthologues catalyze oxygenation of aromatic hydrocarbons and their phenol and dihydrodiol metabolites. For example, oxygenation of benzo[a]pyrene (BP)’ to BP-7,8-oxide and of BP-7,8-diol to BPDE is * T o w h o m correspondence should be addressed at the Vanderbilt University School of Medicine. + W a y n eState University. * Vanderbilt University School of Medicine.
catalyzed most effectively in vitro by P-45OIA1 (eq 1;see Scheme I) (8). However, P-45OIA1 is not a quantitatively important form in human liver, so in this tissue the two oxygenation steps are catalyzed by different orthologues. BP oxidation to BP-7,8-oxide is catalyzed primarily by P-45OIA2 in human liver microsomes whereas BP-7,8-diol oxidation to BPDE is catalyzed by P-450IIIA4 (9). BP-7,8-diol oxygenation to BPDE is also effected by a variety of non-cytochrome P-450 catalysts. Enzyme reactions that generate peroxyl free radicals are particularly efficient in this regard (IO). Peroxyl radicals insert oxygen atoms into the 9,lO-double bond via the stepwise process Abbreviations: BP,benzo[a]pyrene; 8-NF, 8-naphthoflavone;(+)BP-7B-dio1,7(S),B(S)-dihydroxy-7,B-dihydrobenzo[a]pyrene; (-)-BP-7,& diol, 7(R),B(R)-dihydroxy-7,S-dihydrobenzo[a]pyrene; BPDE, 7,B-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; (+)-anti-BPDE, 7~R~,8~S)-dihydroxy-9~S),10(R~-epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrene; (-)-anti-BPDE, 7(S),B(R)-dihydroxy-9(~),lO(S)-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene; (+)-syn-BPDE,7(S),8(R)-dihydroxy-9(S),lO(R)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; (-)-syn-BPDE,7(R),8(S)-dihydroxy-9~R),10(S)-epoxy-7,8,9,lO-tetr~ydrobenzo[a]pyrene; (-)-antiBPDE-An-dC, 7(S),8(R),9(R)-trihydroxy-lO(S)-(An-deoxyguanosyl)7,8,9,10-tetrahydrobenzoalpyrene; (-)-anti-BPDE-M-dA,7(S),B(R),9~R~-trihydroxy-1O~S~-(~-deoxyadenosy~)-7,8,9,lO-tetr~ydrobenzo~a pyrene;
(+)-syn-BPDE-M-dG, 7(S),B(R),S(S)-trihydroxy-lO(R)-(~:
deoxyguanosyl)-7,8,9,lO-tetrahydrobenzo[apyrene; (+)-anti-BPDEAn-dC, 7( R ),B(S) ,9(S)-trihydroxy-10(R)-(~-deoxyguanosy1)-7,8,9,10tetrahydrobenzo[a]pyrene; D M B A , 7,12-dimethylbenz[a]anthracene; DMBA-3,4-diol,3,4-dihydroxy-3,4-dihydrobenz[a]anthracene;PEI,
poly(ethy1enimine); TLC,thin-layerchromatography;RAL,relative adduct labeling;B H A , butylated hydroxyanisole;HPLC,high-performance liquid chromatography; 4-D,four directional.
0893-228x/92/2705-0026$03.00/00 1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 27
Dihydrodiol Epoxide Adducts in Mouse Skin
M
m
-
m
/
i
OH
-
\
(+) BP-7,Edid
Rm\
-w OH
BPDEs that can be used in conjunction with 32P-postlabeling technology (32). This provides a sensitive method for estimating the extent of formation of individual BPDE's in vivo. Application of this technique to the analysis of DNA adducts formed from BP indicated cytochrome P-450 plays the major, if not exclusive, role in generation of BPDE's (32). However, this result may not be applicable to formation of BPDEs from the proximate carcinogen BP-7,8-diol because BP induces certain cytochrome P-450 orthologues whereas BP-7,8-diol does not (31). Thus, skin may display different metabolic capabilities at different times following administration of BP but not after administration of BP-7,8-diol. Therefore, we conducted experiments to evaluate the DNA binding from (+)-BP-7,8-diol in mouse skin. The results establish that peroxyl radicals play a minor role in DNA adduction in vivo.
Materials and Methods
-
(-) anf
Figure 1. Differential stereochemistry of epoxidation of (+)BP-7,8-diol by peroxyl radicals and cytochrome P-450.
depicted in eq 2 (see Scheme I). Peroxyl radical generation is associated with enzyme-catalyzed oxygenation of polyunsaturated fatty acids, NADPH- and ascorbate-dependent lipid peroxidation, peroxidase-dependentoxidation of 8-dicarbonyl compounds, bisulfite, and hydroperoxides, and neutrophil-dependent superoxide generation (11-1 7). BP-7,8-diol is also epoxidized by activated neutrophils in a reaction mediated by myeloperoxidase (18-22). These P-450-independent pathways of polycyclic hydrocarbon oxidation have been demonstrated to occur in vitro, but the extent to which they operate in vivo is uncertain. Stereochemistryhas proven a useful tool for estimating the contribution of peroxyl radicals to epoxidation of (+)-7(S),8(S)-BP-7,8-diol (Figure 1)(23,24). The major product of epoxidation of (+)-BP-7,&diolby P-45OIA1 and -1IIA4 is (+)-syn-BPDE ( 9 , 2 5 , 2 6 ) . This outcome is determined by the orientation of the hydrocarbon at the active site of the protein (27). In contrast, peroxyl radical dependent oxidation of (+)-BP-7,&diolproduces primarily (but not exclusively) (-)-anti-BPDE (23,24). Hydrogen bonding of the 8-hydroxyl group with the approaching peroxyl radical apparently accounts for this result (eq 2). By quantitating the extent of conversion of (+)-BP-7,&diol to BPDE-derived products, it is possible to estimate the relative contribution of cytochrome P-450 and non-cytochrome P-450 processes to epoxidation. This approach has been used to demonstrate non-cytochrome P-450-dependent epoxidation of (+)-BP-7,8-diol in cultured C3H10T1/2 cells, cultured hamster trachea, freshly isolated mouse epidermal cells and epidermal homogenates, and mouse skin in vivo (28-31). In the preceding paper, we described a TLC system to separate 3',5'-bisphosphate adducts of diastereomeric
Chemicals. Unlabeled (+)-BP-7,8-diol, (+)-anti-BPDE, (-)-anti-BPDE, and (h)-syn-BPDEwere obtained from the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention (Bethesda, MD). Standards of BPDE-modified DNA and deoxynucleoside 3',5'-bisphosphates were prepared as previously described (32,33). P-NF was purchased from Aldrich Chemical Co. (Milwaukee, WI). Spleen phosphdesterase (20 units/mg) was from Boehringer Mannheim (Indianapolis, IN). Deoxyribonucleotideswere from Pharmacia (Piscataway, NJ). Micrococcal endonuclease (100 units/mg), potato apyraae, dithiothreitol, spermidine, bovine serum albumin, NADPH, ribonuclease A (bovine pancreas, type 1-A),ribonuclease TI (Aspergillus oryzae, grade IV),and proteinase K (Tn'tirachium album, type XI) were from Sigma Chemical Co. (St. Louis, MO). Calf thymus DNA was obtained from Sigma and purified by proteinase K and ribonuclease A digestion (34).Lysis buffer was purchased from Applied Biosystems (Foster City, CA). Cumene hydroperoxide was obtained from ICN Pharmaceuticals, Inc. (Plainsview, NJ). Polynucleotide kinase was from U.S. Biochemicals (Cleveland, OH). Poly(ethy1enimine) (PE1)-cellulose TLC plates were from Machery and Nagel, FRG, purchased through Brinkmann Instruments (Westbury, NY). They were developed to the top with distilled water before use. [y3?]ATP was prepared using Promega Biotec's Gamma Prep-A kit (Madison, WI). Carrier-free [32P]phosphate (3000 Ci/mmol) was purchased from ICN Radiochemicals (Irvine, CA). Kodak X-Omat AR film was from Fotodyne (New Berlin, WI). Animals. Female Cr1:CD-1 (IRC) BR mice and male Sprague-Dawley rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Mice aged 6-10 weeks were shaved with surgical clippers 48 h prior to treatment (in vivo studies), and only those in the resting phase of the hair growth cycle were used. Rat liver microsomes were prepared as described by Tunek et al. (35). Peroxyl Radical Dependent Adduction of DNA by (+)BP-7,8-diol. (+)-BP-7,8-diol (50pg) was incubated with liver microsomes (1mg of protein from uninduced rats) and calf thymus DNA (500pg) in 1.0 mL of 50 m M Tris.HC1 (pH 7.5). Cumene hydroperoxide (100 pM) was added and the reaction allowed to proceed overnight at 37 "C. The reaction mixture was extracted
Scheme I
28 Chem. Res. Toricol., Vol.5, No.1, 1992
Reddy et al.
twice with ethyl acetate containing 100 pM BHA, and the ethyl acetate was evaporated. The residue was taken up in 50 pL of methanol and analyzed by reversed-phase HPLC for tetrahydrotetraols. DNA in the aqueous phase was precipitated with 0.1 volume of 2 M NaCl and 2 volumes of cold ethanol and then dissolved in 0.5 mL of 10 mM Tris-HC1 (pH 7.5). DNA digestion and postlabeling analysis were as described previously (32). In Vitro Modification of DNA. Purified calf thymus DNA (150 pg) was dissolved in 10 mM Tris.HC1 (pH 7.5), and varying amounts of BPDEs [0,3.2,8,20, and 50 ng each of (-)-anti- and (i)-syn-BPDE in 1 pL of methanol] were added (total volume 250 pL). Reactions were carried out in triplicate at 37 "C for 45 min. The mixture was extracted four times with equal amounts of water-saturated ethyl acetate. Phase separation was performed by centrifugation at 12000 rpm for 2 min. DNA was precipitated by adding 0.1 volume of 2 M NaCl and 2 volumes of cold ethanol. After centrifugation at 12 000 rpm for 10 min, the precipitated DNA was redissolved in 250 pL of 10 mM TrisOHC1 (PH 7.5). DNA (30 pg) was reprecipitated from the solution and then dissolved in 72 pL of 10 mM sodium succinate/5 mM CaClz (pH 6.0). To this solution were added micrococcal endonuclease and spleen phosphodiesterase (15 pL each; 2 pg/pL), and the solution was incubated at 38 "C for 4 h. The digest was diluted to 120 pL with distilled water (0.25 pg of DNA/pL). Twenty microliters of the digest (5 ccg of DNA) was mixed with 6.5 pL of 100 mM ammonium formate (pH 3.5), 6.5 pL of tetrabutylammonium chloride, and 67 pL of water. The solution was extracted twice with watersaturated 1-butanol. The combined extracts were back-extracted with 1-butanol-saturated water and then water (180 pL). The extract was neutralized by adding 3 pL of 200 mM Tris.HC1, pH 9.5, and evaporated in a Speedvac concentrator. The residue was dissolved in 5 pL of water and postlabeled as described (32). The epoxide content of the BPDE's used for this experiment was determined by methanolysis. Separate solutions of (*)syn-BPDE and (-)-anti-BPDE in methanol were prepared a t concentrations of 10,20,40, and 80 pg/mL. They were allowed to sit at ambient temperature for 4 h (anti) or 24 h (syn). The solvent was removed in a stream of Nz and the residue dissolved in 100 pL of methanol for HPLC analysis. HPLC was performed with an Ultrasphere ODS column (5 pm, 4.6 x 250 mm) (Beckman Instruments, Berkeley, CA) eluted at 1.2 mL/min with a methanol/water gradient (45% methanol for 20 min increasing to 50% methanol over 8 min, then increasing to 100% methanol over the next 12 min). Eluting compounds were detected with a Hewlett-Packard 1040A diode m a y detector operated at 344 nm. The retention times of authentic standards were trans-anti-tetrahydrotetraol, 18.9 min; trans-syn-tetrahydrotetraol, 23.2 min; cis-anti-tetrahydrotetraol,26.4 min; cis-syn-tetrahydrotetraol, 33.2 min; trans-anti-methoxytetrahydrotriol,32.0 min; and trans-syn-methoxytetrahydrotriol,36.0 min. The dihydrodiol epoxide content was calculated as the percentage of the methoxytetrahydrotriol relative to the sum of the tetrahydrotetraol and methoxytetrahydrotriol products. The value was 96.6 0.3% for (f)-syn-BPDE and 76 f 6% for (-)-anti-BPDE. In Vivo Modification of DNA. Mice were used 48 h after shaving and were treated with 100 pL of acetone or 100 pL of acetone containing 350 nmol of @NF. Acetone or 0-NF-treated animals were then treated 24 h later with 100 or 200 nmol of (+)-BP-7,8-diol. Typically, two animals were treated for each datum point. After a predetermined time interval (indicated in text), the animals were sacrificed by cervical dislocation and the shaven area of skin was removed (-2 X 2 cm). Epidermal DNA Isolation and Purification. Two different procedures were used for DNA isolation and purification. The first was described by Diamond et al. and modified by Pelling and Slaga (36,37). Skins were floated in a beaker of water for 30 s a t 54 "C and then chilled on ice. The pelts were placed in a chilled Petri plate, and the epidermis was scraped off with a scalpel. The epidermal scrapings were suspended in 2 mL of p-aminosalicylic acid reagent and homogenized with a Polytron homogenizer. The homogenate was phenol extracted and DNA precipitated with 0.1 volume of 2 M NaCl and cold ethanol. The DNA was redissolved and treated with ribonuclease A and proteinase K. After addition of 0.1 volume of NaCl and extraction with CHCl,/isoamyl alcohol, the DNA was precipitated, redissolved, and treated again with ribonuclease A and proteinase K.
*
After this second treatment, the DNA was extracted, precipitated, and dissolved in 10 mM TriseHCl (pH 7.5). The second procedure for DNA isolation and purification involved phenol extraction but only one series of nuclease and proteinase treatments. The treated skin (1.5 X 2 cm) was excised and the epidermis removed as described above. The epidermal scrapings from two animals were combined and homogenized in 4 mL of lysis buffer with 20 passages of a Potter-Elvehjem homogenizer. The suspension was treated with RNase A (170 Kunitz units) and RNase TI (140 units) at 37 "C for 1 h. Proteinase K (40 units) was added and digestion was continued for 2 h at 37 "C and for 16 h at 4 "C. The digested samples were extracted twice with an equal volume of 70% phenol/chloroform/water for 10 min at room temperature followed by a chloroform wash. Phase separation was accomplished by heating at 58 OC for 8 min. After addition of 0.1 volume of 3 M sodium acetate (pH 5.5), DNA was precipitated by addition of ethanol and dissolved in 10 mM Tris.HC1 (pH 7.5). The purity and quantity of DNA were determined spectrophotometrically from the absorbances at 260 and 280 nm. DNA Digestion and Postlabeling Analysis. Epidermal DNA (40 pL of 0.5 pg/pL) was added to 10 pL of 10 mM sodium succinate/5 mM CaC12 (pH 6.0) containing 10 pg each of micrococcal endonuclease and spleen phosphodiesterase. The solution was diluted to 80 pL with distilled water and incubated at 38 "C for 4 h. Forty microliters of the digest was mixed with 6 pL of 100 mM ammonium formate (pH 3.51, 6 pL of tetrabutylammonium chloride, and 47 pL of water. The solution was extracted twice with 1volume of water-saturated 1-butanol (double distilled) in 1.5-mL Eppendorf tubes by vortexing. Phases were separated on a table top centrifuge and the combined organic phases back-extracted twice with 190 pL of water. The butanol extract was neutralized by adding 3 pL of 200 mM Tris.HC1 (pH 9.5) and evaporated in a Speedvac concentrator. The residue was treated as previously described to phosphorylate the 3'-mOnOphosphates and prepare them for chromatographic analysis (32). Chromatographic Analysis. Thirty-five microliters of the labeled solution was applied to the center of a PEI-cellulose TLC plate, 1 pL at a time with intermediate drying. Adducts were resolved by multidirectional solvent developments using the optimized chromatographic conditions described in the preceding paper (32). The solventa used were as follows: D1,l.O M sodium phosphate (pH 6.5); D2,3.0 M ammonium formate (pH 3.5); D3, 4.5 M lithium formate and 7.0 M urea (pH 3.5); D4, 0.5 M Tris.HC1,0.5 M boric acid, 0.5 M magnesium chloride, and 5.0 M urea (pH 8.0); D5, 1.7 M sodium phosphate (pH 6.0). Radioactive s p ~ @ on the chromatograms were located by screenintensified autoradiography. After detection, adduct spots were excised from the plates and their radioactivity was determined by Cerenkov counting. Blank regions adjacent to the adduct spots were also counted and the values subtracted from the adduct counts. Total nucleotides were labeled and adduct levels were calculated from relative adduct labeling (RAL) (38). Recoveries and phosphorylation efficiencies were not determined, so the calculated levels represent minimum values. The reproducibility of RAL values determined from triplicate analysis of selected individual samples was *lo%.
Results Peroxyl Radical Adduction of (+)-BP-?,&diol to Calf Thymus DNA. In the preceding paper, we described a 4-D TLC system that separates deoxynucleoside bisphosphate a d d u c t s of (+)-syn- and (-)-anti-BPDE's. Before conducting in vivo experiments, we tested t o see if the method detects (-)-anti-BPDE-generated adducts by reaction of (+)-BP-'l,&diol with enzymatically generated peroxyl radicals. (+)-BP-7,&diol was incubated with liver microsomes from uninduced Sprague-Dawley rats in t h e presence of calf thymus DNA and cumene hydroperoxide. Addition of cumene hydroperoxide to rat -liver microsomes triggers significant amounts of lipid peroxidation and peroxyl radical generation (15). After incubation at 37 O C , the reaction mixture was extracted with ethyl acetate and t h e D N A precipitated and analyzed by
Dihydrodiol Epoxide Adducts in Mouse Skin
Figure 2. Autoradiogram of the 4 D TLCmap produced following reaction of (+)-BP-7,&diol, rat liver microsomes, and cumene hydroperoxide in the presence of calf thymus DNA. One hundred nanograms of deoxynucleoside 3’,5’-bisphosphates were spotted, and the plates were developed as described (32). Autoradiography performed for 3 h a t -80 “C.
postlabeling. HPLC analysis of the organic extract revealed the presence of tetrahydrotetraol hydrolysis products derived primarily from anti-BPDE, the major product of peroxyl radical oxidation of (+)-BP-7,8-diol. Postlabeling analysis of the DNA reisolated from these incubation mixtures generated the adduct map displayed in Figure 2. The major adduct spot cochromatographedwith a standard of the bisphosphate of (-)-anti-BPDE-W-dG. Minor spots were observed at the positions of the (-)anti-BPDE-W-dA and (+)-syn-BPDE-W-dG. This experiment demonstrated that DNA could be used as a trap for (-)-anti-BPDE generated by reaction of (+)-BP-7,8-diol with biosynthetically produced peroxyl radicals. Relative Efficiency of Reaction of Calf Thymus DNA with (&)-syn-BPDE and (-)-anti-BPDE. Experiments were next conducted to determine the efficiency of reaction of DNA with (-)-anti- and (*)-syn-BPDE. Equimolar amounts of the diastereomeric dihydrodiol epoxideswere reacted with calf thymus DNA in vitro. The actual dihydrodiol epoxide content of each compound was determined by solvolysis in methanol at room temperature. This produces methyl ethers that are separable from tetrahydrotetraolsproduced by hydrolysis of BPDE’s (39). Methyl ethers can only be formed from BPDEs remaining at the time of analysis. Epoxide content was estimated to be 76% for (-)-anti-BPDE and 96% for (*)-syn-BPDE. Postlabeling analysis of DNA reacted with four different concentrationsof the dihydrodiol epoxides indicated that the extent of modification was linearly related to the amount of BPDE added (Figure 3). More binding was observed from (-)-anti-BPDE than (*)-syn-BPDE. Because of the incomplete separation of the spots from the (+)- and (-)-enantiomers of the syn-BPDE, it was not possible to determine the extent of binding for the (+)syn-BPDE alone. Thus, the extent of binding of the (-)-anti-BPDE appears to be approximately twice as great as the extent of binding from the (+)-syn-BPDE. This means our analytical method is somewhat more sensitive to peroxyl radical dependent oxidation than to cytochrome P-450 dependent oxidation. In Vivo Studies of DNA Adduction from (+)-, (-)-, and (*)-BP-7,8-diols. Acetone is commonly used as the vehicle for topical administration of polycyclic hydrocarbons to mouse skin. A control experiment was conducted in which 100 p L of acetone was applied to CD-1
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 29
Figure 3. Dependence of deoxynucleoside 3’,5’-bisphosphate adduct levels on concentration of BPDEs reacted with calf thymus DNA. Reaction of (f)-syn-BPDE and (-)-anti-BPDE with calf thymus DNA and 32P-postlabelinganalysis was carried out as described under Materials and Methods. Spots coeluting with standards of deoxynucleoside 3’,5’-bisphosphate adducts were scraped from the TLC plates, quantitated by Cerenkov counting, and converted to RAL by comparison to the level of unmodified deoxynucleoside 3’,5’-bisphosphates. (m) (-)-anti adducts; (0) (h)-synadducts. mice and epidermal DNA isolated and analyzed for adduct
content. Following postlabeling analysis, no spots were detectable in the autoradiograms of the 4-D TLC plates even after prolonged exposure of the TLC plate to the film. As stated earlier, (+)-BP-7,8-diol is metabolized stereoselectively to (+)-syn-BPDE by the cytochrome P-450 pathway and to (-)-anti-BPDE by the peroxyl radical pathway. (-)-BP-7,8-diol, on the other hand, is metabolized by both systems to (+)-anti-BPDE (23,25,26). An experiment was conducted in which CD-1 mice (2 mice/ group) were treated topically with 200 nmol each of (+)-, (-)-, or (&)-BP-7,&diol. The mice were sacrificed after 3 and 6 h, and the epidermal DNA was extracted and subjected to postlabeling. Figure 4 shows the fingerprint of adducts obtained with 3-h samples of (+)-, (-)-, and (*)-BP-7,8-diol. The 6-h samples showed the same pattern of adducts but with greater intensity, indicatingthat longer exposures led to larger amounts of the same adducts. The (+)-BP-7,8-diol-treated DNA sample showed a single (+)-syn-BPDE-W-dG adduct, and the (-)-BP-7,8-dioltreated mouse skin showed a major (+)-anti-BPDEW-dG adduct and a minor unknown adduct. The unknown adduct was also seen in the reaction of (+)-anti-BPDE with calf thymus DNA in vitro (data not shown). No (-)anti-BPDE-DNA adducts were detected in the (+)-BP7,8-diol-treated mouse skin. Mice treated with (*)-BP7,8-diol showed both the (+)-syn- and (+)-anti-BPDEW-dG adducts and the minor (+)-anti-BPDE-DNA adduct of unknown structure. Again, no (-)-anti-BPDEDNA adducts were detected. Our inability to detect binding from (-)-anti-BPDE was not due to the inability of (-)-anti-BPDE to form adducts in mouse skin. Topical administration of 100 nmol of (-)-anti-BPDE to shaven CD-1 mice generated a postlabeling map with spots cochromatographing with (-)-anti-BPDE-W-dG and (-)anti-BPDE-NG-dA (data not shown). A doseresponse was performed with (+)-BP-7,&diol in amounts of 1-200 nmol. The autoradiographic profiles exhibited only a single (+)-syn-BPDE-DNA adduct, the intensity of which increased with increasing dose of (+)-BP-7,&diol. Figure 5 displays the dependence of adduct level on the amount of (+)-BP-7,&diol applied. The formation of (+)-syn-BPDE-W-dG adducts was linear with dose up to to 100 nmol. A departure from linearity
Reddy et al.
30 Chem. Res. Toxicol., Vol. 5, No.1, 1992
0
100
200
(+)-BP-7,8-diol (nmol)
Figure 5. Dependence of the level of the (+)-syn-BPD&W-dG adduct on the dose of (+)-BP-7,&diol applied to mouse skin. Different doses of (+)-BP-7,8diol were applied in 100 p L of acetone to mouse skin. After 3 h, the animals were sacrificed, DNA was isolated, and the level of (+)-syn-BPDE-W-dG was determined.
0
10
20
30
l i m e (h) Time course of (+)-syn-BPDE-W-dG adduct forFigure 6. mation following administration of (+)-BP-’I,&diol. (+)-BP7,8-diol(200 nmol) was applied to mouse skin, and the animals were sacrificed at various times after administration. DNA was isolated and the level of (+)-syn-BPDE-W-dG determined.
Figure 4. Four-dimensional TLC maps recorded following administration of (+)-,(-)-, and (i)-BP-7,&diolto mouse skin. Two hundred nanomoles of BP-7,Hiol was applied topidly to mouse skin. After 3 h, the animals were sacrificed, and skin DNA was prepared and analyzed for deoxynucleoside 3’,5’-bisphosphates. The same amount of DNA equivalent (10 pg) was applied to each plate. Autoradiography was for 3 h at -80 “C. Plates: top, (+)-BP-’l,&diol;middle, (i)-BP-7,Hiol; bottom, (-)-BP-7,8diol. The arrow in the bottom plate marks the elution position of the (+)-syn-BPDE-W-dG adduct.
was observed at the highest dose (200 nmol) administered. The time course of adduction by (+)-BP-7,8-diol was also determined. (+)-BP-’l,&diol(200nmol/mouse) was applied topically, and the mice were sacrificed 0, 1,3,6, and 24 h later. DNA was extracted and subjected to postlabeling. Autoradiograms at each time point exhibited only a single (+)-syn-BPDE-P-dG adduct. An approximately linear dependence of (+)-syn-BPDEP-dG adduct levels on time was observed (Figure 6). No adducts were detected in DNA samples in which the animals were sacrificed immediately after applying (+)-BP-7,8-diol. Previous investigations from our laboratory demonstrated that topical administration of @-NFto CD-1 mice increases the metabolism of (+)-BP-7,8-diol to (+)-synBPDE (40). The increased formation of (+)-syn-BPDE
could be detected as increased metabolism to tetrahydrotetraol hydrolysis products or as increased levels of deoxynucleosideadducts. An experiment was conducted in which 8-NF was administered topically to CD-1 mice 24 h before administration of (+)-BP-7,8dioL The animals were sacrificed 3 h later, and epidermal DNA was subjected to postlabeling analysis. The pattern of adducts was the same as observed in uninduced animals, but the level of the (+)-syn-BPDE-W-dG adduct was 1.3 times higher in the /3-NF-treated animals. In the experiments described above, a DNA purification scheme was employed that involved two rounds of ribonuclease A and proteinase K treatment before nuclease digestion for postlabeling analysis. The possibility exists that small amounts of adducts were lost during the repeated digestion, washing, and precipitation of DNA. Since the levels of (-)-anti-BPDE-W-dG were very low, losses during handling would be more serious for this adduct than for the (+)-syn-BPDE-W-dG adduct. To investigate thispossibility,a DNA isolation and purification procedure was utilized that involved a single cycle of ribonuclease and proteinase treatment and fewer precipitations and washes. Following topical administration of 200 nmol of (+)-BP-7,8-diol, postlabeling analysis of epidermal DNA revealed the presence of a faint spot cochromatographing with (-)-anti-BPDE-W-dG along with
Dihydrodiol Epoxide Adducts in Mouse Skin
Figure 7. Four-dimensional TLC maps recorded following administration of (+)-BP-7,8diol to mouse skin. Two hundred nanomoles of (+)-BP-’I,Sdiolwas applied topically to mouse skin. After 3 h, the animals were sacrificed, and skin DNA was prepared and analyzed for deoxynucleoside 3’,5’-bisphosphates. An abbreviated procedure for DNA isolation and digestion was employed that is described under Materials and Methods. The major spot on the 4-D TLC map coelutes with (+)-syn-BPDE-W-dG. The minor spot indicated by the arrow coelutes with (-)-antiBPDE-W-dG.
the major spot of (+)-syn-BPDE-W-dG (Figure 7). The ratio of the (-)-anti-BPDE/ (+)-syn-BPDE was approximately 0.2 in this and subsequent experiments. Thus, it appears that a low level of (-)-anti-BPDE-W-dG is produced in mouse skin in vivo. However, its levels are minor when compared to the levels from the (+)-syn-BPDEW-dG.
Discussion We have applied the chromatographic system described in the preceding paper to the 32P-postlabelinganalysis of DNA adducts produced from BP-7,8-diol enantiomers in mouse skin in vivo. Using two separate methods for DNA isolation and purification, the major adduct detected in epidermal DNA following topical administration of (+)BP-7,8-diol was derived from (+)-syn-BPDE. The m a imum amount of adducts derived from (-)-anti-BPDE corresponded to approximately 20% of the adduct derived from (+)-syn-BPDE. The preponderance of the (+)-synBPDE adduct was observed at doses of (+)-BP-7,&diolof 1-200 nmol and at times from 1to 24 h. Analysis of epidermal DNA adducts following administration of (-)BP-7,8-diol revealed the presence of a single major adduct derived from (+)-anti-BPDE. Adduct analysis of (&)BP-7,g-diol-treated skin indicated production of (+)anti-BPDE and (+)-syn-BPDE but, at best, a trace amount of (-)-anti-BPDE. These observations are consistent with metabolism of the BP-7,&diol enantiomersby a cytochrome P-450 enzyme in mouse epidermis. The absence of (-)-anti-BPDEDNA adducts is not due to differential reactivity of the BPDE diastereomers with DNA. In vitro reaction of equal amounts of (-)-anti-BPDE and (i)-syn-BPDE with calf thymus DNA led to comparable levels of adduct formation. This was expected on the basis of previous studies of DNA binding by BPDE diastereomers as assayed by HPLC (41). Furthermore, studies of the extent of DNA binding of (&)-anti-BPDE and (&)-syn-BPDEto whole epidermisand basal cell layers of Sencar mice indicate no dramatic differences between the diastereomers from 3 to 12 h after treatment (37). This suggests that the pattern of DNA adducts detected in the present experiments is not due to preferential adduction
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 31 by (+)-syn-BPDE as opposed to (-)-anti-BPDE in vivo. Likewise, the rather slow time course of BP and DMBA adduct removal in mouse skin makes it unlikely that differential DNA repair exerts a major influence on individual adduct levels during the 3-24-h exposure we employed (42). Earlier work from several laboratories suggested a major role for peroxyl radicals in the epoxidation of BP-7,8-diol in mouse skin homogenates, freshly isolated mouse epidermal cells, and mouse skin in vivo (30,31,43). These studies exploited the same stereochemical approach outlined here except tetrahydrotetraol hydrolysis products of the dihydrodiol epoxides were quantitated instead of DNA adducts. Pruess-Schwartz et al. reported that the major tetrahydrotetraols produced on incubation of mouse skin homogenates with (+)-BP-7,8-diol were derived from anti-BPDE (31). Formation of these products was independent of NADPH and inhibited by phenolic antioxidants but was not inhibited by heat inactivation of the homogenates or inclusion of cytochrome P-450 inhibitors in the incubation mixtures. In contrast, incubation of (+)-BP-7,8-diolwith epidermal homogenates from p-naphthoflavone-pretreated animals produced mainly tetrahydrotetraols derived from syn-BPDE (31). Formation of this epoxide required NADPH, was abolished by heat inactivation of the homogenate, and was inhibited by CYnaphthoflavone with an Im = 2 pM but was not inhibited by phenolic antioxidants. Similar results were obtained by Eling et al. in incubations of (+)-BP-7,8-diol with freshly isolated mouse epidermal cells although the NADPH dependence was not explored (30). These experiments clearly establish that peroxyl radicals are responsible for BP-7,&diol epoxidation in homogenates and cells from untreated animals. The source of the peroxyl radicals was not established, but the time course for tetrahydrotetraol formation correlated with the time course of lipid peroxidation (30). In contrast, cytochrome P-450 appears responsible for epoxidation by homogenates and cells from 8-naphthoflavone-inducedanimals. One can reconcile the results of the experiments with homogenates and cells with the present in vivo results by the observation that disruption of epidermal cells by the procedures used for tissue manipulation induces oxidative stress that can lead to lipid peroxidation. This is obviously the case for epidermal homogenates because of dilution of stores of endogenous antioxidants. However, it is also true for freshly isolated epidermal cells. After isolation, the cells release significant amounts of prostaglandins, which is an indication of oxidative stress.2 In addition, it has recently been found that the trypsin flotation procedure commonly used for epidermal cell isolation causes the cells to release greater than 98% of their glutathione into the medium, which greatly enhances their susceptibility to lipid per~xidation.~ Although one might surmise that peroxyl radical dependent epoxidation of BP-7,8-diol is exclusively the result of cell disruption, Melikian et al. have demonstrated its operation in mouse skin in vivo (43). They applied (+)BP-7,8-diol to shaven female Cr1:CD-I mice and after different times sacrificed the animals and extracted the skin with ethyl acetate. Tetrahydrotetraol metabolites were then analyzed by HPLC. The major tetrahydmtetraol was derived from (-)-anti-BPDE (43). In fact, the HPLC profiles were remarkably similar to those derived from the incubation of (+)-BP-7,8-diol with freshly isolated epidermal cells (30). Furthermore, the formation T. Eling, personal communication. J. Reiners, personal communication.
32 Chem. Res. Toxicol., Vol. 5, No. 1, 1992 of (-)-anti-BPDE in vivo was inhibited by catechol, a phenolic compound that is an efficient radical scavenger (43).
The deoxynucleoside adducts produced in mouse skin in vivo from (+)-BP-7,8-diolwere analyzed by HPLC by both Pruess-Schwartz et al. and Melikian et al. (31,43). Both groups detected an adduct that coeluted with a standard produced by reaction of (-)-anti-BPDE with calf thymus DNA. However, both groups also detected an adduct that coeluted with a standard produced by reaction of (+)-syn-BPDE with DNA. In the Pruess-Schwartz et al. study, the amount of the syn-BPDE adduct was approximately the same as that of the anti-BPDE adduct (31). In the Melikian et al. study, the syn-BPDE adduct contained approximately 6-fold more radioactivity than the anti-BPDE adduct. However, acid hydrolysis of the zone eluting with the syn-BPDE adduct produced mainly a compound that coeluted with BP-7,gquinone (43). This suggests that a substantial portion of the radioactivity eluting with the syn-BPDE adduct was due to another compound or adduct. Whether this unknown is a deoxynucleoside adduct is uncertain. The current study employed the same protocols for animal treatment and DNA isolation as the study by Pruess-Schwartz et al. (31). However, the analytical method for adduct determination differed. It is possible that differencesin the experimental procedures used for stable isotope or postlabeling analysis contributed to the quantitative differences in the relative amounts of (-)-anti and (+)-syn adducts. Alternatively, radioactive compounds that coelute with adducts on HPLC or TLC may have caused an overestimation of adduct amounts. The finding in the present study that the major DNA adduct produced in mouse skin from (+)-BP-7,8-diol is derived from (+)-syn-BPDE is analogous to findings with (3S,4S)-DMBA-3,4dioladministered to cultured NIH fetal mouse cells (44). 32P-Postlabelinganalysis of the deoxynucleoside 3’,5’-bisphosphate adducts 24 h after treatment indicated two major spots that were identified as synDMBA-dihydrodiol epoxide adducts to deoxyadenosine and deoxyguanosine. If adducts derived from antiDMBA-dihydrodiol epoxide were produced, they were present in only trace amounts. The stereochemistry of oxidation of (3S,4S)-DMBA-3,4-diol is similar to that of BP-7,8-diol. It is oxygenated by cytochrome P-450 to form syn-DMBA-dihydrodiol epoxide along with significant amounts of non-dihydrodiol epoxide products, and it reacts with peroxyl radicals to produce mainly anti-DMBA-dihydrodiol epoxide (45,46). The reactivity of DMBA-3,4diol to peroxyl radicals is approximately 3-fold lower than the reactivity of BP-7,8-diol (46). Thus, the probability that DMBA-3,Cdiol reacts with peroxyl radicals in mouse skin or cultured murine cells is significantly lower than for BP-7,8-diol. Postlabeling analysis of the deoxynucleoside bisphosphate adducts produced in mouse skin following topical administration of BP and (+)-BP-7,8-diolas well as analysis of the adducts produced in cultured fetal murine cells with DMBA and (3S,4S)-DMBA-3,4diolindicate a major role for cytochrome P-450 in both oxygenation steps in the metabolic activation of these polycyclic hydrocarbons to their ultimate carcinogenic forms. In the case of BP, the contribution of peroxyl radicals to activation as estimated by postlabeling analysis appears minimal (32), and with BP-7,8-diol the extent of involvement appears to be no greater than 20%. Although cytochromes P-450 appear to be major contributors to BP activation in untreated mouse skin,the low levels of DNA
Reddy et al. adduction via the peroxyl radical dependent pathway may provide an opportunity to test if pharmacological agents increase oxygen radical production in vivo by determining the extent of formation of DNA adducts from BP-7,8-diol by the techniques described.
Acknowledgment. This work was supported by a research grant (CA47479) and training grants (CAN531 and ES05516) from the National Institutes of Health. We are grateful to Karen Canella and Anthony Dipple for authentic standards of (-)-anti-BPDE-W-dG and (-)-antiBPDE-M-dA.
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