Chem. Res. Toxicol. 1992,5, 242-241
242
Metabolism and DNA Binding of 5,6-Dimethylchrysene in Mouse Skin Bijaya Misra, Shantu Amin,* and Stephen
S. Hecht
Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received June 20, 1991
5,6-Dimethylchrysene (5,6-diMeC) is a weaker tumor initiator on mouse skin than 5methylchrysene (5-MeC). To investigate the reasons for the unexpectedly low activity of 5,6diMeC, we have studied its metabolism and DNA binding in mouse skin,particularly with respect to metabolic activation via its anti-1,2-diol3,4-epoxide. The metabolism of 5,6-diMeC was first examined with liver 9OOOg supernatant from Aroclor 1254 pretreated rats. Three major metabolites were identified as 1- or 7-hydroxy-5-(hydroxymethyl)-6-MeC, 1,2-dihydroxy-1,2-dihydro-5,6-diMeC (5,6-diMeC-1,2-diol), and l-hydroxy-5,6-diMeC. The formation of 5,6-diMeC-1,2-diol was then assessed in mouse epidermis, following topical application of [3H]5,6diMeC. Levels of 5,6-diMeC-1,2-diolin epidermis exceeded those of 5-MeC-1,Bdiol formed from 5-MeC under similar conditions. The binding of [3H]5,6-diMeCand that of [3H]5-MeCto mouse epidermal DNA were then compared. 5,6-DiMeC-deoxyribonucleosideadducts were prepared as markers by reaction of anti- and syn-5,6-diMeC-1,2-diol3,4-epoxide with calf thymus DNA. HPLC analysis of enzymatic hydrolysates of mouse epidermal DNA, isolated 18 h after topical treatment with [3H]5,6-diMeC or [3H]5-MeC, demonstrated the formation from [3H]5,6-diMeC of two major adducts produced by reaction of its anti-1,2-diol3,4-epoxidewith deoxyguanosine and deoxyadenosine, respectively, while the major adduct formed from [3H]5-MeC resulted from reaction with deoxyguanosine, in agreement with previous results. Total DNA binding of [3H]5-MeCas well as formation of deoxyguanosine adducts exceeded that of [3H]5,6-diMeCby 3-4-fold. The results of this study demonstrate that the metabolic activation pathway 5,6-diMeC 5,6-diMeC-1,2-diol- anti-5,6-diMeC-1,2-diol3,4-epoxide exists in mouse skin, as in the case of 5-MeC. The results suggest however that the conversion of 5,6-diMeC-1,2-diol to anti-5,6diMeC-1,2-diol 3,4-epoxide in mouse skin does not occur as efficiently as the corresponding conversion of 5MeC-1,2-diol to its anti-1,2-diol3,4-epoxide.The results are discussed with respect to a structurally similar nonplanar compound, benzo[c] phenanthrene, which also has weak tumorigenicity on mouse skin, apparently resulting in part from inefficient metabolism of its 3,4-diol to bay region diol epoxides.
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reversephase Vydac 201TP104 10-pm column (Separations Group, Hesperia, CA) eluted with a linear 20-min gradient from 30% The first paper in this series and two previous studies MeOH to 50% MeOH in H20, followed by a linear 1-h gradient showed that 5,6-diMeC1 is a weaker tumor initiator in to 100% MeOH in H 2 0 a t a flow rate of 1 mL/min. System 2 mouse skin than 5-MeC (1-3).The metabolic activation was the same as system 1but with a semipreparative reversephase of 5-MeC in mouse skin proceeds by the pathway: 5-MeC Vydac 201TP1010 10-pm column and a flow rate of 3 mL/min. System 3 was a 4-mm X 25-cm normal-phase Lichrosorb Si 60 5-MeC-1(R),2 @)-diol 5-MeC-1(R),%(S)-diol3 (5'),45-pm column (EM Reagents, Cincinnati, OH) eluted with 85:15 (R)-epoxide DNA adducts. In this study, we examined hexane/THF at a flow rate of 1mL/min. System 4 was a 4.6" the metabolism and DNA binding of 5,6-diMeC in vitro X 25-cm analytical c18 reverse-phase Beckman Ultrasphere 5-pm and in mouse skin to determine the extent to which its column (Rainin Instruments, Inc., Woburn, MA) eluted with metabolic activation occurs by the analogous pathway, H20/MeOH linear gradients: 15% MeOH to 45% MeOH in 25 illustrated in Figure 1. min, then 45% MeOH to 55% MeOH in 5 min, followed by 55% MeOH to 70% MeOH in 25 min, and finally to 100% MeOH in Experlmental SectIon 5 min. System 5 was a 4.6-mm X 25-cm Pirkle type 1-A 5-pm Apparatus. HPLC was carried out with a Waters Associates column [y-aminopropyl silanized silica to which (R)-N-(3,5-diModel ALC/GPC-204 high-speed liquid chromatograph equipped nitrobenzoy1)phenylglycineis ionically bonded; Regis Chemical with a Model 6000A solvent delivery system, a Model 660 solvent Co., Morton Grove, IL] eluted with 15% 2:l EtOH/acetonitrile programmer, a Model U6K septumless injector, and a Model and 85% hexane a t 2 mL/min. LC-25 UV/visible detector or a Flo-one/Beta radioflow detector UV spectra were run in MeOH on a Hewlett-Packard 8452A (Radiomatic Instruments, Tampa, FL). Systems 1-4 were used diode array spectrophotometer coupled with an IBM X T personal for the analyses. System 1was a 4.6-mm X 25-cm analytical c18 computer. MS were obtained with a Hewlett-Packard Model 5988A instrument. Proton NMR spectra were recorded on a Bruker AM 360 WB spectrometer. * Abbreviations: 5,6-diMeC, 5,6-dimethylchrysene; 5-MeC, 5Chemicals. 5,BDiMeC was prepared as described previously methylchrysene; 5-MeC-l(R),z(R)-diol, l(R),2(R)-dihydroxy-l,2-di(I). [3H]5,6-DiMeCwas synthesized via catalytic halogen-tritium hydro-5-methylchrysene; 5-MeC-l(R),g(S)-diolB(S),a(R)-epoxide,1which was prepared (R),2(s)-dihydroxy-3(s),4(R)-epoxy-1,2,3,4~trahydro-5-methylc~ne; exchange of 12-bromo-5,6-dimethylchrysene 5-CH20H-6-MeC, 5-(hydroxymethyl)-6-methylchrysene; 1-OH-5as follows. A suspension of 5,GdiMeC (512 mg, 2 "01) in glacial CH20H-6-MeC,l-hydroxy-5-(hydroxymethyl)-6-methylchrysene; 5,6-diacetic acid (5 mL) was heated under reflux for 10 min. A solution MeC-l,P-diol, 1,2-dihydro-l,2-dihydroxy-5,6-dimethylchrysene; 1-OHof bromine (160 mg, 2 "01) in 2 mL of acetic acid was then added 5,6-diMeC, l-hydroxy-5,6-dimethylchrysene;anti-5,6-diMeC-1,2-diol 3,4-epoxide;(+)-anti-l,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-5,6-di- dropwise, and heating under reflux was continued for 1 h. The reaction mixture, upon cooling, afforded the product as a colorless methylchrysene; dA, deoxyadenosine; dG, deoxyguanosine; THF, tetracrystalline solid: mp 219-220 "C (45 mg); 'H NMR (CDC13) 6 hydrofuran; BcPh, benzo[c]phenanthrene.
Introductlon
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0893-228x/92/2105-0242$03.00/0
0 1992 American Chemical Society
Dimethylchrysene
Chem. Res. Toxicol., Vol. 5, No. 2,1992 243
Diol Epoxides
CH3
5,6-diMeC
5,6-diMeC-l,2-dIoi
enrCS,6-diMeC-l,2-dioi-3,4-epoxide
Figure 1. Metabolic activation pathway of 5,6-diMeC in mouse epidermis. 8.95 (8, 1H, Hill, 8.6-8.2 (m, 4 H, H1,4,7,10)77.65 (m, 4 H, H2,3,8,9), 3.05 (8, 3 H, CH,), 2.8 (s,3 H, CH,); MS m / z (relative intensity) 336,334 (M+,84),239 (100). The tritium exchange was performed by Amersham Corp. (Arlington Heights, IL). [3H]5-MeC was synthesized from 5-MeC via hydrogen-tritium exchange (Amersham Corp.). [,H]5,6-diMeC and [,H]5-MeC were purified by HPLC using system 2, with a gradient of 70-100% MeOH in H2O in 30 min. Their radiochemical purities were >95% on the basis of analysis by HPLC using the Vydac 201TP104 10-rm column with a linear 30-min gradient from 50% to 100% MeOH in H20. 6-CHzOH-5-MeC and 5-CHzOH-6-MeC were prepared as follows. A suspension of N-bromosuccinimide (10 mg, 0.055 mmol), 5,6-diMeC (15 mg, 0.058 mmol), and benzoyl peroxide (1 mg) in 10 mL of CCl, was irradiated with a UV lamp (250 W, 280-400 nm) for 45 min. The succinimide was filtered a t room temperature and washed with CC14(2 X 5 mL). The filtrate was concentrated to give a solid which was chromatographed on silica gel. Elution with hexane/CHzC12(3070) gave a mixture of 5(bromomethyl)-6-methylchrysene and 6-(bromomethyl)-5methylchrysene. Further elution with hexane/CHzC12(5050) gave pure &(bromomethyl)-bmethylchrysene:'H NMFt (CDCl,) 6 3.20 (8, 3 H, CHJ, 5.45 (8, 2 H, CH2), 7.6-7.75 (m, 4 H, Hz,3,S,g),7.98 (d, 1H, H12, J11,12 = 8.8 Hz), 8.01 (m, 1H, Hi), 8.37 (m, 1 H, H7), 8.6-8.65 (m, 1H, H4),8.68 (d, 1H, Hl1, J1l,lz = 9.05 Hz), 8.75-8.8 (m, 1 H, Hlo). The structural assignment was confirmed by the NOESY spectrum which showed interaction between 6-CH2Br (5.45 ppm) and H7 (8.37 ppm). A mixture of NaOAc (100 mg) and 6-(bromomethyl)-5methylchrysene (7 mg, 0.02 mmol) in 2 mL of HOAc was heated under reflux for 18 h. The reaction mixture was poured into ice-cold H20, and the pH was adjusted to 7. The mixture was extracted with CHzC12(3 X 30 mL), and the extracts were washed with HzO and dried (MgSO,). The solvent was removed to give crude 6(acetoxymethyl)-5-methylchrysenewhich was used without further purification. To a solution of the crude acetate in 20 mL of THF/H20 (3:l) was added NaOH (100 mg, 2.5 "01). The reaction mixture was stirred at 60 "C for 8 h. It was poured into H 2 0 and extracted with EtOAc (2 X 25 mL), and the EtOAc extracts were dried (MgSO,). The solvent was removed to give crude 6-CHzOH-5MeC, which was purified by chromatography on silica gel (1 g) with elution by hexane/CH2Clz (5050): lH NMR (CDC13)6 3.2 (e, 3 H, CH3),5.4 (8, 2 H, CH2),7.67-7.8 (m, 4 H, H2,3,8,9),8.0 (d, 1H, H12,Jil,lz = 8.74 Hz),8.08-8.15 (m, 1H, Hi), 8.43-8.47 (m, 1 H, H7),8.68-8.72 (m, 1H, Hlo), 8.75 (d, 1 H, Hii! J1l,lz = 9.02 Hz), 8.84-8.88 (m, 1 H, H4). The structural assignment was confirmed by the NOESY spectrum which showed interaction between 6-CHzOH (5.4 ppm) and H7 (8.43-8.47 ppm). MS m / z (relative intensity) 272 (M+, 82.5), 239 (100). A mixture of 6-(bromomethyl)-5-methylchryseneand 5-(bromomethyl)-6-methylchrysene(6.0 mg) was converted to the corresponding alcohols as described above and analyzed by HPLC system 1. Two major peaks eluted at 57.6 and 59.6 min. The 57.6-min peak was identified as 6-CHzOH-5-MeCby comparison to the standard. The peak eluting at 59.6 min was 5-CH20H-6MeC: lH NMR 6 (CDClJ 3.01 (s, 3 H, CH3),5.30 ( 8 , 2 H, CHz), 7.60-7.72 (m, 4 H, H2,3,8,g),7.94 (d, 1H, H12, J1l,lz = 8.90 Hz), 7.98 (m, 1H, Hi), 8.27 (m, 1H, H7), 8.66 (d, 1H, Hl1, J11,12 = 9.05 Hz), 8.75-8.8 (m, 1 H, Hioh 9.12 (d, 1 H, H4, J3,4= 8.20 Hz). Other Materials. DNase I from bovine pancreas (EC 3.1.21.1), RNase A type 111-A from bovine pancreas (EC 3.1.27.5), RNase Ti grade IV from Aspergillus oryzae (EC 3.1.27.3), phosphodiesterase I type I1 from Crotalus adamanteus venom (EC 3.1.4.1), alkaline phosphatase type VII-NT from bovine intestinal mucosa (EC 3.1.3.1), protease type XIA from Wtarchium album, NADP+,
glucose 6-phosphate, and phenol (biomolecular grade) were obtained from Sigma Chemical Co., St. Louis, MO. The phenol was mixed with 0.1% &hydroxyquinoline, and the mixture was washed twice with 1.0 M Wi-HC1 (pH 8.0) and then with 0.1 M Tris-HCl (pH 8.0) until the pH of the aqueous washings was 7.4. Aroclor 1254 was procured from Analabs, Inc., Hamden, CT. Animal Treatments for in Vitro Metabolism. Male F344 rats (190-230 g) were obtained from Charles River Breeding Laboratory, Kingston, NY. Rats were treated 5 days prior to s a d i c e with an ip injection of Aroclor 1254 in com oil, 500 mg/kg. Metabolism in Vitro. A mixture of 3.2 mL of 0.4 M MgC12, 3.2 mL of 1.65 M KCl, 0.8 mL of 1.0 M D-glUCOSe 6-phosphate, 6.4 mL of 0.1 M NADP+ in 66.4 mL of phosphate buffer (pH 7.0), and 80 mL of 9OOOg supernatant from the livers of Aroclor 1254 pretreated rats was prepared. It was slowly added to a solution of 20 mg of 5,B-diMeC in 5 mL of DMSO. The resulting mixture was divided into three portions which were incubated a t 37 "C with shaking for 20 min, 1h, or 2 h. The reactions were quenched by adding an equal volume of acetone to each, and the mixtures were extracted three times with 80 mL of EtOAc. The EtOAc layers were combined and dried (MgSO,), and the solvent was removed under reduced pressure. The residue was dissolved in 2 mL of MeOH and was analyzed by HPLC, using systems 1and 2. The quantities of metabolites of 5,6-diMeC isolated were sufficient for spectral characterization. Metabolism in Vivo. Twenty female CD-1 mice (7-8 weeks of age, Charles River Breeding Laboratories, Kingston, NY)were divided into 4 groups of 5 mice each. They were shaved, and 24 h later, 0.15 mL of an acetone solution of [,H]5,6-diMeC (1.49 Ci/mmol, 0.07 pmol per mouse) was applied to the shaved back of each mouse. Groups of mice were sacrificed at 20 min, 1 h, 2 h, and 4 h. The treated areas of the skin were removed, and epidermal layers were isolated and ground in liquid N2,and then suspended in 20 mL of sodium potassium phosphate buffer (pH 7.4) containing 8 mM Na2HP04,1.5 mM KHzP04,145 mM NaCl, and 2.7 mM KC1. The suspension was homogenized, frozen, and then thawed. To the resulting suspension, 20 mL of acetone was added and the mixture was extracted four times with 40 mL of EtOAc. The organic soluble metabolites were analyzed by HPLC, using systems 1 and 3. DNA Binding in Vitro. The syntheses of syn- and anti5,6-diMeC-1,2-diol3,4epoxide are described in the following paper (8). Their reactions with calf thymus DNA were carried out under conditions similar to those described previously (4). One milliliter of a 1mg/mL solution of the diol epoxide in dry THF was added to 10 mL of a 1mg/mL solution of calf thymus DNA in 10 mM Tris-HC1 buffer, pH 7.0. The mixture was incubated a t 37 "C overnight. Tetraols were extracted with 3 X 10 mL of EtOAc. The aqueous layer was cooled to 0 "C, and the DNA was precipitated with cold (-20 "C) EtOH. It was spooled out and washed with 10 mL of 70% EtOH, 10 mL of acetone, and 10 mL of EhO. It was then dried under a stream of N2 and hydrolyzed to deoxyribonucleosides, as follows. One milligram of modified DNA in 1.25 mL of 10 mM Tris-HC1 and 10 mM MgC12, pH 7.0, was incubated overnight at 37 "C with 300 Kunitz units of DNase I, followed by 64-h incubation with 0.5 unit of phosphodiesterase I, type 11, in 1.25 mL of 0.2 M Tris at 37 "C. The ratio of phosphodiesterase I to DNA was chosen to ensure complete hydrolysis of dA adducts (5). The resulting digest was treated with 200 units of alkaline phosphatase for 18 h a t 37 "C. The hydrolysates were analyzed by HPLC system 4. Spectral data were obtained by combining hydrolysates from three reactions. DNA Binding in Vivo. The backs of mice were shaved 24 h prior to the application of the hydrocarbons. Groups of 4 mice were topically treated with acetone solutions (0.15 mL) of 820
244 Chem. Res. Toxicol., Vol.5, No.2, 1992
HI 1-OH-5-
CH20H6-MeCa8*
Table I. Spectral Properties of 5,6-DiMeC Metabolites (A) Proton NMR [Chemical Shifts,6; Coupling Constants, J (Hz)] HB and H2 H3 H4 HI H9 H1o H11 Hi2 CH3 CHZOH 7.10. d 7.43. dd 8.85, d 8.32, dd 7.74, m 8.90, dd 8.72, d 8.41, d 3.05 5.22 J 2 , 3 = 7.69 J 2 , 3 = 7.55 J 3 , 4 = 8.64 J 7 , 8 = 7.37 J g , l o = 7.27 J11,12 = 9.31 Jl1,12 = 9.31 53.4 = 8.45 57.9 = 2.34 J8,Jo= 2.10
5,6-diMeC- 4.65, m 4.55, m l,2-diolc 1-OH-5,6diMeCa
Misra et al.
6.95, d 52.3 7.52
6.12, dd 7.00, dd 8.12, m = 10.20 J3,4 = 10.46 J 2 , 4 = 2.60 52.3 = 2.57
7.60, m
7.40, m
7.65, m
8.70, d
8.75, m
J3,d
8.20, m
J11,12
8.75, m
8.46
=
7.91, d
2.65
8.84 2.75
J11,12
8.65, d 8.32, d 2.73 = 9.21 J11,12 = 9.23 3.01
J11,12
(B)MS and UV m / z (relative intensity)
(MeOH)
,A,
1-OH-5-CH20H-6-MeC 288 (loo), 271 (47.7), 255 (68.8),239 (39.6), 226 (44.0) 274, 310
5,6-diMeC-1,2-diol l-OH-5,6-diMeC
290 (10.2), 272 (50), 244 (5) 272 (loo),257 (38.2),239 (20.6), 226 (le), 215 (17.6)
254 (sh), 267 (sh), 275, 291 (sh), 310 (sh), 322, 334 (sh) 274, 312
In CDCIS. *Data are also consistent with 7-OH-5-CH20H-6-MeC; assignments: H, nmol of [3H]5-MeCor [3H]5,6-diMeC(specific activity 0.41 Ci/mmol) per mouse. The animals were sacrificed after 18 h, the treated areas of the skin were removed, and the epidermal layer was isolated and ground in liquid N2. The DNA from the epidermis was isolated as follows (6). The frozen tissue was thawed in 20 mL of 1%sodium lauryl sulfate/l mM EDTA and homogenized, and the homogenate was incubated with protease type XIA (500 pg/mL). After the addition of 1mL of 1M Tris-HC1 (pH 7.4), the homogenate was successivelyextracted with 1volume each of phenol, a 1:l mixture of phenol/Sevag (chloroform/isoamyl alcohol, 241), and Sevag alone. After addition of 0.1 volume of 5 M NaC1, DNA was precipitated by addition of 2 volumes of cold EtOH. The DNA was dissolved in 50 mM Tris buffer, pH 7.4 (about 1 mg of DNA/mL). Residual RNA was removed by incubation with a mixture of RNase TI (50 units/mL) and RNase A (100 pg/mL) for 30 min at 37 "C. After the extraction of this solution with Sevag, DNA was recovered and was redissolved in 2 mL of 50 mM Tris, pH 7.4. Ita concentration (600-800 pg/ mouse) was estimated spectrophotometricallyusing 20 Azso units/mg. The ratio of the absorption measured at 260280 nm was 1.85. DNA from 4 mice was hydrolyzed enzymatically to deoxyribonucleosides by incubation with enzymes as follows: DNase I (2000 Kunitz unita) and phosphodiesterase I (1.5 unita) for 54 h followed by alkaline phosphatase (500 units) for 24 h. The enzyme digest was analyzed by HPLC system 4.
Results and Dlscusslon The metabolism of 5,6-diMeC was studied with liver
0
= H7, H2 =
I
I
I
10
20
30
H8, H3 = Hg, etc.
I
I
40 50 Time (min)
In acetone-d,.
I
1
I
60
70
80
Figure 2. HPLC trace of ethyl acetate soluble metabolites of 5,6-diMeC formed upon incubation with cofactors and 9000g supernatant from the livers of rats treated with Aroclor 1254.
9OOOg supernatant from Aroclor 1254 pretreated rats to
establish ita general pattern of metabolism and to develop HPLC conditions for separating products. The HPLC separation of EtOAc-soluble metabolites of 5,6-diMeC formed in this system is illustrated in Figure 2. The three major metabolites were identified as I- or 7-OH-5CH20H-6-MeC,5,6-diMeC-1,2-diol,and l-OH-5,6-diMeC, respectively. Spectral data for these metabolites are summarized in Table I. The peak eluting at 45 min had a molecular ion of mlz 288 corresponding to the addition of two oxygens to 5,6diMeC. Its UV spectrum was similar to that of the peak eluting at 67 min which was identified by the data discussed below as l-OH-5,6-diMeC. The NMR data demonstrated the presence of a hydroxymethyl group. Therefore, this compound was a phenolic derivative of either 5-CHzOH-6-MeCor 6-CHzOH-5-MeC. To distinguish between these possibilities, 5-CH,OH-6-MeC and 6-CH20H-5-MeCwere prepared, and each was incubated with 9OOOg supernatant from Aroclor 1254 pretreated rata. Both compounds were extensively metabolized. Analysis of the metabolites by HPLC revealed the presence in each mixture of a peak with identical W spectrum and similar retention time to those of the unknown. However, only
the metabolite formed from 5-CH20H-6-MeC coeluted with the unknown. These data established the position of the hydroxymethyl group. The data summarized in Table I are fully consistent with the structural assignment as 1-OH-5-CHzOH-6-MeC. However, we cannot exclude the possibility that the metabolite is 7-OH-5-CH20H-6MeC, although this is unlikely because metabolism of 5,6-diMeC leads to l-OH-5,6-diMeC and not 7-0H-5,6diMeC as a major product (see below). The peak eluting at 52 min had a molecular ion at mlz 290 corresponding to a dihydrodiol of 5,6-diMeC. Its UV spectrum was similar to that of a methylchrysene 1,2- or 7,8-dihydrodiol (7). Its NMR spectrum and HPLC retention time were the same as those of synthetic 5,6-diMeC-1,2-diol. The syntheses of this compound as well as the corresponding diol epoxides are described in the third paper in this series (8). The peak eluting at 67 min had a molecular ion at mlz 272, consistent with a phenolic metabolite of 5,6-diMeC. Ita UV spectrum was similar to that of 1-hydroxy-5-MeC (9). Its NMR spectrum indicated that the metabolite was either 1-or 7-OH-5,6-diMeCa Standard l-OH-5,6-diMeC was prepared by dehydration of 5,6-diMeC-1,2-diol. Its
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 245
Dimethylchrysene Diol Epoxides
Table 11. Spectral Properties of anti-5,6-DiMeC-1,2-diol3,4-Epoxide-Deoxyribonucleoside Adducts" (A) Downfield Portions of the Proton NMR Spectra HPLC peakb 3 6 7
H1'
6.45, dd J 1 , , 2 p B = 5.98, 6.98 6.48, dd J1t,yu8 6.62, 6.62 6.47, dd J1r,2raB = 6.62, 6.98
H4
H7
6.19, d 8.14, m J 3 , 4 = 2.58 8.13, m 6.63, d J 3 , 4 = 1.84 6.63, d 8.13, m 5 3 4 = 2.58
HB H 9 7.61, m
HlO 8.74, m
7.60, m
8.74, m
7.60, m
8.74, m
Hll
Hl2
8.77, d 8.82 8.77, d J11,12 = 8.81 8.76, d J11,12 = 8.25 J11,12
7.64, d J11,12 = 8.82 7.66, d J l l , l Z = 8.45 7.66, d J11,12 = 8.27
dG,8 8.0, 8
dA,8
dA,2
8.26, s
8.43, s
8.26, s
8.43, s
(B)UV Spectra peak 2 ~
3 6 7 a
(MeOH) 263. 279 Ish). 308 Ish) 262; 277 ish); 307 ish) 263, 278 (sh), 306 (sh) 263, 277 (sh), 306 (sh)
absorbance ratio [ (262-263):(277-279)] 1.9 1.9 1.6 1.5
A,
Spectra were run in methanol-& *Figure 4.
NMR was identical to that of the metabolite, confirming the assignment. Synthetic 5,6-diMeC-1,2-diol was resolved on a chiral stationary-phase HPLC column (system 5) into its (S,S)and (R,R)-enantiomers which eluted a t 18 and 19 min, respectively. Their CD spectra were similar to those published for dihydrodiols of chrysene, 5-MeC, and 6-MeC. In all cases, the (R,R)-enantiomer elutes later and has a negative maximum at approximately 250 nm in its CD spectrum (10, 11). Metabolically formed 5,6-diMeC-1,2diol was analyzed in this HPLC system and was found to be almost exclusively the (R,R)-enantiomer. This is consistent with results obtained with chrysene, 5-MeC, and 6-MeC (10, 11). The in vitro metabolism results indicate that the methyl group at the 6-position inhibits oxidation at the 7- and 8-positions of 5,6-diMeC. We did not detect significant amounts of 5,6-diMeC-7,8-diolor 7-OH-5,6-diMeC in this in vitro system. These observations are consistent with our previous study of 5,lZdiMeC metabolism in which we observed that the methyl group at the 12-position inhibited metabolic oxidation at the 1-and 2-positions (7). The metabolism of 5,6-diMeC was then examined in mouse epidermis in vivo. [3H]5,6-diMeCwas synthesized and applied topically to mice which were sacrificed at various time intervals. The HPLC profile of EtOAc-soluble metabolites extracted from the epidermis 2 h after application of [3H]5,6-diMeCis illustrated in Figure 3A. The pattern was more complex than that shown in Figure 2. Since our purpose was to assess levels of formation of 5,6-diMeC-1,2-diol, we focused only on this metabolite. The peak marked with the asterisk coeluted with standard 5,6-diMeC-1,2-diol. The identities of the other peaks were not investigated. This peak was collected and reanalyzed by normal-phase HPLC (system 3). It also coeluted with standard 5,6-diMeC-1,2-diolin this system, supporting its identity (Figure 3B). Levels of 5,6-diMeC-1,2-diolin mouse epidermis at intervals after application of [3H]5,6-diMeC were as follows (pmol/mg of dry epidermis): 4.0 (20 min), 8.3 (1h), 11.3 (2 h), and 8.1 (4 h). In two previous studies carried out under essentially identical conditions, we found that the levels of 5-MeC-1,Zdiol in mouse epidermis were also maximal 2 h after topical application of [3H]5-MeC and ranged from 1to 4 pmol/mg of dry epidermis in the 20-min-4-h period following treatment (12,13).Thus, it appears that the extent of formation of 5,6-diMeC-1,2-diol in mouse epidermis exceeds that of 5-MeC-1,2-diol. The next series of experiments was designed to determine the extent to which 5,6-diMeC formed DNA adducts in mouse skin by the pathway illustrated in Figure 1. These studies required markers of potential 5,6-diMeC1,2-diol 3,4-epoxide-deoxyribonucleoside adducts.
fA
5,B-diMeC
I ~
2.5
0
20
40
60
Time (min)
Figure 3. HPLC analysis of EtOAc-soluble metabolites of [3H]5,6-diMeC formed in mouse epidermis in vivo. (A) Reversephaae analysis (system 1);the peak marked with the asterisk coeluted with standard 5,6-diMeC-1,2-diol. (B)Normal-phase analysis of the collected 5,6-diMeC-1,2-diol (system 3).
Therefore, anti- and syn-5,6-diMeC-1,2-diol3,4-epoxide were allowed to react with calf thymus DNA. The enzymatic hydrolysates of the DNA from these reactions were analyzed by reverse-phase HPLC (system 4). The chromatogram obtained from anti-5,6-diMeC-1,2-diol3,4-epoxide is illustrated in Figure 4. Peaks 2 and 3 were initially tentatively identified as adducts of dG and peaks 6 and 7 as adducts of dA by comparison of their retention times to standards prepared by reaction of anti-5,6-diMeC-1,2-diol3,4-epoxide with polydeoxyguanylic acid or dA. While we did not attempt to fully characterize the structures of these adducts, the UV and NMR spectral data summarized in Table I1 confirm the identities of peaks 2 and 3 as dG adducts and peaks 6 and 7 as dA adducts. These spectra are consistent with published and -dA spectra of anti-5-MeC-1,2-diol3,4-epoxide-dG adducts. These have been characterized as products of trans opening of the epoxide ring at C-4 by the exocyclic amino group of the purine (14-16). Seven peaks were also observed in the hydrolysates obtained from the reaction of syn-5,6-diMeC-1,2-diol3,4epoxide with calf thymus DNA. The two major products eluted 3.3 and 4.4 min later, respectively, than the major dG and dA adducts (peaks 3 and 6 of Figure 4) of the anti-diol epoxide. Groups of mice were treated topically with either [3H]5-MeC or [3H]5,6-diMeC and sacrificed 18 h later.
246 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
0
20
." .
Figure 4. HPLC trace (system 4) of enzymatic hydrolysate of calf thymus DNA reacted with anti-5,6-diMeC-1,2-diol 3,4-epoxide. Peaks 2 and 3 are dG adducts, and peaks 6 and 7 are dA adducts. See text and spectral data in Table 11.
h,lbd,il
g
0
0.5
10
20
30
40
50
60 0 Time (min)
10
20
30
40
50
60
Figure 5. HPLC analysis of enzymatic hydrolysates of calf thymus DNA reacted with (A) racemic anti-5-MeC-1,2-diol3,4and epoxide or (C) racemic anti-5,6-diMeC-1,2-diol3,4-epoxide of DNA isolated from mouse epidermis 18 h after topical application of (B) [3H]5-MeC or (D) [sH]5,6-diMeC. The peak marked with an "X" in panel D is a contaminant. Table 111. DNA Binding of 5-MeC and 5,6-DiMeC in Mouse
EDidermis"
5-MeC 5,6-diMeC
5
4
5,6-diMeC BcPh Figure 6. Structures of 5,6-diMeC and BcPh.
60
40
Time (min)
Misra et al.
pmol/mg of DNAb total bindine dG adducts dA adducts 5.0 2.5 0.2 1.3 0.7 0 2
Groups of mice were treated topically with 820 nmol of [3H]5MeC or [3H]5,6-diMeCand sacrificed 18 h later. DNA was isolated, enzymatically hydrolyzed, and analyzed by HPLC (see Figure 5). *Each value is the mean of data from 2 groups of 4 mice each, except where noted. Values were within 15% of the mean. "ased on 1 group of 4 mice.
The epidermal DNA was isolated, hydrolyzed to deoxyribonucleosides, and analyzed by HPLC. The results of the HPLC analysis are illustrated in Figure 5. The major radioactive peak eluting at 44 min in Figure 5B coeluted with the major dG adduct formed upon reaction of 5MeC-l(R),2(S)-diol3(S),4(R)-epoxide with DNA (Figure 5A). This adduct has been identified as the product of trans opening of the epoxide ring at C-4 by the exocyclic amino group of dG (4,14-16). The two large radioactive peaks eluting at 46 and 51 min in Figure 5D coeluted with the major dG and dA adducts formed by reaction of anti-5,6-diMeC-1,2-diol 3,4-epoxide with DNA (peaks 3 and 6 of Figure 4), but not with any of the adducts formed from syn-5,6-diMeC-1,2-diol3,4-epoxide. The results of these experiments are summarized in Table 111. Total DNA binding and dG binding of [3H]5-MeC were 3-4 times greater than those of [3H]5,6-diMeC. The data indicate that there is stereoselectivity in the metabolic formation of 5,6-diMeC-1,2-diol3,4-epoxide and in its reactions with DNA. As discussed above, liver 9O0Og supernatant from rats pretreated with Aroclor 1254 produced almost exclusively the (R,R)-enantiomer of 5,6-di-
MeC-1,2-diol. In previous studies, we have observed that mouse epidermis has the same stereoselectivity for production of (R,R)-dihydrodioLsof 5-MeC and 6-MeC as liver 9OoOg supernatant from Aroclor 1254 treated rats (10). These studies also showed that the (R,R)-dihydrodioLswere metabolized to (R,S,S,R)-diolepoxides in mouse skin (17). Therefore, we would expect that the 5,6-diMeC-1,2-diol 3,4-epoxide formed in mouse epidermis is predominantly the (R,S,S,R)-enantiomer. The detection of peaks 3 and 6 of Figure 4 as major DNA adducts of [3H]5,6-diMeCin mouse skin (Figure 5D) is consistent with this proposed stereoselectivity. Peaks 3 and 6 were produced by reaction with DNA. of racemic anti-5,6-diMeC-1,2-diol3,4-epoxide Their CD spectra had positive signs at 265 and 282 nm, respectively, which in this case probably arise by trans ring opening of the (R,S,S,R)-diol epoxide by the exocyclic amino group of the purine (4,15,16,18). Taken together, the data indicate that 5,6-diMeC is stereoselectively meand tabolized in mouse skin to 5,6-diMeC-l(R),2(R)-diol 5,6-diMeC-l(R),2(S)-diol 3(S),4(R)-epoxide. The results of this study demonstrate that the pathway illustrated in Figure 1 occurs in mouse epidermis. The metabolism experiments showed that 5,6-diMeC-1,2-diol was formed. The DNA binding study showed that adducts were produced from anti-5,6-diMeC-1,2-diol3,4-epoxide. Thus, the routes of metabolic activation of the weak tumor initiator 5,6-diMeC and the strong tumor initiator 5-MeC are qualitatively similar in mouse skin. Quantitatively, they are different. The extent of formation of 5,6-diMeC-1,2-diol apparently exceeds that of 5-MeC-1,2-diol, but the level of diol epoxide-DNA adducts formed from 5,6-diMeC is less than that formed from 5-MeC. Therefore, either 5,6-diMeC-1,2-diolis less efficiently oxidized to the corresponding diol epoxide than is 5-MeC or anti5,6-diMeC-1,2-diol3,4-epoxide is more readily detoxified than is the corresponding diol epoxide of 5-MeC. Further metabolism and binding studies as well as bioassays in mouse skin will be necessary to resolve this question. The results described in the following paper (8) demonstrate that anti-5,6-diMeC-1,2-diol 3,4-epoxide is a strong tumorigen in newborn mice, thus suggesting that it is not readily detogied. Therefore, a lower extent of conversion of 5,6-diMeC-1,2-diol than of 5-MeC-l,2-diol to tumorigenic diol epoxides may be the most important factor in the lower tumorigenicity of 5,6-diMeC than 5-MeC. There are interesting similarities between 5,6-diMeC and BcPh (Figure 6). 5,6-diMeC has a sterically congested bay region between carbons 4 and 5, and BcPh has a structurally similar fjord region between carbons 1 and 12. These structural features lead to deformation from planarity, with torsion angles as great as 22' and 16O, respectively (19,20). Both 5,SdiMeC and BcPh are relatively weak tumorigens on mouse skin (1-3, 21, 22). However, the 3,4-diol1,2-epoxides of BcPh are highly tumorigenic on mouse skin, with activities considerably greater than those of either BcPh or BcPh-3,4-diol(22,23). The diol epoxides of BcPh and 5,6-diMeC have conformational similarities, as discussed in the following paper, and both anti-diol epoxides are highly tumorigenic in
Dimethylchrysene Diol Epoxides newborn mice (8, 23). The relatively low tumorigenic activities of BcPh and BcPh-3P-diol in mouse skin have been partially attributed to poor metabolic conversion of BcPh-3,Cdiol to its bay region diol epoxides (22). This has been observed using mouse and rat liver microsomes although studies of the metabolism and DNA binding of BcPh and BcPh-3,4-diol in mouse skin have not been reported (24). Thus, inefficient metabolism of diols to bay region diol epoxides, perhaps due to their molecular shapes, may be largely responsible for the weak tumorigenic activities of both 5,6-diMeC and BcPh.
Acknowledgment. This is paper 139 in the series “A Study of Chemical Carcinogenesis”. This study was supported by Grant CA-44377 from the National Cancer Institute. We thank Nicholas E. Geacintov, Chemistry Department, New York University, for obtaining the CD spectra.
References (1) Amin, S., Balanikas, G., Huie, K., and Hecht, S. S. (1988) Synthe& and tumor initiating activities of dimethylchrysenes. Chem. Res. Toxicol. 1, 349-355. (2) Amin,S. G., Hecht, S. S., Bi Raddo, P., and Harvey, R. G . (1990) Comparative tumor initiating activities of cyclopentano and methyl derivatives of 5-methylchrysene and chrysene. Cancer Lett. 51, 17-20. (3) Amin, S., Desai, D., and Hecht, S. S. (1992) Comparative tumorigenicity of dimethylchrysenes in mouse skin. Chem. Res. Toxicol. (preceding paper in this issue). (4) Melikian, A. A,, Amin, S., Huie, K., Hecht, S. S., and Harvey, R. G. (1988) Reactivity with DNA bases and mutagenicity toward Salmonella typhimurium of methylchrysene diol epoxide enantiomers. Cancer Res. 48, 1781-1787. (5) Dipple, A., and Pigott, M. A. (1987) Resistance of 7,12-dimethylbem[a]anthracene adducts in DNA to hydrolysis by snake venom phosphodiesterase. Carcinogenesis 8, 491-493. (6) Gupta, R. C. (1984) Nonrandom binding of the carcinogen Nhydroxy-2-acetyleminofluoreneto repetitive sequences of rat liver DNA in vivo. h o c . Natl. Acad. Sci. U.S.A. 81, 6943-6947. (7) Amin, S., Camanzo, J., and Hecht, S. S. (1982) Identification of metabolites of 5,ll-dimethylchrysene and 5,12-dimethylchrysene and the influence of a peri-methyl group on their formation. Carcinogenesis 3, 1159-1163. (8) Misra, B., Amin, S., and Hecht, S. S. (1992) Dimethylchrysene diol epoxides: Mutagenicity in Salmonella typhimurium, tumorigenicity in newborn mice, and reactivity with deoxyadenosine in DNA. Chem. Res. Toxicol. (following paper in this issue). (9) Amin, S., Hecht, S. S., and Hoffmann, D. (1981) Synthesis of angular ring methoxy-5-methylchrysenesand Smethylchrysenols. J. Org. Chem. 46, 2394-2398. (10) Amin,S., Huie, K., Balanikas, G., Hecht, S. S., Pataki, J., and Harvey, R. G. (1987) High stereoselectivity in mouse skin metabolic activation of methylchrysenes to tumorigenic dihydrodiols. Cancer Res. 47, 3613-3617. (11) Weems, H. B., Fu, P. P., and Yang, S. K. (1986) Stereoselective metabolism of chrysene by rat liver microsomes. Direct separa-
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 247 tion of diol enantiomers by chiral stationary phase HPLC. Carcinogenesis 7, 1221-1230. (12) Melikian, A. A., LaVoie, E. J., Hecht, S. S., and Hoffmann, D. (1983) 5-Methylchrysene metabolism in mouse epidermis in vivo, diol epoxideDNA adduct persistence, and diol epoxide reactivity with DNA as potential factors influencing the predominance of 5-methylchrysene-l,2-diol-3,4-epoxide-DNA adducts in mouse epidermis. Carcinogenesis 4, 843-849. (13) Shiue, G. H., El-Bayoumy, K., and Hecht, S. S. (1987) Comparative metabolism and DNA binding of 6-nitro-&methylchrysene and 5-methylchrysene. Carcinogenesis 8, 1327-1331. (14) Melikian, A. A., Amin, S., Hecht, S. S., Hoffmann, D., Pataki, J., and Harvey, R. G. (1984) Identification of the major adducts formed by reaction of 5-methylchryseneanti-dihydrodiol epoxides with DNA in vitro. Cancer Res. 44, 2524-2529. (15) Reardon, D. B., Prakash, A. S., Hilton, B. D., Roman, J. M., Pataki, J., Harvey, R. G., and Dipple, A. (1987) Characterization adducts. of 5-methylchrysene-l,2-dihydrodiol-3,4-epoxide-DNA Carcinogenesis 8, 1317-1322. (16) Peltonen, K., Hilton, B. D., Pataki, J., Lee, H., Harvey, R. G., and Dipple, A. (1991) Spectroscopic characterization of syn-5methylchrysene l,2-dihydrodiol3,4-epoxide-deoxyribonucleoside adducts. Chem. Res. Toxicol. 4, 305-310. (17) Hecht, S. S., Amin, S., Huie, K., Melikian, A. A., and Harvey, R. G. (1987) Enhancing effect of a bay region methyl group on tumorigenicity in newborn mice and mouse skin of enantiomeric bay region diol epoxides formed stereoselectively from methylchrysenes in mouse epidermis. Cancer Res. 47, 5310-5315. (18) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, 334-340. (19) Zacharias, D. E., Kashino, S., Glusker, J. P., Harvey, R. G., Amin, S., and Hecht, S. S. (1984) The bay region geometry of some 5-methylchrysenes: steric effects in 5,6- and 5,12-dimethylchrysenes. Carcinogenesis 5, 1421-1430. (20) Hirshfeld, F. L. (1963) The structure of overcrowded aromatic compounds. Part VII. Out of plane deformation in benzo[c]phenanthrene and 1,12-dimethylbenzo[c]phenanthrene. J. Chem. SOC.,2126-2135. (21) International Agency for Research on Cancer (1983) Vol. 32, Polynuclear Aromatic Compounds, Part 1, Chemical, Environmental, and Experimental Data. ZARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, pp 205-209, IARC, Lyon, France. (22) Levin, W., Wood, A. W., Chang, R. L., Ittah, Y., Croisy-Delcey, M., Yagi, H., Jerina, D. M., and Conney, A. H. (1980) Exceptionally high tumor-initiating activity of benzo[c]phenanthrene bayregion diol-epoxides on mouse skin. Cancer Res. 40,3910-3914. (23) Levin, W., Chang, R. L., Wood, A. W., Thakker, D., 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. (24) Thakker, D. R., Levin, W., Yagi, H., Yeh, H. J. C., Ryan, D. E., Thomas, P. E., Conney, A. H., and Jerina, D. M. (1986) Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system. J. Biol. Chem. 261, 5404-5413.
