Chem. Res. Toxicol. 1992,5, 248-254
Dimethylchrysene Diol Epoxides: Mutagenicity in Salmonella typhimurium, Tumorigenicity in Newborn Mice, and Reactivity with Deoxyadenosine in DNA 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 In contrast to 5-methylchrysene and 5,9-dimethylchrysene, 5,6-dimethylchrysene and 5,7dimethylchrysene are weak tumor initiators on mouse skin. In order to investigate the basis for this, we have evaluated the mutagenic activities toward Salmonella typhimurium TA 100 and reactivity with DNA of (f)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetr~ydro-5,6-dimethylchrysene (anti-5,6-diMeC-1,2-diol3,4-epoxide) and anti-5,7- and anti-5,9-diMeC-1,2-diol3,4epoxide. The tumorigenic activities of anti-5,6- and anti-5,7-diMeC-1,2-diol 3,4-epoxides in newborn mice were also investigated. anti-5,9-diMeC-l,2-diol3,4epoxide was the most mutagenic was highly tumorigenic in newborn of the three diol epoxides. anti-5,6-diMeC-l,2-diol3,4-epoxide mouse lung, with activity significantly greater than that of either anti-5-MeC- or anti-5,7-diMeC-1,2-diol3,4-epoxide. Although the amounts of total binding of the diol epoxides to calf thymus DNA were similar, anti-5,6-diMeC-1,2-diol 3,4-epoxide bound extensively to deoxyadenosine residues. High binding to deoxyadenosine is related to the presence of a sterically hindered bay or fjord region as present in 5,6-diMeC, 7,12-dimethylbenz[a]anthracene,benzo[glchrysene, and benzo[c]phenanthrene. The conformations of the anti- and syn-diol epoxides of 5,6-diMeC and benzo[c]phenanthrene were similar, with both having pseudodiequatorial hydroxyl groups, in contrast to less sterically crowded diol epoxides. The high tumorigenicity of anti-5,6-diMeC-1,2-diol 3,4-epoxide in newborn mice is of interest with respect to its high deoxyadenosine binding. However, a relationship between deoxyadenosine binding and tuwas not apparent. The results of this morigenicity among other chrysene-1,2-diol3,4-epoxides study provide further insight into the structural requirements favoring diol epoxide reactivity with deoxyadenosine in RNA and tumorigenicity in newborn mice.
I ntroductlon In the first paper of three in this issue (l), we showed that 5,9-diMeC1and 5-MeC (Figure 1)are equally potent tumor initiators on mouse skin, with activities significantly greater than those of 5,g-diMeC and 5,7-diMeC. In the second paper (2), we demonstrated the existence in mouse epidermis of the 5,g-diMeC 5,6-diMeC-1,2-diol anti-5,6-diMeC-1,2-diol3,4-epoxide metabolic activation pathway. In this study we have evaluated the mutagenicity toward Salmonella typhimurium TA 100 and in vitro DNA binding of anti-5,6-, anti-5,7- and anti-5,g-diMeC1,2-diol3,4-epoxides. We have also assessed the tumorigenic activities of anti-5,6- and anti-5,7-diMeC-1,2-diol 3,4-epoxides in newborn mice. These studies were carried out to evaluate the potential of these diol epoxides as ultimate tumorigens of dimethylchrysenes and thereby gain insight into their roles in dimethylchrysenetumorigenesis.
-
-
Experimental Section Apparatus. Proton NMR spectra were determined in CDC13 with a Bruker AM 360 WB spectrometer, unless noted otherwise. Abbreviations: 5-MeC, 5-methylchrysene; 5,6-diMeC, 5,6-dimethylchrysene; anti-5-MeC-1,2-diol 3,4-epoxide, (&)-anti-l,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-5methylchrysene; anti-5,6-diMeC1,2-diol 3,4-epoxide, (f)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5,6-dimethylchrysene;5,6-diMeC-1,2-diol, 1,2-dihydro-l,Z-dihydroxy-5,6-dimethylchrysene;5,9-diMeC-1,2-dione, 5,g-dimethylchrysene-1,2-dione; dG, deoxyguanosine; dA, deoxyadenosine; BcPh, BgC, benzo[c]phenanthrene; DMBA, 7,12-dimethylbenz[a]anthracene; benzo(g]chrysene, THF,tetrahydrofuran; LAH, lithium aluminum hydride; PCC, pyridinium chlorochromate; DMSO, dimethyl sulfoxide; m-CPBA, m-chloroperoxybenzoic acid.
MS were determined with a Hewlett-Packard Model 5988A instrument and high-resolution MS with a GV 70-250 double-focusing magnetic sector instrument at the Rockefeller University Mass Spectrometric Biotechnology Resource. Chemicals. anti-5-MeC- and anti-5,7-diMeC-1,2-diol3,4-epoxides were synthesized as described ( 3 , 4 ) . The syntheses of the other diol epoxides are summarized in Scheme I and were performed as described below. All chemicals used in these syntheses were obtained from Aldrich Chemical Co., Milwaukee, WI, unless noted otherwise. Polydeoxyguanylic acid was obtained from Sigma Chemical Co. (St. Louis, MO). Synthesis. (A) anti-5,6-DiMeC-l,2-diol3,4-Epoxide (8). ( 1 ) 1-(3,4-Dimethyl-l-naphthyl)-2-(3-methoxyphenyl)ethylene (3). A mixture of l-(chloromethyl)-3,4-dimethylnaphthalene (5) (1.0 g, 5 mmol) and triphenylphosphine (1.31 g, 5 mmol) in benzene (100 mL) was heated at reflux for 6 h, cooled, and filtered to give the phosphonium salt 1 (1.3 g, 56.5%). NaOMe (0.23 g, 4 mmol) was added to a solution of 1 (1.3 g, 3 mmol) and m-anisaldehyde (2, 0.4 g, 3 mmol) in MeOH (150 mL) at room temperature, and the mixture was stirred for 3 h. The contents were diluted with H20 (150 mL) and extracted with CH2C12 The organic layer was washed with H20,dried (MgSOJ, and concentrated in vacuo. Purification of the residue by silica gel column chromatography with 9:l hexane/CH2C1, as eluent yielded 3 (0.45 g, 52%) as a mixture of cis and trans isomers: 'H NMR 6 2.4 (s, CH,), 2.6 (s, CH,), 3.4 (s, OCH3), 3.8 (s, OCH3), 6.6-7.2 (m, 6 H), 7.25 (s, 1 H), 7.3-7.6 (m, 2 H), 7.9-8.2 (m, 2 H); MS m / z (relative intensity) 288 (M+, 100). (2) 2-Methoxy-5,6-diMeC(4). Air was bubbled through a solution of 3 (0.4 g, 1.4 mmol) and I2 (5 mg) in dry benzene (1 L) at room temperature, and it was irradiated with a Pyrex-filtered Havonia 4WW medium-pressure lamp. The reaction was followed by TLC. After 16 h, the benzene was removed under vacuum and the residue was purified by chromatography on a silica gel column with elution by 4:l hexane/CH2C12to yield 4 (0.25 g, 63%): mp 186-188 "C (MeOH); 'H NMR 6 2.8 (s, 3 H, CH,), 3.0 (s, 3 H, CH,), 4.0 (5,3 H, OCHS), 7.25 (dd, 1 H, H3, J 3 , 4 = 10.29 Hz, J3,1
0893-228xf 92 f 2705-0248$03.00 f 0 0 1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 249
Dimethylchrysene Diol Epoxides 43
7
4
6
Figure 1. Chrysene ring system.
= 2.8 Hz), 7.35 (d, 1 H, H,, J1,3 = 2.8 Hz), 7.6-7.68 (m, 2 H, H8 and Hs), 7.82 (d, 1H, H12,J12,11 = 8.97 Hz), 8.18-8.2 (m, 1H, H,), 8.45 (d, 1 H, H4, J 4 , 3 = 10.2 Hz), 8.62 (d, 1 H, Hilt Jl1,lz = 8.96 Hz),8.68-8.75 (m, 1H, Hlo);MS m/z (relative intensity) 286 (M+, loo), 271 (61). (3) 2-Hydroxy-5,6-diMeC (5). Boron tribromide in CHZCl2 (2.5 mL, 1 M) was added to a stirred solution of 4 (0.14 g, 0.5 "01) in CHzClz(100 mL) at 0 OC, under a positive pressure of N2 The addition took 5 min. The reaction mixture was allowed to stir at room temperature for 16 h. The reaction was quenched with HzO,the contents were extracted with CHzCl2(2 X 100 mL), and the combined organic layers were washed with HzO, dried (MgS04),and concentrated in vacuo to yield 5 (90mg, 66%): mp 212-214 "C (CH2C12/hexane);'H NMR S 2.8 ( 8 , 3 H, CH&, 3.0 ( ~ ,H, 3 CHd, 7.1 (dd, 1H, H3, J%4 = 10.1 Hz,J3,1 = 2.6 Hz),7.2-7.3 (m, 2 H, H8 and Hs), 7.4-7.8 (m, 3 H, H1, H7, and Hlz),8.1-8.2 (d, 1 H, HJ, 8.4-8.7 (m, 2 H, Hlo and Hll); MS m / z (relative intensity) 272 (M+, loo), 257 (32). (4) S,G-DiMeC-l,2-diol (7). Adogen 464 [methyltrialkyl(C8-Clo)a"onium chloride, 3 drops] was added to a vigorously Fremy's salt ['ON(S03K)z, stirred mixture of 5 (0.1 g, 0.36 "011, 0.5 g, 2 mmol], and KHzP04 (35 mL, 0.17 M) in 130 mL of CHzClz/benzene (16:84), and the contents were stirred at room temperature for 16 h. The resulting dark colored solution was diluted with HzO (150 mL) and extracted with benzene (3 X 150 mL). The combined organic layers were washed with HzO (2 X 100 mL), dried (MgS04),and concentrated under vacuum to yield crude 5,6-diMeC-1,2-dione(6, 80 mg, 80%). It was used in the next step without further purification. MS m/z (relativeintensity) 286 (M', 30), 244 (18). N&H4 (0.4 g) was added to a stirred suspension of crude 6 (80 me) in EtOH (100 mL) at room temperature. The reaction mixture was stirred for 16 h. The resulting light yellow solution
was poured into H20 (200 mL), and the organic portion was extracted with EtOAc (2 X 100 mL), washed with HzO (2 X 200 mL), dried (MgS04),and concentrated to dryness. The residue was purified by chromatography on a Florid column with elution by CHzCl2/EtOAc(1:l)to give the diol 7 (30 mg, 30%): 'H NMR (acetone-d,) 6 2.6 (s, 3 H, CH3), 2.65 (8, 3 H, CH3), 4.4 (d, 1 H, H2, Jz,l = 11.0 Hz), 4.65 (d, 1H, Hi, J1,z = 11.0 Hz), 6.0 (d, 1H, Hs, J3,4 = 10.1 Hz), 6.91 (d, 1 H, H4, J4,3 = 10.1 Hz), 7.55-7.61 (m, 2 H, Ha and Hs), 7.8 (d, 1 H, H12,J1z,ll = 8.3, Hz), 8.05-8.1 (m, 1 H, H7),8.68-8.8 (m, 2 H, Hlo and Hll); MS m / z (relative intensity) 290 (M+, 30.8), 272 (70.1), 244 (18.8); high-resolution MS, calcd for M+ 290.1307, found 290.1316. (5) aati-5,6-DiMeC-l~-diol3,4-Epoxide (8). A solution of 7 (29 mg, 0.1 mmol) in dry THF (40 mL) was reacted with mCPBA (200 mg) at room temperature. After 5 h, the reaction mixture was diluted with ice-cold Ego (100 mL), and the EgO/THF was washed with 2% aqueous NaOH (3 X 40 mL) followed by HzO (2 X 50 mL). The ethereal solution was dried (KzC03),and EgO was removed in vacuo at room temperature to afford a colorless solid, which was recrystallized from E g o / hexane to give 8 (12 mg, 40%): 'H NMR (acetone-d6)6 2.7 (8, 3 H, CH3),3.01 (s, 3 H, CH3),3.7 (m, 1H, H3), 3.82 (dd, 1 H, Hz, J z , =~ 7.1 Hz, J2,3 = 1.5 Hz), 4.61 (d, 1H, H4, J4,3 = 4.0 Hz), 4.82 (d, 1 H, HI, J1,z= 7.0 Hz), 7.6-7.7 (m, 2 H, HBand H9), 8.0 (d, 1H, H ~ Z , J = ~ 8.1 ~ , Hz), , ~ 8.15 (m, 1H, H,), 8.8-8.9 (m, 2 H, Hlo and Hll); MS m/z (relative intensity) 306 (M+, 30), 272 (100); high-resolution MS, calcd for M+ 306.1256, found 306.1268. (B) syn-5,6-~M&-lf-di013,4-Epoxide. To a stirred solution of 5,6-diMeC-1,2-diol(7) (5.6 mg, 0.020 mmol) in THF (3 mL) and HzO (1mL) at 0 O C under Nz was added N-bromoacetamide (3.2 mg, 0.2 "01). The reaction mixture was stirred at 0 "C for 1h and then at room temperature for 1 h. The reaction mixture was diluted with EtOAc (10 mL), washed with ice-cold HzO (2 X 10 mL), dried (NazS04),and concentrated under reduced pressure to yield the bromohydrin as a colorless solid. The solid was triturated with cold Ego (1mL). The residue showed a single spot on TLC (Rf= 0.5 in EtOAc). This bromohydrin (4 mg) was used in the next step without further purification. To a stirred solution of the bromohydrin (4 mg)in dry THF' (1mL) was added Amberlite-IRA-400 (50 mg, in hydroxide form) under N2 After 1h, the resin was filtered and the filtrate was concentrated under reduced pressure. Trituration of the residue with cold EtzO (2 mL) gave pure syn-5,6-diMeC-l,2-diol 3,4-epoxide (3 mg, 0.01
Scheme I. Synthesis of (A) anti-5,6-DiMeC-1,2-diol3,a-Epoxide (Compound 8) and (B) anti-5,9-DiMeC-1,2-diol3,4-Epoxide (Compound 19)
+
OCH3
NaOMa
3
2
m-CPBA
lHF
CR
-
PhH. 12
4 CY
-
250 Chem. Res. Toxicol., Vol. 5, No. 2, 1992 mmol, R, = 0.75 in EtOAc): 'H NMR (acetone-d,J 6 2.75 (s, 3 H, CH3), 2.80 (9, 3 H, CH3), 3.85 (dd, 1H, H3, J 3 , 4 = 4.10 Hz, J 3 , 2 = 2.08 Hz), 4.05 (dd, 1H, Hz, J 2 , l = 6.88 Hz, 52.3 = 2.08 Hz), 4.50 (d, 1 H, H4,54,3= 4.10 Hz), 4.72 (d, 1 H, Hi, J1,Z = 6.88 Hz), 7.60 = 8.60 Hz), 8.20 (m, (m, 2 H, H8 and H9),7.80 (d, 1 H, Hlz, J1z,ll 1H, H,), 8.80 (m, 1 H, Hlo), 8.85 (d, 1 H, Hll, J1l,lz = 8.60 Hz). The purity of the epoxide was greater than 98% by HPLC analysis [4.6-mm X 25-cm Lichrosorb 5360, 5-pm column, isocratic (1:2 THF/hexane), 2 mL/min, retention time 7.8 min]. The anti-diol epoxide eluted at 10.0 min under these conditions. (C) anti-5,9-DiMeC-lI2-diol3,4-Epoxide (19). (1) Methyl 2 4 6-Methoxy-l-naphthyl)-3-(p -tolyl)propenoate (11). A stirred solution of 6-methoxy-1-naphthylaceticacid (10,4.3 g, 0.02 mol) and p-tolualdehyde (9, 2.4 g, 0.02 mol) in a 1:l mixture of EhN and AQO (20 mL) was heated at 150 O C for 8 h. The reaction mixture was cooled and acidified with HCl (200 mL, 2.5 N). The resulting suspension was extracted with CHzClz(2 X 150 mL), and the combined organic layers were washed with H20 (300 mL), dried (MgSO,), and concentrated to afford the crude acid (2.0 g). This was used in the next step without further purification. MS m/z (relative intensity), 318 (M+, 38.4), 274 (51.1). A mixture of the above acid (2.0 g, 6.6 mmol), KzC03 (1.8 g, 13 mmol), and dimethyl sulfate (1.5 g, 12 mmol) in acetone was heated at reflux for 2 h, after which the reaction mixture was cooled and filtered. The filtrate was partitioned in CH2C12(200 mL) and HzO (200 mL), and the organic layer was separated and dried (MgSO,). The solvent was evaporated in vacuo, and the residue was purified on a silica gel column with elution by hexane/CHzClz(3:l) to give 11 (1.2 g, 54.7%): mp 138-139 "C; 'H NMR 6 2.18 (s,3 H,CH3),3.61(s,3 H,0CH3),3.92(s,3 H,OCHJ, 6.8-7.8 (m, 10 H), 8.0 (8, 1 H); MS m / z (relative intensity) 332 (M+, 61.2), 272 (100). (2) 2-Methoxy-5,9-DiMeC (15). 5-Carbomethoxy-2-methoxy-9-methylchrysene (12) was prepared in 61% yield from 11 (0.7 g, 2 "01) via photolysis as described for 4: mp 156-157 "C; 'H NMR 6 2.65 (8, 3 H, CH3),3.95 (8, 6 H, OCH3,COOCH3),7.25 (dd, 1H, H3,J3,4 = 9.3 Hz, J3,1 = 2.8 Hz), 7.3 (d, 1 H, Hi, J1,3 = 2.8 Hz), 7.45 (dd, 1 H, Hg, J8,7 = 8.1 Hz, J8,1o = 1.8 Hz), 7.9 (d, 1 H, H7,J7,8 = 8.1 Hz), 7.95 (d, 1 H, H1ztJ1z,ll= 9 Hz), 8.08 (d, 1 H, H4, J 4 , 3 = 9.3 Hz), 8.18 (9, 1 H, HB), 8.48 (8, 1 H, Hi,), 8.66 (d, 1H, Hll, Jll,lz = 9.1 Hz); MS m/z (relative intensity) 330 (M+, 56.6), 299 (100). A solution of 12 (0.7 g, 2 "01) in dry THF (25 mL) was added to a suspension of LAH (0.14 g, 4 mmol) in EtzO (50 mL) at 0 "C in 10 min. The reaction mixture was stirred at room temperature for 2 h, hydrolyzed with H 2 0 (200 mL), and extracted with E g o (200 mL). The organic layer was separated and dried (MgSO,), and the EgO was removed under vacuum, yielding 5-(hydroxymethyl)-2-methoxy-9-methylchrysene (13,0.6 g, 100%): mp 153-154 "C; 'H NMR 6 2.6 (8,3 H, CH3),4.0 (e, 3 H, CH,), 5.35 (s, 2 H, CHzOH),7.3-7.38 (m, 2 H, H1 and H3),7.45 (d, 1 H, HE, JS,, = 8.1 Hz), 7.85 (d, 1 H, H7, 57,s= 8.1 Hz), 7.91 (d, 1 H, Hi,, J12,11 = 8.0 Hz), 8.08 (9, 1 H, He), 8.5 (9, 1 H, Hi,), 8.72 (d, 1H, H11,511,12 = 8.0 Hz), 8.92 (d, 1H, H4,J4,3 = 9.1 Hz); MS m / z (relative intensity) 302 (M+, 60.1), 301 (100). The oxidation of 13 (0.6 g, 2 mmol) was carried out with PCC (0.5 g) in CHzC12(150 mL) at room temperature for 3 h. After the usual workup, 2-methoxy-9-methylc~ene-5-carboxaldehyde (14,0.6 g) was isolated and used in the next step without further purification. A mixture containing 14 (0.33 g, 1 mmol), hydrazine (2 mL), and KOH (0.12 g, 3 "01) in diethylene glycol (25 mL) was heated at reflux for 2 h. The contents were brought to room temperature and acidified with HCl(5 mL, 10 N). The organics were extracted with CH2ClZ(2 X 50 mL) and dried (MgS04),and the solvent was removed in vacuo to yield 15 (0.11 g, 38.8%) as a colorless solid: mp 182-184 "C (EtOH); 'H NMR B 2.65 (s,3 H, CH,), 3.2 ( s , 3 H, CH3),4.0 (8, 3 H, OCHJ, 7.25-7.31 (m, 2 H, H1 and H3),7.37 (d, 1 H, HE,J8,, = 8.23 Hz), 7.75 (d + s, 2 H, H6 and H,), 7.88 J1z 11 = 9.1 Hz), 8.49 (s,1 H, Hie), 8.73 (d, 1 H, H11, (d, 1H, J11,12 = 9.1 Hz), 8.9 (d, 1H, H4,J4,3 = 9.42 Hz); MS m / z (relative intensity) 286 (M+, 100). (3) 2-Hydroxy-5,9-DiMeC (16). The title compound (30 mg) was prepared in 55% yield from 15 (57 mg, 2 mmol), following the procedure outlined for 5: 'H NMR 6 2.65 (s, 3 H, CH,), 3.2 (9, 3 H, CH3), 7.2 (dd, 1 H, H3, 5 3 , 4 = 9.29 Hz, 53,' = 2.87 Hz),
Misra et al. 7.3 (d, 1 H, Hi, 51.3 = 2.85 Hz), 7.42 (dd, 1H, HE, J8,7 = 8.16 Hz, J8,10 = 1.18 Hz), 7.75-7.85 (m, 3 H, H,, H,, and H12),8.48 (s, 1 H, Hlo), 8.7 (d, 1 H, Hi,, J113 = 9.0 Hz), 8.9 (d, 1 H, H4, J 4 , 3 = 9.3 Hz); MS m/z (relative intensity) 272 (M+, loo), 243 (31.1). (4) 5,9-DiMeC-1,2-dione (17). Dione 17 (20 mg, 80%) was synthesized from 16 (24 mg) and Fremy's salt (100 mg) using the method described for 6: 'H NMR 6 2.6 (e, 3 H, CH3), 3.0 (8, 3 H, CH3), 6.45 (d, 1 H, H3,53,4 = 10.1 Hz), 7.4 (d, 1 H, Hg, J7,g = 8.1 Hz), 7.65-7.75 (m, 2 H, H6 and H7),8.28 (d, 1 H, H12,J1z,ll = 8.61 Hz), 8.38 (9, 1 H, Hi,), 8.55 (d, 1 H, Hq, J 4 , 3 = 10.1 Hz), 8.8 (d, 1 H, Hll, J11,12 = 8.6 Hz); MS m/z (relative intensity) 286 (M+, 24.2), 258 (30.1). (5) 5,9-DiMeC-1,2-diol (18). Reduction of 17 (18 mg) with NaBH, (100 mg) to diol 18 was carried out by a procedure similar to that used for preparation of 7. The yield after column purification was 70%: 'H NMR 6 2.7 (s,3 H, CH3),2.95 (8,3 H, CH3), 4.4-4.5 (m, 2 H, H2 and OH), 4.75 (d, 2 H, H1 and OH, J13= 10.2 Hz), 6.15 (dd, 1 H, H3, J3,4 = 10.3 Hz, J3,2 = 1.9 Hz), 7.38 (d, 1 H, H4, 5 4 , 3 = 10.3 Hz), 7.42 (d, 1 H, HE, J8,7 = 8.2 Hz), 7.6 (9, 1 H, Hs), 7.7 (d, 1 H, H7,J7,g = 8.2 Hz), 7.98 (d, 1 H, H12, J l z , l l = 8.8 Hz), 8.54 ( 8 , 1 H, Hlo), 8.78 (d, 1 H, H11,511,12 = 8.4 Hz); MS m/z (relative intensity) 290 (M+,10.2),272 (100); high-resolution MS, calculated for M+ 290.1307, found 290.1328. (6)anti-5,g-DiMeC-1,2-diol3,4-Epoxide (19). Diol epoxide 19 was prepared in 61% yield from 18 (10 mg) by m-CPBA oxidation, analogous to the procedure described for 8: 'H NMR (acetone-d6)6 2.6 (s, 3 H, CH3), 3.0 (s, 3 H, CH3),3.78 (dd, 1 H, H3,53,4 = 4.7 Hz, 53.2 = 1.6 Hz), 3.96 (dd, 1 H, HZ, J2,l = 8.4 Hz, J2,3 = 1.7 Hz), 4.79 (d, 1 H, Hi, J1,z = 8.5 Hz), 4.9 (d, 1 H, H4, J3,4 = 4.4 Hz), 7.60-7.65 (m, 3 H, H,, HE,and HB),8.05 (d, 1H, H12, J12,11 = 8.7 Hz), 8.58 (8, 1 H, Hie), 8.85 (41H, Hi,, Ji1,lz = 8.7 Hz); high-resolution MS, calculated for M+ 306.1256, found 306.1234. Mutagenicity Assays. S. typhimurium strain TA 100 was kindly provided by Dr. Bruce N. Ames, University of California, Berkeley. The diol epoxides were dissolved in DMSO, and assays were performed as described without preincubation (6, 7). Reported mutagenicity values are means of triplicate assays. Background revertants have not been subtracted. Bioassay for Tumorigenicity in Newborn Mice. The Pregnant protocol was identical to that described previously (8,9). ICR/Ha mice were obtained from Harlan Sprague-Dawley, Madison, WI. Pups were given ip injections of the appropriate compound in DMSO as follows: 8 nmol in 5 pL on day 1,16 nmol in 10 pL on day 8, and 32 nmol in 20 pL on day 15 of life. The mice were weaned at age 21 days and separated by sex. They were maintained under standard conditions as described and were given NIH-07 diet. They were killed at age 35 weeks. Lung and liver adenomas were counted, and representative samples were confirmed histopathologically. Statistical analyses were carried out by analysis of variance followed by the Newman-Keuls range test. Reactions of Diol Epoxides with DNA and Polydeoxyguanylic Acid. These reactions were carried out as described for anti-5,6-diMeC-1,2-diol 3,4-epoxide in the second paper of three in this issue (2). The methodology was virtually identical to that described in ref 10.
Results The 2-methoxydimethylchrysenes4 and 15 were the key intermediates in t h e syntheses of anti-5,6- and 5,9-diMeC-1,2-diol3,4-epoxidea(Scheme I). They were prepared by standard methods which we have developed for t h e synthesis of methylchrysene derivatives (11). In the case of 4, we used t h e Wittig reaction to prepare the olefin 3, followed b y photolytic ring closure. Both reactions proceeded in good yield. In the case of 15, we used the aldol condensation approach to give t h e carboxylate 11. Photolysis of carboxylates such as 11 t o chrysenes generally proceeds more readily than photolysis of the corresponding methyl-substituted derivatives (11). The 5-substituted chrysene carboxylate 12 was converted to 15 in good yield by our standard reduction, oxidation, reduction sequence (11).
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 251
Dimethylchrysene Diol Epoxides
Table I. Tumorigenicity of Diol Epoxides in Newborn Mice' pulmonary tumors hepatic tumors effective no. % tumortumors per % tumortumors per of mice bearing mice mouse f SD bearing mice mouse f SD compound anti-5-MeC-1,2-diol3,4-epoxide female 35 86 3.3 f 2.7b 3 0.03 f 0.2 38 1.1f 1.8d male 37 59 1.6 f 2.2b total 72 72 2.4 f 2.6b 21 0.6 f 1.4d anti-5,6-diMeC-l,a-diol 3,4-epoxide female 45 89 9.5 f 9.4bJ 2 0.1 f 0.9 22 55 5.5 f 9.4b" 18 0.4 h 0.90 male total 67 78 8.2 f 9.5b" 7 0.2 f 0.90 anti-5,7-diMeC-l,2-diol 3,4-epoxide female 36 75 2.7 f 2.gb 6 0.08 f 0.4 male 36 61 2.0 f 2.7b 42 1.6 f 2.gd 24 0.9 f 2.2d total 72 68 2.4 f 2.4b DMSO control female 38 5 0.05 f 0.2 3 0.03 f 0.2 9 0.09 0.3 male 34 15 0.2 0.6 6 0.06 f 0.02 total 72 10 0.1 f 0.4
*
*
Groups of 72 ICR/Ha mice were given ip injections of 8 nmol of racemic diol epoxide in DMSO on day 1 of life, 16 nmol on day 8, and 32 nmol on day 15. They were weaned at age 21 days, separated by sex, and killed at age 35 weeks. Significantly more active than DMSO control, P < 0.01. cSignificantly more active than all other treatments, P < 0.01. dSignificantly more active than anti-5,6-diMeC-1,2-diol 3,Cepoxide and DMSO control, P < 0.01. e Significantly more active than DMSO control, P < 0.05.
Compounds 4 and 15 were converted to the corresponding 1,2-diols by hydrolysis with BBr3 followed by oxidation with Fremy's salt and reduction with NaBH4. As in previous syntheses of methylchrysene diols, this approach worked smoothly (4,12). The dihydrodiols were reacted with m-CPBA to produce the desired anti-diol epoxides (8 and 19). Previous studies have shown that anti-diol epoxides are produced by m-CPBA oxidation of methylchrysene diols (4, 12). The spectral properties of all compounds were in agreement with the structures illustrated in Scheme I. syn-5,6-DiMeC-1,2-diol3,4-epoxide was also prepared. Interestingly, the coupling constant, J1,2,in the NMR spectrum of this diol epoxide was 6.88 Hz which is similar to Jl,2= 7.10 Hz in the spectrum of the anti-diol epoxide. This indicates that both diol epoxides have conformational equilibria in which the hydroxyl groups are preferentially pseudodiequatorial. Similar observations have been reported in the NMR spectra of the syn- and anti-3,4-diol 1,bepoxides of BcPh, as well as the 11,12-diol 13,14-epoxides of BgC and the 9,lO-diol 11J2-epoxides of benzo[clchrysene,which all have their epoxide rings in sterically crowded fjord regions analogous to the sterically congested bay region of syn- and anti-B,&diMeC-l,2-di013,4epoxides (13-15). The pseudodiaxial conformation of the hydroxyl groups is generally preferred in diol epoxides not having sterically crowded bay regions, although it should be noted that such preferences are solvent dependent. The results of the mutagenicity assay are illustrated in Figure 2. The assay was carried out in S. typhimurium TA 100 without metabolic activation because previous studies have shown that this strain is responsive to methylchrysene diol epoxides (10). anti-5-MeC- and anti5,9-diMeC-1,2-diol3,4-epoxide had equivalent activities up to 0.5 nmol per plate; the former was toxic at higher doses. The mutagenicities of anti-5,7-diMeC- and anti5,6-diMeC-1,2-diol3,4epoxides were comparable, and both were less active than the other two diol epoxides. The relative mutagenic activities of anti-5-MeC- and anti5,7-diMeC-1,2-diol 3,4-epoxides were the same as previously reported (3). The tumorigenic activities of anti-5,SdiMeC- and anti-5,7-diMeC-192-diol3,4-epoxides were compared with that of anti-5-MeC-1,2-diol3,4-epoxide in newborn mice. The newborn mouse model was chosen for this study because it is known to be sensitive to diol epoxide tumorigenesis (16). The results are summarized in Table I. anti-5,6-DiMeC-1,2-diol3,4-epoxide was clearly the most active compound in the induction of pulmonary tumors.
1500
c
1
I
0.5
I
1.o
%.O
nmol/plate Figure 2. Mutagenicity toward S. typhimurium TA 100 of anti-5-MeC-1,2-diol 3,4-epoxide (e), antid,9-diMeC-l,2-diol 3,4-epoxide (A),anti-5,7-diMeC-1,2-diol 3,4-epoxide (O), and anti-5,6-diMeC-1,2-diol 3,4-epoxide (A).
Its activity was approximately 3 times as great as those as of anti-5,7-diMeC- or anti-5-MeC-1,2-diol3,4-epoxide, judged by the number of pulmonary tumors per mouse. The yield of hepatic tumors was lower than the yield of pulmonary tumors, as is typical in this model system. Both anti-5,7-diMeC- and anti-5-MeC-1,2-diol 3,4-epoxide produced higher incidences of hepatic tumors than did anti-5,6-diMeC-1,2-diol 3,4 epoxide. The HPLC traces obtained by analysis of hydrolysates of DNA that had been reacted with the diol epoxides are illustrated in Figure 3. The identities of the dG and dA adducts of anti-5-MeC-1,2-diol 3,4-epoxide have been established previously (17,18);dG and dA adduct assignments for anti-5,6-diMeC-1,2-diol3,4-epoxide were confirmed in the preceding paper (2). The major adducts formed from anti-5,'I-diMeC-and anti-5,9-diMeC-l,2-diol 3,4-epoxide coeluted with those produced by reaction of these diol epoxides with polydeoxyguanylic acid. The NMR spectra of the major adduct formed from each of these diol epoxides confirmed their identities as dG adducts. The UV spectra of the adducts were also consistent with the assignments indicated (18).The extensive reaction of anti-5,6-diMeC-1,2-diol3,4epoxide with dA is immediately obvious by inspection of Figure 3B. The results of the DNA binding studies are summarized in Table 11. The dA/dG ratio for anti-5,6-diMeC-1,2-diol3,4-epoxide was notably higher than those of the other diol epoxides.
252 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
Misra et al.
Table 11. Reactivity of Diol Epoxides with Calf Thymus DNA dA/dG relative total anti-l,2-diol 3,4-epoxide ratio adduct formation 5-MeC 0.24 1.0 5,6-diMeC 1.1 0.9 5,7-diMeC 0.14 0.3 5,9-diMeC 0.12 0.3
A.
a Racemic diol epoxides were allowed to react with calf thymus DNA at 37 "C. The DNA was hydrolyzed enzymatically and analyzed by HPLC with UV detection (254 nm). Extents of formation of adducts were determined by integration of peak areas.
Dlscusslon One goal in this study was to obtain further insights on the metabolic activation of 5,6-diMeC and 5,7-diMeC which would help to explain their unexpectedly low tumorigenic activities on mouse skin. Since our ongoing studies indicated that 5,6-diMeC-1,2-diol and anti-5,6diMeC-1,2-diol3,4-epoxide, the potential proximate and ultimate tumorigens of 5,6-diMeC, were formed in mouse skin, we hypothesized that anti-5,6-diMeC-1,2-diol 3,4epoxide would have low tumorigenic activity. The tumorigenic activities of diol epoxides are known to be sensitive to molecular shape, and it seemed possible that the buttressing effect of the 6-methyl group could lead to lower activity. A similar, but perhaps less severe, steric interaction could occur in anti-5,7-diMeC-l,2-diol3,4-epoxide through interaction of the 7-methyl group with the peri proton at the 6-position, but no such steric crowding would The be expected in anti-5,9-diMeC-1,2-diol3,4-epoxide. results of the mutagenicity assay seemed to support our hypothesis since the mutagenicity of the 5,g-isomer was and comparable to that of anti-5-MeC-1,2-diol3,4-epoxide was greater than those of either the 5,6- or 5,7-isomer. In previous studies of methylchrysene diol epoxides, we have noted a reasonably good correlation between mutagenicity in 5 '. typhimurium TA 100 and tumorigenicity in newborn mice and on mouse skin (3,4,9,10,19-21). However, the results of the tumorigenicity assay clearly show that our hypothesis was incorrect, at least with respect to the newborn mouse system. anti-5,6-DiMeC-1,2-diol3,4-epoxide was a potent pulmonary tumorigen with activity which was greater than those of both anti-5-MeC- and
W
v)
c
0
U
W v)
a
0
Retention Time (min)
Figure 3. HPLC traces obtained upon analysis of hydrolysates of calf thymus DNA that had been reacted with (A) anti-5MeC-1,2-diol3,4epoxide,(B)anti-5,6-diMeC-1,2-diol3,4epoxide, (C) anti-5,7-diMeC-1,2-diol3,4-epoxide, or (D)anti-5,g-diMeC1,2-diol 3,4-epoxide.
anti-5,7-diMeC-1,2-diol 3,4-epoxide. We are presently carrying out bioassays of the diol epoxides in mouse skin to further evaluate their roles in the metabolic activation of 5,6-diMeC and 5,7-diMeC. However, our results suggest that these diol epoxides are not extensively formed from 5,6-diMeC- and 5,7-diMeC-1,2-diol or may readily be detoxified in mouse skin. The results of the tumorigenicity assay prompted us to
Table 111. Chrysene-IJ-diol 3,4-Epoxides: Tumorigenicity in Newborn Mice and Reactivity with DNA
tumorigenicity in tumorigenic newborn mice (lung activity relative to anti-1.2 diol 3.4-e~oxide" tumors Der mouee/56 nmol) anti-5-MeC-1.2-diol 3.4-e~oxide' (1)5-MeC (1R,2S,3S,4R) 13.4* 1.0 (2) 5-MeC (1S,2R,3R,4S) 0.46b 0.03 (3) 6-MeC (lR,2S,3S,4R) 0.09 0.007 (4) 6-MeC (lS,2R,3R,4S) 0.17 0.01 (5) 11-MeC (lR,2S,3S,4R)f O.4Ob 0.03 (6) 11-MeC (1S,2R,3R,4S) 0.31 0.02 (7) 5,6-diMeC 8.2b 3.4 (8)5,7-diMeC 2.4b 1.0
total DNA dA/dG adducts relative to ratiod anti-5-MeC-1.2-diol 3.4-e~oxide' 0.07 1.0 0.27 0.3 0.24 0.3 1.0 0.2 0.09 0.5 0.35 0.1 1.1 0.9 0.14 0.3
Compounds 1-6 were pure enantiomers as indicated in parentheses. Compounds 7 and 8 were racemic mixtures. Statistically significant lung tumor multiplicity compared to controls. Compounds 1-6 were tested in the same assay; the values are relative to anti-5-MeC1,2-diol3,4-epoxide (1R,2S,3S,4R) in that assay (19). Compounds 7 and 8 were tested in a second assay; the values are relative to 2.4 lung (see Table I). dFrom ref 10 for compounds 1-6 and Table I1 for compounds tumors per mouse for racemic anti-5-MeC-1,2-diol3,4-epoxide 7 and 8. 'Values for compounds 1-6 are relative to anti-5-MeC-1,2-diol 3,4-epoxide (1R,2S,3S,4R); Compounds 7 and 8 are relative to racemic anti-5-MeC-1,2-diol 3,4-epoxide. /Equivalent to 7(R),8(S)-dihydroxy-9(S),lO(R)-epoxy-7,8,9,lO-tetr~ydr~5-methylc~ysene,
Chem. Res. Toxicol., Vol. 5, No. 2,1992 263
Dimethylchrysene Diol Epoxides
BcPh
BgC
DMBA
5,WIMeC
5-Mec
Figure 4. Structures of BcPh, BgC, DMBA, 5,6-diMeC, and 5-MeC. Shaded area is the rigid four-carbon chain adjacent to a bay or fjord region associated with extensive deformation from planarity in BcPh, BgC, DMBA, and 5,6-diMeC. This structural feature is not present in 5-MeC.
investigate the DNA interactions of these diol epoxides. The formation of dA adducts was distinctly greater in the reaction of antid,6-diMeC-l,2-diol3,4-epoxide with DNA than in the other reactions. This is shown by the data in Table II. Deformation from planarity appears to be a key factor favoring reaction with dA in DNA. This has been noted earlier in comparisons of the reactions of BcPh, BgC, DMBA, and 5-MeC diol epoxides with DNA (18,22,23). The torsion angles resulting from steric crowding in the bay region of 5,g-diMeC are 1&22O, and similar distortions As illustrated in Figure occur in BcPh and DMBA (a,%). 4, BcPh, BgC, DMBA, and 5,6-diMeC all have a rigid four-carbon chain adjacent to their sterically crowded bay or fjord regions. These features will result in severe deformations from planarity. In contrast, 5-MeC, which has a three-carbon chain in the same relative position, responds to the steric crowding by expanding the bay region angle and by a relatively small deformation from planarity with torsion angles of 5-10’ (24). Presumably, similar interactions occur in the bay region diol epoxides of these hydrocarbons. Other diol epoxides which are not likely to have large torsion angles and do not react extensively with dA in DNA are benzo[a]pyrene-7,&diolO,lO-epoxide(26), dibenz[uj]anthracene-3,4-diol1,2-epoxide and ita 7-methyl derivative (27),7-methylbenz[a]anthracene-3,4-diol 1,2epoxide (28),and several methylchrysene diol epoxides (Table III). It should be noted, however, that deformation from planarity is not the only factor leading to extensive reaction with dA in DNA. Others include an SRRS configuration and 6-substitution in the chrysene system (10, 22, 26). For example, the 1S,2R,3R,4S enantiomer of anti-6-MeC-1,2-diol3,4-epoxide has a high dA/dG ratio, although its overall extent of reaction with DNA is relatively low (see Table 111). syn-Diol epoxides of BcPh and DMBA are also highly reactive with dA in DNA (22,29). Dipple and colleagues have noted a relationship between reactivity with dA in DNA and tumorigenicity of diol epoxides or their parent hydrocarbons (22). Thus, DMBA, one of the most tumorigenic polynuclear aromatic hydrocarbons known, forms extensive dA adducts in DNA of mouse skin via ita syn- and anti-3,4-diol1,2-epoxides (29). The highly tumorigenic BcPh-3,4-diol1,2-epoxides react extensively with dA in DNA, and one of the adducts may be correlated with tumorigenicity (22). These observations suggest that extensive reactivity with dA may be one factor favoring tumorigenicity. The high tumorigenicity of anti-5,6-diMeC-l,2-diol3,4-epoxide seems to fit this pattern. The dA/dG adduct ratios and reactivity with DNA are compared of various anti-chrysene-l,2-diol3,4epoxides to their tumorigenic activities in mouse lung in Table 111. Of the 8 compounds tested, 5 showed significant tumorigenicity; anti-5,6-diMeC-l,2-diol3,4-epoxide was the most active of these, and it has the highest dA/dG ratio. On the other hand, the other 4 tumorigenic compounds-both enantiomers of anti-5-MeC-1,2-diol3,4-epoxide, the RSSR enantiomer of anti-ll-MeC-1,2-diol 3,4-epoxide, and anti-5,7-diMeC-1,2-diol 3,4-epoxide-have low dA/dG ratios. Among the 3 inactive compounds, the dA/dG ratios
range from 0.24 to 1.0. Clearly, reaction with dA is not by itself an indicator of potential tumorigenicity in the chrysene system. This is also indicated by the DNA binding studies of 5-MeC and 5,6-diMeC in mouse skin, described in the preceding paper (2). Nevertheless, reactivity with dA may be one factor favoring tumorigenicity. One structural feature favoring tumorigenicity of antichrysene-1,2-diol 3,4-epoxides in newborn mice is the presence of a methyl group in the same bay region as the epoxide ring, as shown in Table III. This feature is present in the most active compounds-anti-5-MeC-, anti-5,6diMeC-, and anti-5,7-diMeC-l,2-diol3,4-epoxide. Methyl substitution in other parts of the unti-chrysene-l,2-diol 3,4-epoxide system does not lead to high activity, nor is high activity observed when it is unsubstituted or has a 5-ethyl or 5-propyl substituent (21). The 1R,2S,3S,4R configuration is another major determinant of chrysene1,Pdiol 3,4-epoxide tumorigenicity.
Acknowledgment. This is paper 140 in the series “A Study of Chemical Carcinogenesis.” We thank Joanne Braley for her participation in the newborn mouse study and C. X. Wang for histopathologicalanalysis. This study was supported by Grant CA-44377 from the National Cancer Institute.
References (1) Amin, S., Desai, D., and Hecht, S. S. (1992) Comparative tu-
morigenicity of dimethylchrysenes in mouse skin. Chem. Res. Toxicol. (first of three papers in this issue). (2) Misra, B., Amin, S., and Hecht, S. S. (1992) Metabolism and DNA binding of 5,6-dimethylchrysenein mouse skin. Chem. Res. Toxicol. (second of three papers in this issue). (3) Amin, S., Balanikas, G., Huie, K., and Hecht, S. S. (1988) Synthesis and tumor initiating activitiea of dimethylchrpenes. Chem. Res. Toxicol. 1, 349-355. (4) Amin, S., Huie, K., Balanikas, G., and Hecht, S. S. (1988) Synthesis and mutagenicity of 5-alkyl substituted chrysene-l,2-diol3,4-epoxides. Carcinogenesis 9, 2305-2308. (5) Hewett, C. L. (1940) Polycyclic aromatic hydrocarbons. Part XXII. J. Chem. SOC.,293-303. (6) Ames, B. N., McCann, J., and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 347-364. (7) Yahagi, T., Nagao, M., Seino, Y., Mateushima, T., Sugimura, T., and Okada, M. (1977) Mutagenicities of N-nitrosamines in Salmonella. Mutat. Res. 48, 121-130. (8)Amin,S. G., Hecht, S. S., Di 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. (9) Hecht, S. S., Radok, L., Amin, S., Huie, K., Melikian, A. A., Hoffmann, D., Pataki, J., and Harvey, R. G. (1985) Tumorigenicity of 5-methylchrysene dihydrodiols and dihydrodiol epoxides in newborn mice and on mouse skin. Cancer Res. 46,144S1452. (10) 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. (11) Amin, S., Camanzo, J., Huie, K., and Hecht, S. S. (1984) Improved photochemical synthesis of 5-methylchrysene derivatives and ita application to the preparation of 7,8-dihydro-7,8-dihydroxy-5-methylchrysene.J. Org. Chem. 49, 381-384. (12) Harvey, R. G., Pataki, J., and Lee, H. (1986) Synthesis of the dihydrodiol and diol-epoxide metabolite of chrysene and 5methylchrysene. J. Org. Chem. 51, 1407-1412. (13) Sayer, J. M., Yagi, H., Croisy-Delcey, M., and Jerina, D. M. (1981) Novel bay-region diol epoxides from benzo[c]phenanthrene. J. Am. Chem. SOC.103,4970-4972. (14) Glatt, H., Piee, A., Steinbrecher, T., Schrode, R., Oesch, F., and Seidel, A. (1991)Fjord- and bay-region diol-epoxides investigated for stability, SOS induction in Escherichia coli and mutagenicity in Salmonella typhimurium and mammalian cells. Cancer Res. 51, 1659-1667. (15) Bushman, D. R., Grossman, S. J., Jerina, D. M., and Lehr, R. E. (1989) Synthesis of optically active fjord-region llJ2-diol
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13,14-epoxidesand K-region 9,lO-oxide of the carcinogen benzo[glchrysene. J. Org. Chem. 54, 3533-3544. (16) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875-4917. (17) Melikian, A. A., Amin, S., Hecht, S. S., Hoffman, D., Pataki, J., and Harvey, R. G. (1984) Identification of the major adducts formed by reaction of 5-methylchrysene anti-dihydrodiol epoxides with DNA in vitro. Cancer Res. 44, 2524-2529. (18) 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. (19) 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. (20) Amin, S., Huie, K., Hecht, S. S., and Harvey, R. G. (1986) and comparSynthesis of 6-methylchrysene-l,2-diol-3,4-epoxides ison of their mutagenicity to 5-methylchrysene-1,2-diol-3,4-epoxides. Carcinogenesis 7, 2067-2070. (21) Amin, S., Misra, B., Braley, J., and Hecht, S. S. (1991) Comparative tumorigenicity in newborn mice of chrysene- and 5-alkylchrysene-l,2-diol-3,4-epoxides. Cancer Lett. 58, 115-118. (22) Dipple, A,, Pigott, M. A., Agarwal, S. K., Yagi,H., Sayer, J. M., and Jerina, D. M. (1987) Optically active benzo[c]phenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327, 535-536.
(23) Schurdak, M. E., Bekesi, E., Jerina, D. M., Yagi, H., Bushman, D. R., Lehr, R. E., and Wood, A. W. (1990) Evaluation of sitespecific DNA adduct formation by polycyclic aromatic hydrogen diol epoxides in H-ras gene sequences as determined by 32Ppostlabelling analysis. Proc. Am. Assoc. Cancer Res. 31, 90. (24) 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. (25) 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. (26) Cheng, S. C., Hilton, D., Roman, J. M., and Dipple, A. (1989) DNA adducts from carcinogenicand noncarcinogenicenantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, 334-340. (27) Nair, R. V., Gill, R., Cortez, C., Harvey, R. G., and DiGiovanni, J. (1989) Characterization of DNA adducts derived from (*)trans-3,4-dihydroxy-anti-l,2-epoxy-l,2,3,4-tetrahydrodibenz[ ai]anthracene and (f)-7-methyl-tram-3,4-dihydroxy-anti-l,2-epoxy-1,2,3,4-tetrahydrodibenz[aj]anthracene.Chem. Res. Toxicol. 2, 341-348. (28) Peltonen, K., Hilton, B. D., Cortez, C., Harvey, R. G., and Dipple, A. (1990) Adducta formed in the reaction of 7-methylbenz[a]anthracene-3,4-dihydrodiol-1,2 epoxide with DNA. Proc. Am. Assoc. Cancer Res. 31, 89. (29) Bigger, C. A. H., Sawicki, J. T., Blake, D. M., Raymond, L. G., and Dipple, A. (1983) Products of binding of 7,12-dimethylbenz[alanthraceneto DNA in mouse skin. Cancer Res. 43,5647-5651.
Covalent Cross-Linking of Proteins by Carbon Disulfide William M. Valentine,* Venkataraman Amamath, Doyle G. Graham, and Douglas C. Anthony Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 Received September 11, 1991 Carbon disulfide is known to react with amino groups of proteins to generate dithiocarbamates (2). We observed covalent cross-linking of dithiocarbamate-derivatized proteins under physiological conditions which may occur through several mechanisms. Evidence for the structure of these covalent bridges and the reactive intermediate was obtained using 13CNMR spectroscopy in conjunction with specific isotopic labeling. On incubation a t 37 "C oxidative coupling of dithiocarbamates generated bis(thiocarbamoy1) disulfides (3) which were reduced by cysteine. In addition, an electrophilic isothiocyanate (4) was generated from decomposition of the dithiocarbamate. Nucleophilic addition of sulfhydryl and amine moieties to the isothiocyanate produced dithiocarbamate ester (5) and thiourea linkages (6), respectively. Evidence for the presence of inter- and intramolecular cross-links was obtained using denaturing polyacrylamide gel electrophoresis under reducing conditions. The formation of isothiocyanate in neutral solution, through elimination of sulfhydryl ion, was correlated with increased pK, values of the parent amine of amino acids. Dithiocarbamates derived from terminal amino groups of proteins did not appear to generate isothiocyanate or form thiourea or dithiocarbamate ester. Both the thiourea and the dithiocarbamate ester were stable at reduced pH, whereas in alkaline media the thiourea was stable but dithiocarbamate ester was hydrolyzed. Although the disulfide and ester linkages were formed more rapidly than the thiourea, generation of the latter appeared to be irreversible, leading to its gradual accumulation over a longer period of time. Generation of isothiocyanate by CS2-derived dithiocarbamates and subsequent covalent cross-linking of proteins may provide a molecular mechanism for CS2-induced axonopathy. Introduction Clinical abnormalities associated with occupational exposure to CS2have been recognized for over a century (1). *R-J Reynolds Leon Golberg Memorial Fellow in Toxicology. Author to whom correspondence should be addressed.
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Currently CS2 is used in the production of cellophane and rayon (2) and is a decomposition product of dithiocarbamates (3) used as pesticides and therapeutic agents. Although several mechanisms of toxicity based on dithiocarbamate formation by CS2 and amines of biological molecules have been proposed (2), a generally accepted mechanism has not been established. Elaboration of the 1992 American Chemical Society
