Chem. Res. Toxicol. 1992,5, 153-156 mutagenicity of 1,6- and 3,6-dinitrobenzo[a]pyrenes. Chem. Pharm. Bull. 38, 3158-3161. (13) Sera, N., Kai, M., Horikawa, K., Fukuhara, K., Miyata, N., and Tokiwa, H. (1991) Detection of 3,6-dinitrobenzo[a]pyrenein airborne particulates. Mutat. Res. 263, 27-32. (14) Tokiwa, H., Nakagawa, R., and Ohnishi, Y. (1981) Mutagenic assay of aromatic nitro compounds with Salmonella typhimurium. Mutat. Res. 91, 321-325. (15) El-Bayoumy, K., and Hecht, S. S. (1983) Identification and mutagenicity of metabolites of 1-nitropyrene formed by rat liver. Cancer Res. 43, 3132-3137. (16) Chou, M. W., Heflich, R. H., Casciano, D. A., Miller, D. W., Freeman, J. P., Evans, F. E., and Fu, P. P. (1984) Synthesis, spectral analysis, and mutagenicity of 1-,3-, and 6-nitrobenzo[alpyrene. J. Med. Chem. 27, 1156-1161. (17) Pitta, J. N., Jr., Zielinska, B., and Harger, W. P. (1984) Isomeric mononitrobenzo[a]pyrenes: synthesis, identification and mutagenic activities. Mutat. Res. 140, 81-85. (18) Fu, P. P., Chou, M. W., Miller, D. W., White, G. L., Heflich, R. H., and Beland, F. A. (1985) The orientation of the nitro substituent predicts the direct-acting bacterial mutagenicity of nitrated polycyclic aromatic hydrocarbons. Mutat. Res. 143, 173-181. (19) El-Bayoumy, K., Shiue, G.-H., and Hecht, S. S. (1988) Metabolism and DNA binding of 1-nitropyrene and 1-nitrosopyrene in newborn mice. Chem. Res. Toxicol. 1, 243-247.
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(20) Fu, P. P. (1990) Metabolism of nitro-polycyclic aromatic hydrocarbons. Drug Metab. Rev. 22, 209-268. (21) Klopman, G,. Tonucci, D. A,, Holloway, M., and Rosenkranz, H. S. (1984) Relationship between polarographic reduction potential and mutagenicity of nitroarenes. Mutat. Res. 126,139-144. (22) Eddy, E. P., McCoy, W. C., Rosenkranz, H. S., and Mermelstein, R. (1986) Dichotomy in the mutagenicity and genotoxicity of nitropyrenes: apparent effect of the number of electrons involved in nitroreduction. Mutat. Res. 161, 109-111. (23) Debnath, A. K., Lopez de Compadre, R. L., Debnath, G., Shusterman, A. J., and Hansch, C. (1991) Structure-activity relationship of mutagenic aromatic and heteroaromatic nitro compounds. Correlation with molecular orbital energies and hydrophobicity. J. Med. Chem. 34, 786-797. (24) Fukuhara, K., Miyata, N., Matsui, M., Ishidate, M., Jr., and Kamiya, S. (1991) Relationship between electrochemical properties and mutagenicity of 6-substituted 1- and S-nitrobenzo[a]pyrenes. Mutat. Res. 252,86. (25) McCoy, E. C., Rosenkranz, H. S., and Mermelstein, R. (1981) Evidence for the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens. Enuiron. Mutagen. 3,421-427. (26) Mermelstein, R., Kiriazides, D. K., Butler, M., McCoy, E. C., and Rosenkranz, H. S. (1981) The extraordinary mutagenicity of nitropyrenes in bacteria. Mutat. Res. 89, 187-196.
Mouse Skin Tumor Initiating Activity of Fluorinated Derivatives of 1,2,3,4-Tetrahydro-7,12-dimethylbenz[ a ]anthracene Raghunathan V. Nair,t Susan E. Walker,? Pradeep K. Sharma,T Donald T. Witiak,T and John DiGiovanni*?t Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, P.O. Box 389, Smithville, Texas 78957, and Division of Medicinal Chemistry, College of Pharmacy, and Comprehensive Cancer Center, The Ohio State University, 500 West 12th Avenue, Columbus, Ohio 43210 Received November 7, 1991
Introduction The tumor initiating activity of 1,2,3,4-tetrahydro7,12-dimethylbenz[a]anthracene(THDMBA;' Chart I) in Swiss (1) and SENCAR (2) mice, the ability of this polycyclic aromatic hydrocarbon (PAH) to transform human neonatal fibroblast (HNF) cells (343, and the propensity of this PAH to form adducta with cellular DNA (6)are well documented. However, the metabolic and biochemical mechanisms underlying the biological properties of these substances are not well understood. Because the terminal A-ring in THDMBA is partially saturated, direct conversion of this molecule to a bay region diol epoxide is unlikely. As part of our plan to examine alternative modes and sites involved in the metabolic activation of THDMBA, the mouse skin tumor initiating activities of 7,12-dimethylbenz[a]anthracene(DMBA),THDMBA, five site-specificallyfluorinated derivatives of THDMBA (5F, 6F, 8F, 9F, and llF), an A-ring cyclopentano analogue of THDMBA, and a 6-fluoro-12-exo-methylenetautomer of 6F-THDMBA (Chart I) were investigated in one experiment. In a second experiment, the relative tumor initiating activity of 10F-THDMBA also was evaluated. Replacement of hydrogens by fluorine in PAHs is known to alter metabolism, DNA bonding, and carcinogenicity *To whom correspondence and reprint requests should be addressed. 'The University of Texas M. D. Anderson Cancer Center. *The Ohio State University.
Chart I
CHI DMBA
CH¶
Positions of fluorine substitutionin the THDMBA
A"
CH¶ A-ring cyclopentano analogue of THDMBA
6F-IZ-exomethylcne taUtomQ
11F-7-cxomthylene faytomQ
of the PAH (2,9-19). Variations in tumor initiating potencies of site-specifically fluorinated derivatives are anticipated to provide clues concerning the metabolic sites and pathways of significance to THDMBA carcinogenesis. Abbreviations: THDMBA, 1,2,3,4-tetrahydro-7,12-dimethylbenz[alanthracene; HNF, human neonatal fibroblast; DMBA, 7,12-dimethylbenz[a]anthracene; 5F-THDMBA, 5-fluoro-THDMBA; 6FTHDMBA, 6-fluoro-THDMBA;8F-THDMBA,8fluoro-THDMBA;9FTHDMBA, 9-fluoro-THDMBA, 10F-THDMBA, 10-fluoro-THDMBA; 11F-THDMBA, 11-fluoro-THDMBA;PAH, polycyclic aromatic hydrocarbon; A-ring, angular benzo ring in DMBA constituting positions 1-4 of the molecule; TPA, 12-0-tetradecanoylphorbol13-acetate.
0893-228x/92/2705-0153$03.00/00 1992 American Chemical Society
154 Chem. Res. Toxicol., Vol. 5,No. 2, 1992
Communications
The assumption is that aryl fluoro substituent effects on PAH metabolism, in part, reflect steric and electronic influences of the halogen function. Although fluorine is often considered to be isosteric with hydrogen, peri fluoro steric effects are well characterized (7, 8). The 6-fluoro12-exo-methylenederivative provides a compound with a nonplanar tautomeric tricyclic array in place of the planar anthracene system. This compound and an A-ring cyclopentano analogue of THDMBA serve to identify additional structural features important to the carcinogenicity of the THDMBA series.
Materials and Methods Chemicals. DMBA was purchased from Eastman Kcdak Co. (Rochester, NY). 12-0-Tetradecanoylphorboll3-acetate (TPA) was obtained from LC Services (Woburn, MA). Syntheses for the THDMBA analogues used in the present study were carried out as previously reported (7,8). All THDMBA analogues are free of A-ring aromatic contaminants, and their purities were established to be greater than 98% by high-pressure liquid chromatography (H20/CH3CN, 55/45). 'H-NMR and mass spectroscopic analyses showed these compounds to be free of A-ring aromatic contaminants (7,8). Animals. Female SENCAR mice were obtained from the National Cancer Institute, Frederick, MD, and, when 7-9 weeks of age, shaved on the dorsal side. Mice were allowed to stabilize for at least 2 days, and only those mice in the resting phase of the hair growth cycle were subsequently used. All chemicals were applied topically to the shaved area in 0.2 mL of acetone, and control animals were treated with an equal volume of acetone. Tumor Induction Experiments. Each experimental group contained 20 preshaved mice. Mice were initiated with DMBA, THDMBA, and the various THDMBA analogues at the doses indicated. Two weeks after initiation mice began receiving twice-weekly treatments with 3.4 nmol of TPA. The incidence of papillomas was observed and recorded weekly. Promotion was continued in all groups until the average number of papillomas per m o w reached a plateau. Papillomas were removed at random throughout the different experimental groups. Tissues for histological evaluations were prepared using conventional paraffin sections and hematoxyljn-eosin staining. One hundred papillomas from individual mice were verified histologically. Statistical analyses of the difference between mean papilloma response (i.e., papillomas per mouse) were performed using the Wilcoxon rank sum test.
Results and Discussion The mouse skin tumor initiating activities of all evaluated compounds were assessed at two doses, and the data are presented in Table I. The relative tumor initiating potencies are in the order DMBA >> 10F-THDMBA 2 6F-THDMBA > THDMBA > 8F-THDMBA > 5FTHDMBA 9F-THDMBA 11F-THDMBA the 6F-12-exo-methyleneanalogue an A-ring cyclopentano analogue of THDMBA (Table I). This order of activity differs from the order of activity observed for these compounds in the HNF cell transformation assay reported previously (6). Thus, the potent mouse skin tumor initiator, DMBA, does not transform HNF cells whereas the nonmouse skin tumor initiating A-ring cyclopentano derivative of THDMBA is an active transformer of such cells (4,5). However, the order of activity exhibited by the aryl fluoro analogues is roughly similar in both mouse skin tumor initiation and HNF cell transformation assays. Although the 6F-12-exo-methylene compound was not examined in the HNF cell transformation assay, a similar THDMBA analogue not having the planar anthracene ring system (11F-7-exo-methylenecompound) was inactive in HNF cell transformation assays (6). Thus, the planar anthracene system seems to be necessary for THDMBA
-
--
-
Table I. Tumor Initiating Activity of DMBA, THDMBA, and Related Derivatives in SENCAR Mouse Epidermis" % of dose mice with papillomas initiator (nmol) papillomas per mouse Experiment 1 acetone 0.2 mL 12 0.12 f 0.10 DMBA 10 100 11.90 f 2.Ogb THDMBA 10 25 0.40 f 0.14b 100 90 4.20 f 0.43b A-ring cyclopentano 10 5 0.05 f 0.09 analogue to THDMBA 100 15 0.15 f 0.09 5F-THDMBA 10 15 0.15 f 0.09 100 25 0.35 f 0.35 6F-THDMBA 10 30 0.55 f 0.17b 100 90 7.30 f 1.23b 6F-12-exo-methylene 10 15 0.15 f 0.07 tautomer 100 15 0.20 f 0.20 8F-THDMBA 10 5 0.05 f 0.08 100 85 2.15 f 0.49* 9F-THDMBA 10 10 0.15 f 0.07 100 15 0.15 f 0.09 11F-THDMBA 10 0 Of0 100 10 0.10 f 0.10
THDMBA 10F-THDMBA
Experiment 2' 100 10
100
80 30 95
5.85 f 0.66 1.13 f 0.13 12.53 f 1.61d
a G r o u p ~of 20 female SENCAR mice each were initiated with the compound at doses indicated. Two weeks after initiation mice received twice-weekly applications of 3.4 nmol of TPA for 18 weeks. The incidence and multiplicity of papillomas were scored weekly as previously described (2). Survival in all groups was 295% at the termination of the experiment. Significantlygreater (p 5 0.05) than the acetone control group as determined by the Wilcoxon rank sum test. 'Data taken after 16 weeks of promotion. Significantly greater (p 5 0.05) than THDMBA at 100 nmol (experiment 2).
analogue carcinogenicity as defined in both investigated biological models. The differences observed by some of these PAHs examined in the present study with those previously found in the HNF cell transformation assay suggest species differences and multiple modes of metabolic activation. Possible explanations for the observed results are presented in Schemes I and 11. Analysis of the back-resonance electronic effects of fluorine reveal stabilization of carbocation intermediates at the benzylic C-1 and C-12 methyl groups of THDMBA in the active (Table I, experiment 1) 6F- and 8F-THDMBA analogues (Scheme I). In contrast, the inactive (Table I, experiment 1) 5F-, 9F-, and 11FTHDMBA compounds stabilize carbocations at the benzylic C-4 and C-7 methyl groups of THDMBA (Scheme I). Seemingly, stabilization of electrophilic centers at the C-1 and/or C-12 benzylic position leads to activation, whereas stabilization of carbocations at the C-4 and/or C-7 benzylic position leads to inactivation of the THDMBA analogues. To test this hypothesis, the 10F-THDMBA compound, wherein the fluorine atom also may stabilize by resonance with carbocationic centers at C-1 and/or C-12, was evaluated (experiment 2, Table I). As predicted, this compound is a potent tumor initiator in mouse skin. Since 6F-THDMBA, but not BF-THDMBA, possesses tumor initiating activity and the 6F, but not the 5F, function stabilizes a carbocation at C-1, DNA reaction at C-1 rather than at C-4 (Scheme 11) remains a viable requirement for initiation of carcinogenesis in mouse skin. Such a carbocation could easily be formed following solvolysis of a sulfate ester derivative of the C-1 benzylic alcohol (1,20-25). Additionally, concurrent metabolism studies (26),wherein THDMBA, 5F-THDMBA, and 6F-
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 155
Communications Scheme I 6F-THDMBA (active)
0
0
CH,
k
CH,
+? CH,
F+
@
F
CH,
F+
SF-THDMBA (inactive)
c*
MF *3
CHJ+
Scheme I1
@CHI
1
Reaction wiul cytosolic sulfouansferase and PAPS
J
L
@
"&" &: & CH,
CH,OH
0
0
I
CH1
0
0
dH,
I
7
Reaction with cytosolic sulfouansferase and PAPS
J
Bay region dihydrodiol epoxide
3.
Reaction with cellular DNA
0
THDMBA were incubated with NADPH, 02, and rat liver microsomes from phenobarbital-treated animals, reveal monohydroxylation at C-1, (2-7, and C-12 benzylic carbons for THDMBA, C-4, C-7, and (2-12 for 5F-THDMBA7and C-1 and C-12 for 6F-THDMBA. Furthermore, no DMBA or fluoro-DMBA metabolites could be detected under a multitude of experimental conditions (26). These data, together with a consideration of the electronic and steric effects of fluorine and the observed tumor initiating activity for these three compounds in the present study, suggest to us that hydroxylation at benzylic C-1 or (3-12 carbons may be important for the bioactivation of such PAHs to carcinogenic DNA-bonding species. Although hydroxylation at C-1 or C-4 could via a dehydration/oxidation pathway generate DMBA (Scheme 11), such a mechanism appears less likely on the basis of the above metabolism studies in vitro wherein metabolites having a fully aromatic A-ring could not be detected (4, 26). However, the inactivity of the cyclopentano analogue as a tumor initiator in mouse skin might suggest a need for six-membered ring metabolism which could lead to DMBA by dehydration/oxidation pathways (Scheme 11). Thus, the latter mechanism cannot be completely ruled out at present. Further metabolism and DNA adduct studies in mouse epidermal cells are necessary to reconcile these mechanistic possibilities. Finally, since exo-methylene tautomers are inactive, the planar anthracene system, possibly necessary for intercalation, seems to be important. The decreased carcinogenicity observed upon introduction of fluorine at position C-5, (3-9, and C-11 could also, in theory, be a consequence of reduced metabolism at the site of fluorination, but this possibility is unlikely since tumor initiating activity is enhanced by substitution of fluorine proximate to these positions (ie., at (2-6, (2-8,and C-10). Subsequent studies are required to establish whether this approach may be employed to predict regioisomeric metabolic possibilities and DNA bonding positions of significance to carcinogenesis.
Acknowledgment. Support for this work by Grants USPHS CA 36979 (J.D.), ACS FRA-375 (J.D.), and US EPA R-814207-01-0 (D.T.W.) is gratefully acknowledged.
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