Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2746-2750, July 1977 Biochemistry
Mutagenicity and cytotoxicity of benz[a]anthracene diol epoxides and tetrahydro-epoxides: Exceptional activity of the bay region 1,2-epoxides (carbonium ion/polycyclic hydrocarbon carcinogenicity/ultimate carcinogen)
ALEXANDER W. WOOD*, RICHARD L. CHANG*, WAYNE LEVIN*, ROLAND E. LEHRt, MARIA SCHAEFER-RIDDERt, JEAN M. KARLEt, DONALD M. JERINAt, AND ALLAN H. CONNEY* * Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, New Jersey 07110; and tSection on Oxidation Mechanisms, Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014
Communicated by J. J. Burns, April 20,1977
ABSTRACT Three diastereomeric pairs of diol epoxides, two tetrahydro-epoxides, and the K-region oxide of the polycyclic aromatic hydrocarbon benz[alanthracene were evaluated for mutagenic activity in strain TA 100 of Salmonella typhimurium and in line V79-6 of Chinese hamster lung cells. The two diastereomeric 1,2-epoxides of the trans-3,4-dihydrodiol of are 15 to 35 times more mutagenic to the benz[alanthracene bacteria and 65 to 125 times more mutagenic to the mammalian cells than are the diastereomeric pairs of benz[ajanthracene-8,9-diol-10,11-epoxides or benz[alanthracene-10,11-diol8,9-epoxides. 1,2-Epoxy-1,2,3,4-tetrahydrobenzlajanthracene is the most mutagenic and cytotoxic of the nine derivatives and is 5 and 25 times more mutagenic than 3,4-epoxy-1,2,3,4-tein bacterial and mammalian cells, trahydrobenla~anthracene respectively. In either test system, benz[alanthracene 5,6-oxide (K-region oxide) has less than 10% of the activity of any of the 1,2-epoxides derived from benzlajanthracene. The relative stabilities of the derivatives in aqueous solution do not account for the differences in mutagenic activity because the more mutagenic derivatives tend to be less stable. The benz[alanthracene diol epoxides, like the benzo[a pyrene diol epoxides, are refractory to the action of epoxide hydrase. The exceptional mutagenic activity of the 1,2-epoxide derivatives of benz[ajanthracene is consistent with and supportive of the hypothesis that bay region epoxides on saturated, anglar benzo-rings of unsubstituted polycyclic aromatic hydrocarbons are ultimate carcinogens. The diastereomeric pair of benzo[alpyrene (BP)-7,8-diol9,10-epoxides in which the epoxide oxygen is either cis (isomer 1 series) or trans (isomer 2 series) to the benzylic hydroxyl group have high chemical reactivity (1), covalently bind to nucleic acid (2-5), and have exceptional mutagenic and cytotoxic activity (6-9). Both of these diastereomers are metabolically formed from BP-7,8-dihydrodiol (7, 10-12), and one of the diastereomeric diol epoxides (isomer 2) has been shown to be highly carcinogenic (13). These findings and the high carcinogenicity of the two metabolic precursors of the diol epoxides, BP-7,8-dihydrodiol (13, 14) and BP-7,8-oxide (15), strongly suggest that the principal ultimate carcinogenic metabolite of
benzo[ajpyrene is a BP-7,8-diol-9,10-epoxide. A considerable
advance in our understanding of how polycyclic hydrocarbons cause cancer would be made if the key structural and chemical features of the BP-7,8-diol-9,10-epoxides could be identified and generalized to other polycyclic hydrocarbons. As a working hypothesis we have suggested (9, 16, 17) that the unique structural feature of the BP-7,8-diol-9,10-epoxides is the presence of an epoxide on a saturated, angular benzo-ring that forms part of a "bay region"t of the hydrocarbon. Support for this hypothesis comes from a reexamination of the available carcinogenicity data on substituted polycyclic hydrocarbons. The data indicate that substituents that would be expected to block formation of bay region diol epoxides markedly reduce carcinogenicity (16). Furthermore, perturbational molecular orbital calculations (17) for a number of carcinogenic polycyclic hydrocarbons predict more facile formation of chemically reactive benzylic carbonium ions from epoxides on saturated benzo-rings when they form part of a bay region of the hydrocarbon. Confirmation of the bay region hypothesis depends on the demonstration that the appropriate diol epoxides of a number of carcinogenic polycyclic hydrocarbons possess unique biological activity. Toward this end we have undertaken studies to evaluate the mutagenicity of the diol epoxides of benz[ajanthracene (BA). Because the bay region of benz[aJanthracene is located between the 1 and 12 positions of the molecule (Fig. 1), the bay region concept predicts that the 1,2-epoxide of BA-3,4-dihydrodiol should be uniquely active. This prediction differs from the proposal of Grover and Sims and associates (18, 19) that BA-8,9-diol-10,11-epoxide would have high biological activity. Recent studies consistent with our bay region concept have shown that BA-3,4-dihydrodiol is metabolized by a highly purified monooxygenase system to products which are 10 times as mutagenic to Salmonella typhimurium strain TA 100 as are the metabolites of benz[a]anthracene or the four other metabolically possible vicinial trans dihydrodiols of benz[ajanthracene (20). We have now synthesized a number of diol epoxides and tetrahydro-epoxides of benz[a]anthracene and have examined their inherent mutagenic and cytotoxic activities. As predicted from the bay region theory, 1,2-epoxides in a saturated angular benzo-ring of benz[aJanthracene are uniquely mutagenic and cytotoxic in both bacterial and mammalian cells.
Abbreviations: BP, benzo[ajpyrene; BA, benz[ajanthracene; BA1, (+)-3a,4(3-dihydroxy-1l3,2(3-epoxy-1,2,3,4tetrahydrobenz[a lanthracene; BA-3,4-diol-1,2-epoxide 2, (±)-3a, 4(3 - dihydroxy - la, 2a - epoxy - 1, 2, 3, 4 - tetrahydrobenz[ajanthracene; BA-8,9-diol-10,11-epoxides 1 and 2, and BA-10,11-diol-8,9epoxides 1 and 2, other diol epoxides of benz[a]anthracene with analogous stereochemistry; BA-H4-1,2-epoxide, 1,2-epoxy-1,2,3,4tetrahydrobenz[a]anthracene; BA-H4-3,4-epoxide, 3,4-epoxy1,2,3,4-tetrahydrobenz[a]anthracene; BA-5,6-oxide, benz[a]anthracene 5,6-oxide; BP-H4-9,10-epoxide, 9,1O-epoxy-7,8,9,10-tetrahydrobenzolalpyrene; BP-7,8-dihydrodiol, trans-7,8-dihydroxy-7,8-dihydrobenz[a]pyrene; BA-3,4-dihydrodiol, trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene; BA-1,2-, 8,9-, and 10,11-dihydrodiol, other BA-dihydrodiols.
3,4-diol-1,2-epoxide
* A bay region occurs in a polycylic aromatic hydrocarbon when an
angularly fused benzo-ring is present. The simplest example of a bay region is the sterically hindered area between the 4 and 5 positions of phenanthrene. The bay region of benz[a]anthracene is between carbon atoms 1 and 12 (see Fig. 1). In benzo[a]pyrene, the bay region is between positions 10 and 11.
2746
Biochemistry: Bay region
Wood et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
2
%OH. OH
8
7
6
Benz[a]anthracene
HO"9
BA-3,4-diol-1,2-epoxide 1
~HO,H
,
OH
BA-8,9-diol-10,11-epoxide 1
BA-10,11-diol-8,9-epoxide 1
FIG. 1. Structures of benz[a]anthracene (BA) and of the chemically more reactive diastereomers (isomer 1 series) of the diol epoxides derived from the trans-3,4-, .8,9-, and 11,12-dihydrodiols. The corresponding chemically less reactive diastereomers (isomer 2 series) have the benzylic hydroxyl groups at positions 4, 8, or 11 trans to the oxygen of the epoxide ring (1, 21).
MATERIALS AND METHODS Synthesis of Diol Epoxides and Tetrahydro-Epoxides. Diol epoxides of non-K-region dihydrodiols from benz[a]anthracene (see Fig. 1) were synthesized (21) by methods developed for the synthesis of diol epoxides from naphthalene-1,2-dihydrodiol and BP-7,8-dihydrodiol (1, 22). The trans dihydrodiol precursors were synthesized as described (23) using synthetic procedures originally developed for benzo[a 1pyrene dihydrodiols (24, 25). Direct oxidation of the dihydrodiols with peroxyacid produced diol epoxides (isomer 2 series) in which the benzylic hydroxyl group and oxygen of the epoxide ring are trans to each other, while intermediate bromohydrin formation followed by dehydrohalogenation to form the epoxide ring produced the diastereomeric diol epoxides (isomer 1 series) in which these functional groups are cis to each other. These diol epoxides derived from the trans-3,4-, trans-8,9-, and trans10,11-dihydrodiols were free of detectable impurities and free of stereoisomeric cross-contamination as determined by nuclear magnetic resonance spectroscopy and mass spectrometry (21). The procedures utilized in the synthesis of BA-H4-1,2-epoxide and BA-H4-3,4-epoxide were analogous to those used for the synthesis of the tetrahydro-epoxides of benzo[a]pyrene (26) and consisted of cyclization of trans-bromohydrin precursors at the indicated positions with base. BA-5,6-oxide (K-region) was synthesized by the chlorohydrin route as described (27). All of the derivatives were stored in the solid state at -90° and dissolved just before use in anhydrous dimethyl sulfoxide obtained by distillation from calcium hydride. Bacterial Mutagenesis Assay. The ability of the benz[a Janthracene epoxide derivatives to mutate bacteria was evaluated by previously described procedures (9, 28) in strain TA 100 of histidine-dependent Salmonella typhimurium LT-2 (29). Bacteria, obtained from B. Ames, University of California (Berkeley), were grown in Trypticase (Becton, Dickinson & Co.) soy broth containing 1% yeast extract and 0.02% ampicillin (Bristol Laboratories). After an overnight incubation in culture medium, the bacteria were collected by centrifugation and resuspended to a density of 1 X 109 cells per ml in phosphatebuffered saline (5 mM potassium phosphate/150 mM sodium chloride; final pH, 7.0). Epoxides were added in 15 ,ul of dimethyl sulfoxide to 2 X 108 bacteria in a final volume of 0.5 ml of phosphate- buffered saline and the mixture was then incubated for 5 min at 20°. After the 5-min incubation of epoxide with bacteria, 2 ml of top agar was added to the samples, and the entire contents of the culture tube were mixed and poured
2747
into a petri dish containing 15 ml of Vogel-Bonner medium with a 2% agar base. The petri dishes were then placed in the dark at 370 for 2 days, during which time only those bacteria that were mutated from histidine dependence to histidine independence were capable of growth. Mutation frequency was thus assessed by counting the macroscopic colonies of bacteria on the petri dishes at the end of the 2 days. Stability of the epoxides was evaluated by determining the decrease in the number of observed mutant colonies that resulted from preincubating the derivatives in 0.4 ml of phosphate-buffered saline for defined periods of time prior to addition of the bacteria. The samples were then incubated for 5 min prior to the addition of top agar. All experiments were performed in triplicate and coefficients of variation of the colony counts rarely exceeded 15%. Both the bacteria and the mammalian cells described below are devoid of detectable monooxygenase or epoxide hydrase activity and benz[a Janthracene is inactive as a mutagen in both systems. Mutagenesis Assay with Cultured Mammalian Cells. The Chinese hamster cell line V79-6 was kindly provided by E. H. Y. Chu, University of Michigan (Ann Arbor). Resistance to the lethal effects of the purine analog 8-azaguanine was used as the mutagenic marker in these studies. Procedures for culturing the cells, assessing cytotoxicity, and inducing 8-azaguanineresistant cells were adapted from Chu and Malling (30), and the conditions used were as described previously (9, 31) with the exception that 3 rather than 2 days elapsed between treatment of the cells with mutagen and the first addition of 8azaguanine. Derivatives were added in 20 Al of anhydrous dimethyl sulfoxide -to cells which were growing in 5 ml of culture medium. For each determination, four replicate culture dishes, each seeded with 102 cells, were used to evaluate toxicity. For each concentration of hydrocarbon, 16 replicate culture dishes, each seeded with 104 cells, were used to score 8-azaguanineresistant colonies. The mutagenic activity of each diastereomeric pair of epoxides was evaluated in the same experiment.
(i)-7#,8a-Dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydroben-
zo[a ]pyrene (isomer 2 series) (9), at two different concentrations, was included in all experiments as a positive control and gave essentially the same mutation frequency in all experiments (coefficient of variation was 14.9%, n = 5). In addition, all the compounds were tested at one concentration in a single experiment, and the absolute and relative mutagenic activities agreed with the previous experiments. In the studies to evaluate epoxide stability, the epoxides were preincubated in culture medium at 370 prior to addition to the cell cultures in 1.0 ml of medium, as previously described for the assessment of benzo[a 1pyrene diol epoxide stability (9). RESULTS All eight epoxide derivatives of benz[a ]anthracene induced reversions to histidine independence in strain TA 100 of Salmonella typhimurium in direct proportion to the applied dose (Fig. 2). However, there was an almost 400-fold range in mutagenic activities among the compounds. BA-H4-1,2-epoxide, the most mutagenic derivative, induced 2300 revertants per nmol, while BA-10,11-diol-8,9-epoxide 1, the least active derivative, induced 6 revertants per nmol. Diastereomers 1 and 2 of BA-3,4-diol-1,2-epoxide induced 1650 and 850 histidine revertants per nmol, respectively, and were the second and third most active derivatives. BA-H4-3,4-epoxide had 20% of the activity of the isomeric BA-H4-1,2-epoxide and 25% of the activity of BA-3,4-diol-1,2-epoxide 1. BA-8,9-diol-10,11-epoxide 2 and BA-10,11-diol-8,9-epoxide 2, the next most active compounds, both induced 50 revertants per nmol, and thus had 3%
Biochemistry:
2748
Proc. Nati. Acad. Sci. USA 74 (1977)
Wood et al.
R
~
U
2020
0
~~~~~~~~~0
04
0 0
0.2 0.4 0.6 0.8 1.01.2
10
20
Benz[a] anthracene derivative, nmol
FIG. 2. Mutagenicity of benz[a]anthracene epoxides in strain TA 100 of S. typhimurium. Bacteria were treated with the indicated amounts of the compounds as described in Materials and Methods. Each value for histidine-independent revertants represents the average of three replicate determinations and the spontaneous mutation frequency (120 revertants per plate) has been subtracted. BA-3,4diol-1,2-epoxides: *, isomer 1; 0, isomer 2. BA-8,9-diol-10,11-epoxides: A, isomer 1; A, isomer 2. BA-10,11-diol-8,9-epoxides: *, isomer 1; 0, isomer 2; X, BA-H4-1,2-epoxide; v, BA-H4-3,4-epoxide.
150-
' la
'eO
ta
0
io au
0
5o
v
1
2
3
10
20
30
40
Benz[a] anthracene derivative, nmol/ml
FIG. 3. Cytotoxicity (Upper) and mutagenicity (Lower) of
benz[a]anthracene epoxides in Chinese hamster V79 cells. Cells were of the mutagenic activity of BA-3,4-diol-1,2-epoxide 1. Finally, BA-8,9-diol-10,11-epoxide 1 had one-third the activity of its diastereomer and BA-10,11-diol-8,9-epoxide 1 had one-eighth the activity of its diastereomer. When tested at concentrations between 0.3 and 3.0 nmol per plate, BA-5,6-oxide induced approximately 100 revertants per nmol and thus had 6% of the activity of BA-3,4-diol-1,2-epoxide 1. BA-H4-1,2-epoxide was the most mutagenic and cytotoxic derivative in Chinese hamster V79 cells, inducing nearly 200 azaguanine-resistant variants in 105 surviving cells at a concentration of 1.2 nmol/ml and killing all the cells at a concentration of 3 nmol/ml (Fig. 3). Both diastereomeric 1,2-epoxides of BA-3,4-dihydrodiol killed 45% and 100% of the cells at concentrations of 3 and 10 nmol/ml, respectively. Depending upon the concentration, BA-3,4-diol-1,2-epoxide 2 was from 15 to 60% more mutagenic than its chemically more reactive (21) diastereomer BA-3,4-diol-1,2-epoxide 1. At equal concentrations, the diastereomeric BA-3,4-diol-1,2-epoxides induced fewer mutations than BA-H4-1,2-epoxide. However, the BA-3,4-diol-1,2-epoxide diastereomers induced more mutations than BA-H4-1,2-epoxide at equitoxic concentrations. BA-H43,4-epoxide had 10% of the cytotoxic activity and approximately 4% of the mutagenic activity of the isomeric BA-H41,2-epoxide. The mutagenic activities of the diastereoisomeric BA-8,9-diol-10,11-epoxides and BA-10,11-diol-8,9-epoxides were in all cases modest and did not exceed 1% of the mutagenic activities of the BA-3,4-diol-1,2-epoxides. When tested at concentrations between 1 and 15 nmol/ml, BA-5,6-oxide induced no more than 0.2 8-azaguanine-resistant colonies/105 survivors per nmol and thus had less than 2% of the activity of the BA-3,4-diol-1,2-epoxides. Because unstable epoxides might decompose at a rate sufficient to preclude interaction with critical receptors in the tester cells, the biological half-life of the eight epoxides was estimated in both phosphate-buffered saline and tissue culture medium. In phosphate-buffered saline, BA-H4-1,2-epoxide had a 2- to 4-min half-life, the diastereomeric pair of BA-3,4-diol-1,2epoxides had half-lives of 6-7 min, BA-H4-epoxide had a half-life of 8-10 min, and the diastereomers of BA-8,9-diol10,11-epoxide or of BA-10,11-diol-8,9-epoxide were stable,
treated with the indicated amounts of the compounds as described in Materials and Methods and the spontaneous mutation frequency in solvent-treated cultures never exceeded 1.5 8-azaguanine-resistant colonies per 105 surviving cells. See Fig. 2 for key to symbols.
exhibiting no detectable loss of mutagenicity after preincubation in phosphate-buffered saline for up to 60 min. In tissue culture medium, BA-H4-1,2-epoxide and BA-3,4-diol-1,2epoxide 1 had 3-min half-lives, BA-3,4-diol-1,2-epoxide 2 had a 7- to 8-min half-life, BA-H4-3,4-epoxide had a 5- to 8-min half-life, and BA-10,11-diol-8,9-epoxide 2 had a half-life of 7 min. BA-10,11-diol-8,9-epoxide 1 and the diastereomeric BA8,9-diol-10,11-epoxides had insufficient biological activity in V79 cells to evaluate stability in tissue culture medium. These results indicate that the relative stabilities of the derivatives in aqueous solution cannot account for the differences in mutagenic activity because the more mutagenic derivatives tended to be less stable. Microsomal epoxide hydrase catalyzes the trans addition of water to a variety of arene and alkene oxides and preincubation of six units of the highly purified enzyme (32)§ with BA-5,6oxide or BA-H4-3,4-epoxide for 5 min resulted in a complete loss of mutagenic activity of these two compounds toward strain TA 100 of S. typhimurium (data not shown). However, 20 units of the enzyme decreased the mutagenic activity of the six benz[ajanthracene diol epoxides by less than 20% and reduced the mutagenicity of BA-H4-1,2-epoxide by only 50%. Thus, as has been found for the benzo[a]pyrene diol epoxides (9, 10, 33), the diol epoxides of benzo[a ]anthracene are also very poor substrates for purified epoxide hydrase. DISCUSSION Benzo-ring epoxides of non-K-region trans dihydrodiols exist as pairs of diastereomers in which the oxygen of the epoxide ring is either cis (isomer 1 series) or trans (isomer 2 series) to the benzylic hydroxyl group (see Fig. 1). Three of the diaste§ Units of epoxide hydrase activity are defined as nmol of styrene glycol formed from styrene oxide per minute at 370.
Biochemistry: Wood et al. reomeric pairs of benzo-ring diol epoxides derived from the weak carcinogen benz[a janthracene have been evaluated for mutagenic activity in bacterial and mammalian cells. The BA-3,4-diol-1,2-epoxides were highly mutagenic in both systems and were from one to three orders of magnitude more mutagenic than BA-5,6-oxide (K-region oxide) or two pairs of BA diol epoxides with the epoxide ring at the 8,9- or 10,11positions. Estimations of the biological half-lives of the BA diol epoxides excluded the possibility that the low mutagenicity of the BA-8,9-diol-10,11-epoxides and the 10,11-diol-8,9-epoxides was due to decomposition in the test medium. Thus, our results argue against the proposal of Grover and Sims and their associates (18, 19) that the BA-8,9-diol-10,11-epoxides are the important biologically active metabolites derived from benz[a Janthracene. The large differences in mutagenic activities of the various diol epoxides dramatically illustrate the importance of the position of the oxirane ring for biological activity. The chemical reactivity of an epoxide at different positions of a hydrocarbon molecule can be predicted by simple quantum mechanical calculations based on the ease of carbonium ion formation. These perturbational molecular orbital calculations had predicted that the 1,2-epoxides of either benz[a]anthracene-3,4dihydrodiol or of 1,2,3,4,-tetrahydrobenz[a]anthracene would be more mutagenic and cytotoxic than any of the other diol epoxides or tetrahydro-epoxides of benz[a]anthracene (17). The unique structural feature of these highly mutagenic 1,2-epoxide derivatives of benz[a ]anthracene is the presence of an epoxide on a saturated angular benzo-ring which forms part of the bay region of the hydrocarbon. The high mutagenicity of BAH4-1,2-epoxide in the present study and of BP-H4-9,10-epoxide in a previous study (9) illustrates that the hydroxyl groups are not obligatory for the high mutagenic activity of bay region diol epoxides. The critical role of the 3,4-dihydrodiol of benz[a ]anthracene and the 7,8-dihydrodiol of benzo[a]pyrene in the metabolism of these hydrocarbons is that of providing a substrate which can be converted into an aliphatic epoxide as opposed to an arene oxide. The importance of saturation of the benzo-ring is illustrated by the fact that BP-H4-9,10-epoxide is at least 100-fold more mutagenic than the corresponding arene oxide, BP-9,10-oxide, in which the 7,8-double bond is present. In addition to predicting the high mutagenic activity of the BA-3,4-diol-1,2-epoxides relative to the other benz[a]anthracene diol epoxides, the quantum mechanical calculations also indicated that the bay region diol epoxides of the strong carcinogen benzo[a ]pyrene would be more biologically active than the bay region diol-epoxides of the weak carcinogen benz[a ]anthracene (17). Under the same assay conditions, the diastereomeric 7,8-diol-9,10-epoxides of benzo[a]pyrene are severalfold more mutagenic than the diastereomeric 3,4-diol1,2-epoxides of benz[a]anthracene in both bacterial and mammalian cells. Also in accord with these calculations is the lack of significant mutagenic activity for the diastereomeric pair of diol epoxides derived from trans-1,2-dihydroxy-1,2dihydronaphthalene (unpublished observations). The results presented in this communication on the high mutagenicity of the diastereomeric pair of BA-3,4-diol-1,2epoxides, and a previous study demonstrating the marked metabolic activation of BA-3,4-dihydrodiol (20), suggest that one or both of these bay region diol epoxides are the ultimately reactive forms of benz[a]anthracene. Recent studies in our laboratories indicating high tumorogenicity for BA-3,4-dihydrodiol (34) support this conclusion and the bay region hypothesis of hydrocarbon carcinogenesis.
Proc. Natl..Acad. Scd. USA 74 (1977)
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Note Added in Proof. Biological activity of the 3,4-epoxides of BA, 1,2-dihydrodiol has not been described in this study due to technical difficulties in the synthesis and purification of diol epoxides derived from bay region dihydrodiols. However, a small sample of a BA-1,2diol-3,4-epoxide, the nuclear magnetic resonance spectrum of which suggested the isomer 2 series, was obtained, and was found to have mutagenic activity similar to or less than that of the diol epoxides derived from BA-8,9- and 10,11-dihydrodiols. The authors thank Mrs. Arlene Ott for her assistance in the preparation of this manuscript. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Yagi, H., Hernandez, 0. & Jerina, D. M. (1976) J. Am. Chem. Soc. 97,6881-6883. 2. Sims, P., Grover, P. L., Swaisland, A., Pal, K. & Hewer, A. (1974) Nature 252,326-328. 3. Koreeda, M., Moore, P. D., Yagi, H., Yeh, H. J. C. & Jerina, D. M. (1976) J. Am. Chem. Soc. 98, 6720-6722. 4. King, H. W. S., Osborne, M. R., Beland, F. A., Harvey, R. G. & Brooks, P. (1976) Proc. Natl. Acad. Sci. USA 73,2679-2681. 5. Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H. & Nakanishi, K. (1976) Science 193, 592-595. 6. Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., Yagi, H., Hernandez, O., Jerina, D. M. & Conney, A. H. (1976) Biochem. Biophys. Res. Commun. 68, 1006-1012. 7. Huberman, E., Sachs, L., Yang, S. K. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73, 607-611. 8. Newbold, R. F. & Brooks, P. (1976) Nature 261, 52-54. 9. Wood, A. W., Wislocki, P. G., Chang, R. L., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O., jerina, D. M. & Conney, A. H. (1976) Cancer Res. 36, 3358-3366. 10. Thakker, D. R., Yagi, H., Lu, A. Y. H., Levin, W., Conney, A. H. & Jerina, D. M. (1976) Proc. Natl. Acad. Sci. USA 73, 33813385. 11. Yang, S. K., McCourt, D. W., Roller, P. R. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73,2594-2598. 12. Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, A. Y. H., Levin, W., Wood, A. W., Conney, A. H. & Jerina, D. M. (1977) Chem. Biol. Interact. 16,281-300. 13. Kapitulnik, J., Levin, W., Conney, A. H., Yagi, H. & Jerina, D. M. (1977) Nature 266,378-380. 14. Levin, W., Wood, A. W., Yagi, H., Jerina, D. M. & Conney, A. H. (1976) Proc. Natl. Acad. Sci. USA 73,3867-3871. 15. Levin, W., Wood, A. W., Yagi, H., Dansette, P. M., Jerina, D. M. & Conney, A. H. (1976) Proc. Natl. Acad. Sci. USA 73, 243247. 16. Jerina, D. M. & Daly, J. W. (1977) in Drug Metabolism, eds. Parke, D. V. & Smith, R. L. (Taylor and Francis, London), pp. 13-33. 17. Jerina, D. M., Lehr, R. E., Yagi, H., Hernandez, O., Dansette, P. M., Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W. & Conney, A. H. (1976) in In Vitro Metabolic Activation in Mutagenesis Testing, eds. deSerres, F. J., Fouts, J. R., Bend, J. R. & Philpot, R. M. (Elsevier/North Holland Biomedical Press, Amsterdam), pp. 159-177. 18. Swaisland, A. J., Hewer, A., Pal, K., Keysell, G. R., Booth, J., Grover, P. L. & Sims, P. (1974) FEBS Lett. 47,34-38. 19. Malaveille, C., Bartsch, H., Grover, P. L. & Sims, P. (1975) Biochem. Biophys. Res. Commun. 66,693-700. 20. Wood, A. W., Levin, W., Lu, A. Y. H., Ryan, D., West, S. B., Lehr, R. E., Schaefer-Ridder, M., Jerina, D. M. & Conney, A. H. (1976) Biochem. Biophys. Res. Commun. 72,680-686. 21. Lehr, R., Schaeffer-Ridder, M. & Jerina, D. M. (1977) Tetrahedron Lett., 539-542.
22.
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M. (1977) J. Am. Chem. Soc. 99, 1604-1611. 23. Lehr, R. E., Schaeffer-Ridder, M. & Jerina, D. M. (1977) J. Org. Chem. 42,736-744. 24. Gibson, D. T., Mahadevan, V., Jerina, D. M., Yagi, H. & Yeh, H. J. C. (1975) Science 189,295-297. 25. McCaustland, D. J. & Engel, J. F. (1975) Tetrahedron Lett.,
2549-2552. 26. Yagi, H., Holder, G. M., Dansette, P., Hernandez, O., Yeh, H. J. C., LeMahieu, R. A. & Jerina, D. M. (1976) J. Org. Chem. 41, 977-985. 27. Dansette, P. M. & Jerina, D. M. (1974) J. Am. Chem. Soc. 96, 1224-1225. 28. Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., Yagi, H., Hernandez, O., Dansette, P. M., Jerina, D. M. & Conney, A. H. (1976) Cancer Res. 36, 350-3357.
Proc. Nati. Acad. Sci. USA 74 (1977) 29. McCann, J., Spingarn, N. E., Kobori, J. & Ames, B. N. (1975) Proc. Natl. Acad. Sci. USA 72,979-983. 30. Chu, E. H. Y. & Mailing, M. V. (1968) Proc. NatI. Acad. Sci. USA 61, 1306-1312. 31. Wood, A. W., Goode, R. L., Chang, R. L., Levin, W., Conney, A. H., Yagi, H., Dansette, D. M. & Jerina, D. M. (1975) Proc. Natl. Acad. Sci. USA 72,3176-3180. 32. Lu, A. Y. H., Ryan, D., Jerina, D. M., Daly, J. W. & Levin, W. (1975) J. Biol. Chem. 250,8283-8288. 33. Wood, A. W., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O., Jerina, D. M. & Conney, A. H. (1976) J. Biol. Chem. 251, 4882-4890. 34. Wood, A. W., Levin, W., Chang, R. L., Lehr, R. E., SchaeferRidder, M., Karle, J., Jerina, D. M. & Conney, A. H. (1977) Proc. Natl. Acad. Sci. USA 74, in press.
Mutagenicity and cytotoxicity of benz[a]anthracene diol epoxides and tetrahydro-epoxides: Exceptional activity of the bay region 1,2-epoxides (carbonium ion/polycyclic hydrocarbon carcinogenicity/ultimate carcinogen)
ALEXANDER W. WOOD*, RICHARD L. CHANG*, WAYNE LEVIN*, ROLAND E. LEHRt, MARIA SCHAEFER-RIDDERt, JEAN M. KARLEt, DONALD M. JERINAt, AND ALLAN H. CONNEY* * Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, New Jersey 07110; and tSection on Oxidation Mechanisms, Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014
Communicated by J. J. Burns, April 20,1977
ABSTRACT Three diastereomeric pairs of diol epoxides, two tetrahydro-epoxides, and the K-region oxide of the polycyclic aromatic hydrocarbon benz[alanthracene were evaluated for mutagenic activity in strain TA 100 of Salmonella typhimurium and in line V79-6 of Chinese hamster lung cells. The two diastereomeric 1,2-epoxides of the trans-3,4-dihydrodiol of are 15 to 35 times more mutagenic to the benz[alanthracene bacteria and 65 to 125 times more mutagenic to the mammalian cells than are the diastereomeric pairs of benz[ajanthracene-8,9-diol-10,11-epoxides or benz[alanthracene-10,11-diol8,9-epoxides. 1,2-Epoxy-1,2,3,4-tetrahydrobenzlajanthracene is the most mutagenic and cytotoxic of the nine derivatives and is 5 and 25 times more mutagenic than 3,4-epoxy-1,2,3,4-tein bacterial and mammalian cells, trahydrobenla~anthracene respectively. In either test system, benz[alanthracene 5,6-oxide (K-region oxide) has less than 10% of the activity of any of the 1,2-epoxides derived from benzlajanthracene. The relative stabilities of the derivatives in aqueous solution do not account for the differences in mutagenic activity because the more mutagenic derivatives tend to be less stable. The benz[alanthracene diol epoxides, like the benzo[a pyrene diol epoxides, are refractory to the action of epoxide hydrase. The exceptional mutagenic activity of the 1,2-epoxide derivatives of benz[ajanthracene is consistent with and supportive of the hypothesis that bay region epoxides on saturated, anglar benzo-rings of unsubstituted polycyclic aromatic hydrocarbons are ultimate carcinogens. The diastereomeric pair of benzo[alpyrene (BP)-7,8-diol9,10-epoxides in which the epoxide oxygen is either cis (isomer 1 series) or trans (isomer 2 series) to the benzylic hydroxyl group have high chemical reactivity (1), covalently bind to nucleic acid (2-5), and have exceptional mutagenic and cytotoxic activity (6-9). Both of these diastereomers are metabolically formed from BP-7,8-dihydrodiol (7, 10-12), and one of the diastereomeric diol epoxides (isomer 2) has been shown to be highly carcinogenic (13). These findings and the high carcinogenicity of the two metabolic precursors of the diol epoxides, BP-7,8-dihydrodiol (13, 14) and BP-7,8-oxide (15), strongly suggest that the principal ultimate carcinogenic metabolite of
benzo[ajpyrene is a BP-7,8-diol-9,10-epoxide. A considerable
advance in our understanding of how polycyclic hydrocarbons cause cancer would be made if the key structural and chemical features of the BP-7,8-diol-9,10-epoxides could be identified and generalized to other polycyclic hydrocarbons. As a working hypothesis we have suggested (9, 16, 17) that the unique structural feature of the BP-7,8-diol-9,10-epoxides is the presence of an epoxide on a saturated, angular benzo-ring that forms part of a "bay region"t of the hydrocarbon. Support for this hypothesis comes from a reexamination of the available carcinogenicity data on substituted polycyclic hydrocarbons. The data indicate that substituents that would be expected to block formation of bay region diol epoxides markedly reduce carcinogenicity (16). Furthermore, perturbational molecular orbital calculations (17) for a number of carcinogenic polycyclic hydrocarbons predict more facile formation of chemically reactive benzylic carbonium ions from epoxides on saturated benzo-rings when they form part of a bay region of the hydrocarbon. Confirmation of the bay region hypothesis depends on the demonstration that the appropriate diol epoxides of a number of carcinogenic polycyclic hydrocarbons possess unique biological activity. Toward this end we have undertaken studies to evaluate the mutagenicity of the diol epoxides of benz[ajanthracene (BA). Because the bay region of benz[aJanthracene is located between the 1 and 12 positions of the molecule (Fig. 1), the bay region concept predicts that the 1,2-epoxide of BA-3,4-dihydrodiol should be uniquely active. This prediction differs from the proposal of Grover and Sims and associates (18, 19) that BA-8,9-diol-10,11-epoxide would have high biological activity. Recent studies consistent with our bay region concept have shown that BA-3,4-dihydrodiol is metabolized by a highly purified monooxygenase system to products which are 10 times as mutagenic to Salmonella typhimurium strain TA 100 as are the metabolites of benz[a]anthracene or the four other metabolically possible vicinial trans dihydrodiols of benz[ajanthracene (20). We have now synthesized a number of diol epoxides and tetrahydro-epoxides of benz[a]anthracene and have examined their inherent mutagenic and cytotoxic activities. As predicted from the bay region theory, 1,2-epoxides in a saturated angular benzo-ring of benz[aJanthracene are uniquely mutagenic and cytotoxic in both bacterial and mammalian cells.
Abbreviations: BP, benzo[ajpyrene; BA, benz[ajanthracene; BA1, (+)-3a,4(3-dihydroxy-1l3,2(3-epoxy-1,2,3,4tetrahydrobenz[a lanthracene; BA-3,4-diol-1,2-epoxide 2, (±)-3a, 4(3 - dihydroxy - la, 2a - epoxy - 1, 2, 3, 4 - tetrahydrobenz[ajanthracene; BA-8,9-diol-10,11-epoxides 1 and 2, and BA-10,11-diol-8,9epoxides 1 and 2, other diol epoxides of benz[a]anthracene with analogous stereochemistry; BA-H4-1,2-epoxide, 1,2-epoxy-1,2,3,4tetrahydrobenz[a]anthracene; BA-H4-3,4-epoxide, 3,4-epoxy1,2,3,4-tetrahydrobenz[a]anthracene; BA-5,6-oxide, benz[a]anthracene 5,6-oxide; BP-H4-9,10-epoxide, 9,1O-epoxy-7,8,9,10-tetrahydrobenzolalpyrene; BP-7,8-dihydrodiol, trans-7,8-dihydroxy-7,8-dihydrobenz[a]pyrene; BA-3,4-dihydrodiol, trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene; BA-1,2-, 8,9-, and 10,11-dihydrodiol, other BA-dihydrodiols.
3,4-diol-1,2-epoxide
* A bay region occurs in a polycylic aromatic hydrocarbon when an
angularly fused benzo-ring is present. The simplest example of a bay region is the sterically hindered area between the 4 and 5 positions of phenanthrene. The bay region of benz[a]anthracene is between carbon atoms 1 and 12 (see Fig. 1). In benzo[a]pyrene, the bay region is between positions 10 and 11.
2746
Biochemistry: Bay region
Wood et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
2
%OH. OH
8
7
6
Benz[a]anthracene
HO"9
BA-3,4-diol-1,2-epoxide 1
~HO,H
,
OH
BA-8,9-diol-10,11-epoxide 1
BA-10,11-diol-8,9-epoxide 1
FIG. 1. Structures of benz[a]anthracene (BA) and of the chemically more reactive diastereomers (isomer 1 series) of the diol epoxides derived from the trans-3,4-, .8,9-, and 11,12-dihydrodiols. The corresponding chemically less reactive diastereomers (isomer 2 series) have the benzylic hydroxyl groups at positions 4, 8, or 11 trans to the oxygen of the epoxide ring (1, 21).
MATERIALS AND METHODS Synthesis of Diol Epoxides and Tetrahydro-Epoxides. Diol epoxides of non-K-region dihydrodiols from benz[a]anthracene (see Fig. 1) were synthesized (21) by methods developed for the synthesis of diol epoxides from naphthalene-1,2-dihydrodiol and BP-7,8-dihydrodiol (1, 22). The trans dihydrodiol precursors were synthesized as described (23) using synthetic procedures originally developed for benzo[a 1pyrene dihydrodiols (24, 25). Direct oxidation of the dihydrodiols with peroxyacid produced diol epoxides (isomer 2 series) in which the benzylic hydroxyl group and oxygen of the epoxide ring are trans to each other, while intermediate bromohydrin formation followed by dehydrohalogenation to form the epoxide ring produced the diastereomeric diol epoxides (isomer 1 series) in which these functional groups are cis to each other. These diol epoxides derived from the trans-3,4-, trans-8,9-, and trans10,11-dihydrodiols were free of detectable impurities and free of stereoisomeric cross-contamination as determined by nuclear magnetic resonance spectroscopy and mass spectrometry (21). The procedures utilized in the synthesis of BA-H4-1,2-epoxide and BA-H4-3,4-epoxide were analogous to those used for the synthesis of the tetrahydro-epoxides of benzo[a]pyrene (26) and consisted of cyclization of trans-bromohydrin precursors at the indicated positions with base. BA-5,6-oxide (K-region) was synthesized by the chlorohydrin route as described (27). All of the derivatives were stored in the solid state at -90° and dissolved just before use in anhydrous dimethyl sulfoxide obtained by distillation from calcium hydride. Bacterial Mutagenesis Assay. The ability of the benz[a Janthracene epoxide derivatives to mutate bacteria was evaluated by previously described procedures (9, 28) in strain TA 100 of histidine-dependent Salmonella typhimurium LT-2 (29). Bacteria, obtained from B. Ames, University of California (Berkeley), were grown in Trypticase (Becton, Dickinson & Co.) soy broth containing 1% yeast extract and 0.02% ampicillin (Bristol Laboratories). After an overnight incubation in culture medium, the bacteria were collected by centrifugation and resuspended to a density of 1 X 109 cells per ml in phosphatebuffered saline (5 mM potassium phosphate/150 mM sodium chloride; final pH, 7.0). Epoxides were added in 15 ,ul of dimethyl sulfoxide to 2 X 108 bacteria in a final volume of 0.5 ml of phosphate- buffered saline and the mixture was then incubated for 5 min at 20°. After the 5-min incubation of epoxide with bacteria, 2 ml of top agar was added to the samples, and the entire contents of the culture tube were mixed and poured
2747
into a petri dish containing 15 ml of Vogel-Bonner medium with a 2% agar base. The petri dishes were then placed in the dark at 370 for 2 days, during which time only those bacteria that were mutated from histidine dependence to histidine independence were capable of growth. Mutation frequency was thus assessed by counting the macroscopic colonies of bacteria on the petri dishes at the end of the 2 days. Stability of the epoxides was evaluated by determining the decrease in the number of observed mutant colonies that resulted from preincubating the derivatives in 0.4 ml of phosphate-buffered saline for defined periods of time prior to addition of the bacteria. The samples were then incubated for 5 min prior to the addition of top agar. All experiments were performed in triplicate and coefficients of variation of the colony counts rarely exceeded 15%. Both the bacteria and the mammalian cells described below are devoid of detectable monooxygenase or epoxide hydrase activity and benz[a Janthracene is inactive as a mutagen in both systems. Mutagenesis Assay with Cultured Mammalian Cells. The Chinese hamster cell line V79-6 was kindly provided by E. H. Y. Chu, University of Michigan (Ann Arbor). Resistance to the lethal effects of the purine analog 8-azaguanine was used as the mutagenic marker in these studies. Procedures for culturing the cells, assessing cytotoxicity, and inducing 8-azaguanineresistant cells were adapted from Chu and Malling (30), and the conditions used were as described previously (9, 31) with the exception that 3 rather than 2 days elapsed between treatment of the cells with mutagen and the first addition of 8azaguanine. Derivatives were added in 20 Al of anhydrous dimethyl sulfoxide -to cells which were growing in 5 ml of culture medium. For each determination, four replicate culture dishes, each seeded with 102 cells, were used to evaluate toxicity. For each concentration of hydrocarbon, 16 replicate culture dishes, each seeded with 104 cells, were used to score 8-azaguanineresistant colonies. The mutagenic activity of each diastereomeric pair of epoxides was evaluated in the same experiment.
(i)-7#,8a-Dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydroben-
zo[a ]pyrene (isomer 2 series) (9), at two different concentrations, was included in all experiments as a positive control and gave essentially the same mutation frequency in all experiments (coefficient of variation was 14.9%, n = 5). In addition, all the compounds were tested at one concentration in a single experiment, and the absolute and relative mutagenic activities agreed with the previous experiments. In the studies to evaluate epoxide stability, the epoxides were preincubated in culture medium at 370 prior to addition to the cell cultures in 1.0 ml of medium, as previously described for the assessment of benzo[a 1pyrene diol epoxide stability (9). RESULTS All eight epoxide derivatives of benz[a ]anthracene induced reversions to histidine independence in strain TA 100 of Salmonella typhimurium in direct proportion to the applied dose (Fig. 2). However, there was an almost 400-fold range in mutagenic activities among the compounds. BA-H4-1,2-epoxide, the most mutagenic derivative, induced 2300 revertants per nmol, while BA-10,11-diol-8,9-epoxide 1, the least active derivative, induced 6 revertants per nmol. Diastereomers 1 and 2 of BA-3,4-diol-1,2-epoxide induced 1650 and 850 histidine revertants per nmol, respectively, and were the second and third most active derivatives. BA-H4-3,4-epoxide had 20% of the activity of the isomeric BA-H4-1,2-epoxide and 25% of the activity of BA-3,4-diol-1,2-epoxide 1. BA-8,9-diol-10,11-epoxide 2 and BA-10,11-diol-8,9-epoxide 2, the next most active compounds, both induced 50 revertants per nmol, and thus had 3%
Biochemistry:
2748
Proc. Nati. Acad. Sci. USA 74 (1977)
Wood et al.
R
~
U
2020
0
~~~~~~~~~0
04
0 0
0.2 0.4 0.6 0.8 1.01.2
10
20
Benz[a] anthracene derivative, nmol
FIG. 2. Mutagenicity of benz[a]anthracene epoxides in strain TA 100 of S. typhimurium. Bacteria were treated with the indicated amounts of the compounds as described in Materials and Methods. Each value for histidine-independent revertants represents the average of three replicate determinations and the spontaneous mutation frequency (120 revertants per plate) has been subtracted. BA-3,4diol-1,2-epoxides: *, isomer 1; 0, isomer 2. BA-8,9-diol-10,11-epoxides: A, isomer 1; A, isomer 2. BA-10,11-diol-8,9-epoxides: *, isomer 1; 0, isomer 2; X, BA-H4-1,2-epoxide; v, BA-H4-3,4-epoxide.
150-
' la
'eO
ta
0
io au
0
5o
v
1
2
3
10
20
30
40
Benz[a] anthracene derivative, nmol/ml
FIG. 3. Cytotoxicity (Upper) and mutagenicity (Lower) of
benz[a]anthracene epoxides in Chinese hamster V79 cells. Cells were of the mutagenic activity of BA-3,4-diol-1,2-epoxide 1. Finally, BA-8,9-diol-10,11-epoxide 1 had one-third the activity of its diastereomer and BA-10,11-diol-8,9-epoxide 1 had one-eighth the activity of its diastereomer. When tested at concentrations between 0.3 and 3.0 nmol per plate, BA-5,6-oxide induced approximately 100 revertants per nmol and thus had 6% of the activity of BA-3,4-diol-1,2-epoxide 1. BA-H4-1,2-epoxide was the most mutagenic and cytotoxic derivative in Chinese hamster V79 cells, inducing nearly 200 azaguanine-resistant variants in 105 surviving cells at a concentration of 1.2 nmol/ml and killing all the cells at a concentration of 3 nmol/ml (Fig. 3). Both diastereomeric 1,2-epoxides of BA-3,4-dihydrodiol killed 45% and 100% of the cells at concentrations of 3 and 10 nmol/ml, respectively. Depending upon the concentration, BA-3,4-diol-1,2-epoxide 2 was from 15 to 60% more mutagenic than its chemically more reactive (21) diastereomer BA-3,4-diol-1,2-epoxide 1. At equal concentrations, the diastereomeric BA-3,4-diol-1,2-epoxides induced fewer mutations than BA-H4-1,2-epoxide. However, the BA-3,4-diol-1,2-epoxide diastereomers induced more mutations than BA-H4-1,2-epoxide at equitoxic concentrations. BA-H43,4-epoxide had 10% of the cytotoxic activity and approximately 4% of the mutagenic activity of the isomeric BA-H41,2-epoxide. The mutagenic activities of the diastereoisomeric BA-8,9-diol-10,11-epoxides and BA-10,11-diol-8,9-epoxides were in all cases modest and did not exceed 1% of the mutagenic activities of the BA-3,4-diol-1,2-epoxides. When tested at concentrations between 1 and 15 nmol/ml, BA-5,6-oxide induced no more than 0.2 8-azaguanine-resistant colonies/105 survivors per nmol and thus had less than 2% of the activity of the BA-3,4-diol-1,2-epoxides. Because unstable epoxides might decompose at a rate sufficient to preclude interaction with critical receptors in the tester cells, the biological half-life of the eight epoxides was estimated in both phosphate-buffered saline and tissue culture medium. In phosphate-buffered saline, BA-H4-1,2-epoxide had a 2- to 4-min half-life, the diastereomeric pair of BA-3,4-diol-1,2epoxides had half-lives of 6-7 min, BA-H4-epoxide had a half-life of 8-10 min, and the diastereomers of BA-8,9-diol10,11-epoxide or of BA-10,11-diol-8,9-epoxide were stable,
treated with the indicated amounts of the compounds as described in Materials and Methods and the spontaneous mutation frequency in solvent-treated cultures never exceeded 1.5 8-azaguanine-resistant colonies per 105 surviving cells. See Fig. 2 for key to symbols.
exhibiting no detectable loss of mutagenicity after preincubation in phosphate-buffered saline for up to 60 min. In tissue culture medium, BA-H4-1,2-epoxide and BA-3,4-diol-1,2epoxide 1 had 3-min half-lives, BA-3,4-diol-1,2-epoxide 2 had a 7- to 8-min half-life, BA-H4-3,4-epoxide had a 5- to 8-min half-life, and BA-10,11-diol-8,9-epoxide 2 had a half-life of 7 min. BA-10,11-diol-8,9-epoxide 1 and the diastereomeric BA8,9-diol-10,11-epoxides had insufficient biological activity in V79 cells to evaluate stability in tissue culture medium. These results indicate that the relative stabilities of the derivatives in aqueous solution cannot account for the differences in mutagenic activity because the more mutagenic derivatives tended to be less stable. Microsomal epoxide hydrase catalyzes the trans addition of water to a variety of arene and alkene oxides and preincubation of six units of the highly purified enzyme (32)§ with BA-5,6oxide or BA-H4-3,4-epoxide for 5 min resulted in a complete loss of mutagenic activity of these two compounds toward strain TA 100 of S. typhimurium (data not shown). However, 20 units of the enzyme decreased the mutagenic activity of the six benz[ajanthracene diol epoxides by less than 20% and reduced the mutagenicity of BA-H4-1,2-epoxide by only 50%. Thus, as has been found for the benzo[a]pyrene diol epoxides (9, 10, 33), the diol epoxides of benzo[a ]anthracene are also very poor substrates for purified epoxide hydrase. DISCUSSION Benzo-ring epoxides of non-K-region trans dihydrodiols exist as pairs of diastereomers in which the oxygen of the epoxide ring is either cis (isomer 1 series) or trans (isomer 2 series) to the benzylic hydroxyl group (see Fig. 1). Three of the diaste§ Units of epoxide hydrase activity are defined as nmol of styrene glycol formed from styrene oxide per minute at 370.
Biochemistry: Wood et al. reomeric pairs of benzo-ring diol epoxides derived from the weak carcinogen benz[a janthracene have been evaluated for mutagenic activity in bacterial and mammalian cells. The BA-3,4-diol-1,2-epoxides were highly mutagenic in both systems and were from one to three orders of magnitude more mutagenic than BA-5,6-oxide (K-region oxide) or two pairs of BA diol epoxides with the epoxide ring at the 8,9- or 10,11positions. Estimations of the biological half-lives of the BA diol epoxides excluded the possibility that the low mutagenicity of the BA-8,9-diol-10,11-epoxides and the 10,11-diol-8,9-epoxides was due to decomposition in the test medium. Thus, our results argue against the proposal of Grover and Sims and their associates (18, 19) that the BA-8,9-diol-10,11-epoxides are the important biologically active metabolites derived from benz[a Janthracene. The large differences in mutagenic activities of the various diol epoxides dramatically illustrate the importance of the position of the oxirane ring for biological activity. The chemical reactivity of an epoxide at different positions of a hydrocarbon molecule can be predicted by simple quantum mechanical calculations based on the ease of carbonium ion formation. These perturbational molecular orbital calculations had predicted that the 1,2-epoxides of either benz[a]anthracene-3,4dihydrodiol or of 1,2,3,4,-tetrahydrobenz[a]anthracene would be more mutagenic and cytotoxic than any of the other diol epoxides or tetrahydro-epoxides of benz[a]anthracene (17). The unique structural feature of these highly mutagenic 1,2-epoxide derivatives of benz[a ]anthracene is the presence of an epoxide on a saturated angular benzo-ring which forms part of the bay region of the hydrocarbon. The high mutagenicity of BAH4-1,2-epoxide in the present study and of BP-H4-9,10-epoxide in a previous study (9) illustrates that the hydroxyl groups are not obligatory for the high mutagenic activity of bay region diol epoxides. The critical role of the 3,4-dihydrodiol of benz[a ]anthracene and the 7,8-dihydrodiol of benzo[a]pyrene in the metabolism of these hydrocarbons is that of providing a substrate which can be converted into an aliphatic epoxide as opposed to an arene oxide. The importance of saturation of the benzo-ring is illustrated by the fact that BP-H4-9,10-epoxide is at least 100-fold more mutagenic than the corresponding arene oxide, BP-9,10-oxide, in which the 7,8-double bond is present. In addition to predicting the high mutagenic activity of the BA-3,4-diol-1,2-epoxides relative to the other benz[a]anthracene diol epoxides, the quantum mechanical calculations also indicated that the bay region diol epoxides of the strong carcinogen benzo[a ]pyrene would be more biologically active than the bay region diol-epoxides of the weak carcinogen benz[a ]anthracene (17). Under the same assay conditions, the diastereomeric 7,8-diol-9,10-epoxides of benzo[a]pyrene are severalfold more mutagenic than the diastereomeric 3,4-diol1,2-epoxides of benz[a]anthracene in both bacterial and mammalian cells. Also in accord with these calculations is the lack of significant mutagenic activity for the diastereomeric pair of diol epoxides derived from trans-1,2-dihydroxy-1,2dihydronaphthalene (unpublished observations). The results presented in this communication on the high mutagenicity of the diastereomeric pair of BA-3,4-diol-1,2epoxides, and a previous study demonstrating the marked metabolic activation of BA-3,4-dihydrodiol (20), suggest that one or both of these bay region diol epoxides are the ultimately reactive forms of benz[a]anthracene. Recent studies in our laboratories indicating high tumorogenicity for BA-3,4-dihydrodiol (34) support this conclusion and the bay region hypothesis of hydrocarbon carcinogenesis.
Proc. Natl..Acad. Scd. USA 74 (1977)
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Note Added in Proof. Biological activity of the 3,4-epoxides of BA, 1,2-dihydrodiol has not been described in this study due to technical difficulties in the synthesis and purification of diol epoxides derived from bay region dihydrodiols. However, a small sample of a BA-1,2diol-3,4-epoxide, the nuclear magnetic resonance spectrum of which suggested the isomer 2 series, was obtained, and was found to have mutagenic activity similar to or less than that of the diol epoxides derived from BA-8,9- and 10,11-dihydrodiols. The authors thank Mrs. Arlene Ott for her assistance in the preparation of this manuscript. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Yagi, H., Hernandez, 0. & Jerina, D. M. (1976) J. Am. Chem. Soc. 97,6881-6883. 2. Sims, P., Grover, P. L., Swaisland, A., Pal, K. & Hewer, A. (1974) Nature 252,326-328. 3. Koreeda, M., Moore, P. D., Yagi, H., Yeh, H. J. C. & Jerina, D. M. (1976) J. Am. Chem. Soc. 98, 6720-6722. 4. King, H. W. S., Osborne, M. R., Beland, F. A., Harvey, R. G. & Brooks, P. (1976) Proc. Natl. Acad. Sci. USA 73,2679-2681. 5. Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H. & Nakanishi, K. (1976) Science 193, 592-595. 6. Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., Yagi, H., Hernandez, O., Jerina, D. M. & Conney, A. H. (1976) Biochem. Biophys. Res. Commun. 68, 1006-1012. 7. Huberman, E., Sachs, L., Yang, S. K. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73, 607-611. 8. Newbold, R. F. & Brooks, P. (1976) Nature 261, 52-54. 9. Wood, A. W., Wislocki, P. G., Chang, R. L., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O., jerina, D. M. & Conney, A. H. (1976) Cancer Res. 36, 3358-3366. 10. Thakker, D. R., Yagi, H., Lu, A. Y. H., Levin, W., Conney, A. H. & Jerina, D. M. (1976) Proc. Natl. Acad. Sci. USA 73, 33813385. 11. Yang, S. K., McCourt, D. W., Roller, P. R. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73,2594-2598. 12. Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, A. Y. H., Levin, W., Wood, A. W., Conney, A. H. & Jerina, D. M. (1977) Chem. Biol. Interact. 16,281-300. 13. Kapitulnik, J., Levin, W., Conney, A. H., Yagi, H. & Jerina, D. M. (1977) Nature 266,378-380. 14. Levin, W., Wood, A. W., Yagi, H., Jerina, D. M. & Conney, A. H. (1976) Proc. Natl. Acad. Sci. USA 73,3867-3871. 15. Levin, W., Wood, A. W., Yagi, H., Dansette, P. M., Jerina, D. M. & Conney, A. H. (1976) Proc. Natl. Acad. Sci. USA 73, 243247. 16. Jerina, D. M. & Daly, J. W. (1977) in Drug Metabolism, eds. Parke, D. V. & Smith, R. L. (Taylor and Francis, London), pp. 13-33. 17. Jerina, D. M., Lehr, R. E., Yagi, H., Hernandez, O., Dansette, P. M., Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W. & Conney, A. H. (1976) in In Vitro Metabolic Activation in Mutagenesis Testing, eds. deSerres, F. J., Fouts, J. R., Bend, J. R. & Philpot, R. M. (Elsevier/North Holland Biomedical Press, Amsterdam), pp. 159-177. 18. Swaisland, A. J., Hewer, A., Pal, K., Keysell, G. R., Booth, J., Grover, P. L. & Sims, P. (1974) FEBS Lett. 47,34-38. 19. Malaveille, C., Bartsch, H., Grover, P. L. & Sims, P. (1975) Biochem. Biophys. Res. Commun. 66,693-700. 20. Wood, A. W., Levin, W., Lu, A. Y. H., Ryan, D., West, S. B., Lehr, R. E., Schaefer-Ridder, M., Jerina, D. M. & Conney, A. H. (1976) Biochem. Biophys. Res. Commun. 72,680-686. 21. Lehr, R., Schaeffer-Ridder, M. & Jerina, D. M. (1977) Tetrahedron Lett., 539-542.
22.
Yagi, H., Thakker, D., Hernandez, O., Koreeda, M. & Jerina, D.
2750
Biochemistry: Wood et al.
M. (1977) J. Am. Chem. Soc. 99, 1604-1611. 23. Lehr, R. E., Schaeffer-Ridder, M. & Jerina, D. M. (1977) J. Org. Chem. 42,736-744. 24. Gibson, D. T., Mahadevan, V., Jerina, D. M., Yagi, H. & Yeh, H. J. C. (1975) Science 189,295-297. 25. McCaustland, D. J. & Engel, J. F. (1975) Tetrahedron Lett.,
2549-2552. 26. Yagi, H., Holder, G. M., Dansette, P., Hernandez, O., Yeh, H. J. C., LeMahieu, R. A. & Jerina, D. M. (1976) J. Org. Chem. 41, 977-985. 27. Dansette, P. M. & Jerina, D. M. (1974) J. Am. Chem. Soc. 96, 1224-1225. 28. Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., Yagi, H., Hernandez, O., Dansette, P. M., Jerina, D. M. & Conney, A. H. (1976) Cancer Res. 36, 350-3357.
Proc. Nati. Acad. Sci. USA 74 (1977) 29. McCann, J., Spingarn, N. E., Kobori, J. & Ames, B. N. (1975) Proc. Natl. Acad. Sci. USA 72,979-983. 30. Chu, E. H. Y. & Mailing, M. V. (1968) Proc. NatI. Acad. Sci. USA 61, 1306-1312. 31. Wood, A. W., Goode, R. L., Chang, R. L., Levin, W., Conney, A. H., Yagi, H., Dansette, D. M. & Jerina, D. M. (1975) Proc. Natl. Acad. Sci. USA 72,3176-3180. 32. Lu, A. Y. H., Ryan, D., Jerina, D. M., Daly, J. W. & Levin, W. (1975) J. Biol. Chem. 250,8283-8288. 33. Wood, A. W., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O., Jerina, D. M. & Conney, A. H. (1976) J. Biol. Chem. 251, 4882-4890. 34. Wood, A. W., Levin, W., Chang, R. L., Lehr, R. E., SchaeferRidder, M., Karle, J., Jerina, D. M. & Conney, A. H. (1977) Proc. Natl. Acad. Sci. USA 74, in press.
