Carcinogenicity and Mutagenicity of Benz(a)anthracene Diols and … · [CANCER RESEARCH 38,...
Transcript of Carcinogenicity and Mutagenicity of Benz(a)anthracene Diols and … · [CANCER RESEARCH 38,...
[CANCER RESEARCH 38, 1699-1704, June 1978]
Carcinogenicity and Mutagenicity of Benz(a)anthracene Diols andDiol-Epoxides1
Thomas J. Slaga,2 Eliezer Huberman, James K. Selkirk, Ronald G. Harvey, and William M. Bracken
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 [J. J. S., E. H., J. K. S., W. M. B.], and Ben May Laboratory lor CancerResearch, University of Chicago, Chicago, Illinois 60680 ¡R.G. H.]
ABSTRACT
Benz(a)anthracene (BA) and its five possible trans-di-hydrodiols were evaluated for determination of their skintumor-initiating activity and their mutagenic activity inChinese hamster V79 cells. In addition, the skin tumor-initiating abilities of five diol-epoxides of BA were tested.Results showed (±)-trans-3,4-dihydroxy-3,4-dihydrobenz-(a)anthracene (BA 3,4-dihydrodiol) to be approximately 10times more mutagenic than was BA and about 20 timesmore mutagenic than were the other possible dihydrodiolsin the V79 cells cocultivated with irradiated hamster embryo cells. As a skin tumor initiator, BA 3,4-dihydrodiolwas approximately 5 times more active than BA, whereasthe other BA dihydrodiols were all less active tumorinitiators. (±)-frans-3a,40-Dihydroxy-1a,2c*-epoxy-1,2,3,4-tetrahydrobenz(a)anthracene was found to be approximately 20% more active as a tumor initiator than was BA3,4-dihydrodiol, whereas the other diol-epoxides of BAwere less active than BA itself. The results suggest thatthe bay-region diol-epoxide of BA may be the ultimatecarcinogenic and mutagenic form of BA.
INTRODUCTION
The results obtained from in vitro and in vivo binding (2,6, 16, 22, 23, 26, 34), mutagenicity (12, 20, 25, 35, 36),metabolism (27, 33, 41), and carcinogenicity (5, 17-19, 30-32) studies have led us to the conclusion that frans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene is a proximate carcinogenic metabolite of BP3 and that the BP-7ß,8«-diol-9a,10a-epoxide is an ultimate carcinogenic metabolite ofBP. Recent results with the strong carcinogenicity of BP7/3,8a-diol-9«,10a-epoxide in the newborn mouse (15)strongly indicate that it is the ultimate carcinogenic metabolite of BP.
Since diol-epoxides may be responsible for the carcinogenicity of PAH other than that of BP, we have undertakenthe testing of diols and diol-epoxides of some of theseothers for skin tumor initiation in mice and mutagenesis in
1 Supported in part by NIH Grant CA-20076 and by the Department of
Energy under contract with the Union Carbide Corporation.' To whom requests for reprints should be addressed.3 The abbreviations used are: BP, benzo(a)pyrene; BP 7ß,8u-diol-9«,
10a-epoxide, (±)-r/-ans-7/3,8«-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydro-
benzo(a)pyrene; PAH, polyaromatic hydrocarbons; BA, benz(a)anthracene;BA 3,4-dihydrodiol, (±)-frans-3,4-dihydroxy-3,4-dihydrobenzo(a)anthracene;TPA, 12-O-tetradecanoylphorbol-13-acetate; BA 3,4-diol-1,2-epoxide I, (±)-frans-3a,4/3-dihydroxy-1 a,2a-epoxy-1,2,3,4-tetrahydrobenz(a (anthracene;BA 8,9-diol-10,11-epoxide I, (±)-frar7s-8/3,9a-dihydroxy-10a,11a-epoxy-8,9,10,11-tetrahydrobenz(a(anthracene; DMBA, 7,12-dimethylbenz(a(anthracene.
Received January 3, 1978; accepted March 10, 1978.
Chinese hamster V79 cells. In this study we compared theskin tumor initiation in mice and mutagenesis in V79 cellsof several diols and diol-epoxides of BA. During the courseof this investigation, Wood ef al. (37, 39) reported that BA3,4-diol-1,2-epoxides, in which the epoxide forms part ofthe "bay region" (see Chart 3), were more mutagenic to
bacterial cells than were the other diol-epoxides. Wood e?al. (38) also reported that BA-3,4-dihydrodiol was moretumorigenic toward mice than were BA or the 4 other possible frans-dihydrodiols of BA. In our studies we found thatboth the BA 3,4-dihydrodiol and BA 3,4-diol-1,2-epoxide Iare much stronger skin tumor initiators than were BA or theother possible diols and diol-epoxides of BA. Also, BA 3,4-dihydrodiol was found to be a potent mutagen in mammalian cells.
MATERIALS AND METHODS
Chemicals. BP and BA were purchased from AldrichChemical Company, Milwaukee, Wis. and were more than99% pure. TPA was obtained from Dr. Peter Borchert,University of Minnesota, Minneapolis, Minn. The BA diolsand diol-epoxides were synthesized and purified as previously described (8, 9). The BA diol-epoxides were appliedtopically in tetrahydrofuran (distilled over LiAIH, and storedover sodium wire). BP and BA diols were dissolved inspectroquality acetone to give a concentration of 400 nmolof compound per 0.2 ml of solvent. All hydrocarbons wereconsistently prepared in the dark immediately before use.The mice were treated topically with the above compoundsat a dose of 2 ¿u.molunder subdued light. The time lapsebetween preparation of the above solutions and animaltreatment was less than 0.5 hr. TPA was prepared in stocksolutions and kept in a freezer until use. Mice receivedtwice-weekly applications of 10 ^g of TPA 1 week aftertreatment.
Tumor Experiments. Female CD-1 mice were purchasedfrom Charles River Breeding Laboratory, Wilmington, Mass.Mice, 7 to 9 weeks old, were shaved with surgical clippers2 days before treatment, and only those in the resting phaseof the hair cycle were used. In the tumor experiments,groups of 30 animals received a single topical applicationof the test compound, followed 1 week later by twice-weeklyapplications of TPA. The incidence of both papillomas andcarcinomas was recorded weekly, and papillomas and carcinomas were removed at random for histological verification.
Cell Culture. Chinese hamster V79 cells, derived from asubclone of V79-4 cells kindly supplied by E. H. Y. Chu,University of Michigan, Ann Arbor, Mich., and secondary
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cultures from whole golden hamster embryos, which servedas polycyclic hydrocarbon-metabolizing cells, were grownin Dulbecco's modified Eagle's medium with 10% fetal calf
serum (Grand Island Biological Co., Grand Island, N. Y.)and incubated at 37 ±0.5°(S.D.). For each 50-mm tissue
culture Retri dish (Falcon Plastics, Oxnard, Calif.), 5 ml ofmedium were used. The cultures were incubated in ahumidified incubator supplied with a constant amount of10% COo in air. Secondary hamster embryo cells wereirradiated with 5000 R of X-rays, seeded at 2 x 106cells/50-mm Retri dish, and used for experiments 1 day after irradiation. These cells had about an 80% plating efficiency.
Direct and Cell-mediated Mutagenesis Assay. Ouabainresistance was tested by seeding 3 x 105 V79 cells in 4 ml
of medium into a Retri dish without the hamster cells forthe direct assay, or on a 1-day-old monolayer of 5000-R-irradiated normal golden hamster cells seeded at 2 x 106cells/Petri dish for the cell-mediated assay. BP, BA, andderivatives were added 5 hr after the V79 cells were seededin 1 ml of medium, and the cultures were incubated for 2days. The cells were then dissociated with a trypsin-EDTAsolution and seeded at 200 cells/Petri dish in 5 ml ofmedium to determine cloning efficiency and at 105 cells/Petri dish in 4 ml of medium to determine the frequency ofouabain-resistant mutants. For selection of the mutants,ouabain at a final concentration of 1 FTIMwas added in 1 mlof medium 2 days after cell seeding. The colonies werecounted after Giemsa staining. Cloning efficiency was determined by a count of the number of colonies in 6 to 8Petri dishes/point at 7 to 8 days after cell seeding, and thefrequency of ouabain-resistant mutants was determined bycounting 24 to 32 Petri dishes/point at 14 to 16 days aftercell seeding. In the mutagenesis assays, with or withoutcocultivation with the normal golden hamster cells, themutation frequency for resistance to ouabain was calculated per 106survivors, based on the cloning efficiency andthe number of cells seeded for mutant selection. The resultswere based on 2 experiments/point. The number of mutantsafter induction with BP or the f/-ans-3,4-dihydrodiols of BA
varied up to 25%. In all other cases, the number of mutantsin the different experiments varied up to 40%.
RESULTS
Shown in Table 1 are the skin tumor-initiating activities ofBA and the 1,2-, 3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols ofBA. BA 3,4-dihydrodiol was the most active tumor initiatorwhen compared with BA and its other dihydrodiols. After 26weeks of promotion with TPA, BA 3,4-dihydrodiol hadinduced tumors in 85% of the animals, with an average of4.7 papillomas/mouse, whereas BA induced tumors in 57%of the animals, with approximately 1 papilloma/mouse. The1,2- and 8,9-dihydrodiols of BA had weak tumor-initiatingactivity, whereas the other dihydrodiols were extremelyweak. Although BA 3,4-dihydrodiol at a dose level of 2 /umolis a good tumor initiator when compared with BA, its tumor-initiating activity is less impressive when compared with BPat a dose of 0.2 jumol (Table 1).
The incidence of skin tumors in female CD-1 mice after asingle topical application of BA, BA 3,4-diol-1,2-epoxide I,(±)-frans-1/3,2a-dihydroxy-3«,4«-epoxy-1,2,3,4-tetrahydro-benz(a)anthracene, BA 10,11-diol-8,9-epoxide I, BA 8,9-diol-10,11-epoxide I, or (±)-frans-8jO,9«-dihydroxy-10/3,11/3-epoxy-8,9,10,11-tetrahydrobenz(a)anthracene, followed bytwice-weekly applications of TRA, is shown in Table 2. BA3,4-diol-1,2-epoxide I was found to be approximately 5times more active than was BA. This should be noted as afirst example of a reactive intermediate of a PAH that wasmore active as a tumor initiator on mouse skin than was theparent hydrocarbon. BA 8,9-diol-10,11-epoxide I had weaktumor-initiating activity in mouse skin, whereas the otherdiol-epoxides were extremely weak in this regard. The timecourse of tumor induction by BA, BA 3,4-dihydrodiol, andBA 3,4-diol-1,2-epoxide I over a 26-week period of promotion by TPA is illustrated in Chart 1. The tumor-initiatingactivity of BA 3,4-diol-1,2-epoxide I was slightly greater thanthat of BA 3,4-dihydrodiol, especially in terms of the percentage of mice with tumors. All the mice developed tumors
Table 1Skin tumor-initiating activities of BP, BA, and BA dihydrodiols after TPA tumor
promotionAll of the BA compounds were applied at a dose of 2 /¿mol,whereas BP was applied at
0.2 tirnol. Each of these treatments was followed 1 week later by twice-weekly applications of 10 fig of TPA.
Initiator
Total no. of papillo-No. of mice mas/total no. of sur-
surviving at the viving mice26th week after
promotion 15 wk 26 wk
" Treated once with 2 /¿molBA; not followed by promotion.* Treated with TPA only, twice weekly for 26 weeks.
% of surviving micewith papillomas
15 wk 26 wk
BPBABA
1,2-dihydrodiolBA3,4-dihydrodiolBA5,6-dihydrodiolBA
8,9-dihydrodiolBA10,11-dihydrodiolBAcontrols"TPA
control*2930302829302930292.00.70.23.20.10.20.1005.31.20.44.70.20.80.200.160371380101810009257338514461406
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Carcinogenesis and Mutagenesis of BA Diols and Diol-Epoxides
Table 2Skin tumor-initiating activities of BA and BA diol-epoxides after TPA tumor promotion
All of the compounds were applied at a dose of 2 /xmol and were followed 1 week later by twice-weekly applications of 10 pg of TPA.
No. of micesurviving atthe•?£th
uuooliaftorInitiator
promotionBA(±)-frans-1/3,2a-Dihydroxy-3a,4a-epoxy-1
,2,3,4-tetrahydrobenz(a)anthraceneBA
3,4-diol-1 ,2-epoxideIBA8,9-diol-10,11-epoxideI(±)-frans-8/3,9a-Dihydroxy-1
0/3,11 /3-epoxy-8,9, 10,11-tetra-hydrobenz(a)anthraceneBA
10,11-diol-8,9-epoxide I302827292829Total
no. of papillo-mas/total no. of sur
vivingmice15
wk0.504.20.300.126wk1.00.15.00.60.20.2%
of surviving micewithpapillomas15
wk380100250726wk5210100402017
100-
80
B 60
-o-o-o-o-o-o-o-o-o
o BA-3,4-dioi-l,2-epox«Je1/ A BA-3,4-diol
/ •BA
15 20 25
WEEKS OF PROMOTION
Chart 1. The skin tumor-initiating activities of BA, BA 3,4-dihydrodiol, andBA 3,4-diol-1,2-epxoide I. Each group contained 30 mice, and all mice wereinitiated with 2 ¿¿molof one of the above compounds. One week afterinitiation, the mice were promoted twice weekly with 10 /¿gTPA. Top,percentage of mice with papillomas as a function of weeks of tumorpromotion; bottom, average number of papillomas per mouse as a functionof weeks of tumor promotion. •BA; A, BA 3,4-dihydrodiol; O, BA 3,4-diol-1,2-epoxide I.
when initiated with BA 3,4-diol-1,3-epoxide I, whereas 85%developed skin tumors after BA 3,4-dihydrodiol initiation.
The mutagenic activity of BA dihydrodiols was also determined in order to discover whether a correlation existsbetween their carcinogenic and mutagenic activities. Directmutagenicity (i.e., not requiring further metabolism of thehydrocarbons) was determined by testing of BP, BA, andthe 4 possible dihydrodiols for mutagenicity to ouabainresistance in V79 Chinese hamster cells, which do notmetabolize polycyclic hydrocarbons. The compounds included (±)-frans-1,2-dihydroxy-1,2-dihydrobenz(a)anthra-cene, BA 3,4-dihydrodiol, (±)-frans-5,6-dihydroxy-5,6-di-hydrobenz(a)anthracene, and (±)-frans-8,9-dihydroxy-8,9-dihydrobenz(a)anthracene. The results indicated that neither BA, nor BP, nor any of the 4 derivatives were mutagenicfor ouabain resistance in V79 cells at concentrations of up
to about 4 /LiM.Also, none of the tested compounds wascytotoxic at the above concentrations. Since the testedhydrocarbons were not mutagenic in the absence of poly-cyclic hydrocarbon-metabolizing cells, it was important todetermine whether these derivatives would be mutagenic inthe presence of cells capable of metabolizing the hydrocarbons. Therefore, V79 cells cocultivated in the cell-mediatedassay (10, 11) were treated with the above derivatives, andtheir mutagenicity was compared with that of the parenthydrocarbon, BA. The results indicated that BP and BA3,4-dihydrodiol were cytotoxic and mutagenic and that themutagenic response increased as a function of the hydrocarbon concentration (Table 3). It is clearly shown in Chart2 that, although BA 3,4-dihydrodiol was about 10 timesmore potent a mutagen than BA, it still had much less mutagenic activity than BP. The other dihydrodiols of BA hadless activity than had the parent hydrocarbon.
DISCUSSION
The results of this investigation and those recently reported (37-39) indicate that BA 3,4-dihydrodiol may represent the proximate and BA 3,4-diol-1,2-epoxide I may represent the ultimate carcinogenic and mutagenic forms of BA.This is in agreement with the theory proposed by Jerina etal. (13, 14), which predicts that diol-epoxides in the bayregion are the principal determinants of PAH carcinogenic-ity. A bay region occurs in a PAH when an angularly fusedbenzo ring is present (13). The bay region of BA is betweencarbon atoms 1 and 12 (Chart 3). Recent studies by Woodef al. (40) on the high mutagenicity of frans-1,2-dihydrodiolsof chrysene provide additional support to the bay-regiontheory of PAH.
This is the first report wherein both a proximate and anultimate carcinogenic form of a PAH were found to be muchmore active on mouse skin than was the parent hydrocarbon. However, BA is generally considered to be noncarcin-ogenic in most/n vivo animal tumorigenesis models (28), aswell as in in vitro cell transformation systems (4, 7). Wepreviously reported that, although not a complete skincarcinogen, BA is a weak skin tumor initiator in a 2-stagetumorigenesis system (28).
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Table 3Induction of mutagenicity in the cell-mediated assay by BP and BA and its trans-
d/hydrodiolsThe polycyclic hydrocarbons tested were added 5 hr after 3 x 105V79 cells were seen
on 2 x 106 irradiated golden hamster cells. Two days after treatment, the cells weredissociated and seeded to determine the cloning efficiency of the V79 cells and thefrequency of ouabain-resistant mutants. At the time when the cells were seeded forcloning efficiency and selection of mutation, there were in the control and in thehydrocarbon-treated cultures from 1.5 to 2.7 x 106V79 cells/Petri dish.
HydrocarbonControlBABA
1,2-dihydrodiolBA
3,4-dihydrodiolBA
5,6-dihydrodiolBA
8,9-dihydrodiolBPConcen
tration(MM)4.444.04.141.01.2
4.112.041.04.141.04.141.00.4
1.34.0Cloning
efficiency79737476
7765
60523164
6571
6159
3823No.
of ouabain-resistant mutants/106
survivors"0.70.9
2.10.8
0.91.0
2.38.2
23.00.8
0.90.9
1.211.0
25.099.0
" The maximum percentage of S.D. was less than 25%.
Chart 2. Induction of mutagenicity in V79 cells by use of the cell-mediatedassay with BP, BA, and BA 3,4-dihydrodiol. The PAH were added 5 hr after 3x 10" V79 cells were seeded on 2 x 106irradiated golden hamster cells. Twodays after treatment, the cells were dissociated and seeded for determinationof the cloning efficiency of the V79 cells and the frequency of ouabain-resistant mutants. CONCN, concentration.
If one compares the skin tumor-initiating activity of BP,BA, and their possible cellular metabolites, some veryimportant differences emerge. BP is between 50 and 100times more potent as a tumor initiator than is BA. The (±)-frans-7,8-dihydrodiol of BP has approximately the sametumor-initiating activity as BP, whereas (±)-frans-3,4-dihy-drodiol of BA has about 5 times the activity of BA. The bay-
BENZWâNTHRACENE BA-3.4-DIOL-1, 2 EPOXIDEI
Chart. 3. Structures of BA and BA 3,4-diol-1.2-epoxide I.
region diol-epoxide of BP was found to be about one-thirdas active as BP, while the bay-region diol-epoxide of BAwas at least 5 times more active than BA. The skin tumor-initiating activities of BA 8,9-dihydrodiol and BA 8,9-diol-10,11-epoxide I correspond more closely to the activities offrans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene and BP7/3,8«-diol-9a,10«-epoxide.
There are several possible reasons for the above differences in skin tumor-initiating activities of BP, BA, and theirpossible cellular metabolites: (a) the proportion of BPmetabolized to the bay-region diol-epoxide may be greaterthan is that of BA; (b) BA may not be metabolized in the bayregion in target cells but may be metabolized mainly to the8,9-dihydrodiol and then to the 8,9-diol-10,11-epoxide; (c)the ultimate carcinogenic metabolite of BP may not havebeen found, or there may be several metabolites contributing to the activity of BP. Since BA is a very weak tumorinitiator (28), the 8,9-dihydrodiol has been detected as acellular metabolite (1, 3), and the BA 8,9-diol-10,11-epoxidehas been implicated in BA binding to DMA (1); the secondpossibility appears to be the most likely reason for thenoted differences.
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Carcinogenesis and Mutagenesis of BA Diols and Diol-Epoxides
The extremely potent carcinogenic activity of DMBA maybe related to its metabolism to a bay-region diol-epoxide.The presence of the 7,12-dimethyl groups may favor attackat the bay region, whereas BA without the methyl groupsis predominantly metabolized at the 8,9,10,11 positions.We have recently found that the 8,9-dihydrodiol and the8,9-diol-10,11-epoxide I of DMBA are very weak tumorinitiators and that a fluoro or methyl addition to the 1 and 2positions completely destroys DMBA tumor-initiating activity.4 In addition, Moschel er al. (24) have presented fluorescence data that implicate the bay-region diol-epoxide ofDMBA in the interaction of DMBA with cellular DMA.
The mutagenesis studies in the V79 cells indicate that BA3,4-diol-1,2-epoxide I, not BA 3,4-dihydrodiol itself, is responsible for the mutagenicity of BA 3,4-dihydrodiol. BAand the 4 possible ffans-dihydrodiols of BA were all foundto be without mutagenic activity in the direct mutagenesisassay. However, in the presence of irradiated golden hamster embryo cells, which are capable of metabolizing PAH,BA 3,4-dihydrodiol was mutagenic to V79 cells, whereas BAand the other dihydrodiols of BA were either negative orextremely weak. The mutagenic activities of BP, BA, andBA dihydrodiols revealed a good correlation with their skintumor-initiating activities. In general, there is both a qualitative and a quantitative correlation between the skin tumor-initiating activities of many PAH and PAH derivatives andtheir mutagenic activities in V79 cells cocultivated withirradiated hamster embryo cells (10-12, 30-32, 35). There ismuch less of a correlation between PAH mutagenesis andcarcinogenesis when mutagenic activity in various strainsof Salmonella typhimurium (30-32, 35, 36, 39) is comparedwith skin tumor-initiating activity. This is especially truewhen comparing the skin tumor-initiating activities in miceand the mutagenic activities in Salmonella of DMBA todibenz(a,c)anthracene and BP 7/3,8a-diol-9a,10a-epoxideto (±)-frans-7/3,8a-dihydroxy-9/3,10)3-epoxy-7,8,9,10-tetra-hydrobenzo(a)pyrene (21, 29, 30, 35, 36). DMBA is a verypotent and dibenz(a,c)anthracene a very weak skin tumorinitiator, while in S. typhimurium dibenz(a,c)anthracene ismore mutagenic than DMBA (21, 29). A similar situationexists for the diol-epoxides of BP (30, 36). However, theirmutagenic activities in V79 cells correlate with their skintumor-initiating activity.
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)anthracene DiolsaCarcinogenicity and Mutagenicity of Benz(
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