Carcinogenesis vol 12 no.l pp.65-69, 1991
Novel coumarins as potential anticarcinogenic agents
Raghunathan V.Nair, Edward P.Fisher, Steven H.Safe1, Cecilia Cortez2, Ronald G.Harvey2 and John DiGiovanni3 The University of Texas, M.D.Anderson Cancer Center, Department of Carcinogenesis, Science Park-Research Division, PO Box 389, Smithville, TX 78957, 'Department of Physiology and Pharmacology, School of Veterinary Medicine, Texas A&M University, College Station, TX 77843 and 2The Ben May Institute, University of Chicago, 5841 Maryland Avenue, Chicago, IL 60637, USA 3
To whom correspondence should be addressed
Introduction The two-stage model of mouse skin tumorigenesis represents an ideal test system for identification of potential anticarcinogenic agents. In this model system, the process of carcinogenesis involves the tumor initiation and subsequent promotion phases leading to the development of visible tumors (1,2). Thus, a topically applied carcinogenic xenobiotic such as 7,12-dimethylbenz[a]anthracene (DMBA*) is metabolically transformed to highly reactive intermediates capable of reacting with cellular macromolecules such as the DNA. Modification of cellular DNA •Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; TPA, 12-0-tetradecanoylphorbol-13-acetate; B[a]P, benzo[a]pyrene; AHH, aryl hydrocarbon hydroxylase; DB[a,c]A, 1,2,3,4-dibenzanthracene; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; HEDG buffer, 25 mM HEPES, 1.5 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 10% (v/v) glycerol; PAH, polycyclic aromatic hydrocarbon; 5,6-BF, 5,6-benzoflavone. © Oxford University Press
1 , R=H, Coumarin 2, R=CH3, 4-Methylcoumarln
3, R=OH, R1=R2=H; Umbelliferone 4, R=OH, R 1= OCH 3 , R2=H; Scopoletln 5, R=R2=OCH3, R,=H; Limettln
6, R=R 1= H 7, R=CH3, R,=H 8, R=H, R,=CH 3
9, R=R 2 =H,R 1 =CH 3 10, R=CH3> R,=R2=H
11,DB[a,c]A
Fig. 1. Chemical structures of coumarins and novel coumarins relevant to the present study. The chemical structure of DB[a,c]A, a potent inducer of mouse epidermal AHH, is also presented.
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The potential anticarcinogenic properties of several novel coumarin derivatives whose structures are based on polycyclic aromatic hydrocarbons (PAHs) were examined in the multistage model of mouse skin tumorigenesis. The test compounds were evaluated for their affinity to bind competitively with rat cytosolic Ah-receptor in rat hepatic cytosol, their effects on mouse epidermal aryl hydrocarbon hydroxylase (AHH) after topical application, and for their effects on the levels of hydrocarbon-DNA adducts formed in vivo. All compounds showed good correlations between cytosolic Ah-receptor binding and their ability to induce epidermal AHH activity. Among the derivatives evaluated the coumarin (8-methyl-9f/-10-oxabenzo[a]pyren-9-one) exhibited the highest affinity for the Ah-receptor and was also the most potent inducer of epidermal AHH activity. This compound also effectively inhibited the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to epidermal DNA when given either 5 min or 24 h prior to application of [3H]DMBA. This novel coumarin derivative significantly inhibited skin tumor initiation by DMBA in SENCAR mice when given at a dose of 200 nmol, 5 min (69% inhibition) or 24 h (76% inhibition) prior to initiation. The results of these studies suggest that this class of compounds shows considerable promise for future development as potential inhibitors of PAH-mediated tumor initiation on mouse skin. Potential mechanism(s) for the anti-initiating action of these compounds are discussed.
by such reactive metabolites is thought to lead to the process of tumor initiation. Following initiation, subsequent repeated exposure to promoting agents such as 12-O-tetradecanoylphorbol13-acetate (TPA) results in the formation of tumors. This model provides for facile identification of anticarcinogenic agents effective at the initiation and/or promotion phases of the carcinogenic process. A wide variety of naturally occuring as well as synthetic chemicals are known to inhibit chemical carcinogenesis in various experimental animal tumor models (reviewed in 3). Coumarins are widely distributed in nature and form the natural constituents of several fruits and vegetables (4,5). Earlier reports (6 — 8) have identified the inhibitory effects of coumarin (1), 4-methylcoumarin (2), and the naturally occurring derivatives 3—5 (Figure 1) in DMBA-induced mammary tumors in rats and benzo[a]pyrene (B[a]P)-induced neoplasia of the forestomach in mice. However, the structure—activity relationships and the mechanism(s) by which coumarins inhibit the carcinogenic process is unknown. In order to gain further insight into the mode of chemopreventive action of various coumarin derivatives and to apply this knowledge for development of more potent chemical entities, we have undertaken to screen a variety of structurally diverse coumarin analogs for their effects on the initiation or promotion phases of mouse skin carcinogenesis. In this report, we present initial results examining the affinity of novel coumarin
R.V.Nair et al.
derivatives 6 - 9 to bind to the Ah-receptor in rat hepatic cytosol and, further, the ability of coumarins 6 - 1 0 to induce aryl hydrocarbon hydroxylase (AHH, EC 1.14.14.2) activity in mouse epidermis. Based upon these results, we further examined the effect of coumarins 7 and 9 on covalent binding of topically applied [3H]DMBA to mouse epidermal DNA, and ultimately the effect of compound 7 on mouse skin tumor initiation by DMBA. The results demonstrate that these novel coumarins may have the potential to be effective chemopreventive agents and are worthy of more in-depth study. Materials and methods
Animals Female SENCAR mice 7 - 9 weeks old were purchased from the National Cancer Institute, Frederick, MD. Mice were shaved on the dorsal side using surgical clippers 2 days prior to treatment and only those mice in the resting phase of the hair growth cycle were used in the biochemical and tumor experiments. All chemicals were applied topically to the shaved dorsal skin in 0.2 ml acetone, 24 h prior to killing the animals unless otherwise specified. AHH enzyme assay Groups of four mice were used for each experimental group. Epidermal homogenates were prepared according to published procedures (10,11). Mice were killed by cervical dislocation and a dipilatory agent was applied to the shaved dorsal skin for ~ 5 min (Nair, Carter-Wallace, NY). Following washing with cold water, the skins were removed and stored on ice. The epidermis, collected by scraping skins placed dermis side down on a cold glass plate, was placed in 1 ml of 0.25 M sucrose—0.05 M Tris buffer, pH 7.5, and homogenized using a polytron FT 10 homogenizer for three 15 s intervals at setting 6. The crude epidermal homogenate was used as the enzyme source for AHH assays. The assays for AHH activity were conducted under subdued light. The total volume of 1 ml of the assay mixture contained 50 jimol Tris-HCl buffer, pH 7.5, 0.4 mg NADPH, 3/imol MgCl2, 0.2-0.4 ml epidermal homogenate having 2—4 mg protein, 7.4 ^mol glucose-6-phosphate, 2 units glucose-6phosphate dehydrogenase and 100 nmol B[a]P. The assay mixtures were incubated at 37°C for 60 min. The reaction was terminated by addition of ice-cold acetone (1 ml) and n-hexane (3 ml). Samples were extracted vigorously and then spun at 2000 g to separate the phases. A portion of the organic phase (2 ml) was then extracted with 0.5 N NaOH (2 ml). The phenolic metabolites extracted into the alkaline phase were estimated by fluorescence measurement using excitation and emission settings of 396 and 522 nm respectively. 3-Hydroxybenzo[a]pyrene was used to prepare the standard curve. The protein content of the epidermal homogenate was estimated by the method of Lowry et al. (12). The specific activity expressed as pmol 3-hydroxybenzo[a]pyrene formed during 60 min incubation per mg protein was determined in duplicate and recorded as a percentage of the acetone control. Ah-receptor binding assay Rat hepatic cytosol was prepared according to procedures described in Bandiera et al. (13). Hepatic cytosol ( — 3—5 mg/ml) was incubated with 10 nM [2,3.7,8- 3 H]TCDD with or without different concentrations of unlabelled coumarin analogs for 1.5 h at 20°C as previously described (14). Each assay was performed in duplicate. An hydroxylapatite slurry (Bio Rad Lab; 3 parts gel in 5 parts phosphate or HEDG buffer) was freshly prepared. The phosphate buffer was 50 mM Tris-HCl, 1 mM KH 2 PO 4 . After incubation, a 200 ^1 aliquot of the cytosol was transferred to a second tube containing 200 jtl of the hydroxy-lapatite slurry. The tubes were incubated on ice for 30 min with shaking every 10 min. The mixture was then resuspended in 2 ml phosphate buffer and centrifuged at 800 g for 2 min. The hydroxylapatite pellets were washed three additional times with phosphate buffer (2.0 ml buffer) and then resuspended in 1.0 ml absolute ethanol. The hydroxylapatite/ethanol solution was vortexed and transferred to scintillation vials and any remaining hydroxylapatite was washed
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DNA binding For each experiment, groups of 6 - 8 mice were used. Following pretreatments with test compounds at indicated times and dose levels, mice were treated with 10 nmol [3H]DMBA. Epidermal DNA was isolated according to established procedures (15). DNA content was estimated according to the method of Burton (16) and DNA associated radioactivity was measured using a Beckman LS 1800 liquid scintillation counter. Binding is expressed as pmol of [ H]DMBA per mg epidermal DNA. Tumor initiation experiments Each experimental group consisted of 30 preshaved SENCAR mice. Mice were initiated by application of 10 nmol DMBA two weeks prior to commencement of promotion with TPA. Twice-weekly applications of 3.4 nmol TPA were continued for 14 weeks. Papilloma formation was recorded weekly and results are presented as the average number of papillomas per mouse at 14 weeks of promotion. Statistical analyses of the differences between mean papilloma responses were determined using Student's t -test.
Results The effects of single topical applications of 200 nmol coumarins 6—10 and DB[a,c]A (11) on the induction of mouse epidermal AHH activity are shown in Table I. Data shown in Table II summarize the EC50 values for compounds 6 - 9 to displace competitively [3H]TCDD from the rat hepatic cytosolic Ah receptor. Coumarin 7 was a good inducer of mouse epidermal AHH and an excellent ligand for the Ah-receptor in rat hepatic cytosol. At a dose of 200 nmol per mouse, this compound produced an —700% increase in epidermal AHH activity 24 h following treatment. A group treated with 200 nmol DB[a,c]A Table I. Effect of novel coumarins on mouse epidermal AHH activity8 Compound
Dose
SA (pmol 3-OH-B[a]P/mg/60 min) % of acetone control
Acetone DB[a,c]A Coumarin Coumarin Coumarin Coumarin Coumarin
0.2 ml 200 nmol 200 nmol 200 nmol 200 nmol 200 nmol 200 nmol
100 848 191 663 95 168 133
6 7 8 9 10
a
Data represent an average of two separate experiments run in duplicate. Maximum variation for a given group between the two experiments was 18%.
b
Most potent coumarin derivative tested.
Table II. Ah-receptor binding in rat hepatic cytosol of various novel coumarin derivatives3 Compound
Ah-receptor binding (EC 50 values, M)
B[a]P TCDD Coumarin Coumarin Coumarin Coumarin
7.94 x 1.0 x 1.17 x 1.3 x 2.88 x >10"4
6 7 8 9
10" 9 10" 9 10 " _ b 10 9 10
a Values in the table are presented as EC 50 (i.e. the concentration producing a 50% inhibition of [3H]TCDD binding). ECso values were obtained from a minimum of five concentrations of compound and represent an average of duplicate experiments using the same cytosol. Maximum variation for a given value between the two determinations was 16%. Most potent coumarin derivative tested.
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Chemicals 9//-Pyreno[ 1,2-b]pyran-9-one (6) and its methyl substituted derivatives (7,8) and 2//-anthra [ 1,2-fo]pyran-2-one derivatives (9,10) were prepared according to published procedures (9). DMBA, 1,2,3,4-dibenzanthracene (DB[a,c]A), NADPH, glucose-6-phosphate and glucose-6-phosphate dehydrogenase (type Xl.torula yeast) were purchased from Sigma Chemical Co., St Louis, MO. 3-Hydroxybenzo[a]pyrene was obtained from the Chemical Carcinogen Repository, National Cancer Institute. B[a]P was purchased from Aldrich Chemical Co., Milwaukee, WI. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Dow Chemical Co., Midland, MI. TPA was purchased from Chemicals for Cancer Research, Eden Prairie, MN. [3H]DMBA was supplied by Amersham, Arlington Heights, IL, and [3H]TCDD was obtained from KOR Isotopes, Cambridge, MA.
into the vial with an additional 1 ml of ethanol; 10 ml of scintillation cocktail was added and samples were counted for radioactivity. The dose-response displacement of the [2,3,7,8-3H]TCDD was determined for all the ligands and the EC5o^values were graphically estimated (i.e. the concentration of unlabelled ligand which competitively displaces 50% of the radioligand in this assay procedure).
Novel coumarins as potential anticarcinogenic agents
Table III. Effect of novel coumarins on covalent binding of [3H]DMBA to mouse epidermal DNAa Compound, dose(nmol)
Pretreatment time
SA (pmol/mg DNA)
Acetone (0.2 ml) 9,200 9,400 9,200 9, 400
5 min 5 min 5 min 24 h 24 h
2.59 1.36 1.45 2.63 2.28
— 48 44 0 12
7, 200 7, 400 7, 200 7,400
5 min 5 mm 24 h 24 h
1.35 1.29 1.42 1.19
48 50 45 54
Inhibition
a
Binding (i.e. specific activity or SA) is expressed as the pmol of hydrocarbon bound per mg epidermal DNA. Similar results were obtained in a repeat experiment.
of 200 or 400 nmol per mouse, coumarin 7 significantly (P < 0.05) inhibited skin tumor initiation by DMBA when applied either 5 min or 24 h before treatment with DMBA (Table IV). Compound 7 did not show any tumor-initiating activity at the dose of 400 nmol per mouse after 14 weeks of promotion. Discussion In order to identify potential inhibitors of chemically induced tumor initiation among selected coumarin derivatives, we examined the ability of several novel compounds to induce mouse epidermal AHH. In addition, the affinity of these compounds to bind to the rat cytosolic Ah-receptor in a competitive binding assay was also evaluated. Previous studies have indicated good correlations between Ah-receptor binding and the relative potency of AHH induction by a variety of chemicals (20—23). Accordingly, following the interaction of the inducer with the cytosolic receptor, the complex translocates into the nucleus, interacts with specific segments of DNA upstream from the CYP1A1 gene and induces transcription of this gene (24). Thus, using the induction and binding assays, coumarin derivative 7 was a good inducer of mouse epidermal AHH (Table I) and an excellent ligand for the cytosolic Ah-receptor (Table II). All evaluated compounds showed good correlation between their affinities for the Ah-receptor and their potencies as inducers of mouse epidermal AHH activity. Numerous studies with PAHs have shown good correlations between levels of hydrocarbon-DNA binding and the tumorinitiating potencies of these hydrocarbons (25). Such interactions of activated xenobiotics with cellular DNA is considered as a critical event leading to the process of tumor initiation in mouse skin, as well as in other tissues (25-28). In this context, many inhibitors of PAH-induced mouse skin tumors produce a parallel reduction in the level of PAH-DNA binding in this tissue (reviewed in 3). In similar experiments designed to assess the effect on the covalent binding of [3H]DMBA to mouse epidermal DNA, 24 h pretreatment with coumarin 7 inhibited the level of DMBA-DNA binding - 5 0 % , whereas similar pretreatment with compound 9 had little or no effect (Table IE). These results were consistent both with the affinity of compounds 7 and 9 for the Ah-receptor as well as their ability to induce epidermal AHH activity. However, coumarin derivative 9 was found to inhibit DMBA-DNA binding (Table III) when applied 5 min prior to [3H]DMBA treatment. This result was in contrast to the data obtained with 9 in the AHH induction and Ah-receptor binding assays and possibly relates to the existence of selective or multiple mechanisms of action by these coumarin derivatives. The data suggest that under certain conditions coumarin 9 may also have anti-initiating activity. The results also point out the
Table IV. Effect of coumarin 7 on mouse skin tumor initiation by DMBAa Pretreatment, dose (nmol)
Pretreatment time
Initiator, dose (nmol)
Papillomas per mouse
Acetone. 0.2 ml Acetone, 0.2 ml 7, 200 7,400 7, 200
5 min 5 min 5 min 5 min 24 h
DMBA, 7, 400 DMBA, DMBA, DMBA,
8.9 0.14 2.8 3.3 2.1
10 10 10 10
± ± ± ± ±
2.01 0.12b 0.7 c l.l c 0.9 1
Inhibition — 69 63 76
"Thirty mice were used for each experimental group. All mice were alive and healthy at 14 weeks. b Not significantly different (P > 0.5) than the group receiving acetone'(0.2 ml) at initiation followed by twice-weekly applications of 3.4 nmol TPA (0.07 ± 0.05 papillomas per mouse). c Significantly (P < 0.05) lower than the DMBA-TPA control group.
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served as a positive control in this experiment (Table I) (17,18). In the Ah-receptor binding assay, compound 7 was only slightly less potent than unlabelled TCDD [EC50 = 1 X 10"' M] as a competitive ligand. Other test compounds (Tables I and II) showed considerably weaker activities in both the induction and binding assays. Taken together, data obtained with compound 7 suggested that this coumarin derivative contained structural characteristics favorable for inhibition of polycyclic aromatic hydrocarbon (PAH)-induced skin tumor initiaton in mice (17-19). To explore further the potential anti-initiating activity of these novel coumarins, the effects of several derivatives on covalent binding of DMBA to epidermal DNA was examined. Topical application of 200 or 400 nmol per mouse of coumarin 7 afforded nearly 50% inhibition of covalent binding of [3H]DMBA to epidermal DNA when applied either 5 min or 24 h prior to [%]DMBA treatment (Table III). In contrast, although the coumarin derivative 9 was equally effective when applied 5 min prior to DMBA treatment, this compound had little effect when applied 24 h before treatment with DMBA. The effects on DMBA-DNA binding observed with the 24 h pretreatment of mice using coumarins 7 and 9 correlated well with their potencies as inducers of mouse epidermal AHH activity and their affinities for the rat cytosolic Ah receptor. However, the effect of coumarin 9 on DMBA—DNA binding when given 5 min prior to [3H]DMBA was not predicted by either the binding or induction assays. The anti-initiating activity of coumarin 7 was tested using a standard initiation-promotion protocol with DMBA as the initiator. These results are summarized in Table IV. At dose levels
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mechanisms of inhibition of skin tumor initiation by coumarin derivatives. In summary, the results presented in this paper form the basis for the evolution of a structure—activity study involving coumarin derivatives of both natural and synthetic origin. The coumarins used in the present investigation are novel in that they are based on the structures of B[a]P and several benz[a]anthracenes. This structural feature may contribute to their ability to bind to the Ah-receptor and/or to binding sites on specific cytochrome(s) P450. Therefore, these compounds may represent 'targeted' anticarcinogenic agents. Experiments currently in progress in our laboratory will further characterize the structural features necessary for optimal anti-initiating activity on mouse skin. In addition, we are examining the spectrum of anticarcinogenic activity of these derivatives toward other PAH skin tumor initiators. Such studies will hopefully provide for the development of more potent and efficacious compounds in this class of inhibitors of chemical carcinogenesis. Acknowledgements The authors wish to thank Mary Anne Denomme for her technical assistance and Joyce Mayhugh for her excellent secretarial skills in preparing this manuscript. Research supported by American Cancer Society grant FRA-375 (J.D.), the Council for Tobacco Research grant number 1608 (R.G.H.) and the National Institutes of Health grant ES 03843 (S.S.).
References 1. Bourwell.R.K. (1964) Some biological aspects of skin carcinogenesis. Prog. Exp. Tumor Res., 4, 207-250. 2. Boutwell.R.K. (1974) The function and mechanism of promoters of carcinogenesis. CRC Crit. Rev. Toxicoi, 2, 419—443. 3. DiGiovanniJ. (1990) Inhibition of chemical carcinogenesis. In Cooper,C.S. and Grover,P.L. (eds), Handbook of Experimental Pharmacology. SpringerVerlag, New York, Vol. 94/n, pp. 159-223. 4. Dean,F.M. (1963) Naturally Occurring Oxygen Ring Compounds. Butterworth, London, pp. 176-219. 5. Robinson,T. (1963) The Organic Constituents of Higher Plants. Burgess, Minneapolis, MN, pp. 4 5 - 6 9 . 6. Feuer,G. and Kellen.J.A. (1974) Inhibition and enhancement of mammary tumorigenesis by 7,12-dimethylbenz[a]anthracene in the female Sprague—Dawley rat. Int. J. Clin. Pharmacol., 9, 62—69. 7. Feuer.G., KellenJ.A. and Kovacs.K. (1976) Suppression of 7,12-dimethylbenz[a]anthracene-induced breast carcinoma by coumarin in the rat. Oncology, 33, 35-39. 8.Wattenberg,L.W., Lam,L.K.T. and Fladmoe.A.V. (1979) Inhibition of chemical carcinogen-induced neoplasia by coumarins and a-angelicalactone. Cancer Res., 39, 1651-1654. 9. Harvey,R.G., Cortez.C, Ananthanarayan,T.P. and Schmolka.S. (1988) A new coumarin synthesis and its utilization for the synthesis of polycyclic coumarin compounds with anticarcinogenic properties. J. Org. Chem., 53, 3936-3943. 10. SlagaJ.J., Thompson,S., Berry,D.L., DiGiovanni.J., Juchau,M.R. and Viaje.A. (1977) The effects of benzoflavones on polycyclic hydrocarbon metabolism and skin tumor initiation. Chem.-Biol. Interactions, 17, 297—312. 11. Bowden.G.T., SlagaJ.J., Shapas,B.G. and Boutwell.R.K. (1974) The role of aryl hydrocarbon hydroxylase in skin tumor initiation by 7,12-dimethylbenz[a]anthracene and 1,2,5,6-dibenzanthracene using DNA binding and thymidine-3H incorporation into DNA as criteria. Cancer Res., 34, 2634-2642. 12. Lowry.O.H., Rosebrough,N.J., Farr.A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. /. Biol. Chem., 193,265-275. 13. Bandiera.S., Safe.S. and Okey,A.B. (1982) Binding of polychlorinated biphenyls classified as either phenobarbitone-, 3-methylcholanthrene- or mixedtype inducers tocytosolic Ah receptor. Chem.-Biol. Interactions, 39, 259—277. 14. GasiewiczJ.A. and Neal,R.A. (1982) The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxyapatite. Anal. Biochem., VIA, 1-11. 15. Ashurst,S.W., Cohen.G.M., Nesnow,S., DiGiovanniJ. and SlagaJ.J. (1983) Formation of benzo[a]pyrene/DNA adducts and their relationship to tumor initiation in mouse epidermis. Cancer Res., 43, 1024 — 1029.
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importance of using several assay systems for screening potential anti-initiating agents for a given group of compounds. Although we have not yet tested coumarin 9 for anti-initiating activity, coumarin 7 was an effective inhibitor of skin tumor initiation by DMBA in SENCAR mice when given 5 min or 24 h prior to initiation (Table IV). Furthermore, when evaluated as a tumor initiator at dose levels of 400 nmol per mouse, coumarin 7 was inactive. Thus, the anti-initiating effects of coumarin 7 correlated well with the ability of this compound to lower the level of DMBA-DNA adducts. Inhibition of chemically induced tumor initiation may involve inhibition and/or induction of the enzymes involved in both metabolic activation and detoxication in addition to other potential mechanisms (reviewed in 3). In this regard, induction of monooxygenase enzymes has been proposed as a mechanism by which certain flavones [e.g. 5,6-benzoflavone (5,6-BF)] inhibit PAH carcinogenesis in mouse skin (reviewed in 3) as well as other tissues (29,30). The mechanism(s) by which increased monooxygenase activity leads to inhibition of tumor initiation is not clearly understood. However, the existence and selective induction of multiple isozymes for the cytochrome(s) P450 is well documented (31—34). These mixed-function oxidases are capable of both activating and detoxifying various xenobiotics, including PAH. Thus, the relative induction of specific P450 isozymes involved in activation/detoxification pathways may contribute to the overall effect of an inhibitor of chemically induced tumor initiation. In addition, many compounds that induce monooxygenase enzymes through interaction with the Ah-receptor also induce the coordinate expression of certain other enzymes (reviewed in 35), including UDP-glucuronosyltransferase, NADPH:menadione oxidoreductase and glutathione-S-transferase. The coordinate induction or elevation in both monooxygenase as well as conjugative or phase II enzyme activities may ultimately be responsible for the anti-initiating activity of enzyme inducers. The anti-initiating activity exhibited by coumarin 7, when given 24 h prior to DMBA, may be related to the induction of enzymes involved in the ultimate detoxification of this PAH. The ability of coumarin 7 also to inhibit DMBA—DNA binding and skin tumor initiation when administered 5 min before the carcinogen seemed also to parallel similar effects produced by 5,6-BF (10,11) and DB[a,c]A (17,18) on skin tumor initiation by DMBA. These studies reported that both DB[a,c]A and 5,6-BF inhibited AHH activities in mouse epidermal homogenates and inhibited tumor initiation when given 5 min before the initiator. Our present data suggest that coumarin 7 (or one of its metabolites) may also inhibit skin tumor initiation by competing for binding sites on specific cytochrome P450 species, thereby altering the metabolic activation of DMBA. Coumarin 9 appeared to act either by this latter pathway and/or by functioning as an Ah-receptor antagonist, since it was a comparatively weak inducer of AHH activity but an effective inhibitor of DMBA—DNA binding when given 5 min before [3H]DMBA. 7,8-Benzoflavone, a potent inhibitor of epidermal AHH activity and tumor initiation by DMBA (3), has recently been shown to be an effective Ah-receptor antagonist (36). One or both of these mechanisms could possibly play a role in the ability of coumarin 9 to inhibit the covalent binding of DMBA to epidermal DNA. In general, those compounds that possess both AHH inducing properties as well as inhibiting properties appear to be much more effective inhibitors of skin tumor initiation by different PAHs (3). While further work is necessary to prove these hypotheses, the availability of a large number of structural analogs should allow us to delineate the
Novel coumarins as potential anticarcinogenic agents
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16. Burton,K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of DNA. Biochem. J., 62, 315-323. 17. DiGiovanni.J., Rymer.J., Slaga.T.J. and Boutwell.R.K. (1982) Anticarcinogenic and cocarcinogenic effects of benzo[e]pyrene and dibenz[a,c]anthracene on skin tumor initiation by polycyclic hydrocarbons. Carcinogenesis, 3, 371-375. 18. Slaga,T.J. and Boutwell,R.K. (1977) Inhibition of the tumor-initiating ability of the potent carcinogen 7,12-dimethylbenz[a]anthracene by the weak tumorinitiator, 1,2,3,4-dibenzanthracene. Cancer Res., 37, 128—133. 19. DiGiovanni,J., Berry,D.L., Gleason.G.L., Kishore.G.S. and Slaga.T.J. (1980) Time-dependent inhibition by 2,3,7,8-tetrachlorodibenzo-/Miioxin of skin tumorigenesis with polycyclic hydrocarbons. Cancer Res., 40, 1580—1587. 20. Poland.A. and Glover.E. (1976) Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J. Biol. Chem., 251, 4936-4946. 21. Poland,A. and Knutson.J.C. (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol., 22, 517—554. 22. Poland.A., Glover,E., DeCamp,M., Giandomenico.C.M. and Kende.A.S. (1976) 3,4,3',4'-Tetrachloro azoxybenzene and azobenzene: potent inducers of aryl hydrocarbon hydroxylase. Science, 194, 627—630. 23. Safe,S. (1986) Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol. Toxicol., 26, 371-399. 24. Whitlock,J.P. (1986) The regulation of cytochrome P-450 gene expression. Annu. Rev. Pharmacol. Toxicol., 26, 333—369. 25.Dipple,A., Moschel,R.C, Bigger,C.A.H. (1984) Polynuclear aromatic hydrocarbons. In Searle.C.E. (ed.), Chemical Carcinogenesis, ACS Monograph 182. American Chemical Society, Washington, DC, Vol. 1, pp. 41-163. 26. Heidelberger.C. (1975) Chemical carcinogenesis. Annu. Rev. Biochem., 44, 79-121. 27. Phillips,D.H. and Sims.P. (1979) Polycyclic aromatic hydrocarbon metabolites: their reactions with nucleic acids. In Grover.P.L. (ed.), Chemical Carcinogens and DNA. CRC Press, Boca Raton, FL, Vol. 2, pp. 29-57. 28. Sims,P. (1980) The metabolic activation of chemical carcinogens. Br. Med. Bull., 36, 11-18. 29. Wattenberg,L.W. and Leong,J.L. (1968) Inhibition of the carcinogenic action of 7,12-dimethylbenzo[a]anthracene by beta-napthoflavone. Proc. Soc. Exp. Biol. Med., 128, 940-943. 30. Wattenberg.L.W.and Leong,J.L. (1970) Inhibition of the carcinogenic action of benzo[a]pyrene by flavones. Cancer Res., 30, 1922-1925. 31.Coon,M.J. and Vatsis.K.P. (1978) Biochemical studies on chemical carcinogenesis: role of multiple forms of cytochrome P-450 in the metabolism of benzo[a]pyrene and other foreign compounds. In Gelboin,H.V. and Ts'o.P.O.P. (eds), Polycyclic Hydrocarbons and Cancer: Environment, Chemistry, and Metabolism. Academic Press, New York, Vol. 1, pp. 336-360. 32. Lu.A.Y.H. and West.S.B. (1980) Multiplicity of mammalian microsomal cytochromes P-450. Pharmacol. Rev., 31, 277-295. 33. Holder.G., Yagi.H., Dansette.P., Jerina.D.M., Levin.W., Lu.A.Y.H. and Conney.A.H. (1974) Effects of inducers and epoxide hydrase on the metabolism of benzo[a]pyrene by liver rrucrosomes and a reconstituted system: analysis by high pressure liquid chromatography. Proc. Nail. Acad. Sci. USA, 71, 4356-4360. 34. Rasmussen.R.E. and Wang.I.Y. (1974) Dependence of specific metabolism of benzo[a]pyrene on the inducer of hydroxylase activity. Cancer Res., 34, 2290-2295. 35. Nebert,D.W. and Gonzalez,F.J. (1987) P^50 genes: structure, evolution, and regulation. Annu. Rev. Biochem., 56, 945-993. 36. Merchant.M., Arellano,L. and Safe,S. (1990) The mechanisms of action of a-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo-p-dioxininduced CYP1A1 gene expression. Arch. Biochem. Biophys., 281, 84—89. Received on July 9, 1990; revised on September 27, 1990; accepted on October 1, 1990
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Novel coumarins as potential anticarcinogenic agents
Raghunathan V.Nair, Edward P.Fisher, Steven H.Safe1, Cecilia Cortez2, Ronald G.Harvey2 and John DiGiovanni3 The University of Texas, M.D.Anderson Cancer Center, Department of Carcinogenesis, Science Park-Research Division, PO Box 389, Smithville, TX 78957, 'Department of Physiology and Pharmacology, School of Veterinary Medicine, Texas A&M University, College Station, TX 77843 and 2The Ben May Institute, University of Chicago, 5841 Maryland Avenue, Chicago, IL 60637, USA 3
To whom correspondence should be addressed
Introduction The two-stage model of mouse skin tumorigenesis represents an ideal test system for identification of potential anticarcinogenic agents. In this model system, the process of carcinogenesis involves the tumor initiation and subsequent promotion phases leading to the development of visible tumors (1,2). Thus, a topically applied carcinogenic xenobiotic such as 7,12-dimethylbenz[a]anthracene (DMBA*) is metabolically transformed to highly reactive intermediates capable of reacting with cellular macromolecules such as the DNA. Modification of cellular DNA •Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; TPA, 12-0-tetradecanoylphorbol-13-acetate; B[a]P, benzo[a]pyrene; AHH, aryl hydrocarbon hydroxylase; DB[a,c]A, 1,2,3,4-dibenzanthracene; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; HEDG buffer, 25 mM HEPES, 1.5 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 10% (v/v) glycerol; PAH, polycyclic aromatic hydrocarbon; 5,6-BF, 5,6-benzoflavone. © Oxford University Press
1 , R=H, Coumarin 2, R=CH3, 4-Methylcoumarln
3, R=OH, R1=R2=H; Umbelliferone 4, R=OH, R 1= OCH 3 , R2=H; Scopoletln 5, R=R2=OCH3, R,=H; Limettln
6, R=R 1= H 7, R=CH3, R,=H 8, R=H, R,=CH 3
9, R=R 2 =H,R 1 =CH 3 10, R=CH3> R,=R2=H
11,DB[a,c]A
Fig. 1. Chemical structures of coumarins and novel coumarins relevant to the present study. The chemical structure of DB[a,c]A, a potent inducer of mouse epidermal AHH, is also presented.
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The potential anticarcinogenic properties of several novel coumarin derivatives whose structures are based on polycyclic aromatic hydrocarbons (PAHs) were examined in the multistage model of mouse skin tumorigenesis. The test compounds were evaluated for their affinity to bind competitively with rat cytosolic Ah-receptor in rat hepatic cytosol, their effects on mouse epidermal aryl hydrocarbon hydroxylase (AHH) after topical application, and for their effects on the levels of hydrocarbon-DNA adducts formed in vivo. All compounds showed good correlations between cytosolic Ah-receptor binding and their ability to induce epidermal AHH activity. Among the derivatives evaluated the coumarin (8-methyl-9f/-10-oxabenzo[a]pyren-9-one) exhibited the highest affinity for the Ah-receptor and was also the most potent inducer of epidermal AHH activity. This compound also effectively inhibited the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to epidermal DNA when given either 5 min or 24 h prior to application of [3H]DMBA. This novel coumarin derivative significantly inhibited skin tumor initiation by DMBA in SENCAR mice when given at a dose of 200 nmol, 5 min (69% inhibition) or 24 h (76% inhibition) prior to initiation. The results of these studies suggest that this class of compounds shows considerable promise for future development as potential inhibitors of PAH-mediated tumor initiation on mouse skin. Potential mechanism(s) for the anti-initiating action of these compounds are discussed.
by such reactive metabolites is thought to lead to the process of tumor initiation. Following initiation, subsequent repeated exposure to promoting agents such as 12-O-tetradecanoylphorbol13-acetate (TPA) results in the formation of tumors. This model provides for facile identification of anticarcinogenic agents effective at the initiation and/or promotion phases of the carcinogenic process. A wide variety of naturally occuring as well as synthetic chemicals are known to inhibit chemical carcinogenesis in various experimental animal tumor models (reviewed in 3). Coumarins are widely distributed in nature and form the natural constituents of several fruits and vegetables (4,5). Earlier reports (6 — 8) have identified the inhibitory effects of coumarin (1), 4-methylcoumarin (2), and the naturally occurring derivatives 3—5 (Figure 1) in DMBA-induced mammary tumors in rats and benzo[a]pyrene (B[a]P)-induced neoplasia of the forestomach in mice. However, the structure—activity relationships and the mechanism(s) by which coumarins inhibit the carcinogenic process is unknown. In order to gain further insight into the mode of chemopreventive action of various coumarin derivatives and to apply this knowledge for development of more potent chemical entities, we have undertaken to screen a variety of structurally diverse coumarin analogs for their effects on the initiation or promotion phases of mouse skin carcinogenesis. In this report, we present initial results examining the affinity of novel coumarin
R.V.Nair et al.
derivatives 6 - 9 to bind to the Ah-receptor in rat hepatic cytosol and, further, the ability of coumarins 6 - 1 0 to induce aryl hydrocarbon hydroxylase (AHH, EC 1.14.14.2) activity in mouse epidermis. Based upon these results, we further examined the effect of coumarins 7 and 9 on covalent binding of topically applied [3H]DMBA to mouse epidermal DNA, and ultimately the effect of compound 7 on mouse skin tumor initiation by DMBA. The results demonstrate that these novel coumarins may have the potential to be effective chemopreventive agents and are worthy of more in-depth study. Materials and methods
Animals Female SENCAR mice 7 - 9 weeks old were purchased from the National Cancer Institute, Frederick, MD. Mice were shaved on the dorsal side using surgical clippers 2 days prior to treatment and only those mice in the resting phase of the hair growth cycle were used in the biochemical and tumor experiments. All chemicals were applied topically to the shaved dorsal skin in 0.2 ml acetone, 24 h prior to killing the animals unless otherwise specified. AHH enzyme assay Groups of four mice were used for each experimental group. Epidermal homogenates were prepared according to published procedures (10,11). Mice were killed by cervical dislocation and a dipilatory agent was applied to the shaved dorsal skin for ~ 5 min (Nair, Carter-Wallace, NY). Following washing with cold water, the skins were removed and stored on ice. The epidermis, collected by scraping skins placed dermis side down on a cold glass plate, was placed in 1 ml of 0.25 M sucrose—0.05 M Tris buffer, pH 7.5, and homogenized using a polytron FT 10 homogenizer for three 15 s intervals at setting 6. The crude epidermal homogenate was used as the enzyme source for AHH assays. The assays for AHH activity were conducted under subdued light. The total volume of 1 ml of the assay mixture contained 50 jimol Tris-HCl buffer, pH 7.5, 0.4 mg NADPH, 3/imol MgCl2, 0.2-0.4 ml epidermal homogenate having 2—4 mg protein, 7.4 ^mol glucose-6-phosphate, 2 units glucose-6phosphate dehydrogenase and 100 nmol B[a]P. The assay mixtures were incubated at 37°C for 60 min. The reaction was terminated by addition of ice-cold acetone (1 ml) and n-hexane (3 ml). Samples were extracted vigorously and then spun at 2000 g to separate the phases. A portion of the organic phase (2 ml) was then extracted with 0.5 N NaOH (2 ml). The phenolic metabolites extracted into the alkaline phase were estimated by fluorescence measurement using excitation and emission settings of 396 and 522 nm respectively. 3-Hydroxybenzo[a]pyrene was used to prepare the standard curve. The protein content of the epidermal homogenate was estimated by the method of Lowry et al. (12). The specific activity expressed as pmol 3-hydroxybenzo[a]pyrene formed during 60 min incubation per mg protein was determined in duplicate and recorded as a percentage of the acetone control. Ah-receptor binding assay Rat hepatic cytosol was prepared according to procedures described in Bandiera et al. (13). Hepatic cytosol ( — 3—5 mg/ml) was incubated with 10 nM [2,3.7,8- 3 H]TCDD with or without different concentrations of unlabelled coumarin analogs for 1.5 h at 20°C as previously described (14). Each assay was performed in duplicate. An hydroxylapatite slurry (Bio Rad Lab; 3 parts gel in 5 parts phosphate or HEDG buffer) was freshly prepared. The phosphate buffer was 50 mM Tris-HCl, 1 mM KH 2 PO 4 . After incubation, a 200 ^1 aliquot of the cytosol was transferred to a second tube containing 200 jtl of the hydroxy-lapatite slurry. The tubes were incubated on ice for 30 min with shaking every 10 min. The mixture was then resuspended in 2 ml phosphate buffer and centrifuged at 800 g for 2 min. The hydroxylapatite pellets were washed three additional times with phosphate buffer (2.0 ml buffer) and then resuspended in 1.0 ml absolute ethanol. The hydroxylapatite/ethanol solution was vortexed and transferred to scintillation vials and any remaining hydroxylapatite was washed
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DNA binding For each experiment, groups of 6 - 8 mice were used. Following pretreatments with test compounds at indicated times and dose levels, mice were treated with 10 nmol [3H]DMBA. Epidermal DNA was isolated according to established procedures (15). DNA content was estimated according to the method of Burton (16) and DNA associated radioactivity was measured using a Beckman LS 1800 liquid scintillation counter. Binding is expressed as pmol of [ H]DMBA per mg epidermal DNA. Tumor initiation experiments Each experimental group consisted of 30 preshaved SENCAR mice. Mice were initiated by application of 10 nmol DMBA two weeks prior to commencement of promotion with TPA. Twice-weekly applications of 3.4 nmol TPA were continued for 14 weeks. Papilloma formation was recorded weekly and results are presented as the average number of papillomas per mouse at 14 weeks of promotion. Statistical analyses of the differences between mean papilloma responses were determined using Student's t -test.
Results The effects of single topical applications of 200 nmol coumarins 6—10 and DB[a,c]A (11) on the induction of mouse epidermal AHH activity are shown in Table I. Data shown in Table II summarize the EC50 values for compounds 6 - 9 to displace competitively [3H]TCDD from the rat hepatic cytosolic Ah receptor. Coumarin 7 was a good inducer of mouse epidermal AHH and an excellent ligand for the Ah-receptor in rat hepatic cytosol. At a dose of 200 nmol per mouse, this compound produced an —700% increase in epidermal AHH activity 24 h following treatment. A group treated with 200 nmol DB[a,c]A Table I. Effect of novel coumarins on mouse epidermal AHH activity8 Compound
Dose
SA (pmol 3-OH-B[a]P/mg/60 min) % of acetone control
Acetone DB[a,c]A Coumarin Coumarin Coumarin Coumarin Coumarin
0.2 ml 200 nmol 200 nmol 200 nmol 200 nmol 200 nmol 200 nmol
100 848 191 663 95 168 133
6 7 8 9 10
a
Data represent an average of two separate experiments run in duplicate. Maximum variation for a given group between the two experiments was 18%.
b
Most potent coumarin derivative tested.
Table II. Ah-receptor binding in rat hepatic cytosol of various novel coumarin derivatives3 Compound
Ah-receptor binding (EC 50 values, M)
B[a]P TCDD Coumarin Coumarin Coumarin Coumarin
7.94 x 1.0 x 1.17 x 1.3 x 2.88 x >10"4
6 7 8 9
10" 9 10" 9 10 " _ b 10 9 10
a Values in the table are presented as EC 50 (i.e. the concentration producing a 50% inhibition of [3H]TCDD binding). ECso values were obtained from a minimum of five concentrations of compound and represent an average of duplicate experiments using the same cytosol. Maximum variation for a given value between the two determinations was 16%. Most potent coumarin derivative tested.
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Chemicals 9//-Pyreno[ 1,2-b]pyran-9-one (6) and its methyl substituted derivatives (7,8) and 2//-anthra [ 1,2-fo]pyran-2-one derivatives (9,10) were prepared according to published procedures (9). DMBA, 1,2,3,4-dibenzanthracene (DB[a,c]A), NADPH, glucose-6-phosphate and glucose-6-phosphate dehydrogenase (type Xl.torula yeast) were purchased from Sigma Chemical Co., St Louis, MO. 3-Hydroxybenzo[a]pyrene was obtained from the Chemical Carcinogen Repository, National Cancer Institute. B[a]P was purchased from Aldrich Chemical Co., Milwaukee, WI. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Dow Chemical Co., Midland, MI. TPA was purchased from Chemicals for Cancer Research, Eden Prairie, MN. [3H]DMBA was supplied by Amersham, Arlington Heights, IL, and [3H]TCDD was obtained from KOR Isotopes, Cambridge, MA.
into the vial with an additional 1 ml of ethanol; 10 ml of scintillation cocktail was added and samples were counted for radioactivity. The dose-response displacement of the [2,3,7,8-3H]TCDD was determined for all the ligands and the EC5o^values were graphically estimated (i.e. the concentration of unlabelled ligand which competitively displaces 50% of the radioligand in this assay procedure).
Novel coumarins as potential anticarcinogenic agents
Table III. Effect of novel coumarins on covalent binding of [3H]DMBA to mouse epidermal DNAa Compound, dose(nmol)
Pretreatment time
SA (pmol/mg DNA)
Acetone (0.2 ml) 9,200 9,400 9,200 9, 400
5 min 5 min 5 min 24 h 24 h
2.59 1.36 1.45 2.63 2.28
— 48 44 0 12
7, 200 7, 400 7, 200 7,400
5 min 5 mm 24 h 24 h
1.35 1.29 1.42 1.19
48 50 45 54
Inhibition
a
Binding (i.e. specific activity or SA) is expressed as the pmol of hydrocarbon bound per mg epidermal DNA. Similar results were obtained in a repeat experiment.
of 200 or 400 nmol per mouse, coumarin 7 significantly (P < 0.05) inhibited skin tumor initiation by DMBA when applied either 5 min or 24 h before treatment with DMBA (Table IV). Compound 7 did not show any tumor-initiating activity at the dose of 400 nmol per mouse after 14 weeks of promotion. Discussion In order to identify potential inhibitors of chemically induced tumor initiation among selected coumarin derivatives, we examined the ability of several novel compounds to induce mouse epidermal AHH. In addition, the affinity of these compounds to bind to the rat cytosolic Ah-receptor in a competitive binding assay was also evaluated. Previous studies have indicated good correlations between Ah-receptor binding and the relative potency of AHH induction by a variety of chemicals (20—23). Accordingly, following the interaction of the inducer with the cytosolic receptor, the complex translocates into the nucleus, interacts with specific segments of DNA upstream from the CYP1A1 gene and induces transcription of this gene (24). Thus, using the induction and binding assays, coumarin derivative 7 was a good inducer of mouse epidermal AHH (Table I) and an excellent ligand for the cytosolic Ah-receptor (Table II). All evaluated compounds showed good correlation between their affinities for the Ah-receptor and their potencies as inducers of mouse epidermal AHH activity. Numerous studies with PAHs have shown good correlations between levels of hydrocarbon-DNA binding and the tumorinitiating potencies of these hydrocarbons (25). Such interactions of activated xenobiotics with cellular DNA is considered as a critical event leading to the process of tumor initiation in mouse skin, as well as in other tissues (25-28). In this context, many inhibitors of PAH-induced mouse skin tumors produce a parallel reduction in the level of PAH-DNA binding in this tissue (reviewed in 3). In similar experiments designed to assess the effect on the covalent binding of [3H]DMBA to mouse epidermal DNA, 24 h pretreatment with coumarin 7 inhibited the level of DMBA-DNA binding - 5 0 % , whereas similar pretreatment with compound 9 had little or no effect (Table IE). These results were consistent both with the affinity of compounds 7 and 9 for the Ah-receptor as well as their ability to induce epidermal AHH activity. However, coumarin derivative 9 was found to inhibit DMBA-DNA binding (Table III) when applied 5 min prior to [3H]DMBA treatment. This result was in contrast to the data obtained with 9 in the AHH induction and Ah-receptor binding assays and possibly relates to the existence of selective or multiple mechanisms of action by these coumarin derivatives. The data suggest that under certain conditions coumarin 9 may also have anti-initiating activity. The results also point out the
Table IV. Effect of coumarin 7 on mouse skin tumor initiation by DMBAa Pretreatment, dose (nmol)
Pretreatment time
Initiator, dose (nmol)
Papillomas per mouse
Acetone. 0.2 ml Acetone, 0.2 ml 7, 200 7,400 7, 200
5 min 5 min 5 min 5 min 24 h
DMBA, 7, 400 DMBA, DMBA, DMBA,
8.9 0.14 2.8 3.3 2.1
10 10 10 10
± ± ± ± ±
2.01 0.12b 0.7 c l.l c 0.9 1
Inhibition — 69 63 76
"Thirty mice were used for each experimental group. All mice were alive and healthy at 14 weeks. b Not significantly different (P > 0.5) than the group receiving acetone'(0.2 ml) at initiation followed by twice-weekly applications of 3.4 nmol TPA (0.07 ± 0.05 papillomas per mouse). c Significantly (P < 0.05) lower than the DMBA-TPA control group.
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served as a positive control in this experiment (Table I) (17,18). In the Ah-receptor binding assay, compound 7 was only slightly less potent than unlabelled TCDD [EC50 = 1 X 10"' M] as a competitive ligand. Other test compounds (Tables I and II) showed considerably weaker activities in both the induction and binding assays. Taken together, data obtained with compound 7 suggested that this coumarin derivative contained structural characteristics favorable for inhibition of polycyclic aromatic hydrocarbon (PAH)-induced skin tumor initiaton in mice (17-19). To explore further the potential anti-initiating activity of these novel coumarins, the effects of several derivatives on covalent binding of DMBA to epidermal DNA was examined. Topical application of 200 or 400 nmol per mouse of coumarin 7 afforded nearly 50% inhibition of covalent binding of [3H]DMBA to epidermal DNA when applied either 5 min or 24 h prior to [%]DMBA treatment (Table III). In contrast, although the coumarin derivative 9 was equally effective when applied 5 min prior to DMBA treatment, this compound had little effect when applied 24 h before treatment with DMBA. The effects on DMBA-DNA binding observed with the 24 h pretreatment of mice using coumarins 7 and 9 correlated well with their potencies as inducers of mouse epidermal AHH activity and their affinities for the rat cytosolic Ah receptor. However, the effect of coumarin 9 on DMBA—DNA binding when given 5 min prior to [3H]DMBA was not predicted by either the binding or induction assays. The anti-initiating activity of coumarin 7 was tested using a standard initiation-promotion protocol with DMBA as the initiator. These results are summarized in Table IV. At dose levels
R.V.Nair et al.
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mechanisms of inhibition of skin tumor initiation by coumarin derivatives. In summary, the results presented in this paper form the basis for the evolution of a structure—activity study involving coumarin derivatives of both natural and synthetic origin. The coumarins used in the present investigation are novel in that they are based on the structures of B[a]P and several benz[a]anthracenes. This structural feature may contribute to their ability to bind to the Ah-receptor and/or to binding sites on specific cytochrome(s) P450. Therefore, these compounds may represent 'targeted' anticarcinogenic agents. Experiments currently in progress in our laboratory will further characterize the structural features necessary for optimal anti-initiating activity on mouse skin. In addition, we are examining the spectrum of anticarcinogenic activity of these derivatives toward other PAH skin tumor initiators. Such studies will hopefully provide for the development of more potent and efficacious compounds in this class of inhibitors of chemical carcinogenesis. Acknowledgements The authors wish to thank Mary Anne Denomme for her technical assistance and Joyce Mayhugh for her excellent secretarial skills in preparing this manuscript. Research supported by American Cancer Society grant FRA-375 (J.D.), the Council for Tobacco Research grant number 1608 (R.G.H.) and the National Institutes of Health grant ES 03843 (S.S.).
References 1. Bourwell.R.K. (1964) Some biological aspects of skin carcinogenesis. Prog. Exp. Tumor Res., 4, 207-250. 2. Boutwell.R.K. (1974) The function and mechanism of promoters of carcinogenesis. CRC Crit. Rev. Toxicoi, 2, 419—443. 3. DiGiovanniJ. (1990) Inhibition of chemical carcinogenesis. In Cooper,C.S. and Grover,P.L. (eds), Handbook of Experimental Pharmacology. SpringerVerlag, New York, Vol. 94/n, pp. 159-223. 4. Dean,F.M. (1963) Naturally Occurring Oxygen Ring Compounds. Butterworth, London, pp. 176-219. 5. Robinson,T. (1963) The Organic Constituents of Higher Plants. Burgess, Minneapolis, MN, pp. 4 5 - 6 9 . 6. Feuer,G. and Kellen.J.A. (1974) Inhibition and enhancement of mammary tumorigenesis by 7,12-dimethylbenz[a]anthracene in the female Sprague—Dawley rat. Int. J. Clin. Pharmacol., 9, 62—69. 7. Feuer.G., KellenJ.A. and Kovacs.K. (1976) Suppression of 7,12-dimethylbenz[a]anthracene-induced breast carcinoma by coumarin in the rat. Oncology, 33, 35-39. 8.Wattenberg,L.W., Lam,L.K.T. and Fladmoe.A.V. (1979) Inhibition of chemical carcinogen-induced neoplasia by coumarins and a-angelicalactone. Cancer Res., 39, 1651-1654. 9. Harvey,R.G., Cortez.C, Ananthanarayan,T.P. and Schmolka.S. (1988) A new coumarin synthesis and its utilization for the synthesis of polycyclic coumarin compounds with anticarcinogenic properties. J. Org. Chem., 53, 3936-3943. 10. SlagaJ.J., Thompson,S., Berry,D.L., DiGiovanni.J., Juchau,M.R. and Viaje.A. (1977) The effects of benzoflavones on polycyclic hydrocarbon metabolism and skin tumor initiation. Chem.-Biol. Interactions, 17, 297—312. 11. Bowden.G.T., SlagaJ.J., Shapas,B.G. and Boutwell.R.K. (1974) The role of aryl hydrocarbon hydroxylase in skin tumor initiation by 7,12-dimethylbenz[a]anthracene and 1,2,5,6-dibenzanthracene using DNA binding and thymidine-3H incorporation into DNA as criteria. Cancer Res., 34, 2634-2642. 12. Lowry.O.H., Rosebrough,N.J., Farr.A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. /. Biol. Chem., 193,265-275. 13. Bandiera.S., Safe.S. and Okey,A.B. (1982) Binding of polychlorinated biphenyls classified as either phenobarbitone-, 3-methylcholanthrene- or mixedtype inducers tocytosolic Ah receptor. Chem.-Biol. Interactions, 39, 259—277. 14. GasiewiczJ.A. and Neal,R.A. (1982) The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxyapatite. Anal. Biochem., VIA, 1-11. 15. Ashurst,S.W., Cohen.G.M., Nesnow,S., DiGiovanniJ. and SlagaJ.J. (1983) Formation of benzo[a]pyrene/DNA adducts and their relationship to tumor initiation in mouse epidermis. Cancer Res., 43, 1024 — 1029.
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importance of using several assay systems for screening potential anti-initiating agents for a given group of compounds. Although we have not yet tested coumarin 9 for anti-initiating activity, coumarin 7 was an effective inhibitor of skin tumor initiation by DMBA in SENCAR mice when given 5 min or 24 h prior to initiation (Table IV). Furthermore, when evaluated as a tumor initiator at dose levels of 400 nmol per mouse, coumarin 7 was inactive. Thus, the anti-initiating effects of coumarin 7 correlated well with the ability of this compound to lower the level of DMBA-DNA adducts. Inhibition of chemically induced tumor initiation may involve inhibition and/or induction of the enzymes involved in both metabolic activation and detoxication in addition to other potential mechanisms (reviewed in 3). In this regard, induction of monooxygenase enzymes has been proposed as a mechanism by which certain flavones [e.g. 5,6-benzoflavone (5,6-BF)] inhibit PAH carcinogenesis in mouse skin (reviewed in 3) as well as other tissues (29,30). The mechanism(s) by which increased monooxygenase activity leads to inhibition of tumor initiation is not clearly understood. However, the existence and selective induction of multiple isozymes for the cytochrome(s) P450 is well documented (31—34). These mixed-function oxidases are capable of both activating and detoxifying various xenobiotics, including PAH. Thus, the relative induction of specific P450 isozymes involved in activation/detoxification pathways may contribute to the overall effect of an inhibitor of chemically induced tumor initiation. In addition, many compounds that induce monooxygenase enzymes through interaction with the Ah-receptor also induce the coordinate expression of certain other enzymes (reviewed in 35), including UDP-glucuronosyltransferase, NADPH:menadione oxidoreductase and glutathione-S-transferase. The coordinate induction or elevation in both monooxygenase as well as conjugative or phase II enzyme activities may ultimately be responsible for the anti-initiating activity of enzyme inducers. The anti-initiating activity exhibited by coumarin 7, when given 24 h prior to DMBA, may be related to the induction of enzymes involved in the ultimate detoxification of this PAH. The ability of coumarin 7 also to inhibit DMBA—DNA binding and skin tumor initiation when administered 5 min before the carcinogen seemed also to parallel similar effects produced by 5,6-BF (10,11) and DB[a,c]A (17,18) on skin tumor initiation by DMBA. These studies reported that both DB[a,c]A and 5,6-BF inhibited AHH activities in mouse epidermal homogenates and inhibited tumor initiation when given 5 min before the initiator. Our present data suggest that coumarin 7 (or one of its metabolites) may also inhibit skin tumor initiation by competing for binding sites on specific cytochrome P450 species, thereby altering the metabolic activation of DMBA. Coumarin 9 appeared to act either by this latter pathway and/or by functioning as an Ah-receptor antagonist, since it was a comparatively weak inducer of AHH activity but an effective inhibitor of DMBA—DNA binding when given 5 min before [3H]DMBA. 7,8-Benzoflavone, a potent inhibitor of epidermal AHH activity and tumor initiation by DMBA (3), has recently been shown to be an effective Ah-receptor antagonist (36). One or both of these mechanisms could possibly play a role in the ability of coumarin 9 to inhibit the covalent binding of DMBA to epidermal DNA. In general, those compounds that possess both AHH inducing properties as well as inhibiting properties appear to be much more effective inhibitors of skin tumor initiation by different PAHs (3). While further work is necessary to prove these hypotheses, the availability of a large number of structural analogs should allow us to delineate the
Novel coumarins as potential anticarcinogenic agents
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