The Effects of Antioxidants on Skin Tumor Initiation and Aryl Hydrocarbon Hydroxylase1

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[CANCER RESEARCH 37, 1631-1635, June 1977] The Effects of Antioxidants on Skin Tumor Initiation and Aryl Hydrocarbon Hydroxylase1

Thomas J. Slaga2 and William M. Bracken Cancerand ToxicologyProgram,BiologyDivision,OakRidgeNationalLaboratory,OakRidge,Tennessee37830fT.J. S.), and EastTennesseeCancer ResearchCenter,Knoxville,Tennessee37919fT.J. S., W.M. B.J

SUMMARY antioxidants, as well as selenium and vitamins C and E, have been shown to exert a protective function against liver Butylated hydroxytoluene, butylated hydroxyanisole, and damage, aging, carcinogen-induced chrornosornal break vitamins C and E are effective inhibitors of 7,12-dimethyl age, and chemical carcinogenesis in experimental animals benz(a)anthracene tumor initiation in a two-stage system of (11, 14, 21, 29-31). tumorigenesis. These antioxidants did not significantly in When added to the food of rodents, BHA, BHT, and duce epidermal aryl hydrocarbon [benzo(a)pyrene]hydrox ethoxyquin inhibited the carcinogenic effect of dietary ylase, nor did they have any effect when added directly to DMBA or BP on the forestomach of the mouse and the the in vitro aryl hydrocarbon [benzo(a)pyrenejhydroxylase mammary gland of the rat by 50% or greater (29). Addition assay. However, butylated hydroxytoluene and butylated of BHA to the diet protected against pulmonary neoplasm hydroxyanisole, when applied topically to mice, inhibited resulting from acute exposures to DMBA, BP, urethan, or the in vitro , epidermally mediated, covalent binding of uracil mustard (31). The addition of BHA to diets containing radioactive benzo(a)pyrene and 7,12-dirnethylbenz(a)an DMBA, 7-hydroxy-1 2@rnethyIbenz(a)anthracene, or 1,2,5,6- thracene to DNA. When butylated hydroxytoluene and bu DBA likewise inhibited pulmonary tumor formation (31). tylated hydroxyanisole were added in vitro , they did not Administration of BHA i.p. caused a decreased number of inhibit the epidermally mediated covalent binding of the pulmonary adenornas resulting from s.c administration of hydrocarbons to DNA. The inhibition of polycyclic hydro diethylnitrosarnine and 4-nitroquinoline N-oxide (30). Sele carbon tumorigenesis by antioxidants may be related to the nium and BHT were effective inhibitors against N-2-fluoren ability of antioxidants to prevent the in vivo activation of ylacetamide-induced liver and mammary tumors (12, 28). hydrocarbons to carcinogenic epoxides and/or other Clayton and Baurnann (5) observed a lower incidence of electrophilicintermediatesormay be relatedtotheirabilitydirnethylarninoazobenzene-induced liver tumors in animals to increase detoxification of the reactive intermediate that fed diets with added sodium selenite. Other antioxidants, requires intact cells to be operational. In any event, the such as 4-rnethyl-2,6-di-tert-butylphenol, inhibited hepatic results suggest that the antioxidants have an indirect effect tumor formation in rats fed p-dimethylaminoazobenzene on the epidermal metabolizing system which leads to a de (8). crease in covalent binding to DNA. The antioxidants selenium (19), vitamin E (22), and ascor bic acid (20), when applied to mouse skin, significantly reduced tumor formation by DMBA initiation in a 2-stage INTRODUCTION system of turnorigenesis. This is a report of further studies on the inhibitory effect of antioxidants on mouse skin BHA,3 BHT, and propyl gallate are antioxidants that are tumorigenesis and the role of epidermal AHH in their inhibi commonly added to foods to maintain freshness and pre tion. vent spoilage by oxidation. They are frequently used in dried cereals, cooking oils, canned goods, and various ani mal foods. The average daily intake of phenolic antioxidants MATERIALS AND METHODS by man has been estimated at 2 mg (6). They are not readily excreted and tend to accumulate in the body (6). These Animals. Female Charles River CD-i mice were pur chased from Charles River Breeding Laboratories, Wilming

I Supported by NIH Grant CA-17605 and by the Energy Research and ton , Mass. Mice, 7 to 9 weeks old , were carefully shaved Development Administration under contract with the Union Carbide Corpora with surgical clippers 2 days before treatment, and only tion. By acceptance of this article, the publisher or recipient acknowledges those mice in the resting phase of the hair cycle were used the right of the U. S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. in biochemical and tumor experiments. The incidence of 2 To whom requests for reprints should be addressed, at Biology Division, both papillomas and carcinomas were recorded weekly, Oak Ridge, National Laboratory, Post Office Box V. Oak Ridge, Tenn. 37830. and papillomas and carcinomas were removed at random 3 The abbreviations used are: BHA. butylated hydroxyanisole; BHT, buty lated hydroxytoluene; DMBA; 7,12-dimethylbenz(a)anthracene; BP, for histological verification. benzo(a)pyrene; DBA, dlbenzanthracene; AHH, aryl hydrocarbon hydroxyl Chemicals. Benz(a)anthracene, 7,8-BF, BP, and 1,2,3,4- ase; BF, benzoflavone; 3-OHBP, 3-hydroxybenzo(a)pyrene; PAH, polycyclic aromatic hydrocarbon. DBA were obtained from Aldrich Chemical Company, Inc., Received July 19, 1976; accepted February 24, 1977. Milwaukee, Wis. DMBA, BHA, BHT, and vitamins C and E

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1977 American Association for Cancer Research. T.J.S!agaand W. M. Bracken were purchased from Sigma Chemical Company, St. Louis, obtained very similar results with respect to both the magni Mo. 3-OHBP was a generous gift from Dr. H. V. Gelboin, tude and order of inhibition of DMBA-initiated tumors by National Cancer Institute, Bethesda, Md. 12-O-tetradeca BHT and BHA. In this report the tumor response is pre noyl-phorbol-13-acetate was prepared as previously de sented as the average number of mice with papillomas and scribed (1) and was purified by preparative thin-layer chro the percentage of mice with papillomas. We consistently matography. [3H]BP (specific activity, 5 Ci/mmole) was pur used 30 mice/group because this number makes our tumor chased from Schwarz/Mann, Orangeburg, N. Y., and data very reproducible when comparing like groups. In Ta [3H]DMBA (specific acitivity, 7 Ci/mmole) was from Arner sham/Searle, Arlington Heights, Ill. AHH Enzyme Assay. The epidermis was isolated as pre viously described (24), and the epidermal material from 3 mice was homogenized with a Polytron PT-i0 homogenizer. This epidermal homogenate was the source of enzyme for the AHH assays. The assay was performed as described by Bowden et a!. (2) with the modifications as detailed in a previous report (26). The specific activity was expressed as pmoles of 3-OHBP formed in 30 mm of incubation per mg of protein. In most cases, the specific activities were ex pressed as a percentage of the acetone control groups, and each time point tested represented an average of 2 to 5 groups containing 3 mice each. The average standard de viation for the control groups for each experiment was less than 20%. Determination of PAH Covalent Binding to DNA. This assay system is based on the one originally described by Gelboin (9) and Grover and Sims (10) with modifications as previously described (4, 27). The specific activity of binding is expressed as pmoles of hydrocarbon bound per @gof DNA per mg of protein per 15 minutes of incubation. A zero incubation value was subtracted from each specific activity, and each experiment was done in triplicate. In most cases each specific activity represents the average of 2 to 3 exper iments.

6 8 10 12 14 16 18 20 RESULTS WEEKS OF PROMOTION Chart 1. Inhibition of DMBA tumor initiation by BHA and BHT. Each group BHA and BHT were found to inhibit DMBA-initiated tu consisted of 30 mice. All animals were initiated with 2.56 @.tgofDMBA and promoted with 10 @.tgof12-O-tetradecanoyl-1 3-acetate twice weekly starting mors in mouse skin, although inhibition by BHT was greater 1 week after initiation. BHA (1 mg) and BHT (1 mg) were given 5 mm before (Chart 1). In a repeat of the experiment shown in Chart 1, we DMBA initiation. 0 DMBA (control); â€¢BHA+ DMBA; t@BHT + DMBA.

1Effect Table initiationThirty of several antioxidants and BF's on DMBA skin tumor DMBAandmice were used per experiment, and all mice were initiated with 2.56 @gof perweek.%promoted 1 wk later with 10 @gof12-O-tetradecanoyl-phorbol-13-acetate 2 times

of surviv miceExpeni- ing Modifier treat- No. of Papillomas/ with papillo mentmas1 mentâ€• Dose/mouse (pg) miceâ€• mousec 822 None 30 3.4 48â€˜3 7,8-BF 25 30 0.9 224 7,8-BF 25 (x 3)d 30 0.4 655 5,6-BF 100 29 2.6 606 5,6-BF 1000 26 2.2 577 BHT 1000 30 1.6 358 BHT 1000 (x 3)â€• 29 0.9 569 VitaminC 1000 30 1.9 Vitamin E 1000 29 2.1 68

a The modifiers were applied 5 mm before the initiator. b Surviving at the 28th week of observation.

C Total number of papillomas/total number of surviving mice. d 7,8-BF (25 /Lg) applied 6 hr before, 5 mm before, and 6 hr after initiation.

P BHT (1000 @g) applied 6 hr before, 5 mm before, and 6 hr after initiation.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1977 American Association for Cancer Research. Antioxidants and Tumor !nitiation ble 1 the effects of several antioxidants and BF's on DMBA Next the effect of adding different antioxidants to the in skin tumor initiation are shown. Even at doses of 25 @g,7,8- vitro AHH assay was determined. As shown in Table 4, the BF was found to be a potent inhibitor of DMBA tumor antioxidants at equimolar or twice the molar amount of the initiation, whereas 5,6-BF inhibited tumorigenesis to a BP substrate had very little effect on the assay. 7,8-BF, a lesser degree. All the antioxidants studied inhibited DMBA strong in vitro inhibitor of the epidermal AHH activity, is tumor initiation. When BHT was applied topically at a dose shown in Table 4 as a means of comparison. The above level of 1 mg at 6 hr before, 5 mm before, and 6 hr after assays were monitored fluorometrically by the conversion DMBA initiation, it inhibited tumorigenesis quite effectively. of BP to 3-OHBP. In general the inhibition of turnorigenesis by the various BHA and BHT, when applied topically to mice, inhibited in antioxidants was greater and more consistent when ex vitro, epidermally mediated , covalent binding of radioactive pressed as the average number of papillomas per mouse BP and DMBA to DNA (Table 5). The inhibition was dose than as the percentage of mice with papillomas. Also, the dependent and also was greater at 3 hr than at 12 hr after inhibitory effect of the antioxidants on the carcinoma mci topical treatment. In general, BHT was more effective than dences correlated better with the average number of papil was BHA in inhibiting the binding of both BP and DMBA to lomas per mouse. DNA. This finding correlates well with the skin tumorigene Table 2 demonstrates the effects of topical application of several antioxidants on mouse epidermal AHH activity. In 4In Table general, the antioxidants tested had either no effect or only vitro effect of antioxidantsfrommouse and 7,8-BF on AHH activity epidermisThe a slight inducing effect on AHH activity in vivo. Also shown mice were pretreated topically with0.5 mg of benz(a) are the results of topical treatment with the strong inducer anthracene 17 hr before being killed.Benz(a)anthracene 1,2,3,4-DMBA, that increased AHH activity by 1100 and 716% of controls at 12 and 24 hr, respectively. We initially induced specific ac suspected that antioxidants had an inducing effect on epi Concentration (xtivity (% of con dermal AHH that could consequently be responsible for the M)trols)â€•BHAIn vitroâ€• 10-i tumor inhibition. In order to confirm this, 1 of the antioxi 11242106BHT dants was randomly selected for further study. As Table 3 reveals, i-mg doses of vitamin E had only a minimal effect on epidermal AHH activity from 2 to 36 hr after topical 1110299Vitamin treatment. Varied doses of vitamin E (25, 50, 125, 250, and 500 @g)showed only slight differences from control values C 1105 12 hr after treatment. 107Vitamin

11022997,8-BFE Table 2 Effect of a single topica application of antioxidants on mouse activityCompound@'SpecificepidIermal AHH 1724 controls)12activityb(% of a The compounds in acetone were added directly to the incuba hrBHA174 hr 24 tion tubes at the final concentrations indicated. The substrate concentration was 10@BP. The average specific activity for the 108BHT154 85VitaminC120 benz(a)anthracene-treated mice was 356 pmoles of 3-OHBP formed per mg of protein. 134Vitamin b pmoles of 3-OHBP formed per mg of protein. Each value repre 1061,2,3,4-DBA1100E131 sents 2 experiments.S.D. for each figure was lessthan 12%. 716 a All compounds were applied at a concentration of 500 @g/0.2 5Effect Table ml of acetone. invitroof a single topical application of either BHT or BHA on the b pmoles of 3-OHBP formed per mg of protein. Each value repre covalent bindingbyepidermal of (3HJBPand (3HJDMBAto DNA sents2 experimentswith duplicate determinationsper experiment. homogenatesThe S.D. for each figure was less than 18%. hrbeforemice were treated topically with BHA or BHT either 3 or 12 killing.Specific Table 3 104)6[3H]BP activity (x Effectmouseepidermal of a single topical application of 1mg of vitamin E on AHH activity as a functioninductionSpecific of time after [3H]DMBATreatment activityâ€• Time after treatment hrControl 3 hr 12 hr 3 hr 12 (hr)E2 Acetone control Vitamin 4.5BHA (acetone) 1.56 1.30 5.0 356 44 3.8BHA(0.5 mg) 0.81 0.92 2.5 3612 43 3.5BHT(1.0 mg) 0.72 0.74 2.2 4718 42 3.2BHT(1.Omg)(0.5 mg) 0.74 0.76 2.3 3924 44 0.52 0.68 1.8 2.6 5636 54 39_ 37 a pmoles bound per @gof DNA per mg of protein per 15 mm of incubation at 37Â°in the dark. Each value represents 2 experiments a pmoles of 3-OHBP formed per mg of protein. Each value repre with triplicate determinations per experiment.S.D. for each figure sents duplicate determinations. was lessthan 22%.

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6The Table action against PAH-, nitrosarnine-, and dimethylhydrazine ofradioactivein vitro effect of BHA and BHT on the covalent binding induced neoplasia. Disulfirarn and dirnethyldithiocarba homogenatesTheBP and DMBA to DNA by epidermal mate, when added to the diet, inhibited DMBA-induced unlabeledBPmicewere pretreatedtopically with 200nmolesof mammary tumor formation and adrenal necrosis in female or DMBA18 hr before killing.Specific rats (32). In the mouse, disulfirarn prevented the occurrence inducedcontrols)â€•In activity (% of of tumors of the forestomach that resulted from BP in the diet (32). Wattenberg (33) also showed that disulfiram, vitro[3H]DMBABHA additionâ€•Concentration [3H]BP added to the diet of female mice, inhibited large bowel 1205xlx 106 neoplasia produced by repeated s.c. administrations of di 107BHT 112 rnethylhydrazine. In general, the antioxidants may protect against chemical carcinogenesis by inhibiting the fo@rnation 1075xlx 94 of the carcinogenic electrophile in a number of structurally 88 123 diverse chemical compounds. a The compounds in acetone were added directly to the incuba The enzyme system AHH is part of the microsomal mixed tion tubes at either equal or 5 times the concentration of the BP or function oxidases that convert PAH to ultimate carcinogens DMBAsubstrateconcentration. that are thought to be reactive epoxides. These electro 6 pmoles bound per @g of DNA per mg of protein. Each value represents 2 experiments with triplicate determinations per experi philes can then be converted to trans-dihydrodiols by the mont. S.D. foreach figurewas lessthan 15%. action of microsornal epoxide hydrase(s), rearranged spon taneously to phenols, conjugated with glutathione, or corn sis studies. Table 6 shows that when BHA and BHT were bined covalently with cellular nucleophiles (3, 7, 13, 15, 17, added in vitro they did not inhibit the epidermally mediated 18, 23). In a recent investigation, using epidermal hornoge covalent binding of radioactive BP and DMBA to DNA. Even nates and NADPH as the electrophile-generating system at 5 times the DMBA or BP substrate concentration used in (24), we showed that a strong correlation exists between the the in vitro binding assay, no inhibition was noted. tumor-initiating ability of several PAH's and their ability to bind covalently to DNA in vitro. To explain chemical carcinogenesis, Miller (16) has pro DISCUSSION posed a general theory that has had a significant impact on cancer research. He suggested that: (a) most chemical car The results of this study show that the inhibition of PAH cinogens that are not themselves chemically reactive must tumorigenesis by antioxidants may be related to their ability be converted metabolically into a chemically reactive form; to prevent the in vivo activation of PAH to carcinogenic (b) the activated metabolite is an electrophilic reagent; and epoxides and/or other electrophilic intermediates or may be (c) this activated metabolite reacts with nucleophilic groups related to their ability to increase detoxification of the reac in cellular macromolecules to initiate carcinogenesis. The tive intermediate. Increased epoxide hydrase and/or gluta fact that all reactive forms thus far characterized are elec thione S-transferase activity in vivo could effectively lead to trophilic represents a generalization that is probably the a decrease in covalent binding to DNA. However, in either only connection among the structurally diverse chemical case, this can occur only in vivo. Speier and Wattenberg carcinogens. Antioxidants, because of their effectiveness in (25) reported that incubation of radioactive BP with DNA inhibiting neoplasia induced by a wide variety of carcino and liver microsomes from BHA-fed mice resulted in sign ifi gens in many different tissues, may become useful cherno cantly less covalent binding of BP to DNA than with control prophylactic agents against environmental carcinogens. microsomes. They also reported that BHA feeding did not change the liver AHH activity (25). As revealed in Tables 2 REFERENCES and 3, the antioxidants tested had neither an effect on inducing epidermal AHH nor any significant effect when I . Baird, W. M., and Boutwell, A. K. Tumor-Promoting Activity of Phorbol added directly to the in vitro assay or to the in vitro binding and Four Diesters of Phorbol in Mouse Skin. 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Thomas J. Slaga and William M. Bracken

Cancer Res 1977;37:1631-1635.

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