[CANCER RESEARCH 40, 2367-2371. July 1980]0008-5472/80/0040-OOOOS02.00

Inhibition of Skin Tumor Promotion by Retinoic Acid and Its Metabolite5,6-Epoxyretinoic Acid1

Ajit K. Verma, Thomas J. Slaga, Phillip W. Wertz, Gerald C. Mueller, and R. K. Boutwell2

McArdle Laboratory for Cancer Research. The University of Wisconsin, Madison, Wisconsin 53706 ¡A.K. V.. P W. W.. G. C. M., R. K. B.I, and Biology Division,Oak Ridge National Laboratory, Oak Ridge. Tennessee 37830 ¡TJ. S.¡

ABSTRACT

The ability of 5,6-epoxyretinoic acid, a biologically activemetabolite of retinole acid, to inhibit both the induction ofornithine decarboxylase (ODC) activity and skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was evaluated. Application of 5,6-epoxyretinoic acid either concurrentlywith or 1 hr after each application of TPA to the initiated mouseskin inhibited the formation of skin tumors as effectively as didretinoic acid. 5,6-Dihydroretinoic acid, which is a poor substrate for epoxidation, also inhibited skin tumor promotion. 5,6-Epoxyretinoic acid, 5,6-dihydroretinoic acid, and retinoic acidwere equally effective in inhibiting the induction of ODC activityby TPA. Insect juvenile hormones inhibited neither the inductionof ODC activity nor skin tumor promotion by TPA.

These results indicate that (a) epoxidation of retinoic acid atthe 5,6-position is not a rate-limiting modification for the anti-

promoting activity of retinoic acid and that (b) inhibition of theinduction by TPA of mouse epidermal ODC activity may be asimple test for screening the potential prophylactic activities ofnew retinoids.

INTRODUCTION

The chemical induction of cancer in mouse skin can bedivided into at least 2 defined stages, initiation and promotion(1-3, 30, 44). Application of a single subcarcinogenic dose ofa carcinogen leads to the process of initiation. This treatmentwith the carcinogen alone, however, will not lead to the development of visible tumors during the life span of the animal.Visible tumors do develop when the initiated skin is subjectedto repeated exposure to a second chemical known as a promoter. Among several known tumor promoters, TPA,3 a com

ponent of croton oil, is the most potent (3).Application of TPA to mouse skin leads to numerous biolog

ical and biochemical changes in the epidermis (3, 4, 31 ); theseinclude a dramatic increase (about 200-fold) in activity of

epidermal ODC (EC 4.1.1.17) (5, 33). Recently, we reportedthat the application of retinoic acid and certain related congeners 1 hr before the treatment of mouse skin with TPA inhibitsthe induction of epidermal ODC activity by TPA and that theability of various retinoids to inhibit ODC induction correlateswell with their ability to inhibit skin tumor promotion by TPA(40, 41). Furthermore, the activity of retinoids in the mouse

1The work was supported by NIH Grants CA-07175 and CA-22484; National

Cancer Institute Contract Y01CP70227; and the Office of Health and Environmental Research. United States Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.

2 To whom requests for reprints should be addressed.3 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;

ODC, ornithine decarboxylase; DMBA, dimethylbenz(a)anthracene; B(a)P,benzo(a)pyrene.

Received February 5, 1980; accepted April 15, 1980.

skin system with few exceptions correlates with their activityas retinoids in various in vitro test systems (6, 7, 17, 22, 23,35-38, 43). Thus, the property of retinoids to inhibit ODC

induction and skin tumor promotion by TPA may provide asimple test for screening the potential prophylactic activities ofnew retinoids.

Several recent reports (28, 29, 32, 34, 42) provide evidencethat 5,6-epoxyretinoic acid may be an active metabolite ofretinoic acid. In addition 5,6-epoxyretinoic acid has beenshown to promote the growth of vitamin A-deficient rats (16).In this paper, we provide evidence that 5,6-epoxyretinoic acid

is also active in mouse skin where it inhibits both the inductionof ODC activity and skin tumor promotion by TPA. This activityof 5,6-epoxyretinoic acid is compared with that of 2 otheractive agents, retinoic acid and 5,6-dihydroretinoic acid as well

as the relatively inactive juvenile hormones.

MATERIALS AND METHODS

Materials. Female Charles River CD-1 mice or female Oak

Ridge Sencar mice were used for experimentation at 7 to 9weeks of age. TPA was obtained from Dr. Peter Borchert, EdenPrairie, Minn. DMBA was purchased from Eastman OrganicChemicals, Rochester, N. Y. Retinoic acid (all-frans) and juve

nile hormones were from Sigma Chemical Co., St. Louis, Mo.5,6-Epoxyretinoic acid and 5,8-oxyretinoic acid were preparedas previously reported (42). 5,6-Dihydroretinoic acid was supplied by Dr. B. A. Pawson, Hoffmann-La Roche, Inc., Nutley,N. J. DL-{1-'4C]Ornithine hydrochloride (specific activity, 49.9

mCi/mmol) was purchased from New England Nuclear, Boston,Mass.

Treatment of Mice. All mice were housed 10/cage in stainless steel cages, and food and water were available ad libitum.Mice were kept in a normal rhythm of 12-hr light and 12-hr

dark periods. The dorsal skin of the mice was shaved 3 to 4days before treatment, and only those mice in the resting phaseof the hair cycle were used for experimentation. The solutionsof DMBA, B(a)P, TPA, and retinoids were prepared in acetoneand were delivered to the shaved areas of individual mice in avolume of 0.2 ml. Control mice were treated with the samevolume of acetone.

Tumor Induction Experiments. Tumors were initiated in allmice by a single application of either B(a)P or DMBA in 0.2 mlof acetone. Two weeks later, all mice were treated twice weeklywith TPA for the duration of the experiment. Mice were treatedwith the retinoid either concurrently with or 1 hr after eachapplication of TPA. Controls were treated with solvent only (0.2ml acetone). There were at least 30 mice in each treatmentgroup. The incidence of papillomas was recorded weekly.

ODC Assay. At appropriate times after treatment, mice werekilled by cervical dislocation, and the epidermis from individual

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A. K. Verma et al.

mice was separated by a brief heat treatment (57°for 30 sec)

(27). The epidermal preparations from 2 to 3 mice were pooled,homogenized in 50 FTIMsodium phosphate buffer (pH 7.2)containing 0.1 HIMpyridoxal phosphate and 0.1 rtiM EDTA, andcentrifuged at 30,000 x g for 30 min to give a soluble supernatant (39).

ODC activity in the clear supernatant was determined bymeasuring the release of 14CO2from DL-[1-14C]ornithine hydro-

chloride. The assay mixture contained 40 nriM sodium phosphate (pH 7.2), 0.2 rriM pyridoxal phosphate, 4 rriM dithiothre-itol, 1 HIM EDTA, 0.4 HIM L-ornithine containing 0.5 /iCi of DL-[1-14C]ornithine hydrochloride, and 100 /il (0.1 to 0.3 mg

protein) of epidermal extract in a final volume of 0.25 ml. Afterincubation at 37°for 30 min in 15-ml Corex centrifuge tubes

equipped with rubber stoppers and center well assemblies, thereaction was stopped by the addition of 0.5 ml of 2 M citricacid. The incubation was continued for at least another 1-hrperiod to ensure complete absorption of 14CO2by the ethanol-

amine and methoxyethanol (0.2 ml; 2:1, v/v) contained in thecenter well. Finally, the center well and its contents weretransferred to a vial containing 10 ml of toluene-based scintil

lation fluid and 2 ml of ethanol. The radioactivity was measuredin a Packard Tri-Carb liquid scintillation spectrometer with an

80% efficiency. Assays were always carried out in duplicate.Blank assays contained no enzyme or boiled enzyme.

The protein content of the epidermal extracts was determined by the procedure of Lowry et al. (24).

RESULTS

Inhibition by Retinólas of Skin Tumor Promotion by TPA.The ability to inhibit skin tumor promotion by TPA is notconfined to retinole acid only. A number of natural as well assynthetic retinoids have been shown to inhibit skin tumorpromotion by TPA (39-41). In order to compare the relativeprophylactic property of 5,6-epoxyretinoic acid to retinoic acid

and other congeners, their effect on skin tumor promotion byTPA was determined. The results are shown in Chart 1. In thisexperiment, 17 nmol of each of the retinoids were appliedtopically 1 hr after each application of 5 nmol of TPA to theinitiated skin of CD-1 mice. Retinoic acid, 5,6-epoxyretinoicacid, and 5,6-dihydroretinoic acid inhibited the number of

papillomas per mouse by 93, 95, and 80% and mice bearingpapillomas by 70, 80, and 40%, respectively.

A more detailed dose effect of retinoic acid and 5,6-epoxyretinoic acid on skin tumor promotion in Sencar mice is shownin Chart 2. Application of 0.35 nmol of either of the retinoidsdid not significantly inhibit skin tumor promotion by 1.6 nmol ofTPA; 3.5 nmol of retinoic acid or 5,6-epoxyretinoic acid in

hibited the number of papillomas per mouse by 52 and 48%and the incidence of carcinomas by 45 and 40%, respectively.Application of 70 nmol of retinoic acid inhibited the number ofpapillomas per mouse by 92% and completely inhibited theincidence of carcinomas (Chart 2).

The Effect of Retinoids on Skin Tumor Initiation. Thepossibility that the retinoids may inhibit formation of skin tumorsby interference with tumor initiation was explored. The resultsare shown in Table 1. Topical application of as much as 333nmol of either retinoic acid or 5,6-epoxyretinoic acid either 6hr or 5 min before or 6 hr after the single application of theinitiator (0.2 /¿molof B(a)P) to Sencar mice did not significantly

80

2 60

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88 20

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ORA

A5,6-epoxy RA

. B5,6-dihydroRA

20

WEEKS OF PROMOTION

Chart 1. Inhibition of skin tumor promotion by the retinoids. CD-1 mice wereinitiated with 0.2 /imol of DMBA in 0.2 ml of acetone. Beginning 14 days afterinitiation, all mice were treated twice weekly with 5 nmol of TPA for the durationof the experiment. Mice were treated with 17 nmol of the retinoids in 0.2 ml ofacetone 1 hr after each treatment with 5 nmol of TPA. Control mice were treatedwith acetone only. There were 30 mice in each treatment group, and the incidenceof papillomas was recorded weekly. RA, retinoic acid; 5.6-epoxy RA, 5.6-epox-yretinoic acid; 5,6-dihydro RA, 5,6-dihydroretinoic acid.

IllOZLUQO60

5 40

30

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r~1 RETINOIC ACID

5,6-EPOXYRETINOIC ACID

175 3.5 175 35None 0.35 3.5 35 70 0 35 IDOSE (nmol)

Chart 2. The effect of retinoid dose on skin tumor promotion by TPA. Thesencar mice were initiated with 10 nmol of DMBA and promoted twice weeklywith 1.6 nmol of TPA plus the retinoid for the duration of the experiment. Bottom,papillomas mouse at 16 weeks after promotion; fop, carcinoma incidence (percentage of mice with carcinomas) at 45 weeks after promotion.

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5,6-Epoxyretinoic Acid Inhibits Tumor Promotion by TPA

affect the yield of skin tumors elicited by subsequent applications of 3.2 nmol of TPA. In addition, it has been shownpreviously (41) that retinoic acid does not interfere with skintumor initiation of CD-1 mice with DMBA.

Correlation of the Inhibition by Retinoids of the Inductionof Epidermal ODC Activity and of Skin Tumor Promotion byTPA. In order to further document the correlation between theability of a retinoid to inhibit the induction of ODC activity andskin tumor promotion by TPA (39, 41), the effect of 5,6-epox-yretinoic acid and of 5,6-dihydroretinoic acid on the induction

of ODC activity by TPA was determined, and the results areshown in Table 2. In this experiment, 17 nmol of each retinoidwere applied 1 hr after treatment with 17 nmol of TPA. Allretinoids tested inhibited both induction of ODC activity as wellas skin tumor promotion by TPA (Table 2). Furthermore, retinoids applied either 1 hr before or 1 or 2 hr after TPA treatmentdid not differ in their ability to inhibit ODC induction by TPA(Chart 3). In addition, the dose of the retinoid required to inhibitby 50% the induction of ODC activity by TPA for either retinoicacid, 5,6-epoxyretinoic acid, or 5,6-dihydroretinoic acid was

0.17 nmol.Effect of Juvenile Hormones on Skin Tumor Promotion by

TPA. Insect juvenile hormone (10,11 -epoxymethylfarnesoate),

which bears an epoxide moiety at a position corresponding tothe 5,6-double bond in the ionone ring of retinoic acid, inhibits,as does retinoic acid, the TPA-enhanced thymidine incorpora

tion into DNA and the induction of ODC activity of bovinelymphocytes (18, 19). We studied the effect of juvenile hormones I, II, and III on mouse skin tumor promotion by TPA inSencar mice. The results are shown in Table 3. None of the

Table 1Effects of retinoic acid and 5,6-epoxyretinoic acid on skin tumor initiation

Sencar mice were initiated with 0.2 ¿imolof B(a)P and promoted 1 week laterwith twice-weekly applications of 3.2 nmol of TPA. Retinoic acid or 5.6-epoxyretinoic acid at a dose of 333 nmol was applied either before or after initiationwith B(a)P.

TreatmentNone

Retinoic acid 5 min before B(a)P5,6-Epoxyretinoic acid 5 min before B(a)P5,6-Epoxyretinoic acid 6 hr before B(a)P5,6-Epoxyretinoic acid 6 hr after B(a)PPapillomas/mouse

at 16wk5.6

5.86.04.95.2

Table 2

Inhibition by retinoids of the induction of ODC activity and skin tumor promotionby TPA

CD-1 mice were initiated with 0.2 nmol of DMBA in 0.2 ml of acetone.Beginning 2 weeks after initiation, mice were treated twice weekly with 5 nmol ofTPA for the duration of the experiment (22 weeks). Mice were treated withretinoids in 0.2 ml of acetone 1 hr after each treatment with TPA. Control micewere treated with acetone only. To test for ODC induction, separate groups ofmice were used which were treated only once with the retinoid 1 hr aftertreatment with 5 nmol of TPA and were killed for ODC assay 4.5 hr after TPAtreatment.

TreatmentAcetone

Retinoic acid5,6-Epoxyretinoic

acid5,6-Dihydroretinoic

acidDose

(nmol)17

1717ODC

activity(nmol CO2/30

min/mgprotein)2.92±0.11a

0.50 ±0.200.48 ±0.060.72

±0.25Papillo-mas/

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TIME RELATIVE TO TREATMENT WITH TPA (hr)

Chart 3. Effect of treatment with retinoid on the induction of epidermal ODCactivity by TPA. Groups of CD-1 mice were treated with 17 nmol of the retinoidin acetone at the indicated times before and after the application of 5 nmol ofTPA (Time 0). The mice were killed 4.5 hr after TPA treatment, and ODC activityfrom the soluble epidermal extracts was determined. Each point represents themean ±S.E. of determinations of enzyme activity from soluble epidermal extractsprepared from 3 groups of mice with 3 mice/group. ODC activity in the epidermalextracts prepared from mice treated with 5 nmol of TPA only was 2.47 ±0.47nmol of CO2 per 30 min per mg protein, and it did not significantly change whenacetone was applied either 1 hr before or 1 or 2 hr after application of 5 nmol ofTPA. In addition, the results of the effect of retinoid on the induction of ODCactivity by TPA in sencar mice were virtually similar to those of CD-1 mice (notshown).

Table 3

Effect of juvenile hormones on skin tumor promotion

Sencar mice were initiated with 10 nmol of DMBA and promoted 1 week laterwith twice weekly applications of 1.6 nmol of TPA plus 10 /ig of one of thejuvenile hormones.

ModifierPapillomas/mouse

at 16 wk

NoneJuvenile hormone IJuvenile hormone IIJuvenile hormone IIIJuvenile hormone IIIa

6.87.27.45.80.0

Mean ±S.E. of determinations carried out from 3 groups of mice with 3mice/group.

The mice were initiated with 10 nmol of DMBA and promoted twice weeklywith only 10 /ig of juvenile hormone III for 16 weeks (no TPA).

juvenile hormones inhibited the formation of skin tumors promoted by TPA.

DISCUSSION

Retinoic acid is the major metabolite of retinol or retinylesters in several tissues of the rat (8-14). It is required to

maintain normal growth (21, 25, 26, 45) and epithelial differentiation (9), but it cannot replace retinal to support the visualfunction and reproduction (11). Retinoic acid is rapidly metabolized, it is not stored, and its oxidation products are rapidlyexcreted in the bile (14, 15, 20). Recently, 5,6-epoxyretinoicacid has been detected in the intestinal mucosa, kidney, liver,testes, and serum of vitamin A-deficient rats given [3H]retinoic

acid (28, 29). It appears that 5,6-epoxyretinoic acid is a bio

logically active metabolite of retinoic acid (28, 29, 32, 34, 42).It can support growth (16), and it is as active as all-frans-retinoic acid in the reversal of keratinization in trachéalorganculture (28). Furthermore, 5,6-epoxyretinoic acid, like retinoic

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A. K. Verma et al.

acid, antagonizes certain effects of TPA in the bovine lymphocyte system (42). We now report that 5,6-epoxyretinoic acid is

as active as retinoic acid in its ability to inhibit both inductionof ODC activity and mouse skin tumor promotion by TPA.

The results (Charts 1 and 2) indicate that the application of5,6-epoxyretinoic acid either concurrently with or 1 hr after

treatment with TPA inhibits the formation of both papillomasand carcinomas; 5,6-epoxyretinoic acid was as active as retinoic acid. Like retinoic acid (41), 5,6-epoxyretinoic acid did

not affect the initiation of skin carcinogenesis. Inhibition of skintumor formation was observed only when it was applied duringpromotion with TPA (Charts 1 and 2).

Our results concerning the effects of 5,6-epoxyretinoic acid

on skin tumor promotion by TPA do not support the conceptthat 5,6-epoxyretinoic acid is the active form of retinoic acid

required to inhibit skin tumor promotion. Rather, it appears thatepoxidation of retinoic acid at the 5,6-position is not a rate-

limiting modification for the antipromoting activity of retinoicacid and related species. It remains to be determined in mouseepidermis whether retinoic acid is metabolized to 5,6-epoxyretinoic acid and related products.

A number of retinoids with diverse structures inhibit skintumor promotion by TPA (41). As shown in Chart 1 and Table2, 5,6-dihydroretinoic acid, which has a saturated cyclohexyl

ring and is a poor substrate for epoxidation, retains prophylactic activity. Furthermore, 5,8-oxyretinoic acid, an isomer of 5,6-epoxyretinoic acid which is generated from 5,6-epoxyretinoic

acid upon acid treatment, was also able to inhibit skin tumorformation by TPA (data not shown). Juvenile hormone III, aneffective antagonist of phorbol ester-mediated comitogenesisin the lymphocyte system (18, 19), was ineffective in its abilityto inhibit either induction of ODC activity (data not shown) orskin tumor promotion by TPA (Table 3). These results supportthe concept that epoxidation of retinoic acid may not be required for inhibition by retinoic acid of skin tumor promotion byTPA.

Again, a correlation was found to exist between the ability ofa retinoid to inhibit induction of ODC activity and skin tumorpromotion by TPA. This strengthens the concept that inhibitionof the induction by TPA of mouse epidermal ODC activity maybe a simple, rapid in vivo test for antipromoting properties ofretinoids (40).

In conclusion, 5,6-epoxyretinoic acid, a biologically active

metabolite of retinoic acid (28, 29, 32, 34, 42), is a potentinhibitor of skin tumor promotion by TPA, but the results of theeffects of 5,6-dihydroretinoic acid presented here and of other

retinoids reported previously (40) suggest that epoxidation maynot be a necessary modification for the antipromoting properties of a retinoid.

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1980;40:2367-2371. Cancer Res   Ajit K. Verma, Thomas J. Slaga, Phillip W. Wertz, et al.   Metabolite 5,6-Epoxyretinoic AcidInhibition of Skin Tumor Promotion by Retinoic Acid and Its

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