Vol. 76 No. 5 December 1993

Editor: &. A!. Eversole

Vitamin G enhances the development of carcinomas in the hamster buccal pouch experimental mode

Joel Schwartz, DMD, DMSC,~ Gerald Shklar, DDS, MS,b and Diane Trickler, HT/HTL,C Boston, Mass. DEPARTMENT OF ORAL MEDICINE AND ORAL PATHOLOGY, HARVARD SCHOOL OF DENTAL

MEDICINE

Forty young adult male Syrian hamsters (Mesocricetus auratus) were divided into four groups of IO animals. The animals in group 1 (tumor control) had the right buccai pouches painted three times a week with a 0.5% solution of 7,12 dimethylbenz(a)anthracene in heavy mineral oil USP with the use of a number 4 sable brush. The animals in group 2 (experimental group) had the right buccal pouches painted with the same solution as group 1. In addition, they received 1 mg ascorbic acid in 0.5 ml mineral oil three times a week on days alternating with the other application. The ascorbic acid was administered by mouth with the use of a pipette. The animals in group 3 received 1 mg ascorbic acid in 0.5 ml mineral oil three times weekly, and the animals in group 4 were untreated controls. The animals were killed after 14 weeks. Tumors were counted and measured. Both right and left (control) pouches were photographed, excised, fixed in formalin, sectioned in paraffin, and studied histologically. The animals that received the ascorbic acid (vitamin C) had significantly larger tumors in the right buccal pouch, although actual numbers of gross tumors were only slightly increased. The figures for tumor burden in the animals in groups 1 and 2 were 364 versus 648 mm3. Histologic study revealed that the animals in group 2 had more anaplastic tumors and a significantly greater number of areas of dysplastic leukoplakia than the animals in group 1. (ORAL SURC ORAL MED

ORAL PATHOL 1993;76:718-22)

There has been controversy on the possible role of vi- tamin C as a potential cancer inhibitory or preventive agent. Several early studies had discussed the possi- ble anticancer properties of vitamin C.‘, 2A controlled trial found no benefit of high-dose vitamin C in patients with advanced cancer.3 Because many recent studies have demonstrated the cancer inhibitory or preventive action of a number of antioxidant nutrients such as retinoids,4, 5 carotenoids,6, 7 and tocopheroLs, 9 it was of interest to test the cancer inhibitory effect of vitamin C, another potent antioxidant.102 l1 An ex- periment was designed with the use of the hamster buccal pouch carcinoma model, an excellent carcino- genesis system in which epidermoid carcinomas are developed as a result of a three times a week painting with the potent carcinogen, 7,12 dimethylbenz[ alan-

aAssociate Professor of Oral Pathology. bCharles A. Brackett Professor of Oral Pathology. CResearch Assistant in Oral Pathology. Copyright a 1993 by Mosby-Year Book, Inc. 0030-4220/93/$1.00 + .I0 7/14/50245

718

thracene (DMBA). In this experimental model, pre- cancerous leukoplakia develops within 6 to 8 weeks, carcinoma in situ within 8 to 10 weeks, epidermoid carcinomas within 10 to 12 weeks, and frankly inva- sive and proliferative large tumors at 12 to 14 weeks.12T I3

MATERIAL AND METHODS Forty male, young adult Syrian golden hamsters

(Mesocricetus auratus) served as experimental ani- mals and were divided into equal groups of 10 animals. The animals were randomly bred (Lakeview Strain LVG, Charles River Breeding Laboratories, Wilmington, Mass.), housed five per cage, and fed standard Purina Laboratory pellets (Ralston Purina, St. Louis, MO.) and water ad libitum. The animals were maintained in a controlled environment under standardized conditions of temperature (24” C) and humidity (35%) with an alternating 12-hour light and dark cycle. The animals were 60 to 90 days old and weighed 96 to 102 gm at the beginning of the exper- imental period.

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Schwartz, Shklar, and Trickier 719

Fig. 1. L.arge epidermoid carcinoma and several small carcinomas of hamster buccal pouch in animal in group 1 (t.umor control).

Fig. 2. Several moderately sized carcinomas and areas of leukoplakia in buccal pouch of animal in group 1.

The animals in group 1 (tumor control group) had the right buccal pouches painted three times per week with a 0.5% solution of DMBA in heavy mineral oil USP, with a number 4 sable brush. Each application leaves approximately 0.6 mg of DMBA on the mucosal surface of the pouch as determined by the use of radioactive 14C DMBA.

The animals in group 2 (experimental vitamin C group) had the right pouches painted with DMBA as in group 1. In addition., they received 1 mg vitamin C (L-ascorbic acid, Sigma Chemical Co., St. Louis, MO.) in 0.5 ml mineral oil delivered orally by pipette

three times a week on days alternating with the DMBA painting.

The animals in group 3 (vitamin C control) re- ceived only the vitamin C as in group 2. And the an- imals in group 4 (untreated control) received neither DMBA painting nor orally administered vitamin C.

Animals were observed weekly for tumor develop- ment and killed in a carbon dioxide chamber after 14 weeks. Tumors of right buccal pouches were counted and measured. The right and left buccal pouches were excised, fixed in 10% formalin, sectioned in paraffin, and stained with hematoxylin and eosin for micro-

72% Schwartz, Shklar, and Trickier ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December I993

Fig. 3. Extensive proliferative lesion of buccal pouch with surface necrosis in animal in group 2.

Fig. 4. Large buccalpouch carcinoma has invaded through cheek to appear as massive tumor on skin surface in animal in group 2.

scopic study. Cervical lymph nodes and salivary glands were also excised and similarly prepared for histologic study.

Figures for tumor burdenI were developed by multiplying the number of tumors in each group by

the mean tumor volume in mm (4/3 7rr3 where r rep- resents the tumor radius in mm). Statistical analysis that compared tumor burden in the animals in groups 1 and 2 was carried out using the Student’s t test. Measurements of tumors were carried out grossly at autopsy with the use of a micrometer. The measure- ments for tumor burden were based on the assumption that the tumors were usuaily spherical in overall morphology. Because not all tumors are spherical the figures for tumor burden represent a reasonable approximation of the true figures.

RESULTS The animais in group 2 (DMBA + vitamin C)

demonstrated a slight increase in the number of tumors when compared with the animals in group 1 (DMBA only). However, the tumors were substan- tially larger and appeared to be more invasive with fixation of the pouch tissue so that many pouches could not be easily everted (Figs. 1 to 3). In two an- imals the carcinomas had actually penetrated through the buccal mucosa and appeared as large lesions on the skin surface (Fig. 4). The tumors were epidermoid carcinomas (Fig. 5).

Mean tumor burden was 364 mm3 in the DMBA control group and 648 mm3 in the vitamin C exper- imental group (Fig. 6). Statistical significance was r0.001 by the Student’s t test. Histologic study revealed epidermoid carcinomas in both groups with more anaplastic lesions in the group that was given vitamin C. There was a greater number of areas of dysplastic leukoplakia in the animals given vitamin C, compared with the tumor control animals. Metasta- sis to cervical lymph nodes had not yet occurred in ei-

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Schwartz, Shklar, and Trickier 721

Fig. 5. I’hotomicrograph of moderately differentiated epidermoid carcinoma in animal in group 2. (Hema- toxylin-eosin stain; original magnification X200.)

ther group by the 14-week termination of the exper- iment.

The animals in group 3 (vitamin C but no carcin- ogen) and group 4 (no carcinogen) represented con- trols for groups 1 and 2. There were no tumors of the right buccal pouches and no other evidence of disease in these control groups.

DISCUSSION Ascorbic acid has been reported to be associated

with the suppression of carcinogenesis,15, l6 but un- fortunately there is a lack of information about the action of ascorbic acid during oral carcinogenesis. Furthermore, thle hydroxyl radical of ascorbate has been associated with the progression of carcinogene- sis in a rat bladder tumor model.r7 Therefore the role of vitamin C in carcinogenesis and tumor progression requires further 8clarification.

The endogenous production of ascorbic acid is found in many mammals including the rodents.18 Unfortunately, the human being is not one of these speciest9, 2o and .requires exogenous dietary vitamin C. The ability to produce endogenous ascorbic acid becomes critical in this discussion of the possible pro- duction of hydroxyl radicals of ascorbic acid during the process of oral carcinogenesis.2’

Specifically, in the hamster, lwhich endogenously produces ascorbic acid, L-ascorbic acid has been noted to inhibit squamous metaplasia in tracheal ex- plants when administered with asbestos or ben-

600

T U

m

0

r

B 400

U

ii e

n

200

(mm3)

0

Tumor Control Vitamin C

Experimental Groups

Fig. 6. Graphic representation of tumor burden in ani- mals in group 1 and group 2.

722 Schwartz, Shklar, and Trickier ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 1993

zo[ alpyrene-coated asbestos. i5 In another study, vi- tamin C was orally administered for 12 days in a sat- urated fat (20% lard or 5% lard) diet, after the last carcinogenic treatment of N-nitrosobis(2-oxyopropy- 1)amine. The results indicated that vitamin C (L- ascorbic acid) in the 5% lard diet significantly reduced the presence of ductal carcinomas in the pancreas of the hamster.22

5.

6.

the induction of tumors of forestomach and cervix in the Syr- ian hamster by carcinogenic polycyclic hydrocarbons. Cancer Res 1965;25:884-95. Bollag W. Therapeutic effects of an aromatic retinoic acid an- alog on chemically induced skin papillomas and carcinomas. Eu; J Cancer 1974;10:731-7. _ - Suda D. Schwartz J. Shklar 6. Inhibition of exoerimental oral carcinogenesis by topical beta carotene. Carcinogenesis 1986; 7:711-5.

7.

These results are in contrast with the results we present here, which related oral administration of L-ascorbic acid dissolved in heavy mineral oil to increased oral carcinoma size development in the hamster buccal pouch. We reconcile the differences in tumor development to the following differences in our protocol: (1) the carcinogen used to develop the tumor, (2) the vehicle used to administer the vitamin C, and (3) the type of tumor induced.

The carcinogen, DMBA, becomes metabolized to a complete carcinogen through its oxidation to 1,2 diol epoxide.23 This oxidant binds to and damages DNA.23 The metabolism of DMBA is known to occur in the microsomes of the liverz4 but would also take place in the well-oxygenated buccal pouch. This metabolic oxidation process oxidizes L-ascorbate to the ascorbic hydroxyl radical. This oxygen reactive radical could produce DNA changes (mutations and deletions) that enhance tumorigenesis. Other polyaromatic hydro- carbons would also enhance the oxidation of ascorbic acid as a byproduct of their own oxidation to epoxides that damage and intercalate into DNA.

The use of different vehicles to administer vitamin C must also be considered. The use of a saturated fat to administer vitamin C may significantly change its oxidative dynamics.

8.

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15,

16.

17.

Schwartz .I, Shklar G. Regression of experimental oral carci- nomas by local injection of beta carotene and canthaxanthin. Nutr Cancer 1988;11:35-40. Shklar, G. Inhibition of oral mucosal carcinogenesis by vita- min E. J Nat1 Cancer Inst 1982;68:791-7. Shklar G, Schwartz JL, Trickier DP, Niukian K. Regression by vitamin E of experimental oral cancer. J Nat1 Cancer Inst 1987;78:987-92. Niki E, Saito T, Kamiya Y. The role of vitamin C as an an- tioxidant. Chem Lett 1983;631-2. Doba T, Burton GW, Ingold KU. Antioxidant and co-antiox- idant activity of vitamin C: the effect of vitamin C either alone or in the presence of vitamin E, or a watersoluble vitamin E analogue upon the peroxidation of aqueous multi-lamellar phospholipid liposomes. Biochem Biophys 1985;835:298-303. Salley JJ. Experimental carcinogenesis in the cheek pouch of the Syrian hamster. J Dent Res 1954;33:253-8. Shklar 6. Experimental oral pathology in the Syrian hamster. Prog Exp Tumor Res 1972;16:518-38. Niukian K, Schwartz J, Shklar 6. Effect of onion extract on the development of hamster buccal pouch carcinomas as expressed in tumor burden. Nutr Cancer 1987;9:171-6. Holtz G, Bresnick E. Ascorbic acid inhibits the squamous metaplasia that results from treatment of tracheal explants with asbestos or benzo(a)pyrene-coated asbestos. Cancer Lett 1988;42:23-8. Liehr JG, Roy D, Gladek A. Mechanism of inhibition of estrogen-induced renal carcinogenesis in male Syrian hamsters by vitamin C. Carcinogenesis 1989;10:1983-8. Fukushima S, Kurata Y, Hasegawa R, Asamoto M, Shibata MA, Tamano S. L-ascorbate acid amplification of bladder carcinogenesis promotion by K2 C03. Cancer Res 1991; 51:2548-51.

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It is also evident that cancer cell implants and the observed inhibition of their seeding and growth is dif- ferent from the host chemically induced system used in the hamster oral cancer studies presented here.

A possible role for vitamin C in reducing the risk of several types of cancers is suggested by epidemiologic evidence. Unfortunately, this evidence is inconsistent possibly because of the oxidation properties of ascorbic acid forming a hydroxyl radical and other chemopre- ventive influences that remain to be elucidated.

Scott ML. Environmental influences on ascorbic acid require- ments in animals. Ann N Y Acad Sci 1975;2§8:151-5. Hornig D. Distribution of ascorbic acid, metabolites and an- alogues in man and animals. Ann N Y Acad Sci 1975;258:103- 117.

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Wang YH, Dhariwal KR, Levine M. Ascorbic acid bioavail- ability in humans. Ann N Y Acad Sci 1992;669:383-6. Sies H, Stahl W, Sundquist AR. Antioxidant function of vita- mins: vitamin E and beta-carotene, and other carotenoids. Ann N Y Acad Sci 1992;669:7-20. Woutersen RA, Hetmer A. Inhibition of dietary fat-promoted development of (pre) neoplastic lesions in exocrine pancreas of rats and hamsters by suppiemental vitamin A, C, and E. Can- cer Lett 1988;41:179-89. Miller EC. Some current perspectives on chemical carcino,- genesis in humans and experimental animals. Cancer Res 1979;38:1479-96. Oesch HR, Vogel GK, Petrovic SP, Platt KL. Dihydrodiol de- hydrogenase: a new level of control by both sequestration of proximate and inactivation of ultimate carcinogen. In: Greim H, Kramer JM, Marquardt H, Oesch F, eds. Biochemical ba- sis of chemical carcinogenesis. New York: Raven Press, 1984;23-31.

Reprint requests: Gerald Shklar

3. Cregan ET, Moertel CG, O’Fallon JR, et al. Vitamin C Department of Oral Medicine and Oral Pathology (ascorbic acid) therapy to benefit patients with advanced can- Harvard School of Dental Medicine cer: a controlled trial. N Engl J Med 1979;301:687-90. 188 Longwood Avenue

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