Effects of Vitamin A on 3-Methylcholanthrene-lnduced Squamous Metaplasias and Early Tumors in the Respiratory Tract of Rats 1,2

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Effects of Vitamin A on 3-Methylcholanthrene-lnduced Squamous Metaplasias and Early Tumors in the Respiratory Tract of Rats 1,2 M. Virginia Cone and P.

1 Effects of Vitamin A on 3-Methylcholanthrene-lnduced Squamous Metaplasias and Early Tumors in the Respiratory Tract of Rats 1,2 M. Virginia Cone and P. NeHesheim/ Carcinogenesis Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee SUMMARY-The influence of high levels of vitamin A on the susceptibility of respiratory-tract epithelium to the metaplastogenic and tumorigenic effects of 3-methylcholanthrene (MCA) was tested in rats. Fischer- 344 rats were maintained on a vitamin A-Free diet and were given either 1740 J.Lg or 2 X J.Lg retinyl acetate twice a week intragastrically. At 9 weeks of age each rat received 2 intratracheal injections of 5 mg MCA. Groups of 4-5 rats were killed 2,3,5,8, and 12 weeks after carcinogen administration. Vitamin A storage in the liver, assessed at 3 different times, corresponded to the vitamin A dose. The number of microscopic hyperplastic and metaplastic lesions and grossly visible tumor nodules per rat lung was determined. The development of squamous metaplasias and squamous cell tumors was significantly reduced in rats receiving a high dose of retinyl acetate. Our experiments confirm earlier findings suggesting an inhibitory effect of vitamin A on the development of respiratory-tract tumors in hamsters.-j Natl Cancer Inst 50: , IT HAS BEE~ RECOGNIZED for many years that vitamin A is required to maintain cellular differentiation of certain cpithelial tissues. ~umerous studies have demonstrated the relationship between vitamin A deficiency and keratinization of normally nonkeratinizing epithelia. These include epithelia of the para-ocular and salivary glands, larynx, trachea, bronchi, and urogenital tract in rats (1, 2); similar effects are noted for the guinea pig (3). Studies of in vitro systems have shown that vitamin A prevents keratinization of epithelial tissues (4-6). Addition of excess vitamin A also produces mucous metaplasia (7-). Such investigations have aroused interest in the effects of vitamin A on metaplastic and neoplastic lesions (11-13). Of particular interest to us was the induction, by polycyclic hydrocarbons, of squamous metaplasias in the respiratory tract and their progression to squamous cell carcinomas. In vitro induction of hyperplasias and metaplasias in hamster tracheas by benzo[ajpyrene was inhibited by vitamin A (14). Saffiottietal. (15) demonstrated that vitamin A fed to hamsters after intratracheal carcinogen administration reduced the incidence of squamous metaplasias and malignancies in the respiratory tract. In this laboratory, intratracheal instillation of 3-methylcholanthrene (MCA) caused rapid development of squamous cell carcinomas in the rat. A typical sequence of morphologic events was observed in the peripheral segments of the bronchial tree: hyperplasia; squamous metaplasia; nodular squamous-cell lesion; and finally, squamous cell carcinoma. The first hyperplastic and metaplastic lesions developed 2-4 weeks after carcinogen administration, and numerous macroscopic squamous-cell tumors became visible at 8-12 weeks. Invasive and metastasizing carcinomas developed several months later (16). The present investigation was to determine if the effect of vitamin A on the repiratory-tract tumor response of hamsters, demonstrated by Saffiotti et al. (15), could be reproduced in the rat lungtumor system. Several recent publications have described an enhancement of tumor development by vitamin A in various tissues (17, 18), including mouse lung (19). We also wanted to find out what 1 Received January 5,1973; accepted February, Supported by Public Health Service contract E72208 from the National Cancer Institute and by the Atomic Energy Commission under contract with the Union Carbide Corporation. 3 We thank Anna S. Hammons for her technical assistance: 1599

1600 CONE AND NETTESHEIM stages, if any, in the morphogenesis of lung squamous-cell carcinomas would be altered by vitamin A and how. MATERIALS AND METHODS Treatment of animals.

2 1600 CONE AND NETTESHEIM stages, if any, in the morphogenesis of lung squamous-cell carcinomas would be altered by vitamin A and how. MATERIALS AND METHODS Treatment of animals.-specific-pathogen-free Fischer-344 (F-344) rats were raised and maintained in a barrier facility (20). They were given food and water ad libitum. Pregnant females were fed a diet low in vitamin A [00 IV vitamin A/kg food (autoclavable pellets, TD from General Biochemicals, Chagrin Falls, Ohio)] for the last days of gestation. At weaning, 83 male and female offspring received vitamin A-free diet (TD-69389, General Biochemicals). Of these rats, 63 were given intragastric (ig) administrations of Jlg retinyl acetate (RA) per rat, twice a week. This is a normal maintenance level for rats (21). The remaining 20 rats (group 4), were given 17.4 Jlg twice weekly. All animals were weighed weekly. At 6 weeks of age, the 63 rats were divided into 3 groups: in group 1 the twice-weekly RA dose was raised to 1740 Jlg, and groups 2 and 3 were continued on Jlg RA twice a week. At 9 weeks of age, rats in groups I and 2 (given 1740 and Jlg RA, respectively) received 2 intratracheal injections of 5 mg MCA each, 24 hours apart (Aldrich Chemical Co., Milwaukee, Wis.). The experimental protocol is outlined in table 1. Carcinogen.-The MCA (50 mg/ml) was ground in a mortar, then suspended by ultrasonic vibration in a sterile solution of 0.2% gelatin in physiologic saline. The rats were anesthetized with Metofane (Pitman-Moore, Fort Washington, Pa.) (22), and given injections intratracheally (23) with 0.1 ml carcinogen suspension. Preparation and storage of vitamin A.-Crystalline vitamin A (all trans-ra, 2.88 IV / Jlg), obtained from Eastman Kodak Company, Rochester, New York, was stored under nitrogen gas at C in the dark. Solutions were prepared every 5 days; the desired amount of RA was weighed, dissolved in chloroform, and brought to volume with cottonseed oil (General Biochemicals). The final amount of chloroform did not exceed 1.2% (vol/vol), and each RA dose was delivered ig in a volume of 0.1 mi. Solutions were stored under nitrogen gas at 4 0 C in the dark. Tissue preparation.-from each of the 4 groups, 2 males and 2 females, were killed by intraperitoneal injection of 0.5 ml Diabutal (Diamond Laboratories, Des Moines, Iowa), 2, 3, 5, 8, and 124 weeks after carcinogen administration. Lungs were fixed intravenously with 1.25% glutaraldehyde in 0.06M sym-collidine buffer (ph 7.4) and then, with trachea and esophagus, immersed in fixative. After examination under the dissecting scope to determine size and number of tuulors, the lung lobes were sectioned parallel to the long axes of the primary bronchi and prepared for paraffin embedding. Sections of lung, trachea, and esophagus were stained with hematoxylin and eosin. Sections from all lobes of lungs from each animal were used for histologic studies; 3 sections 50Jl apart were taken from each block. We counted the hyperplastic and metaplastic lesions and the number of longitudinally sectioned bronchioles in these sections. The data are expressed as number of hyperplastic and/or metaplastic lesions per 0 bronchioles, since these lesions are found in the bronchiolo-alveolar regions, and it is not always possible to section through comparable areas of lung. Livers, to be used for vitamin A determination, were frozen in tightly capped vials and stored at C. Their vitamin A content was determined by the colorimetric method with trifluoroacetic acid (24). RESULTS TABLE 1.-Experimental protocol Throughout the experiment, none of the animals exhibited signs of impaired health. There were no symptoms of either vitamin A deficiency or hypervitaminosis as indicated by the body weight gains, which were not noticeably affected by the vitamin A doses. Text-figure 1 shows growth curves of the groups given MeA. Body weights became stationary in all groups between 8 and 12 weeks after carcinogen administration. The amount of vitamin A stored in the liver was determined for 2 rats per group at ages 11, 14, and 21 weeks (table 2). Differences in amounts of vitamin A detected corresponded roughly with 4 At the 12-week time point, 3 males and 2 females were killed. Group No. From age 3 to 6 wk RA dose* (Jlg) From age 6 wk to death Carcinogen at age 9 wk Number of animals mg MCAt 5 mg MCAt None None Administered twice a week. t 2 injections, 24 hours apart. JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 EFFECT OF VITAMIN A ON LUNG TUMOR INDUCTION /.< /~A '00 ',//~!Ii HIGH RA o 1 DOSE BEGUN WEANING 3WK / ~ '/ / //,;::';::/. Group No. MeA AGE IN WEEKS -_ TABLE 2.-Liver vitamin A* TEXT-FIGURE I.-Growth of rats maintained on different vitamin A intakes and given MCA at 9 weeks of age. There were -11 rats per group at the start of the experiment. D: ri', 1740 p.g RA twice a week;.: ri', p.g RA twice a week; 0:.., 1740 p.g RA twice a week;.:.., p.g RA twice a week. Arrow indicates time of death after MCA application. Age 11 wk Age 14 wk Age 21 wk Itat l:t Itat 2:t 'Values are I'g vitamin Ajg liver. tfor dosage, see table 1; 2 rats per group (total, 24 rats) were tested at each time point. differences in dose levels on which the groups had been maintained (table 1). In addition, a difference in liver storage of vitamin A occurred between carcinogen-treated and normal animals that received the same amounts of the vitamin (cf. groups 2, 3). The carcinogen-treated animals stored consistently lower levels of vitamin A than did their controls. As described previously (16), MeA injected intratracheally into F-344 rats induced peripheral hyperplastic and metaplastic lesions originating at bronchiolo-alveolar junctions in the lung. To ascertain whether time of appearance, frequency, and relative proportions of these lesions were influenced by the amounts of vitamin A intake in the different groups, we measured 2 parameters: 1) The number and size of tumor nodules on the surface of fixed lung lobes were determined with a dissecting scope, and the largest diameters of these surface tumors (fig. 1) were measured with an ocular micrometer; 2) the numbers of microscopically identifiable hyperplastic and/or metaplastic lesions per 0 longitudinally sectioned bronchioles were scored in the various lung lobes. These microscopically detectable lesions will be described first, since they developed first. Histologic counts were not made at 12 weeks, because at that time the number and size of tumors occupying large areas of lung parenchyma (fig. 2) were too large for quantitative histologic evaluation. The number of lesions at 2, 3, 5, and 8 weeks is summarized in text-figure 2. At 2 and 3 weeks after MeA injection, epithelial hyperplasias in the terminal and respiratory bronchioles (figs. 3-5) were observed with similar frequency in the lungs of all animals. In both groups the hyperplastic response decreased at 3 weeks, then increased at 5 weeks. (The earliest hyperplastic lesions may indicate the toxic effects of MeA.) A few metaplastic lesions appeared at 2 and 3 weeks and occurred less frequently in the groups low in vitamin A. At 5 weeks, the first substantial number of microscopic metaplastic lesions appeared (figs. 6-8). Both at 5 and at 8 weeks, the number of bronchiolar lesions was reduced in the high vitamin A group. The first macroscopically identifiable lung tumors appeared 5 weeks after carcinogen injection. The counts and volumes of lung surface VOL. 50, NO.6, JUNE 1973

4 1602 CONE AND NETTESHEIM j 0 50 I u 30 HYPERPlASIAS 40 I- - z - METAPlASIAS - 0 cr <D 0 Q 5 "- if> 20 z I- - Q if> W -' - m - o J1lJJb TIME AFTER MeA IN WEEKS TEXT-FIGURE 2.-Effect of vitamin A on the number of hyperplastic and metaplastic lesions per 0 longitudinally sectioned bronchioles at 2, 3, 5, and 8 weeks after carcinogen administration..: Rats receiving 1740 J.Lg RA twice a week; 0: rats receiving J.Lg RA twice a week. Values are means derived from 4 to 5 male and female rats per time point with I SE of the mean. nodules 5, 8, and 12 weeks after MeA administration are summarized in table 3. The high and low vitamin A groups differed markedly in tumor number and size, especially at 5 and 8 weeks (fig. 1). At 12 weeks the differences appear less striking. All carcinogen-treated animals killed between 5 and 12 weeks had 1 or more lung tumors except the rats in group 1 (high RA dose) at 5 weeks, when only 1 of 4 animals had developed a Tumors Total per group Mean No. per animal Total volume (mm 3 ) per group 11_ Mean volume (mm 3 ) per animal I tumor. Thus high levels of vitamin A suppressed the development of early microscopically identifiable metaplastic lesions and the formation of macroscopic nodular lesions induced by MeA. Both microscopic and the macroscopic lesions developing in the low and high vitamin A groups were histologically indistinguishable. No adenomas or adenomatous lesions were seen. At no time were microscopic metaplasias or macroscopic nodular lesions observed in controls (groups 3, 4), which received and 17.4 p,g RA twice a week, respectively. Infrequently, basal cell hyperplasias in bronchial and bronchiolar epithelia were seen in animals of group 4. DISCUSSION Our experiments show that high, but nontoxic, dose levels of vitamin A partially protected the respiratory-tract epithelium against the metaplastic and tumorigenic activity of high doses of MeA. The incidence of carcinogen-induced microscopic squamous metaplasias and macroscopic squamous cell tumors was significantly reduced in rats receiving high doses of vitamin A at all 5 time points at which animals were examined for carcinogen-induced lesions. The persistence of this phenomenon during the entire 12 weeks provided strong evidence for this inhibitory effect of vitamin A, even though the number of observations (4-5 rats) at any given time was small. Similar inhibitory effects of vitamin A on the action of chemical carcinogens (J 2-15, 25, 26) and carcinogenic viruses (27) have been reported. We used the high carcinogen dose because it induced metaplasias and multiple squamous cell tumors in TABLE 3.-Numbers and volumes of tumors 5 wk after MeA * 8 wk after MeA * Group It (±0.25) (±. 13) Group 2t Group It (±0.63) ( ± 2. 2) (±3.0) (±40.9) Group 2t (±3.8) (±54.0) 12 wk after MeAt Group It Group 2t ( ± 3. 2) (±3.9) (±96.9) (±96.9) "Data are for 4 animals per group. t Data are for 5 animals per group. ~Dosage for group 1: 1740 I'g RA twice a week; for group 2: 1'g RA twice a week. Values iu parentheses are 1 SE. II Diameters were measured and volumes calculated from the formula v=4.189 rl. JOURNAL OF THE NATIONAL CANCER INSTITUTE

EFFECT OF VITAMIN A ON LUNG TUMOR INDUCTION 1603 0% of the rats in a short time [see (16) for histogenesis]. Thus results could be obtained in a short time.

5 EFFECT OF VITAMIN A ON LUNG TUMOR INDUCTION % of the rats in a short time [see (16) for histogenesis]. Thus results could be obtained in a short time. However, the true protective potential of vitamin A may be underestimated, since the system is overloaded with carcinogen; thus a "dose-reduction factor" cannot be derived from the present study. For this purpose, a combined, serial sacrifice and survival study of >400 rats, given different carcinogen doses at different levels of vitamin A, is under way. Data obtained during the first 12 weeks confirm the previous findings (28). No squamous metaplasias were observed in the 2 control groups receiving or 17 J.lg RA twice a week. (Only occasional focal basal-cell hyperplasia was seen in the bronchi of rats in the lowest vitamin A control group.) Neither was there evidence that the high vitamin A dose had toxic effects-the growth rate of these animals was not different from that of the low vitamin A dose group. Thus the protective effect observed in this study occurred within a vitamin A dose range that caused no symptoms of either vitamin A deficiency or toxicity. At 5-8 weeks after intratracheal application of MCA, the microscopic metaplasias developed into macroscopic squamous cell nodules (16). By 12 weeks they often occupied 30-50% of a lung lobe (fig. 2). We classified these "nodular lesions" as tumors for the following reasons: 1) They rapidly grow into the lung parenchyma; 2) they become invasive and metastasize several months later; and 3) they can be transplanted to isogenic hosts (16). Their biologic behavior is thus similar to that of pulmonary and adrenal adenomas, which eventually progress to adenocarcinomas and are recognized as tumors before they show frank signs of malignancy (29, 30). The present study does not explain whether the vitamin A effect occurs on the level of carcinogen metabolism or on the level of cellular differentiation (and thus prevents or reduces development of preneoplastic and neoplastic squamous cell lesions). Saffiotti et al. (15) administered high vitamin A doses after carcinogen applications intratracheally to hamsters and showed a reduced respiratorytract tumor incidence. This suggests that at least one mechanism of action is related to the effect of vitamin A on cell replication and differentiation. In our experiments, vitamin A inhibited the metaplastogenic and tumorigenic activity of MCA (possibly by making potential target cells less susceptible to the effect of carcinogen), but it did not detectably alter cell differentiation in developing tumors. We found no histologic evidence of a shift toward mucus production in tumors in rats maintained on high levels of vitamin A, as might have been expected (7-, 12, 31). Our results support and extend findings by other investigators (11-15, 25-27), who have shown inhibition of carcinogenesis by vitamin A. However, vitamin A has been reported to enhance tumorigenesis in several different tumor systems (17-19). The reason for these contradictory results is presently unclear. REFERENCES (1) MORI S: Changes in the para-ocular glands which follow the administration of diets low in fat-soluble A; with notes of the effect of the same diets on the salivary glands and the mucosa of the larynx and trachea. Bull Hopkins Hosp 33 : , 1922 (2) WOLBACH SB, HOWE PR: Tissue changes following deprivation of fat-soluble A vitamin. j Exp Med 42: ,1925 (3) ---: Vitamin A deficiency in the guinea pig. Arch Pathol 5 : , 1928 (4) KAHN RH: Effect of oestrogen and of vitamin A on vaginal cornification in tissue culture. Nature (Lond) 174:317,1954 (5) LASNITZKI I: Effect of excess vitamin A on the normal and oestrone-treated mouse vagina grown in chemically defined medium. Exp Cell Res 24:37-45, 1961 (6) AYDELOTTE MB: The effects of vitamin A and citral on epithelial differentiation in vitro. I. The chick tracheal epithelium. j Embryol Exp Morphol II : , 1963 (7) FELL HB, MELLANBY E: Metaplasia produced in cultures of chick ectoderm by high vitamin A. J Physiol 119: , 1953 (8) FELL HB: The effect of excess vitamin A on cultures of embryonic chick skin explanted at different stages of differentiation. Proc R Soc Lond [BioI} 146: , 1957 (9) WEISS P, JAMES R: Skin metaplasia in vitro induced by brief exposure to vitamin A. In Development and Differentiation. Exp Cell Res Suppl 3, 1955, pp () LASNITZKI I: The effect of excess vitamin A on tht embryonic rat oesophagus in culture. j Exp Med 118:1-5,1963 VOL. 50, NO.6, JUNE 1973

6 1604 CONE AND NETTESHEIM (11) ROWE NH, GRAMMER FC, WATSON FR, et al: A study of environmental influence upon salivary gland neoplasia in rats. Cancer 26: , 1970 (12) PRUTKIN L: Modification of the effect of vitamin A acid on the skin tumor keratoacanthoma by applications of actinomycin D. Cancer Res 31 :80-86, 1971 (13) SHAMBERGER RL: Inhibitory effect of vitamin A on carcinogenesis. J Nat! Cancer Inst 47: , 1971 (14) CROCKER TT, SANDERS LL: Influence of vitamin A and 3,7-dimethyl-2,6-octadienal (citral) on the effect of benzo[ajpyrene on hamster trachea in organ culture. Cancer Res 30: , 1970 (15) SAFFIOTTI U, MONTESANO R, SELLAKUMAR AR, et al: Experimental cancer of the lung. Inhibition by vitamin A of the induction of tracheobronchial squamous metaplasia and squamous cell tumors. Cancer 20: , 1967 (16) SCHREIBER H, NETTESHEIM P, MARTIN DH: Rapid development of bronchiolo-alveolar squamous cell tumors in rats after intratracheal injection of 3- methylcholanthrene. J Nat! Cancer Inst 49 : , 1972 (17) LEVI] IS, POLLIACK 'MB: Potentiating effect of vitamin A on 9, IO-dimethyl-I,2-benzanthracene carcinogenesis in the hamster cheek pouch. Cancer 22 : , 1968 (J 8) POLLIACK A, LEVI] IS: The effect of topical vitamin A on papillomas and intraepithelial carcinomas induced in hamster cheek pouches with 9,- dimethyl-i,2-benzanthracene. Cancer Res 29: , 1969 (19) SMITH WE, YAZDI E, MILLER L: Carcinogenesis in pulmonary epithelia in mice on different levels of vitamin A. Environ Res 5: , 1972 (20) BRICK JO, NEWELL RF, DOHERTY DG: A barrier system for a breeding and experimental rodent colony: Description and operation. Lab Anim Care 19 :92-97, 1969 (21) Dietary standards for laboratory rats and mice. In Laboratory Animal Handbook (Coates ME, O'Donoghue TN, Payne PR, Ward RJ, eds.), vol 2. London, Laboratory Animals Ltd., 1969, P 17 (22) SCHREIBER H, NETTESHEIM P: A new method for pulmonary cytology in rats and hamsters. Cancer Res 32 : , 1972 (23) SAFFIOTTI U, CEFIS F, KOLB LH: A method for the experimental induction of bronchogenic carcinoma. Cancer Res 28:4-124,1968 (24) NEELD JB, PEARSON WN: Macro- and micro-methods for the determination of serum vitamin A using trifluoroacetic acid. J Nutr 79:54-62, 1963 (25) DAVIES RE: Effect of vitamin A on 7,12-dimethylbenz [ajanthracene-induced papillomas in rhino mouse skin. Cancer Res 27: , 1967 (26) CHU EW, MALMGREN RA: An inhibitory effect of vitamin A on the induction of tumors of forestomach and cervix in the Syrian hamster by carcinogenic polycyclic hydrocarbons. Cancer Res 25 : , 1965 (27) McMICHAEL H: Inhibition of growth of Shope rabbit papilloma by hypervitaminosis A. Cancer Res 25: , 1965 (28) KIM JC, SNYDER CM, NETTESHEIM P: Unpublished data (29) STEWART HL, DUNN TB, SNELL KC: Pathology of tumors and non-neoplastic proliferative lesions of the lungs of mice; In Morphology of Experimental Respiratory Carcinogenesis (Nettesheim P, et ai, eds.). Oak Ridge, Tenn., USAEC (DTIE Symp Ser 21), 1970, pp (30) DUNN T: Panel discussion: Recommendations for classification of lung tumors in animals. In Morphology of Experimental Respiratory Carcinogenesis (Nettesheim P, et ai., eds.). Oak Ridge, Tenn., USAEC (DTIE Symp Ser 21), 1970, pp (31) LEVINSON SS, WOLF G: The effect of vitamin A acid on glycoprotein synthesis in skin tumors (keratoacanthomas). Cancer Res 32 : , 1972

g RA twice a week); bottom lobe was from an animal in group 1 (I740 p.g RA twice a week). X 2.

7 FIGURE I.-Lung lobes from female rats killed 8 weeks after carcinogen administration. Top lobe was from an animal in group 2 ( p.g RA twice a week); bottom lobe was from an animal in group 1 (I740 p.g RA twice a week). X 2.5 FIGURE 2.-Squamous cell tumors in lung of female rat maintained on RA twice a week. 12 weeks after MeA. X 11 CONE AND NETTESHEIM 1605

FIGURES 3-S.-Typical microscopic lesions in lungs of rats 2-S weeks after intratracheal carcinogen administration. Examples are from a male rat (group 2) 5 weeks after MeA. FIGURES 3-5.

8 FIGURES 3-S.-Typical microscopic lesions in lungs of rats 2-S weeks after intratracheal carcinogen administration. Examples are from a male rat (group 2) 5 weeks after MeA. FIGURES 3-5.-Hyperplastic bronchiolar lesions (X 200, 312, and 312, respectively). FIGURES 6, 7.-Metaplastic lesions of bronchioles and adjacent alveoli (X 200 and 312, respectively). FIGURE S.-Microscopic nodular metaplasia with keratinization. X CONE AND NETTESHEIM

Respiratory Tract Carcinogenesis in Hamsters Induced by Different Numbers of Administrations of Benzo( a)pyrene and Ferric Oxide1

[CANCER RESEARCH 32, 1073-1081, May 1972] Respiratory Tract Carcinogenesis in Hamsters Induced by Different Numbers of Administrations of Benzo( a)pyrene and Ferric Oxide1 Umberto SaffÂ otti, Buggero Montesano,3