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[CANCER RESEARCH 32, 1442-1445, July 1972]
Lymphatic Leukemia and Pulmonary Tumors in Female SwissMice Fed Bracken Fern (Pteris aquilina)1
A. M. Pamukcu, E. Erturk, J. M. Price, and George T. Bryan2
Department of Pathological Anatomy, College of Veterinary Medicine, University of Ankara, Ankara, Turkey [A. M. P., E. E.]; ScientificDivisions, Abbott Laboratories, North Chicago, Illinois 60064 /J. M. P.]; and Division of Clinical Oncologv, University of Wisconsin MedicalSchool, Madison, Wisconsin 53706 [G. T. B./
SUMMARY
Bracken fern (Pteris aquilina) mixed with a grain mixture(1:3 by weight) was fed to a group of 40 female 6-week-oldSwiss mice intermittently every other week for a totalexperimental period of 60 weeks. Thirty-three mice survived30 or more weeks and all developed lymphatic leukemia withmultiple organ involvement. Additionally, 5 mice developedmultiple pulmonary tumors. No urinary bladder or intestinaltumors, common in rats fed a similar bracken fern dietmixture, were found. No tumors were detected in 38 controlmice that survived for 30 to 60 weeks while ingesting the grainmixture.
Bracken fern has an inhibitory effect on myeloid tissue incows (12, 19), causing a progressive diminution in the numberof white blood cells and platelets in the peripheral blood.These alterations maximally coincide with evidence of clinicaltoxicity due to bracken fern. The leukocytic depression isassociated with a drastic diminution in the number ofpolymorphonuclear leukocytes. This inversion of the ratio ofneutrophils to lymphocytes suggests that bracken fern severelydamages the bone marrow. However, the effect, if any, ofbracken fern on the lymphoreticular tissue of rodents is notknown. The objective of this study was to investigate thepossible murine leukemogenic and carcinogenic effects ofchronic p.o. administration of bracken fern.
INTRODUCTION
The potent carcinogenic activity of bracken fern has beendemonstrated for many species of animals. Several factorsinfluence the pattern, location, and incidence of neoplasticlesions induced by bracken fern; i.e., species and age ofanimals, duration of bracken fern feeding, thiaminesupplementation, and induced microsomal enzyme activity.The target organs vary with the species but include the smallintestine, colon, and urinary bladder in rats and quail (5, 6, 8,13, 19); the urinary bladder and hemopoietic system in cows(12, 19); the urinary bladder in guinea pigs; and the lung inmice (22). Age dependence at the time of initial exposure tothe fern has been shown for rats (5), Le., young rats appear tobe more susceptible than older ones, but not for cows (12, 19).The duration of bracken fern feeding determined theoccurrence of simultaneous tumors at various sites in rats.Intermittent feeding to young rats produced only intestinaltumors (6), while continuous administration for 12 monthsresulted in the development of coexisting intestinal andurinary bladder tumors (13, 17, 19). Thiaminesupplementation increased the incidence of urinary bladdercarcinomas in rats fed bracken fern (15, 17). Induction ofmicrosomal activity by chronic phenothiazine administrationreduced the incidence of intestinal and urinary bladderneoplasms by more than 50% in bracken fern-fed rats (16).
1Supported in part by Grant CA-08254 from the National Cancer
Institute, USPHS, and by a grant from the Turkish Scientific andTechnical Research Council.
1Career Development Awardee of the National Cancer Institute,USPHS (1-K4-CA-8245).
Received February 18, 1972; accepted March 23, 1972.
MATERIALS AND METHODS
Bracken fern (P. aquilina) was collected in June 1969 fromfarms in the Bolu Province, Turkey, where the incidence ofbovine urinary bladder cancer, associated with a dietcomposed of a substantial portion of bracken fern, is high(10-12, 19). The bracken was dried in the shade to preserveits natural dark green color and was then milled and mixedwith a basic grain mixture, the composition of which wasdescribed previously (17), in the ratio of 1 part of powderedbracken to 2 parts of grain diet. By the use of steam andcompression, the basic diet and bracken fern-containing dietwere molded into pellets that were immediately dried to avoidmold growth. These pellets were fed to mice during theexperiment.
Female 6-week-old Swiss mice (Institute of Bacteriology,Elazig, Turkey), free from "spontaneous" leukemia, were
housed in screen-bottomed metal cages, 6 mice/cage, and werefed their diets and water ad libitum. A total of 80 mice weredivided equally in 2 groups. Group 1 received the brackenfern-containing diet. Because of the acute toxicity of this diet,manifested by failure of the mice to gain weight, it was fedintermittently every other week. On alternate weeks, theanimals received the basic diet. This feeding schedule wascarried out for 60 weeks. Group 2, a negative control group,was fed only the basic grain diet. No thiamine supplement wasadministered to either group of animals as was done inprevious studies with rats ( 17).
Mice that died or were killed were subjected to necropsy.The urinary bladders were distended with Bouin's fixative
injected through the urethra. Representative histologicalsections of intestine, stomach, liver, spleen, kidneys, adrenals,
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lungs, heart, thymus, lymph nodes, and urinary bladder wereprepared and stained with hematoxylin and eosin.
RESULTS
The daily dose of bracken fern ingested by the mice was 1.5g/mouse with a mean maximal cumulative dose of 315g/mouse/60 weeks. The mice tolerated the bracken fern verywell; only 7 test mice died during the period of theexperiment. Two control mice died during the llth and 18thexperimental weeks. The bracken fern administration wasrelated to the development of lymphatic leukemia in all 33test animals that survived more than 30 weeks (Table 1). Theleukemia produced was grossly characterized by markedenlargement of the spleen and lymph nodes but not of thethymus. The spleen was increased to about 5 times its normalsize and on section had an accentuated follicular pattern. Thelymph nodes were enlarged 2- to 3-fold and were soft andwhite. In addition, 5 of 33 mice had multiple primary lungtumors ranging in size from microscopic to 1.5 cm in diameter.The cut surface showed a pinkish-gray, medullary tumor withan undefined periphery. Other organs including the thymusappeared normal in gross appearance.
Microscopic examination of the organs revealed that theprincipal changes were in the spleen, lymph nodes, liver,kidney, and lungs. The incidence of lymphatic leukemia and alist of organs that were infiltrated with lymphoid cells aregiven in Table 1. Enlargement of lymph nodes and spleen wasdue to a marked proliferation of lymphoid or reticular cells.The usual leukemic cells were of the large lymphocyte variety.Invasion of the capsule by lymphoid cells was constant. Theleukemic cells varied in size. They might closely resemble anormal lymphocyte and have a deeply basophilic, small, roundnucleus with a scant rim of clear basophilic cytoplasm, or theymight be much larger than the normal lymphocyte and have around or slightly indented vesicular nucleus that was lessdeeply basophilic, with one or more prominent nucleoli. Thecytoplasm might be clear or distinctly basophilic.
In the spleen, leukemic cells were present in both folliclesand pulp (Fig. 1). In many instances of marked involvement,the demarcation of the follicles and pulp was lost and almostthe entire spleen was replaced by leukemic cells. The spleenwas rich in megakaryocytes. In all cases (33/33), the lymphnodes showed a marked infiltration by lymphoblasts. In 27/33instances, perivascular and intracapillary collections ofleukemic cells were found in the kidneys (Fig. 2). In 17/33cases, the liver was involved in a similar manner. The sinusoidswere widely distended by large numbers of lymphoblasts, andthere was marked atrophy of the hepatic cords (Fig. 3). In the
Bracken Fern Leukemogenicity in Mice
lung (9/33 cases), there was a widespread infiltration of thealveolar walls with characteristic lymphoblasts. The alveolarspaces were diminished in size, and occasional tumor cellscould be seen lying free in the blood vessels. In addition, in5/33 instances, there were pulmonary epithelial tumors withhistological features of adenoma (3 cases) and adenocarcinoma(2 cases) (Fig. 4). Surprisingly, no urinary bladder or intestinaltumors were found. No neoplastic lesions were present in thecontrol mice.
DISCUSSION
Bracken fern contains a potent leukemogen for mice and,when chronically fed to them, results in a 100% incidence oflymphatic leukemia in this species. The lymphatic leukemiawas characterized by splenomegaly, accompanied byenlargement of lymph nodes and leukemic infiltration of thekidneys, liver, and lungs. Noteworthy was the absence ofenlargement of the thymus, and apparently the involvement ofthe thymus was not essential for induction of murinelymphocytic leukemia with bracken fern. Studies in the mouseindicate that the thymus may be the primary site ofdevelopment of lymphatic leukemia in certain chemical (1,2,4, 7, 20) and viral-induced (3, 9, 21) murine leukemias. Thefundamental histological changes in these murine lymphaticleukemias begin in the thymus. The histological patterns oflymphatic leukemia do not appear to differ in their essentialnature. Further studies on the pathogenesis of brackenfern-induced leukemia in Swiss mice are needed to clarify therole of the thymus in this system.
Pulmonary adenoma and adenocarcinoma occurred in 5animals with lymphatic leukemia, with an incidence of 15%.Lung tumors associated with lymphatic leukemia weredemonstrated in 5-week-old Swiss mice fed formic acid2-[4-(5-nitro-2-furyl)-2-thiazolyl]hydrazide (1) orjV-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (2). A similarassociation was noted in newborn Swiss mice administered7 ,1 2-dimethylbenz(a)anthracene, benz(a)pyrene,3-methylcholanthrene, or urethan (9, 18, 20). The incidenceof pulmonary adenomas and their associated malignanthemopoietic counterpart ranged from 59 to 96% dependingupon the carcinogen used (18).
The duration of bracken fern feeding determined theoccurrence of simultaneous lymphatic leukemia andpulmonary tumors. Continuous feeding appears necessary forthe induction of these associated neoplasms in mice. Widdop(22) produced only lung adenomas in 3-month-old mice byfeeding them an ethanol extract of bracken fern for a total
Table 1Survival, lymphatic leukemia, and pulmonary tumors in female Swiss mice fed bracken fern
No. of mice aliveatTreatmentgroupsControlBracken
fernStart4040Week303833Week603833No.
of mice with leukemia in the following organsitesSpleen033Lymphnodes033Kidney027Liver017Lung09other
tumorsofthelung05
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Pamukcu, Ertürk,Price, and Bryan
period of 44 days. He did not observe leukemic changes inanimals sacrificed 10 months after the start of feeding.
The absence of intestinal or urinary bladder carcinomas,neoplasms that are commonly found in rats fed a similarbracken fern diet mixture (13), may be due to differences inmetabolism of the bracken fern carcinogen in the 2 species ofanimal. Conversely, it may be due to differences in the bindingsites of the carcinogen in the 2 species of rodents. It appearsthat the target organs in rats are the intestine and urinarybladder, depending upon the amount of bracken fernadministered. In mice, lymphoreticular tissue is primarilyinvolved in cancer. However, this does not necessarily rule outthe possibility of excretion of the carcinogen in murine bile orurine, but the amount excreted may be insufficient to induceneoplasms in the intestine or urinary bladder or both.Continuation of the feeding of the fern to mice beyond 60weeks might also alter the sites of neoplasm formation. It wasshown that topical application of bracken fern extracts to themouse urinary bladder epithelium by the pellet implantationtechnique produced a high incidence of bladder carcinoma(14). These data suggest that mouse urinary bladderepithelium is responsive to the carcinogenic activity of certainfern extracts. The absence of urinary bladder tumors in micein the present experiment may suggest that the carcinogenpresent in bracken fern was destroyed, perhaps in the smallintestine or liver, suggesting that only a small amount of activematerial reached the intestine or urinary bladder. This amountcould not initiate cancer in either organ, but it was enough tocause lymphatic leukemia. Further investigation concerningthe interesting differences in species susceptibility to thebracken fern carcinogen must await chemical identification ofthis substance.
REFERENCES
1. Cohen, S. M., Ertürk,E., and Bryan, G. T. Carcinogenicity ofFormic Acid 2-(4-(5-Nitro-2-furyl)-2-thiazolyl]hydrazide in SwissMice. Cancer Res., 30: 906-912, 1970.
2. Cohen, S. M., Ertürk,E., and Bryan, G. T. Production of Leukemiaand Stomach Neoplasms in Swiss, RF, BALB/c, and C3H FemaleMice by Feeding JV-[4-(5-Nitro-2-furyl)-2-thiazolyl)acetamide.Cancer Res., 30: 2320-2325, 1970.
3. Dunn, T. B., Moloney, J. B., Green, A. W., and Arnold, B.Pathogenesis of a Virus-induced Leukemia in Mice. J. Nati. CancerInst., 26: 189-221,1961.
4. Erturk, E., Cohen, S. M., and Bryan, G. T. Urinary BladderCarcinogenicity of jV-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide inFemale Swiss Mice. Cancer Res., 30: 1309-1311, 1970.
5. Evans, I. A. The Radiomimetic Nature of Bracken Toxin. CancerRes., 28: 2252-2261, 1968.
6. Evans, 1. A., and Mason, J. Carcinogenic Activity of Bracken.Nature, 205:913-914,1965.
7. Goodman, S. B., and Block, M. H. The Histogenesis of Gross's
Viral Induced Mouse Leukemia. Cancer Res., 23: 1634-1640,1963.
8. Hirono, I., Shibuya, C., Fushimi, K., and Haga, M. Studies onCarcinogenic Properties of Bracken, Pteridium aquilinum. ¡.Nati.Cancer Inst., 45: 179-188, 1970.
9. Ito, T., Hoshino, T., and Sawauchi, K. Pathogenesis of ThymicLymphoma Induced by Urethan in Mice. Z. Krebsforsch., 66:267-273, 1964.
10. Pamukcu, A. M. Investigations on the Pathology of EnzooticBovine Haematuria in Turkey. Zentr. Veterinaermed., 2: 409-429,
1955.11. Pamukcu, A. M. Epidemiologie Studies on Urinary Bladder Tumors
in Turkish Cattle. Ann. N. Y. Acad. Sci., 108: 938-947, 1963.12. Pamukcu, A. M., Göksoy,S. K., and Price, J. M. Urinary Bladder
Neoplasms Induced by Feeding Bracken Fern (Pteris aquilina) toCows. Cancer Res., 27: 917-924, 1967.
13. Pamukcu, A. M., and Price, J. M. Induction of Intestinal andUrinary Bladder Cancer in Rats by Feeding Bracken Fern (Pterisaquilina). J. Nati. Cancer Inst., 43: 275-281, 1969.
14. Pamukcu, A. M., Price, J. M., and Bryan, G. T. Assay of Fractionsof Bracken Fern (Pteris aquilina) for Carcinogenic Activity.Cancer Res., 30: 902-905, 1970.
15. Pamukcu, A. M., Price, J. M., and Bryan, G. T. Altered Urinary andIntestinal Carcinogenic Activity of Bracken Fern in Rats. Proc.Am. Assoc. Cancer Res., 12: 7, 1971.
16. Pamukcu, A. M., Wattenberg, L. W., Price, J. M., and Bryan, G. T.Phenothiazine Inhibition of Intestinal and Urinary Bladder TumorsInduced in Rats by Bracken Fern. J. Nati. Cancer Inst., 47:155-159, 1971.
17. Pamukcu, A. M., Yalciner, S., Price, J. M., and Bryan, G. T. Effectsof the Co-administration of Thiamine on the Incidence of UrinaryBladder Carcinomas in Rats Fed Bracken Fern. Cancer Res., 30:2671-2674, 1970.
18. Pietra, G., Rappaport, H., and Shubik, P. The Effects ofCarcinogenic Chemicals in Newborn Mice. Cancer, 14: 308-317,
1961.19. Price, J. M., and Pamukcu, A. M. The Induction of Neoplasms of
the Urinary Bladder of the Cow and the Small Intestine of the Ratby Feeding Bracken Fern (Pteris aquilina). Cancer Res., 28:2247-2251, 1968.
20. Rappaport, H., and Baroni, C. A Study of the Pathogenesis ofMalignant Lymphoma Induced in the Swiss Mouse by7,12-Dimethylbenz[a) anthracene Injected at Birth. Cancer Res.,22: 1067-1074, 1962.
21. Siegler, R., Geldner, J., and Rich, M. A. Histogenesis of ThymicLymphoma Induced by a Murine Leukemia Virus (Rich). CancerRes., 24: 444-459, 1964.
22. Widdop, B. Separation and Characterisation of VariousToxicological Components of Bracken. Ph.D. Thesis, University ofWales, Bangor, Wales, 1967.
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Fig. 1. Massive splenic replacement by primitive neoplastic cells. H & E, X 260.Pig. 2. Neoplastic lymphoblasts diffusely infiltrating between the renal tubules. H & E, X 260.I?ig. 3. Neoplastic lymphoblasts infiltrated around the central vein of the liver. H & E, X 240.
Fig. 4. Adenocarcinorna of the lung. H & E, X 105.
JULY 1972 1445
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1972;32:1442-1445. Cancer Res A. M. Pamukcu, E. Ertürk, J. M. Price, et al.
)Pteris aquilinaMice Fed Bracken Fern (Lymphatic Leukemia and Pulmonary Tumors in Female Swiss
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