Association of Bovine Papillomavirus Type 2 and Bracken Fern with Bladder Cancer in Cattle

[CANCER RESEARCH 52, 6898-6904, December 15, 1992] Association of Bovine Papillomavirus Type 2 and Bracken Fern with Bladder Cancer in Cattle

M. Saveria Campo, 2 William F. H. Jarrett, Ron Barron, Brian W. O'Neil, and Kenneth T. Smith 3 The Beatson Institute for Cancer Research, CRC Beatson Laboratories [M. S. C., K. T. S.] and Department of Veterinary Pathology [IV. F. H. J., R. B., B. W. 0.], Glasgow University Veterinary School, Garscube Estate, Glasgow G61 1BD, Scotland

ABSTRACT MATERIALS AND METHODS

The bladder cancer syndrome that often accompanies chronic en- Experimental Plan. These experiments were primarily designed to zootic hematuria in cattle grazing on pastures infested by bracken fern reproduce in controlled conditions the synergism between BPV-4 and has been experimentally reproduced in animals fed a diet of bracken. bracken as observed in naturally occurring carcinogenesis of the upper The experimentally induced tumors were histologically and pathologi- alimentary canal, and for this reason the animals were injected with cally indistinguishable from the naturally occurring ones and comprised BPV-4, the papillomavirus specific for the mucous epithelium of the two main types: (a) carcinoma of the urothelium identical to that seen in alimentary tract (7). humans; and (b) hemangioendotheliomas of the subjacent capillaries. Thirty-six young animals, ages approximately 3-5 months and born Often the two types of tumor occurred together in the same bladder. In of papillomatosis-free mothers, were divided into eight groups (Table animals experimentally immunosuppressed with azathioprine "bracken 1): group 1 (animals 1-6) was made up of six Ayrshire bull calves which type" hemangiomas developed in the bladder lining. DNA of bovine were inoculated with BPV-4; group 2 (animals 7-12) was made up of papillomavirus (BPV) type 2 was found in 46% (7 of 15) of the natural cancer cases and in 69% (9 of 13) of the experimentally induced lesions, five Ayrshire bull calves and one Friesian heifer; these animals were independently of histological type and including the hemangiomas of the inoculated with BPV-4 and treated with the immunosuppressant aza- azathioprine-treated animals, suggesting a close association between thioprine; group 3 (animals 13-16) was made up of four Friesian bull BPV and bovine bladder neoplasia. Moreover, BPV-2 DNA was found calves treated only with azathioprine; group 4 (animals 17-20) was in experimental animals that had not been inoculated with BPV at all or made up of four bull calves of mixed breed and was used as control; had been inoculated with a different BPV type and had been kept in group 5 (animals 21-26) was made up of six bull calves of mixed breed isolation, suggesting that BPV can persist in a latent state and be which were fed a diet of bracken fern; group 6 (animals 27-32) was activated when the animal is exposed to the bracken cocarcinogens and made up of six bull calves of mixed breed inoculated with BPV-4 and to immunosuppressants. fed with bracken; group 7 (animals 33-34) was made up of two bull calves given quercetin, the flavonoid present in bracken which has been shown to be mutagenic in both prokaryotic (9, 10) and eukaryotic INTRODUCTION (11-13) cells and reported to be carcinogenic in experimental animals Chronic enzootic hematuria is a disease of cattle in various (14); group 8 (animals 35-36) was made up of two bull calves inoculated localized areas of the world (1), which is often associated with with BPV-4 and given quercetin. neoplasia of the urinary bladder (2), involving the epithelium The groups were housed in semiisolation in separate, clean, well and the mesenchyme. The disease has been associated with the ventilated pens. The animals were cared for in complete accordance presence of bracken fern in the diet of the animals, either as with the directives of the Home Office of Great Britain. fresh fronds in the pasture or as dried leaves in hay (3). The BPV-4 Inoculation. BPV-4 was isolated and purified from a single plant is known to contain both mutagens and carcinogens (4) case of esophageal frond papilloma as previously described (7), and its genome was typed by restriction enzyme analysis (7). The designated and immunosuppressants (5). In the Scottish Highlands the animals were sedated with Rompun, 0.5-0.75 ml/kg body weight. The high frequency of neoplasia of the upper and lower alimentary virus was inoculated by shallow injection beneath the epithelium at the canal of cattle is epidemiologically associated with infection by right side of the dorsum of the tongue and the right site of the soft BPV-44 and ingestion of bracken fern (6, 7). Thirty % of the palate. Immediately after inoculation the pharynx was scarified in all animals presenting with alimentary cancers are also affected by calves by means of a bristle bottle brush. lesions of the urinary bladder, which include hemangiomas, Treatment with Azathioprine. The azathioprine solution was pre- hemangiosarcomas, fibromas, transitional cell carcinomas, and pared by dissolving 2 g azathioprine powder (Calmic Medical Division, adenocarcinomas (6). The Wellcome Foundation, Ltd.) in 90 ml sterile saline solution by In August 1979 we started an experiment designed to repro- dropwise addition of 7.24 ml 1 N NaOH. In some cases a slight excess duce in controlled conditions the cocarcinogenic action of virus of alkali was required to ensure full dissolution of the powder. The and bracken and to distinguish between the effects of the solution was prepared up to 3 days in advance and stored at 4~ It was bracken mutagens and immunosuppressants. Partial results administered daily to the designated calves by s.c. injection at a dose of have been reported (8). Here we describe in detail the results 2 mg/kg body weight. concerning carcinogenesis in the bladder and compare them to Bracken Fern Feeding. Fresh bracken was collected daily, and only those obtained with natural bladder cancers. the upper softer parts of the plant were used. Between 20 and 25 kg of bracken were divided between the two pens housing the designated animals every day from the beginning of June to the end of September, Received 6/26/92, accepted 10/6/92. with an interval of 3 weeks after the first 6 weeks. During the winter The costs of publication of this article were defrayed in part by the payment of months the animals were fed on hay. The bracken feeding cycle was page charges. This article must therefore be hereby marked advertisement in accord- repeated every year to the end of the experiment. The quantities eaten ance with 18 U.S.C. Section 1734 solely to indicate this fact. by each calf are unknown because ad libitum feeding was used. Thanks are due to the Cancer Research Campaign for continuous financial support. Treatment with Quercetin. Quercetin (5,7,3',4'-tetrahydroxyflavone; 2 CRC Fellow. To whom requests for reprints should be addressed. Sigma) was dissolved in dimethyl sulfoxide and ethanol and adminis- 3 Present address: Quality Biotech, West of Scotland Science Park, Kelvin Cam- tered p.o. to the designated calves at a dose of I g/calf/day for 5 months. pus, Garscube Estate, Glasgow, Scotland. 4 The abbreviations used are: BPV-4, bovine papillomavirus type 4, PBL, Thereafter the dose was increased to 20 g/calf/day. Treatment was peripheral blood leukocyte(s). suspended for a month every 4 months. 6898 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1992 American Association for Cancer Research. BLADDER CANCER, BPV-2, AND BRACKEN FERN

Table 1 Experimentalplan cemia. This is the well described veterinary syndrome of acute Azathio- bracken poisoning. Acute bracken poisoning occurred in all of Group Animal BPV-4 prine Bracken Quercetin Lesion" our bracken-fed animals. As soon as it was detected hemato- 1 1 + .... logically, bracken feeding was stopped; in the following week or 2 + .... 3 + .... two the polymorphonuclear cell count rose to normal levels and 4 + .... bracken was restored to the diet. This was a cyclic pattern 5 + .... 6 + .... through the summer months when bracken was available (Fig. IA). The second effect of bracken feeding is a chronic drop in 2 7 + + - - He circulating lymphocytes. After approximately 34 weeks from 8 + + - - He 9 + + - - He 10 + + - - He 11 + + - - He 12 + + - - He PBLs x 10-9/L lO 3 13 - + - - He 14 - + - - He 15 - + - - He 16 - + - - He

4 17 ..... 18 ..... 19 ..... 20 .....

5 21 - - + - HS 22 - - + - HEn, Ca, M 23 - - + - HS, HEn, Ca 24 - - + - He, Ca 1 25 - - + - Ca / 26 - - + - Ca 2 i i i ~1 i i i i [ i i i i ( i i i 9 [ i [ t i .l~l t i i i ~ ~J 2 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 6 27 + - + - HS 28 + - + - HS, HEn months A 29 + - + - sa 30 + - + - Ca, M LYMPHOCYTES x 10-9/L 31 + - + - He, Ca 32 + - + - HS, Ca, Po, M

7 33 - - - + - 34 - - - + - 5 i ,~ ., 8 35 + - - + - 36 + - - + - a He, hemangioma; HS, hemangiosarcoma; HEn, hemangioendothelioma; Ca, transitional cell carcinoma; Po, polyps; M, metastases; sa, still alive. + and - indicate presence or absence, respectively.

Collection of Blood Samples. Blood samples were collected from every animal at monthly intervals by jugular venepuncture for hema- tology, biochemistry, serum preparation, and PBL estimation. 2 i L _ i .... Collection of Naturally Occurring Bladder Cancers. Cancers were 0 5 10 15 20 25 30 35 obtained from diseased animals referred to the veterinary school by local farmers and veterinarians from bracken-infested areas. One half of weeks B each specimen was used for histopathology and the other one half was PBLs x 10-9/L frozen in liquid N2 as soon as possible after excision and then stored at 7 -70~ until needed for DNA analysis. Collection of Experimentally Induced Bladder Cancers. Cancers were obtained at autopsy and processed as above. Collection of Normal Bladder Tissue. Normal bladder tissue was obtained from the local abattoir. BPV Sequences in Bladder Tissue. The isolation and purification of ! tissue DNA, restriction enzyme analysis, and Southern blot hybridization were as described (15). The hybridization probes were DNA from recombinant BPV-1, BPV-2, and BPV-4 (16) radioactively labeled either by nick-translation (17) or by random priming (18).

RESULTS

Effect of Bracken Eating on the Immune Status. Bracken 1 2 3 4 5 6 7 8 9 10 11 12 feeding caused two marked hematological changes which are months c associated with immunosuppression. The first of these was a Fig. I..4, cyclic drop in PBL count with bracken feeding in animal 31. The dramatic fall in polymorphonuclear leukocytes. If unchecked, yearly drop took place throughout the course of the experiment. B, lymphocyte this leads to severe acute immunosuppression with invasion of count in animal 31. C, drop in PBL count with azathioprine treatment in animal 9. These animals are representative of groups 5 and 6 and groups 2 and 3, the bloodstream by alimentary bacteria and death from septi- respectively. 6899 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1992 American Association for Cancer Research. BLADDER CANCER, BPV-2, AND BRACKEN FERN the beginning of bracken feeding the lymphocyte count was very bladder was covered with transitional cell carcinoma. Histology low and remained so throughout the experiment (Fig. 1B). showed that this ranged through all grades and stages. It had Effect of Azathioprine on the Immune Status. Administra- invaded the whole bladder wall penetrating the surrounding tion of azathioprine produced a marked drop in PBL in all tissues and the lymphatic channels where it caused marked treated animals. An example is given in Fig. 1C: in this partic- fibrosis and had infiltrated the lymph nodes. The ureter had a ular animal (group 2, number 9), PBL dropped from 6.7 x normal epithelium but its wall near the point of entry into the 10a/liter to 2.2 x 109/liter over the course of 1 year. Because of bladder had been heavily infiltrated by anaplastic tumor cells. the severe immunosuppression and continuous hematuria, the Animal 31 was killed in November 1989. The whole surface azathioprine treated animals were killed after 1 year of treat- of the bladder was covered with several hundred hemangiomas ment. ranging from 3 cm in diameter to pinpoint size. Several were Effect of Quercetin on the Immune Status. No effect on the bleeding and several were mixed with transitional cell carci- immune status of the animals in groups 7 and 8 was noticed; the noma. The carcinomas ranged from one which was completely number of PBL remained constant and within normal limits. papillomatous with a very thin stalk to several which were cecal Bladder Cancers in Bracken-fed Animals. All animals in- and firmly attached to the epithelium. Histology showed no jected with BPV-4 developed alimentary canal papillomas. The evidence of penetration of the bladder wall and no lymph node effects of bracken, azathioprine, and quercetin on the deve- involvement. lopment and progression of these tumors will be described Animal 24 was killed in March 1990. The bladder had several elsewhere. hemangiomas largely scattered over the fundus and covering The two groups of bracken-fed animals (group 5, animals the whole area. Also spread all over the fundus were grade I 21-26, bracken diet only; and group 6, animals 27-32, bracken transitional cell carcinomas, ranging from plaque-like lesions to plus BPV-4) will be considered together because there were no protruding papilliform tumors with many intermediate cecal differences between them regarding tumor development in the tumors. Histology showed no penetration of the tumors into urinary bladder. the bladder wall. Animals 21, 22, 25, 27, and 28 were killed in November 1981 Animal 29 died in October 1992. The bladder had hemor- because of continuous hematuria. Visual inspection showed rhagic papillomas, transitional cell carcinoma covering the en- that the fundus of the bladder of these animals was covered in tire mucosal surface and infiltrating the bladder wall, and hem- neoplastic folds with markedly thickened urothelium and sev- angiosarcomas. eral hemangiomas, ranging from 2 mm to 6 mm in diameter Bladder Abnormalities in Azathioprine-treated Animals. (Fig. 2,4). Histological examination revealed that several of the Group 2 (animals 7-12, azathioprine plus BPV-4) and group 3 hemangiomas were in fact hemangiosarcomas (Fig. 2B) or he- (animals 13-16, azathioprine only) will be described together. mangioendotheliomas in animals 21, 22, 27, and 28. Animal 22 These animals were killed in September 1981 because of con- had, in addition, carcinoma of the transitional epithelium (Fig. tinuous hematuria. 2C), which had metastasized to the local lymph nodes. Animal In all cases there were pinpoint hemorrhagic lesions on the 25 had transitional cell carcinoma. bladder surface, which in animal 12 extended over an area of Animal 23 was killed in January 1983. It had been passing approximately 10 x 10 cm in the distal part of the bladder. large amounts of blood during the previous week and over the Histological examination revealed that these lesions consisted last 2 days had developed urinary retention. The main lesion of areas of subepithelial edema with focal proliferation of cap- was a grossly distended bladder full of blood clots blocking the illaries. These caused small corrugations of the bladder lining, urethra. The whole internal surface of the bladder was covered typical of the early lesions in areas surrounding "bracken type" with large hemangiomas, 20 of them greater than 1 cm in hemangiomas (Fig. 2D). The base of some of the folds con- diameter; there were also carcinomas. Histologically, these tained small lymphoid aggregates. were confirmed as hemangiosarcomas, hemangioendothelio- Urinary Bladder Status in Group 1 and 4 Animals. In mas, and transitional cell carcinomas, respectively. marked contrast to the animals in groups 2, 3, 5, and 6, no Animals 32 was killed in March 1985 and Animal 30 in bladder lesions were observed in these animals, which either September 1985. In Animal 32, the wall of the urinary bladder had been infected with BPV-4 only (group 1) or had received no was thickened with a mat of fronded epithelium approximately treatment (group 4). Histological examination revealed normal 7 mm deep, with several discrete polyps, the biggest of which bladder lining and walls. was 2 cm in diameter attached by a stalk 3 cm in length and 0.5 Animals in Groups 7 and 8. Neither group 7 (quercetin cm thick. There were a few isolated hemangiomas measuring 3 alone) nor group 8 (quercetin and BPV-4) animals showed any mm in diameter. There was a nodular sublumbar mass approx- sign of hematuria during the course of the experiment; they imately 40 cm in diameter, which had extended to the left ureter were no different from the control animals in groups 4 and 1 and to the aorta and iliac arteries with penetration to the lumen and were not histologically examined at postmortem. The an- in each case. Histological examination confirmed the presence imals in group 8 were no different from animals in group 1 also of transitional cell carcinoma with massive infiltration of the with respect to p.o. papillomatosis, which was confined to the sublumbar lymph nodes. Within the carcinoma there were areas injection sites (not shown). of glandular metaplasia and squamous metaplasia. There were Bladder Cancers in Field Cases. The 15 cases of naturally frequent areas of collision between carcinoma and hemangiosa- occurring bladder tumors (animals A-O; Fig. 2E) comprised rcoma. Animal 30 presented with similar lesions. In this case, transitional epithelium carcinomas (Fig. 2F), papillary carcino- the carcinoma had infiltrated the right media iliac, the left mas (Fig. 2G), hemangiomas (Fig. 2H), hemangiosarcomas, media iliac, and the superficial inguinal lymph nodes. fibromas, and polyps. Six animals had more than one type of Animal 26 was killed in December 1987. Although some bladder tumor (Table 2); in addition, two animals had rumen parts of the bladder were heavily vascularized, there was no fibropapillomas containing BPV-2 DNA (19), three animals obvious hemangiomatous tissue. However, the surface of the had BPV-4 papillomas at several sites in the upper alimentary 6900 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1992 American Association for Cancer Research. BLADDER CANCER, BPV-2, AND BRACKEN FERN

Fig. 2. Experimental and naturally occurring bladder cancers. A-C, animal 22 from group 5. ,4, bladder with carcinoma and hemangioma. B, hemangiosarcoma. C, transitional epithelium carcinoma. D, hemangioma in bladder of animal 9 from group 2. E-H, naturally occurring cancers. E, carcinoma and hemangioma. F, transitional epithelium carcinoma invading the wall of the bladder. G, papillary carcinoma. H, hemangioma. A and E, one-third original size; B-D, G, and H, • 245; F, • 115.

6901 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1992 American Association for Cancer Research. BLADDER CANCER, BPV-2, AND BRACKEN FERN canal (7), three animals had skin warts containing either BPV-1 Table 2). The remaining cancer biopsies were negative. As for or BPV-2 (20), and five animals had also malignancies of the the experimental cancers, no virus or viral antigens were de- upper alimentary canal and/or the bowels (Table 2). The tongue tected in the BPV-2 DNA-positive cancers. Eight of the normal carcinoma of animal F was the only alimentary tract cancer that bladder biopsies were negative; of the remaining two, one was contained BPV-4 DNA (15). positive for BPV-2 DNA and the other was positive for an Viral DNA in Bladder Lesions from Bracken-fed and Azathi- unidentified papillomavirus (data not shown). oprine-treated Animals. Genomic DNA from bladder lesions of animals 7, 8, 12 (group 2, BPV-4 plus azathioprine), 21, 22, 23, 25, 26 (group 5, bracken diet), 27, 28, 30, 31, and 32 (group DISCUSSION 6, BPV-4 plus bracken diet) was digested with several restric- Urinary Bladder Cancer in Cattle Is Associated with Papil- tion enzymes and Southern blotted. Animals 22, 23, 28, 30, 31, lomavirus and Bracken Fern. The association between papillo- and 32 had multiple cancer types (Table 1); DNA was analyzed mavirus and cancer of the urinary bladder in cattle was first from the hemangioendothelioma and transitional cell carci- proposed by Olson et al. (21). These authors had shown that noma of animal 22, the transitional cell carcinomas of animals extracts of bovine cutaneous warts could induce cancer when 23 and 30, both cancer types of animals 28 and 31, and all cancers of animal 32, including the metastatic lymph nodes but injected into the urinary bladder of cows (21). Vice versa sus- excluding the hemangiosarcoma. Hybridization of the blots to pension of spontaneous, bracken-associated bovine bladder tu- BPV-I, BPV-2, and BPV-4 DNA probes showed that the ma- mors produced warts of the skin and vagina as well as polyps jority of the biopsies contained multiple copies of episomal and fibromas in the bladder of test calves (22). These observa- BPV-2 DNA (Fig. 3,4); the exceptions were cases 23, 26, 30, tions strongly suggested the presence of papillomavirus in nat- and 31, which were consistently negative for all the probes. In urally occurring bladder cancers. However, when these experi- the cases with multiple tumor types, the tumors were either all ments were performed, the existence of multiple types of BPV positive or all negative. No virus or viral antigens were detected was not known (20, 23), and the identity of the virus was not in the bladder lesions (data not shown), indicating that the confirmed. Moreover, the relationship between bladder malig- bladder epithelium is nonpermissive for virus replication. nancies and bracken fern, suggested by the experiments of Pa- Viral DNA in Natural Bladder Cancers. Genomic DNA mukcu et aL (2), was not further investigated; and the exact role from 15 naturally occurring bladder cancer biopsies (A-O) and of virus and bracken and the possible synergism between viral, from 10 histologically normal bladder epithelium biopsies was chemical, and immunological factors in the genesis of bladder digested with several restriction enzymes and Southern blotted. cancer were not clear. The results of our long-term experiments The blot was hybridized to the BPV-1, BPV-2, and BPV-4 confirm that both virus and bracken fern are involved in bladder probes and this showed that seven of the cancer biopsies (D, F, carcinogenesis and that the fern is indeed instrumental in the G, H, J, N, and O) were positive for BPV-2 DNA (Fig. 3B; process.

A B a b c d e a b c d

r I Fig. 3. BPV-2 DNA, in experimental and S naturally occurring bladder cancers. ,4, animal 32; total DNA (10 #g) was undigested (a) or digested with EcoRI (b), HindIII (c), BamHI (d), and HincII (e). B, animal G; total DNA was undigested (a) or digested with HindIII (b), EcoRI (c), and BamHI (d). The probe was s. 4 nick-translated BPV-2 DNA. s, supercoiled circles: r, relaxed circles; l, linear molecules.

Table 2 Naturally occurring tumors activated in immunocompromised animals. The points to con- Animal Bladdera Alimentary Tract Skin sider are the following: (a) the young calves were obtained from A Ca + He papillomatosis-free mothers and were papillomatosis-free B Ca + He themselves; (b) the calves were kept in isolated pens, and to C Ca Warts (BPV-I/2)b D Ca (BPV-2) Rumen fibropapilloma Warts(BPV-1/2) minimize risk of cross-infection, care was taken by the stock- (BPV-2) men in changing protective clothing and boots when moving Esophageal carcinoma from pen to pen; (c) the calves were experimentally infected E Ca Colon adenoma F Ca + He (BPV-2) Tonguecarcinoma Warts (BPV-1/2) with well characterized BPV-4 or with no virus at all; (d) most (BPV-4) of the bladder lesions, whether the animal had been injected G Ca (BPV-2) Palate papilloma (BPV-4) with BPV-4 or not, contained BPV-2 DNA; and finally (d) Colon adenoma some, but not all, immunosuppressed animals developed skin H Ca + HS (BPV-2) Rumenfibropapilloma warts harboring BPV-1 or BPV-2 at sites of damaged skin (8). 5 (BPV-2) I Ca Cardia papilloma The animals that developed bladder lesions and in which pap- (BPV-4) illomavirus was activated were all immunosuppressed, either by J HS (BPV-2) treatment with azathioprine or by a diet of bracken fern. This K Ca Palate papilloma (BPV-4) suggests that, as in humans (26-28), immunosuppression reac- L He tivates latent papillomavirus, leading to both benign and ma- M Ca + He + F Tongue carcinoma Pericardium carcinoma lignant tumors. Indeed, reports of papillomavirus presence in N Ca + Po (BPV-2) urinary bladder tumors in immunocompromised humans have O Ca (BPV-2) recently been published. Akin to our findings is the detection of a Ca, transitional cell carcinoma; He, hemangioma; HS, hemangiosarcoma; F, HPV-I 1 DNA in bladder tumors of immunosuppressed renal fibroma; Po, polyps. The virus in the skin warts was not genotyped. transplant recipients (29, 30) and of HPV-16 DNA in the bladder carcinoma of a patient with mild immunodeficiency (31). BPV-2 DNA in Urinary Bladder Cancers. The virus impli- The bladder lesions in the azathioprine-treated animals were cated is most often BPV-2; its DNA has been detected in 69% premalignant hemangiomas, whereas the lesions in the brack- of experimental cancers and in 46% of naturally occurring ones en-fed animals were frank cancers. We speculate that immun- by conventional Southern blotting. In contrast, only 20% of the osuppression activates papillomavirus, but for full malignant control animals had detectable BPV DNA in their bladder; in progression of viral lesions the chemical mutagens of bracken one case this was BPV-2 and in the other it was an unidentified are needed. It must be pointed out, however, that the azathio- papillomavirus. It is possible that a higher percentage of ani- prine-treated animals had to be killed much earlier than the mals would have scored positive if the much more sensitive bracken-fed ones for humanitarian reasons, and it is possible polymerase chain reaction technique had been used. These fig- that their bladder hemangiomas might have progressed to can- ures are similar to those reported for papillomavirus-associated cer had the animals lived longer. genital cancer in humans, where approximately 80% of the BPV-2 DNA Is Present in Naturally Occurring Bladder Can- cancers contain viral DNA detectable by Southern blotting hut cers. The animals presenting with naturally occurring bladder only approximately 20% of normal asymptomatic controls (24). cancers were all from bracken-infested areas and immunosup- The high degree of association between bladder cancer and pressed. Often they had other papillomavirus-related tumors, BPV-2 reported here and Olson's (21) early experiments show- both benign and malignant. These comprised BPV-1/2 skin ing induction of cancer by injection in the bladder of wart warts (20); fibropapillomas of the rumen, containing BPV-2 extracts suggest that BPV-2 is a factor in bladder oncogenesis. DNA (19); BPV-4 papillomas at several sites in the alimentary However, the presence of the closely related BPV-I in the wart canal, and malignancies of the upper and lower alimentary extracts of Olson et al. (2 I) cannot be excluded. BPV-2 is the tract, including a tongue carcinoma which was the only alimen- virus inducing fibropapillomas of the head, neck, and shoulder tary canal cancer containing BPV-4 DNA (15). Some of the of cattle (20), and also fibropapillomas of the upper alimentary animals therefore were additionally infected with BPV-2 at sites canal, although the latter lesions are the result of abortive in- other than the urinary bladder, and the presence of BPV-2 fection and are nonproductive for viral progeny (19). Likewise, DNA in the bladder malignancies might be explained as an the bladder lesions are nonproductive. The viral DNA is pre- opportunistic infection. However, not all the animals positive sumably still infectious and capable of initiating a replicative for BPV-2 DNA in their bladder cancers had other BPV-2 cycle in the permissive environment of the skin, as inferred tumors, at least at the time of presentation, and this reinforces from the results of Olson et al. (22). the argument that the virus is a causal factor in bladder car- Immunosuppression Activates Latent Papillomavirus. Blad- cinogenesis. der cancer and hematuria in cattle are associated also with Approximately one-half of the natural bladder cancers, but ingestion of bracken fern (3), and indeed animals experimen- only approximately 30% of the experimental ones, were nega- tally fed bracken fern similarly developed hematuria and blad- tive for viral DNA. This may be due to lower amounts of viral der tumors, both in our experiment and in the early ones of DNA in the natural cancers, below the relatively low sensitivity Price and Pamukcu (25) and Pamukcu et al. (2). However, of Southern blot hybridization, or, possibly, to the involvement papillomavirus presence in the tumors was not investigated by of a BPV type unrelated to the types used as molecular probes. those authors and there was no report of papillomatosis affect- The route of infection of the urinary bladder by BPV-2 is a ing their animals, thus leaving open the question of virus in- matter for speculation. As it has been postulated for human volvement in bracken-related bladder carcinogenesis. The re- papillomavirus in human bladder tumors (29-31), the virus sults presented here and in a preliminary report (8) suggest that may spread from genital sites, although in the British Isles latent papillomavirus can be transmitted from mother to calf at and after birth and during nursing; the virus then becomes 5 M. S. Campo et aL, manuscript in preparation. 6903 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1992 American Association for Cancer Research. BLADDER CANCER, BPV-2. AND BRACKEN FERN tumors and papillomas of the genital area are most often asso- 10. Maruta A., Enaka, K., and Umeda, M. Mutagenicity of quercetin and ciated with BPV-I (20). kaempferol on cultured mammalian cells. Gann, 70: 273-276, 1979. 11. Amacher, D. E., Paillet, S., and Ray, V. A. Point mutations at the thymidine Quercetin Alone Does Not Affect the Outcome of Papilloma- kinase locus in L5178Y mouse iymphoma cells. Mutat. Res., 64: 391-406, virus Infection. Quercetin is reportedly one of the most potent 1979. 12. Nakayasu, M., Sakamoto, H., Terada, M., Nagao, M., and Sugimura, T. mutagens found in bracken fern (see Ref. 32 for review); nev- Mutagenicity of quercetin in Chinese hamster lung cells in culture. Mutat. ertheless, administration of quercetin to the experimental Res., 174: 79-83, 1986. calves had no detectable effect. This contrasts with the earlier 13. Ishikawa, M., Okada, F., Hamada, J., Hosokawa, M., and Kobayashi, A. Changes in the tumourigenic and metastatic properties of tumour cells finding of Pamucku et al. (14) that administration of quercetin treated with quercetin or 5-azacytididne. Int. J. Cancer, 39: 338-342, 1987. induces intestinal and bladder cancer in experimental rats. It is 14. Pamukcu, A. M., Yalciner, S., Hatcher, J. F., and Bryan, G. T. Quercetin, a conceivable that rats, being homogastric, metabolize quercetin rat intestinal and bladder carcinogen present in bracken fern. Cancer Res., 40: 3468-3472, 1980. differently from cattle and this may explain the difference be- 15. Campo, M. S., Moar, M. H., Sartirana, M. L., Kennedy, I. M., and Jarrett, tween Pamukcu's results and ours. However, our quercetin- W. F. H. The presence of bovine papillomavirus type 4 DNA is not required treated calves were not immunosuppressed and it would appear for the progression to or the maintenance of the malignant state in cancers of the alimentary canal in cattle. EMBO J., 4: 1819-1825, 1985. that immunosuppression is a critical step, necessary but per- 16. Campo, M. S., and Coggins, L. W. Molecular cloning of bovine papilloma- haps not sufficient, for carcinogenesis in cattle. Treatment of virus genomes and comparison of their sequence homology by heteroduplex animals with azathioprine and quercetin should resolve this mapping. J. Gen. Virol., 63: 255-264, 1982. 17. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. Labelling deox- point. Recent experiments (33) have shown that full malignant yribonucleic acid to high specific activity in vitro by nick-translation with transformation of BPV-4-infected primary bovine cells is DNA polymerase I. J. Mol. Biol., 98." 503-517, 1975. achieved only when the cells are pretreated with quercetin, 18. Feinberg, A. P., and Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., stressing the importance of the mutagens present in the fern in 132: 6-13, 1983. carcinogenesis in cattle. 19. Jarrett, W. F. H., Campo, M. S., Blaxter, M. L., O'Neil, B. W., Laird, H. M., In summary, our observations support the view that immun- Moar, M. H., and Sartirana, M. L. Alimentary fibropapilloma in cattle: a spontaneous tumor, nonpermissive for papillomavirus replication. J. Natl. osuppression activates latent papillomavirus giving rise to pre- Cancer Inst., 73: 499-504, 1984. malignant lesions and that the synergism between virus and 20. Campo, M. S., Moar, M. H., Laird, H. M., and Jarrett, W. F. H. Molecular environmental cocarcinogens results in cancer progression. heterogeneity and lesion site specificity of cutaneous bovine papillomavi- ruses. Virology, 113: 323-335, 1981. 21. OIson, C., Pamukcu, A. M., Brobst, D. F., Kowalczyk, T., Satter, E. J., and ACKNOWLEDGMENTS Price, J. M. A urinary bladder tumor induced by a bovine cutaneous papilloma agent. Cancer Res., 19: 779-783, 1959. 22. OIson, C., Pamukcu, A. M., and Brobst, D. F. Papilloma-like virus from Thanks are due to all the colleagues that contributed to this work: to bovine urinary bladder tumors. Cancer Res., 25: 840-849, 1965. Drs. M. L. Sartirana, M. H. Moar, and M. L. Blaxter for their initial 23. Jarrett, W. F. H., Campo, M. S., O'Neil, B. W., Laird, H. M., and Coggins, analysis of viral DNA in bladder cancers; to the stockmen of the vet- L. W. A novel bovine papillomavirus (BPV-6) causing true epithelial papil- erinary school for their expert handling of the animals; and to Dr. M. lomas of the mammary gland skin: a member of a proposed new BPV sub- group. Virology, 136: 255-264, 1984. E. Jackson and Professor J. A. Wyke for critical reading of the manu- 24. zur Hausen, H. Papillomavirus in anogenital cancer: the dilemma of epide- script. miological approaches. J. Natl. Cancer Inst., 81: 1680-1682, 1989. 25. Price J. M., and Pamukcu, A. M. 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6904 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1992 American Association for Cancer Research. Association of Bovine Papillomavirus Type 2 and Bracken Fern with Bladder Cancer in Cattle

M. Saveria Campo, William F. H. Jarrett, Ron Barron, et al.

Cancer Res 1992;52:6898-6904.

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