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Effect of Schistosoma haematobium and N-butyl-N-(4-hydroxybutyl)nitrosamine on the development of urothelial neoplasia in the baboon.

Experiments were conducted to determine whether bladder cancer would develop in primates (Papio sp.) infected with S. haematobium and concurrently exposed to low initiating doses of the bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). To control for the systemic effects of schistosomiasis, 5 baboons were infected with S. mansoni, which does not lay its eggs in the bladder wall; to control for the effect of the carcinogen alone, 5 others were treated with BBN alone at the rate of 5 or 50 mg/kg per week for the duration of the experiment. Five animals were infected with S. haematobium and had no further treatment, and the main experimental group of 10 baboons was infected with S. haematobium and also treated weekly with 5 mg/kg BBN for up to 2½ years. Four of the 10 animals in the last group, but none in the three control groups developed neoplastic disease of the urothelium. Four animals with S. haematobium plus BBN treatment developed in situ carcinoma in the bladder (3 latent adenomatous lesions and 1 more advanced papillary tumour) and 2 of these animals plus 1 other had slightly dysplastic urothelial endophytic papillary growths of the ureter which penetrated the muscle layer. By contrast, none of the control animals developed urothelial carcinomas, though 4/5 of those with S. haematobium infection alone had inflamed bladders with polypoid lesions, and one individual had endophytic papillary hyperplasia of the ureter. The animals were killed after 2½ years while still relatively immature or adolescent, and it is possible that had they been allowed to survive longer some of the BBN-only group would have developed bladder cancer, and more of the latent lesions seen in the BBN + schistosomiasis group would have progressed to invasive carcinoma. It is postulated that, in this model for human bilharzial bladder cancer, schistosomiasis supplies the proliferative stimulus necessary to accelerate cancer growth from latent tumour foci produced by exposure to low doses of the bladder carcinogen. In areas of endemic schistosomiasis, carcinogenesis might be initiated, for example, by low doses of nitrosamines produced in the urinary tract during bouts of bacteriuria. ImagesFig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11Fig. 12Fig. 13Fig. 14Fig. 15Fig. 16Fig. 17Fig. 18Fig. 19Fig. 20Fig. 21Fig. 22Fig. 23Fig. 24Fig. 25Fig. 26Fig. 27Fig. 28Fig. 29Fig. 30Fig. 31Fig. 32

it is possible that had they been allowed to survive longer some of the BBN-only group would have developed bladder cancer, and more of the latent lesions seen in the BBN + schistosomiasis group would have progressed to invasive carcinoma. It is postulated that, in this model for human bilharzial bladder cancer, schistosomiasis supplies the proliferative stimulus necessary to accelerate cancer growth from latent tumour foci produced by exposure to low doses of the bladder carcinogen. In areas of endemic schistosomiasis, carcinogenesis might be initiated, for example, by low doses of nitrosamines produced in the urinary tract during bouts of bacteriuria.
THE CLINICAL and pathological consequences of S. haematobium infection vary, and may be related directly to the prevalence and intensity of the infection. Thus in Egypt and Mozambique, where both prevalence and intensity are high, the close association of schistosomiasis and bladder cancer has been emphasized by many workers (Goebel, 1905;Hashem, 1961;Gillman & Prates, 1962;Prates & Torres, 1965;Brand, 1979) whereas in other areas with a lower endemicity the association is less evident (e.g. Higginson & Oettle, 1962;Dodge, 1962;Houston, 1964). Another possibility is that the connection between schistosomiasis and bladder cancer is more indirect.
There is now good experimental evidence from animal models that the biogenesis of bladder cancer is a multistage phenomenon, involving initiation by a carcinogen followed by promotion and SCHISTOSOMIASIS, A NITROSAMINE AND BLADDER CANCER IN BABOONS 731 propagation of tumour growth by other factors which are not necessarily carcinogens per se (Hicks, 1980). All proven bladder carcinogens identified so far are chemicals which act on the urothelium from its urinary face, and they include a number of N-nitroso compounds which may be formed in the urine during bouts of bacterial infection of the urinary tract (Hill & Hawksworth, 1972;Brookes et al., 1972). Preliminary investigations demonstrated that nitrosamines are indeed formed in the urine in some individuals in Egypt who have both S. haematobium and bacterial infection of the urinary tract (Hicks et al., , 1978. In Egypt, the incidence of bacteriuria is generally low in adults, but a significantly high level of infection (10-37%) was found in village boys and was associated more with poor standards of hygiene than with S. haematobium infection (Carter et al., 1970). More recently, a 5-1% incidence of bacteriuria was found in 390 Egyptian boys aged 5-16 years in regions of endemic S. haematobium infection, and this was 10 times that in areas non-endemic for schistosomiasis (Laughlin et al., 1978). It is thus theoretically possible that the urothelia of a significant percentage of juveniles are intermittently exposed to nitrosamines during bouts of bacterial cystitis. Bladder cancer has a long, clinically "silent" asymptomatic latent period, and in Egypt the peak prevalence (i.e. diagnosis) is in middle age: 50 + 5 years. It is possible that urine-borne nitrosamines produced in childhood or adolescence during episodes of bacteriuria may initiate neoplastic change in the urothelium, and that in areas where schistosomiasis is endemic the development of bladder cancer is subsequently accelerated by chronic irritation of the bladder wall by erupting or calcified ova.
One attraction of this theory is that it accounts satisfactorily for the observed association of high bladder-cancer incidence with S. haematobium but not with S. mansoni infections. S. mansoni, unlike S. haematobium, does not deposit its eggs in the bladder wall, and thus does not provide a direct irritant stimulus to accelerate the development of any previously induced neoplastic lesion. On the other hand, the adults of both species live within the host's vascular system and, if carcinogens were excreted or secreted by the worms per se, the bladder should be equally at risk from blood-borne and/or urine-borne carcinogens in both S. haematobium and S. mansoni infections. A further argument in favour of the above theory, involving initiation of tumour growth by a carcinogen from an alternative source, is that though both S. haematobium and S. mansoni deposit their eggs in the submucosa of the bowel, there is no elevation of cancer of the bowel associated with these infections. Only bladder cancer is elevated, and that only in the S. haematobium-infected population, where the ova are in the bladder wall in a position to promote tumour growth if the urothelium has been initiated by some environmental, bladder-specific carcinogen.
The work reported here was designed to investigate whether in baboons the process of carcinogenesis initiated in the urinary bladder by low, threshold doses of a urine-borne carcinogen is accelerated by the proliferative changes in the urothelium which develop in response to S. haematobium infection. It was not designed as a classic initiation/promotion trial in which the initiating agent is given first and then later is followed by a promoting regime. Instead, in order to simulate the human condition, the S. haematobium infection was established first and small pulses of nitrosamines were administered over a long period of time. There was thus concurrent exposure of the urothelium to schistosomiasis and to the carcinogenic urinary metabolites. In this system, the carcinogen could theoretically initiate neoplastic change at the cellular level and also convert initiated cells to small foci of latent tumour cells. It was postulated that the schistosomiasis might supply the proliferative stimulus necessary to accelerate the development of ongoing tumours from such latent foci.

Carcinogen
Nbutyl -N(4 -hydroxybutyl)nitrosamine (BBN) was synthesized for this experiment by Cambrian Chemicals Ltd., Beddington Farm Road, Croydon, by the method of Druckrey et al. (1964). The redistilled BBN was shown to be chromatographically pure by the method of Preussmann et al. (1964). It was stored undiluted in the dark at -20°C, and used as required for injection as a 25 or 2.5%o solution in saline.

Animals
Twenty-five young male baboons (Papio sp.) weighing 3-8 kg on arrival were divided into 5 experimental treatment groups as follows: Animal Group 1.-The 5 animals in this group were infected with S. mansoni using the abdominal-pouch method illustrated by Webbe & James (1971). The animals were necropsied 7-8 months after infection.
Group 2.-Three baboons in this group were given i.m. injections of 50 mg/kg BBN and the other 2 were given one tenth of this dose, namely 5 mg/kg BBN per week.
Group 3.-The 5 baboons in this group were infected with the Ghanaian strain of S. haematobium.
Group 4. There were 10 animals in this group, infected as for Group 3 with S. haematobium and also given weekly injections of the lower dose of 5 mg/kg BBN. The injections were started one week after infection with S. haematobium and continued until necropsy.
The baboons were kept for 2years before necropsy, with the exception of 2 animals, Nos 94 and 95, which were necropsied prematurely at 12 and 14 months respectively, and No. 96 which died during the acute stage, 3 months after infection.
After infection, the cereariae develop into sexually mature adults in 8-10 weeks, and eggs are excreted in the faeces and urine from S. haematobium animals from then on (Webbe et al., 1974). Both the urinary egg count and the faecal egg count reflect the progress and intensity of the infection, and give some indication of the probable onset of inflammatory hyperplasia of the urothelium in response to the infection. The progress of the infection in Group 3 animals was monitored throughout the experiment by weekly urinary and faecal egg counts, using the Bell filtration technique (Bell, 1963). The faeces and urines of Group 4 animals were also sporadically tested for the presence of eggs, but regular handling of the exereta was avoided because of possible contact with excreted carcinogen. Egg-excretion graphs were drawn, using the 3-point moving average method (Thompson et al., 1962). All baboons were weighed monthly, and at the same time blood samples were taken and packed cell volumes (PCV) determined. Three times a year the animals were examined radiologically with a Watson portable MX2 X-ray machine (Webbe et al., 1974). At necropsy, the bladder was removed and instantly fixed for histology and electron microscopy. The baboons were then perfused using the method of Smithers & Terry (1965). Tissue egg counts and routine histopathology were performed as described by Webbe et al. (1976).

Histology and electron microscopy of the bladder and ureters
The bladder was inflated with phosphatebuffered formalin, then immediately opened and small areas, including the mucosal surface but discarding most of the muscle, were taken for electron microscopy. These were fixed in 10% osmium tetroxide buffered to pH 7-4 with 0-1M cacodylate for 1 h at 4°C before being dehydrated through graded alcohols and embedded in Spurr resin. Sections were cut at 2 Ftm and stained with toluidine blue for high-resolution light microscopy. Thin sections were cut on an LKB or a Porter-Blum microtome, contrast-stained with lead and uranyl salts and examined by transmission electron microscopy in a JEOL lOOB or Phillips 200 electron microscope.
After removing areas for electron micros-copy samples of the whole thickness of the bladder wall were taken for histology. These were fixed for a further 1-3 days in phosphatebuffered formalin before wax embedding, sectioning and staining with haematoxylin and eosin. Ureters were immerse-fixed in formalin or osmium tetroxide and processed for histology or electron microscopy as described above.

Parasitology
The faecal egg output showed an early rise, then a fall after about one year ( Fig.  1) which is characteristic for S. haematobium infections in the baboon (Webbe et al., 1974). A summary of the infection data is given in Table I. As previously reported (Webbe et al., 1974) the infection rate, expressed as percentage of worms recovered in relation to the number of penetrating cercariae, was very variable. Both the experimental Group 4 and the infection-control Group 3 included baboons with infection rates as low as 1-2%. One animal (No. 94) had an exceptionally high infection rate of 48% and had to be necropsied prematurely, and No. 96 died 3 months after infection, during the acute stage.

Body weight and packed cell volumes (PC V)
The relative weight changes of baboons during the period of the experiment are shown in Figs 2-5. The normal average monthly gain for a 4kg baboon is 0 7 kg (Millier, 1976). This was not affected by carcinogen treatment alone or, with the exception of Baboon No. 101, by infection alone (Fig. 2). By contrast, the weight gain of baboons in experimental Group 4 receiving both BBN and infection was severely affected. Nos 94-99 actually lost weight (-16.7% in the first 10 months after patency) (Fig. 3), and only Nos 89 and 93 showed a normal weight gain.
The normal PCV value of 40-45 remained constant in Group 2, despite BBN treatment, but showed a severe depression in all infected animals in Groups 3 and 4, coincident with the time of maximum egg output (Figs 2 & 3). As the infection became chronic, the PCV values rose with the exception of that of the heavily infected animal No. 94 which was necropsied early with a PCV of 11. Radiology and gross pathology tion of the liver and spleen. Extensive The gross pathological changes of haemorrhages were seen in the small and animals in Control Group 1 were typical large intestines. The bladder and ureters ofS. mansoni infection (Sadun et al., 1966). of these animals were, however, essentially These were spotting and brown discoloranormal. The infected animals in Group 3 all had gross pathological changes typical of S. haematobium infection, including greatly dilated ureters, lung adhesions, enlarged lymph nodes, epididymitis, and sandy patches on the bladder wall (Webbe et al., 1974). Similar lesions were seen in Group 4 animals receiving both the BBN and S. haematobium, but in addition the ureters were severely damaged in Baboons Nos 91-94, being nodular and granulomatous as well as grossly dilated. In Nos 95-97 the bladder wall was markedly thickened, and No. 97 also had an enlarged right kidney which appeared as hydronephrosis on the radiograph.  (Hicks, 1975). 98. This field also shows deep invaginations of the surface urothelium, each with its lumen, and one of these processes is seen to lie below the circular muscle (arrow). The urothelium lining both the invaginated processes and the luminal face of the ureter appears to be well-differentiated. Toluidine-blue-stained, epon-embedded section.

Histopathology of bladders and ureters
x 136. were mostly heavily loaded with ova both deep in the wall and in the lamina propria.
The infection was associated with the characteristic inflammatory response by the host, and numerous polymorphs, scattered lymphocytes and lymphoid follicles were seen in the tissues. In one individual, # 103, the urothelium was normal despite considerable inflammation, but no ova were found in the submucosa. Three animals, Nos 100, 101 and 102, had mild cystitis cystica and polypoid hyperplasia ( Fig. 9) with heavily inflamed lamina propria between the epithelial surfaces. In these animals the mucosa was slightly thickened but well differentiated, there were no down-growths through the muscle and the few small endophytic processes into the submucosa mainly had a visible lumen. The ureters were minimally infected and their urothelium was normal. The ureters of the 5th animal, No. 98, were severely infected, grossly inflamed with heavy egg deposition and marked polypoidal hyperplasia of the urothelium showing mild dysplasia (Fig. 10). One small endophytic process of urothelium with a lumen had penetrated the muscle in the wall of one ureter, but the cells in this area were well differentiated (Fig. 11). The bladder of this animal was also severely infected and inflamed, and again there 740 was polypoidal hyperplasia and cystitis cystica (Fig. 12). However, the bladder urothelium was well differentiated, there was no dysplasia and electron-microscopical examination showed the surface cells to be normal, slightly immature superficial cells (Fig. 13). The histopathology of Group 3 bladders and ureters is summarized in Table II. The urothelia lining the ureters and bladders of animals in Group 4, which were infected with S. haematobium and given weekly injections of the lower-dose BBN (5 mg/kg), showed more florid proliferative changes and more dysplastic histology than animals in Group 3, which were infected with S. haematobium but did not receive the carcinogen. Table II summarizes the histopathological findings in the ureters and bladders of these animals in relation to egg deposition and inflammation of the walls of the ureters and bladder.

Ureters
The ureters of Baboon No. 96, which was found dead, were not sampled. Baboon No. 99 was the only animal in which the ureteral urothelium was mainly of normal thickness and well differentiated. No schistosomal ova were found in the ureters of this animal, even though moderate egg deposition accompanied by reactive hyperplasia of the urothelium was found in its bladder. In the ureters of one other individual (No. 90) there was a thickened hyperplastic urothelium but a normal growth pattern with normal cell differentiation; very few ova were found in the submucosa, though the ureters were slightly inflamed. The remaining 7 animals all had moderate to severe inflammation of the ureters, variable numbers of eggs being deposited deep in the ureter wall and sometimes superficially in the submucosa. In 5 animals (Nos 89, 91, 93, 95 and 97) there were endophytic processes of urothelium clearly located within or beneath the circular muscle in the wall of the ureter. In two of these (Nos 91 and 93) cell differentiation appeared normal, and comparable to that in the S. haematobium control animal shown in Fig. 11. One of the others No. 95) had numerous downgrowths, many of which were well-differentiated (Fig. 14), but others were slightly dysplastic (Fig. 15). There were dysplastic down-growths in Nos 89 and 97 showing differential growth patterns, irregularity in size and shape of nuclei, and failure of  normal transitional-cell differentiation (Fig. 16). Occasional groups of adenomatous cells were also located around a single central lumen oir around several small lumina, each containing mucoid material (Fig. 16). The diagnostic assessment of the ureters in these Group 4 animals is shown in Table III.

Bladders
The pathology of the bladders was comparable to that of the ureters (Table II). Heavy egg deposition was found in the bladder wall of 6 animals and inflammatory changes in all of them. The urothelium in Baboon No. 91 was essentially normal, apart from some mild polypoidal hyperplasia, even though there was polypoidal and papillary hyperplasia of the urothelium in the ureters of this animal, plus well differentiated endophytic processes within and below the circular musle wall. Baboons Nos 90, 92, 93, 95 and 99 all had polypoidal hyperplasia and cystitis cystica of varying degrees of severity (Figs 17 & 18). In most of these animals, the superficial cells on the urinary face were normally differentiated (Fig. 19), though some patches of mucoussecreting cells were also found (Fig. 20). More severe cell atypia was seen in Baboon No. 95, particularly in the endophytic growths (Figs. 18 & 21) and some of the surface cells were poorly differentiated, being neither well developed mucous cells nor transitional cells; their free surfaces were covered with numerous atypical short microvilli (Fig. 22). The urothelium of Nos 89, 94 and 97 showed marked glandular and trabecular growth patterns, extending deeply into the lamina propria and passing between strands of the superficial muscle (e.g. Fig.  23). Many of these glands were composed of an orderly bilayer of small basal cells plus mucus-secreting columnar surface cells with the characteristic subcellular features of mucous metaplasia. In Baboon No. 89, however, in some of the downgrowths there was no ordered orientation of the cells between base and lumen, and there was cell atypia, with piling and crowding, and the nuclei were deeply indented and of irregular size and shape (Figs 24 & 25). The same differential growth patterns, atypia and crowding were seen in the endophytic glandular extensions of the urothelium of Baboon No. 94, and in addition there were many mitotic figures. Another feature in Baboon No. 94 were the numerous sharp tongues small clusters of highly atypical epithelial cells which extended in random directions in the submucosa and which just penetrated the most superficial muscle layer (Fig. 26). The cells at the urinary surface of the bladder epithelium in this animal had a variable complement of microvilli varying considerably inlength and covered with a prominent glycocalyx (Fig. 27 plus inset). All the features described for Baboons Nos 89 and 94 were also seen in No. 97, bands of the superficial muscle layer being interposed between the endophytic glandular down-growths. Again, irregularly arranged sharp tongues and small isolated groups of highly atypical epithelial cells invaded the submucosa. The urinary face of the most superficial cells in the bladder epithelium were almost uniformly covered with numerous, highly irregular, long-branched microvilli with a fine glycocalyx (Fig. 28 plus inset). The remaining animal, No. 96, was found dead, and the preservation of its bladder was inadequate for electron microscopy. Histological examination showed gross polypoidal and papillary carcinoma . In many areas, there were numerous mitoses and the urothelium was grossly dysplastic (Fig. 30). In places, irregular tongues of dysplastic urothelial cells with no apparent limiting basal lamina extended into the heavily inflamed submucosa (Figs 31 & 32). Their appearance was similar to those seen in Baboons Nos 94 and 97, but the tongues of cells did not penetrate the muscle layers. In this animal there was no mucous metaplasia or cystitis cystica. The diagnostic assessment of the pathology of the bladders of the animals in Group 4 is shown in Table III. DISCUSSION A number of investigations designed to determine whether S. haematobium infections alone could induce bladder cancer in various primate species have been published (Jordan et al., 1967;Vogel, 1967;Sadun et al., 1970;Kuntz et al., 1971Kuntz et al., , 1972. In general, schistosomal infections have produced extensively inflamed polyps in the bladder rather than atypical proliferation of the urothelium. However, Kuntz et al. (1972) found well differentiated papillary carcinoma in the bladder of one capuchin and a talapoin monkey, and in the ureter in one of 21 baboons infected with S. haematobium, plus other proliferative lesions of the urothelium in 2/7 capuchins and 2/5 squirrel monkeys. These lesions were all non-invasive, and even the carcinomas were relatively well differentiated. Subsequent experiments with S. haematobiuminfected capuchin monkeys showed similar well differentiated papillary lesions with restricted growth potential, which failed to survive transplantation (Kuntz et al., 1978). Furthermore, the epithelial lesions regressed as the infection resolved in the 3rd and 4th years after infection. This raises doubts about the malignant potential of these S. haematobium-induced well differentiated tumours of the urothelium.
It is notoriously difficult to differentiate histologically between inflamed proliferative hyperplasias of the urothelitum and early, well differentiated carcinomas. In this study of control and S. haenmatobiuininfected baboons, we have examined the bladders and ureters both histologically and by electron microscopy. This has demonstrated that the normal cell structure of the urothelium lining the baboon ureters and bladders is the same, both histologically and at the subeellular level, as that of man and other mammals (Hicks, 1975;Newman & Hicks, 1977). In particular, the baboon urothelium is limited by large, flat superficial cells with a well characterized surface structure, typical of all mammalian species studied. It is now widely accepted, as a result both of numerous experimental studies and of observations of human biopsy specimens, that malignant transformation of the urothelium is accompanied by loss of normally differentiated urothelial cells and their replacement by smaller cells covered with long pleiomorphic microvilli (Arai et al., 1974;Hicks et al., 1974;Fulker et al., 1971;Hicks, 1976Hicks, , 1977Hicks & Wakefield, 1976;Hodges et al., 1976;Jacobs et al., 1976;Newman & Hicks, 1977;Friedell et al., 1977). In the present study, where pleiomorphic microvilli have been observed by electron microscopy in urothelia which appeared to be hyperplastic or dysplastic by simple histological criteria, we have reclassified the lesions as latent carcinomas. Clearly, any differential diagnosis based on morphological criteria alone cannot be unequivocal, but the presence of microvilli is a useful indication that the urothelial response to insult has progressed beyond reversible hyperplasia and that initiated cells have been promoted and converted to foci of neoplastic cells. In rodents, it is experimentally demonstrable that these early-stage, latent cancers are capable of progressing to invasive carcinoma. The rate at which they progress from latent carcinomas to a mass large enough to be clinically diagnosable as cancer is con-trolled by the presence or absence of further propagating factors which accelerate the rate of tumour growth by increasing the rate of cell turnover.
In this study, 4/5 baboons infected with S. haemnatobiurn but given no further treatment developed inflamed polyps of the bladder plus mild polypoidal hyperplasia with cystitis cystica. Both the surface epithelium and the few small endophytic processes into the submucosa were well differentiated, and few mitoses could be found. Interestingly, there was one deep endophytic process through the muscle wall of the ureter in one baboon in which the infection was particularly heavy and where the ureter wall was grossly inflamed. As in the papillary growths seen by Kuntz et al. (1972Kuntz et al. ( , 1978, the epithelium of this process was well differentiated as judged by both conventional histology and electron microscopy. These findings confirm that infection with S. haematobium alone can produce papillomas of the urothelium which from their growth pattern might be classified as carcinomas but which histologically are benign. Accordingly we have classified the endophytic papillary lesion seen in the ureter of one of the 5 baboons infected with S. haematobium in these experiments as papillary hyperplasia with both an exophytic and endophytic growth pattern. Both in humans and in experimental animals, the only proven direct-acting carcinogens known for the bladder are chemicals excreted in the urine which act on the bladder mucosa from its urinary face. The nitrosamine BBN is known to be an organotropic carcinouen for the bladder of several species including the mouse, rat and golden hamster (Druckrey et al., 1964;Ito et al., 1969;Bertram & Craig, 1972;Hirose et al., 1976;Becci et al., 1979) and its mode of action in rodents has been investigated in several laboratories. In this study, low doses of BBN were adnministered to baboons at weekly intervals throughout the experiment, to provide regular, intermittent exposure of the urothelium to the metabolites of this carcinogen which are known to be excreted in the urine. None of the 5 baboons treated with BBN alone developed bladder cancer, although a high percentage of carcinoma of the urothelium developed in hamsters treated concurrently with the same batch of carcinogen. Although the BBN treatment thus appeared to be subcarcinogenic for the baboons in the 2 2 years' duration of these experiments, it would be imprudent to conclude that primates are immune to the effect of BBN. Two and a half years represents only a quarter or less of the normal life-span of this animal, while 18 months is closer to the normal life-span of the hamster. Primates are also known to be slow to respond to chemical carcinogens; it required 33 months before the earliest bladder cancers developed in rhesus monkeys treated with 2-naphthylamine (Conzelman et al., 1969), and the average latency before bladder cancer develops in humans after exposure to this chemical is 20 years (Case, 1966). It is therefore quite possible that if the BBN-treated baboons had been maintained for 5 years or more bladder cancer would have developed in these animals also. Undoubtedly BBN has some effect on the bladder mucosa, for the proliferative response of the urothelium in animals given BBN as well as S. haematobium infection was far greater than in animals given either treatment alone. If carcinogenesis in the baboon bladder is a multistage process, as it is in other species (Hicks, 1980) it is highly probable that when necropsied the bladders of these animals were in the long latent period between initiation and promotion by BBN of neoplastic change within individual cells of the urothelium, and the development of visible, latent tumour foci or ongoing tumours.
There was indeed a striking difference between the pathological changes of the S. haematobium-infected animals in Groups 3 and 4. Those in Group 4 (which had additionally received BBN) had a relatively poor weight gain and far worse bladder lesions, despite comparable levels of infection. This is particularly noteworthy, since on average egg deposition in the bladder was actually less in the BBN-treated animals than in the infection-only group. Despite this, 3/10 baboons in Group 4 developed gross adenomatous lesions of the bladder which, on the basis of their ultrastructure, were classified as latent adenocarcinomas. A fourth animal had a histologically diagnosable papillary carcinoma, classified as Plb by the WHO system. All but one of the remaining animals in this group had polypoidal hyperplasia and cystitis cystica of varying degrees of severity, whereas only 1 of the 5 infected baboons in Group 3 had a comparatively inflamed, hyperplastic urothelium.
The urothelial lesions seen in the ureters in 5 of the Group 4 and 1 of the Group 3 animals are interesting but difficult to interpret. Undoubtedly, in all these animals urothelial processes were clearly visible lying deep in the ureter wall below the band of circular muscle. It is possible, however, that these processes were trapped below the muscle after widespread disruption of muscle and connective tissue during the acute phase of infection. If so, they do not indicate invasive growth of the urothelium, but only hyperplasia, and the well differentiated normal substructure of the endophytic processes in 3 animals (1 in Group 3 and 2 in Group 4) supports this suggestion. On the other hand, definite signs of cell atypia and abnormal subcellular differentiation were present in the endophytic processes of 3/4 BBN-treated animals. We suggest, therefore, that the increased cell turnover of the urothelium during the acute phase of infection may be sufficient to account for the abnormal papillary growth patterns when the animals were necropsied, but that such proliferative lesions may well have a limited growth potential (see Kuntz et al., 1978). If, however, the urothelial cells are additionally initiated by exposure to a low dose of a carcinogen, the accelerated cell turnover provoked by the infection will in-crease the rate at which any new, abnormal and possibly neoplastic phenotype can be expressed, as demonstrated by the cell atypia in the endophytic processes of 3 of the Group 4 animals. Taking into consideration the state of the bladder urothelium in these animals, we have therefore tentatively classified the 3 lesions with atypia as latent carcinomas, by contrast with the well differentiated lesions which we have referred to as hyperplasias. This diagnosis based on morphological criteria only must be equivocal.
Overall, the results from this study support the postulate that infection with S. haematobiunt supplies the proliferative stimulus necessary to accelerate the development of visible, latent tumour foci from cells initiated and converted by exposure to low doses of bladder carcinogens. It might therefore be expected to increase the incidence of clinically symptomatic bladder cancer in exposed populations for any particular age group. In these experiments we used a nitrosamine to initiate neoplastic change, since we had detected nitrosamines in the urines of both Egyptians and Europeans with bacteriuria (Hicks et al., , 1978. The doses of BBN used in the current experiments with baboons were high, but then so are the potential levels of exposure of patients with chronic bacteriuria. Brookes et al. (1972) calculated that an American patient with episodic Proteus mirabilis infection of the urinary tract, whose urine contained 0 5mM N-nitrosodimethylamine, would have been exposed to about 166 mg of the nitrosamine during the 3 days before the infection was reduced by treatment. A sufficiently high level of bacterial infection of the urinary tract is present in Egyptian boys (Carter et al., 1970;Laughlin et al., 1978) to suggest either chronic or regular intermittent exposure of the urothelium to nitrosamines during childhood and adolescence. This represents only one possible source of low doses of urine-borne carcinogens, and others may be encountered which are related to local variations in diet, drugs, water supply, smoking habits, etc. In the absence of schistosomiasis, a low background of bladder cancer is still to be expected, and this background incidence will reflect the potency of and exposure to individual environmental carcinogens. It may be suggested that better control of secondary bacterial infection of the lower urinary tract, particularly in juveniles in areas of endemic schistosomiasis, could well lead to a reduction in later years of the incidence of bladder cancer superimposed on the bilharzial bladder syndrome.