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Purpose: Little is known regarding differences in childhood growth between somatic and heritable retinoblastoma (Rb) populations. We aimed to compare childhood growth parameters between somatic and heritable Rb cohorts at birth and at time of diagnosis with Rb., Methods: A multinational, longitudinal cohort study was conducted with patients from 11 centers in 10 countries who presented with treatment naïve Rb from January to December 2019. Variables of interest included age, sex, and size characteristics at birth and at time of presentation, as well as germline mutation status. After Bonferroni correction, results were statistically significant if the P value was less than 0.005., Results: We enrolled 696 patients, with 253 analyzed after exclusion criteria applied. Between somatic (n = 39) and heritable (n = 214) Rb cohorts, with males and females analyzed separately, there was no significant difference in birth weight percentile, weight percentile at time of diagnosis, length percentile at time of diagnosis, weight-for-length percentile at time of diagnosis, or change of weight percentile from birth to time of diagnosis. Patients with heritable Rb had a smaller mean weight percentile at birth and smaller mean weight and length percentiles at time of diagnosis with Rb, although this difference was not statistically significant. All cohorts experienced a slight negative change of weight percentile from birth to time of diagnosis. No cohort mean percentiles met criteria for failure to thrive, defined as less than the 5th percentile., Conclusions: Children with Rb seem to have normal birth and childhood growth patterns. There is no definitive evidence that somatic or heritable Rb has a biological or environmental impact on childhood growth parameters.

Background: Invasive dental treatment in patients exposed to antiresorptive and antiangiogenic drugs can cause medication-related osteonecrosis of the jaw (MRONJ). Currently, the exact pathogenesis of this disease is unclear., Methods: In March 2022, Medline (Ovid), Embase (Ovid), Scopus, and Web of Science were screened to identify eligible in vitro studies investigating the effects of antiresorptive and antiangiogenic compounds on orally derived cells., Results: Fifty-nine articles met the inclusion criteria. Bisphosphonates were used in 57 studies, denosumab in two, and sunitinib and bevacizumab in one. Zoledronate was the most commonly used nitrogen-containing bisphosphonate. The only non-nitrogen-containing bisphosphonate studied was clodronate. The most frequently tested tissues were gingival fibroblasts, oral keratinocytes, and alveolar osteoblasts. These drugs caused a decrease in cell proliferation, viability, and migration., Conclusions: Antiresorptive and antiangiogenic drugs displayed cytotoxic effects in a dose and time-dependent manner. Additional research is required to further elucidate the pathways of MRONJ.

Extensively drug-resistant tuberculosis (XDR TB) has extremely poor treatment outcomes in adults. Limited data are available for children. We report on clinical manifestations, treatment, and outcomes for 37 children (<15 years of age) with bacteriologically confirmed XDR TB in 11 countries. These patients were managed during 1999-2013. For the 37 children, median age was 11 years, 32 (87%) had pulmonary TB, and 29 had a recorded HIV status; 7 (24%) were infected with HIV. Median treatment duration was 7.0 months for the intensive phase and 12.2 months for the continuation phase. Thirty (81%) children had favorable treatment outcomes. Four (11%) died, 1 (3%) failed treatment, and 2 (5%) did not complete treatment. We found a high proportion of favorable treatment outcomes among children, with mortality rates markedly lower than for adults. Regimens and duration of treatment varied considerably. Evaluation of new regimens in children is required.

Background: An estimated 32,000 children develop multidrug-resistant tuberculosis (MDR-TB; Mycobacterium tuberculosis resistant to isoniazid and rifampin) each year. Little is known about the optimal treatment for these children., Methods and Findings: To inform the pediatric aspects of the revised World Health Organization (WHO) MDR-TB treatment guidelines, we performed a systematic review and individual patient data (IPD) meta-analysis, describing treatment outcomes in children treated for MDR-TB. To identify eligible reports we searched PubMed, LILACS, Embase, The Cochrane Library, PsychINFO, and BioMedCentral databases through 1 October 2014. To identify unpublished data, we reviewed conference abstracts, contacted experts in the field, and requested data through other routes, including at national and international conferences and through organizations working in pediatric MDR-TB. A cohort was eligible for inclusion if it included a minimum of three children (aged <15 years) who were treated for bacteriologically confirmed or clinically diagnosed MDR-TB, and if treatment outcomes were reported. The search yielded 2,772 reports; after review, 33 studies were eligible for inclusion, with IPD provided for 28 of these. All data were from published or unpublished observational cohorts. We analyzed demographic, clinical, and treatment factors as predictors of treatment outcome. In order to obtain adjusted estimates, we used a random-effects multivariable logistic regression (random intercept and random slope, unless specified otherwise) adjusted for the following covariates: age, sex, HIV infection, malnutrition, severe extrapulmonary disease, or the presence of severe disease on chest radiograph. We analyzed data from 975 children from 18 countries; 731 (75%) had bacteriologically confirmed and 244 (25%) had clinically diagnosed MDR-TB. The median age was 7.1 years. Of 910 (93%) children with documented HIV status, 359 (39%) were infected with HIV. When compared to clinically diagnosed patients, children with confirmed MDR-TB were more likely to be older, to be infected with HIV, to be malnourished, and to have severe tuberculosis (TB) on chest radiograph (p < 0.001 for all characteristics). Overall, 764 of 975 (78%) had a successful treatment outcome at the conclusion of therapy: 548/731 (75%) of confirmed and 216/244 (89%) of clinically diagnosed children (absolute difference 14%, 95% confidence interval [CI] 8%-19%, p < 0.001). Treatment was successful in only 56% of children with bacteriologically confirmed TB who were infected with HIV who did not receive any antiretroviral treatment (ART) during MDR-TB therapy, compared to 82% in children infected with HIV who received ART during MDR-TB therapy (absolute difference 26%, 95% CI 5%-48%, p = 0.006). In children with confirmed MDR-TB, the use of second-line injectable agents and high-dose isoniazid (15-20 mg/kg/day) were associated with treatment success (adjusted odds ratio [aOR] 2.9, 95% CI 1.0-8.3, p = 0.041 and aOR 5.9, 95% CI 1.7-20.5, p = 0.007, respectively). These findings for high-dose isoniazid may have been affected by site effect, as the majority of patients came from Cape Town. Limitations of this study include the difficulty of estimating the treatment effects of individual drugs within multidrug regimens, only observational cohort studies were available for inclusion, and treatment decisions were based on the clinician's perception of illness, with resulting potential for bias., Conclusions: This study suggests that children respond favorably to MDR-TB treatment. The low success rate in children infected with HIV who did not receive ART during their MDR-TB treatment highlights the need for ART in these children. Our findings of individual drug effects on treatment outcome should be further evaluated., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: DF, MG, and MvDB are staff members of the World Health Organization (WHO). AJGP's institution, Stellenbosch University, has received funds from the US National Institutes of Health for an observational study of the pharmacokinetics and safety of key second-line TB medications in children, for which AJGP is the Principal Investigator. AJGP's institution has also received funds from Otsuka Pharmaceuticals for implementation of pediatric trial of the novel TB drug delamanid; AJGP is the PI for his site for this trial.

he comparative teratogenicity of nine retinoids in Wistar rats was investigated. The compounds studied and dose levels tested (mg/kg) were: all-trans-retinoic acid (TRA), 6.25, 12.5, 25, 50, 100; etretinate (ETR), 25, 50; acitretin (ACIT), 25, 50; 13-cis-retinoic acid (13CRA), 100, 200; and five retinamides, each at 300 and 600 mg/kg, N-(4-hydroxyphenyl)-retinamide (4HPR); N-tetrazol-5-ylretinamide (TZR); N-butylretinamide (NBR); N-ethylretinamide (NER); 13-cis-N-ethylretinamide (13CNER). Retinoids were administered by oral intubation on days 10 and 11 post coitum (p.c.). Dams were killed on day 22 p.c. and examinations carried out to assess teratogenic potential. TRA, ETR, ACIT, 13CRA and 4HPR increased the incidence of resorptions. The incidence of abnormal fetuses, irrespective of the specific abnormalities induced, was markedly increased (50-100%) by TRA, ETR, ACIT, 13CRA and 4HPR, whereas TZR and NBR caused moderate increases (20-50%), and NER and 13CNER induced mild increases (10-20%). The incidences of CNS, craniofacial and urinogenital defects were generally high with TRA, ETR, ACIT and 13CRA. Cardiac vessel defects were markedly increased by 4HPR. Using a number of criteria, a generalized ranking order of the toxicity of the compounds was drawn up: TRA > ETR > ACIT > 13CRA > 4HPR > TZR identical to NBR > NER identical to 13CNER. The ranked order of relative in-vivo teratogenicity for the nine retinoids is compared with a previously reported in-vitro assessment of the compounds using a rat whole embryo culture technique.

Several multivariate statistical methods are available which can alleviate the problems of analysing the large volumes of data generated from toxicological experiments. One such technique, principal components analysis, provides a method for exploring the relationships between a number of variables (such as blood parameters) and for eliminating redundant data if strong correlations exist between the characters. It also provides a method for clustering individuals, which may reveal similarities between animals in a treatment group or highlight individual 'outliers'. The application of principal components analysis to a set of haematological data from a trial evaluating the efficacy of a synthetic retinoid against carcinogen-induced bladder cancer in the rat has clearly shown, in two bivariate plots, that while some animals in the carcinogen-treated groups were normal, others were anaemic and that animals fed the synthetic retinoid and killed at 1 year had a microcytic anaemia. A full exploration of the data using conventional univariate statistical analysis would have involved at least 28 graphic representations of the data, as well as the interpretation of more than 130 means and SDs. Principal components analysis provides a valuable additional tool for the statistical analysis and exploration of toxicological data, but it must be used in conjunction with univariate or other multivariate methods if hypothesis testing is required. The use of multivariate techniques in toxicology may best be assessed by their practical application to toxicological data, and this paper presents such an evaluation with the aim of encouraging further exploration of the usefulness of principal components analysis. The raw data on which most analyses have been carried out are given.

Three nephrectomy specimens with transitional cell carcinoma (TCC) of the renal pelvis were thoroughly examined by both light and scanning electron microscopy. The tumours as well as the urothelium of the upper urinary tract were studied. In all three cases, extensive areas of the urothelium, even in places remote from the tumours, were found by scanning electron microscopy (SEM) to be covered by pleomorphic microvilli. This suggests that there is a widespread failure of differentiation of the urothelium to a much greater extent than can be appreciated by conventional light microscopy.

This study examines the effects of in-vivo immune regulation by vitamin A acetate (VAA) and 13-cis-retinoic acid (13-CRA) on in-vitro accessory cell function. Mice were fed a control diet, or diet containing VAA or 13-CRA, and monitored by body weight gains and diet consumptions at weekly intervals. At 4, 7 and 12 weeks mice were killed, differential blood counts performed and accessory cells isolated from lymphomedullary tissues. Histology confirmed that the chief feature of the lymphomedullary organs of the VAA-fed animals was an expansion of the splenic marginal zone and the paracortical region of the lymph nodes. There was an increase in the number of accessory cells present, and this included both dendritic cells and macrophages. The accessory cell function of these cells was also increased, as evidenced by both alloproliferative and allocytotoxic responses in vitro. In 13-CRA-fed animals the effects were similar to those seen with VAA, but were less pronounced. We suggest that the primary effects of these compounds on in-vivo immunoregulation could be due to their promotion of accessory cell function.

As little comprehensive baseline data are available on age-related haematological changes in genetically-defined rat strains, the haematology of female F344 rats is described in animals sampled at 2, 4, 8, 20, 66 and 121 weeks of age. Values for Hb, RBC and PCV increased from 2 weeks of age to reach adult levels at 8 weeks, whereas MCV, MCH and reticulocyte counts were high initially but decreased to reach the adult range at 8 weeks. Between 66 and 121 weeks, reticulocyte counts were significantly increased and values for MCHC significantly decreased. Lymphocytes were the predominant white cell type in each age group. The absolute numbers of neutrophils and lymphocytes showed slight variations between 2 and 66 weeks and both cell types increased significantly between 66 and 121 weeks. Platelet counts showed no overall age-related trends. Fibrinogen values increased from 2 weeks of age to reach the adult level at 8 weeks. One animal of the 14 sampled at 121 weeks showed changes in the blood, liver and spleen consistent with a diagnosis of lymphoid leukaemia.

Experiments were conducted to determine the dose response of rat bladder urothelium to a range of different single and fractionated intravesicular doses of the carcinogen, N-methyl-N-nitrosourea (MNU). A dose-related response of bladder-tumour incidence to single graded doses of MNU was found, and a threshold does suitable for use of multistage carcinogenesis experiments was derived from these data. For any given total dose of MNU, the tumour incidence was greater if the MNU had been administered in several small fractions than if it had been administered in fewer larger ones. Extending the interval between doses did not reduce the tumour incidence. It is argued that these results support the multistage theory of carcinogenesis. The histopathology and cell-surface alterations which characterize the development of MNU-induced bladder cancer are described and the contribution of hyperplasia and calculi are discussed.

Single toxic doses of 1,2-dimethylhydrazine induced mild centrilobular necrosis of the liver in rats and mice. Ultrastructural studies showed hepatic nuclear changes including nucleolar microsegregation and changes in the endoplasmic reticulum and mitochondria. 1-Methylhydrazine caused little morphological change in the liver. Tumours of the colon and kidney and also massive cystic hyperplasia of the liver were found in some of the rats and tumours of the anal margin and kidney in some of the mice, following single doses of 1,2-dimethylhydrazine. Incorporation of amino acids into rat liver proteins was inhibited by 1,2-dimethylhydrazine, which also caused disaggregation of hepatic polysomes. No effects on hepatic protein synthesis by 1,1-dimethylhydrazine or 1-methylhydrazine were observed. Similarities between the effects of 1,2-dimethylhydrazine, cycasin and dimethylnitrosamine are discussed.

Normal adult human bladder obtained at cystoscopy has been maintained in long-term organ culture. Several media were tested for their ability to maintain viability and normal tissue morphology. The optimum medium was Ham's F-12 nutrient mixture, supplemented with 10% fetal calf serum, hydrocortisone (1 microgram/ml), and FeSO4, (0.45 microgram/ml). During the first 28 days in vitro, epithelial damage incurred at biopsy and during preparation of the cultures was repaired, and epithelialization of cut stromal surfaces occurred. A wave of cell proliferation was identified by [3H]thymidine autoradiography, 24-h labeling indices rising to a peak of up to 50% on the cut sides of the cultures between 7 and 21 days and falling to 0 to 5% by 21 to 28 days. The regenerating epithelium showed all the normal features of urothelial cell differentiation when examined by scanning and transmission electron microscopy. From 28 days, histology and scanning and transmission electron microscopy showed the cultured urothelium in most cultures to resemble closely that in the normal bladder in vivo, and in this mature state cultures were maintained for 100 days. Urothelium derived from certain patients, although showing normal surface maturation, developed enlarged intercellular spaces or intraepithelial mucin-containing acini. A study of the cytology of cells shed into the medium at different stages in culture showed that culture viability and epithelial differentiation could be monitored easily in long-term culture by this nondestructive means.

Some biochemical and ultrastructural changes induced in the livers of rats treated with N-nitrosomorpholine are described and compared with parallel observations in rats given dimethyl- or diethylnitrosamine. Hepatotoxic doses of the nitrosamines caused inhibition of incorporation of [14C]leucine into hepatic proteins, accompanied by progressive disaggregation of polysomes which paralleled the known time course of metabolism of each compound. Dimethylnitrosamine (DMN) and N-nitrosomorpholine (NM) inhibited incorporation of [14C]orotate into liver RNA but diethylnitrosamine (DEN) caused a slight stimulation of orotate incorporation. Electron microscopy revealed similar hepatic cytoplasmic changed induced by each nitrosamine, including dilation and degranulation of the rough surfaced endoplasmic reticulum and subsequent increase of the smooth endoplasmic reticulum. Nuclear changes differed with each compound, N-nitrosomorpholine having more marked effects than either dialkyl compound. The results are discussed with particular reference to the metabolism of N-nitrosomorpholine in the liver.

A sensitive rat model has been designed to detect potential weak bladder carcinogens or co-carcinogens. The test compound is given to animals which have received a single initiating, but non-carcinogenic, dose of N-methyl-N-nitrosourea (MNU). The model has been used to investigate two compounds currently under suspicion as weak bladder carcinogens, namely sodium saccharin and sodium cyclamate, and one compound known to be cytotoxic but not carcinogenic for the bladder epithelium namely cyclophosphamide. For comparison, these three compounds were also tested as solitary carcinogens in animals not pre-treated with MNU. At the very high dose levels used, sodium saccharin and sodium cyclamate were weak solitary carcinogens producing 4/253 and 3/228 bladder tumours respectively, and the first of these tumours did not appear for more than 80 weeks. When tested in the MNU/rat model more than half the animals receiving either sodium saccharin or sodium cyclamate developed bladder tumours from 10 weeks onwards. By contrast, cyclophosphamide failed to produce any tumours when tested either as a solitary carcinogen or in the MNU/rat model. It must be emphasized that the doses of saccharin and cyclamate used were far higher than those consumed by man, including diabetics, and these results should not be directly extrapolated to man without careful consideration of many other factors including negative epidemiological findings. The theoretical basis of the model is discussed and also the relevance, in terms of environmental human exposure, of detecting compounds which have a synergistic effect with other known bladder carcinogens. It appears that this model can be used to detect a carcinogenic or co-carcinogenic potential in compounds which are organotropic for the bladder more rapidly and with fewer animals than if the compounds are tested as solitary carcinogens by more conventional methods. It is suggested that it could be used to detect those compounds which require further investigation.

The widely held belief that 2-naphthylamine is not carcinogenic for the rat has been re-examined. Twenty female Wistar rats were dosed by gastric intubation weekly for 57 weeks with 2-naphthylamine, 300 mg/kg body wt, in arachis oil and 20 controls were given arachis oil alone. Animals which became moribund were killed during the course of the experiment and the remainder after 100 weeks. A 2-naphthylamine-treated animal died at 21 weeks; all others survived 57 weeks or longer. The urinary tracts of all but two 2-naphthylamine-treated animals, which were found dead and cannibalized, were examined histologically.No neoplastic disease of the urinary tract was present in control animals. In 10 of the 2-naphthylamine-treated rats there was neither neoplasia nor hyperplasia of the urothelium, but 4 of the 18 examined histologically had large, macroscopically visible bladder cancers; one of these also had bilateral transitional cell tumours of the kidney calyces and multiple tumours in both ureters. Another animal had bilateral urothelial cancers in the ureters. The histology and ultrastructure of these urothelial cancers were comparable to those of rat transitional-cell carcinomas experimentally induced with other chemical carcinogens.These results, considered in the context both of early and more recently published biochemical studies of 2-naphthylamine metabolism in the rat, support the possibility that production of the active carcinogenic metabolite in this species may be influenced by a pH-dependent, non-enzymic mechanism in the urine, which could account for individual, strain- and diet-related variations in response in the rat.

A review of various in vivo and in vitro studies indicates that saccharin has second-stage promoting activity in the rat bladder. In vitro, its effect on the human bladder is comparable to that on the rat bladder, and it produces marked hyperplasia of the urothelium. It is clear from the evidence available that the role of saccharin in carcinogenesis of the urinary bladder in vivo is complex. Given before initiation it may possibly act as a co-carcinogen and influence the response of the urothelium to initiating carcinogens. Post-initiation it can cause clonal expansion of preneoplastic cells, thus providing a large population of susceptible cells, which must still undergo some further genetic modification before they can express their full malignant potential. It is possible that saccharin, if present in sufficiently high concentration for a long enough time, may also catalyse this final malignant conversion. Nevertheless, saccharin is not a powerful co-carcinogen or promoting agent by comparison with classical skin promoters such as TPA, and it has been shown to affect tumour prevalence only if present in high concentrations over a prolonged period of time.

Mid-gestation rat conceptuses were cultured for 48 h in serum containing the retinoids all-trans-retinoic acid (TRA), 13-cis-retinoic acid (13-CRA), etretinate (ETR), etretin or one of six retinamides at concentrations ranging from 0.5 to 400 micrograms/ml. TRA was toxic at a concentration of 0.5 microgram/ml. 13-CRA and etretin caused abnormal development at 1.0 microgram/ml. However, the six retinamides were less toxic and adverse developmental effects were only evident at concentrations of 50 or 100 micrograms/ml. ETR was without effect at 100 micrograms/ml, the highest dose level of this compound tested. In vivo, TRA, 13-CRA and ETR are highly teratogenic. In this culture system, TRA and 13-CRA caused abnormal development at very low concentrations but in contrast, ETR was non-toxic at 100 micrograms/ml. Therefore these findings indicate that in vivo, maternal pharmacokinetics, and bioactivation in particular, play a major role in inducing abnormal development. Cis/trans isomerization was not a major determinant of toxicity. However, there appeared to be a relationship between abnormal development and the actual or estimated pKa values of the 10 retinoids tested.

The ability of organ cultures of normal human and rat bladder to metabolize the polycyclic hydrocarbon, benzo(a)pyrene (BP), and the arylamine, 2-acetylaminofluorene, has been studied. Cultures were maintained for 0 to 6 days in a chemically defined medium before incubation with [3H]BP (0.3 to 0.5 microM) or 2-[14C]acetylaminofluorene (18 to 25 microM) for 24 hr. Ethyl acetate-soluble and water-soluble metabolites were produced from both compounds by both species. The ethyl acetate extracts from [3H]BP-treated human cultures contained 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, and 3-hydroxybenzo(a)pyrene. Rat bladder cultures produced similar metabolites but in slightly different proportions. Ethyl acetate-soluble products of 2-[14C]acetylaminofluorene from human cultures contained 7-hydroxy-2-acetylaminofluorene, 9-hydroxy-2-acetylaminofluorene, 2-aminofluorene, and N-hydroxy-2-aminofluorene. Rat bladder cultures produced similar metabolites, but 2-aminofluorene was found in relatively higher proportion. Hydrolysis by beta-glucuronidase of the water-soluble products produced from both carcinogens gave ethyl acetate-extractable derivatives. These hydrolyzable glucuronide conjugates were relatively more abundant following metabolism of the carcinogens by the rat than by the human cultures. Covalent binding to DNA occurred with [3H]BP in both human (19.7 +/- 13 pmol/mg DNA) and rat cultures (22.8 +/- 8.6 pmol/mg DNA). As with other human tissues, considerable variation (50-fold) was observed between individuals. The results demonstrate that both human and rat bladder epithelium can metabolize known potent carcinogens and, in the case of BP, can effect covalent binding between the products of metabolism and the urothelial cell DNA. In theory, carcinogenesis in the urinary bladder could thus be initiated by carcinogens produced or excreted in the urine without the necessity for their prior metabolism elsewhere in the body.

The carcinogenic activity of the alkylating agent methyl methanesulphonate (MMS) was investigated in the F344 rat bladder, both untreated and pretreated with a single threshold dose of N-methyl-N-nitrosourea (MNU). On its own, 6 doses of 2.5 mg MMS produced a 7% incidence of bladder cancer. After a single intravesical instillation of MNU, the same MMS treatment produced a bladder cancer incidence of 56%. This was significantly higher than the incidence (24%) observed after treatment with MNU alone, and greater than the sum of the lesions produced by either treatment alone. By reference to the mouse skin multistage carcinogenesis model, it is argued that MMS is a complete, albeit weak carcinogen with little initiating but powerful late-stage activity. Its promoting activity is most probably attributable to its potent mitogenic action and in this model it is analogous to a stage 2, rather than a stage 1 skin promoter.

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.

Normal mammalian urothelium has a complex pattern of morphogenesis and cell differentiation. The luminal face of the polyploid, superficial cells is limited by a morphologically unique plasma membrane with a highly ordered substructure. This membrane is a convenient marker for normal differentiation in the urothelium. In reversible, benign hyperplasia, following cytotoxic damage, the luminal face of the most superficial layer of cells is limited by an undifferentiated membrane which is comparable to the plasma membrane of normal basal cells. When the hyperplasia regresses, normal differentiation of the urothelium is reestablished, and the normal, specialized surface membrane is again produced. In both preneoplastic hyperplasia and early neoplasia induced by various carcinogenic regimens, a novel form of differentiation affecting the surface membrane is seen in some superficial cells. Microvilli form on their free luminal face and are limited by a membrane with an unusual, structured glycocalyx, which is not found in normal urothelium or benign hyperplasia. This glycocalyx also develops on some cells in transitional cell carcinoma of the bladder in man. This novel glycocalyx may be a morphological marker for neoplastic transformation in the urothelium.

The finding of cells with pleomorphic microvilli in urinary sediments has been proposed as an indicator for urothelial neoplasia. Recently, in addition to such cells, others with less bizarre, non-pleomorphic microvilli have also been found in urothelial cancers, and these cells are similar in appearance to others detected in the urinary sediments of healthy people. When using scanning electron microscopy as a diagnostic tool, these cells are a possible source of confusion. The entire lower urinary tracts from people free of urothelial neoplasia have therefore been examined to delineate the normal surface appearance of all cell types which could appear in the urine. There are 4 predominant cell types: the large, flat squamous cells of the urethral meatus which have abundant microridges; cells with mucus-coated, short, stubby microvilli lining the urethra and renal papilla; immature urothelial cells with chains and ridges of bleb-like processes in the ureters and bladder; and, also in the ureters and bladder, mature urothelial cells with microridges or ruffles. The lining epithelia of the normal urethra and renal papilla may thus contribute cells with non-pleomorphic stubby microvilli to urine sediments, which cannot be differentiated by scanning electron microscopy alone from similar cells derived from urothelial neoplasms. However, the normal complement of cells lining the adult lower urinary tract does not include any with prolific, long, pleomorphic microvilli such as characterize transitional-cell carcinomas of the urothelium.

Effects of X-irradiating the urinary bladder of female F344 rats with a single dose of 20 Gy were studied by light and electron microscopy. The animals were killed 1 week-20 months post-irradiation, and all tissues of the bladder wall were found to be affected by the irradiation. In the urothelium, damage was initially restricted to the basal cells but slowly extended to intermediate cells, and by 6 months post-irradiation the urothelium was focally hyperplastic. Twenty months post-irradiation, transitional-cell carcinomas were found in 10 of the surviving 17 animals (59%). The blood vessels in the bladder wall showed damage to both the endothelial cells and the smooth muscle. The fibroblasts in the connective tissue of the bladder wall appeared to show increased secretion after irradiation, and there was abundant collagen deposition, resulting in severe fibrosis of the bladder wall. After a latent period of a few months, focal degeneration and extensive necrosis of the smooth muscle cells were seen, leading to severe destruction and disorganization of the muscular coats of the bladder wall. Thus, a single dose of irradiation of 20 Gy was sufficient to produce severe fibrosis of the bladder wall with smooth muscle degeneration and to induce carcinoma of the urothelium in most of the treated animals within 20 months.

Male and female Wistar rats were administered sodium saccharin for life (2 yr) either in the drinking water or diet. The maximum palatable dose of saccharin in the drinking water was found to be 2 g/kg/day and, even then, there was some voluntary restriction of fluid intake in the males. By contrast, double this dose--namely 4 g/kg/day, was palatable in the diet. A control group of rats of both sexes received saccharin-free diet and drinking water. Mild urothelial hyperplasias developed from 85 weeks in rats of both sexes receiving saccharin either in the drinking water or diet; the incidence was statistically significant in both the bladders and kidneys of rats receiving the higher dose of saccharin in the diet, but in the kidneys only of rats receiving the lower dose of saccharin in the drinking water. Telangiectasia of the vasa recta was significant in saccharin-treated rats of both sexes at both doses. A very low incidence of bladder tumours, exclusively in males receiving the higher saccharin dose in the diet was seen from 95 weeks. No consistent relationship between bladder epithelial hyperplasias and crystalluria could be demonstrated, although all 3 bladder tumours were associated with some form of mineralisation. Results suggest a particular susceptibility of males to saccharin treatment. The possibility that saccharin may promote, or enhance, the development of latent tumour cells already present in the experimental population, rather than initiate carcinogenesis per se is considered.

Multistage models of carcinogenesis proposed to account for the observed patterns of tumor development in the skin, liver, lung, bladder and other organs involve initiation of neoplastic change in a few cells by a threshold dose of carcinogen followed by conversion of these latent tumor cells into an autonomous cancer by further doses of the same and/or other carcinogens, and/or noncarcinogenic promoting agents. In the rat urinary bladder, neoplastic change can be initiated by a few weeks treatment with low doses of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) or N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or by a single, low dose, intravesicular instillation of N-methyl-N-nitrosourea (MNU). Very few animals treated thus will develop bladder cancer unless exposed subsequently to some further regime which will promote tumor growth from the initiated cells. Many different factors will stimulate tumor growth in the initiated rat bladder, including further low doses of complete bladder carcinogens, dietary factors such as metabolites of tryptophan or deficiency of vitamin A, the food additives saccharin and cyclamate and some alkylating agents such as cyclophosphamide and methylmethane sulfonate. New and published evidence is reviewed which supports the belief that these and other factors are promoters or later stage carcinogens in the bladder. The difficulties of defining a promoter and of identifying markers of promotion, i.e., of distinguishing the second from the later stages of carcinogenesis in the urinary bladder, are discussed with reference to the action of promoters in the mouse skin initiation/promotion model. However, in terms of their effect on an initiated population on the risk of developing cancer, it is suggested that such a distinction is largely irrelevant. Since both second and later stage carcinogens accelerate tumor development in an initiated urothelium, they both have the potential to lower the age at which bladder cancer becomes symptomatic. They are thus as important as are initiating carcinogens in determining the patterns of age-related neoplastic disease in any population.

It is now well established that the interaction of multiple environmental factors may increase the incidence of some human cancers more than exposure to a single carcinogen. With an in vivo experimental rat model, we have demonstrated a synergistic effect in bladder carcinogenesis between a subcarcinogenic dose of the strong bladder carcinogen, N-methyl-N-nitrosourea, and saccharin- or cyclamate-containing diets. If these artificial sweeteners are capable of interacting with other environmental bladder carcinogens, their potential for increasing the incidence of human bladder cancer is greater than many more potent chemical carcinogens, because of their wide distribution as food additives to all sections of the population. Retrospective epidemiology shows no evidence of such risk from saccharin at current levels of consumption. No comparable studies are available for cyclamate, which was consumed in greater quantities but for relatively few years. It is emphasized that it is possible for interaction between multiple factors to contribute to the incidence of human bladder cancer as it does in other human organs and in other animal species.

The early and late morphological changes induced in rat bladder urothelium by intravesicular administration of the alkylating agents methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) are described. In the short-term, both compounds produced dose-related toxic damage followed by a regenerative hyperplasia of the urothelium. At any given dose-level, the effects of MMS were more severe than those of EMS. Two years after administration of multiple doses of 2.5 mg MMS or 7.5 mg EMS the majority of animals had dose-related simple urothelial hyperplasias with occasional mild dysplasia. However, in three MMS-treated animals the hyperplasias had progressed to well-differentiated transitional-cell carcinomas. No bladder neoplasms were seen in EMS-treated animals. The urothelial response of the rat to MMS and EMS is discussed with reference to the known chemical reactivity of these compounds. It is concluded that EMS is a mitogen for the urothelium and that the few carcinomas which develop following topical exposure of the bladder to MMS do not necessarily reflect any initiating potential in this compound. Rather it is argued that the results are consistent with MMS acting as a promoter in cells which have either been previously initiated or which carry a latent oncogene.

The importance of the contaminant OTS in the promoting activity of commercial saccharin on rat bladder neoplasia was investigated. OTS, OTS-free and OTS-contaminated saccharin were administered in the drinking water or diet for 2 years to groups of rats pretreated with an intravesical instillation of MNU; OTS alone and OTS-free saccharin were also given to groups of rats not pretreated with MNU. Administration of OTS was not associated with changes in urinary pH, crystalluria or calculus formation, had no effect on the histology of normal rat bladder, and did not increase the incidence of bladder hyperplasia or neoplasia elicited by pretreatment with MNU. No differences could be found between the effect of OTS-free or OTS-contaminated saccharin on bladders of rats pretreated with MNU. These results indicate that OTS contamination played no part in the reported promoting activity of saccharin on the rat bladder. Administration of saccharin did not increase urinary pH, crystalluria or calculus formation, and failed to promote bladder neoplasia after a carcinogenic dose of MNU, though the numbers of proliferative lesions in the bladder were increased.

The superficial squamous cells of rat transitional epithelium are limited, on their luminal face, by an asymmetrically thickened membrane. Patches of similar thick membrane are found in the walls of the Golgi cisternae and it is suggested that the Golgi system is the site of assembly of the thick plasma membrane. This implies membrane flow from the Golgi apparatus to the cell surface, and there is indirect evidence that the membrane is transported in the form of fusiform vacuoles, derived from the Golgi cisternae, which fuse with, and become part of, the free cell membrane. Uptake of injected Imferon shows that similar, large, thick-walled vacuoles may be formed by invagination of the free cell surface. Some of these vacuoles are subsequently transformed into multivesicular bodies and autophagic vacuoles. The formation of other large heterogeneous bodies is described, and some of these are shown to have acid phosphatase activity.

Discrete localization of stain in pericanalicular granules was found in 10 micro frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 micro thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 micro slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 micro frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.

A study has been made of the preservation of fine structure, phospholipids, and the activity of acid phosphatase and esterase in rat liver fixed in various solutions containing 4 per cent formaldehyde. Examination of methacrylate-embedded preparations shows that calcium-containing fixatives result in poor preservation of fine structure, whereas veronal-treated or phosphate-buffered formalin gives excellent results if the tonicity of the solutions is suitably adjusted by addition of sucrose. Formol-phosphate, to which Versene has been added, causes deterioration of cellular morphology. Phospholipids are retained almost quantitatively in tissue fixed in formol-calcium, and in phosphate-, collidine-, or triethanolamine-buffered formalin. About 50 per cent of the activity of acid phosphatase and esterase are preserved after 24 hours exposure to these fixatives at 0-2 degrees C, and the distributions of the enzymes and of phospholipids, as judged by cytochemical staining results, are not altered by any of these formalin solutions. Consideration of the morphological and biochemical integrity of the fixed tissue suggests that 4 per cent formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, and containing 7.5 per cent sucrose, is the most suitable of the fixatives for combined cytochemical staining and electron microscopical studies.