Development of urinary bladder preneoplasia by injection of Schistosoma haematobium eggs and administration of chemical carcinogen in mice

Abstract

Background: Human schistosomiasis is a chronic disease caused by the blood flukes belonging to the genus Schistosoma including Schistosoma haematobium (Sh). According to International Agency for Research on Cancer (IARC), S. haematobium is categorized as group 1 biocarcinogen of human urinary bladder (UB). The present study investigated developing UB cancer mouse model by injecting Sh eggs into the bladder wall and introduction of chemical carcinogens. The association of experimental Sh eggs injection into the mouse bladder wall and co-administration of N-nitrosodimethylamine (NDMA) for bladder cancer induction has not been studied yet. Methods: A total of 72 female ICR mice were divided into 6 group with different combinations of Sh eggs, NDMA, and N-butyl n-(4-hydroxybutyl) nitrosamine (BBN): control group (Group 1)

Sh eggs (Group 2)

NDMA (Group 3)

BBN (Group 4)

Sh eggs + NDMA (Group 5)

Sh eggs + BBN (Group 6). Group 2, 5 and 6 animals underwent anaesthesia by isoflurane and after midline lower abdominal incision was made 1000 Sh eggs in 50 µL saline were injected submucosally into the anterior aspect of the bladder dome. The control group was injected with 50 µL saline. NDMA and BBN, prepared in drinking water with a concentration of 12.5 part per million (ppm) and 0.05%, respectively were provided to mice ad libitum throughout the experimental process. Two mice from each group were sacrificed after 4, 12, 20, 28 and 36 weeks post injection of Sh eggs and NDMA or BBN treatment. Histopathological and immunohistochemical (IHC) analyses of mouse bladder tissue, relative expression of tumor suppressor and epithelial mesenchymal transition (EMT) marker genes by qRT-PCR were studied. Mouse cytokines and IgG subtypes analyses were also investigated. Results: Significant reduction (20.5%) of body weight was observed in Sh eggs + BBN group (36.26 ± 7.2 g

P < 0.01) and Sh eggs + NDMA group with 11.6% weight loss (40.33 ± 8.5 g

P < 0.01), compared to the control group (45.61 ± 8.3 g). The histopathological findings of the bladder wall showed variable degrees of abnormalities ranging from mild hyperplasia to epithelial vacuolar change, squamous metaplasia and dysplasia. In particular, squamous metaplasia and dysplasia, urinary schistosomiasis related pre-neoplastic changes were observed in Sh eggs + NDMA group at week 12 but not in the Sh eggs group. Moreover, few S. haematobium eggs were observed from the mouse bladder tissue sections but there were no inflammation or granuloma surrounding the eggs. IHC revealed that Ki-67 expression intensity for urothelial epithelial cells was significantly high for the Sh eggs + BBN group at week 20 while weak or no staining for the remaining groups. With the exception for IgG2b, the mean serum levels of IgG1, IgG2a and IgG3 of the Sh eggs and Sh eggs + NDMA group showed more or less strong significant difference compared to those of control in week 4, 12 and 20. IgG1 also showed significant increase for the Sh eggs + BBN group at week 4 and 12 while IgG2b only for the Sh eggs + NDMA group at week 12. Results from qRT-PCR of Sh eggs group showed strong relative mRNA expression of p53 gene at week 4 compared to the rest group. Expression level of p53 gene in the week 20 showed a slight significant variation for the treated group but the remaining group did not show significant variation over the remaining weeks. Relative mRNA expression of E-cadherin among Sh eggs + BBN group at week 12, 20 and 28 and all treated groups was downregulated whereas vimentin level was upregulated for Sh eggs + BBN group at week 12 and 20. Conclusions: Sh eggs and nitrosamines may transform UB cells into squamous metaplasia and dysplasia in correlation with increased expression of Ki-67, p53, and vimentin with decreased E-cadherin. Such inverse expressions of E-cadherin and vimentin mainly in Sh eggs + BBN group may be an indication for EMT. The present study provides a possibility of the mouse model for bladder cancer study.