MyD88 Signaling Accompanied by Microbiota Changes Supports Urinary Bladder Carcinogenesis.

Urinary bladder cancer (BC) inflicts a significant impairment of life quality and poses a high mortality risk. Schistosoma haematobium infection can cause BC, and the urinary microbiota of BC patients differs from healthy controls. Importantly, intravesical instillation of the bacterium Bacillus Calmette-Guerin stands as the foremost therapy for non-muscle invasive BC. Hence, studying the receptors and signaling molecules orchestrating bacterial recognition and the cellular response in the context of BC is of paramount importance. Thus, we challenged Toll-like receptor 4 (Tlr4) and myeloid differentiation factor 88 (Myd88) knock-out (KO) mice with N-butyl-N-(4-hydroxylbutyl)-nitrosamine (BBN), a well-known urinary bladder carcinogen. Gut microbiota, gene expression, and urinary bladder pathology were followed. Acute exposure to BBN did not reveal a difference in bladder pathology despite differences in the animal's ability to recognize and react to bacteria. However, chronic treatment resulted in reduced cancer invasiveness among Myd88KO mice while the absence of functional Tlr4 did not influence BC development or progression. These differences correlate with a heightened abundance of the Faecalibaculum genus and the lowest microbial diversity observed among Myd88KO mice. The presented data underscore the important role of microbiota composition and MyD88-mediated signaling during bladder carcinogenesis.

Aging induces changes in cancer formation and microbial content in a murine model of bladder cancer.

Bladder cancer (BCa) incidence is tightly linked to aging. Older patients with BCa present with higher grade tumors and have worse outcomes on Bacillus-Calmette-Guerin (BCG) immunotherapy. Aging is also known to result in changes in the gut microbiome over mammalian lifespan, with recent studies linking alterations in the gut microbiome to changes in tumor immunity. There is limited information on the microbiome in BCa models though, despite known links to aging and immunotherapy. The purpose of this study was to evaluate how aging impacts tumor formation, inflammation, and the microbiome of mice given the model BCa carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We hypothesized old animals would have larger, more inflamed tumors and a shift in their fecal microbiome compared to their younger counterparts. Young (~8-week-old) or old (~78-week-old) C57Bl/6J animals were administered 0.05% BBN in drinking water for 16 weeks and then euthanized or allowed to progress for an additional 4 weeks. After 16 weeks of BBN, old mice had higher bladder to body weight ratio than young mice, and also muscle invasive tumors, which were not seen in their young counterparts. Old animals also had increased innate immune recruitment, but CD4+/CD8+ T cell recruitment did not appear different. BBN dramatically altered the microbiome in both sets of animals as measured by ß-diversity, including changes in multiple genera of bacteria. These data suggest old mice have a differential response to BBN-induced BCa. Given the median age of patients with BCa, understanding how the aged phenotype interacts with BCa is imperative.

Chemopreventive Efficacy of Thymoquinone in Chemically Induced Urinary Bladder Carcinogenesis in Rat.

The effects of thymoquinone (TQ) in a carcinogen-based models of urinary bladder cancer were evaluated, using 45 male rats in five groups. In negative control (n = 10), only tap water was given. In positive control (n = 10), the rats received 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in drinking water for 9 weeks. In preventive groups with 25 mg/kg (n = 10) and 50 mg/kg (n = 10), oral TQ was concurrently given with 0.05% BBN for 9 weeks and continued for one more week after cessation of BBN. Preventive-treatment group (n = 5) received 50 mg/kg TQ orally for 20 weeks. Five rats from each group were sequentially sacrificed in two phases: the induction phase at 12th week (except the last group) and the rest in postinduction phase at 20th week. The bladders were examined macroscopically for lesion formation, and the masses were submitted for histopathological evaluation. Markers for total oxidant status (TOS), inflammation (nuclear factor kappa B (NF-κB)), and angiogenesis (vascular endothelial growth factor (VEGF)) were also assessed. There was a reduced number of bladder lesions in the TQ groups versus the carcinogen group at both phases. Histopathological findings demonstrated a significant improvement in the abnormal morphological changes in the urothelium of the TQ-treated groups. Thymoquinone exerted a significant antioxidant and anti-inflammatory effect by a decrease in serum level of TOS and NF-κB at week 12 which was maintained low in phase two at week 20. The serum level of VEGF was also alleviated in the induction phase at week 12 and maintained low in postinduction period. In TQ preventive-treatment approach, a nonsignificant elevation of serum level of TOS and NF-κB and slight reduction in VEGF were observed at the end of the experiment. These data suggest that TQ may be effective in preventing bladder carcinogenesis, and the suggested mechanisms might be related to antioxidant, prooxidant, and anti-inflammatory properties of TQ.

Persistent γ-H2AX Formation and Expression of Stem Cell Markers in N-Butyl-N-(4-Hydroxybutyl)Nitrosamine-Induced Bladder Carcinogenesis in Rats.

We investigated γ-H2AX formation, a biomarker of DNA damage, and expression of stem cell markers (SCMs), including cytokeratin 14, aldehyde dehydrogenase 1A1 (ALDH1A1), and CD44, in the development of rat bladder tumors induced by short-term administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Histopathological examination showed that diffuse simple hyperplasia of the bladder urothelium induced by BBN recovered to the normal-appearing urothelium after withdrawal, whereas focal proliferative lesions were newly developed and subsequently progressed to benign papilloma and carcinoma. Immunohistochemical analysis revealed that BBN-induced γ-H2AX formation and ALDH1A1 and CD44 expression persisted at higher levels in the normal-appearing urothelium than those in the control group for long periods after withdrawal. Since persistent chronic inflammation was observed even after withdrawal, targeted gene expression analysis of inflammation-related factors revealed 101 genes, including Stat3 and Myc, that showed persistent high expression. Pathway analysis suggested that Stat3 and/or Myc activation may be associated with SCM expression. We focused on hepatocyte growth factor (Hgf), one of the genes predicted in relation to Stat3/Myc, and confirmed that HGF-positive cells increased by BBN persisted in the normal-appearing urothelium after withdrawal and colocalized with γ-H2AX and SCMs. These results suggested that the long-term persistence of γ-H2AX formation and SCM expression, which occurred during the early stages of bladder tumorigenesis, is not a transient response to exposure and might contribute to bladder tumorigenesis. Although further studies are needed, BBN-induced rat bladder tumors may originate from focal hyperplasia arising from SCM-positive cells via activation of the STAT3/MYC pathway after DNA damage involving γ-H2AX formation.

Chemopreventive Potential of Myrtenal against Nitrosamine-Initiated, Radiation-Promoted Rat Bladder Carcinogenesis.

The present study was undertaken to evaluate the chemopreventive activity of myrtenal, a natural monoterpene, against bladder carcinoma in rats induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and promoted with γ-ionizing radiation (γ-IRR) as well as to assess the involvement of inflammation, apoptosis and oxidative damage in tumor development. Histopathological examination of rat bladder revealed the presence of noninvasive papillary transitional cell carcinoma (Grade 2) in sections from BBN group indicating the credibility of the applied carcinogenesis model. Myrtenal treatment caused improvement in urinary bladder mucosa with cells more likely in Grade 1. Administration of myrtenal to BBN-treated rats exhibited downregulation in the expressions of COX-2, NF-kB and STAT-3 associated with suppression of inflammatory cytokines levels of TNF-α and IL-6 as well as biomarkers of oxidative damage (MDA & NO). In addition, myrtenal treatment caused a significant increase in caspase-3 activity and Bax/Bcl-2 ratio. Data obtained suggested that the anti-inflammatory effect and the induction of apoptosis contributed largely to the beneficial antitumor effects of myrtenal in rats with BBN/γ-IRR-induced bladder carcinoma. Present findings, in addition to benefits described in other pathologies, indicated myrtenal as a potential adjuvant natural compound for the prevention of tumor progression of bladder cancer.

Metabolomic credentialing of murine carcinogen-induced urothelial cancer.

Bladder cancer (BCa) is the most common malignancy of the urinary system with increasing incidence, mortality, and limited treatment options. Therefore, it is imperative to validate preclinical models that faithfully represent BCa cellular, molecular, and metabolic heterogeneity to develop new therapeutics. We performed metabolomic profiling of premalignant and non-muscle invasive bladder cancer (NMIBC) that ensued in the chemical carcinogenesis N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model. We identified the enriched metabolic signatures that associate with premalignant and NMIBC. We found that enrichment of lipid metabolism is the forerunner of carcinogen-induced premalignant and NMIBC lesions. Cross-species analysis revealed the prognostic value of the enzymes associated with carcinogen-induced enriched metabolic in human disease. To date, this is the first study describing the global metabolomic profiles associated with early premalignant and NMIBC and provide evidence that these metabolomic signatures can be used for prognostication of human disease.

mRNA translation is a therapeutic vulnerability necessary for bladder epithelial transformation.

Using genetically engineered mouse models, this work demonstrates that protein synthesis is essential for efficient urothelial cancer formation and growth but dispensable for bladder homeostasis. Through a candidate gene analysis for translation regulators implicated in this dependency, we discovered that phosphorylation of the translation initiation factor eIF4E at serine 209 is increased in both murine and human bladder cancer, and this phosphorylation corresponds with an increase in de novo protein synthesis. Employing an eIF4E serine 209 to alanine knock-in mutant mouse model, we show that this single posttranslational modification is critical for bladder cancer initiation and progression, despite having no impact on normal bladder tissue maintenance. Using murine and human models of advanced bladder cancer, we demonstrate that only tumors with high levels of eIF4E phosphorylation are therapeutically vulnerable to eFT508, the first clinical-grade inhibitor of MNK1 and MNK2, the upstream kinases of eIF4E. Our results show that phospho-eIF4E plays an important role in bladder cancer pathogenesis, and targeting its upstream kinases could be an effective therapeutic option for bladder cancer patients with high levels of eIF4E phosphorylation.

BBN-driven urinary bladder cancer mouse model.

Around 3% of new cancer diagnoses and 2% of all cancer deaths every year are caused by urinary bladder cancer (BC). This indicates a great need for intensive studying of BC by using different approaches including indispensable mice models. The most common preclinical mouse model of bladder carcinogenesis relies on the use of a nitrosamine compound, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) which causes high-grade, invasive tumors in the urinary bladder. BBN-induced bladder cancer in mice recapitulates the histology and manifests genetic alterations similar to human muscle-invasive bladder cancer. Here we present a detailed protocol for the induction of BC in mice which is based on the administration of 0.05%-0.1% BBN in drinking water. Six-to-eight-week-old mice are treated orally with BBN for 12weeks and tumors are expected 8weeks after the termination of BBN regimen. Histopathologic examination of the lesions should be routinely assessed after hematoxylin and eosin staining by an experienced pathologist and it can vary from urothelial dysplasia to invasive bladder cancer with glandular and squamous divergent differentiation, the incidence of which might depend on the mouse strain, gender, BBN concentration and the timeline of the protocol. Utilizing half of the urinary bladder tissue for the isolation and analysis of RNA, DNA and proteins provides a comprehensive insight into the biology of BC and reduces the number of mice per study. Finally, the successful use of the BC model can facilitate fundamental biomedical discoveries leading to novel diagnostic and therapeutic approaches with clinical benefits.

Alterations in detrusor contractility in rat model of bladder cancer.

Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with cancer are not known. Here by using tensiometry on detrusor smooth muscle (DSM) strips from normal rats and rats with bladder cancer induced by known urothelial carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), we show that bladder cancer is associated with considerable changes in DSM contractility. These changes include: (1) decrease in the amplitude and frequency of spontaneous contractions, consistent with the decline of luminal pressures during filling, and detrusor underactivity; (2) diminution of parasympathetic DSM stimulation mainly at the expense of m-cholinergic excitatory transmission, suggestive of difficulty in bladder emptying and weakening of urine stream; (3) strengthening of TRPV1-dependent afferent limb of micturition reflex and TRPV1-mediated local contractility, promoting urge incontinence; (4) attenuation of stretch-dependent, TRPV4-mediated spontaneous contractility leading to overflow incontinence. These changes are consistent with the symptomatic of bladder dysfunction in bladder cancer patients. Considering that BBN-induced urothelial lesions in rodents largely resemble human urothelial lesions at least in their morphology, our studies establish for the first time underlying reasons for bladder dysfunction in bladder cancer.

Dose dependency of γ-H2AX formation in the rat urinary bladder treated with genotoxic and nongenotoxic bladder carcinogens.

We previously reported that immunostaining for γ-H2AX, a biomarker of DNA damage, in the rat urinary bladder is useful for early detection of bladder carcinogens in 28-day toxicity studies. Here, we aimed to examine the dose dependency of γ-H2AX formation in the urinary bladder of rats. Male F344 rats (aged 6 weeks) were orally administered N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN; 0%, 0.0001%, 0.001%, 0.01%, 0.02%, or 0.05% in drinking water), a genotoxic bladder carcinogen, and melamine (0%, 0.3%, 1.0%, or 3.0% in the diet), a nongenotoxic bladder carcinogen, for 2 days or 4 weeks. Immunohistochemical analysis showed that γ-H2AX- and Ki67-positive epithelial cells in the bladder urothelium were significantly increased, with a clear dose dependency, in both BBN- and melamine-treated groups. Additionally, γ-H2AX formation was detected from the lower-dose group, without increased Ki67 expression or histopathologic findings. The ratios of γ-H2AX-positive cells at week 4 in both BBN- and melamine-treated groups were higher than those on day 2, indicating the time-dependent increase in γ-H2AX formation. Immunofluorescence double-staining revealed that γ-H2AX single-positive cells without Ki67 expression were often found in the urothelium of BBN-treated rats, whereas most γ-H2AX-positive cells were Ki67-positive in the melamine group. Our results demonstrated that γ-H2AX formation in the urinary bladder increased in a clear dose-dependent manner and that γ-H2AX immunostaining has the potential to detect bladder carcinogens after a 2-day administration. Furthermore, the association of genotoxic mechanisms in bladder carcinogenesis could be determined by analyzing the colocalization of γ-H2AX and Ki67 in the urothelium.

Luteolin suppresses bladder cancer growth via regulation of mechanistic target of rapamycin pathway.

Luteolin is a natural flavonoid with strong anti-oxidative properties that is reported to have an anti-cancer effect in several malignancies other than bladder cancer. In this study, we describe the effect of luteolin on a human bladder cancer cell line, T24, in the context of the regulation of p21, thioredoxin-1 (TRX1) and the mechanistic target of rapamycin (mTOR) pathway. Luteolin inhibited cell survival and induced G2/M cell-cycle arrest, p21 upregulation and downregulation of phospho(p)-S6, which is downstream of mTOR signaling. Luteolin also upregulated TRX1 and reduced intracellular reactive oxygen species production. In a subcutaneous xenograft mouse model using the rat bladder cancer cell line, BC31, tumor volumes were significantly decreased in mice orally administered luteolin compared to control. Immunohistochemical analysis revealed that increased p21 and decreased p-S6 expression were induced in the luteolin treatment group. Moreover, in another in vivo N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat bladder cancer model, the oral administration of luteolin led to a trend of decreased bladder tumor dimension and significantly decreased the Ki67-labeling index and p-S6 expression. Furthermore, the major findings on the metabolism of luteolin suggest that both plasma and urine luteolin-3'-O-glucuronide concentrations are strongly associated with the inhibition of cell proliferation and mTOR signaling. Moreover, a significant decrease in the squamous differentiation of bladder cancer is attributed to plasma luteolin-3'-glucuronide concentration. In conclusion, luteolin, and in particular its metabolized product, may represent another natural product-derived therapeutic agent that acts against bladder cancer by upregulating p21 and inhibiting mTOR signaling.

Combination of Erlotinib and Naproxen Employing Pulsatile or Intermittent Dosing Profoundly Inhibits Urinary Bladder Cancers.

Daily dosing of either NSAIDs or EGFR inhibitors has been shown to prevent bladder cancer development in a N-butyl-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced rat model. However, these inhibitors cause gastrointestinal ulceration and acneiform rash, respectively, limiting their continuous use in a clinical prevention setting. We studied chemopreventive efficacy of pulsatile dosing of EGFR inhibitor erlotinib (42 mg/kg BW, once/week) combined with intermittent or continuous low doses of the NSAID naproxen (30 mg/kg BW/day, 3 weeks on/off or 128 ppm daily in diet) in the OH-BBN induced rat bladder cancer model. The interventions were started either at 1 or 4 weeks (early intervention) or 3 months (delayed intervention) after the last OH-BBN treatment, by which time the rats had developed microscopic bladder lesions. All combination regimens tested as early versus late intervention led to the reduction of the average bladder tumor weights (54%-82%; P < 0.01 to P < 0.0001), a decrease in tumor multiplicity (65%-85%; P < 0.01 to P < 0.0001), and a decrease in the number of rats with large palpable tumors (>200 mg; 83%-90%; P < 0.01 to P < 0.0001). Levels of signal transduction markers, Ki-67, cyclin D1, IL1ß, pSTAT3, and pERK, were significantly (P < 0.05 to P < 0.001) reduced in the treated tumors, demonstrating their potential utility as predictive markers for efficacy. These findings demonstrate that significant chemopreventive efficacy could be achieved with alternative intervention regimens designed to reduce the toxicity of agents, and that starting erlotinib and/or naproxen treatments at the time microscopic tumors were present still conferred the efficacy.

Promotion effects of acetoaceto-o-toluidide on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder carcinogenesis in rats.

Recent epidemiological studies have indicated that occupational exposure to the aromatic amine acetoaceto-o-toluidide (AAOT) was associated with a marked increase in urinary bladder cancers in Japan. However, little is known about the carcinogenicity of AAOT. To evaluate the urinary bladder carcinogenicity of AAOT, male and female F344 rats were treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks followed by dietary administration of 0, 0.167, 0.5, or 1.5% AAOT for 31 weeks. The incidences and multiplicities of bladder tumors were significantly increased in the 0.5 and 1.5% groups of male and female rats in a dose-response manner. AAOT and seven downstream metabolites were detected in the urine of the male and female rats administered AAOT with levels increasing in a dose-dependent manner. The most abundant urinary metabolite of AAOT was the human bladder carcinogen o-toluidine (OTD), which was at least one order of magnitude higher than AAOT and the other AAOT metabolites. In a second experiment, male F344 rats were administered 0, 0.167, or 1.5% AAOT for 4 weeks. Gene expression analyses revealed that the expression of JUN and its downstream target genes was increased in the urothelium of male rats treated with 1.5% AAOT. These results demonstrate that AAOT promotes BBN-induced urinary bladder carcinogenesis in rats and suggest that overexpressed of JUN and its downstream target genes may be involved the bladder carcinogenicity of AAOT. In conclusion, AAOT, like other carcinogenic aromatic amines, is likely to be a carcinogen to the urinary bladder, and OTD metabolized from AAOT is the ultimate carcinogen.

Intravesical treatment of chemotherapeutic agents sensitizes bacillus Calmette­Guerin by the modulation of the tumor immune environment.

Intravesical treatment with bacillus Calmette­Guerin (BCG) is the most common treatment for preventing progression and recurrence of non­muscle invasive bladder cancer. Our previous study using the N­butyl­N­(4­hydroxybutyl) nitrosamine (BBN)­induced orthotopic bladder cancer model demonstrated that intravesical treatment with mitomycin C (MMC) and adriamycin (ADM) suppressed pro­tumoral immunity, including the aggregation of tumor­associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. Previous evidence supports the association of resistance to intravesical treatment of BCG with TAMs and Tregs. In the present study, we investigated the antitumoral efficacy of sequential intravesical treatments with chemotherapeutic agents and BCG in a BBN­induced orthotopic bladder cancer model. Thirty­six C57BL/6J mice bearing bladder cancer were randomly divided into six treatment groups as follows: control, BCG, MMC, ADM, MMC­BCG and ADM­BCG. Intravesical treatment was performed once a week for six weeks. One week after the completion of intravesical treatment, bladder and blood were harvested. MMC­BCG and ADM­BCG were more effective antitumor activities than BCG monotherapy. Bladders were subjected to immunohistochemical analysis and revealed that intravesical BCG treatment combined with MMC/ADM promoted the local recruitment of NK cells to the bladder as effectively as BCG monotherapy and reduced TAMs and Tregs in the bladder. Interleukin (IL)­17 and granulocyte­colony stimulating factor (G­CSF) in serum were analyzed by enzyme­linked immunosorbent assay and these levels were revealed to be elevated in mice treated with sequential treatments similar to levels following monotherapy with MMC and ADM. Our findings indicated that intravesical sequential treatment could suppress the resistance to BCG through the enhancement of antitumor immunity (induction of NK cells) and inhibition of pro­tumoral immunity (reduction of TAMs and Tregs). Systemic changes in IL­17 and G­CSF may be involved in topical immunomodulation. Further studies including clinical trials may be required to establish an appropriate strategy based on the immunomodulation of the tumor microenvironment.

Sulforaphane Normalizes Intestinal Flora and Enhances Gut Barrier in Mice with BBN-Induced Bladder Cancer.

SCOPE: Gut microbiota imbalance, inflammation, and gut barrier deficiency play an important role in carcinogenesis. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, has been proven to be highly effective in inhibiting cancer. The objective of this study is to investigate the potential roles of the gut microbiota in the inhibition of BBN-induced bladder cancer by SFN. METHODS AND RESULTS: N-butyl-N-(4-hydroxybutyl)-nitrosamine is used to induce bladder cancer in male C57BL/6 mice, with or without SFN for 23 weeks. SFN ameliorates the histological changes characteristic of bladder cancer, resulting in fewer submucosal capillaries. SFN normalizes gut microbiota dysbiosis in mice with BBN-induced bladder cancer with a significant increase in Bacteroides fragilis and Clostridium cluster I. SFN also increases butyric acid levels in the mouse colon, and repairs the injury to the mucosal epithelium of the colon and cecum through the upregulation of the expression of tight junction proteins and GLP2. SFN greatly decreases the release of cytokines (IL-6) and secretory immunoglobulin A in the mice with bladder cancer. CONCLUSION: These results suggest that SFN protects against chemical-induced bladder cancer through normalizing the composition of gut microbiota and repairing the physiological destruction of the gut barrier, as well as decreasing inflammation and the immune response.

Implementation of Humane Endpoints in a Urinary Bladder Carcinogenesis Study in Rats.

BACKGROUND/AIM: This study aimed to evaluate the utility of several biological parameters for the prediction of tumor development and animal welfare in a rat model of urinary bladder cancer. MATERIALS AND METHODS: The control group (n=9) received tap water while the test group (n=12) received the carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water. A score sheet with biological variables was used to monitor animals' welfare. Body weight, food and drink consumption and rectal temperature were measured weekly. Blood and urine samples were collected. RESULTS: Animals from the control group exhibited a slightly higher body weight and body weight gain. The final urine volume was higher in BBN group (p<0.05). All animals from the BBN group exhibited macroscopic hematuria at 35th week. Four animals were anemic in the last week of the experiment. CONCLUSION: The routine control of hematuria was a useful non-invasive biomarker of disease progression that may be used as a potential earlier humane endpoint. Animals did not show clinical signs of suffering that justified their sacrifice before the end of the study.

Biodistribution of gold nanoparticles in BBN-induced muscle-invasive bladder cancer in mice.

Bladder-sparing options are being developed for muscle-invasive bladder cancer in place of radical cystectomy, including the combination of chemotherapy and radiation therapy. We reasoned that improving the radiotherapy component of chemoradiation could improve the control of locally advanced disease. Previously, we showed that gold nanoparticles (AuNPs) are potent enhancers of radiation therapy. We hypothesized that if AuNPs were to preferentially localize to bladder tumors, they may be used to enhance the radiation component of muscle-invasive bladder tumor therapy. Mice were treated with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 17, 20, and 22 weeks - long enough to induce muscle-invasive tumors. Mice were then anesthetized and injected intravenously with 1.9 nm AuNPs of which most were rapidly cleared from the blood and excreted after a 30-50 minute residence time in the bladder. We found AuNPs distributed throughout the bladder wall, but most of the AuNPs were associated with the stroma surrounding the tumor cells or extracellular keratin produced by the tumor cells. There were relatively few AuNPs in the tumor cells themselves. The AuNPs therefore localized to tumor-associated stroma and this tumor specificity might be useful for specific X-ray dose enhancement therapy of muscle-invasive bladder carcinomas.

The interaction of arsenic and N-butyl-N-(4-hydroxybutyl)nitrosamine on urothelial carcinogenesis in mice.

The bladder is an important organ for the storage of excreted water and metabolites. If metabolites with carcinogenic characteristics are present in urine, the urothelial lining of the bladder could be damaged and genetically altered. In this study, we analyzed the interaction of arsenic and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) on mouse bladder carcinogenesis. Our previous study found that arsenic affects BBN-altered urothelial enzymatic activity, protein expression, DNA oxidation and global DNA CpG methylation levels. In this study, two mouse models were used. First, after administering a co-treatment of BBN and arsenic for 20 weeks, BBN alone led to a urothelial carcinoma formation of 20%, and arsenic promoted a BBN-induced urothelial carcinoma formation of 10%. The protein expression of GSTM1, GSTO1, NQO1, and p21 did not change by arsenic along with the BBN co-treatment, but the Sp1 expression increased. In the second mouse model, BBN was a pretreatment promoter; arsenic dose-dependently deteriorated BBN-promoted dysplasia by 10% and 40% at 10 ppm and 100 ppm, respectively. Conversely, BBN pretreatment also accelerated arsenic-induced dysplasia by 30%. The urothelial carcinogenic effect reversed after ceasing BBN for a period of 20 weeks. In summary, three conclusions were drawn from this study. The first is the mutual promotion of arsenic and BBN in bladder carcinogenesis. Second, arsenic dosages without bladder carcinogenicity (10 ppm) or with slight carcinogenicity (100 ppm) promote BBN-induced mice bladder cancer progression. Finally, the dysplastic urothelium had reverted to near-normal morphology after ceasing BBN intake for 20 weeks, providing a good suggestion for people who want to quit smoking.

Effect of lupeol on antioxidants and xenobiotic enzymes in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in experimental rats.

OBJECTIVE: Urothelial carcinoma of the bladder is a common malignancy ranked 9th with an estimated 356,600 new cases diagnosed annually worldwide. The study showed the protective effects of Lupeol in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in in vivo experimental model. Forty male healthy wistar rats were selected randomly divided into four groups. Group I rats served as healthy control. Group II rats were treated with BBN (150 mg/gavage/twice a week) for 8 weeks. Group III rats were treated with BBN + Lupeol [ Lupeol (50 mg/kg bw/day) treatment was started 1 week prior to the BBN treatment, and it was orally administered for 8 weeks]. Group IV rats were treated with Lupeol alone (50 mg/kg bw/day) for 8 weeks. All the experimental rats were maintained and euthanized at 32nd week. Serum and bladder tissues were collected and examined for biochemical parameters, serum markers and histopathological evaluation. Preventive (BBN + Lupeol) group modulates the activity of antioxidant enzymes such as Superoxide dismutase, Catalase, Reduced glutathione, Glutathione Peroxidase, Thiobarbituric acid reactive substances (TBARS) and drug metabolizing enzymes such as Cytochrome P450, Cytochrome b5, NADPH Cytochrome c reductase, NADPH- Quinone Oxidoreductase 1 and Glutathione-S-transferase when compared to BBN treated rats. Serological markers such as Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) were significantly (P&#60;0.05) decreased in preventive lupeol treated groups. Lupeol supplementation protects BBN induced bladder carcinogenesis in experimental rats by its antioxidant, anti-inflammatory and antiproliferative properties.

Enhanced Susceptibility of Ogg1 Mutant Mice to Multiorgan Carcinogenesis.

The role of deficiency of oxoguanine glycosylase 1 (Ogg1) Mmh homolog, a repair enzyme of the 8-hydroxy-2'-deoxyguanosine (8-OHdG) residue in DNA, was investigated using the multiorgan carcinogenesis bioassay in mice. A total of 80 male and female six-week-old mice of C57BL/6J background carrying a mutant Mmh allele of the Mmh/Ogg1 gene (Ogg1-/-) and wild type (Ogg1+/+) mice were administered N-diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), N-bis (2-hydroxypropyl) nitrosamine (DHPN) and 1,2-dimethylhydrazine dihydrochloride (DMH) (DMBDD) to induce carcinogenesis in multiple organs, and observed up to 34 weeks. Significant increase of lung adenocarcinomas incidence was observed in DMBDD-treated Ogg1-/- male mice, but not in DMBDD-administered Ogg1+/+ animals. Furthermore, incidences of lung adenomas were significantly elevated in both Ogg1-/- males and females as compared with respective Ogg1-/- control and DMBDD-treated Ogg1+/+ groups. Incidence of total liver tumors (hepatocellular adenomas, hemangiomas and hemangiosarcomas) was significantly higher in the DMBDD-administered Ogg1-/- males and females. In addition, in DMBDD-treated male Ogg1-/- mice, incidences of colon adenomas and total colon tumors showed a trend and a significant increase, respectively, along with significant rise in incidence of simple hyperplasia of the urinary bladder, and a trend to increase for renal tubules hyperplasia in the kidney. Furthermore, incidence of squamous cell hyperplasia in the forestomach of DMBDD-treated Ogg1-/- male mice was significantly higher than that of Ogg1+/+ males. Incidence of small intestine adenomas in DMBDD Ogg1-/- groups showed a trend for increase, as compared to the wild type mice. The current results demonstrated increased susceptibility of Ogg1 mutant mice to the multiorgan carcinogenesis induced by DMBDD. The present bioassay could become a useful tool to examine the influence of various targets on mouse carcinogenesis.