Ectonucleotidase Activity in Smooth Muscle Tissues of Rats with a Valproate Model of Autism.

Ectonucleotidases play an important role in regulating the level of extracellular nucleotides and nucleosides and are an important part of the regulation of the effects of adenosine and ATP on adenosine and P2 receptors, respectively. We have previously established the ambiguous effect of P2 receptor agonists on the contractile activity of smooth muscle tissue in rats with the valproate model of autism. In this work, HPLC was used to evaluate the activity of ectonucleotidases in the smooth muscle tissues of the internal organs of rats with a valproate model of autism. The activity of ectonucleotidases was significantly higher in the smooth muscle tissues of the duodenum, vas deferens, and bladder, but lower in the ileum and uterus. The results obtained make it possible to compare the activity of ectonucleotidases identified here with changes in P2 receptor-mediated contractility of smooth muscle tissues revealed in our previous experiments.

Lower urinary dysfunction as a long-term effect of childhood vincristine treatment, with potential influences by sex and dose.

Vincristine (VCR) is one of the most widely used chemotherapy agents in treating pediatric cancer. Nonetheless, it is known to cause dose-dependent neurotoxicity which can impact virtually every organ system. Despite its widespread use, the precise impact of VCR on the lower urinary tract (LUT) remains inadequately elucidated. Our initial clinical and translational investigations suggest a sex-specific influence of childhood VCR exposure on LUT function. Thus, the current study aimed to investigate the late effects of systemic VCR exposure on LUT physiology and the underlying mechanisms, focusing on dosage and male-sex, employing juvenile CD-1 mice as a model. Male mice subjected to VCR exhibited augmented functional bladder capacity accompanied by frequent non-void contractions during awake cystometry, alongside mast cell accumulation within the bladder, compared to the saline-treated control group. Noteworthy functional changes were observed in bladder strips from the VCR group, including decreased nerve-mediated contraction, heightened contractile responses to cholinergic and purinergic agonists, enhanced responsiveness to histamine-primarily via histamine receptor 1 (Hrh1)-and an augmented relaxation effect with compound 48/80 (a mast cell degranulator), relative to the control group. Significant changes in gene expression levels associated with neuroinflammation and nociception were observed in both the bladder and lumbosacral dorsal root ganglia (Ls-DRG) of the VCR group. These findings suggest that VCR exposure during childhood, particularly in males, triggers neuroimmune responses in the bladder and Ls-DRG, amplifying responsiveness to neurotransmitters in the bladder, thereby contributing to LUT dysfunction characterized by a mixed bladder phenotype as a late effect during survivorship.

Immunomodulatory imide drugs inhibit human detrusor smooth muscle contraction and growth of human detrusor smooth muscle cells, and exhibit vaso-regulatory functions.

BACKGROUND: The immunomodulatory imide drugs (IMiDs) thalidomide, lenalidomide and pomalidomide may exhibit therapeutic efficacy in the prostate. In lower urinary tract symptoms (LUTS), voiding and storage disorders may arise from benign prostate hyperplasia, or overactive bladder. While current therapeutic options target smooth muscle contraction or cell proliferation, side effects are mostly cardiovascular. Therefore, we investigated effects of IMiDs on human detrusor and porcine artery smooth muscle contraction, and growth-related functions in detrusor smooth muscle cells (HBdSMC). METHODS: Cell viability was assessed by CCK8, and apoptosis and cell death by flow cytometry in cultured HBdSMC. Contractions of human detrusor tissues and porcine interlobar and coronary arteries were induced by contractile agonists, or electric field stimulation (EFS) in the presence or absence of an IMID using an organ bath. Proliferation was assessed by EdU assay and colony formation, cytoskeletal organization by phalloidin staining, RESULTS: Depending on tissue type, IMiDs inhibited cholinergic contractions with varying degree, up to 50 %, while non-cholinergic contractions were inhibited up to 80 % and 60 % for U46619 and endothelin-1, respectively, and EFS-induced contractions up to 75 %. IMiDs reduced viable HBdSM cells in a time-dependent manner. Correspondingly, proliferation was reduced, without showing pro-apoptotic effects. In parallel, IMiDs induced cytoskeletal disorganization. CONCLUSIONS: IMiDs exhibit regulatory functions in various smooth muscle-rich tissues, and of cell proliferation in the lower urinary tract. This points to a novel drug class effect for IMiDs, in which the molecular mechanisms of action of IMiDs merit further consideration for the application in LUTS.

Divergent histopathological and molecular patterns in chemically induced interstitial cystitis/bladder pain syndrome rat models.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a complex chronic pain disorder with an elusive etiology and nonspecific symptoms. Although numerous animal models with phenotypes similar to human disease have been established, no available regimen can consistently alleviate clinical symptoms. This dilemma led us to question whether current animal models adequately represent IC/BPS. We compared four commonly used IC/BPS rat models to determine their diverse histopathological and molecular patterns. Female rats were given single treatments with hydrochloric acid (HCL), acetic acid (AA), protamine sulfate plus lipopolysaccharide (PS + LPS), or cyclophosphamide (CYP) to induce IC/BPS. Bladder sections were stained for histopathologic evaluation, and mRNA expression profiles were examined using next-generation sequencing and gene set analyses. Mast cell counts were significantly higher in the HCL and AA groups than in the PS + LPS, CYP, and control groups, but only the AA group showed significant collagen accumulation. The models differed substantially in terms of their gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our observations suggest that none of these rat models fully reflects the complexity of IC/BPS. We recommend that future studies apply and compare multiple models simultaneously to fully replicate the complicated features of IC/BPS.

Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

Arsenic activated GLUT1-mTORC1/HIF-1α-PKM2 positive feedback networks promote proliferation and migration of bladder epithelial cells.

Arsenic (As) is recognized as a potent environmental contaminant associated with bladder carcinogenesis. However, its molecular mechanism remains unclear. Metabolic reprogramming is one of the hallmarks of cancer and is as a central feature of malignancy. Here, we performed the study of cross-talk between the mammalian target of rapamycin complex 1 (mTORC1)/ Hypoxia-inducible factor 1 alpha (HIF-1α) pathway and aerobic glycolysis in promoting the proliferation and migration of bladder epithelial cells treated by arsenic in vivo and in vitro. We demonstrated that arsenite promoted N-methyl-N-nitrosourea (MNU)-induced tumor formation in the bladder of rats and the malignant behavior of human ureteral epithelial (SV-HUC-1) cell. We found that arsenite positively regulated the mTORC1/HIF-1α pathway through glucose transporter protein 1 (GLUT1), which involved in the malignant progression of bladder epithelial cells relying on glycolysis. In addition, pyruvate kinase M2 (PKM2) increased by arsenite reduced the protein expressions of succinate dehydrogenase (SDH) and fumarate hydratase (FH), leading to the accumulation of tumor metabolites of succinate and fumarate. Moreover, heat shock protein (HSP)90, functioning as a chaperone protein, stabilized PKM2 and thereby regulated the proliferation and aerobic glycolysis in arsenite treated SV-HUC-1 cells. Taken together, these results provide new insights into mTORC1/HIF-1α and PKM2 networks as critical molecular targets that contribute to the arsenic-induced malignant progression of bladder epithelial cells.

Strategies for Safe Transurethral Injections of Botulinum Toxin into the Bladder Wall.

Introduction: Transurethral injections into the bladder wall with botulinum toxin are an established treatment for refractory overactive bladder or detrusor overactivity. With the current injection technique, an average of approx. 18% and up to 40% of botulinum toxin is injected next to the bladder wall, potentially causing reduced efficacy or non-response. The article aims to evaluate the reasons for incorrect injections and propose strategies for complete delivery of the entire botulinum toxin fluid into the bladder wall. Material and Methods: Unstructured literature search and narrative review of the literature. Results: Incorrect injection of botulinum toxin fluid next to the bladder wall is caused by pushing the injection needle too deep and through the bladder wall. Bladder wall thickness decreases with increasing bladder filling and has a thickness of less than 2 mm beyond 100 mL in healthy individuals. Ultrasound imaging of the bladder wall before botulinum toxin injection can verify bladder wall thickness in individual patients. Patient movements during the injection therapy increase the chance of incorrect placement of the needle tip. Conclusions: Based on the literature search, it is helpful and recommended to (1) perform pretreatment ultrasound imaging of the bladder to estimate bladder wall thickness and to adjust the injection depth accordingly, (2) fill the bladder as low as possible, ideally below 100 mL, (3) use short needles, ideally 2 mm, and (4) provide sufficient anesthesia and pain management to avoid patient movements during the injection therapy.

Adrenergic relaxations in an in situ urinary bladder model evoked by stimulation of sensory pelvic and pudendal nerves in the rat.

Urinary bladder dysfunction might be related to disturbances at different levels of the micturition reflex arc. The current study aimed to further develop and evaluate a split bladder model for detecting and analysing relaxatory signalling in the rat urinary bladder. The model allows for discrimination between effects at the efferent and the afferent side of the innervation. In in vivo experiments, the stimulation at a low frequency (1 Hz) of the ipsilateral pelvic nerve tended to evoke relaxation of the split bladder half (contralateral side; -1.0 ± 0.4 mN; n = 5), in contrast to high frequency-evoked contractions. In preparations in which the contralateral pelvic nerve was cut the relaxation occurred at a wider range of frequencies (0.5-2 Hz). In separate experiments, responses to 1 and 2 Hz were studied before and after intravenous injections of propranolol (1 mg/kg IV). The presence of propranolol significantly shifted the relaxations into contractions. Also, electrical stimulation of the ipsilateral pudendal nerve evoked relaxations of similar magnitude as for the pelvic stimulations, which were also affected by propranolol. In control in vitro experiments, substances with ß-adrenoceptor agonism, in contrast to a selective α-agonist, evoked relaxations. The current study shows that the split bladder model can be used for in vivo studies of relaxations. In the model, reflex-evoked sympathetic responses caused relaxations at low intensity stimulation. The involvement of ß-adrenoceptors is supported by the sensitivity to propranolol and by the in vitro observations.

Pirfenidone improves voiding function by suppressing bladder fibrosis in underactive bladder rats.

Underactive bladder (UAB), characterized by a complex set of symptoms with few treatment options, can significantly reduce the quality of life of affected people. UAB is characterized by hyperplasia and fibrosis of the bladder wall as well as decreased bladder compliance. Pirfenidone is a powerful anti-fibrotic agent that inhibits the progression of fibrosis in people with idiopathic pulmonary fibrosis. In the current study, we evaluated the efficacy of pirfenidone in the treatment of bladder fibrosis in a UAB rat model. UAB was induced by crushing damage to nerve bundles in the major pelvic ganglion. Forty-two days after surgery, 1 mL distilled water containing pirfenidone (100, 300, or 500 mg/kg) was orally administered once every 2 days for a total of 10 times for 20 days to the rats in the pirfenidone-treated groups. Crushing damage to the nerve bundles caused voiding dysfunction, resulting in increased bladder weight and the level of fibrous related factors in the bladder, leading to UAB symptoms. Pirfenidone treatment improved urinary function, increased bladder weight and suppressed the expression of fibrosis factors. The results of this experiment suggest that pirfenidone can be used to ameliorate difficult-to-treat urological conditions such as bladder fibrosis. Therefore, pirfenidone treatment can be considered an option to improve voiding function in patient with incurable UAB.

Specialized pro-resolution mediators in the bladder: effects of resolvin E1 on diabetic bladder dysfunction in the type 1 diabetic male Akita mouse model.

BACKGROUND: One of the most common, but least studied, diabetic complication is diabetic bladder dysfunction. Current therapies include glucose control and symptom-based interventions. However, efficacy of these therapies is mixed and often have undesirable side effects. Diabetes is now known to be a chronic inflammatory disease. Specialized pro-resolving mediators are a class of compounds that promote the resolution of inflammation and have been shown to be effective in treating chronic inflammatory conditions. In this study we examine the ability of resolvin E1 to improve signs of diabetic bladder dysfunction. METHODS: Male Akita mice (Type 1 diabetic) develop hyperglycemia at 4 weeks and signs of bladder underactivity by 15 weeks. Starting at 15 weeks, mice were given one or two weeks of daily resolvin E1 and compared to age-matched wild type and untreated Akita mice. RESULTS: Resolvin E1 did not affect diabetic blood glucose after one week, although there was a slight decrease after two weeks. Diabetes decreased body weight and increased bladder weights and this was not affected by resolvin E1. Evan's blue dye extravasation (an indirect index of inflammation) was dramatically suppressed after one week of resolvin E1 treatment, but, surprisingly, had returned to diabetic levels after two weeks of treatment. Using cystometry, untreated Akita mice showed signs of underactivity (increased void volumes and intercontraction intervals). One week of resolvin E1treatment restored these cystometric findings back to control levels. After two weeks of treatment, cystometric changes were changed from controls but still significantly different from untreated levels, indicating a durable treatment effect even in the presence of increased inflammation at 2 weeks. CONCLUSIONS: Resolvin E1 has a beneficial effect on diabetic bladder dysfunction in the type 1 diabetic male Akita mouse model.

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Time-dependent progress of lower urinary tract dysfunction in streptozotocin-induced diabetic rats with or without low-dose insulin treatment.

Objectives: To examine time-dependent functional and structural changes of the lower urinary tract in streptozotocin-induced diabetic rats with or without low-dose insulin treatment and explore the pathophysiological characteristics of insulin therapy on lower urinary tract dysfunction (LUTD) caused by diabetes mellitus (DM). Methods: Female Sprague-Dawley rats were divided into five groups: normal control (NC) group, 4 weeks insulin-treated DM (4-DI) group, 4 weeks DM (4-DM) group, 8 weeks insulin-treated DM (8-DI) group and 8 weeks DM (8-DM) group. DM was initially induced by i.p. injection of streptozotocin (65 mg/kg), and then the DI groups received subcutaneous implantation of insulin pellets under the mid dorsal skin. Voiding behavior was evaluated in metabolic cages. The function of bladder and urethra in vivo were evaluated by simultaneous recordings of the cystometrogram and urethral perfusion pressure (UPP) under urethane anesthesia. The function of bladder and urethra in vitro were tested by organ bath techniques. The morphologic changes of the bladder and urethra were investigated using Hematoxylin-Eosin and Masson's staining. Results: Both 4-and 8-weeks diabetic rats have altered micturition patterns, including increased 12-h urine volume, urinary frequency/12 hours and voided volume. In-vivo urodynamics showed the EUS bursting activity duration is longer in 4-DM group and shorter in 8-DM group compared to NC group. UPP change in 8-DM were significantly lower than NC group. While none of these changes were found between DI and NC groups. Organ bath showed the response to Carbachol and EFS in bladder smooth muscle per tissue weights was decreased significantly in 4- and 8-weeks DM groups compared with insulin-treated DM or NC groups. In contrast, the contraction of urethral muscle and maximum urethral muscle contraction per gram of the tissue to EFS stimulation were significantly increased in 4- and 8-weeks DM groups. The thickness of bladder smooth muscle was time-dependently increased, but the thickness of the urethral muscle had no difference. Conclusions: DM-induced LUTD is characterized by time-dependent functional and structural remodeling in the bladder and urethra, which shows the hypertrophy of the bladder smooth muscle, reduced urethral smooth muscle relaxation and EUS dysfunction. Low-dose insulin can protect against diuresis-induced bladder over-distention, preserve urethral relaxation and protect EUS bursting activity, which would be helpful to study the slow-onset, time-dependent progress of DM-induced LUTD.

Effects of oral administration of nonselective Trk inhibitor on bladder overactivity in rodent models of prostatic inflammation.

BACKGROUND: Our research focused on the assessment of the impact of systemic inhibition of Trk receptors, which bind to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), on bladder hypersensitivity in two distinct rodent models of prostatic inflammation (PI). METHODS: Male Sprague-Dawley rats were divided into three groups (n = 6 each): the control group (no PI, vehicle administration), the untreated group (PI, vehicle administration), and the treated group (PI, nonselective Trk inhibitor, GNF 5837, administration). PI in rats was induced by a intraprostatic injection of 5% formalin. Posttreatment, we carried out conscious cystometry and a range of histological and molecular analyses. Moreover, the study additionally evaluated the effects of a nonselective Trk inhibitor on bladder overactivity in a mouse model of PI, which was induced by prostate epithelium-specific conditional deletion of E-cadherin. RESULTS: The rat model of PI showed upregulations of NGF and BDNF in both bladder and prostate tissues in association with bladder overactivity and inflammation in the ventral lobes of the prostate. GNF 5837 treatment effectively mitigated these PI-induced changes, along with reductions in TrkA, TrkB, TrkC, and TRPV1 mRNA expressions in L6-S1 dorsal root ganglia. Also, in the mouse PI model, GNF 5837 treatment similarly improved bladder overactivity. CONCLUSIONS: The findings of our study suggest that Trk receptor inhibition, which reduced bladder hypersensitivity and inflammatory responses in the prostate, along with a decrease in overexpression of Trk and TRPV1 receptors in sensory pathways, could be an effective treatment strategy for male lower urinary tract symptoms associated with PI and bladder overactivity.

Protective effect of equol intake on bladder dysfunction in a rat model of bladder outlet obstruction.

OBJECTIVES: This study evaluates the impact of equol, a metabolite of soy isoflavone, on bladder dysfunction in rats with bladder outlet obstruction (BOO). In addition, we investigate its potential as a neuroprotective agent for the obstructed bladder and discuss its applicability in managing overactive bladder (OAB). METHODS: Eighteen male Sprague-Dawley rats were divided into three groups (six rats per group) during the rearing period. The Sham and C-BOO groups received an equol-free diet, while the E-BOO group received equol supplementation (0.25 g/kg). At 8 weeks old, rats underwent BOO surgery, followed by continuous cystometry after 4 weeks of rearing. The urinary oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde) were measured, and the bladder histology was analyzed using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining (neurofilament heavy chain for myelinated nerves, peripherin for unmyelinated nerves, and malondialdehyde). RESULTS: Equol reduced BOO-induced smooth muscle layer fibrosis, significantly prolonged the micturition interval (C-BOO: 193 s, E-BOO: 438 s) and increased the micturition volume (C-BOO: 0.54 mL, E-BOO: 1.02 mL) compared to the C-BOO group. Equol inhibited the increase in urinary and bladder tissue malondialdehyde levels. While the C-BOO group exhibited reduced peripherin alone positive nerve fibers within the smooth muscle layer, equol effectively attenuated this decline. CONCLUSIONS: Equol reduces lipid peroxidation and smooth muscle layer fibrosis in the bladder and exhibited neuroprotective effects on bladder nerves (peripheral nerves) and prevented the development of bladder dysfunction associated with BOO in rats. Consumption of equol is promising for the prevention of OAB associated with BOO.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

Novel pharmacotherapeutic avenues for bladder storage dysfunction in men.

INTRODUCTION: Bladder storage dysfunction is associated with low quality of life in men and remains a challenging field in pharmacotherapy because of low persistence followed by patient-perceived lack of efficacy and adverse effects. The persistent desire for the development of novel pharmacotherapy is evident, leading to numerous research efforts based on its pathophysiology. AREAS COVERED: This review describes the pathophysiology, current pharmacotherapeutic strategies, and emerging novel drugs for male bladder storage dysfunction. The section on emerging pharmacotherapy provides an overview of current research, focusing on high-potential target molecules, particularly those being evaluated in ongoing clinical trials. EXPERT OPINION: As pharmacotherapies targeting alpha-adrenergic, beta-adrenergic, and muscarinic receptors - the current primary targets for treating male bladder storage dysfunction - have demonstrated insufficient efficacy and side effects, researchers are exploring various alternative molecular targets. Numerous targets have been identified as central to regulating bladder afferent nerve activity, and their pharmacological effects and potential have been evaluated in animal-based experiments. However, there is a limited number of clinical trials for these new pharmacotherapies, and they have not demonstrated clear superiority over current treatments. Further research is needed to develop new effective pharmacotherapies for bladder storage dysfunction in men.

A pilot study exploring time- and dose-dependent DNA damage and chromosomal instability caused by benzo[a]pyrene in two urothelial cell types.

Environmental and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health effects in humans. Uncertainty exists regarding the causation of urinary bladder cancer by benzo[a]pyrene (B[a]P) due to a lack of sufficient data. In this work, we focused on in-vitro DNA damage and the formation of micronuclei and chromosomal aberrations as predictors of cancer risk, applying a wide range of dosages and time periods to quantify the onset, intensity, and duration of the response. We chose two urothelial cell types to compare susceptibility and the ability to increase the malignity of a pre-existing bladder cancer: a cancer cell line (T24) and a pooled sample of primary urinary bladder epithelia cells (PUBEC) from pigs. The highest level of DNA damage assessed by comet assay was observed following 24-h treatment in both cell types, whereas PUBEC cells were clearly more susceptible. Even 4-h treatment induced DNA damage in PUBEC cells with benchmark doses of 0.0027 µM B[a]P and 0.00023 µM after 4-h and 24-h exposure, respectively. Nearly no effect was observed for periods of 48 h. The frequency of micronucleus formation increased more markedly in T24 cells, particularly with 24-h treatment. In PUBEC cells, 48-h exposure notably induced the formation of nucleoplasmic bridges and nuclear buds. Even though only one biological replicate was studied due to the sophisticated study design, our results give a strong indication of the potential of B[a]P to induce and increase malignity in human-relevant cell types.

Predicting muscarinic receptor occupancy in human bladder mucosa from urinary concentrations of antimuscarinic agents for overactive bladder.

To assess the pharmacologically relevant and selective muscarinic receptor occupancy in the bladder mucosa, we considered not only plasma drug concentrations but also urinary drug concentrations. The purpose of this study was to predict muscarinic receptor occupancy in the human bladder mucosa based on urinary concentrations in response to clinical dosages of antimuscarinic agents used to treat overactive bladder. The calculated mean plasma or serum unbound steady state concentrations were 0.06-11 nM in clinical dosages of five antimuscarinic agents. Urinary concentrations calculated from the mean plasma or serum and renal clearance ranged between 19 nM and 2 µM, which were >10-fold higher than the Ki values for bladder muscarinic receptors excluding propiverine. Bladder mucosal muscarinic receptor occupancy estimated from the urinary concentrations and the Ki values was >90 % at a steady state in clinical dosages of five antimuscarinic agents. The bladder muscarinic receptor occupancy was higher than that in the parotid gland calculated based on the mean plasma or serum unbound concentrations and Ki values for muscarinic receptors in the parotid gland. These results suggest that sufficient and selective muscarinic receptor occupancy by antimuscarinic agents, to exert pharmacological effects, in the bladder mucosa can be predicted using urinary concentrations.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.