Effects of male sexual maturity of reproductive endpoints relevant to DART studies in Wistar Hannover rats.

Wistar Hannover rats have been utilized as one of major strains in regulatory toxicology studies. This study was performed to verify the appropriate age of male sexual maturity in the development and reproductive toxicity (DART) study in Wistar Hannover rats (RccHan:WIST) by comparing reproductive endpoints between 8, 10 and 12 weeks of ages. Although fertility showed a tendency toward decrease in 8-week-old males, copulation index was not different among three ages. Testis weights reached a plateau at 10 weeks of age, whereas weights of other reproductive organs developed until 12 weeks of age. Indices of spermatogenesis (sperm motility, number of sperm in the epididymis and testis and contents of morphologically abnormal sperm) showed age-related progress and did not fully develop except for 12-week-old. For histology, epididymal tubules in 8-week-old animals showed immaturity with tall epithelium. At cesarean section, dams mated with 8-week-old males showed high incidence of preimplantation loss and the number of live fetuses was less than 10. In conclusion, although reproductive performance attained maturity by age of 10 weeks, spermatogenesis was not fully established at 10-week-old, which could result in a low fertility index. Therefore, we recommend that Wistar Hannover male rats at 12-week-old or older are used to conduct DART study properly and evaluate any adverse effects on dams and embryo-fetal development.

Extramedullary hematopoiesis in pyogenic granuloma.

Extramedullary hematopoiesis (EMH) suggests the presence of hematopoietic stem cells (HSC) outside bone marrow. EMH has been reported, albeit rarely, in pyogenic granuloma (PG), a polypoid lobular capillary hemangioma. However, statistical data have hitherto been lacking on the actual incidence of EMH in PG. Therefore, we here reviewed 157 consecutive cases using routine diagnostic surgical slides and found unequivocal EMH in 17 (10.8%). This indicates that EMH is a rather common finding in PG, which could thus have strong potential to be an important resource for the study of HSC.

An intrapelvic extraintestinal gastrointestinal stromal tumor of undetermined origin: diagnosis by prostate needle biopsy.

We herein report a case of intrapelvic gastrointestinal stromal tumor (GIST) of undetermined origin in a 48-year-old male who presented with dysuria. An enlarged tumor was detected on digital rectal examination. Imaging studies showed a solid and lobular homogenous tumor of 7.0 cm in diameter. The tumor was attached to the right dorsal aspect of the prostate with compression of the seminal vesicles and rectum. It was considered that the tumor had arisen from the prostate, although the patient's serum prostate-specific antigen level was low (0.436 ng/mL). The histological diagnosis by prostate needle biopsy was a spindle cell tumor. At cystoprostatectomy, the tumor was confirmed to be separated from the prostate by a fibrous band, and showed spindle cells with a fascicular growth pattern, but without necrotic areas. Mitotic figures were noted in 12 of 50 high-power fields. The tumor cells were immunoreactive for the KIT protein (CD117), CD34, Discovered on GIST-1 (DOG-1), and vimentin. In contrast, they were negative for desmin, α-smooth muscle actin, pancytokeratin (AE1/AE3), and S100 protein. The Ki-67 labeling index was 5%. The genetic analyses targeting the c-kit gene revealed a point mutation at codon 559 (GTT→GAT). The diagnosis of GIST was confirmed on the basis of the morphological features, immunoprofile, and results of the molecular analyses. Since extraintestinal GIST can resemble a prostatic tumor clinically, KIT (CD117) and DOG-1 should be considered for inclusion in the immunohistochemical panel for spindle cell tumors obtained by prostate needle biopsy.

Comparison of the effects of four α1-adrenoceptor antagonists on ejaculatory function in rats.

OBJECTIVE: To compare the effects of four α(1)-adrenoceptor (AR) subtype-selective antagonists on ejaculatory function in rats to investigate whether the differences in their modes of action-based on their selectivities for the α(1A)-AR subtype-would be related to the prevalence of ejaculation disorder (EjD). METHODS: The effects of α(1)-AR antagonists on noradrenaline-induced contractions were studied in rat isolated seminal vesicles, vas deferens, bladder trigone, and prostate. Male rats were given α(1)-AR antagonists orally and, 1 hour after the drug administration they were cohoused in pairs for 1 hour with untreated female rats certified to be in estrus. The number of copulatory plugs (NP) present after mating was measured as a marker of EjD. Drug effects on ejaculatory function (ie, on NP) were compared with those on the prostatic urethra (ie, phenylephrine-induced increase in intraurethral pressure [IUP]). RESULTS: All α(1)-AR antagonists concentration-dependently inhibited noradrenaline-induced contraction in all 4 tissues, and there were no differences in the rank order of potencies (tamsulosin > silodosin > alfuzosin > naftopidil) among the tissues. All α(1)-AR antagonists dose-dependently decreased NP and inhibited the phenylephrine-induced increase in IUP. There was little difference in the dose ratio ID(50) value (dose required to produce 50% inhibition) for NP/ID(50) value for IUP response among the four drugs. Drug potencies associated NP and IUP correlated closely with affinities for the human α(1A)-AR. CONCLUSION: α(1)-AR antagonists cause EjD as a class effect that depends on affinity for α(1A)-AR. Differences in α(1A)-AR selectivity would be unlikely to be related to the incidence of EjD.

Bladder selectivity of the novel ß3-agonist ritobegron (KUC-7483) explored by in vitro and in vivo studies in the rat.

We performed in vitro and in vivo experiments to evaluate the pharmacological profile of ritobegron and its effects on the bladder in rats. ß(3)-AR selectivity was assessed using CHO cells expressing various subtypes of the human ß-adrenoceptor (AR). Effects on isolated organs were evaluated using the organ-bath method. Effects on intravesical pressure, heart rate, and mean blood pressure were evaluated in urethane-anesthetized rats. Ritobegron increased cAMP accumulation in a concentration-dependent manner in CHO cells expressing any one of three human ß-AR, its selectivity for ß(3)-AR being 301-fold and 32-fold higher versus ß(1)-AR and ß(2)-AR, respectively. Ritobegron decreased the resting tension of the isolated bladder in a concentration-dependent manner (EC(50), 7.7 × 10(-8) mol/L; maximal relaxation, 97.0 %), and the ß(3)-AR antagonist SR58894A produced a parallel rightward-shift of this concentration-response curve without altering the maximal response [pK(B) value, 6.43]. Ritobegron concentration-dependently increased atrial rate and decreased myometrial contractions in vitro, and its selectivity for the bladder was 2,078-fold higher versus the atria and 14-fold higher versus the uterus. In vivo, ritobegron induced a dose-dependent decrease in intravesical pressure (ED(50) 0.4 mg/kg), without affecting heart rate and only slightly lowering mean blood pressure. Thus, ritobegron displayed potent and selective ß(3)-AR agonistic activity toward transfected human ß-AR and exhibited a high selectivity for the bladder versus other organs in rats. Moreover, it decreased intravesical pressure with minimal effects on the cardiovascular system in anesthetized rats. These results suggest that ritobegron shows promise as a potential agent for the treatment of overactive bladder.

Effects of ritobegron (KUC-7483), a novel selective ß3-adrenoceptor agonist, on bladder function in cynomolgus monkey.

We evaluated the pharmacological profile of ritobegron [KUC-7483; (-)-ethyl 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)-2,5-dimethylphenyloxy]acetate monohydrochloride] and its effects on the bladder in cynomolgus monkeys by in vitro and in vivo experiments. In vitro, ritobegron decreased the resting tension of the isolated bladder in a concentration-dependent manner (EC(50) 8.2 ± 2.3 × 10(-7) M; maximal relaxation 88.7 ± 3.7%). The ß(3)-adrenoceptor (AR) antagonist 3-(2-allylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol hydrochloride (SR58894A) produced a rightward shift of this concentration-response curve without altering the maximal response (pK(B) value 6.56 ± 0.35). In isolated atria, ritobegron increased the atrial rate only at high concentrations (EC(50) 6.5 ± 1.2 × 10(-5) M). Ritobegron had no effect on tracheal contraction at concentrations from 10(-9) to 10(-4) M, and even at the highest concentration tested, 10(-3) M, the maximal relaxation it induced was only 26.7 ± 8.1%. Tests of the selectivity of ritobegron for the bladder gave values of 79.3- and 1200-fold higher versus atria and trachea, respectively. In the in vivo study ritobegron significantly decreased intravesical pressure (ED(50) 1.44 mg/kg) without affecting either mean blood pressure or heart rate. In conclusion, ritobegron displayed potent and selective ß(3)-AR agonistic activity and relaxed the monkey isolated bladder, and in vivo it decreased intravesical pressure without affecting cardiovascular parameters. These results suggest that ritobegron may be a promising potential agent for the treatment of overactive bladder.

[A case of plasmacytoid urothelial carcinoma of the bladder: rapid progression after transurethral resection].

An 85-year-old man complained of macroscopic hematuria and painful urination. Cytoscopy revealed a non-papillary tumor at the bladder neck extending to the trigone. Abdominal computed tomography revealed thickening of the bladder wall in the same area but did not reveal lymph node swelling. Urinary cytology was class IIIb. We conducted a transurethral resection of the bladder tumor (TURBT) after which a histopathological examination showed urothelial carcinoma, G3, INFγ, pT2. From 6 days after TURBT, severe fever persisted despite the administration of various antibiotics and his general condition deteriorated. He died of acute myocardial infarction at 37 days after TURBT. Histopathological examination at autopsy revealed extensive urothelial carcinoma, a plasmacytoid variant, of the bladder which had invaded into the entire body including the lungs, liver, kidneys, adrenal glands, and veins, although tumor cells were not identified in lymph nodes. We review the literature and report this rare case of urothelial carcinoma, a plasmacytoid variant, of the bladder.

Effects of ritobegron (KUC-7483), a novel ß3-adrenoceptor agonist, on both rat bladder function following partial bladder outlet obstruction and on rat salivary secretion: a comparison with the effects of tolterodine.

The objective of this study was to investigate the effects of the ß3-adrenoceptor (AR) agonist ritobegron on rat bladder function following partial bladder outlet obstruction and on rat salivary secretion. In addition, the effects of ritobegron were compared with those of the anti-muscarinic agent tolterodine. After a 6-week partial bladder outlet obstruction (BOO), drug effects on bladder functions were evaluated using cystometrography. Effects on carbachol (CCh)-induced salivary secretion were evaluated in urethane-anesthetized rats. Ritobegron significantly decreased the frequency of non-voiding contractions (NVC), while both ritobegron and tolterodine each significantly decreased the amplitude of NVC. Ritobegron had no effect on either the micturition pressure (MP) or the residual volume (RV). In contrast, tolterodine dose-dependently decreased MP and increased RV. Ritobegron had no effect on CCh-induced salivary secretion, whereas tolterodine dose-dependently decreased it. Ritobegron decreased both the frequency and amplitude of NVC, which is similar to its effect on the contractions associated with detrusor overactivity (DO) in patients with an overactive bladder (OAB), without affecting MP, RV, or CCh-induced salivary secretion. Although tolterodine reduced the amplitude of NVC, it also markedly increased RV and significantly inhibited CCh-induced salivary secretion. These results suggest that use of ritobegron, a ß3-AR agonist, is unlikely to lead to the residual urine and dry mouth symptoms that are associated with anti-muscarinic drugs, and that ritobegron may hold promise as a safe and effective agent for OAB treatment.

[Successful treatment with rituximab in a patient with primary thymic MALT lymphoma complicated with acquired von Willebrand syndrome and Sjögren syndrome].

A 53-year-old female developed epigastric discomfort and back pain in 2007. Diagnostic imaging studies demonstrated a soft tissue tumor with heterogeneous enhancement in the anterior mediastinum and multiple nodules in the right lung. She underwent expanded thymectomy with subtotal resection of the right lung. The pathological diagnosis was primary thymic mucosa-associated lymphoid tissue (MALT) lymphoma. The patient complained of ocular discomfort, oral dryness and continuous nasal bleeding in 2007. Detailed examination led to a diagnosis of Sjögren syndrome and acquired von Willebrand syndrome. Rituximab treatment for residual disease achieved not only a reduction of the lung MALT lymphoma but also clinical and hematological remission of both syndromes. This is, to our knowledge, the first reported case of primary thymic MALT lymphoma accompanied by Sjögren and acquired von Willebrand syndromes.

Gd-EOB-DTPA-enhanced MR imaging findings of hepatocellular adenoma: correlation with pathological findings.

We report a case of a 28-year-old woman with hepatocellular adenoma and correlate findings of pathology and magnetic resonance (MR) imaging with gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) enhancement. In the hepatobiliary phase, the peripheral region of the tumor that corresponded with proliferating hepatocytes with steatosis showed slight hypointensity compared with the surrounding liver parenchyma, and the central region of the tumor that corresponded with cellular areas showed isointensity.

Carcinosarcoma of the Sigmoid Colon: Report of a Case.

Our case was a 65-year-old male, with the chief complaints of diarrhea and abdominal distention. Three years earlier, the patient had undergone transcatheter arterial embolization and radiofrequency treatment based on a diagnosis of hepatocellular carcinoma due to hepatitis B by another doctor. In October 2007, the patient developed diarrhea and increased abdominal distention. In December, CT examination conducted by the previous doctor revealed a 20-cm tumor within the pelvis. The patient was diagnosed with sigmoid colon cancer based on barium enema examination using gastrografin, and was introduced to our hospital for treatment. He was diagnosed with low-differentiated carcinoma by biopsy of the colon during endoscopy and underwent sigmoidectomy based on a diagnosis of sigmoid colon cancer. The tumor had infiltrated the bladder, and a tumorectomy was conducted through partially combined resection. The tumor was a huge lesion occupying the inside of the lumen, and histopathological findings revealed that the tumor, the main part of which lay beneath the mucous membrane, had a transitional image composed of both spindle-shaped atypical cells and sarcomatoid shape. The result of immunostaining was CK7(+), CK20(-), AFP(-), and the patient was diagnosed as having carcinosarcoma of the colon. Carcinosarcoma of the colon is a malignant tumor with poor prognosis, and the mean survival period in past reports was approximately 6 months. The patient was treated with FOLFIRI+Bevacizumab therapy according to chemotherapy for colon cancer, but he was refractory to the therapy.

Effects of four different alpha(1)-adrenoceptor antagonists on alpha-adrenoceptor agonist-induced contractions in isolated mouse and hamster ureters.

PURPOSE: To compare the efficacy of the selective alpha(1A)-adrenoceptor antagonist silodosin with those of doxazosin, terazosin, and alfuzosin against alpha-adrenoceptor agonist-induced contractions in mouse and hamster ureters. METHODS: The four alpha(1)-adrenoceptor antagonists were evaluated against norepinephrine-induced phasic contractions in mouse isolated ureteral preparations and against phenylephrine-induced sustained contractions in hamster isolated ureteral preparations using a functional experimental technique. RESULTS: In mouse ureters, silodosin (a selective alpha(1A)-adrenoceptor antagonist), doxazosin (a nonselective alpha(1)-adrenoceptor antagonist), terazosin (a nonselective alpha(1)-adrenoceptor antagonist), and alfuzosin (a nonselective alpha(1)-adrenoceptor antagonist) all shifted the norepinephrine concentration-response curve to the right. The rank order of potencies (pK(B) value) was silodosin (9.47 +/- 0.16) > doxazosin (8.62 +/- 0.15) > terazosin (8.39 +/- 0.16) > alfuzosin (8.03 +/- 0.12). In hamster ureters, all four antagonists shifted the phenylephrine concentration-response curve to the right, the rank order of potencies being silodosin (10.09 +/- 0.13) > doxazosin (8.22 +/- 0.16) > terazosin (7.75 +/- 0.15) > alfuzosin (7.70 +/- 0.10). In each case, silodosin was much more potent than the other three drugs. CONCLUSION: In this study, silodosin suppressed both mouse and hamster ureteral contractions more potently than doxazosin, terazosin, or alfuzosin. Hence, this alpha(1A)-adrenoceptor antagonist warrants further study as a potentially very useful medication for stone passage in urolithiasis patients.

Salivation triggered by pilocarpine involves aquaporin-5 in normal rats but not in irradiated rats.

1. Using rats, we examined the muscarinic receptor subtype mediating pilocarpine-induced parotid salivary secretion and the contributions of ion transporter systems (effluxes of K+ and Cl(-)) and aquaporin-5 (AQP5) translocation to this response in parotid glands in irradiated-induced xerostomia. 2. Salivary secretion was significantly lower in irradiated compared with sham-irradiated (normal) rats. In xerostomia rats, 0.4 and 0.8 mg/kg pilocarpine significantly increased parotid salivary secretion, although the salivary volume was still significantly less than in normal rats after the same dose of pilocarpine. 3. Pirenzepine (1 x 10(-6) to 1 x 10(-1) mol/L), AF-DX 116 (3 x 10(-6) to 3 x 10(-2) mol/L) and N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP; 1 x 10(-8) to 1 x 10(-2) mol/L) dose-dependently displaced radioligand binding to M(1), M(2) and M(3) receptors, respectively, in parotid membranes from both normal and irradiated rats. In each group of rats, 4-DAMP had the highest binding affinity. Pretreatment with 4-DAMP or pirenzepine dose-dependently inhibited pilocarpine-induced parotid secretion in both normal and irradiated rats, with 4-DAMP being markedly more potent than pirenzepine. 4. Normal and irradiated-rat parotid cells did not differ significantly in terms of pilocarpine-induced changes in [Ca2+](i), [K+](i) and [Cl(-)](i). Pilocarpine markedly increased the amount of AQP5 in the apical plasma membrane of parotid cells isolated from normal but not irradiated rats. 5. Thus, pilocarpine induces parotid salivary secretion mainly via the M(3) receptor subtype in both irradiated and normal rats. The reduction in this pilocarpine-induced secretion seen in irradiated rats is due not to disturbances of intracellular Ca2+ mobilization or ion transporter systems, but rather to a disturbance of AQP5 translocation, which may be involved in the pathogenesis of X-ray irradiation-induced xerostomia.

Mechanical function and gene expression of alpha(1)-adrenoceptor subtypes in dog intravesical ureter.

OBJECTIVES: To characterize the contractile functions and gene expression of the alpha(1)-adrenoceptor (AR) subtypes present in the dog intravesical ureter. METHODS: In a functional study, alpha(1)-AR antagonists were evaluated against phenylephrine (alpha(1)-AR agonist)-induced contractions in dog isolated intravesical ureteral preparations. The quantitative expression of alpha(1)-AR subtype mRNA in this tissue was determined using real-time quantitative reverse transcriptase-polymerase chain reaction. RESULTS: In the isolated intravesical ureter, prazosin (nonselective alpha(1)-AR antagonist), silodosin (selective alpha(1A)-AR antagonist), naftopidil (selective alpha(1D)-AR antagonist), and BMY-7378 (selective alpha(1D)-AR antagonist) all shifted the concentration-contractile response curve for phenylephrine to the right. The rank order of potencies (pK(B) value) was silodosin (9.45 +/- 0.14), prazosin (8.16 +/- 0.08), naftopidil (7.39 +/- 0.19), and BMY-7378 (6.78 +/- 0.20). The alpha(1A)-AR antagonist silodosin was much more potent than the 2 alpha(1D)-AR antagonists. The rank order of mRNA expression levels among the alpha(1)-AR subtypes was alpha(1d) (72.68%), alpha(1a) (24.14%), and alpha(1b) (3.18%). CONCLUSIONS: In the dog intravesical ureter, alpha(1A)-AR plays a major role in contraction, despite the prevalence of alpha(1D)-AR.

Sergliflozin etabonate, a selective SGLT2 inhibitor, improves glycemic control in streptozotocin-induced diabetic rats and Zucker fatty rats.

The low-affinity sodium glucose cotransporter (SGLT2) is responsible for most of the glucose reabsorption in the kidney and has been highlighted as a novel therapeutic target for the treatment of diabetes. We discovered sergliflozin etabonate, a novel selective SGLT2 inhibitor, and found that selective inhibition of SGLT2 increased urinary glucose excretion and consequently decreased plasma glucose levels. In this report, we examined the antihyperglycemic effects of sergliflozin etabonate in normal and diabetic rats in comparison with those of a sulfonylurea (gliclazide) and an alpha-glucosidase inhibitor (voglibose). Sergliflozin etabonate increased urinary glucose excretion in a dose-dependent manner, and inhibited the increase in plasma glucose after sucrose loading independently of insulin secretion in normal rats. Sergliflozin etabonate also improved postprandial hyperglycemia in neonatal streptozotocin-induced diabetic rats; whereas gliclazide did not improve it. In rats with mild or moderate streptozotocin-induced diabetes, the degree of the antihyperglycemic effects of sergliflozin etabonate correlated with the severity of the diabetic condition. Sergliflozin etabonate did not affect the plasma glucose level of normal rats as seen with gliclazide. Chronic treatment with sergliflozin etabonate reduced the levels of glycated hemoglobin and fasting plasma glucose, and improved the glycemic response after glucose loading in Zucker fatty rats. In addition, sergliflozin etabonate did not affect the body weight or food intake. These data indicate that sergliflozin etabonate could improve glycemic control without its use resulting in insulin secretion, hypoglycemia, and body weight gain, and may provide a unique approach to the treatment of diabetes.

Collaborative work on evaluation of ovarian toxicity. 2) Two- or four-week repeated dose studies and fertility study of mifepristone in female rats.

In order to assess ovarian pathological changes and their relationship to changes in female fertility parameters, mifepristone, a progesterone receptor antagonist, was selected as the test article and was administered orally to female rats at dose levels of 0, 0.8, 4, 20 and 100 mg/kg for 2 or 4 weeks in repeated dose-toxicity studies and in a female fertility study at dose levels of 0, 0.8, 4 and 20 mg/kg from > 2 weeks before copulation to postcoital day 7. In the repeated dose toxicity studies, persistent estrus was seen in the vaginal smears, and multiple cysts in the ovaries at necropsy, increases in luteinized cysts and hypertrophy of previously formed corpora lutea were observed in the histopathological examination of ovaries in rats receiving 20 mg/kg or more for 2 or 4 weeks. In female fertility studies, persistent vaginal cornification was also observed at 20 mg/kg and the precoital interval was significantly shortened. All of the animals were completely infertile when dosed with 20 mg/kg during the post-coital period. An increase in pre-implantation losses was observed in the animals treated with 20 mg/kg during the pre-coital phase, while treatment with 4 mg/kg mifepristone during the post-coital phase induced an increase in post-implantation losses. These results suggested that a 2-week administration period would be sufficient to detect the ovarian toxicity of mifepristone in repeated dose toxicity study and the pathological findings in the ovaries would reflect the alterations in female reproductive endpoints in the female fertility study.

Gene expressions and mechanical functions of α1-adrenoceptor subtypes in mouse ureter.

PURPOSE: This study was performed to characterize the α1-adrenoceptor subtypes in mouse ureters as regards gene expressions and contractile functions. METHODS: The mRNAs for these subtypes were quantified by the real-time quantitative reverse transcription polymerase chain reaction. In a functional study, α1-adrenoceptor antagonists were evaluated against the noradrenaline-induced contraction in mouse isolated ureteral preparations. RESULTS: In mouse ureter, the relative mRNA expression levels for α1a-, α1b- and α1d-adrenoceptors were 74.5, 14.3 and 11.2%, respectively. Adrenaline and noradrenaline each produced a concentration-dependent phasic contraction (pD 2 values, 5.73±0.05 and 5.69±0.06, respectively). Prazosin (non-selective α1-adrenoceptor antagonist), silodosin (selective α1A-adrenoceptor antagonist), and BMY-7378 (selective α1D-adrenoceptor antagonist) all shifted the concentration­response curve for noradrenaline to the right, the rank order of potencies (apparent pA 2 values) being silodosin (9.32±0.11)>prazosin (8.55±0.10)>BMY-7378 (6.06±0.15). The α1A-adrenoceptor antagonist silodosin was thus much more effective than the α1D-adrenoceptor antagonist BYM-7378. CONCLUSIONS: Our results demonstrate that in mouse ureters: the mRNA for the α1A-adrenoceptor was more prevalent than those for the α1B- and α1D-adrenoceptors, and that among these subtypes, the α1A-adrenoceptor plays the major role in noradrenaline-induced contraction.

A liquid chromatography/tandem mass spectrometry method for detecting UGT-mediated bioactivation of drugs as their N-acetylcysteine adducts in human liver microsomes.

The detection of the reactive metabolites of drugs has recently been gaining increasing importance. In vitro trapping studies using trapping agents such as glutathione are usually conducted for the detection of reactive metabolites, especially those of cytochrome P450-mediated metabolism. In order to detect the UDP-glucuronosyltransferase (UGT)-mediated bioactivation of drugs, an in vitro trapping method using N-acetylcysteine (NAC) as a trapping agent followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed in this study. After the test compounds (diclofenac and ketoprofen) had been incubated in human liver microsomes with uridine diphosphoglucuronic acid (UDPGA) and NAC, the NAC adducts formed through their acyl glucuronides were analyzed using LC/MS/MS with electrospray ionization (ESI). The NAC adduct showed a mass shift of 145 units as compared to its parent, and the characteristic ion fragmentations reflected the parent. This is a concise and high-throughput method for evaluating reactive metabolites by UGT-mediated bioactivation.

Embryonic mortality and intrauterine growth retardation (IUGR) associated with placental alterations in pregnant rats treated with methyl methanesulfonate (MMS) at the peri-implantation stage.

Embryonic mortality and intrauterine growth retardation (IUGR) are induced by exposure of rodents to xenobiotic agents during the pregastrulation period of development. We examined the time course of the effects of methyl methanesulfonate (MMS), an alkylating agent, on conceptus development in order to clarify the relative roles of the embryo and the placenta in their induction. Pregnant rats were treated orally with a single dose of MMS (200 mg/kg) in the morning of gestation day (GD) 6 (peri-implantation stage). Embryonic mortality was increased on GD12 and thereafter by MMS treatment, with newly dead embryos showing placental hypoplasia at GD12. Embryo or fetal weight was also smaller for MMS-treated dams than for control dams from GD14 to GD20. The labyrinth zone and junctional zone (JZ) of the placenta were thinner in MMS-treated rats from GD12 to GD17 and from GD12 to GD20 (except for GD17), respectively. Furthermore, MMS-treated dams showed a smaller number of glycogen cells in the JZ on GD14. In contrast, the placental glycogen concentration was higher and the expression of glucose transporter 1 in the JZ remained at GD20. These results indicate that exposure of pregnant rats to MMS at the peri-implantation stage of embryogenesis affects placental development and growth. The placental impairment induced by MMS was likely responsible for the embryonic death observed 6 days after exposure of dams to this agent as well as for the IUGR of surviving embryos or fetuses throughout the gestation period.

Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model, and suppresses fibrogenic response induced by transforming growth factor-beta1 in a cultured stellate cell line.

AIM: The aim of this study was to investigate the preventive actions of bezafibrate against non-alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model. METHODS: Male KK-A(y)/TaJcl (KK-A(y)) mice were fed a methionine and choline-deficient (MCD) diet or a MCD diet containing bezafibrate or pioglitazone for 7 weeks, after which biochemical parameters, pathological changes, and hepatic mRNA levels were assessed. An in vitro HSC model was designed by using a previously described RI-T cell line stimulated by transforming growth factor-beta1 (TGF-beta1). RESULTS: MCD diet-fed KK-A(y) mice developed hepatic steatosis, oxidative stress, inflammation, and hepatic fibrosis. Bezafibrate markedly decreased the hepatic content of triglyceride accumulation of fatty droplets within hepatocytes, and increased the expression of hepatic fatty acid beta-oxidative genes in MCD diet-fed KK-A(y) mice. Bezafibrate markedly inhibited the increases in the plasma alanine aminotransferase level and hepatic content of thiobarbituric acid-reactive substances in this model. Moreover, it dramatically reduced hepatic inflammatory changes and fibrosis concomitantly with marked reductions in the mRNA levels for inflammatory cytokine, chemokine, and profibrogenic genes. Importantly, both bezafibrate and pioglitazone markedly reduced the mRNA levels of profibrogenic and fibrogenic genes in TGF-beta1-stimulated cells. CONCLUSION: Bezafibrate improved hepatic steatosis and potently prevented inflammation, oxidative stress, HSC activation, and fibrogenesis in the liver. Moreover, this study was the first to demonstrate that bezafibrate directly inhibits hepatic fibrogenic response induced by TGF-beta1 in vitro. Hence bezafibrate may be a new therapeutic strategy against NASH and hepatic fibrosis.