Role of CD44 expressed in renal tubules during maladaptive repair in renal fibrogenesis in an allopurinol-induced rat model of chronic kidney disease.

The kidney is a major target organ for the adverse effects of pharmaceuticals; renal tubular epithelial cells (TECs) are particularly vulnerable to drug-induced toxicity. TECs have regenerative capacity; however, maladaptive repair of TECs after injury leads to renal fibrosis, resulting in chronic kidney disease (CKD). We previously reported the specific expression of CD44 in failed-repair TECs of rat CKD model induced by ischemia reperfusion injury. Here, we investigated the pathophysiological role of CD44 in renal fibrogenesis in allopurinol-treated rat CKD model. Dilated or atrophic TECs expressing CD44 in fibrotic areas were collected by laser microdissection and subjected to microarray analysis. Gene ontology showed that extracellular matrix (ECM)-related genes were upregulated and differentiation-related genes were downregulated in dilated/atrophic TECs. Ingenuity Pathway Analysis identified CD44 as an upstream regulator of fibrosis-related genes, including Fn1, which encodes fibronectin. Immunohistochemistry demonstrated that dilated/atrophic TECs expressing CD44 showed decreases in differentiation markers of TECs and clear expression of mesenchymal markers during basement membrane attachment. In situ hybridization revealed an increase in Fn1 mRNA in the cytoplasm of dilated/atrophic TECs, whereas fibronectin was localized in the stroma around these TECs, supporting the production/secretion of ECM by dilated/atrophic TECs. Overall, these data indicated that dilated/atrophic TECs underwent a partial epithelial-mesenchymal transition (pEMT) and that CD44 promoted renal fibrogenesis via induction of ECM production in failed-repair TECs exhibiting pEMT. CD44 was detected in the urine and serum of APL-treated rats, which may reflect the expression of CD44 in the kidney.

Comparison of the sensitivity of histopathological and immunohistochemical analyses and blood hormone levels for early detection of antithyroid effects in rats treated with thyroid peroxidase inhibitors.

Although measurements of blood triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) levels in rodent toxicity studies are useful for detection of antithyroid substances, assays for these measurements are expensive and can show high variability depending on blood sampling conditions. To develop more efficient methods for detecting thyroid disruptors, we compared histopathological and immunohistochemical findings in the thyroid and pituitary glands with blood hormone levels. Six-week-old male and female Sprague-Dawley rats (five rats per group) were treated with multiple doses of the thyroid peroxidase inhibitors propylthiouracil (PTU) and methimazole by gavage for 28 days. Significant decreases in serum T3 and T4 and increases in TSH were observed in the ≥1 mg/kg PTU and ≥3 mg/kg methimazole groups. An increase in TSH was also detected in male rats in the 0.3 mg/kg PTU group. Histopathological and immunohistochemical analyses revealed that follicular cell hypertrophy and decreased T4 and T3 expressions in the thyroid gland were induced at doses lower than doses at which significant changes in serum hormone levels were observed, suggesting that these findings may be more sensitive than blood hormone levels. Significant increases in thyroid weights, Ki67-positive thyroid follicular cell counts, and TSH-positive areas in the pituitary gland were detected at doses comparable with those at which changes in serum T4 and TSH levels were observed, indicating that these parameters may also be useful for evaluation of antithyroid effects. Combining these parameters may be effective for detecting antithyroid substances without relying on hormone measurements.

Promoter methylation levels of microRNA-124 in non-neoplastic rectal mucosa as a potential biomarker for ulcerative colitis-associated colorectal cancer in pediatric-onset patients.

PURPOSE: To determine the methylation level of the miR-124 promoter in non-neoplastic rectal mucosa of patients with pediatric-onset ulcerative colitis (UC) to predict UC-associated colorectal cancer (UC-CRC). METHODS: Between 2005 and 2017, non-neoplastic rectal tissue specimens were collected from 86 patients with UC, including 13 patients with UC-CRC; cancer tissues were obtained from the latter group. The methylation status of the miR-124 promoter was quantified using bisulfite pyrosequencing and compared between pediatric- and adult-onset UC patients. RESULTS: Patients with pediatric-onset UC experienced a significantly shorter disease duration than those with adult-onset UC. The levels of miR-124 promoter methylation in non-neoplastic rectal mucosa were positively correlated with the age at the diagnosis and duration of UC. The rate of increase in miR-124 methylation was accelerated in patients with pediatric-onset UC compared to those with adult-onset UC. Furthermore, the miR-124 methylation levels in non-neoplastic rectal mucosa were significantly higher in patients with UC-CRC than in those with UC alone (P = 0.02). A receiver operating characteristic analysis revealed that miR-124 methylation in non-neoplastic tissue discriminated between patients with pediatric-onset UC with or without CRC. CONCLUSION: miR-124 methylation in non-neoplastic rectal mucosa may be a useful biomarker for identifying patients with pediatric-onset UC who face the highest risk of developing UC-CRC.

CD44 expression in renal tubular epithelial cells in the kidneys of rats with cyclosporine-induced chronic kidney disease.

Renal tubular epithelial cell (TEC) injury is the most common cause of drug-induced kidney injury (DIKI). Although TEC regeneration facilitates renal function and structural recovery following DIKI, maladaptive repair of TECs leads to irreversible fibrosis, resulting in chronic kidney disease (CKD). CD44 is specifically expressed in TECs during maladaptive repair in several types of rat CKD models. In this study, we investigated CD44 expression and its role in renal fibrogenesis in a cyclosporine (CyA) rat model of CKD. Seven-week-old male Sprague-Dawley rats fed a low-salt diet were subcutaneously administered CyA (0, 15, or 30 mg/kg) for 28 days. CD44 was expressed in atrophic, dilated, and hypertrophic TECs in the fibrotic lesions of the CyA groups. These TECs were collected by laser microdissection and evaluated by microarray analysis. Gene ontology analysis suggested that these TECs have a mesenchymal phenotype, and pathway analysis identified CD44 as an upstream regulator of fibrosis-related genes, including fibronectin 1 (Fn1). Immunohistochemistry revealed that epithelial and mesenchymal markers of TECs of fibrotic lesions were downregulated and upregulated, respectively, and that these TECs were surrounded by a thickened basement membrane. In situ hybridization revealed an increase in Fn1 mRNA in the cytoplasm of TECs of fibrotic lesions, whereas fibronectin protein was localized in the stroma surrounding these tubules. Enzyme-linked immunosorbent assay revealed increased serum CD44 levels in CyA-treated rats. Collectively, these findings suggest that CD44 contributes to renal fibrosis by inducing fibronectin secretion in TECs exhibiting partial epithelial-mesenchymal transition and highlight the potential of CD44 as a biomarker of renal fibrosis.

Mucosal damage and γ-H2AX formation in the rat urinary bladder induced by aromatic amines with structures similar to o-toluidine and o-anisidine.

Although aromatic amines are widely used as raw materials for dyes, some, such as o-toluidine and o-anisidine, have shown concerning results regarding carcinogenicity in the urinary bladder. We have recently developed a short-term detection method for bladder carcinogens using immunohistochemistry for γ-H2AX, a DNA damage marker. Here, using this method, we evaluated aromatic amines with structures similar to o-toluidine and o-anisidine for bladder mucosal damage and potential carcinogenicity. In total, 17 aromatic amines were orally administered to male F344 rats for 28 days, and histopathological examination and γ-H2AX immunostaining of the urinary bladder were performed. Histopathological analysis revealed that seven aromatic amines, including 4-chloro-o-toluidine (4-CT), o-aminoazotoluene, 2-aminobenzyl alcohol (ABA), o-acetotoluidine (o-AT), 3,3'-dimethoxybenzidine, 4-aminoazobenzene (AAB), and 4,4'-methylenedianiline (MDA), induced various bladder lesions, such as hemorrhage, necrosis, and urothelial hyperplasia. The morphological characteristics of mucosal damage induced by these substances were divided into two major types: those resembling o-toluidine and those resembling o-anisidine. Six of these aromatic amines, excluding MDA, also caused significant increases in γ-H2AX formation in the bladder urothelium. Interestingly, 4-CT did not cause mucosal damage or γ-H2AX formation at the lower dose applied in previous carcinogenicity studies. These results showed for the first time that o-AT and ABA, metabolites of o-toluidine, as well as AAB caused damage to the bladder mucosa and suggested that they may be bladder carcinogens. In addition, 4-CT, which was thought to be a noncarcinogen, was found to exhibit bladder toxicity upon exposure to high doses, indicating that this compound may contribute to bladder carcinogenesis.

Extraction of a metallic susceptor after accidental ingestion of the heated tobaccostick TEREA™: a case report.

BACKGROUND: Tobacco ingestion is widely known to cause nicotine toxicity, which may result in severe symptoms. Two heated tobacco sticks, called TEREA™ and SENTIA™, were launched in 2021 by Philip Morris International (New York, NY, USA), and their ingestion is associated with a risk of bowel injury because they contain a partially pointed metallic susceptor. However, this risk is not well known to the general public or healthcare providers. To increase awareness of this risk, we herein report a case involving extraction of a metallic susceptor after ingestion of the heated tobacco stick TEREA™. CASE PRESENTATION: A 7-month-old girl presented to the emergency department of a nearby hospital because she was suspected to have accidentally swallowed heated tobacco. Although she presented with no symptoms related to nicotine poisoning, abdominal X-ray examination revealed a metal object in her stomach. According to a statement released by the Japan Poison Information Center, the TEREA™ heated tobacco stick contains a metallic susceptor with a rectangular shape and sharp corners. The patient was transferred to our department because of the risk of bowel injury, and upper gastrointestinal endoscopy was performed. No cigarettes were found by endoscopic observation; however, a metallic susceptor was located in the second part of the duodenum. We grasped it with biopsy forceps and carefully removed it using an endoscope with a cap attached to the tip. The post-endoscopic course was uneventful. CONCLUSIONS: Some patients who ingest heated tobacco sticks might be exposed not only to the effects of nicotine but also to physical damage caused by a metallic susceptor. Infants and toddlers especially could swallow these sticks, therefore tobacco companies need to make the problem more public. Clinicians also should alert the problem, and pay attention to this risk in the clinical setting.

Preoperative myopenia and myosteatosis and their impact on postoperative complications in children with inflammatory bowel disease.

PURPOSE: To assess the severity of preoperative myopenia and myosteatosis in pediatric patients with inflammatory bowel disease (IBD) and examine their impact on postoperative complications. METHODS: The subjects of this retrospective study were 30 pediatric patients with IBD (22 with ulcerative colitis (UC) and 8 with Crohn's disease (CD)) and 67 age-matched controls. Preoperative body mass index (BMI), psoas muscle index (PMI), and intramuscular adipose tissue content were compared between the patient groups, to investigate their association with postoperative complications. RESULTS: BMI and PMI were significantly lower in the IBD patients than in the controls (p < 0.0001, p < 0.0001, respectively). CD was associated with significantly lower BMI and PMI (p = 0.01, p = 0.01, respectively) than UC. Intramuscular adipose tissue content was comparable between the IBD patients and the controls and between the UC and CD patients. There were no significant differences among the three indices in relation to the presence or absence of postoperative complications in patients with IBD. When limited to surgical site infection (SSI), only PMI was significantly lower in the patients with SSI than in those without SSI (p = 0.04). CONCLUSIONS: Although BMI and PMI were lower preoperatively in pediatric IBD patients than in controls, only myopenia seemed to affect the development of SSI.

o-Anisidine Dimer, 2-Methoxy-N4-(2-methoxyphenyl) Benzene-1,4-diamine, in Rat Urine Associated with Urinary bladder Carcinogenesis.

Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

In vivo mutagenicity and tumor-promoting activity of 1,3-dichloro-2-propanol in the liver and kidneys of gpt delta rats.

1,3-Dichloro-2-propanol (1,3-DCP), a food contaminant, exerts carcinogenic effects in multiple organs, including the liver and kidneys, in rats. However, the underlying mechanisms of 1,3-DCP-induced carcinogenesis remain unclear. Here, the in vivo mutagenicity and tumor-promoting activity of 1,3-DCP in the liver and kidneys were evaluated using medium-term gpt delta rat models previously established in our laboratory (GPG and GNP models). Six-week-old male F344 gpt delta rats were treated with 0 or 50 mg/kg body weight/day 1,3-DCP by gavage for 4 weeks. After 2 weeks of cessation, partial hepatectomy or unilateral nephrectomy was performed to collect samples for in vivo mutation assays, followed by single administration of diethylnitrosamine (DEN) for tumor initiation. One week after DEN injection, 1,3-DCP treatment was resumed, and tumor-promoting activity was evaluated in the residual liver or kidneys by histopathological analysis of preneoplastic lesions. gpt mutant frequencies increased in excised liver and kidney tissues following 1,3-DCP treatment. 1,3-DCP did not affect the development of glutathione S-transferase placental form-positive foci in residual liver tissues, but enhanced atypical tubule hyperplasia in residual kidney tissues. Detailed histopathological analyses revealed glomerular injury and increased cell proliferation of renal tubular cells in residual kidney tissues of rats treated with 1,3-DCP. These results suggested possible involvement of genotoxic mechanisms in 1,3-DCP-induced carcinogenesis in the liver and kidneys. In addition, we found that 1,3-DCP exhibited limited tumor-promoting activity in the liver, but enhanced clonal expansion in renal carcinogenesis via proliferation of renal tubular cells following glomerular injury.

Expression of stem cell markers as useful complementary factors in the early detection of urinary bladder carcinogens by immunohistochemistry for γ-H2AX.

We previously demonstrated that immunohistochemistry for γ-H2AX, a biomarker of DNA damage, is useful for early detection of urinary bladder carcinogens in rats. In a 28-day repeated-dose study, γ-H2AX was shown to have high sensitivity for detection of bladder carcinogens. However, no reports have evaluated whether a combination of multiple biomarkers may further improve sensitivity. Accordingly, in this study, we aimed to evaluate the applicability of bladder tissue and cancer stem cell markers, including cytokeratin 14 (KRT14), aldehyde dehydrogenase 1A1 (ALDH1A1), and cluster of differentiation 44 (CD44), as complementary markers for early detection of bladder carcinogens. Bladder samples obtained from male F344 rats orally treated with 14 bladder carcinogens and five nonbladder carcinogens for 28 days were used for immunohistochemical analysis of stem cell markers. In the bladder carcinogen-treated rats, increases in KRT14, ALDH1A1, and CD44 expression were observed in 9, 10, and 10 out of 14 groups, respectively, whereas the five nonbladder carcinogens did not cause upregulation of these markers. Although most epithelial cells with KRT14 or ALDH1A1 expression were also positive for CD44, KRT14 and ALDH1A1 expression were mutually exclusive. Twelve bladder carcinogens showed increases in at least one of the three markers, indicating that the combined evaluation showed higher sensitivity than the use of individual markers alone. Importantly, two of three bladder carcinogens that did not induce γ-H2AX immunostaining showed stem cell marker expression. Our results demonstrated that these stem cell markers may be useful as complementary markers for γ-H2AX in evaluation of bladder carcinogens.

Specific expression of survivin, SOX9, and CD44 in renal tubules in adaptive and maladaptive repair processes after acute kidney injury in rats.

Acute kidney injury (AKI) is thought to be a reversible condition; however, growing evidence has suggested that AKI may be associated with subsequent development of chronic kidney disease. Although renal tubules have intrinsic regeneration capacity, disruption of the regeneration mechanisms leads to irreversible interstitial fibrosis. In this study, we investigated immunohistochemical markers of renal tubules in adaptive and maladaptive repair processes to predict AKI reversibility. Histopathological analysis demonstrated that regenerative tubules and dilated tubules were observed in the kidneys of AKI model rats after ischemia/reperfusion (I/R). Regenerative tubules gradually redifferentiated after I/R, whereas dilated tubules exhibited no tendency for redifferentiation. In fibrotic areas of the kidney in renal fibrosis model rats subjected to I/R, renal tubules were dilated or atrophied. There results suggested that the histopathological features of renal tubules in the maladaptive repair were dilation or atrophy. From microarray data of regenerative tubules, survivin, SOX9, and CD44 were extracted as candidate markers. Immunohistochemical analysis demonstrated that survivin and SOX9 were expressed in regenerative tubules, whereas SOX9 was also detected in renal tubules in fibrotic areas. These findings indicated that survivin and SOX9 contributed to renal tubular regeneration, whereas sustained SOX9 expression may be associated to fibrosis. CD44 was expressed in dilated tubules in the kidneys of AKI model rats and in the tubules of fibrotic areas of renal fibrosis model rats, suggesting that CD44 was expressed in renal tubules in maladaptive repair. Thus, these factors could be useful markers for detecting disruption of the regenerative mechanisms of renal tubules.

A 13-week subchronic toxicity study of 2-(l-menthoxy)ethanol in F344 rats.

2-(l-Menthoxy)ethanol has been frequently employed as a flavoring agent; however, data regarding 2-(l-menthoxy)ethanol toxicity remain limited. We performed a 13-week subchronic toxicity study of 2-(l-menthoxy)ethanol in male and female F344 rats, with doses of 0, 15, 60, or 250 mg/kg body weight (BW)/day orally administered by gavage using corn oil as the vehicle. No significant toxicological changes in general condition, body weight, or food intake were observed in any groups. The hematological assessment showed decreased hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin and increased platelet count in the male 250 mg/kg group. Serum biochemistry revealed elevated total cholesterol in the 250 mg/kg group of male and female rats, reduced triglyceride in the female 250 mg/kg group, and increased total protein in the male 250 mg/kg group, indicating effects on lipid metabolism and protein synthesis. For organ weights, absolute and relative weights of the liver and adrenal glands were increased in the 250 mg/kg group of both sexes and the male 250 mg/kg group, respectively. Histopathological analysis showed chronic nephropathy in the male 15 mg/kg or higher groups, with increased absolute and relative kidney weights, as well as elevated serum creatinine, in the male 60 and 250 mg/kg groups. However, eosinophilic granules containing α2u-globulin were identified in proximal tubules, suggesting α2u-globulin nephropathy specific to male rats and without toxicological significance. These results indicated that the no-observed-adverse-effect level of 2-(l-menthoxy)ethanol was 60 mg/kg BW/day for both sexes.

Neuromuscular and vascular hamartoma of the small intestine in an F344 rat.

An intestinal mass was found in the border area of the jejunum and ileum of a 110-week-old male F344 rat. Histopathologically, the mass protruded into the lumen and was covered with intestinal epithelium, exhibiting a normal architecture. The lesion was located in the submucosa and consisted of loose connective tissue, smooth muscle, scattered ganglion cells, and blood vessels of various sizes. Although these components showed an irregular and disordered structure, no cellular atypia, increased proliferation activity, or invasive growth to adjacent tissues were detected. Immunohistochemical analyses revealed that smooth muscle, ganglion, and endothelial cells were positive for α-smooth muscle actin and vimentin, S-100, and CD34 and von Willebrand factor, respectively, indicating maturation of these cells. Thus, the mass was diagnosed as a neuromuscular and vascular hamartoma of the small intestine. To the best of our knowledge, this is the first report of this type of lesion in rodents.

Background data of 2-year-old male and female F344 gpt delta rats.

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.

Novel o-Toluidine Metabolite in Rat Urine Associated with Urinary Bladder Carcinogenesis.

o-Toluidine (o-Tol), a monocyclic aromatic amine, causes bladder cancer in humans and experimental animals and is therefore classified as a Group 1 carcinogen (IARC) in which the carcinogenicity of o-Tol is involved in metabolic activation, DNA damage, and DNA adduct formation. In the DNA adduct formation mechanism, o-Tol is metabolized by N-hydroxylation, N-acetoxylation, and then deacetoxylation to produce an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8. Therefore, dG-C8-o-Tol is thought to be a plausible DNA adduct of o-Tol exposure. However, direct detection of dG-C8-o-Tol in biological samples has not been reported yet. Here, we show that a novel o-Tol metabolite, 2-methyl-N1-(2-methylphenyl)benzene-1,4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in o-Tol-exposed rat urine. MMBD was also detected in a reaction of o-Tol and S9 mix, indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD showed a potent stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains,and cytotoxicity in human bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells compared with o-Tol. Furthermore, a DNA adduct (m/z 478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA with rat urine containing MMBD, and also in hepatic DNA of rats treated with o-Tol. These results therefore suggested that o-Tol-induced bladder carcinogenesis could be at least partly attributed to MMBD formation. The possible dimerization of monocyclic aromatic amines should be considered in the evaluation of the risk of bladder carcinogenesis after exposure.

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.

Comprehensive expression analysis of mRNA and microRNA for the investigation of compensatory mechanisms in the rat kidney after unilateral nephrectomy.

Compensation is a physiological response that occurs during chemical exposure to maintain homeostasis. Because compensatory responses are not usually considered adverse effects, it is important to understand compensatory mechanisms for chemical risk assessment. Although the kidney is a major target organ for toxicity, there is controversy over whether hyperplasia or hypertrophy contributes to the compensatory mechanism, and there is limited information to apply for chemical risk assessment. In the present study, compensatory mechanisms of the kidney were investigated in a unilateral nephrectomy (UNx) model using adult male and female F344 rats. In residual kidneys of male and female rats after UNx, 5-bromo-2'-deoxyuridine-labeling indices and mRNA expression of cell cycle-related genes were increased, although there were no fluctuations in mRNA expression of transforming growth factor-ß1, which contributes to hypertrophy in renal tubules. Pathway analysis using mRNA expression data from a complementary DNA (cDNA) microarray revealed that canonical pathways related to cell proliferation were mainly activated and that forkhead box M1 (FOXM1) was an upstream regulator of compensatory cell proliferation in residual kidneys of male and female rats. cDNA microarray for microRNAs (miRNAs) demonstrated that nine miRNAs were downregulated in residual kidneys, and mRNA/miRNA integrated analysis indicated that miRNAs were associated with the expression of factors downstream of FOXM1. Overall, these results suggested that FOXM1-mediated hyperplasia rather than hypertrophy contributed to compensatory mechanisms in the kidney and that miRNAs regulated downstream FOXM1 signaling. These results will be beneficial for evaluating nephrotoxicity in chemical risk assessment and for developing new biomarkers to predict nephrotoxicity.

Safety of double-balloon enteroscopy in postoperative pediatric patients.

BACKGROUND: The aim of the present study was to investigate the efficacy and safety of double-balloon enteroscopy (DBE) in postoperative pediatric patients. METHODS: This was a retrospective analysis of pediatric patients 18 years and younger referred to Mie University Hospital. Twenty procedures in 11 children occurred postoperatively; 29 children (42 procedures) had not undergone surgery. RESULTS: Among postoperative patients, five DBE procedures were performed via the oral route, 12 via the anal route, and three via a stomal route. Among nonoperative patients, 14 DBE procedures were performed via the oral route and 28 via the anal route. Four postoperative patients and two nonoperative patients had difficult pleating via the transanal route because of adhesions or thickening of the intestinal wall resulting from inflammation (P = 0.02). Excluding patients with stenosis, the mean length of endoscopic insertion for transanal procedures was significantly shorter among postoperative patients than among nonoperative patients (73.6 cm vs 160.5 cm, P < 0.01). There were no major complications in either group. CONCLUSIONS: Insertion difficulty was encountered in postoperative pediatric patients. However, our findings indicate that DBE is a safe procedure in postoperative pediatric patients.

Risk factors and intraoral breast milk application for methicillin-resistant Staphylococcus aureus colonization in surgical neonates.

BACKGROUND: Our previous study identified methicillin-resistant Staphylococcus aureus (MRSA) colonization as an independent risk factor for neonatal surgical site infection. Here we introduce intraoral breast milk application (IBMA) during a fasting state to prevent MRSA colonization. We aimed to evaluate both the risk factors for MRSA colonization and the efficacy of IBMA in neonatal surgical patients. METHODS: A retrospective review was performed using admission data from 2007 to 2016. Neonatal patients who underwent surgery and were tested periodically for MRSA colonization were evaluated for an association between MRSA colonization and perinatal or perioperative factors. RESULTS: The overall incidence of MRSA colonization for the 159 patients enrolled in this study was 16.4%. Univariate analysis showed that MRSA colonization was significantly more frequent in the following patients: those with Down syndrome, those admitted on their day of birth, those in need of fasting immediately after birth, and those not receiving IBMA. Multivariate analysis showed that comorbid Down syndrome was an independent risk factor (hazard ratio: 4.6; 95% confidence interval: 1.2-19.5, P = 0.03) and implementation of IBMA was an independent preventive factor for MRSA colonization (hazard ratio: 0.4; 95% confidence interval: 0.1-0.9, P = 0.04). MRSA-positive patients admitted significantly earlier and stayed longer preoperatively than MRSA-negative patients. CONCLUSIONS: In neonates undergoing surgery, and patients with Down syndrome, early diagnosis after birth and a long waiting period before operation may be associated with MRSA colonization. Intraoral breast milk application may be beneficial for preventing MRSA colonization.

Predictors for Pouchitis After Ileal Pouch-Anal Anastomosis for Pediatric-Onset Ulcerative Colitis.

BACKGROUND: The predictive factors for the development of pouchitis after ileal pouch-anal anastomosis (IPAA) in pediatric-onset ulcerative colitis (UC) have not been well investigated. The present study aimed to determine the predictive factors for the development of pouchitis after IPAA in the pediatric UC population. METHODS: The data from 54 patients with pediatric-onset UC who underwent IPAA in Mie University Hospital between 2000 and 2017 were retrospectively reviewed. A modified pouchitis disease activity index of ≥5 was defined as pouchitis. Potential preoperative, intraoperative, and postoperative predictors for pouchitis including various demographic and clinical variables were analyzed using Cox regression analysis, Students' t-tests, Mann-Whitney U tests, and Kaplan-Meier curves. The optimal cutoff value for continuous variables was determined using the receiver operating characteristic curve analysis. RESULTS: Pouchitis was identified in 17 (31.5%) patients within 5 y of follow-up. In multivariable analysis, the independent predictors for pouchitis were preoperative cumulative steroid dose of >10,000 mg (P = 0.0056) and >65% neutrophils just before IPAA (P = 0.032). Multivariate analysis revealed that the independent predictors of pouchitis were a total steroid dose of >10,000 mg (P = 0.0002) and a neutrophil percentage of >65% (P = 0.0078). No patient for whom both of these independent predictors were negative developed pouchitis, whereas >40% of patients who had one or both predictors developed pouchitis. CONCLUSIONS: In pediatric patients with UC, the predictive factors for pouchitis development are a greater cumulative total dose of steroids and a greater percentage of neutrophils before IPAA.