Evaluation of circulating miR-216a and miR-217 as biomarkers of pancreatic damage in the L-arginine-induced acute pancreatitis mouse model.

We investigated the usefulness of circulating miR-216a-5p and miR-217-5p that are pancreas-enriched micro RNAs (miRNAs) as biomarkers of acute pancreatic damage, and compared them with conventional pancreatic biomarkers in L-arginine-induced acute pancreatitis mouse model. As the results, amylase and lipase levels apparently increased and peaked on Day 3 when acute pancreatitis including acinar cell degeneration/necrosis and inflammatory cell infiltration reached its peak. In contrast, miR-216a-5p and miR-217-5p increased from Day 1 when histopathological findings in the acinar cells were limited to decreased zymogen granules, and the increases in ratios were much higher than those of amylase and lipase. The miRNAs remained at high levels until Day 5 when the pseudo-tubular complex and replacement of inflammatory cells and fibrotic cells were apparent instead of necrosis, whereas amylase and lipase levels decreased to the control levels. Furthermore, we examined the relationship between biomarker levels and histopathological degeneration/necrosis scores in the acinar cells. miR-216a-5p and miR-217-5p levels increased depending on the score of degeneration/necrosis, and all individual miRNAs exceeded the control levels from a score of 2 (focal necrosis), whereas all individual amylase and lipase levels exceeded the control levels at scores of 4 (lobular necrosis) and 3 (sublobular necrosis), respectively. In conclusion, we demonstrated that circulating miR-216a-5p and miR-217-5p could detect pancreatic damage earlier with greater magnitude, and the sensitivity to detect acinar cell degeneration/necrosis was superior to that of conventional biomarkers in the L-arginine-induced acute pancreatitis mouse model.

Spontaneous carcinosarcoma originating from the renal pelvis ina rat.

Carcinosarcoma is a rare neoplasm composed of malignant epithelial and stromal elements, and, for rats, carcinosarcomas in the kidney have not been reported. In a long-term study to gather background data, we encountered a spontaneous carcinosarcoma originating from the renal pelvis with metastasis to the lung. At necropsy, a mass was observed in the abdominal cavity, and white nodules were scattered in lung lobes. Microscopically, there was polypoid hyperplasia of the urothelium accompanied by hyperplasia of spindle stromal cells in the pelvis. The intra-abdominal tumor was composed of epithelial and stromal elements; in the lung, the tumor cells invaded along alveoli/bronchi and occasionally invaded the parenchyma from the blood vessels. Immunohistochemical and electron microscopic examinations revealed that the epithelial element consisted of transitional epithelial cells and that the stromal element consisted of lipoblasts. The tumor was diagnosed as a carcinosarcoma originating from the renal pelvis, and this is the first report of a carcinosarcoma originating from the renal pelvis in a rat.

Oscillatory potentials in electroretinogram as an early marker of visual abnormalities in vitamin A deficiency.

Vitamin A deficiency (VAD) caused by malnutrition and certain intestinal diseases induces visual impairments, including night blindness and photoreceptor cell dysfunction as indicated by reduced a­ and b­waves in an electroretinogram (ERG). The effects of VAD on the inner retinal layer cells, including amacrine and ganglion cells, remain to be elucidated. The functions of these cells are reflected in oscillatory potentials (OPs), another component of the ERG. The present study investigated inner retinal layer cell function in VAD rats by analyzing OPs. In the present study, VAD was induced by feeding Brown Norway rats a vitamin A deficient diet for 10 weeks. A reduced body weight and peri­papillary opacification indicative of papilledema without histopathological alterations were observed, which are considered early symptoms of VAD. At this stage, the ERG revealed reduced OPs as well as a­ and b­waves at various intensities of light stimulation. Further analysis indicated that the ratio of the alterations in OPs was more significant than those of a­ and b­waves. After 5 weeks of recovery, these changes returned to control levels. These results suggest that OPs are the most sensitive and early marker of VAD­associated visual impairment in the ERG.

E2012-induced cataract and its predictive biomarkers.

E2012, a gamma secretase modulator without affecting Notch processing, aimed at Alzheimer's disease by reduction of amyloid ß-42, induced cataract following repeated doses in the rat. Cataract appeared first at week 10-11 of treatment as a posterior subcapsular area with granular/punctate opaque or shiny dots along the suture line, characterized histologically as lenticular fiber degeneration, which eventually coalesced to form a triangular or circular opacity. It was associated with prolonged and sustained elevation of lenticular desmosterol (24-dehydrocholesterol), the final precursor of cholesterol, and decrease in lenticular cholesterol. In vitro studies to investigate the effect of E2012 on cholesterol metabolism demonstrated that E2012 inhibits 3ß-hydroxysterol Δ24-reductase (DHCR24) at the final step in the cholesterol biosynthesis. In vivo lenticular concentration of E2012 after 13-week repeated dose with cataract was well above those where inhibition was observed in vitro. There was no cataract formation at doses where desmosterol did not accumulate in the lens. The elevation of desmosterol and decreased cholesterol levels were also seen in the liver and plasma and preceded those in the lens. These results demonstrate that E2012 induces cataract in the rat by inhibiting DHCR24 at the final step of cholesterol synthesis with associated elevation in desmosterol within the lens, preceded by desmosterol changes that would serve as a predictive safety biomarker for lenticular opacity.

Study for collecting background data on Wistar Hannover [Crl:WI(Han)] rats in general toxicity studies--comparative data to Sprague Dawley rats.

The purpose of the present study was to collect background data from repeated dose toxicity studies in Wistar Hannover [Crl:WI(Han)] (hereafter Wistar Han) rats with dosing periods of 4, 13 and 26 weeks from four safety research facilities of pharmaceutical companies and contract research organizations participating in the International Genetic Standardization (IGS) rat forum supported by Charles River Laboratories Japan, Inc. The data from Wistar Han rats were compared with those from Sprague Dawley Crl:CD(SD) rats. In addition, the effects of restricted feeding of SD rats were also investigated by one facility. As a result, body weights and food consumption in Wistar Han rats were lower than those of SD rats. White blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were almost half of those noted for SD rats and platelet counts were almost 20% less than those in SD rats. Minimal strain differences were noted in several biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase, total cholesterol, triglyceride and phospholipids, and in thymus, ovary and testis weights. Ophthalmologic or histopathologic examinations revealed a higher incidence of corneal opacities or corneal mineralization in Wistar Han rats. Restricted feeding of SD rats resulted in intermediate values for body weights and food consumption between the ad libitum fed SD and Wistar Han rats, and WBC and AST were lower than those in the ad libitum fed SD rats. Based on these results, some strain differences might be ascribable to reduced food consumption and associated body weight changes in Wistar Han rats.

Spontaneous gingivitis related to hair penetration in rats.

Maxillary gingivae from male and female Crl:CD(SD) rats at 12, 16, 21, and 34 weeks of age were examined histologically. The incidence of gingivitis was approximately 40%, with no age or sex predilection, and was most frequent between the first and second molar. Lesions were characterized by acute focal neutrophilic infiltration into the gingival mucosa, occasionally with inflammatory exudate. In severe cases, inflammation extended to the periodontal ligament with abscess formation, and adjacent alveolar bone destruction/resorption. The most characteristic finding was the presence of hair shafts associated with the lesion, which was observed in approximately 80% of the rats with gingivitis. These findings suggest that molar gingivitis occurs in rats from an early age and persists thereafter, and that the main cause of gingivitis in rats is hair penetration into the gingiva. It would be prudent to keep these background lesions in mind as potential modifiers in toxicity studies.

Malformation spectrum induced by ketoconazole after single administration to pregnant rats during the critical period - comparison with vitamin A-induced malformation spectrum.

Azole derivatives have teratogenic effects in rodents. In the present study, malformations and their sensitive windows induced by high-dose ketoconazole (KCZ), an azole derivative, without maternal toxicity were investigated. In addition, the malformation spectrum determined was compared with that induced by vitamin A palmitate (VAP). Pregnant rats were administered a single dose of KCZ by oral gavage on specific individual days from gestational days 8 to 15 (GDs 8-15). Maternal animals were subjected to necropsy on GD 20, and the obtained fetuses were examined for external, visceral and skeletal malformations. The malformation spectrum of VAP was identified from available published data (Noda, Sato, and Udaka, 1982) and a complementary study (single administration of VAP at 1 200 000 IU kg(-1) ). Embryonic lethality was observed in dams given KCZ on GDs 9-12 with peak incidence on GDs 10 and 11 with complete resorption. KCZ induced major malformations included cleft palate, digital anomalies, misshapen limbs and unique discontinuous ribs, and the sensitive window for each was identified. Compared with the malformations induced by VAP, unique malformations (e.g. discontinuous ribs by KCZ, neural tube defects by VAP), similar malformations with similar sensitive windows (e.g. digital and limb malformations) and similar malformations with different sensitive windows (e.g. embryonic lethality and cleft palate) were distinguished, suggesting that the mechanisms of several of the types of KCZ-induced malformation are related to excessive vitamin A.

Pharmacokinetics, metabolism and excretion of 14C-monoethyl phthalate (MEP) and 14C-diethyl phthalate (DEP) after single oral and IV administration in the juvenile dog.

The pharmacokinetics, metabolism and excretion of monoethyl phthalate (MEP) and diethyl phthalate (DEP) were compared after intravenous or oral administration of [(14)C]MEP or [(14)C]DEP in juvenile beagle dogs. Four male juvenile beagle dogs were treated with a single oral or bolus intravenous dose of either [(14)C]MEP or [(14)C]DEP (164 µg/kg). The absorption, metabolism, excretion and pharmacokinetics of [(14)C]MEP and [(14)C]DEP were nearly identical. [(14)C]DEP was rapidly and nearly completely metabolized to [(14)C]MEP following either intravenous or oral administration. [(14)C]MEP and[(14)C]DEP were rapidly absorbed, the elimination half-life was estimated to be 1 hour. Approximately 90%-96% of the dose was excreted in urine with 2%-3% of the dose in faeces. MEP accounted for the majority of the dose in plasma and urine; in addition, three minor metabolites (M1, M2 and M3) were detected. The minor metabolites were neither phthalic acid nor glucuronide/sulfate conjugates. The nearly identical metabolism and pharmacokinetics of MEP and DEP in juvenile dogs justifies the use of DEP toxicity data in the risk assessment of MEP exposure.

Rapid induction of colonic adenocarcinoma in mice exposed to benzo[a]pyrene and dextran sulfate sodium.

Previously, we reported that the mutation frequency was markedly increased in the colon after the oral treatment of mice with an environmental mutagen/carcinogen, benzo[a]pyrene (BP); however this was not followed by tumor development. The reasons for this are as yet unresolved. The purpose of the present study is to explore the mechanisms why a high frequency of mutations induced by BP in the colon is not associated with subsequent tumor development. We show in this study that oral administration of BP to CD2F(1) mice at 125 mg/kg/day for 5 days can lead to adenocarcinomas in the mouse colon both at Weeks 4 (5/8 mice) and 11 (100% of mice), but only in the presence of inflammation induced by 4% dextran sulfate sodium (DSS) in the drinking water for up to 2 weeks. These data indicate that, in this DSS model, BP induced mutagenic events lead to tumors in the mouse colon, a tissue which is not a BP target organ. DSS-induced inflammation in a tissue primed with mutagenic risk is a key to the induction of tumors in this model. This study provides a novel, rapid and useful colon carcinogenesis model (BP/DSS model).

Histopathological characteristics of luteal hypertrophy induced by ethylene glycol monomethyl ether with a comparison to normal luteal morphology in rats.

Ethylene glycol monomethyl ether (EGME) is a known reproductive toxicant that induces luteal hypertrophy in rat ovaries. In this study, we characterized the histopathological features of corpora lutea (CL) from EGME-treated rats and compared them with normal CL formation and regression. Normally cycling female Sprague-Dawley rats were treated with 5-bromo-2'-deoxyuridine (BrdU) intraperitoneally on the morning of estrus and their ovaries were examined 1 (metestrus), 4 (estrus), 8 (estrus), or 12 (estrus) days later to observe the transition of BrdU-labeled cells within in the CL. CL at each time point of estrus stage were classified into 4 types: Type I (newly formed CL), Type II (mature CL), Type III (regressing CL), and Type IV (residual CL). CL almost fully regressed within 4 estrus cycles. In contrast, in female rats given EGME orally (30, 100, or 300 mg/kg for 2 or 4 weeks), luteal cells were hypertrophic with abundant cytoplasm. Although the size of CL varied, all CL in EGME-treated rats had histological features similar to Type II CL, but they were more hypertrophic with less apoptosis. These results suggest that EGME has a luteal hypertrophic effect on all CL phases, including regression.

Proteomic analysis for neuronal vacuolation induced by MK-801 in rat retrosplenial cortex.

Proteomic analysis was carried out for neuronal vacuolation in rat retrosplenial cortex (RSC) induced by MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist. Female rats were given a single subcutaneous (sc) injection of either MK-801 (9 mg/kg in saline) or saline. Comparison of changes in proteins in the RSC region between MK-801- and saline-treated groups revealed that MK-801 induced changes in six proteins involved in vesicular transport (vesicle-fusing ATPase) and glycolysis (fructose-bisphosphate aldolase C, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase).

Identification of urinary biomarkers useful for distinguishing a difference in mechanism of toxicity in rat model of cholestasis.

This (1)H nuclear magnetic resonance metabonomics study was aimed to determine urinary biomarkers of cholestasis resulting from inhibition of biliary secretion of bile or obstruction of bile flow. To inhibit biliary secretion of bile, cyclosporine A was administered to male Sprague-Dawley rats. Obstruction of bile flow was induced by administration of 4,4'-methylene dianiline, alpha-naphthylisothiocyanate or bile duct ligation. Clinical pathological and histopathological examinations were performed to confirm cholestatic injury and (1)H nuclear magnetic resonance spectral data for urine samples were analysed to determine similarities and differences in profiles of metabolites using the Spotfire. In cyclosporine A-treated groups, serum total bilirubin and bile acid were significantly increased but no remarkable hepatic histopathological-changes were observed. In 4,4'-methylene dianiline-, alpha-naphthylisothiocyanate- and bile duct ligation-treated groups, serum alkaline phosphatase, gamma-glutamyltranspeptidase and total bilirubin levels increased significantly, and hepatic histopathological-changes were observed. On urinary (1)H nuclear magnetic resonance spectral analysis, area intensities derived from 0.66 to 1.90 ppm were decreased by cyclosporine A, whereas they were increased by other treatments. These metabolites were identified using the NMR suite as bile acids, branched-chain amino acids, n-butyrate, propionate, methyl malonate and valerate. These metabolites were further investigated by K-means clustering analysis. The cluster of these metabolites is considered to be altered by cholestasis. We conclude that bile acids, valine and methyl malonate have a possibility to be urinary cholestatic biomarkers, which distinguish a difference in mechanism of toxicity. (1)H nuclear magnetic resonance metabonomics thus appears to be useful for determining the mechanisms of toxicity and can be front-loaded in drug safety evaluation and biomarker discovery.

A comparative investigation of fetal skeletal anomalies in rats induced by acetylsalicylic acid with single- and double-staining techniques.

Acetylsalicylic acid (ASA) at single doses of 125, 250, and 500mg/kg was administered to pregnant rats on Gestation Day (GD) 10, and skeletal changes in fetuses harvested on GD 20 and pups on post-natal (PN) Day 21 were evaluated. Changes in cartilage and ossified bones identified by Alizarin Red S single-staining were compared with Alizarin Red S and Alcian Blue double-staining. By the single-staining technique, skeletal abnormalities including fused rib, incomplete ossification of the cervical arch, absent/hemicentric body of thoracic or lumbar vertebra, deformation of lumbar arch, and absent sacral arch were demonstrated in at 250 and 500mg/kg ASA on GD 20. The double-staining technique facilitated identification of additional cartilaginous changes in the vertebrae, paws, and ribs: including discontinuous rib cartilage, fused carpus, and split cartilage of thoracic centrum at same doses. Discontinuous rib cartilage and fused carpus persisted in pups until PN Day 21 demonstrating that these changes were irreversible. With use of the double-staining technique, the incidence of abnormalities at 250mg/kg were dramatically increased, thus this technique was more sensitive for identifying fetal cartilaginous and ossified skeletal changes.

Collaborative work on evaluation of ovarian toxicity. 11) Two- or four-week repeated-dose studies and fertility study of ethylene glycol monomethyl ether in female rats.

The objective of this study was to determine the optimal period of administration for detection of ovarian toxicity in rat repeated-dose toxicity studies. A well-known ovarian toxicant, ethylene glycol monomethyl ether (EGME), was administered to female rats at dose levels of 0, 30, 100, or 300 mg/kg for 2 or 4 weeks (repeated-dose toxicity studies). The same doses were administered to female rats for 2 weeks prior to mating, during mating, and until Day 6 of pregnancy (fertility study). In the repeated-dose toxicity studies, continuous diestrus was observed at > or = 100 mg/kg regardless of period of administration. The alterations of ovarian morphology observed at > or = 100 mg/kg after 2 or 4 weeks of administration were characterized by hypertrophy of the corpora lutea with decreased cellular debris indicating apoptosis, and increased proliferating cell nuclear antigen (PCNA)-negative large atretic follicles. The finding that newly-formed basophilic corpora lutea were scarce in affected animals exhibiting continuous diestrus suggested suppression of ovulation due to hypertrophic corpora lutea. In the fertility study, irregular estrous cycles, prolonged mating periods, lower pregnancy rates and decreased corpora lutea of pregnancy were observed at > or = 100 mg/kg. The irregularities of estrous cycle observed in some animals at 30 mg/kg were minimal. The ovarian histopathological changes in repeated-dose toxicity studies correlated well with impairment of female fertility found in the fertility study. It is concluded that a repeated-dose toxicity study with a treatment period for 2 weeks or longer is sufficient for evaluation of ovarian toxicity induced by EGME.

A metabonomics study of the hepatotoxicants galactosamine, methylene dianiline and clofibrate in rats.

The efficacy of high-resolution (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy-based metabonomics was studied in a model of rat liver toxicity. Hepatotoxicities were induced in male rats using methylene dianiline, clofibrate and galactosamine. Twenty-four-hr urine from days 1 to 5 after treatment were subjected to (1)H-NMR evaluation of the biochemical effects. Blood were also taken at Days 2, 3 and 5 to examine biochemical changes associated with hepatotoxicities, and histopathological changes were evaluated at termination. Increases in liver enzymes were observed in animals treated with methylene dianiline or galactosamine, and histopathological analysis revealed changes associated with hepatobiliary damage and hepatocellular necrosis in methylene dianiline- and galactosamine-treated animals, respectively. Principal component analysis and statistical Spotfire analyses were used to visualize similarities and differences in urine biochemical profiles produced by (1)H-NMR spectra. The biochemical effects of methylene dianiline and galactosamine were characterized by elevated levels of glucose, fructose, beta-hydroxybutyrate, alanine, acetoacetate, lactate and creatine and decreased levels of hippurate, 2-oxoglutarate, citrate, succinate, trimethylamine-N-oxide, taurine and N-acetylglutamate in rat urine. Clofibrate treatment elevated the levels of N-methylnicotinamide and 3,4-dihydroxymandelate and decreased the levels of 2-oxoglutarate and N-acetylaspartate. This work shows that combinations of (1)H-NMR and pattern recognition are powerful tools in the evaluation of the biochemical effects of xenobiotics in liver.