Showing papers in "Journal of Toxicologic Pathology in 2015"

Therapeutic antibodies: their mechanisms of action and the pathological findings they induce in toxicity studies.

Abstract: Antibodies can swiftly provide therapeutics to target disease-related molecules discovered in genomic research. Antibody engineering techniques have been actively developed and these technological innovations have intensified the development of therapeutic antibodies. From the mid-1990’s, a series of therapeutic antibodies were launched that are now being used in clinic. The disease areas that therapeutic antibodies can target have subsequently expanded, and antibodies are currently utilized as pharmaceuticals for cancer, inflammatory disease, organ transplantation, cardiovascular disease, infection, respiratory disease, ophthalmologic disease, and so on. This paper briefly describes the modes of action of therapeutic antibodies. Several non-clinical study results of the pathological changes induced by therapeutic antibodies are also presented to aid the future assessment of the toxic potential of an antibody developed as a therapeutic.

Autopsy report for a caffeine intoxication case and review of the current literature

Abstract: Caffeine (1,3,7-trimethylxanthine) is a popular mild central nervous system stimulant found in the leaves, seeds and fruits of various plants and in foodstuffs such as coffee, tea, and chocolate, among others. Caffeine is widely used and is not associated with severe side effects when consumed at relatively low doses. Although rarely observed, overdoses can occur. However, only a few fatal caffeine intoxication cases have been reported in the literature. Herein, we report the pathological examination results and information on caffeine concentrations in the blood, urine and main organs in a fatal caffeine intoxication case. Even though high caffeine concentrations were found in the systemic organs, no caffeine-related pathological changes were detected.

Characteristics of structures and lesions of the eye in laboratory animals used in toxicity studies

Abstract: Histopathology of the eye is an essential part of ocular toxicity evaluation. There are structural variations of the eye among several laboratory animals commonly used in toxicity studies, and many cases of ocular lesions in these animals are related to anatomical and physiological characteristics of the eye. Since albino rats have no melanin in the eye, findings of the fundus can be observed clearly by ophthalmoscopy. Retinal atrophy is observed as a hyper-reflective lesion in the fundus and is usually observed as degeneration of the retina in histopathology. Albino rats are sensitive to light, and light-induced retinal degeneration is commonly observed because there is no melanin in the eye. Therefore, it is important to differentiate the causes of retinal degeneration because the lesion occurs spontaneously and is induced by several drugs or by lighting. In dogs, the tapetum lucidum, a multilayered reflective tissue of the choroid, is one of unique structures of the eye. Since tapetal cells contain reflecting crystals in which a high level of zinc has been demonstrated chemically, drug-induced tapetum degeneration is possibly related to zinc chelation. The eye of the monkey has a macula similar to that of humans. The macula consists only of cones with a high density, and light falls directly on the macula that plays an important role in visual acuity. Macular degeneration occurring in monkeys resembles histopathologically that of humans. Hence, the eye of the monkey is a suitable model to investigate macular degeneration and to assess drug-induced macular lesions.

Practical approaches for evaluating adrenal toxicity in nonclinical safety assessment.

Abstract: The adrenal gland has characteristic morphological and biochemical features that render it particularly susceptible to the actions of xenobiotics. As is the case with other endocrine organs, the adrenal gland is under the control of upstream organs (hypothalamic-pituitary system) in vivo, often making it difficult to elucidate the mode of toxicity of a test article. It is very important, especially for pharmaceuticals, to determine whether a test article-related change is caused by a direct effect or other associated factors. In addition, antemortem data, including clinical signs, body weight, food consumption and clinical pathology, and postmortem data, including gross pathology, organ weight and histopathologic examination of the adrenal glands and other related organs, should be carefully monitored and evaluated. During evaluation, the following should also be taken into account: (1) species, sex and age of animals used, (2) metabolic activation by a cytochrome P450 enzyme(s) and (3) physicochemical properties and the metabolic pathway of the test article. In this review, we describe the following crucial points for toxicologic pathologists to consider when evaluating adrenal toxicity: functional anatomy, blood supply, hormone production in each compartment, steroid biosynthesis, potential medulla-cortex interaction, and species and gender differences in anatomical features and other features of the adrenal gland which could affect vulnerability to toxic effects. Finally practical approaches for evaluating adrenal toxicity in nonclinical safety studies are discussed.

Dietary curcumin supplementation attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in C57BL mice

Abstract: Studies in vivo and in vitro suggest that curcumin is a neuroprotective agent. Experiments were conducted to determine whether dietary supplementation with curcumin has neuroprotective effects in a mouse model of Parkinson’s disease (PD). Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly induced the loss of dopaminergic cells in the substantia nigra and deletion of dopamine in the striatum, which was attenuated by long-term (7 weeks) dietary supplementation with curcumin at a concentration of 0.5% or 2.0% (w/w). Although curcumin did not prevent the MPTP-induced apoptosis of neuroblasts in the subventricular zone (SVZ), it promoted the regeneration of neuroblasts in the anterior part of the SVZ (SVZa) at 3 days after MPTP treatment. Furthermore, curcumin enhanced the MPTP-induced activation of microglia and astrocytes in the striatum and increased the expression of glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-β1 (TGFβ1) in the striatum and SVZ. GDNF and TGFβ1 are thought to play an important role in protecting neurons from injury in the central and peripheral nervous systems. These results suggest that long-term administration of curcumin blocks the neurotoxicity of MPTP in the nigrostriatal dopaminergic system of the mouse and that the neuroprotective effect might be correlated with the increased expression of GDNF and TGFβ1. Curcumin may be effective in preventing or slowing the progression of PD.

Predictive modes of action of pesticides in uterine adenocarcinoma development in rats.

Abstract: Endometrial adenocarcinoma in the uterine corpus is a malignant cancer that occurs in menopausal women and aged rodents. Because of the similarities in pathogenesis and morphology of endometrial adenocarcinoma in rodents and humans, prediction of the modes of action (MOA) in uterine carcinogenesis is important for extrapolation of rodent data to humans. Three MOAs have been accepted as major pathways for uterine carcinogenesis in rodents: 1) estrogenic activity, 2) increased serum 17beta-estradiiol (E2) to progesterone (P4) ratio and 3) modulation of estrogen metabolism to produce 4-hydroxyestradiol via P450 induction. Inhibition of estrogen excretion and increased aromatase in situ in the tumor are also a potential pathway. Here, chemicals showing uterine carcinogenicity were chosen from approximately 300 pesticides evaluated in Japan within the past decade, and their mechanisms were predicted using parameters from mechanistic and toxicity studies. Seven pesticides increased uterine tumor formation in rats, and the pathways of 4 pesticides could be predicted based on various mechanistic studies. The MOAs of cyenopyrafen and benthiavalicarb-isopropyl were predicted to be modulation of estrogen metabolism, while those of pyriminobac-methyl and spirodiclofen were predicted to be increased E2 to P4 ratio. The driven pathways of metazosulfuron and isopyrazam could not be predicted using several mechanistic studies. No mechanistic studies have been reported for sedaxane, which has a chemical structure and toxicological profile similar to isopyrazam. Our results indicated that appropriate mechanistic studies are useful for mechanism prediction in risk assessment. From this analysis, a flowchart showing a decision tree for predictive MOAs in uterine carcinogenesis was proposed.

Sodium bicarbonate protects uranium-induced acute nephrotoxicity through uranium-decorporation by urinary alkalinization in rats

Abstract: To evaluate the effectiveness of sodium bicarbonate (SB) in removing uranium and protecting animals from uranium toxicity, we intramuscularly administered 1 mg/kg of uranyl nitrate to 8-wk-old male SD rats, and 20 min after administration of uranyl nitrate, the animals were given a single oral administration of SB at 0.1, 0.3 or 1 g/kg. The SB treatment at a dose of 0.3 g/kg or more raised the pH of the rats' urine until 4 h after treatment, and it significantly reduced the uranium amounts in the kidneys at 1 day after treatment. In another experiment, rats were intramuscularly administered 1 mg/kg of uranyl nitrate, and 20 min later, the animals were treated with sodium bicarbonate (0.1 or 1 g/kg). The rats were autopsied at 1, 3 and 7 days after uranium treatment. High-dose SB resulted in a significant increase in urinary uranium excretion in the first 24 h and a reduction of uranium deposition in the kidneys and femurs, and it also significantly suppressed uranium-induced renal toxicity, as shown by both histopathology and clinical chemistry at 3 days after uranium treatment. Low-dose SB did not show such marked effects. Our findings demonstrated that the uranium decorporation effect of sodium bicarbonate was observed at the dosage showing urine alkalinization in rats and that decorporation effect of sodium bicarbonate might be beneficial if it is administered immediately after incorporation of soluble uranium.

Comparison of changes in urinary and blood levels of biomarkers associated with proximal tubular injury in rat models.

Abstract: To investigate useful biomarkers associated with proximal tubular injury, we assessed changes in levels of a focused set of biomarkers in urine and blood. Male rats administered a single dose or four doses of gentamicin (GM, 240 mg/kg/day) or a single dose of cisplatin (CDDP, 5 mg/kg) were euthanized on days 2 (the day after initial dosing) 5, or 12. At each time point, histopathological examination of the kidney and immunohistochemistry for biomarkers, kidney injury molecule-1 (Kim-1), lipocalin (NGAL), clusterin (CLU), cystatin C (CysC) and β2-microglobulin (β2M) were performed. Biomarker levels were measured in urine and blood. In both treatment groups, degenerated/necrotic proximal tubules and regenerated tubules were mainly observed on days 5 and 12, respectively. At the same time as these tubular injuries, urinary Kim-1, CysC and β2M levels were increased. Moreover, urinary levels of CysC and β2M in GM-treated animals and Kim-1 in CDDP-treated animals increased (on day 2) prior to tubular injury on day 5. This was considered to reflect the characteristics of drug toxicity. Although almost all of the biomarkers in blood were not sufficiently sensitive to detect proximal tubular injury, urinary and plasma β2M levels simultaneously increased. Therefore, in addition to urinary Kim-1, CysC and β2M levels, plasma β2M levels were also considered useful for detecting proximal tubular injury.

Time course of the incidence/multiplicity and histopathological features of murine colonic dysplasia, adenoma and adenocarcinoma induced by benzo[a]pyrene and dextran sulfate sodium.

Abstract: Benzo[a]pyrene (BP) is mutagenic but noncarcinogenic in the murine colon. Recently, we reported rapid induction of colonic tumors by treatment of CD2F1 mice with BP (125 mg/kg for 5 days) followed by a colitis inducer, dextran sulfate sodium (DSS) (4% in drinking water for 1 or 2 weeks). However, there are no reports on detailed time course and histopathological features of colonic proliferative lesions in this model. Here, we show the detailed time course of colonic dysplasia, adenoma and adenocarcinoma induced by treatment with BP, DSS, and a combination of the two (BP/DSS). In the colon of mice exposed to BP/DSS, 14.6 dysplastic foci per mouse were present one week after DSS treatment (week 4). The number of dysplastic foci decreased with time to 3.1 at week 9 and thereafter remained almost constant. At week 4, 1.5 adenocarcinomas were also observed, with a marked increase in numbers with time, reaching 29.3 at week 14. In contrast, the number of dysplastic foci induced by DSS alone showed a time course similar to that following BP/DSS treatment; however, only a few tumors appeared. Neither dysplastic foci nor neoplastic lesions were induced by BP only. In mice exposed to BP/DSS, β-catenin was demonstrated immunohistochemically in the nucleus and/or cytoplasm of the tumor cells, and this translocation from the cell membrane was evident in subsets of dysplastic foci. In dysplastic foci induced by DSS alone, β-catenin was absent in the nucleus/cytoplasm. These finding suggest that aberrant β-catenin accumulation in dysplastic foci is associated with tumor progression in this BP/DSS model.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) progress to date and future plans.

Abstract: The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) has been operational since 2005. A Global Editorial Steering Committee (GESC) manages the overall objectives of the project and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups (OWG), drawing upon experts from North America, Europe and Japan.Great progress has been made with 9 systems published to date – Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands in Toxicologic Pathology and the Integument and Soft Tissue and Female Reproductive System in the Journal of Toxicologic Pathology as supplements and on a web site – www.goreni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photo-micrographs of morphologic changes, information regarding pathogenesis, and key references. During 2012, INHAND GESC representatives attended meetings with representatives of the FDA Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and the National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to begin incorporation of INHAND terminology as preferred terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA. The interest in utilizing the INHAND nomenclature, based on input from industry and government toxicologists as well as information technology specialists, suggests that there will be wide acceptance of this nomenclature. The purpose of this publication is to provide an update on the progress of INHAND.

Expression and activation of EGFR and STAT3 during the multistage carcinogenesis of intrahepatic cholangiocarcinoma induced by 3′-methyl-4 dimethylaminoazobenzene in rats

Abstract: The purpose of this study was to investigate whether the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription-3 (STAT3) signal pathway contributes to the carcinogenesis of intrahepatic cholangiocarcinoma (ICC) induced by 3’-methyl-4 dimethylaminoazobenzene (3’Me-DAB) in rats. EGFR, TGFα, STAT3 and p-STAT3 in different stages of carcinogenesis were detected by immunohistochemistry (IHC). In situ hybridization (ISH) was applied to investigate the expression of STAT3 mRNA. Oval cells were verified by the immunohistochemical staining of alpha-fetoprotein (AFP), CD133 and epithelial cell adhesion molecules (EpCAM). Sequential development of necrosis, oval cell proliferation, cholangiofibrosis (CF) and ICC was observed in the liver of rats administered 3’Me-DAB. Oval cells showed positive expression of AFP, CD133 and EpCAM. The expression of EGFR was significantly higher in the ICC than in oval cells, CF or normal bile ducts (p<0.05), but there was no difference in EGFR expression between the other groups. The highest expression of p-STAT3 and TGFα was observed in CF. The expression of these two molecules in the ICC and oval cells was significantly higher than in normal bile ducts (p<0.05). Elevation of STAT3 mRNA was detected during carcinogenesis as shown by ISH, strong intensity was observed in the ICC and moderate intensity was observed in oval cells and CF. These observations suggest that the EGFR and STAT3 signal pathway contributes to the carcinogenesis of ICC. High activity of STAT3 during the carcinogenesis of ICC may be the result of high activity of EGFR triggered by TGFα.

Histopathological evaluation of the ocular-irritation potential of shampoos, make-up removers and cleansing foams in the bovine corneal opacity and permeability assay

Abstract: The bovine corneal opacity and permeability (BCOP) assay is an alternative method to the in vivo Draize eye test in rabbits for evaluating eye irritation in vitro. Here, we compared the numerical results of the BCOP assay with the corresponding histopathology for three different corneas for each test substance, including commercially available shampoos, make-up removers and cleansing foams that contained surfactants and other ingredients. The histopathological score was defined based on the severity of lesions in the corneal epithelium. The histopathological findings and scores of the three sections for each test substance were comparable. The in vitro irritancy score (IVIS) generally corresponds to the corneal irritant potential of the test substances assigned on the basis of the histopathological findings in this study. In the present study, we characterized the histopathology of the corneal epithelium and stroma and especially showed that the corneal epithelial injury caused by test substances might be important in assessment of test substances that are mild eye irritants (category 2B) as classified by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS), as corneal lesions suggestive of classification into category 2B were localized on the border between the corneal epithelium and stroma, which contained cell elements related to assessment of prognosis of an in vivo eye injury. Histopathological assessment might be useful in predicting in vivo ocular irritation, particularly for test substances with an IVIS >3.1 but ≤25 that are classified as mild irritants (category 2B) according to the UN GHS.

Lipopolysaccharide induces expression of collagen VI in the rat lung.

Abstract: The involvement of the lung during the septic systemic inflammatory response elicited by administration of lipopolysaccharide (LPS) was investigated. Eight-week-old male Sprague–Dawley rats were injected i.p. with 15 mg/kg LPS. After 24 h, the lungs were excised to evaluate the cellular responses to LPS. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis revealed that type VI collagen (ColVI) was extremely upregulated during sepsis in the rat lung within the first 24 h of LPS administration. Upregulation of ColVI protein and its mRNA was demonstrated by Western blot analysis, real time PCR, and immunohistochemistry. To the best of our knowledge, this is the first report demonstrating the activation of ColVI in the rat lung at the early stage of systemic inflammation. Activation of ColVI might be involved in sepsis-mediated lung fibrosis at an early stage.

Responses to the Standard for Exchange of Nonclinical Data (SEND) in non-US countries.

Abstract: The Standard for the Exchange of Nonclinical Data (SEND), adopted by the US FDA, is part of a set of regulations and guidances requiring the submission of standardized electronic study data for nonclinical and clinical data submissions. SEND is the nonclinical implementation of SDTM (Study Data Tabulation Model), the standard electronic format for clinical regulatory submissions to FDA. SEND, SDTM, and the associated Controlled Terminology have been developed by CDISC (Clinical Data Interchange Standards Consortium). In order to successfully implement SEND, interdisciplinary contributions between sponsors and CROs, need a model for task allocation. This is being undertaken by the Pharmaceutical Users Software Exchange (PhUSE). Because SEND is currently the preferred submission format of the US FDA only and will become required by it starting in December 2016, only American academic societies and companies are actively involved. An exception to this is the INHAND initiative, which leads the way in standardizing terminology for toxicological pathology. On the other hand, international globalization of other clinical and nonclinical practices is not feasible because there are substantial differences between the US and non-US countries in CRO involvement in drug development. Thus, non-US countries must consider and develop approaches to SEND that meet their needs. This paper summarizes the activities of the major organizations involved in SEND development and implementation, discusses the effective use of SEND, and details a compliance scheme (research material of the Showa University School of Medicine) illustrating how pharmaceutical companies can complete a large amount of work up to an FDA application with the effective utilization of CROs and solution providers.

A spontaneously occurring malignant pituicytoma in a male sprague dawley rat.

Abstract: Pituicytoma is an extremely rare neoplasm derived from pituicytes, which are glial cells in the posterior lobe of the pituitary gland. A malignant pituicytoma was found in the intracranial cavity of a 55-week-old male Sprague-Dawley rat. Macroscopically, the tumor was located on the sphenoid bone and involved the pituitary gland. The tumor was composed of sheets of fusiform cells with spindle- or pleomorphic-shaped nuclei and abundant eosinophilic cytoplasms. The cells were arranged in a whirling or irregular growth pattern. Some tumor cells were bizarre multinucleated giant cells with cytoplasmic eosinophilic hyaline droplets. Many tumor cells were strongly positive for vimentin and glial fibrillary acidic protein, and some cells were positive for ED-1 and S-100. These findings closely resembled those of a giant cell glioblastoma derived from the pituitary gland, suggesting anaplastic pituicytoma. From our review of the literature, we believe this is the first report of a spontaneous malignant pituicytoma in a rodent.

Evaluation of the teratogenic effects of three traditional Chinese medicines, Si Jun Zi Tang, Liu Jun Zi Tang and Shenling Baizhu San, during zebrafish pronephros development

Abstract: The aim of this study was to evaluate the teratogenic effects of three common Chinese medical prescriptions, Si Jun Zi Tang (SJZT), Liu Jun Zi Tang (LJZT) and Shenling Baizhu San (SLBS), during zebrafish pronephros development. We used the transgenic zebrafish line Tg(wt1b:EGFP) to assess the teratogenic effects using 12 different protocols, which comprised combinations of 4 doses (0, 25, 250, 1,250 ng/mL) and 3 exposure methods [methods I, 12-36 hours post fertilization (hpf), II, 24-48 hpf, and III, 24-36 hpf]. As a result, few defects in the kidneys were observed in the embryos exposed to 25 ng/mL of each medical prescription. The percentage of kidney malformation phenotypes increased as the exposure concentrations increased (25 ng/mL, 0-10%; 250 ng/mL, 0-60%; 1,250 ng/mL, 80-100%). Immunohistochemistry for α6F, which is a basolateral and renal tubular differentiation marker, revealed no obvious defective phenotypes in either SJZT- or LJZT-treated embryos, indicating that these Chinese medical prescriptions had minimal adverse effects on the pronephric duct. However, SLBS-treated embryos displayed a defective phenotype in the pronephric duct. According to these findings, we suggest (1) that the Chinese medical prescriptions induced kidney malformation phenotypes that are dose dependent and (2) that the embryonic zebrafish kidney was more sensitive to SLBS than SJZT and LJZT.

Induction of cell proliferation in the rat liver by the short-term administration of ethyl tertiary-butyl ether.

Abstract: In the present study, in continuation of our previous experiment in order to investigate the mode of action (MOA) of ethyl tertiary-butyl ether (ETBE) hepatotumorigenicity in rats, we aimed to examine alterations in cell proliferation, that are induced by short-term administration of ETBE. F344 rats were administered ETBE at doses of 0, and 1,000 mg/kg body weight twice a day by gavage for 3, 10, 17 and 28 days. It was found that the previously observed significant increase of P450 total content and hydroxyl radical levels after 7 days of ETBE administration, and 8-OHdG formation at day 14, accompanied by accumulation of CYP2B1/2B2, CYP3A1/3A2, CYP2C6, CYP2E1 and CYP1A1 and downregulation of DNA oxoguanine glycosylase 1, was preceded by induction of cell proliferation at day 3. Furthermore, we observed an increase in regenerative cell proliferation as a result of ETBE treatment at day 28, followed by induction of cell cycle arrest and apoptosis by day 14. These results indicated that short-term administration of ETBE led to a significant early increase in cell proliferation activity associated with induction of oxidative stress, and to a regenerative cell proliferation as an adaptive response, which could contribute to the hepatotumorigenicity of ETBE in rats.

Application of a compact magnetic resonance imaging system for toxicologic pathology: evaluation of lithium-pilocarpine-induced rat brain lesions.

Abstract: Magnetic resonance imaging (MRI) is a useful noninvasive tool used to detect lesions in clinical and veterinary medicine. The present study evaluated the suitability of a new easy-to-use compact MRI platform (M2 permanent magnet system, Aspect Imaging, Shoham, Israel) for assisting with preclinical toxicologic pathology examination of lesions in the rat brain. In order to induce brain lesions, male Sprague-Dawley rats were treated once with lithium chloride (127 mg/kg, intraperitoneal [i.p.]) followed by pilocarpine (30 mg/kg, i.p.). One week after dosing, the perfused, fixed brains were collected, analyzed by the MRI system and examined histopathologically. MRI of the brain of treated rats revealed areas of high T1 and middle to low T2 signals, when compared with the controls, in the piriform cortex, lateral thalamic nucleus, posterior paraventricular thalamic nucleus and posterior hypothalamic nucleus of the cerebrum. The altered MRI signal areas were consistent with well-circumscribed foci of neuronal cell degeneration/necrosis accompanied by glial cell proliferation. The present data demonstrated that quick analysis of fixed organs by the MRI system can detect the presence and location of toxicologic lesions and provide useful temporal information for selection of appropriate sections for histopathologic examination before routine slide preparation, especially in complex and functionally heterogeneous organs such as the brain.

Histological dermal changes caused by preparation and application procedures in percutaneous dose toxicity studies in dogs, rabbits and rats

Abstract: We reevaluated histological slides of dorsal skin in control animals from past percutaneous dose toxicity studies using dogs, rabbits and rats to provide background data concerning histological changes related to preparation and application procedures and vehicles or embrocations of every variety. Acanthosis, dermal or perifollicular inflammatory cell infiltration in dogs; hyperkeratosis, acanthosis, dermal inflammatory cell infiltration or hemorrhage in rabbits; and acanthosis, dermal inflammatory cell infiltration, crust or foreign body granuloma in rats were present as procedure-related underlying histological changes in the control animals. Four mechanical acts, (1) rubbing with gauze to remove an administered substance for reapplication, (2) use of a taut bandage to avoid slipping from the application site, (3) peeling a patch off as a preparation procedure for reapplication, and (4) clipping or shaving, were considered to cause injury to the skin. The degree of influence of the various application procedures was found to be as follows: sham, lotion < cream < ointment and tape in dogs; untreated control, sham < lotion < tape and poultice in rabbits; and sham, sodium carboxymethylcellulose < olive oil and lotion < ointment and tape in rats. The degree of ointment influence on rabbits is equivocal.

Spontaneous necrotizing sialometaplasia of the submandibular salivary gland in a Beagle dog.

Abstract: A single mass was found on the left submandibular salivary gland at necropsy of a 15-month-old male commercially bred laboratory Beagle dog from a control dose group from a repeat toxicity study. Microscopically, the mass was composed of a well-demarcated area of coagulative necrosis surrounded and separated from the normal salivary gland tissue by a thick fibrovascular capsule. Necrosis was admixed with areas of hemorrhage, fibrin, edema, fibrinoid necrosis of the vascular tunica media, and thrombosis of small and large vessels. Within the necrotic tissue, there was marked ductal hyperplasia, and squamous metaplasia of duct and acinar epithelium. The mass was diagnosed as necrotizing sialometaplasia of the submandibular gland. Hyperplastic ductal elements and squamous metaplasia can be mistaken microscopically with squamous cell carcinoma. Therefore, pathologists should be aware of this lesion as to avoid errors in the diagnosis of this benign pathologic condition.

Spontaneous and bilateral necrosis of the femoral head in a young experimental beagle dog.

Abstract: This report describes the pathological characterizations of a rare case of necrosis of the femoral head that was spontaneous, bilateral, avascular and nontraumatic. A 14-month-old beagle dog was presented with pain in the hind limbs. At necropsy, the articular surface in the bilateral femoral head was markedly irregular. There were no gross abnormalities other than in the hip joints. Microscopically, a wide range of trabecular bone necrosis localized in the subchondral area was observed in both femoral heads. In the right femoral head, fibrosis and proliferative vessels were noted in the subchondral area. The articular cartilage was thickened irregularly, but there was no evidence of cartilage necrosis. The bone marrow adjacent to the affected area showed severe depression. In the metaphysis, atrophic bone marrow, but not bone necrosis, was observed. This was a rare case of spontaneous necrosis of the femoral head in an experimental beagle dog.

Histopathology and oxidative stress analysis of concomitant misoprostol and celecoxib administration.

Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs), non-selective or selective inhibitors of cyclooxygenase (COX-1 and -2), reduce pain and inflammation associated with arthritic diseases. Celecoxib, a COX-2-selective inhibitor providing decreased gastric injury relative to non-selective NSAIDs, is commonly prescribed. Misoprostol, a prostaglandin analog, supplements NSAID-inhibited prostaglandin levels. As concomitant celecoxib and misoprostol administration has been shown to intensify renal adverse effects, this article examined the influence of concomitant administration on hepatic histopathology, oxidative stress, and celecoxib concentration. On days 1 and 2, rat groups (n = 6) were gavaged twice daily (two groups with vehicle and two groups with 100 μg/kg misoprostol). From day 3 to day 9, one celecoxib dose (40 mg/kg) replaced a vehicle dose of one group and one group received celecoxib in addition to misoprostol. Livers were harvested on day 10. No hepatic abnormalities were observed denoting a lack of influence by either drug. Also no change in mean biomarker levels was detected. The changes in hepatic celecoxib concentration in the misoprostol-receiving group compared to control were not significant. Thus misoprostol does not influence hepatic celecoxib effects in terms of histopathology, oxidative stress, or celecoxib concentration level at the dosage and duration examined.

Autopsy results of a case of ingestion of sodium hydroxide solution.

Abstract: Sodium hydroxide is a strongly corrosive alkali. We describe herein a case of suicide by ingestion of sodium hydroxide. A man in his 80s was found dead with a mug and a bottle of caustic soda. Macroscopically, liquefaction and/or disappearance of esophagus, trachea and lung tissue and a grayish discoloration of the mucosa of the stomach were seen along with blackish brown coloration of the skin, mouth, and oral cavity. The contents of the gastrointestinal tract showed a pH level of 7-8 on pH indicator strips. Histopathologically, liquefactive necrosis of remnant lung tissue and the stomach were seen. As biological reactions such as vasodilatation and inflammation were not detected in these organs, only a short number of hours must have passed between ingestion and death. This human case provides valuable information concerning the direct irritation induced by systemic exposure to corrosive substances.

Establishment of a syngeneic orthotopic model of prostate cancer in immunocompetent rats

Abstract: We previously established 3 cell lines (PLS10, PLS20 and PLS30) from a chemically-induced prostate carcinoma in F344 rats, and demonstrated high potential for metastasis in nude mice. In the present study, we investigated the feasibility of establishing an orthotopic model using the 3 rat prostate cancer cell lines in immunocompetent rats with the aim of resolving species-mismatch problems and defects of immune systems. The PLS10, PLS20 and PLS30 cell lines were injected into the ventral prostates of 6-week-old rats, which were then sacrificed at experimental weeks 4 and 8. Tumor mass formation was found in rats with PLS10, but not in those with PLS20 or PLS30. Additionally, metastatic carcinomas could be detected in lymph nodes and lungs of PLS10-inoculated rats. Genetic analysis demonstrated K-ras gene mutations in PLS10 and PLS20, but not in PLS30 cells. There were no mutations in p53 and KLF6. In conclusion, we established a syngeneic orthotopic model for prostate cancer in immunocompetent rats simulating human castration-resistant prostate cancer (CRPC), which should prove useful for development and validation of therapeutic agents, especially with immunotherapy.

The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA microarray analysis with laser-capture microdissection samples

Abstract: Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3’ and 5’ primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.

Lipomatosis of the canine parotid gland: case report with a literature review.

Abstract: In this report, we describe a case of lipomatosis in the left parotid gland of an eight-year-old female Shetland sheepdog and review the relevant literature. Preoperative diagnosis of lipomatosis with Tru-Cut biopsy presented difficulties in this case. The postoperative diagnosis was based on the gross appearance of a non-infiltrative, circumscribed swollen mass in the parotid gland and the histological appearance of normal adipocytes that infiltrated into the parotid gland without involving surrounding structures. Additionally, flotation of the whole parotid gland in formalin solution together with well-maintained residual lobulation and a well-maintained configuration on the cut surface were identified as subsidiary characteristics of lipomatosis of the parotid gland. Lipomatosis of the parotid gland has been rarely reported in dogs and is a poorly understood condition.

Modifications of azoxymethane-induced carcinogenesis and 90-day oral toxicities of 2-tetradecylcyclobutanone as a radiolytic product of stearic acid in F344 rats.

Abstract: A 90-day oral toxicity test in rats was performed to evaluate the toxicity of 2-tetradecylcyclobutanone (2-tDCB), a unique radiolytic product of stearic acid. Six-week-old male and female F344 rats (n=15/group) were given 2-tDCB at concentrations of 0, 12, 60 and 300 ppm in a powder diet for 13 weeks. Slight dose-dependent increases in serum total protein and albumin in male rats were found, but these changes were not considered to be a toxic effect. The fasting, but not non-fasting, blood glucose levels of the male rats in the 300 ppm group and female rats in the 60 and 300 ppm groups were lower than those of the controls. Gas chromatography-mass spectrometry analysis showed dose-dependent accumulation of 2-tDCB in adipose tissue, notably in males. Next, we performed an azoxymethane (AOM)-induced two-stage carcinogenesis study. After injection of 6-week-old male F344 rats (n=30/group) once a week for 3 weeks, the animals received 2-tDCB at concentrations of 0, 10, 50 and 250 ppm in a powder diet for 25 weeks. The incidences of colon tumors for the 2-tDCB dosages were 34%, 45%, 40% and 37%, respectively, and were not statistically significant. These data suggest that 2-tDCB shows no toxic or tumor-modifying effects under the present conditions, and that the no-observed-adverse-effect level for 2-tDCB is 300 ppm in both sexes, equivalent to 15.5 mg/kg b.w./day in males and 16.5 mg/kg b.w./day in females.

Spontaneous early-onset glomerulonephritis in a 8-week-old male Crj:CD1 (ICR) mouse

Abstract: Glomerular lesions including membranoproliferative glomerulonephritis occur spontaneously in aged mice, but they are rare in young animals. In our laboratory, spontaneous glomerulonephritis was observed in an 8-week-old male Crj:CD1 (ICR) mouse. Macroscopically, the bilateral kidneys were discolored, but no edema or ascites was observed. Glomerular lesions were characterized by a thickening of capillary walls, a double-contoured basement membrane and mesangial expansion due to increased amounts of matrix. Ultrastructurally, mesangial interposition in the capillary wall and subendothelial deposition of basement membrane-like material were observed. No evidence of immune complex deposition or amyloid was found. On the basis of the observed clinical pathology and histopathology, a secondary form of glomerular lesion was excluded. The glomerular lesion was compatible with glomerulonephritis in a young Crj:CD1 (ICR) mouse.

Obstructive nephropathy induced with DL-potassium hydrogen tartrate in F344 rats.

Abstract: We experienced obstructive nephropathy in F344 rats treated with DL-potassium hydrogen tartrate (PHT) in a 13-week oral repeated dose toxicity study. Six-week-old male and female F344/DuCrj rats were fed a diet containing up to 2.0% PHT for 13 weeks. Microscopical findings including irregular dilation of the distal tubule lumen, foreign body giant cells, inflammatory cell infiltration, and regeneration of renal tubules were observed focally or multifocally in the renal cortex and/or medulla in the 0.5% and higher dosage groups of both sexes. The severity of these lesions increased in a dose-dependent manner. In the urinalysis, an increase in protein and white blood cells or the concentration of tartaric acid was detected in the 0.5% PHT and higher dosage groups of both sexes or males, respectively, though conventional blood biochemical analysis did not indicate failure of renal function. These results indicate that the PHT induces obstructive nephropathy in rats. There were no other treatment-related changes in other organs.

Mead acid supplementation does not rescue rats from cataract and retinal degeneration induced by N-methyl-N-nitrosourea

Abstract: Fatty acids and their derivatives play a role in the response to ocular disease. Our current study investigated the effects of dietary mead acid (MA, 5,8,11-eicosatrienoic acid) supplementation on N-methyl-N-nitrosourea (MNU)-induced cataract and retinal degeneration in Sprague-Dawley rats. Experiment 1 was designed to inhibit cataract formation, with the dams fed a 2.4% MA or basal (<0.01% MA) diet during lactational periods. On postnatal day 7, male pups received a single intraperitoneal (ip) injection of 50 mg/kg MNU or vehicle. Lens opacity and morphology were examined 7 and 14 days after the MNU injection. Experiment 2 was designed to inhibit retinal degeneration and was performed with female postweaning rats. In this experiment, dams were fed the 2.4% MA or basal diet during the lactational periods. Thereafter, the female pups were continuously fed the same diets during their postweaning periods. On postnatal day 21 (at weaning), pups received a single ip injection of 50 mg/kg MNU. Retinal morphology was examined 7 days after the MNU injection. In experiment 3, six-week-old female rats were fed the 2.4% MA or basal diet starting at one week before the MNU injection and were then continuously fed the same diets until sacrifice. Rats at 7 weeks of age were given a single ip injection of 40 mg/kg MNU, and the retina was then examined morphologically one week after the MNU injection. In experiment 1, mature cataract was found in all of the MNU-treated groups, with or without MA supplementation. In experiments 2 and 3, atrophy of both the peripheral and central outer retina occurred in all rats exposed to MNU, with or without MA supplementation, respectively. The severities of the cataracts and retinal atrophy in the rats were similar regardless of MA supplementation. Dietary mead acid, which is used as a substitute in essential fatty acid deficiency in the body, does not modify MNU-induced cataract and retinal degeneration in rat models.