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A 6-month-old female beagle dog, assigned to the low-dose group in a toxicity study, was evaluated for compound toxicity, and spontaneous hyperadrenocorticism was suspected. The animal had an externally apparent distended abdomen on clinical examination upon arrival. Pre-dose clinical pathology showed slightly higher erythroid parameters and stress leukogram on hematology; plasma biochemistry showed higher total protein, gamma-glutamyl transferase, total cholesterol, and triglyceride levels than the reference data. On necropsy, a prominent increase in adipose tissues of the subcutis and abdomen and increased weight of the adrenal gland and liver were observed. Histopathology revealed diffuse hyperplasia of adrenocortical cells in the zona fasciculata and reticularis, cortical atrophy of the thymus, and abundant glycogen accumulation in the hepatocytes. These findings were incidental and not test-substance-related. Electron microscopy of the adrenocortical cells in the zona fasciculata revealed decreased typical translucent lipid droplets, increased electron-dense lipid droplets, and abundant smooth endoplasmic reticulum and lysosomes. Additionally, increased numbers of various sizes and forms of mitochondria with tubular, vesicular, or lamellar cristae compared to that of normal animals were observed. These ultrastructural characteristics of the adrenocortical cells suggested hyperfunction. The pre-dose plasma cortisol levels were slightly higher than those of other females assigned to the toxicity study, while plasma adrenocorticotropic hormone levels were within the normal range. These findings indicate that hyperadrenocorticism is a possible cause of the systemic changes in this case.

Objectives Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) and a devastating worldwide health concern. Development of safe and effective treatments is not only important for interventions during the current pandemic, but also for providing general treatment options moving forward. We have developed ensitrelvir, an antiviral compound that targets the 3C-like protease of SARS-CoV-2. In this study, a delayed-treatment mouse model was used to clarify the potential in vivo efficacy of ensitrelvir. Methods Female BALB/cAJcl mice of different ages were infected with the SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021) or mouse-adapted SARS-CoV-2 MA-P10 and then 24 h post-infection orally administered various doses of ensitrelvir or vehicle. Viral titres and RNA levels in the lungs were quantified using VeroE6/TMPRSS2 cells and RT–qPCR, respectively. Body weight loss, survival, lung weight, cytokine/chemokine production, nucleocapsid protein expression and lung pathology were evaluated to investigate the in vivo efficacy of ensitrelvir. Results Based on infectious viral titres and viral RNA levels in the lungs of infected mice, ensitrelvir reduced viral loads in a dose-dependent manner. The antiviral efficacy correlated with increased survival, reduced body weight loss, reduced pulmonary lesions and suppression of inflammatory cytokine/chemokine levels. Conclusions This was the first evaluation of the in vivo anti-SARS-CoV-2 efficacy of ensitrelvir in a delayed-treatment mouse model. In this model, ensitrelvir demonstrated high antiviral potential and suppressed lung inflammation and lethality caused by SARS-CoV-2 infection. The findings support the continued clinical development of ensitrelvir as an antiviral agent to treat patients with COVID-19.

The present report describes an adrenal dysplasia in which developmental abnormality was observed in the adrenal gland of a six-week-old male Crl:CD(SD) rat. Microscopically, a localized lesion composed of mildly vacuolated adrenal fasciculata cells with a slightly disturbed cord structure and containing areas with high cell density was observed in a unilateral adrenal gland; no macroscopical changes were detected in the organ. The areas with high cell density consisted of two cell types. One type included small cells with a round nucleus and acidophilic cytoplasm, and the cells were positive for steroidogenic factor-1 (SF-1) but negative for nestin. The other type of cells had a spindle to polygonal shape, clear nucleus, and a cytoplasm with an obscure boundary; the cells were positive for nestin but negative for SF-1, neuronal nuclear antigen, and chromogranin A. These results suggested that the former type of cells were adrenal cortex cells and that the latter were immature neuronal cells. Considering that immature adrenal cortex cells and neural crest cells (future adrenal medulla) are mixed during a stage in rat adrenal gland development, we concluded that the observed lesion was caused by developmental abnormality. To the best of our knowledge, this is the first report to describe dysplasia in rat adrenal glands.

We herein investigated the histopathological features, including proliferative activity and immunoexpression, of pancreatic islet cell tumors (ICTs) in male SD rats induced by streptozotocin (STZ) and nicotinamide (NA), and discussed their relevance to biological behaviors and prognoses. A total of 70 and 43% of rats developed ICTs 37-45 weeks after the treatment with STZ (50 or 75mg/kg, i.v.) and NA (350mg/kg, twice, p.o.), respectively. Among the islet tumors observed in the STZ/NA-treated groups, 75% were adenomas, while 25% were carcinomas. Most STZ/NA-induced carcinomas were characterized by well-differentiated tumor cells with/without local invasion into the surrounding tissues, and weak proliferative activity. No outcome such as distance metastasis and death was noted. All of the ICTs strongly expressed insulin, part of which had hormone productivity; however there were no hypoglycemia-related clinical signs such as convulsion in these rats 36 weeks after the treatment. These results suggested that rat ICTs induced STZ/NA have small impact on biological activity or prognosis. STZ/NA treatment significantly increased of focal proliferative lesions in the kidney, liver and adrenal glands other than pancreatic islets. Of the STZ/NA-induced kidney tumors, more than 60% were renal cell adenomas, and many of them were basophilic type. The incidence of eosinophilic or clear cell type of tumors was less than 10%, respectively. Immunohistochemical analyses revealed that many of the STZ/NA-induced basophilic type of renal tumors were derived from proximal tubules, whereas the clear cell and eosinophilic types were derived from collecting tubules.

To identify the molecular profiles of islets from alloxan (ALX)- and streptozotocin (STZ)-treated rats, a microarray-based global gene expression analysis was performed on frozen islets isolated via laser capture microdissection. At 6 weeks old, rats were injected with ALX (40 mg/kg) or STZ (50 or 100 mg/kg) and then euthanized 24 hr later. Histopathological analysis showed β-cell necrosis, macrophage infiltration, and islet atrophy. The extent of these changes was more notable in the STZ groups than in the ALX group. Transcriptome analysis demonstrated a significant up- or downregulation of cell cycle arrest–related genes in the p53 signaling pathway. Cyclin D2 and cyclin-dependent kinase inhibitor 1A, mediators of G1 arrest, were remarkably altered in STZ-treated rats. In contrast, cyclin-B1 and cyclin-dependent kinase 1, mediators of G2 arrest, were remarkably changed in ALX-treated rats. Genes involved in the intrinsic mitochondria-mediated apoptotic pathway were upregulated in the ALX and STZ groups. Moreover, heat-shock 70 kDA protein 1A ( Hspa1a), Hsp90ab1, and Hsph1 were upregulated in ALX-treated rats, suggesting that ALX treatment injures β cells via endoplasmic reticulum stress. These results contribute to a better understanding of gene expression in the pathogenesis of islet toxicity.

Epertinib (S-222611) is a potent, reversible, and selective tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), human EGFR2 (HER2), and human EGFR4. We developed experimental brain metastasis models by intraventricular injection (intraventricular injection mouse model; IVM) of HER2-positive breast cancer (MDA-MB-361-luc-BR2/BR3) or T790M-EGFR-positive lung cancer (NCI-H1975-luc) cells. After a single oral administration, epertinib and lapatinib concentrations in brain metastatic regions were analyzed by quantitative imaging mass spectrometry. In the NCI-H1975 lung cancer IVM, the concentration of epertinib in brain metastasis was comparable to that of lapatinib. However, in the MDA-MB-361 breast cancer IVM, the concentration of epertinib in brain metastasis was >10 times higher than that of lapatinib. Furthermore, the epertinib tumor-to-normal brain ratio was ~4 times higher than that of lapatinib. Blood-tumor barrier (BTB) permeability was assessed in each brain metastatic region. In the lung cancer model, fluorescently labeled dextran was more highly detected in brain metastatic regions than in brain parenchyma. However, in breast cancer models, dextran fluorescence intensity in brain metastatic regions and brain parenchyma were comparable, suggesting that the BTB remained largely intact. Epertinib would be promised as a therapeutic agent for HER2-positive breast cancer with brain metastasis.

Traditional biomarkers such as alanine and aspartate aminotransferase (ALT, AST) and total bilirubin (TBIL) have been widely used for detecting drug-induced liver injury (DILI). Although the Food and Drug Administration (FDA) proposed standardized thresholds for human as Hy's law, those for animals have not been determined, and predictability of these biomarkers for future onset of hepatic lesions remains unclear. In this study, we investigated these diagnostic and predictive performance of 10 traditional biomarkers for liver injury by receiver-operating characteristic (ROC) curve, using a free-access database where 142 hepatotoxic or non-hepatotoxic compounds were administrated to male rats (n=5253). Standardization of each biomarker value was achieved by calculating the ratio to control mean value, and the thresholds were determined under the condition of permitting 5% false positive. Of these 10 biomarkers, AST showed the best diagnostic performance. Furthermore, ALT and TBIL also showed high performance under the situation of hepatocellular necrosis and bile duct injury, respectively. Additionally, the availability of the diagnostic thresholds in difference testing facility was confirmed by the application of these thresholds to in-house prepared dataset. Meanwhile, incorrect diagnosis by the thresholds was also observed. Regarding prediction, all 10 biomarkers showed insufficient performance for future onset of hepatic lesions. In conclusion, the standardized diagnostic thresholds enable consistent evaluation of traditional biomarkers among different facilities, whereas it was suggested that novel biomarker is required for more accurate diagnosis and prediction of DILI.

Cardiotoxicity is one of the major safety concerns in drug development. Therefore, detecting and monitoring cardiotoxicity throughout preclinical and clinical studies is important for pharmaceutical companies. The present study was conducted in order to explore a plasma miRNA biomarker for cardiotoxicity in rats. As organ specificity is an important factor for a biomarker, we analyzed the miRNA microarray dataset in 55 organs/tissues in normal male rats. Based on this analysis, 5 miRNAs consisting of miR-208 (heart-specific), miR-1, miR-133a, miR-133b (heart and skeletal muscle-specific) and miR-206 (skeletal muscle-specific) were selected. Next, we evaluated the usefulness of those 5 miRNAs as circulating biomarkers in rats administered with single-dose isoproterenol or doxorubicin. Plasma miR-208 was consistently increased through 24 h after dosing in rats administered with isoproterenol, whereas plasma concentrations of cardiac troponin (cTn) showed transient elevation. In contrast, the plasma levels of miR-1, miR-133a, miR-133a and miR-206 were elevated after treatment with doxorubicin, probably as a result of skeletal muscle toxicity. Additionally, the plasma miR-208 level was elevated even after repeat-dose administration (once daily for 7 days) of isoproterenol under which the pathological condition proceeded to the sub-chronic phase such as fibrosis. Thus, our data suggest that miR-208 is a promising plasma biomarker for cardiotoxicity in rats. Monitoring of plasma miR-208 levels in rats may lead to more accurate evaluation of cardiotoxicity in preclinical studies.

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver-operating characteristic (ROC) analysis using more cardiotoxic and non-cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi-gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi-gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi-gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs. Copyright © 2013 John Wiley & Sons, Ltd.

BACKGROUND The present study was conducted to evaluate the developmental toxicity in the endometrium and placenta due to GW501516 administration by gavage to pregnant rats. METHODS GW501516 was orally administered repeatedly to pregnant rats from gestation day (GD) 6 to 17 at a dose of 0, 30, and 100 mg/kg/day. In next study, GW501516 was also orally administered to pregnant rats on GD 7, 8, 9, 10, or 11 at a single dose of 275 or 350 mg/kg. In these studies, caesarean section was performed to examine the pregnancy outcome on GD21. Additionally, GW501516 was orally administered to pregnant rats on GD 10 at a single dose of 275 mg/kg. Placentae were subjected for temporal histological examinations on GD 11, 13, 15, or 17. RESULTS Placental malformation was induced by repeated administration of GW501516 at a dose of 100 mg/kg/day. Single oral administration of GW501516 at a dose of 275 and/or 350 mg/kg on GD 8, 9, 10, or 11 induced placental malformation, whereas GW501516 administered on GD 10 was the most effective for increasing placental malformation. Histopathologically, single oral administration of GW501516 on GD 10 induced cystic degeneration associated with cellular lysis of glycogen cells started from GD 15 in the basal zone. CONCLUSIONS High frequency of placental malformation was observed by the administration of GW501516. From GD 8 to 11, especially GD 10, is more sensitive period to induce the placental malformation.

Recent efforts have been made to identify useful urinary biomarkers of nephrotoxicity. Furthermore, the application of urine to the other toxicities as new biomarker source has been recently expanded. Meanwhile, correction of urinary biomarker concentrations according to fluctuations in urine flow rate is required for adequate interpretation of the alteration. The urinary biomarker-to-creatinine ratio (UBCR) is widely used because of the convenience, while the urinary biomarker-excretion rate is regarded as the gold standard corrective method. Because creatinine is a catabolite in energy production in muscles, we hypothesized that altered muscle mass could affect creatinine kinetics, ultimately affecting UBCR. However, no study has examined this hypothesis. In this study, we examined the influence of muscle mass gain on UBCR, using male Sprague-Dawley rats during the growth phase, 6–12-week old. Both plasma creatinine and excretion of urinary creatinine (Ucr excretion) showed increases with muscle mass gain in rats, in which the alterations of UBCR were lowered. The renal mRNA level of the organic cation transporter-2 (Oct2), a creatinine transporter, showed an age-related increase, whereas the mRNA level of multidrug and toxin extrusions-1 (Mate1) remained constant. Multiple regression analysis showed that the increase in creatinine clearance highly contributed to the age-related increase in Ucr excretion compared to the mRNA levels of Oct2 and Mate1. This suggested that the age-related increase in Ucr excretion may be attributable to the increased transglomerular passage of creatinine. In conclusion, the results suggest that muscle mass gain can affect creatinine kinetics, leading to underestimation of UBCR. Therefore, it is important to understand the characteristics of the corrective method when using urinary biomarker, the failure of which can result in an incorrect diagnosis.

Cardiotoxicity and musculoskeletal toxicity can be life-threatening, and thus have strong impact on both the development and marketing of drugs. Because the conventional biomarkers such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) have low detection power, there has been increasing interest in developing biomarkers with higher detection power. The current study examined the usefulness of several promising biomarkers, cardiac and skeletal muscle troponins (cTnI, cTnT and sTnI), fatty acid binding protein 3 (FABP3) and myosin light chain 3 (MYL3), and compared the obtained data to AST, LDH and CK in rat models treated with various myotoxic and non-myotoxic compounds (isoproterenol, metaproterenol, doxorubicin, mitoxantrone, allylamine, cyclosporine A, cyclophosphamide, aminoglutethimide, acetaminophen, methapyrilene, allylalcohol and α-naphthylisothiocyanate). These promising biomarkers were found to be superior to the conventional biomarkers, as they had a specific and abundant distribution within the heart and/or skeletal muscles; exhibited a positive correlation between the amplitude of increases and the degree of pathological alterations; had higher diagnostic accuracy for detecting pathological alterations; and had the additive effect of improving the diagnostic accuracy of conventional biomarkers. However, these promising biomarkers have several drawbacks including a rapid clearance, the fact that they are affected by renal dysfunction, and different reactivity to the mode of action of individual myotoxicants. In conclusion, the promising biomarkers cTnI, cTnT, FABP3, MYL3, and sTnI demonstrated sensitivity and specificity for cardiac and skeletal myotoxicity that was superior to those of conventional biomarkers, while we should pay attention to the drawbacks of these biomarkers when used in toxicity studies.

Recent research has revealed several useful urinary biomarkers of renal dysfunction such as acute kidney injury (AKI). For adequate evaluation of altered urinary biomarkers, it is necessary to consider the influence of varied urine flow rate (UFR). Calculation of the excretion rate of a urinary biomarker (UFR-correction) is the gold standard for the correction of UFR variation. An alternative method that is widely used is to calculate the ratio of the biomarker level to urinary creatinine (Ucr-correction). To date, the equivalence between these two methods has been examined only in a steady state situation such as diabetic nephropathy, and the urinary biomarkers examined have been limited to proteinuria and albuminuria. Therefore, we comprehensively addressed the relationship between Ucr-correction and UFR-correction of ten urinary biomarkers N-acetyl-β-d-glucosaminidase (NAG), lactate dehydrogenase (LDH), total protein, albumin, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β(2)-microglobulin, cystatin-c and glutathione S-transferase-α in non-steady state situations such as AKI. All ten urinary biomarkers showed larger amplitude increases in AKI by Ucr-correction than by UFR-correction in linear regression analysis. Moreover, receiver operating characteristic curves analysis suggested that, at least for the biomarkers NAG and LDH, Ucr-correction had higher diagnostic power than UFR-correction. We observed a decrease in the Ucr excretion in AKI that was accompanied by a reduction in creatinine clearance and reduced mRNA expression of the renal organic cation transporter-2, which is known to function as a transporter for creatinine. These results may provide a mechanistic explanation for the phenomena obtained in Ucr-correction. In conclusion, while Ucr-correction could overestimate the degree of AKI, it could also provide higher diagnostic power for AKI than UFR-correction. We should take into consideration of these backgrounds when using the Ucr-correction.

Cardiotoxicity represents one of the most serious side effects of new drugs. It is essential for pharmaceutical companies to detect potential cardiotoxicity of candidate drugs in non-clinical studies during the early stages of drug development. In this study, we aimed to detect potential genomic biomarkers of rat cardiotoxicity using a toxicogenomics approach. In order to achieve this, we induced cardiac lesions in rats following treatment with the three prototypical cardiotoxic compounds isoproterenol, doxorubicin and carbofuran. We then undertook histopathological examination and microarray analysis at 8 or 24 h after single dosing. Using statistical and cluster analysis, we extracted 36 probe sets commonly up-regulated by the three cardiotoxic compounds. GO analysis revealed that these genes were functionally associated with either chemotaxis, tissue regeneration, positive regulation of cell proliferation, cellular organization and morphogenesis events in accordance with the degeneration of myocardium and inflammation observed in the histopathology analysis. Most of selected genes showed transient up-regulation at different time point for each compound. However, among these genes, Spp1, Fhl1, Timp1, Ccl7 and Reg3b revealed a sustained up-regulation with high expression levels at both time points for all three compounds. In conclusion, even though definitive validation studies are required for the establishment of their usefulness and reliability, these identified genes may prove to be the most promising candidate genomic biomarkers of cardiotoxicity in rats.

Sprague-Dawley rats received once daily tail-vein injections of 360 mM dibasic sodium phosphate solution at 8 mL/kg for fourteen or twenty-eight days. Clinical examination revealed persistent proteinuria from three days after the first dosing and thereafter severe proteinuria from eight days or later in the phosphate-treated groups. Proteinuria developed without remission even after fourteen-day withdrawal in the fourteen-day dosed group. Phosphate-treated animals developed lipemia, hypercholesterolemia, anemia, higher serum fibrinogen levels, and lower serum albumin/globulin ratios on day 29. Renal weight increased significantly compared with control animals, and the kidneys appeared pale and enlarged with a rough surface. Histopathologically, glomerular changes consisted of mineralization in whole glomeruli, glomerular capillary dilatation, partial adhesion of glomerular tufts to Bowman’s capsule, and mesangiolysis. Ultrastructural lesions such as an increased number of microvilli, effacement of foot processes, and thickening of the glomerular basement membrane, and immunocytochemical changes in podocytes, mainly decreased podoplanin-positive cells and increased desmin expression, were also conspicuous in the phosphate-treated rats for twenty-eight days. Marked tubulointerstitial lesions were tubular regeneration and dilatation, protein casts, mineralization in the basement membrane, focal interstitial inflammation, and fibrosis in the cortex. These clinical and morphological changes were similar to features of human nephrotic syndrome.

To investigate the early changes involved in glomerular calcification, 360 mM Na2HPO4 was administered to rats at 8 mL/kg once daily via the tail vein singly or repeatedly. Urinalysis was carried out on days 1, 3, 5 and 8 of dosing, and rats were sacrificed on days 2, 4 and 9 for histopathological and electron microscopic examination of the kidneys. Following single dosing, there were no gross or histological findings, but electron microscopy revealed a number of vacuoles scattered within the Bowman's space. On day 4, minimal and focal mineralization was observed within the parietal epithelial cells. On day 9, mineralization was minimal to mild and localized within the parietal epithelial cells and glomerular basement membrane. Hypertrophy and increased mitotic figures were also frequently observed in the parietal epithelial cells. Low-density lamellar structures with effacement of podocytes, an increased number of microvilli and large amounts of debris filling the Bowman's space were the main electron microscopic changes on days 4 and 9. Increased urinary protein excretion correlated well with the glomerular changes. Immunohistochemically, increased expression of desmin and decreased expression of podoplanin were evident in glomeruli on day 9. The numbers of PCNA-positive podocytes and parietal epithelial cells showed a tendency to increase on day 9. These results suggest that the onset of glomerular calcification is preceded by primary podocyte damage.

Nedaplatin (NDP) is a second-generation antineoplastic platinum complex, with reduced nephrotoxicity. Two experiments were conducted to characterize the time course of changes of its nephrotoxicity and to further evaluate whether hydration is useful for amelioration of nephrotoxicity. In the first experiment, 8-week-old male rats treated with 6 or 9 mg kg(-1) NDP at a single intravenous dose were killed 2, 4, 7 and 14 days after dosing. In the second experiment, nonhydrated (Nhyd) or hydrated (Hyd) rats, treated with a single intravenous dose of 20 mg kg(-1) NDP, were killed 7 days after dosing. Besides renal function and histopathological examinations, the urinary excretion of platinum was measured. Histopathologically, NDP-induced nephrotoxicity was initially characterized by single cell and/or focal necrosis in the epithelium of distal tubules and collecting ducts as well as proximal tubules. In the later stage, subsequent cystic dilatation and regeneration occurred in these affected tubules, but incomplete tissue repair was still observed in the kidney 14 days after dosing. However, NDP-induced nephrotoxicity was dramatically reduced by hydration, while it had no clear effects on myelotoxicity. Measurement of urinary platinum excretion revealed that the total amount of platinum excretion was significantly higher in Hyd-NDP rats than that in Nhyd-NDP rats. In terms of urinary concentration, Hyd-NDP rats showed a lower concentration compared with that in Nhyd-NDP rats. The current results suggest that NDP has the potential risk to cause nephrotoxicity at a human therapeutic dose without hydration and that pre- and post-hydration at dosing can ameliorate this nephrotoxicity.

To elucidate the mechanism of nephrotoxicity caused by anti-neoplastic platinum complex, nedaplatin (NDP), treatment with a particular focus on the renal papillary toxicity, we analysed the gene expression profiles of two renal regions, the cortex (RC) and the papilla (RP) in rat kidneys. Male Wistar rats received a single administration of 10 mg/kg intravenous NDP or vehicle alone (5% xylitol solution) and were sacrificed six days later. The kidneys were dissected into the RC and RP and used for histopathological and microarray analyses. Histopathologically, NDP caused characteristic renal lesions, such as necrosis, single cell necrosis (with TUNEL TdT-mediated dUTP-biotin nick end labelling-positive) and regeneration/hyperplasia of the epithelial cells in both renal regions. Global gene expression analysis revealed that several genes involved in various functional categories were commonly deregulated in both renal regions, such as apoptosis, cell cycle regulation, DNA metabolism, cell migration/adhesion and cytoskeleton organization or genes induced as a perturbation of oxidative status and calcium homeostasis. Comparative analysis of gene expression between RC and RP revealed that genes encoding several subtypes of cytokeratins were identified as being specifically overexpressed in RP by the NDP treatment. Differential expression patterns of these selected genes observed by microarray analysis were further confirmed by quantitative real time RT-PCR and immunohistochemistry, which demonstrated increased expression of cytokeratins (CKs) 14 and 19 at the epithelium covering RP and/or collecting duct epithelium. Overall, the results contribute to understanding the renal molecular events of NDP-induced nephrotoxicity including novel potential biomarker genes encoding CKs 14 and 19 that may serve as indicators of renal papillary toxicity. Human & Experimental Toxicology (2007) 26, 767—780

The current experiment was conducted to evaluate whether prolongation of infusion time is useful for the amelioration of nedaplatin (NDP)-induced nephrotoxicity. Eight-week-old male rats were treated with 12 mg/kg NDP with the following dosing protocols, bolus injection, 1- or 4-hour continuous infusions (1hIF or 4hIF, respectively), and sacrificed 3 days after dosing. Urinary parameters were monitored on the day of the sacrifice, and the kidneys and femurs were removed for histopathological examination and bone marrow analysis. In the Bolus-NDP group, urine pH and specific gravity were decreased compared with the corresponding control group and glucosuria and occult blood were detected, while no abnormalities were noted in these urine parameters in the 4hIF-NDP group. Histopathological examination revealed slight to moderate renal lesions, such as single cell and/or focal necrosis in the epithelium of cortical tubules and collecting ducts in the renal papilla in the Bolus-NDP group. In the 1hIF- and 4hIF-NDP groups, there were clear reductions in the severity of renal tubular damage compared with the Bolus-NDP group, and the severity of renal tubular damage tended to reduce with increased infusion time. A TUNEL assay revealed that the numbers of TUNEL-positive cells in the 1hIF- and 4hIF-NDP groups were significantly lower than that in the Bolus-NDP group. There was a significant decrease in the number of TUNEL-positive cells in the 4hIF-NDP group compared with that in the 1hIF-NDP group. However, prolongation of the infusion time had no clear effect on the myelotoxicity of NDP. These results provide new evidence that prolongation of the infusion time is effective at minimizing the nephrotoxicity of NDP.

We determined the acute hypotensive effect of a single administration and the prophylactic effect of chronic treatment with Irbesartan, an angiotensin II receptor antagonist, on the development of end-organ damage in stroke-prone spontaneously hypertensive rats (SHRSP). The acute hypotensive effect was determined by a telemetrical method in SHRSP fed a normal diet. The prophylactic effect was examined by biochemical, histopathological and immunohistochemical methods in SHRSP fed a high-salt and low-protein diet. Irbesartan (3, 10, 30 and 100 mg/kg) reduced blood pressure in a dose-dependent manner without affecting heart rate. Irbesartan (3, 10 and 30 mg/kg) increased the survival rate in SHRSP fed a high-salt and low-protein diet. Furthermore, Irbesartan ameliorated the appearance of stroke symptoms in dose-dependent manner showing association with the prevention of microscopic lesions. Irbesartan ameliorated the increases in urinary protein excretion and N-acetyl-D-glucosamidase activity by preventing nephrosclerosis, as judged by microscopic observations, and ameliorated the increases in the expression of collagen IV and fibronectin in the kidney. These findings demonstrate that Irbesartan is a potent antihypertensive drug offering a protective effect on the development of hypertension-induced end-organ damages in SHRSP. Thus, Irbesartan is useful for the therapy of hypertension with end-organ damage.

Allelic loss of chromosome 2 is associated with radiation-induced murine acute myeloid leukemia. However, the gene, which contributes mainly to the leukemogenesis in a tumor suppression manner, has not been identified, yet. Expecting predisposition to acute myeloid leukemia, a radiation leukemogenensis experiment was performed with Pax6Sey3H, one of the small eye mutants. Deletion mapping of Pax6Sey3H indicated that the deleted segment extended from 106.00 to 111.47 Mb from the centromere with a length of 5.47 Mb on chromosome 2. Six known and seventeen novel genes were located in the segment. Pax6Sey3H mutants crossed back into C3H/He did not develop hematopoietic tumors spontaneously, but they did after exposure to γ-rays. The final incidence of hematopoietic tumor in mutants (45.2%) was higher than that in normal sibs (26.2%), and the survival curve of mutants shifted toward the left (p

Naturally occurring collagenofibrotic glomerulonephropathy was found in an intact male cynomolgus monkey at 4 years and 5 months of age. Clinical examination showed mild proteinuria and hypoproteinemia, and the kidneys were macroscopically pale and granular on the surface. Histopathology revealed diffuse panglomerular swelling with the accumulation of eosinophilic amorphous material that was negative for amyloid staining and periodic acid-Schiff, but positive for collagen fibers by Mallory-azan staining in the glomerular capillary wall and mesangial area. Immunohistochemistry revealed a strong positive reaction with anti-type III collagen antibody in the glomeruli. The mesangial area was widened, but there was no increase in the number of mesangial cells. No lesion was observed in the efferent/afferent arterioles or other arteries. Mild tubular degeneration was observed in the cortex, occasionally with cholesterin crystals and interstitial lymphocytic infiltration. Electron microscopy revealed various-sized collagen fibrils with typical banding periodicity in the subendothelial space and the mesangial area. The present case offers information on spontaneous and life-threatening primary glomerular fibrosis in a young monkey suggesting an idiopathic familial disease similar to those observed in humans.

Goniodysgenesis, malformation of the filtration angle, was observed in a New Zealand white rabbit supplied with 100 g/day rabbit chow containing 0.2% cholesterol for 10 months. Histopathology revealed cupping of the optic disc, atrophy of the retina and hyalinization of the ciliary body in the bilateral eyeballs. These findings corresponded with histopathological features caused by glaucoma. On the basis of these findings, we diagnosed this lesion as glaucoma, and classified it as primary glaucoma because of the presence of developmental defects of the filtration angle. In this case, hypercholesterolemia-induced changes, such as aggregation of lipid-laden macrophages and cholesterin clefts in the sclera or choroid, might cause deterioration of the lesions in glaucoma.

S-222611 is an oral, reversible tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), human EGFR2 (HER2) and human EGFR4 (HER4) with antitumor activities in animal models expressing these proteins. In phase I trials, S-222611 have shown to be well-tolerated with efficacy against HER2-positive tumors, including breast cancer metastatic to brain. The purpose of this study is to evaluate the mode of action for S-222611 treatment in brain metastasis using experimental mice models. At first, we examined the effect of S-222611 on HER2-positive breast cancer cell “MDA-MB-361” intracranial implantation model (ICM). Once daily oral administration of S-222611 significantly reduced the tumor volume at doses ranging from 25 to 100 mg/kg and the ED50 value was 21.2 mg/kg. Furthermore, S-222611 showed the survival prolonging activity in the ICM and the activity was significantly superior to that of lapatinib. It is known that patients with breast cancers having brain metastases display heterogeneous blood-tumor barrier (BTB) permeability that affects drug delivery and efficacy, also BTB integrities were reported to be disrupted in the ICM. To understand the mode of action for S-222611 in patient whose BTB was intact, we have developed the experimental brain metastases model by serial in vivo passage (MDA-MB-361 intraventricular injection model; IVM). BTB integrities in IVM were confirmed to be largely remaining intact by fluorescently labeled dextran incorporation study, and we examined the distribution of S-222611 on this model after single oral administration at the clinical relevant dose (50 mg/kg). By imaging mass spectrometry analysis, S-222611 concentration in the brain metastases of the IVM was significantly higher than that of lapatinib. The concentrations of S-222611 in the brain metastases of the IVM showed 5.2 µmol/L, which was remarkably higher than the in vitro IC50 value (26.5 nmol/L) of S-222611 for growth inhibition of MDA-MB-361, indicating that S-222611 could have potent antitumor activity in the brain metastases even though the intact BTB was remained. In fact, once daily oral administration of S-222611 significantly reduced the tumor volume in the brain at 50 mg/kg on the IVM. Taken together, in vitro/in vivo antitumor studies for MDA-MB-361 supported that S-222611 could be effective against brain metastases which have both intact and permeable BTB. In conclusion, S-222611 is expected to be useful for the treatment of patients with HER2-positive breast cancers, including those with brain metastases. Citation Format: Michinari Hirata, Yukari Tanaka, Satomi Shinonome, Tomomi Yamada, Mikinori Torii, Ken-ichi Nezasa, Hidekazu Tanaka. Antitumor activities of EGFR/HER2/HER4 kinase inhibitor S-222611 in the experimental brain metastases of HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4073. doi:10.1158/1538-7445.AM2017-4073

S-222611 is a potent and selective reversible tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), human EGFR2 (HER2), and human EGFR4 developed in Shionogi. Greater potency of anti-tumor activity than lapatinib was demonstrated in vitro and in animal models. In the intracranial implantation mice model implanted the HER2-positive cell line (MDA-MB-361-luc-2), S-222611 showed more potent anti-tumor activity than lapatinib. On the basis of higher penetration in brain of S-222611 than lapatinib indicated by the preclinical pharmacokinetic studies, we demonstrated the application of S-222611 to brain metastases with breast cancer. In order to enhance the clinical predictability of S-222611 to the treatment of brain metastases, we have developed the experimental brain metastases models in mice of HER2-positive breast cancer (MDA-MB-361-luc-2-BR2/BR3) or T790M-EGFR expressing lung cancer (NCI-H1975-luc). After a single oral administration of S-222611 or lapatinib, the concentrations of S-222611 and lapatinib in the brain metastatic regions of these mice models were analyzed by quantitative imaging mass spectrometry (IMS). In the NCI-H1975 lung cancer model (intraventricular injection model), the concentrations of S-222611 in brain metastases quantified by IMS were comparable to those of lapatinib. In contrast, in the MDA-MB-361 breast cancer model (intraventricular injection model), the concentrations of S-222611 in brain metastases were over 10 times higher than those of lapatinib, and the tumor-to-normal brain ratio of S-222611 was approximately 4 times higher than that of lapatinib. IMS revealed that the concentrations in brain metastases and tumor-to-normal brain ratio of S-222611 were significantly higher than those of lapatinib in the breast cancer mice model. In addition, the blood-tumor barrier (BTB) permeability in each brain metastatic region using these mice models was assessed simultaneously. In the lung cancer model, fluorescently labeled dextran was highly detected in the brain metastatic regions than brain parenchyma. However, in the breast cancer models, fluorescence intensities for dextran in the brain metastatic regions and brain parenchyma were comparable, indicating that the BTB was remained to be largely intact in brain metastases of the breast cancer model but disrupted in the lung cancer model. These results show that S-222611 is expected to be useful for the prevention and the prompt treatment of patients with HER2-positive breast cancer having brain metastases. Citation Format: Yukari Tanaka, Michinari Hirata, Satomi Shinonome, Mitsunobu Matsumoto, Wataru Nogami, Mikinori Torii, Ken-ichi Nezasa, Hidekazu Tanaka. Distribution analysis of S-222611 in brain metastases of HER2-positive breast cancer by quantitative imaging mass spectrometry: prospect for antitumor activity of EGFR/HER2/HER4 kinase inhibitor S-222611 against brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4085. doi:10.1158/1538-7445.AM2017-4085

Traditional biomarkers such as alanine and aspartate aminotransferase (ALT, AST) and total bilirubin (TBIL) have been widely used for detecting drug-induced liver injury (DILI). Although the Food and Drug Administration (FDA) proposed standardized thresholds for human as Hy's law, those for animals have not been determined, and predictability of these biomarkers for future onset of hepatic lesions remains unclear. In this study, we investigated these diagnostic and predictive performance of 10 traditional biomarkers for liver injury by receiver-operating characteristic (ROC) curve, using a free-access database where 142 hepatotoxic or non-hepatotoxic compounds were administrated to male rats (n=5253). Standardization of each biomarker value was achieved by calculating the ratio to control mean value, and the thresholds were determined under the condition of permitting 5% false positive. Of these 10 biomarkers, AST showed the best diagnostic performance. Furthermore, ALT and TBIL also showed high performance under the situation of hepatocellular necrosis and bile duct injury, respectively. Additionally, the availability of the diagnostic thresholds in difference testing facility was confirmed by the application of these thresholds to in-house prepared dataset. Meanwhile, incorrect diagnosis by the thresholds was also observed. Regarding prediction, all 10 biomarkers showed insufficient performance for future onset of hepatic lesions. In conclusion, the standardized diagnostic thresholds enable consistent evaluation of traditional biomarkers among different facilities, whereas it was suggested that novel biomarker is required for more accurate diagnosis and prediction of DILI.

The kidney has a capacity to recover from ischemic or toxic insults that result in cell death, and timely tissue repair of affected renal tubules may arrest progression of injury, leading to regression of injury and paving the way for recovery. To investigate the roles of neutrophil gelatinase-associated lipocalin (NGAL/lcn2) and osteopontin (OPN/spp1) during renal regeneration, the expression patterns of NGAL and OPN in the cisplatin-induced rat renal failure model were examined. NGAL expression was increased from day 1 after injection; it was seen mainly in the completely regenerating proximal tubules of the cortico-medullary junction on days 3-35; however, the expression was not seen in abnormally dilated or atrophied renal tubules surrounded by fibrotic lesions. On the other hand, OPN expression was increased from day 5 and the increased expression developed exclusively in the abnormal renal tubules. NGAL expression level well correlated with the proliferating activity in the regenerating renal epithelial cells, whereas OPN significantly correlated with the α-smooth muscle actin-positive myofibroblast appearance, expression of transforming growth factor (TGF)-β1, and the number of CD68-positive macrophages. Interestingly, rat renal epithelial cell line (NRK-52E) treated with TGF-β1 decreased NGAL expression, but increased OPN expression in a dose-dependent manner. Because increases of TGF-β1, myofibroblasts and macrophages contribute to progressive interstitial renal fibrosis, OPN may be involved in the pathogenesis of fibrosis; on the contrary, NGAL may play a role in tubular regeneration after injury. Expression analysis of NGAL and OPN would be useful to investigate the tubule damage in renal-toxicity.

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver-operating characteristic (ROC) analysis using more cardiotoxic and non-cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi-gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi-gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi-gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs.

The effects of estrogen on liver sinusoidal cells were investigated immunohistochemically and morphometrically. Male AKR/n mice were orally administered 30mg/kg/day of mestranol suspended in sesame oil for 7 days. Some of the mice were splenectomized before mestranol administration. Relative liver weight significantly increased from day l to day 7 in mestranol-administered mice with or without spleen. Relative spleen weight also significantly increased from day 3 to day 5 in these mice. Histologically, cellular increase was observed in both the sinusoid of the liver and the red pulp of the spleen. Almost all cells that increased in the sinusoid of the liver were positive for F4/80, and cells positive for PCNA also increased. These changes were observed in mestranol-administered mice with or without spleen. In the morphometrical study, the peak number of F4/80 positive cells in the liver was observed from day 5 and day 7 in normal and splenectomized mice administered mentranol, respectively. On day 5 and day 7, the number of F4/80 positive cells observed in the mestranol-administered splenectomized mice was significantly higher than that in mestranol-administered normal mice. The peak number of PCNA positive cells in the liver was observed on day 3 in mestranol-administered splenectomized mice, and on day 5 in mestranol-administered normal mice. The number of PCNA positive cells was significantly higher on day 3 in splenectomized rather than normal mice administered mestranol. These results suggest that the liver disturbances observed in women taking estrogen for a long time may be closely related to local proliferation of Kupffer cells in the sinusoid.

The present study was conducted to evaluate the developmental toxicity in the endometrium and placenta due to GW501516 administration by gavage to pregnant rats.GW501516 was orally administered repeatedly to pregnant rats from gestation day (GD) 6 to 17 at a dose of 0, 30, and 100 mg/kg/day. In next study, GW501516 was also orally administered to pregnant rats on GD 7, 8, 9, 10, or 11 at a single dose of 275 or 350 mg/kg. In these studies, caesarean section was performed to examine the pregnancy outcome on GD21. Additionally, GW501516 was orally administered to pregnant rats on GD 10 at a single dose of 275 mg/kg. Placentae were subjected for temporal histological examinations on GD 11, 13, 15, or 17.Placental malformation was induced by repeated administration of GW501516 at a dose of 100 mg/kg/day. Single oral administration of GW501516 at a dose of 275 and/or 350 mg/kg on GD 8, 9, 10, or 11 induced placental malformation, whereas GW501516 administered on GD 10 was the most effective for increasing placental malformation. Histopathologically, single oral administration of GW501516 on GD 10 induced cystic degeneration associated with cellular lysis of glycogen cells started from GD 15 in the basal zone.High frequency of placental malformation was observed by the administration of GW501516. From GD 8 to 11, especially GD 10, is more sensitive period to induce the placental malformation.

The present study aimed to establish candidate biomarker genes for the early detection of nephrotoxicity in mice, with a particular focus on nephrotoxicity caused by polyene macrolides. Comprehensive gene expression changes were evaluated using microarrays in a mouse model in which acute nephrotoxicity was induced by amphotericin B deoxycholate, trade name Fungizone. The upregulated genes identified through microarray analysis of kidney tissue of Fungizone-treated mice included several genes that have been reported as nephrotoxicity biomarkers in rats, and 14 genes were selected as candidate nephrotoxicity biomarkers. The usefulness of these genes as nephrotoxicity biomarkers in mice was evaluated further through expression profiling under several experimental conditions using real time RT-PCR. Expression of genes encoding kidney injury molecule 1, lipocalin 2, tissue inhibitor of metalloproteinase 1, and secreted phosphoprotein 1 was highly upregulated by Fungizone, nystatin, natamycin, amphotericin B methyl ester, and liposomal amphotericin B, and their area under the ROC curve values were more than 0.95. These genes were more sensitive at detecting nephrotoxicity than traditional clinical chemistry and histopathology parameters. This study provides novel evidence that these nephrotoxicity biomarker genes identified are translatable to mice, and that they are useful for early and sensitive detection of nephrotoxicity.

To study the membrane changes in the myocardium, the sarcolemmal permeability of 16-week-old SHR and age-matched WKY myocardium was examined morphologically using horseradish peroxidase (HRP) on the first and fourth day after doxorubicin administration. No HRP was seen in either saline-treated controls or doxorubicin-treated WKY on the first day. On the fourth day, the doxorubicin-treated WKY showed, HRP around the myocytes due to an increase in the vascular permeability, whereas, the HRP-reactant product was not seen within the sarcoplasm. Electron micros-copy revealed osmiophilic, fine granular HRP-reactant products in the extracellular space of the WKY myocardium on the fourth day. On the other hand, in doxorubicin-treated SHR myocardium, HRP was seen in a portion of the papillary muscle or myocardial cells in the anterior wall on the first day and HRP-positive myocytes were scattered in the anterio-lateral wall at the fourth day. Cross striations were still visible within some HRP-positive myocytes of SHR. Electron microscopy also showed HRP-reactant products, not only in sarcotubules of normal-appearance myocytes, but also in myofibrils and/or mitochondria of highly degenerated myocytes. These results demonstrate the increase in the mem-brane permeability in the SHR myocardium, probably due to genetical disadvantage of the defense system and a proneness for membrane lipid peroxidation.

Heat shock protein 25 (Hsp25) is a molecular chaperone playing roles in cytoprotection. We investigated the distribution and localization of Hsp25 expression in CCl4-induced rat hepatic lesions; liver samples were obtained from 3 h to 10 days after a single oral administration of CCl4. Immunohistochemically, Hsp25-positive hepatocytes started to appear in the perivenular area at 6 h after CCl4 administration. Their number and strength increased till day 1. Expression of Hsp25 mRNA significantly increased after 3 h and proceeded to increase with time till day 1. Apoptotic hepatocytes were detected around the perivenular area after 6 h. The area where Hsp25-positive hepatocytes were observed till day 1 corresponded to the area where apoptotic hepatocytes were seen. On days 2 and 3, degenerative and/or necrotic hepatocytes in the perivenular area were replaced by macrophages reacting to ED1 (for CD68) and ED2 (for CD163); Hsp25 expression was seen in hepatocytes around the perivenular area and there was a close relationship of reactive macrophages with Hsp25-positive hepatocytes, suggesting a potential role for Hsp25 in suppressing injury by inflammation. The mRNA expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and osteopontin, which can be produced by infiltrating macrophages, corresponded to that of Hsp25 from day 1 to day 3; these factors might be related to the induction of Hsp25 expression. The shift of the Hsp25 expression pattern in the liver lesion might have depended on microenvironmental conditions evoked by interactions between necrobiotic hepatocytes and infiltrating macrophages. Thus, Hsp25 expression analyses should be beneficial for evaluations of hepatotoxicants.

Peroxisome proliferator-activated receptors (PPARs) play an important role in different compartments of the female reproductive system in rodents and humans. However, expressional profiles and physiological functions of PPARs in the endometrium prior to the placentation are not well understood. In this study, we determined expressional profiles of the PPARs during early pregnancy. Immunocytochemistry revealed that both PPARα and PPARβ/δ were strongly detected in the endometrial stroma on days 4.5–6.5 of pregnancy, which is just a starting time of implantation. Delayed implantation animal model showed that the expressions of PPARα and PPARβ/δ occurred after the initiation of implantation in the endometrial stroma. Moreover, an in vitro decidualization model further revealed that the expression of PPARα increased in the cultured rat endometrial stromal cells at 24 h after the decidualization treatment, but the expression of PPARβ/δ was delayed and increased at 48 h after the treatment. PPARγ was expressed in the endometrial stroma and its expression decreased significantly at 2.5 days post-coitum and maintained a low level of expression during the period of implantation. These results indicate that PPARα is expressed and induced by the initiation of implantation, prior to the expression of PPARβ/δ in decidualized endometrium. Increasing expression of PPARγ during fertilization and its decline during the period of implantation further suggest that PPARs may play important roles during early pregnancy.

Myelosuppressive anemia is a serious side effect associated with several drugs. Thus, there is an increasing demand for sensitive biomarkers for the early detection of myelosuppressive anemia during toxicological studies. We applied a toxicogenomic approach to identify useful biomarker genes reflecting myelosuppressive anemia in the rat liver. Expression of the hemoglobin beta chain complex (Hbb), aminolevulinic acid synthase 2 (Alas2), and cell division cycle 25 homolog B (Cdc25b) genes changed as a result of anemia induced by the myelosuppressive agents linezolid, cisplatin, and carboplatin, suggesting that these genes may be suitable biomarkers. Moreover, evaluation of perfused and unperfused livers indicated that changes in the expression of these genes originate in circulating reticulocytes in the liver. Erythroid differentiation-associated changes in expression of the Hbb, Alas2, and Cdc25b genes were confirmed in vitro using Friend leukemia cells. In conclusion, our current research provides novel evidence that gene expression in circulating reticulocytes contained in the liver changes dramatically under myelosuppressive conditions. While further large-scale validation studies are needed, our results indicate that the genes we identified might be useful biomarkers for the sensitive detection of myelosuppressive anemia in rats.

The antineoplastic platinum complexes cisplatin and its analogues are widely used in the chemotherapy of a variety of human malignancies, and are especially active against several types of cancers. Nedaplatin is a second-generation platinum complex with reduced nephrotoxicity. However, their use commonly causes nephrotoxicity due to a lack of tumor tissue selectivity. Several recent studies have provided significant insights into the molecular and histopathological events associated with nedaplatin nephrotoxicity. In this review, we summarize findings concerning the renal histopathology and molecular pathogenesis induced by antineoplastic platinum complexes, with a particular focus on the comparative nephrotoxicity of cisplatin and nedaplatin in rats.

Cataract is one of the most serious drug-induced side effects that can terminate the development of drug candidates, and pharmaceutical companies consider it important to evaluate cataract-inducing potential in the early phases. Metabonomics is expected to be a powerful approach for the safety evaluation of drug candidates. In this study, we conducted a toxicological characterization of N-methyl-N-nitrosourea (MNU)-induced cataract in rats by LC/MS-based metabonomic analysis. MNU was intraperitoneally administered once to 15-day old rats at 70 mg kg−1. After that, animals were kept for 3 weeks waiting for cataract formation. Lens samples for metabonomic analysis were collected on 7, 14 and 21 days after MNU administration. Comprehensive analyses of lens metabolites were conducted using an LC/MS system, and multivariate data for each sample were compared by principal component analysis (PCA) to find any changes in lens metabolites. Lens opacity was confirmed by ophthalmoscopy 14 days after dosing, and even by gross observation 21 days after dosing. PCA of the lens samples for the control and MNU-treated groups revealed that the metabolite profiles of lens differed from each other, and several lens metabolites, such as lots of α-amino acids and gluthathione, decreased after MNU treatment. In conclusion, metabonomic analysis enabled us to identify new marker candidates for cataract and provided a better understanding of the mechanism related to MNU-induced cataract. It was considered that metabonomics is a useful approach for the characterization of drug-induced toxicity. Copyright © 2011 John Wiley & Sons, Ltd.

Since nephrotoxicity affects the development of drug candidates, it is important to detect their toxicity at an early stage of drug development. In this study, we measured twelve urinary nephrotoxic biomarkers [total protein, albumin, kidney injury molecule-1 (KIM-1), clusterin, beta2-microglobulin, cystatin-c, alpha-glutathione S-transferase, mu-glutathione S-transferase, N-acetyl-beta-d-glucosaminidase, lactate dehydrogenase (LDH), aspartate aminotransferase and neutrophil gelatinase-associated lipocalin (NGAL)] and two conventional blood nephrotoxic biomarkers (creatinine and blood urea nitrogen) in rat models treated intravenously with puromycin aminonucleoside (PAN) or cisplatin (CDDP), which are known to induce glomerular injury or proximal tubular injury, respectively, and evaluated their usefulness by receiver operating characteristic analysis. In the PAN-treated rats, urinary albumin and (NGAL) were dramatically increased, which were thought to be caused by the dysfunction of proximal tubule in addition to glomerular injury. Conversely, based on its early and time-dependent increase, its large magnitude of alteration and its high accuracy and sensitivity of detection, (KIM-1) in urine appeared to be the best biomarker for detection of CDDP-induced proximal tubular injury. Moreover, (LDH) was considered useful for broad detection of damaged nephrons, because of its broad distribution along the nephron. Therefore, combinatorial measurement of these biomarkers may be a powerful tool for highly effective screening of nephrotoxicity.

Heat shock protein 25 (Hsp25), which has anti-inflammatory activity, was examined for the relationship of its expression to macrophage appearance in thioacetoamide (TAA)-induced rat acute hepatic lesions. TAA-induced lesions, consisting of hepatocyte coagulation necrosis and reactive macrophages, developed in the centrilobular areas. Macrophages immuno-reacting to ED1 (CD68; exudative macrophages) were mainly seen within the lesions, whereas macrophages reacting to ED2 (CD163; resident macrophages and Kupffer cells), which have abundant cytoplasm, appeared mainly in the periphery of the lesions. Hsp25-immunopositivity was seen in hepatocytes around the lesions in relation to ED1- and ED2-positive macrophages in and around the centrilobular lesions, respectively. Because macrophages appearing in early stages of hepatic lesions produce various pro-inflammatory factors, mRNA expressions of tumor necrosis factor-α (TNF-α), monocyte chemoattractant factor-1 (MCP-1) and osteopontin (OPN) were examined in relation to Hsp25 mRNA expression. Hsp25 mRNA expression generally was correlated with TNF-α, MCP-1 and OPN expressions, suggesting their direct or indirect association with Hsp25 expression. Thus, Hsp25 might have a cytoprotection function against macrophages appearing in hepatic lesions, and factors produced by macrophages in the very early stages of hepatic lesions may influence Hsp25 expression. Hsp25 expression should be useful as an index of anti-inflammatory action for evaluation of hepatotoxicants in vivo.

1. Antiperoxidation ability and lipid peroxidation in myocardium were examined in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 6 and 16 weeks of age. 2. Glutathione peroxidase activity was higher in SHR at 6 weeks of age, but lower at 16 weeks compared to that in WKY. alpha-Tocopherol content was lower in SHR at both 6 and 16 weeks of age than in WKY. 3. In vitro formation of free malondialdehyde was more pronounced in SHR myocardium than in WKY. 4. Coincidence of lower antiperoxidation ability and higher peroxidation of membrane phospholipid indicate myocardial cell vulnerability in SHR hypertrophied myocardium.

Morphine, oxycodone, and fentanyl are clinically prescribed drugs for the management of severe pain. We investigated whether these opioids possess different efficacy profiles on several types of pain in mouse pain models. When the three opioids were tested in the femur bone cancer model, all of them significantly reversed guarding behavior, whereas the effects on limb-use abnormality and allodynia-like behavior differed among the opioids. Particularly, although oxycodone (5 – 20 mg/kg) and fentanyl (0.2 mg/kg) significantly reversed limb-use abnormality, not even a high dose of morphine (50 mg/kg) could reverse it. When the effects of these opioids were examined in a sciatic nerve ligation (SNL) model of neuropathic pain, oxycodone was the most effective, producing an antinociceptive effect without affecting the withdrawal threshold of sham-treated animals. When the effects of these opioids were examined with the tail-flick test using naive animals, oxycodone, morphine, and fentanyl exhibited antinociceptive effects on thermal nociception. These results show that the three opioids exhibit different efficacy outcomes in multiple pain models and that the efficacy profile of oxycodone does not overlap those of morphine and fentanyl. Keywords:: oxycodone, morphine, fentanyl, neuropathic pain-like state, bone cancer pain

Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis. In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER to identify gene expression alterations closely associated with CER-induced nephrotoxicity. Male Fischer 344 rats were intravenously injected with CER at three different dose levels (150, 300, and 600 mg/kg) and sacrificed after 24 h. Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and slight renal dysfunction. Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following CER exposure, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners. The genes upregulated dose dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress. In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury. Of the genes modulated in both manners, we found many genes reported to be associated with renal toxicity by other nephrotoxicants. We could also predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and the E2F family. Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity. In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understanding of the molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity.

In order to ascertain the pathogenesis of myocardial cell vulnerability in spontaneously hypertensive rats (SHR), several enzyme activities were examined by using subcellular fractions of myocardium and compared to those in Wistar-Kyoto rats (WKY). In the normotensive WKY heart, both 5'-nucleotidase and Na+/K(+)-ATPase, which are plasma membrane associated enzymes, increased with age. But in the SHR heart, both enzymes were lower at 16 weeks than they were at 10 weeks of age. Moreover, at 16 weeks of age they were lower in SHR than in WKY. On the other hand, NADP(+)-isocitrate dehydrogenase activity, a mitochondria associated enzyme, was higher in SHR than in WKY at 6 weeks, but lower at 10 and again at 16 weeks of age. The activities of both acid phosphatase and N-acetyl-beta-glucosaminidase, which are lysosomal enzymes, decreased with age in SHR but not in WKY. These results suggest that an enzymatic alteration in the plasma membrane and mitochondria may be one of important factors behind myocardial vulnerability in the SHR heart.

Pirfenidone, a broad-spectrum antifibrotic agent, is known to have efficacy in certain fibrotic disease models, and is under clinical trials in patients with idiopathic pulmonary fibrosis. We investigated the antifibrotic effect of pirfenidone, and its regulatory effect on various pulmonary cytokines, in bleomycin-induced lung fibrosis in mice at the protein level, using prednisolone as a reference agent. Pirfenidone attenuated the bleomycin-induced pulmonary fibrosis at a minimum effective dose of 30 mg/kg/day t.i.d. from the analysis of lung hydroxyproline content. Both pirfenidone (30, 100 mg/kg/day t.i.d) and prednisolone (3, 15 mg/kg/day q.d.) suppressed lung inflammatory edema; however, prednisolone failed to suppress pulmonary fibrosis, which was significantly suppressed only by pirfenidone. Both pirfenidone and prednisolone suppressed the increase in lung interleukin (IL)-1beta, IL-6, IL-12p40 and monocyte chemoattractant protein (MCP)-1 levels induced by bleomycin. On the other hand, pirfenidone prevented the bleomycin-induced decrease in lung interferon (IFN)-gamma levels, while prednisolone had no such effect. Furthermore, pirfenidone suppressed elevation of lung basic-fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1 levels, but prednisolone had no such effect. The increases in lung stroma cell derived factor (SDF)-1alpha and IL-18 were also suppressed. These findings suggest that pirfenidone exerts its antifibrotic effect through regulation of lung IFN-gamma, bFGF and TGF-beta1 levels during the development of bleomycin-induced pulmonary fibrosis in mice. The effect on SDF-1alpha and IL-18 levels may also be related to the antifibrotic effects of pirfenidone.

Hemolytic anemia is a serious adverse effect of therapeutic drugs that is caused by increased destruction of drug-damaged erythrocytes by macrophages in the spleen and liver. We previously applied a toxicogenomic approach to the toxicity by analyzing microarray data of the liver of rats dosed with two hemolytic agents: phenylhydrazine and phenacetin. In the present study, we analyzed gene expression profiles in the spleen, the primary organ for destruction of damaged erythrocytes, of the same models in order to identify splenic gene expression alterations that could be used to predict the hematotoxicity. Microarray analyses revealed hundreds of genes commonly deregulated under all severe hemolytic conditions, which included genes related to splenic events characteristic of the hematotoxicity, such as proteolysis and iron metabolism. Eleven upregulated genes were selected as biomarker candidates, and their expression changes were validated by quantitative real-time PCR. The transcript levels of most of these genes showed strong correlation with the results of classical toxicological assays (e.g., histopathology and hematology). Furthermore, hierarchical clustering analysis suggested that altered expression patterns of the 11 genes sensitively reflected the erythrocyte damage even under a condition that caused no decrease in erythrocyte counts. Among the selected genes, heme oxygenase 1 was one of the most promising biomarker candidates, the upregulation of which on the protein level was confirmed by immunohistochemistry. These results indicate that altered splenic expression of a subset of genes may allow detection of drug-induced hemolytic anemia, with better sensitivity than that of erythrocyte counts in the blood.

Iron lactate was given to Sprague–Dawley rats intravenously at the dosage of 10 mg/kg/day and the early effects on the parathyroid gland were examined ultrastructurally along with the blood level of parathyroid hormone (PTH) after single, 3-day or 6-day administration. Blood levels of electrolytes and other parameters related to osteoclast dynamics were also measured by blood chemistry and histopathology. The plasma parathyroid hormone (PTH) level was elevated in the single and 3-day dosing group but was reduced in the 6-day dosing group. Histopathologically, an increase of osteoclasts in the primary spongiosa was observed in the 3- and 6-day dosing groups. Image analysis of the parathyroid gland revealed that the average area of the storage granule decreased during a experimental period, with the number of storage granules decreasing in the 3- and 6-day dosing group. The chief cells of the parathyroid gland were moderately atrophied in the 6-day dosing group. These results demonstrate that iron lactate immediately promotes discharge of PTH from the storage granules after the treatment and induces an increase of osteoclasts in the primary spongiosa. The findings collectively suggest a pathophysiological mechanism of iron lactate-induced osteopenia in rats.

To elucidate the nephrotoxicity of phosphate, dibasic sodium phosphate solution was given to Sprague—Dawley rats by daily bolus intravenous administration at concentrations of 0, 1, 25, 250, or 360 mM (0, 1, 28, 284, or 408 mg/kg Na2HPO4)for 14 days, and the kidneys were pathologically examined. There were no remarkable changes in blood chemistry values; however, urinalysis revealed mild to moderate proteinuria in the 250 and 360 mM groups. The kidneys from the 360 mM group were macroscopically pale. Histopathology revealed panglomerular deposition of basophilic dense granules, which were positive for von Kossa's staining, accompanied by dose-dependent degeneration of the glomerular epithelium and parietal epithelium in the 250 and 360 mM groups. Electron microscopic examination showed fusion of podocytes and increased microvilli, with large amounts of debris in the Bowman's space. Low-density lamellar structures were present not only in the glomerular epithelium, basement membrane, mesangial matrix and parietal epithelium but also within the Bowman's space depending on the severity of the glomerular lesion. Phosphorus and calcium were detected by X-ray microanalysis as fine particles admixed with lamellar structures. These results suggest that high-dose phosphate used in this study transiently overloads the glomerular epithelium during filtration through glomerular capillaries and produces insoluble calcium salt and glomerular lesions, resulting in proteinuria.

Osteomalacia was induced in rats fed a diet containing 50,000 ppm (5%) iron lactate for 13 weeks. The histopathology and histomorphometrical dynamics of osteoblasts under this condition were examined. Bone histomorphometry of the proximal tibial metaphysis revealed that the osteoblast surface, osteoid volume, osteoid surface and labeled surface ratio, which are the parameters of bone formation had increased. The blood chemistry revealed the greatest elevation in the osteocalcin level; however, the parathyroid hormone (PTH) secretion and inorganic phosphorus level were very low. From the serum biochemical, histopathological and histomorphometrical findings, the bone lesion in iron lactate-overloaded rats was considered to be similar to low turnover osteomalacia showing decreased trabeculae in secondary spongiosa and increased lamellar osteoid. Furthermore, an iron-positive reaction was detected at the interface between osteoid and mineralized bone. In the bone lesions induced by chronic iron overload, osteoblast recruitment exceeded that of mineralization or, alternatively, the iron within osteoblasts along the trabecular bone suppressed the remodeling and led to an increase in osteoid thickness.