Outer retinal involvement in N-methyl-D-aspartate-induced inner retinal injury in rabbits assessed by optical coherence tomography.

This study was aimed to investigate morphological alteration of the retina with N-methyl-D-aspartate (NMDA)-induced injury in rabbits by optical coherence tomography (OCT). The right and left eyes of a total of 12 rabbits received single-intravitreal injection of vehicle and NMDA, respectively. Four out of the 12 animals underwent OCT and quantification of plasma microRNA repeatedly (4, 48, and 168 hr after dosing), followed by ocular histopathology at the end of the study. Ocular histopathology was also conducted in the eyes collected 4 or 48 hr after dosing from 4 animals at each time period. OCT revealed hyper-reflective ganglion cell complex and thickened inner retina in NMDA-treated eyes 4 hr after dosing; the inner retina shifted to thinning at later time points. The eyes given NMDA also exhibited greater thickness of the outer retina, which contains photoreceptors, after treatment, and thickened and obscured ellipsoid zone 168 hr after dosing. The plasma levels of miR-182 and miR-183, which are known to be highly expressed in photoreceptors, were higher 4 hr after dosing than pre-dosing values. Histopathologically, NMDA-induced inner retinal damage was confirmed: single-cell necrosis was observed in the ganglion cell layer and the inner nuclear layer 4 hr after dosing, the incidence of which decreased thereafter. At 168 hr after dosing, reduced number of ganglion cells was noted. No change was histopathologically observed in the outer retina. In conclusion, our results suggest involvement of photoreceptors in NMDA-induced inner retinal injury. Additionally, OCT revealed acute inner retinal findings suggestive of temporary edema.

Fluoroquinolones suppress gluconeogenesis by inhibiting fructose 1,6-bisphosphatase in primary monkey hepatocytes.

Dysglycemia is one of the most serious adverse events associated with the clinical use of certain fluoroquinolones. The purpose of this study was to investigate the effects of the representative fluoroquinolones moxifloxacin and gatifloxacin on hepatic gluconeogenesis using primary monkey hepatocytes. Glucose production was induced after the cells were incubated for 4 h with 10 mM sodium lactate and 1 mM sodium pyruvate as gluconeogenic substrates. Under these conditions, moxifloxacin and gatifloxacin dose-dependently suppressed gluconeogenesis at concentrations of 100 µM or higher. Transcriptome analysis of rate-limiting enzymes involved in hepatic gluconeogenesis revealed that moxifloxacin and gatifloxacin at a concentration of 1000 µM did not affect the expression of key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase, glucose 6-phosphatase, and fructose 1,6-bisphosphatase. Furthermore, metabolome analysis, in vitro glucose production assay using additional gluconeogenic substrates, and fructose 1,6-bisphosphatase assay using the cell extracts showed that fluoroquinolones enzymatically suppressed hepatic gluconeogenesis by inhibiting fructose 1,6-bisphosphatase. These inhibitory effects may involve in the clinically relevant dysglycemia associated with fluoroquinolones in human.

Functional and Morphological Characteristics of Pancreatic Islet Lesions Induced by Quinolone Antimicrobial Agent Gatifloxacin in Rats.

We characterized pancreatic islet lesions induced by several quinolones using functional and morphological examinations of the pancreatic islets in male rats orally administered gatifloxacin, lomefloxacin, or levofloxacin at 300 mg/kg for 14 consecutive days. Consequently, in contrast to lomefloxacin or levofloxacin, gatifloxacin increased serum glucose and glycosylated albumin on day 14 and elevated serum glucose tended to decrease insulin in the intravenous glucose tolerance test. Microscopically, only gatifloxacin induced cytoplasmic vacuoles containing eosinophilic homogenous contents in islet cells. Immunohistochemical examination revealed that vacuolated islet cells were positively stained for insulin, demonstrating they were pancreatic ß cells. Electron microscopy showed that the cytoplasmic vacuoles represented dilated cisterna of the rough endoplasmic reticulum filled with electron-lucent materials in pancreatic ß cells. Moreover, insulin secretory granules were drastically decreased in vacuolated islet cells, suggesting impaired insulin synthesis and/or transport. This gatifloxacin-induced pancreatic toxicity in rats was considered to be associated with high pancreatic drug distribution. These results demonstrated that gatifloxacin provoked functional and morphological pancreatic ß cell alteration associated with impaired insulin synthesis and/or transport, leading to hyperglycemia.

[A case of basaloid cell carcinoma of the anal canal in which complete response was obtained by chemoradiation therapy].

The patient was a 69-year-old woman with a chief complaint of melena, who had a palpable firm mass and tenderness on digital rectal examination. Colonoscopy (CS) showed a depressed hemorrhagic lesion 2cm in diameter on the left anterior wall of the anal canal near the dentate line. Biopsy examination yielded a diagnosis of basaloid cell carcinoma. On the basis of a diagnosis of clinical stage I (T1N0M0) anal canal cancer, chemoradiotherapy was performed. After treatment, no tumor cells were detected on biopsy. Basaloid cell carcinoma, accounting for about 1.6% of anal canal cancers, is rare. Only 4 among 62 reported cases (including our patient) received chemoradiation therapy.

Effect of body-weight loading onto the articular cartilage on the occurrence of quinolone-induced chondrotoxicity in juvenile rats.

The effect of body-weight loading onto the articular cartilage on the occurrence of chondrotoxicity was investigated in male juvenile Sprague-Dawley rats given ofloxacin (OFLX) orally once at 900 mg/kg. Just after dosing of OFLX, hindlimb unloading was performed for 0, 2, 4, or 8 h by a tail-suspension method. Animals were sacrificed at 8h post-dose, and then the distal femoral articular cartilage was subjected to a histological examination and an investigation for gene expression of tumor necrosis factor receptor superfamily, member 12a (Tnfrsf12a); prostaglandin-endoperoxide synthase 2 (Ptgs2); plasminogen activator, urokinase receptor (Plaur); and matrix metalloproteinase 3 (Mmp3) by qRT-PCR analysis. As a result, cartilage lesions and up-regulations of these 4 genes that were seen in rats without the tail suspension were not observed in rats with the 8-h tail suspension, and a tendency to decrease in the incidence of the cartilage lesions and the gene expression was noted in a tail-suspension time dependent manner. Our results clearly indicate that body-weight loading onto the cartilage is necessary to induce cartilage lesions and gene expression of Tnfrsf12a, Ptgs2, Plaur, and Mmp3 in juvenile rats treated with OFLX.

Effect of Hypertension on the Occurrence of Micro-hemorrhage in the Pancreatic Islet of Dahl Salt-sensitive Rats.

The effect of hypertension on the occurrence of micro-hemorrhage in the pancreatic islet, known to be observed in Sprague-Dawley (SD) rats spontaneously, and endothelial markers were investigated in male Dahl-Iwai salt-sensitive (DIS, derived from SD rats), salt-resistant (DIR), and SD rats. DIS and DIR rats were fed 8% NaCl-containing diet to induce hypertension, with blood pressure measurement once a week, euthanized at 6, 8, or 12 weeks of age, and subjected to the measurement of plasma nitric oxide (NO) and von Willebrand factor (vWF) concentrations combined with histopathological examinations and immunohistochemical detections of vWF in the pancreas and kidney. As a result, hypertension was observed from 7 through 12 weeks of age in DIS rats. At 12 weeks of age, only DIS rats showed decreased plasma NO and increased vWF, indicating endothelial abnormality in the body. Histopathologically, micro-hemorrhage in the islet was observed with a similar incidence and severity in SD and DIS rats aged 12 weeks, and vWF was immunohistochemically localized in the islet endothelium with similar reactivity between age-matched SD rats. On the other hand, in the kidney, glomerular sclerosis was observed in DIS rats aged 12 weeks and accompanied broad stainability of vWF in the sclerotic glomerulus, including endothelium. In conclusion, there was no enhancement/exaggeration in the micro-hemorrhage in the pancreatic islet of hypertensive DIS rats in comparison with that in SD rats under the present experimental conditions. It is suggested that hypertension is not related to the occurrence of islet micro-hemorrhage, spontaneously observed in SD rats.

Chondrotoxicity and toxicokinetics of novel quinolone antibacterial agents DC-159a and DX-619 in juvenile rats.

Quinolone antibacterial agents are extensively utilized in antimicrobial chemotherapy. However, they have been reported to induce arthropathy in juvenile animals, and the mechanism has not been clarified. Recently, we have demonstrated that Dusp1, Tnfrsf12a, Ptgs2, Fos, Mt1a, Plaur, Mmp3, Sstr1 and Has2 genes change in the articular cartilage of juvenile rats with a single oral administration of ofloxacin (OFLX), suggesting that these genes are involved in the induction of OFLX-induced chondrotoxicity. In the present study, to compare the chondrotoxic potential between new synthesized quinolones DC-159a and DX-619, and OFLX, they were orally administered by gavage at a dose level of 300 or 900 mg/kg/day to male juvenile Sprague-Dawley (SD) rats, 3 weeks of age, for 7 days. Then the distal humerus and femur were subjected to microscopic examination. Moreover, concentrations of these quinolones in the femoral articular cartilage were measured in male juvenile SD rats following a single oral administration at 100, 300 or 900 mg/kg. Furthermore, gene expression of Dusp1, Tnfrsf12a, Ptgs2, Fos, Mt1a, Plaur, Mmp3, Sstr1 and Has2 was investigated in the articular cartilage of the distal femur in male juvenile SD rats treated with 900 mg/kg of DC-159a or DX-619 by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis. In a microscopic examination, no changes in the articular cartilage were observed in any animal administered DC-159a or DX-619. On the contrary, cavity formation and chondrocyte cluster in the cartilage of distal humerus and femur were noted in animals receiving OFLX at 300 mg/kg/day or more. In toxicokinetic analysis, the maximum concentration (C(max)) value in the articular cartilage (cartilage C(max)) of DC-159a or DX-619 at 900 mg/kg was lower than that of OFLX at 300 mg/kg. However, the area under the cartilage concentration-time curve (cartilage AUC)(0-24h) value of DC-159a or DX-619 at 900 mg/kg was higher than that of OFLX at 300 mg/kg. In qRT-PCR analysis, up-regulated Dusp1, Fos and Mt1a, and down-regulated Sstr1 and Has2 genes were seen in the femoral articular cartilage of rats given DX-619 or DC-159a at 900 mg/kg. However, Tnfrsf12a, Ptgs2, Plaur and Mmp3 genes, which were up-regulated in the distal femoral articular cartilage exposed to OFLX, did not increase or slightly increased. In conclusion, the penetration of DC-159a or DX-619 into the cartilage was low compared with that of OFLX, and no obvious changes in Tnfrsf12a, Ptgs2, Plaur and Mmp3 genes were observed in the articular cartilage of juvenile rats treated with DC-159a or DX-619, which was likely to be responsible for non-chondrotoxic potentials of DC-159a and DX-619.

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.

Gene expression profiles in the articular cartilage of juvenile rats receiving the quinolone antibacterial agent ofloxacin.

Quinolone antibacterial agents are extensively utilized in antimicrobial chemotherapy. However, they have been reported to induce arthropathy in juvenile animals, and the mechanism has not been clarified. In the present study, to investigate the molecular details of the chondrotoxicity of the quinolone ofloxacin (OFLX), it was orally administered by gavage at a dose level of 900 mg/kg once to male juvenile Sprague-Dawley rats, 3 weeks of age. Then gene expression profiles in the articular cartilage of the distal femur were analyzed at 2, 4, 8 and 24h post-dose. In the GeneChip analysis, the expression of 134 gene probes in the OFLX-treated group showed statistically significant differences with at least 1.5-fold difference from the control. Among them, intracellular signaling cascade- and stress response-related genes changed at 2h post-dose; cell death- and inflammatory response-related genes at 4 and 8h post-dose; basic-leucine zipper transcription factor and stress response-related genes at 8 and 24h post-dose; stress response-, proteolysis- and glycoprotein-related genes at 24h post-dose. In a quantitative real-time reverse transcription-polymerase chain reaction analysis, up-regulated Dusp1 (intracellular signaling cascade-related gene), Tnfrsf12a (cell death-related gene), Ptgs2, Fos (inflammatory response-related genes), Mt1a, Plaur (stress response-related genes) and Mmp3 (proteolysis-related gene) and down-regulated Sstr1 and Has2 (glycoprotein-related genes) were observed with dose dependency in the articular cartilage of juvenile rats treated with OFLX at 100, 300 and 900 mg/kg. The expression of Tnfrsf12a, Ptgs2, Plaur and Mmp3 was also noted in chondrocytes around the cartilage lesions by in situ hybridization. In conclusion, our results suggest that cytokines, chemokines and/or proteases produced by up-regulation of cell death-, inflammatory response-, stress response- and proteolysis-related genes play a important role in the onset of OFLX-induced chondrotoxicity in juvenile rats.

Species and sex differences in susceptibility to olfactory lesions among the mouse, rat and monkey following an intravenous injection of vincristine sulphate.

Species and sex differences in susceptibility to vincristine sulphate (VCR)-induced olfactory epithelial lesions were investigated among the BALB/c mice, Crj: CD(SD) IGS rats and common marmoset monkeys following a single intravenous administration on day 1. As dosage levels, the 0.17-fold LD10, 0.6-fold LD10 and LD10 were used for mice and rats, and a maximum tolerated dose (MTD) was chosen only for monkeys. The order of strength of VCR action on peripheral neuropathic signs, body weight gain, and hematological parameters was mice > rats > monkeys, without clear sex differences. Histopathologically, on day 2, single cell death in the olfactory epithelium and vomeronasal organ was observed only in male mice at LD10, and in female mice at 0.6-fold LD10 or more. On day 5, the olfactory epithelium in these mice showed regenerative proliferation suggesting a sign of recovery. On day 10, axonopathy and demyelination in the sciatic and trigeminal nerves were noted in mice of both sexes at 0.6-fold LD10 or more. In rats and monkeys of either sex, however, no morphological changes were observed at any dose level. In conclusion, mice, particularly females, were shown to be more susceptible to VCR-induced apoptosis in the olfactory epithelium than rats and monkeys.