Combination of pathological, biochemical and behavioral evaluations for peripheral neurotoxicity assessment in isoniazid-treated rats.

In drug development, assessment of non-clinical peripheral neurotoxicity is important to ensure human safety. Clarifying the pathological features and mechanisms of toxicity enables the management of safety risks in humans by estimating the degree of risk and proposing monitoring strategies. Published guidelines for peripheral neurotoxicity assessment do not provide detailed information on which endpoints should be monitored preferentially and how the results should be integrated and discussed. To identify an optimal assessment method for the characterization of peripheral neurotoxicity, we conducted pathological, biochemical (biomaterials contributing to mechanistic considerations and biomarkers), and behavioral evaluations of isoniazid-treated rats. We found a discrepancy between the days on which marked pathological changes were noted and those on which biochemical and behavioral changes were noted, suggesting the importance of combining these evaluations. Although pathological evaluation is essential for pathological characterization, the results of biochemical and behavioral assessments at the same time points as the pathological evaluation are also important for discussion. In this study, since the measurement of serum neurofilament light chain could detect changes earlier than pathological examination, it could be useful as a biomarker for peripheral neurotoxicity. Moreover, examination of semi-thin specimens and choline acetyltransferase immunostaining were useful for characterizing morphological neurotoxicity, and image analysis of semi-thin specimens enabled us to objectively show the pathological features.

Metabolomic profiling of urine-derived extracellular vesicles from rat model of drug-induced acute kidney injury.

Extracellular vesicles (EVs) are lipid bilayer particles that are released by various cells and provide a real-time snapshot of the state of these cells in tissue in a noninvasive manner. EVs contain components, including mRNA, miRNAs, proteins, and metabolites. Therefore, EVs hold promise for the discovery of liquid biopsy-based biomarkers for disease diagnosis. In the present study, metabolome analysis of urine EVs in rats with kidney injury caused by cisplatin and puromycin aminonucleoside was performed using liquid chromatography/mass spectrometry to identify candidate biomarkers that reflect the type and extent of injury in drug-induced nephrotoxicity. A total of 396 metabolites were detected in urine EVs, of which 65 were identified as potential biomarkers in urine EVs of drug-induced nephrotoxicity. Pathway analysis revealed that these metabolites may reflect changes occurring within damaged cells during kidney injury, suggesting that metabolomics of urine EVs could be a useful informative tool.

Spontaneous ovarian choriocarcinoma in a young ICR mouse.

This paper describes the spontaneous ovarian choriocarcinoma observed in a young female Crl:CD1 (ICR) mouse. The mouse was sacrificed at 8 weeks of age after oral administration of a compound for 2 weeks. The left ovary was found to be cystically enlarged with dark red hemorrhaging. The cystic mass contained abundant blood plasma and erythrocytes. At the peripheral regions of the mass, large pleomorphic tumor cells with bizarre shaped nuclei were detected. Tumor cells contained a single large nucleus and abundant eosinophilic to amphophilic cytoplasm. Histopathology of the tumor cells resembled that of trophoblastic giant cells. Therefore, the observed ovarian lesion was diagnosed as a choriocarcinoma. No microscopic lesions were observed in the right ovary or other reproductive organs. Ovarian choriocarcinoma was considered to be of non-gestational origin. This is the first report of ovarian choriocarcinoma in a young ICR mouse.

Karnovsky's fixative prevents artifacts appearing as vacuolation derived from tissue processing in kidneys treated with antisense oligonucleotide.

Antisense oligonucleotide (ASO) therapies have been identified as a new treatment modality for intractable diseases. In kidneys treated with ASOs, vacuoles, in addition to basophilic granules, are often observed in the proximal tubules. Some reports have described that these vacuoles are likely to be a secondary phenomenon resulting from the extraction of ASOs during tissue processing. In this study, we compared renal morphology after fixation with Karnovsky's fixative or 4% paraformaldehyde phosphate buffer (PFA) with that of 10% neutral-buffered formaldehyde solution (NBF). Female Sprague-Dawley rats, intravenously treated four times with 50 mg/kg locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) for 1 or 2 weeks, were examined. Microscopically, vacuoles and basophilic granules in the proximal tubules were observed in the kidneys fixed with NBF. Basophilic granules are indicative of the accumulation of ASOs. Moreover, some of the vacuoles also contained faint basophilic granules, suggesting that the vacuoles were relevant to the accumulation of ASOs. Although moderate vacuolation was observed in the proximal tubules, the majority of the vacuolated epithelia were negative for kidney injury molecule-1 on immunohistochemical staining. Vacuoles in the proximal tubules were not observed in samples subjected to Karnovsky's fixation, although basophilic granules were observed. In samples subjected to PFA fixation, vacuoles and basophilic granules were observed in the proximal tubules, similar to those in samples subjected to NBF fixation. Overall, our findings demonstrated the possibility of overestimation of vacuolation due to artifacts during tissue processing when using conventional NBF fixation. Karnovsky's fixative is considered a useful alternative for distinguishing artificial vacuoles from true nephrotoxicity.

Spontaneous hyaline glomerulopathy in a young Slc:ICR mouse.

Hyaline glomerulopathy is a type of glomerular lesion that occurs in aging mice. Spontaneous hyaline glomerulopathy is rare in young mice. Here, we report spontaneous hyaline glomerulopathy in a young adult (15-week-old) ICR mouse. Necropsy revealed discoloration and roughness of the kidney surface. Microscopically, diffuse glomerular lesions were prominent. Amorphous, eosinophilic materials were deposited globally in the glomeruli. The mesangial region was expanded; however, the mesangial cells showed no proliferation. Thickening of the Bowman's capsule with proliferation of parietal epithelial cells was observed. Glomerular deposits were strongly positive for anti-IgM, anti-IgG, and periodic acid-Schiff stain and were stained red by Masson's trichrome stain. The deposits were negative for anti C3 and stained negatively with Congo red stain. Periodic acid methenamine silver and electron microscopy revealed glomerular deposits limited to intraglomerular capillaries. Based on the histological features, we diagnosed this lesion as hyaline glomerulopathy. This case could improve our understanding of spontaneous lesions in toxicological and pharmacological studies.

Imaging mass spectrometry for toxicity assessment: a useful technique to confirm drug distribution in histologically confirmed lesions.

To evaluate the usefulness of imaging mass spectrometry (IMS) technology for assessing drug toxicity, we analyzed animal tissues in an amiodarone (AMD)-induced phospholipidosis model by IMS and confirmed the relationship between the distribution of AMD, its metabolites, and representative phospholipids (phosphatidylcholine, PC) and histological changes. AMD was administered to rats for 7 days at 150 mg/kg/day. The lung, spleen, and mesenteric lymph node were histologically examined and analyzed using IMS. The detection intensities of AMD, its metabolites, and typical PCs were higher in regions infiltrated by foamy macrophages compared with normal areas. This tendency was common in all three organs analyzed in this study. For the spleen, signals for AMD, its metabolites, and typical PCs were significantly more intense in the marginal zone, where foamy macrophages and vacuolated lymphocytes are abundant, than in the other areas. These results indicate that AMD, its metabolites, and PCs accumulate together in foamy or vacuolated cells, which is consistent with the mechanism of AMD-induced phospholipidosis. They also indicate that IMS is a useful technique for evaluating the distribution of drugs and biological components in the elucidation of toxicity mechanisms.

Asbestosis in a Japanese Macaque (Macaca fuscata).

Asbestosis is a chronic lung disease caused by inhalation of asbestos, a fibrous mineral. It is one of the most severe diseases resulting from environmental contamination. We found asbestosis in a female Japanese macaque over 25 years of age that died from senility. Clear needle-like crystals were deposited throughout the lung lobes, particularly in the perivascular areas. Asbestos bodies were observed in some of these crystals. Fibrosis without inflammation was observed in the periarterial and peribronchiolar regions. The crystals were identified as tremolite, and a total of 16,633,968 asbestos bodies and 465,334,411 tremolite fibers were observed in 1 g of dry lung tissue. No tumors or pleural adhesions were seen. This is the first report of spontaneous asbestosis in a nonhuman animal.

The first report of peritoneal tetrathyridiosis in squirrel monkey (Saimiri sciureus).

This report describes a case of peritoneal larval cestodiasis caused by tetrathyridia of Mesocestoides sp. in an adult female squirrel monkey. The monkey had lived in a zoological garden in Japan and had a clinical history of wasting. At necropsy, numerous whitish oval masses were found in the liver and peritoneal cavity. These masses contained larval cestodes. Morphological observation and molecular analyses of the mitochondrial 12S ribosomal RNA gene and cytochrome c oxidase subunit 1 gene sequences allowed us to identify the larva as the tetrathyridium of Mesocestoides sp. This is the first report of Mesocestoides larvae in a squirrel monkey in Japan.