Urothelial hyperplasia with calculi (papillomatosis) in the urinary bladder of a male spontaneous diabetic Torii rat.

A 40-week-old male spontaneous diabetic Torii rat, an animal model of type 2 diabetes mellitus, was found to have marked urinary calculi with hematuria in the urinary bladder on necropsy. Histological findings in the urinary bladder included a papillary growth pattern with a fibrovascular stroma without atypia. Fine granular materials in the bladder lumen were positive for Von Kossa staining but negative for periodic acid-Schiff or Gram staining, indicating no apparent bacterial infection in the urinary bladder. Scanning electron microscopy revealed that the urinary calculi were magnesium ammonium phosphate crystals (struvite). On the basis of the results, the lesion was diagnosed as urothelial hyperplasia with calculi (papillomatosis). Chronic inciting stimuli by struvite crystals were considered the primary cause of the bladder findings.

The Comprehensive Characterization of B7-H3 Expression in the Tumor Microenvironment of Lung Squamous Cell Carcinoma: A Retrospective Study.

Lung squamous cell carcinoma (LSCC) is refractory to various therapies for non-small cell cancer; therefore, new therapeutic approaches are required to improve the prognosis of LSCC. Although immunotherapies targeting B7 family molecules were explored as treatments for several cancer types, the expression and significance of B7-H3 in the tumor microenvironment (TME) and its relationship with other immune checkpoint molecules have not yet been investigated in detail. We used high-throughput quantitative multiplex immunohistochemistry to examine B7-H3 expression in the TME. We investigated the relationship between B7-H3 expression and prognosis as well as changes in the TME with B7-H3 expression using 110 surgically resected pathological specimens retrospectively. We examined the correlation between B7-H3 and programmed cell death-ligand 1 (PD-L1) expression in single cells. High B7-H3 expression in tumor cells was associated with a better prognosis and a significant increase in the number of CD163+PD-L1+ macrophages. Quantitative analysis revealed that there is a positive correlation between B7-H3 and PD-L1 expression in tumor and stromal cells, as well as in intratumoral tumor-infiltrating lymphocytes and tumor-associated macrophages in the same cells. CD68+, CD163+, and CK+ cells with PD-L1+ phenotypes had higher B7-H3 expression compared to PD-L1- cells. Our findings demonstrate a correlation between B7-H3 and PD-L1 expression in the same cells, indicating that therapies targeting B7-H3 could provide additional efficacy in patients refractory to PD-L1-targeting therapies.

A spontaneous ganglioneuroma in the adrenal medulla of a young Wistar Hannover rat.

A nodule was observed in the adrenal medulla of a twenty-week-old male Wistar Hannover rat. The nodule was predominantly (over 80%) composed of neural components, with ganglion cells scattered in sparse supporting tissue containing nerve fibers and Schwann cells. In the peripheral area of the tumor, atypical chromaffin cells were also observed. Accumulation of eosinophilic serous fluid was also noted in the stromal tissue. There were neither mitotic figures in the ganglion cells nor necrotic foci. In immunohistochemistry, the ganglion cells were positive for neuronal nuclei (NeuN), and negative for proliferating cell nuclear antigen, S-100, and chromogranin A. There were some NeuN-positive small cells in the peripheral area of the tumor. These findings indicate that this tumor was a ganglioneuroma. This seems to be an extremely rare case, as the spontaneous occurrence of ganglioneuroma in rats is very low, even in two-year carcinogenicity studies.

Disruption of fetal eye development caused by insulin-induced maternal hypoglycemia in rats.

We previously revealed that insulin-induced severe and long-lasting maternal hypoglycemia in rats caused anophthalmia and microphthalmia in fetuses; however, it remained unclear whether hypoglycemia-induced eye anomalies were developmental retardation or disruption, and when and how they developed. Hence, we induced hypoglycemia in pregnant Sprague-Dawley rats by injecting insulin from Days 6 to 11 of pregnancy and performed periodical histopathological examination of fetal eyes from embryonic days (E)10 to 20. On E10, optic vesicle had developed normally both in the control and insulin-treated group; however, on E11, optic cup (OC) had developed in the control group but not in the insulin-treated group. On E12, neural retina (NR), retinal pigmented epithelium (RPE), lens, and presumptive cornea had been observed in the control group. In contrast, lens pit and OC with remaining space between RPE and NR had developed in the insulin-treated group. From E13 to E15, developmental disruption characterized by defects, hypoplasia, and degeneration in the retina, lens, and cornea was observed in the insulin-treated group, resulting in anophthalmia or microphthalmia on E20. Moreover, the expression of MITF and chx10, which are essential for early eye development by expressing in the presumptive retina and lens and regulating each other's expression level, was ectopic and suppressed on E11. In conclusion, insulin-induced maternal hypoglycemia caused developmental disruption, but not simple developmental retardation of fetal eyes, and its trigger might be a failure of presumptive retina and lens to interact on E11.

DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody-Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models.

B7-H3 is overexpressed in various solid tumors and has been considered as an attractive target for cancer therapy. Here, we report the development of DS-7300a, a novel B7-H3-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and its in vitro profile, pharmacokinetic profiles, safety profiles, and in vivo antitumor activities in nonclinical species. The target specificity and species cross-reactivity of DS-7300a were assessed. Its pharmacologic activities were evaluated in several human cancer cell lines in vitro and xenograft mouse models, including patient-derived xenograft (PDX) mouse models in vivo. Pharmacokinetics was investigated in cynomolgus monkeys. Safety profiles in rats and cynomolgus monkeys were also assessed. DS-7300a specifically bound to B7-H3 and inhibited the growth of B7-H3-expressing cancer cells, but not that of B7-H3-negative cancer cells, in vitro. Additionally, treatment with DS-7300a and DXd induced phosphorylated checkpoint kinase 1, a DNA damage marker, and cleaved PARP, an apoptosis marker, in cancer cells. Moreover, DS-7300a demonstrated potent in vivo antitumor activities in high-B7-H3 tumor xenograft models, including various tumor types of high-B7-H3 PDX models. Furthermore, DS-7300a was stable in circulation with acceptable pharmacokinetic profiles in monkeys, and well tolerated in rats and monkeys. DS-7300a exerted potent antitumor activities against B7-H3-expressing tumors in in vitro and in vivo models, including PDX mouse models, and showed acceptable pharmacokinetic and safety profiles in nonclinical species. Therefore, DS-7300a may be effective in treating patients with B7-H3-expressing solid tumors in a clinical setting.

Datopotamab Deruxtecan, a Novel TROP2-directed Antibody-drug Conjugate, Demonstrates Potent Antitumor Activity by Efficient Drug Delivery to Tumor Cells.

Trophoblast cell surface antigen 2 (TROP2) is highly expressed on various epithelial tumors and correlates with poor prognosis. We developed the novel TROP2-directed antibody-drug conjugate (ADC), datopotamab deruxtecan (Dato-DXd, DS-1062a), with a potent DNA topoisomerase I inhibitor (DXd), and evaluated its antitumor activity and safety profiles in preclinical models.The pharmacologic activity and mechanism of action of Dato-DXd were investigated in several human cancer cell lines and xenograft mouse models including patient-derived xenograft (PDX) models. Safety profiles were also assessed in rats and cynomolgus monkeys.Dato-DXd bound specifically to TROP2 and was internalized into tumor cells followed by intracellular trafficking to lysosome and DXd release, which induced DNA damage and apoptosis in TROP2-expressing tumor cells in vitro. Dato-DXd exhibited in vivo antitumor activity with DNA damage induced by the accumulated DXd in TROP2-expressing xenograft tumors, but neither isotype control IgG-ADC nor anti-TROP2 antibody had this effect. Dato-DXd also showed potent antitumor activity with tumor regression in several TROP2-expressing xenograft tumors including NSCLC PDX models. Safety profiles of Dato-DXd in rats and cynomolgus monkeys were acceptable.Dato-DXd demonstrated potent antitumor activity against TROP2-expressing tumors by efficient payload delivery into tumors and acceptable safety profiles in preclinical models. These results suggest Dato-DXd could be a valuable treatment option for patients with TROP2-expressing tumors in the clinical setting.

Methemoglobinemia induced by 1,2-dichloro-4-nitrobenzene in mice with a disrupted glutathione S-transferase Mu 1 gene.

A specific substrate to Mu class glutathione S-transferase (GST), 1,2-dichloro-4-nitrobenzene (DCNB), was administered to mice with a disrupted GST Mu 1 gene (Gstm1-null mice) to investigate the in vivo role of murine Gstm1 in toxicological responses to DCNB. A single oral administration of DCNB at doses of 500 and 1000 mg/kg demonstrated a marked increase in blood methemoglobin (MetHB) in Gstm1-null mice but not in wild-type mice. Therefore, Gstm1-null mice were considered to be more predisposed to methemoglobinemia induced by a single dosing of DCNB. In contrast, 14-day repeated-dose studies of DCNB at doses up to 600 mg/kg demonstrated a marked increase in blood MetHB in both wild-type and Gstm1-null mice. However, marked increases in the blood reticulocyte count, relative spleen weight, and extramedullary hematopoiesis in the spleen were observed in Gstm1-null mice compared with wild-type mice. In addition, microarray and quantitative reverse transcription-polymerase chain reaction analyses in the spleen showed exclusive up-regulation of hematopoiesis-related genes in Gstm1-null mice. These changes were considered to be adaptive responses to methemoglobinemia and attenuated the higher predisposition to methemoglobinemia observed in Gstm1-null mice in the single-dose study. In toxicokinetics monitoring, DCNB concentrations in plasma and blood cells were higher in Gstm1-null mice than those in wild-type mice, resulting from the Gstm1 disruption. In conclusion, it is suggested that the higher exposure to DCNB due to Gstm1 disruption was reflected in methemoglobinemia in the single-dose study and in adaptive responses in the 14-day repeated-dose study.

N-Methyl-N-Nitrosourea-Induced Acute Alteration of Retinal Function and Morphology in Monkeys.

PURPOSE: The purpose of this study was to investigate both functional and morphologic alteration of the retina acutely induced by N-methyl-N-nitrosourea (MNU) in monkeys. METHODS: The MNU was administered intravenously at a single dose of 40 mg/kg to six cynomolgus monkeys, and standard full-field electroretinograms (ERGs) were recorded 1, 3, and 7 days after dosing. In addition, the rod and cone a-waves in response to high-intensity flashes were analyzed by the a-wave fitting model (a-wave analysis). The photopic negative response (PhNR) was also recorded at the same time points. Furthermore, the retinas of two animals each were examined histopathologically 1, 3, or 7 days after dosing. RESULTS: The MNU attenuated all the standard full-field ERGs including the rod-driven and cone-driven responses; in the combined rod-cone response, the b-wave was more affected than the a-wave. In the a-wave analysis, the sensitivity parameters (S) of the rod and cone a-waves had decreased on the day after dosing and remained unchanged thereafter. The maximum response parameter (Rmax) of the rod a-wave gradually decreased. On the other hand, the Rmax in the cone a-wave transiently increased on the day after dosing and decreased thereafter; the PhNR amplitude showed a similar time course change. Histopathologically, the retinal lesion on the day after dosing mainly consisted of pyknosis and karyorrhexis in the photoreceptor nucleus. Depletion of some photoreceptor nuclei, and shortening and disorientation of the photoreceptor segments became prominent at 3 and 7 days after dosing. Localization of degenerated photoreceptors was consistent with that of rhodopsin-positive photoreceptors, resulting in a well-preserved central fovea. CONCLUSIONS: Our results indicated that MNU acutely induced rod-dominant photoreceptor degeneration in monkey retinas, but the photoreceptor function was impaired in both the rods and cones. Functional involvement of the postreceptoral components was also indicated.

Role of connexin 32 in acetaminophen toxicity in a knockout mice model.

Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300mg/kg, and in Cx32KO mice given 100mg/kg or more. At 200mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.

Retinal function and morphology in monkeys with ethambutol-induced optic neuropathy.

PURPOSE: Ethambutol-induced optic neuropathy is a well recognized adverse ocular event. However, abnormalities of the retina in this optic neuropathy are not fully understood. Therefore, the purpose of the present study was to investigate both functional and morphological alterations of the retina induced by ethambutol in monkeys. METHODS: Ethambutol was orally administered to three cynomolgus monkeys, initially at 400 mg/kg/day followed by 800 mg/kg/day, for a maximum of 39 weeks. Full-field electroretinograms (ERGs) were recorded at intervals of approximately one month. The protocol included standard ERG responses to white flashes obtained under dark-adapted conditions (rod, combined rod-cone, oscillatory potentials) or with a white background (single-flash cone, 30 Hz flicker). In addition, we measured the ERG elicited with red flashes under blue background light (single-flash cone response [R/B]). All the ethambutol-treated monkeys were euthanized, and the retinae and various other nervous system tissues were examined histopathologically. RESULTS: No obvious changes were observed in the standard full-field ERGs. On the other hand, selective attenuation of the photopic negative response (PhNR) of the single-flash cone response (R/B) was observed in two out of three ethambutol-treated monkeys at week 22 or 28. Histopathology of these two monkeys revealed single cell necrosis of the retinal ganglion cells (RGCs), decreased RGCs in the parafovea and increased microglial cells in the nerve fiber layer in the retina, in addition to demyelination and glial reaction in the optic nerve, chiasm and tracts. CONCLUSIONS: The attenuated PhNR and histopathology of the retina indicated that RGCs were markedly damaged, both functionally and morphologically in monkeys with ethambutol-induced optic neuropathy. These results implied that RGCs are predominantly affected in the retina of patients with ethambutol-induced optic neuropathy.

Resistance to acetaminophen-induced hepatotoxicity in glutathione S-transferase Mu 1-null mice.

We investigated the role of glutathione S-transferases Mu 1 (GSTM1) in acetaminophen (APAP)-induced hepatotoxicity using Gstm1-null mice. A single oral administration of APAP resulted in a marked increase in plasma alanine aminotransferase accompanied by hepatocyte necrosis 24 hr after administration in wild-type mice, but its magnitude was unexpectedly attenuated in Gstm1-null mice. Therefore, it is suggested that Gstm1-null mice are resistant to APAP-induced hepatotoxicity. To examine the mechanism of this resistance in Gstm1-null mice, we measured phosphorylation of c-jun N-terminal kinase (JNK), which mediates the signal of APAP-induced hepatocyte necrosis, by Western blot analysis 2 and 6 hr after APAP administration. A marked increase in phosphorylated JNK was observed in wild-type mice, but the increase was markedly suppressed in Gstm1-null mice. Therefore, it is suggested that suppressed phosphorylation of JNK may be a main mechanism of the resistance to APAP-induced hepatotoxicity in Gstm1-null mice, although other possibilities of the mechanism cannot be eliminated. Additionally, phosphorylation of glycogen synthase kinase-3ß and mitogen-activated protein kinase kinase 4, which are upstream kinases of JNK in APAP-induced hepatotoxicity, were also suppressed in Gstm1-null mice. A decrease in liver total glutathione 2 hr after APAP administration, which is an indicator for exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of APAP, were similar in wild-type and Gstm1-null mice. In conclusion, Gstm1-null mice are considered to be resistant to APAP-induced hepatotoxicity perhaps by the suppression of JNK phosphorylation. This study indicates the novel role of GSTM1 as a factor mediating the cellular signal for APAP-induced hepatotoxicity.

Mixed Type of Malignant Mesothelioma in an Aged Male ICR Mouse.

Multiple whitish nodules in the thoracic cavity at the site of the thymus were observed in a 101-week-old male ICR mouse. In a histopathological examination, the neoplastic cells were predominantly fusiform in shape and proliferated in sarcomatoid growth patterns. Some neoplastic cells showed epithelial growth patterns, such as the ductal structures. Mitotic figures were frequently seen, and small necrotic foci and invasion to adjacent thoracic organs were noted. In Alcian blue staining, bluish materials were observed between fusiform-shaped cells and in some of the lumens of the ductal structures. In immunohistochemistry, both fusiform-shaped and ductal structure-forming cells were positive for vimentin and weakly positive to positive for cytokeratin. Based on the aforementioned findings, the thoracic nodules were diagnosed as a mixed type of malignant mesothelioma. This case was thought to be rare because of the very low occurrence of spontaneous mesothelioma in mice.

Twenty-six-Week carcinogenicity study of chloroform in CB6F1 rasH2-transgenic mice.

The carcinogenic potential of chloroform was evaluated in a short-term carcinogenicity testing system using CB6F1 rasH2-Tg (rasH2-Tg) mice. Chloroform was administered to rasH2-Tg males at doses of 28, 90, or 140 mg/kg and rasH2-Tg females at 24, 90, or 240 mg/kg by oral gavage for 26 weeks. Wild-type (non-Tg) male and female mice received doses of 140 mg/kg and 240 mg/kg, respectively. N-methyl-N-nitrosourea was administered to rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. In both the rasH2-Tg and non-Tg mice, there was no significant increase in the incidence of neoplastic lesions by chloroform treatment. The incidence of hepatocellular foci in the rasH2- and non-Tg females receiving 240 mg/kg was increased. Forestomach tumors and malignant tumors occurred in most of the rasH2-mice in the positive control group. Swelling or vacuolation of hepatocytes, a toxic change induced by chloroform, occurred in both the rasH2-Tg and non-Tg mice. It is concluded that chloroform, a putative human noncarcinogen, did not show evidence of carcinogenic potential in the present study using rasH2-Tg mice. This study suggests that the rasH2-Tg mouse model may not be appropriate for detecting nongenotoxic carcinogens. However, the sensitivity of rasH2-Tg mice to nongenotoxic carcinogens should be assessed with consideration of the results from the other ILSI-HESI project studies.

Interlaboratory comparison of short-term carcinogenicity studies using CB6F1-rasH2 transgenic mice.

In order to evaluate a short-term carcinogenicity testing system using CB6F1 -Tg rasH2 (rasH2-Tg) mice carrying a human prototype c-Ha-ras gene, 26-week studies were conducted in 12 different facilities as a part of an International Life Science Institute Health and Environmental Science Institute (ILSI HESI) international collaborative project. In each study N-methyl-N-nitrosourea (MNU) was administered to a separate group of rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. We herein have summarized the mortality, body weight change, and neoplastic and nonneoplastic lesions detected in these positive control groups as representative historical positive control data. Also, we performed an interlaboratory comparison of the response of rasH2-Tg mice to MNU based on the data of 11 positive control groups from these studies. Although the body weight of rasH2-Tg mice showed lower values than that of non-Tgmice during the experimental period, body weight gain in the rasH2-Tg mice was similar to that in non-Tg mice. The mortality of rasH2-Tg mice during the study period was very low, the same as for the non-Tg mice. Incidences of spontaneous alveolar/bronchiolar adenomas and splenic hemangiomas/hemangiosarcomas were also low in the rasH2-Tg mice. Nonneoplastic lesions detected in the rasH2-Tg mice were similar to those in non-Tg mice, excluding the incidence of myopathy. There were interlaboratory differences in mortality and incidence of some lesions in the MNU-treated groups. However, the causes of death were common among the 11 laboratories and almost all the MNU-treated rasH2-Tg mice developed forestomach squamous cell papillomas/carcinomas or malignant lymphomas. This suggests that there is no appreciable difference in the response of the rasH2-Tg mouse to MNU used as a positive control. Therefore, it is concluded that MNU would be an adequate positive control compound in this testing system.