Comparison of Acute Gene Expression Profiles of Islet Cells Obtained via Laser Capture Microdissection between Alloxan- and Streptozotocin-treated Rats.

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 ß-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 ß cells via endoplasmic reticulum stress. These results contribute to a better understanding of gene expression in the pathogenesis of islet toxicity.

Characterization of pancreatic islet cell tumors and renal tumors induced by a combined treatment of streptozotocin and nicotinamide in male SD rats.

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.

Historical control data on prenatal developmental toxicity studies in rabbits.

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994-2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter-laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Inhibiting the teratogenicity of the immunosuppressant leflunomide in mice by supplementation of exogenous uridine.

Leflunomide is an immunosuppressant drug displaying teratogenicity in mice, rats, and rabbits. Its immunosuppressive effect occurs via inhibition of dihydroorotate dehydrogenase (DHODH) and tyrosine kinases. In this study, we coadministered Leflunomide and uridine, a precursor substance of pyrimidine nucleotides, to pregnant CD-1 mice, and examined whether or not a decreased level of intracellular pyrimidine nucleotides with inhibition of DHODH is related to the teratogenicity of Leflunomide. Then we examined the alteration of the nucleotide level in fetal tissue by Leflunomide and the effect of coadministered uridine. We administered Leflunomide with or without uridine to pregnant mice on gestation day 10, and used the vehicle of Leflunomide as a control. Leflunomide caused multiple malformations in all fetuses, but coadministration with uridine inhibited most of its teratogenicity. Leflunomide decreased the concentration of pyrimidine nucleotides, not purine nucleotides, whereas uridine coadministered with Leflunomide partially restored the level of pyrimidine nucleotides. These results indicate that the inhibitory effect of DHODH activity is related to the teratogenicity of Leflunomide.

Critical periods for the teratogenicity of immune-suppressant Leflunomide in mice.

Leflunomide has inhibitory effects on dihydroorotate-dehydrogenase activity and protein tyrosine kinase activity. In the present study, a single dose of 50 mg/kg Leflunomide was administered to pregnant mice on one of gestation days (GD)6-11. Characteristic external malformations were craniofacial defects following dosing on GD7, cleft palate on GD9, cleft palate and limb and tail deformities on GD10, and limb deformities on GD11. Skeletal examination revealed cervical to caudal vertebral malformations after treatment on GD7, GD8, GD9 or GD10. In the viscera, cardiovascular deformities were observed in the GD7 and GD9 Leflunomide-treated groups. These results demonstrate that multiple malformations were seen in various organs and most of the malformations observed appeared to be developmental stage-specific responses to Leflunomide treatment.

Teratogenicity study of the dihydroorotate-dehydrogenase inhibitor and protein tyrosine kinase inhibitor Leflunomide in mice.

Leflunomide is an immunosuppressive agent that inhibits de novo synthesis of pyrimidine nucleotides and the activity of protein tyrosine kinase. This study examined the teratogenicity of Leflunomide in mice. Pregnant mice were treated orally with Leflunomide at a dose of 10, 30 or 70 mg/kg/day from day 6 to 15 of pregnancy. At 70 mg/kg, all embryos were resorbed and no live fetuses were detected. At 30 mg/kg, Leflunomide reduced fetal viability, and increased the incidence of multiple external, skeletal and visceral malformations. Characteristic external malformations were neural tube defects, cleft palate and tail deformities. Limb malformations were observed in a small number of fetuses. Skeletal examinations revealed malformations of cervical to sacral vertebrae, ribs and sternebrae. In the viscerae, the main anomalies were membranous ventricular septum defect and persistent truncus arteriosus. The results of this study indicate that Leflunomide administered at 30 mg/kg on days 6 to 15 of pregnancy can induce craniofacial malformations and deformities of the axial skeleton, heart and great vessels in mice.