Comparative gene and protein expression analyses of a panel of cytokines in acute and chronic drug-induced liver injury in rats.

Drug-induced liver injury (DILI) is a significant safety issue associated with medication use, and is the major cause of failures in drug development and withdrawal in post marketing. Cytokines are signaling molecules produced and secreted by immune cells and play crucial roles in the progression of DILI. Although there are numerous reports of cytokine changes in several DILI models, a comprehensive analysis of cytokine expression changes in rat liver injury induced by various compounds has, to the best of our knowledge, not been performed. In the past several years, we have built a public, free, large-scale toxicogenomics database, called Open TG-GATEs, containing microarray data and toxicity data of the liver of rats treated with various hepatotoxic compounds. In this study, we measured the protein expression levels of a panel of 24 cytokines in frozen liver of rats treated with a total of 20 compounds, obtained in the original study that formed the basis of the Open TG-GATEs database and analyzed protein expression profiles combined with mRNA expression profiles to investigate the correlation between mRNA and protein expression levels. As a result, we demonstrated significant correlations between mRNA and protein expression changes for interleukin (IL)-1ß, IL-1α, monocyte chemo-attractant protein (MCP)-1/CC-chemokine ligand (Ccl)2, vascular endothelial growth factor A (VEGF-A), and regulated upon activation normal T cell expressed and secreted (RANTES)/Ccl5 in several different types of DILI. We also demonstrated that IL-1ß protein and MCP-1/Ccl2 mRNA were commonly up-regulated in the liver of rats treated with different classes of hepatotoxicants and exhibited the highest accuracy in the detection of hepatotoxicity. The results also demonstrate that hepatic mRNA changes do not always correlate with protein changes of cytokines in the liver. This is the first study to provide a comprehensive analysis of mRNA-protein correlations of factors involved in various types of DILI, as well as additional insights into the importance of understanding complex cytokine expression changes in assessing DILI.

Effect of PPARß/δ agonist on the placentation and embryo-fetal development in rats.

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.

Predictive genomic biomarkers for drug-induced nephrotoxicity in mice.

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.

Expression of peroxisome proliferator-activated receptor isoforms in the rat uterus during early pregnancy.

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 PPARß/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 PPARß/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 PPARß/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 PPARß/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.

Microarray analysis of Leflunomide-induced limb malformations in CD-1 mice.

The immuno-suppressant Leflunomide, a potent inhibitor of dihydroorotate dehydrogenase (DHODH) and tyrosine kinases, is teratogenic in laboratory animals. To better understand its teratogenic mechanism, pregnant mice (CD-1) received a single dose of 70mg/kg Leflunomide, or vehicle control, by gastric intubation on gestation day 10. Gene expression was evaluated in the pooled fore- and hindlimb buds of embryos 4 and 24h post-treatment. The down-regulation of cholesterol biosynthesis-related genes was observed but could not be correlated with teratogenicity, since Leflunomide did not alter cholesterol concentration in limb bud. Leflunomide inhibited the mitosis of limb mesenchymal cells, which may be linked to DHODH inhibition as well as a potential effect on tyrosine kinases that mediate cytokine and growth factor signaling and that may be responsible for the Leflunomide's teratogenicity.

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.

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.

Bicarbonate-induced phosphorylation of p270 protein in mouse sperm by cAMP-dependent protein kinase.

Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility.

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.