Decreased hepatic phosphorylated p38 mitogen-activated protein kinase contributes to attenuation of thioacetamide-induced hepatic necrosis in diet-induced obese mice.

We previously reported that thioacetamide (TA)-induced hepatocellular necrosis was attenuated in mice fed a high-fat diet (HFD mice) compared with mice fed a normal rodent diet (ND mice). In this study, we investigated whether p38 mitogen-activated protein kinase (p38 MAPK) was involved in this attenuation. Western blot analysis revealed that hepatic phosphorylated p38 MAPK protein decreased at 8 and 24 hours (hr) after TA dosing in the HFD mice, while it decreased only at 24 hr in the ND mice in comparison to the time- and diet-matched, vehicle-treated mice. p38 MAPK regulates various biological functions including inflammation, therefore, hepatic metabolomics analysis focusing on pro-inflammatory lipid mediators was performed. At 24 hr after TA dosing, only one pro-inflammatory mediator, 12-hydroxyeicosatetraenoic acid (HETE), was higher in the HFD mice. On the other hand, in addition to 12-HETE, 15-HETE and 12-hydroxyeicosapentaenoic acid (HEPE) were higher and omega-3/omega-6 polyunsaturated fatty acids ratios were lower in the ND mice at 24 hr. These results of metabolomics indicated that less pro-inflammatory state was seen in HFD mice than in ND mice at 24 hr. Finally, to confirm whether the observed decrease in phosphorylated p38 MAPK could attenuate TA-induced hepatocellular necrosis, we showed that SB203580 hydrochloride, an inhibitor of p38 MAPK, partially attenuated TA-induced hepatic necrosis in ND mice. Collectively, these results suggest that a prompt decrease in phosphorylation of p38 MAPK after TA administration is one of the factors that attenuate TA-induced hepatic necrosis in HFD mice.

Localization of connexin 32 in spontaneous liver lesions of mice.

We examined the localization of connexin 32 (Cx32), a component of gap junctions, in 24-month-old male B6C3F1 mice with spontaneously occurring hepatocellular altered foci or tumors. Immunohistochemically, Cx32-staining intensity in cell-to-cell membranes of altered hepatocytes was decreased in eosinophilic foci and increased in basophilic foci as compared to those in intact hepatocytes. These alterations were enhanced in adenomas and carcinomas with both eosinophilic and basophilic cytoplasm. In cell membranes facing on the sinusoidal portions, the intensities increased in all lesions. Image analyses confirmed that the spot areas of Cx32 were decreased in eosinophilic foci, but increased in basophilic foci, adenomas and carcinomas. These results demonstrate that Cx32 shows different expression in different types of hepatic lesions.

Hepatic glutathione contributes to attenuation of thioacetamide-induced hepatic necrosis due to suppression of oxidative stress in diet-induced obese mice.

We previously reported that hepatic necrosis induced by thioacetamide (TA), a hepatotoxicant, was attenuated in mice fed a high-fat diet (HFD mice) in comparison with mice fed a normal rodent diet (ND mice). In this study, we focused on investigation of the mechanism of the attenuation. Hepatic content of thiobarbituric acid reactive substances (TBARS), an oxidative stress marker, significantly increased in ND mice at 24 and 48 hr after TA administration in comparison to that in vehicle-treated ND mice. At these time points, severe hepatic necrosis was observed in ND mice. Treatment with an established antioxidant, butylated hydroxyanisole, attenuated the TA-induced hepatic necrosis in ND mice. In contrast, in HFD mice, hepatic TBARS content did not increase, and hepatic necrosis was attenuated in comparison with ND mice at 24 and 48 hr after TA dosing. Metabolomics analysis regarding hepatic glutathione, a biological antioxidant, revealed decreased glutathione and changes in the amount of glutathione metabolism-related metabolites, such as increased ophtalmate and decreased cysteine, and this indicated activation of glutathione synthesis and usage in HFD mice. Finally, after treatment with L-buthionine-S,R-sulfoxinine, an inhibitor of glutathione synthesis, TA-induced hepatic necrosis was enhanced and hepatic TBARS contents increased after TA dosing in HFD mice. These results suggested that activated synthesis and usage of hepatic GSH, which suppresses hepatic oxidative stress, is one of the factors that attenuate TA-induced hepatic necrosis in HFD mice.

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.

Thioacetamide-induced Hepatocellular Necrosis Is Attenuated in Diet-induced Obese Mice.

To assess modification of thioacetamide-induced hepatotoxicity in mice fed a high-fat diet, male C57BL/6J mice were fed a normal rodent diet or a high-fat diet for 8 weeks and then treated once intraperitoneally with thioacetamide at 50 mg/kg body weight. At 24 and 48 hours after administration, massive centrilobular hepatocellular necrosis was observed in mice fed the normal rodent diet, while the necrosis was less severe in mice fed the high-fat diet. In contrast, severe swelling of hepatocytes was observed in mice fed the high-fat diet. In addition, mice fed the high-fat diet displayed more than a 4-fold higher number of BrdU-positive hepatocytes compared with mice fed the normal rodent diet at 48 hours after thioacetamide treatment. To clarify the mechanisms by which the hepatic necrosis was attenuated, we investigated exposure to thioacetamide and one of its metabolites, the expression of CYP2E1, which converts thioacetamide to reactive metabolites, and the content of glutathione S-transferases in the liver. However, the reduced hepatocellular necrosis noted in mice fed the high-fat diet could not be explained by the differences in exposure to thioacetamide or thioacetamide sulfoxide or by differences in the expression of drug-metabolizing enzymes. On the other hand, at 8 hours after thioacetamide administration, hepatic total glutathione in mice fed the high-fat diet was significantly lower than that in mice fed the normal diet. Hence, decreased hepatic glutathione amount is a candidate for the mechanism of the attenuated necrosis. In conclusion, this study revealed that thioacetamide-induced hepatic necrosis was attenuated in mice fed the high-fat diet.

Neutrophil gelatinase-associated lipocalin, a sensitive urinary biomarker of acute kidney injury in dogs receiving gentamicin.

A sensitive urinary biomarker for acute kidney injury (AKI) was investigated in beagle dogs with nephrotoxicity induced by gentamicin. Gentamicin sulphate at 25 or 50 mg/kg was injected (s.c.) for 9 days, and conventional urinalysis, ELISA assay of neutrophil gelatinase-associated lipocal (NGAL) in urine, blood chemistry, and pathological examinations were performed. The dog given gentamicin at 25 mg/kg only showed slight deposition of lysosomal granules in the proximal tubular epithelium of the kidneys without any other significant changes even though urinary NGAL was elevated on Day 10 (day of necropsy). In the dog receiving gentamicin at 50 mg/kg, increases in urinary NGAL were observed on Days 3 and 5, and absence of urination, marked increases in serum urea nitrogen and creatinine, enlargement and discoloration of the kidneys with marked necrosis, and swelling of proximal epithelium were observed. In conclusion, urinary NGAL is considered to be a candidate as a sensitive predictable biomarker of AKI in the gentamicin-induced nephrotoxicity model in dogs.

Background lesions during a 24-month observation period in connexin 32-deficient mice.

Connexin 32 (Cx32) is a major gap junction protein in the liver. Neoplastic and non-neoplastic lesions were examined in Cx32-deficient (Cx32KO) mice maintained for 24-month, and compared with those in wild-type mice as a corresponding control. In neoplastic lesions, hepatocellular carcinoma increased significantly only in male Cx32KO mice, suggesting that Cx32 deficiency may be related to their pathogenesis. For females, the incidence of pituitary adenoma in the pars distalis of Cx32KO mice was lower than that of wild-type mice. No non-neoplastic lesions related to Cx32-deficiency were observed in the Cx32KO mice. In conclusion, these results demonstrate that the incidence of hepatocellular carcinoma increases only in male Cx32KO mice, presumably due to enhanced tumor promotion and progression signals associated with Cx32 deficiency.

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.

Toxicogenomic investigation on rat testicular toxicity elicited by 1,3-dinitrobenzene.

Rats were treated with a single oral dose of 10, 25 and 50mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli-germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity.

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.

Recommendations for pathology peer review.

Pathology peer review verifies and improves the accuracy and quality of pathology diagnoses and interpretations. Pathology peer review is recommended when important risk assessment or business decisions are based on nonclinical studies. For pathology peer review conducted before study completion, the peer-review pathologist reviews sufficient slides and pathology data to assist the study pathologist in refining pathology diagnoses and interpretations. Materials to be reviewed are selected by the peer-review pathologist. Consultations with additional experts or a formal (documented) pathology working group may be used to resolve discrepancies. The study pathologist is solely responsible for the content of the final pathology data and report, makes changes resulting from peer-review discussions, initiates the audit trail for microscopic observations after all changes resulting from peer-review have been made, and signs the final pathologist's report. The peer-review pathologist creates a signed peer-review memo describing the peer-review process and confirming that the study pathologist's report accurately and appropriately reflects the pathology data. The study pathologist also may sign a statement of consensus. It is not necessary to archive working notes created during the peer-review process.

Comprehensive analysis of hepatic gene and protein expression profiles on phenobarbital- or clofibrate-induced hepatic hypertrophy in dogs.

In order to characterize the hepatic effects of phenobarbital (PB) and clofibrate (CPIB) in dogs, PB and CPIB were administered to male beagle dogs for 14 days, and biochemical and histopathological examinations and comprehensive genomic and proteomic analyses, including GeneChip analysis and proteomics analysis using the 2-dimension difference gel electrophoresis (2D-DIGE) technique, were performed. Both compounds caused centrilobular hepatocellular hypertrophy, which were related to smooth endoplasmic reticulum (SER) proliferation in PB-treated dogs and to mitochondrial proliferation in CPIB-treated dogs. In the PB-treated dogs, drug-metabolizing enzyme induction was observed by Western blot and genomic analyses. CYP proteins could not be detected by the 2D-DIGE analysis, but increases in several endoplasmic reticulum (ER)-related proteins were observed. In the CPIB-treated dogs, drug-metabolizing enzyme induction was not clearly observed by any of Western blot, genomic and proteomic analyses. Genomic and proteomic analyses revealed that mitochondrial genes and proteins, including carnitine palmytoiltransferase II, acyl-CoA deheydrogenase and hydroxyacyl-CoA dehydrogenase, pyruvate carboxylase and ATP synthase beta chain were induced. There is a relatively good correlation among the morphology and the genomic and proteomic data, but some differences exist between the genomic and proteomic data. Comprehensive evaluation using these techniques in addition to morphological evaluation may provide a useful tool for safety assessment of the liver.

Neutralization of IL-10 exacerbates cycloheximide-induced hepatocellular apoptosis and necrosis.

Cycloheximide (CHX)-induced liver injury in rats has been characterized by hepatocellular apoptosis and necrosis. We previously reported that Kupffer cell inactivation causes a reduction of IL-10 production, resulting in the exacerbation of CHX-induced liver injury. In this study, we directly evaluate the role of IL-10 in liver injury by a pretreatment with anti-IL-10 neutralizing antibody (IL-10Ab). Rats were given goat IgG or IL-10Ab before being treated with CHX (CHX group or IL-10Ab/CHX group). In the CHX group, the CHX treatment markedly induced hepatic mRNA and serum protein levels of IL-10. The up-regulation of IL-10 was significantly suppressed in the IL-10Ab/CHX group. Blocking IL-10 in the IL-10Ab/CHX group led to greater increases in hepatic mRNA and serum levels of proinflammatory cytokines, such as TNF-alpha and IL-6. The IL-10Ab/CHX group developed more severe hepatocellular apoptosis, neutrophil transmigration, and necrotic change of hepatocytes compared with the CHX group. The caspase activities and mRNA levels of Cc120, LOX-1, and E-selectin in the livers were significantly higher in the IL-10Ab/CHX group than the CHX group. These results demonstrate that IL-10 plays an important role in counteracting the effect of proinflammatory cytokines, such as a TNF signaling cascade, and in attenuating the CHX-induced liver injury.

Collaborative work on evaluation of ovarian toxicity. 8) Two- or four-week repeated-dose studies and fertility study of Anastrozole in female rats.

The main focus of this study was to determine the optimal administration period in terms of toxic effects on ovarian morphological changes. To assess the morphological and functional changes induced by anastrozole in ovaries, the compound was administered to female rats at dose levels or 0, 0.01, 0.1 and 50 mg/kg for 2 or 4 weeks in the repeated dose toxicity study and at levels of 0, 0.01, 0.1 and 5 mg/kg from 2 weeks prior to mating to Day 7 or pregnancy in the female fertility study. In the repeated dose toxicity study, large abnormal atretic follicles, follicular cysts, a decrease in corpus luteum and depletion of developing corpus luteum were observed in the 1 and/or 50 mg/kg groups of both the 2-week and 4-week studies in a histopathological examination of the ovaries. In the female fertility study, the pregnancy rate was decreased in the 5 mg/kg group. Irregular estrous cycles, such as an extended cycle or no cycle, were observed in the 0.1 and 5 mg/kg groups. At necropsy, decreased numbers of implantations, corpora lutea and live fetuses were noted in the 1 and/or 5 mg/kg groups. Based on these findings, histopathological changes in the ovary are important endpoints for the evaluation of drugs inducing ovarian damage. We conclude that a 2-week administration period is sufficient to detect ovarian toxicity of anastrozole in a repeated dose toxicity study.

Influence of Kupffer cell inactivation on cycloheximide-induced hepatic injury.

In our previous study, we found that cycloheximide (CHX) induces hepatocellular necrosis as well as hepatocellular apoptosis. This article evaluates the role of Kupffer cells on cycloheximide-induced hepatic injury using gadolinium chloride (GdCl(3)) for the inhibition of Kupffer cells. One group of rats was treated with CHX (CHX group), and another was treated with GdCl(3) before being treated with the same dose of CHX (GdCl(3)/CHX group). The necrotic change in the GdCl(3)/CHX group was exacerbated under the induction of hepatocellular apoptosis by the CHX treatment. A substantial diminution of the number of ED1- or ED2-positive cells was demonstrated in the GdCl(3)/CHX group compared to the CHX group. In addition, the degree of decrease in ED2-positive cells was more apparent than that in ED1-positive cells. Increases in the mRNA levels of IL-10 and Stat3 were observed in the CHX group, but not in the GdCl(3)/CHX group. On the other hand, the hepatic mRNA levels of chemokines and adhesion molecules such as Ccl20, LOX-1, and E-selectin were significantly increased only in the GdCl(3)/CHX group. Thus, Kupffer cell inactivation by the GdCl(3) treatment leads to a loss of the capacity to produce IL-10, supposedly resulting in the enhancement of pro-inflammatory cytokine activities such as tumor necrosis factor (TNF) signaling. These events are suggested to be a factor of the inflammatory exacerbation in the livers of the GdCl(3)/CHX group. In conclusion, Kupffer cells may play a role in protecting hepatic necroinflammatory changes by releasing anti-inflammatory cytokines following the hepatocellular apoptosis resulting from CHX treatment.

Resistance to the skeletal muscle injury expressed by repeated treatment with compound A that has HMG-CoA reductase inhibitory activity.

It has been noted that chemical-induced initial insult is sometimes no longer detected in examinations after additional consecutive treatments, suggesting that the target organs acquire resistance to the chemical toxicity. In this study, whether acquired resistance to the skeletal muscle toxicity is observed during repeated treatment of a toxic dose of Compound A that has a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitory activity was examined. F344 male rats (7-weeks old) were given a mixed diet with 0.12% Compound A (corresponding to approximately 100 mg/kg/day) for up to 56 days. Blood samples were obtained from the tail vein periodically during the dosing period, and utilized for the measurement of creatine kinase (CK) as a marker of skeletal muscle injury. In the necropsies on Days 4, 8, 11, 28, 42 and 56, the skeletal muscles from the rectus femoris were removed for histopathology or gene expression analysis. A satellite group was provided to measure the plasma concentrations of Compound A and M1, the active metabolite of Compound A. CK levels increased from Day 9 and reached approximately 30 times those of the controls on Day 12. Histopathology of the skeletal muscle on Day 11 revealed severe necrosis of the muscle fibers. However, in spite of continuous treatments to the damaged rats, the CK levels decreased after that and returned to normal levels on Day 18. No skeletal muscle injury was observed on Days 42 and 56. There were no marked differences in the exposure levels of Compound A and M1 between Days 8 (prior to CK elevation) and 28 (post CK elevation). As for the most significant changes in the gene expression analysis for the skeletal muscle on Days 42 and 56, the probe for IkappaBa, which is known as an inhibitor for nuclear factor-kappaB (NF-kappaB), increased 2-fold compared to the control. Furthermore, an increased probe for CCAAT/enhancer-binding protein (C/EBP) delta, a transcriptional factor, and a decreased probe for cAMP-response element-binding protein (CBP)/p300, a transcriptional coactivator, were also noted significantly on Day 56. These changes in the gene expression analysis suggested suppressed NF-kappaB-mediated transactivation, which was responsible for the protective effects on the muscle injury. Based on the present findings, the resistance to skeletal muscle injury observed in this study may be attributable to the suppressed NF-kappaB-mediated transactivation, but not to the decreased exposure to toxicants.

Toxicoproteomic investigation of the molecular mechanisms of cycloheximide-induced hepatocellular apoptosis in rat liver.

C/EBP homologous protein (CHOP) is a transcriptional factor and is induced under conditions such as the unfolded protein response or amino acid starvation. A previous study showed that the transcriptional level of CHOP was highly increased in rat liver in which hepatocellular apoptosis was induced by cycloheximide (CHX) treatment. Here, we investigated the relationship between hepatocellular apoptosis and CHOP-mediated apoptotic pathway, and studied the mechanisms of induction of CHOP gene in the liver of rats treated with CHX. Male F344 rats were treated intravenously with 6mg/kg CHX, and sacrificed at 1, 2 and 6h after the treatment. In the gene expression assay using quantitative RT-PCR, the genes related to CHOP-mediated apoptosis such as the C/EBPbeta, ATF3 and ATF4 genes were significantly increased corresponding to the induction of hepatocellular apoptosis in rats treated with CHX. However the GRP78/Bip gene, which serves as a representative marker for the unfolded protein response, did not change after the treatment. Toxicoproteomics using two-dimensional difference gel electrophoresis and mass spectrometry indicated that GRP78/Bip was inactivated by the CHX treatment. Furthermore, the CHX-treated animals exhibited a significant decrease of phosphorylated Akt/PKB (protein kinase B). These results indicate that the protein synthesis inhibition by CHX induces the CHOP gene through a pathway similar to that of amino acid starvation, and that Akt/PKB inactivation enhances the CHOP-mediated hepatocellular apoptosis.