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.

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.

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.

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.

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.

Microarray analysis of T-2 toxin-induced liver, placenta and fetal liver lesions in pregnant rats.

Pregnant rats on day 13 of gestation were treated orally with 2 mg/kg of T-2 toxin and sacrificed at 1, 3, 6, 9 and 12 h after the treatment (HAT). Histopathologically, the number of apoptotic cells was increased in the liver, placenta and fetal liver (peaked at 6, 12 and 9-12 HAT, respectively). To examine the gene expression profiles, we performed microarray analysis of these tissues at two selected time points based on the results of the TdT-mediated dUTP nick end labeling (TUNEL) staining. Increased expression of oxidative stress- and apoptosis-related genes was detected in the liver of dams, placenta and fetal liver of pregnant rats treated with T-2 toxin at the peak time point of apoptosis. Decreased expression of lipid metabolism- and drug-metabolizing enzyme-related genes was also detected in these tissues. The results suggested that the mitogen-activated protein kinase (MAPK) pathway might be involved in the mechanism of T-2 toxin-induced apoptosis. In addition, increased expression of the c-jun gene was consistently observed in these tissues. Our results suggest that the mechanism of T-2 toxin-induced toxicity in pregnant rats is due to oxidative stress followed by the activation of the MAPK pathway, finally inducing apoptosis. The c-jun gene may play an important role in T-2 toxin-induced apoptosis.

Characterization of a potential animal model of an idiosyncratic drug reaction: nevirapine-induced skin rash in the rat.

Idiosyncratic drug reactions are difficult to study in humans due to their unpredictability. Unfortunately, this characteristic also hinders the development of animal models needed for mechanistic studies. Nevirapine, used to treat human immunodeficiency virus (HIV) infections, results in a severe idiosyncratic skin rash in some patients. We found that nevirapine can also cause a significant rash in some strains of rats. At a dose of 150 mg/kg/day, the incidence in female Sprague-Dawley rats was 6/28 (21%), in female Brown Norway rats 32/32 (100%), and in female Lewis rats 0/6 (0%) while no male Sprague-Dawley or Brown Norway rats developed a rash. Female SJL mice 0/7 also did not develop nevirapine-induced skin lesions. The first sign of a reaction in Brown Norway rats was red ears at days 7-10 followed by a rash with scabbing mainly on the back; this was a shorter time to onset than in Sprague-Dawley rats. Light microscopy of the skin revealed a primarily mononuclear inflammatory infiltrate and lesions typical of self-trauma. Immunohistochemistry results suggest that the infiltrate was composed of CD4 and CD8 T cells as well as macrophages. A lower dose of either 40 or 75 mg/kg/day did not lead to a rash and, in fact, 2 weeks of the lower doses induced tolerance to the 150 mg/kg/day dose in female Brown Norway rats. A dose of 100 mg/kg/day resulted in rash in 2/4 (50%) of female Brown Norway rats. Rechallenge of Brown Norway rats that had been allowed to recuperate after a nevirapine-induced rash led to red ears in less than 24 h followed by hair loss and occasional skin lesions. Although the skin rash was less evident on rechallenge, microscopically, the cellular infiltrate was more prominent, especially surrounding the hair follicles. Moreover, there were lesions of interface dermatitis with apoptosis and satellitosis, indicative of a cell-mediated immune attack on the epidermis. While systemic signs of illness did not accompany the rash on primary exposure, on rechallenge, the animals appeared generally unwell and this forced sacrifice after 2 weeks or less of treatment. Importantly, splenocytes isolated from rechallenged animals were able to transfer susceptibility to nevirapine-induced skin rash to naïve female Brown Norway recipients, which was illustrated by a faster time to onset of rash in the recipients. The characteristics of this adverse reaction are similar to that seen in humans; that is, it is idiosyncratic in that it only occurs in some strains of animals, is delayed in onset, is more common in females, is dose-dependent, and appears to be immune-mediated. Therefore, it may represent a good animal model for the study of idiosyncratic drug reactions.