Inflammation of the cardiac coronary artery in ICR mice.

Inflammation of the cardiac coronary artery in ICR mice is occasionally observed in toxicity studies; however, this has not been well explored histologically. Herein, we investigated the detailed histology of the associated lesions in 6-8-week-old ICR mice. Coronary artery inflammation in the right ventricular wall was observed in 10 of 142 mice (7.0%). Histopathological examination revealed hypertrophy of the vascular smooth muscle cells and perivascular infiltration of macrophages in mild cases. In moderate to marked cases, single-cell necrosis of vascular smooth muscle cells, hemorrhage of the tunica media, and fibrinoid necrosis of the vessel wall were observed, in addition to the changes seen in mild cases. Electron microscopic examination of moderate cases revealed a discontinuous internal elastic lamina suggestive of rupture, and vascular smooth muscle cells beneath the elastic lamina showed degeneration and necrosis. These findings suggest that the lesions developed as a rupture of the internal elastic lamina and necrosis of vascular smooth muscle cells, while leaked plasma components caused vascular and perivascular inflammation. In ICR mice, dystrophic calcinosis (DCC) is known to occur rarely in the right ventricle. DCC is defined as focal calcification in necrotic myocardial fibers, the pathogenesis of which is considered to involve ectopic calcification. Since calcification was not observed in any part of the heart, including the inflammation region, the pathophysiology of cardiac arterial inflammation seen in our ICR mice was considered to differ from that of DCC.

A case of spontaneous rete testis adenoma in a Sprague-Dawley rat.

A 104-week-old male CD (SD) rat exhibited enlargement of the left testis. Microscopically, this mass was demarcated from the testis by fibrous connective tissue and characterized by cystic dilatation with single-layered columnar cells and papillary proliferation connected to the solid growth area without clear boundaries. In the solid growth area, cells were dissected into irregular alveolar nests by scant fibrous tissue with small blood vessels. The nuclei of proliferating cells were variable in size and round- to oval-shaped, and their cytoplasm was pale or eosinophilic and sometimes contained vacuoles or eosinophilic granules. Immunohistochemically, the tumor cells were positive for vimentin and cytokeratin (CK) 7. Since CK7 was exclusively positive in the rete testis epithelium of the naïve rat, it was valuable to diagnose this tumor as rete testis-originated. Based on these results and the lack of apparent pleomorphism, mitotic figures, and metastasis, the present case was diagnosed as rete testis adenoma.

Microarray-based gene expression analysis combined with laser capture microdissection is beneficial in investigating the modes of action of ocular toxicity.

The retina consists of several layers, and drugs can affect the retina and choroid separately. Therefore, investigating the target layers of toxicity can provide useful information pertaining to its modes of action. Herein, we compared gene expression profiles obtained via microarray analyses using samples of target layers collected via laser capture microdissection and samples of the whole globe of the eye of rats treated with N-methyl-N-nitrosourea. Pathway analyses suggested changes in the different pathways between the laser capture microdissection samples and the whole globe samples. Consistent with the histological distribution of glial cells, upregulation of several inflammation-related pathways was noted only in the whole globe samples. Individual gene expression analyses revealed several gene expression changes in the laser capture microdissection samples, such as caspase- and glycolysis-related gene expression changes, which is similar to previous reports regarding N-methyl-N-nitrosourea-treated animals; however, caspase- and glycolysis-related gene expressions did not change or changed unexpectedly in the whole globe samples. Analyses of the laser capture microdissection samples revealed new potential candidate genes involved in the modes of action of N-methyl-N-nitrosourea-induced retinal toxicity. Collectively, our results suggest that specific retinal layers, which may be targeted by specific toxins, are beneficial in identifying genes responsible for drug-induced ocular toxicity.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Dog.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the societies of toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in most tissues and organs from the dog used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Interstitial pneumonitis related to trastuzumab deruxtecan, a human epidermal growth factor receptor 2-targeting Ab-drug conjugate, in monkeys.

Trastuzumab deruxtecan (T-DXd: DS-8201a) is an anti-human epidermal growth factor receptor 2 (HER2) Ab-drug conjugated with deruxtecan (DXd), a derivative of exatecan. The objective of this study was to characterize T-DXd-induced lung toxicity in cynomolgus monkeys. Trastuzumab deruxtecan was injected i.v. into monkeys once every 3 weeks for 6 weeks (10, 30, and 78.8 mg/kg) or for 3 months (3, 10, and 30 mg/kg). To evaluate the involvement of DXd alone in T-DXd-induced toxicity, DXd monohydrate was given i.v. to monkeys once a week for 4 weeks (1, 3, and 12 mg/kg). Interstitial pneumonitis was observed in monkeys given T-DXd at 30 mg/kg or more. The histopathological features of diffuse lymphocytic infiltrates and slight fibrosis were similar to interstitial lung diseases (ILD)/pneumonitis related to anticancer drugs in patients, with an incidence that was dose-dependent and dose-frequency-dependent. Monkeys receiving DXd monohydrate did not suffer lung toxicity, although the DXd exposure level was higher than that of DXd in the monkeys given T-DXd. The HER2 expression in monkey lungs was limited to the bronchial level, although the lesions were found at the alveolar level. Immunohistochemical analysis confirmed that T-DXd localization was mainly in alveolar macrophages, but not pulmonary epithelial cells. These findings indicate that monkeys are an appropriate model for investigating T-DXd-related ILD/pneumonitis. The results are also valuable for hypothesis generation regarding the possible mechanism of T-DXd-induced ILD/pneumonitis in which target-independent uptake of T-DXd into alveolar macrophages could be involved. Further evaluation is necessary to clarify the mechanism of ILD/pneumonitis in patients with T-DXd therapy.

The Impact of the Timing of Dosing on the Severity of UNC569-Induced Ultrastructural Changes in the Mouse Retina.

Mer proto-oncogene tyrosine kinase (MerTK), expressed in the retinal pigment epithelium (RPE), regulates the phagocytosis of shed photoreceptor outer segments. To investigate the influence of dosing time on MerTK inhibitor UNC569-induced retinal toxicity, UNC569 at 100 mg/kg was orally administered to male mice at 2 different Zeitgeber times (ZT5.5 or ZT22) for 28 days. Electron microscopy was conducted at ZT2 after the final dosing. Additionally, the visual cycle components (11-cis-retinal, all-trans-retinal, all-trans-retinol, and 11-cis-retinol), which play an important role in maintaining retinal homeostasis, were quantified by liquid chromatography/mass spectrometry/mass spectrometry. Under electron microscopic examination, the number of phagosomes and phagolysosomes in the RPE increased in both the ZT5.5 and ZT22 administered groups, while endoplasmic reticulum dilatation in the RPE and chromatin aggregation of photoreceptor nuclei were observed only in the ZT22 administered group. No change was observed in any of the visual cycle components. These results suggest that the timing of the dosing in relation to the physiological MerTK phosphorylation affected the severity of changes in the RPE, leading to the apoptosis of the photoreceptor cells.

Host cell proteins induce inflammation and immunogenicity as adjuvants in an integrated analysis of in vivo and in vitro assay systems.

INTRODUCTION: Host cell proteins (HCPs) are contaminated proteins remaining after purification of biopharmaceuticals. Recent reports revealed clinical implications of HCPs in anti-drug antibody (ADA) development in patients without any inflammatory effects. Therefore, we evaluated the inflammatory effects and immunogenicity of HCPs in an in vivo study by intravitreal administration to rabbits and an in vitro THP-1 cells assay. METHODS: Escherichia coli-derived HCPs at 200 ng/eye with or without ranibizumab at 0.25 mg/eye were administrated intravitreally to rabbits. For in vitro examination, differentiated THP-1 cells were stimulated with HCPs at 0.17 to 10.88 µg/mL with or without ranibizumab at 0.2 mg/mL. RESULTS: Co-administration of HCPs with ranibizumab, but not HCPs alone, induced ocular inflammation. Presence of ADA (anti-ranibizumab) was detected in the vitreous fluid of rabbits in which HCPs and ranibizumab were co-administered. HCPs increased cytokine release and upregulated cell surface markers involved in the antigen presentation in the THP-1 cell assay, which was enhanced by co-stimulation with ranibizumab. DISCUSSION: These finding suggests that HCPs may induce inflammation and immunogenicity as an adjuvant. Furthermore, integrated analyses by an in vivo rabbit model and in vitro assay system using THP-1 cells would be useful to evaluate the immunological risk of HCPs.

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.

[Pharmacological, pharmacodynamics, and clinical profile of mirogabalin besylate (Tarlige® tablets 2.5†mg∙5†mg∙10†mg∙15†mg)].

Mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium channels, has been approved for the treatment of peripheral neuropathic pain including painful diabetic peripheral neuropathy (DPNP) and postherpetic neuralgia (PHN) in Japan. Mirogabalin showed potent and selective binding affinities for the α2δ subunits, and slower dissociation rates for the α2δ-1 subunit than for the α2δ-2 subunit. It also showed potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for the central nervous system side effects. A pharmacological study using mutant mice demonstrated that the analgesic effects of mirogabalin were mediated by binding of the drug to the α2δ-1 subunit, not the α2δ-2 subunit. The pharmacological properties of mirogabalin can be associated with its unique binding characteristics. The bioavailability of mirogabalin is high and its plasma exposure increases dose-proportionally. Mirogabalin is mainly excreted via the kidneys in an unchanged form, thus, mirogabalin has a low possibility of undergoing drug-drug interaction, while dose adjustment based on the creatinine clearance level is specified in patients with renal impairment. In double-blind, placebo-controlled phase 3 studies in Asian patients with DPNP and PHN, mirogabalin showed significant and dose-dependent pain relief, and all tested doses of mirogabalin were well tolerated. In summary, mirogabalin has a balanced efficacy versus safety profile, and can provide an alternative therapeutic option for the treatment of peripheral neuropathic pain.

Pathological examination of spontaneous vacuolation of pancreatic acinar cells in mice.

Pancreatic acinar cell vacuolation is spontaneously observed in mice; however, the lesion is rare and has not been well documented. Herein, we present a detailed pathological examination of this lesion. Vacuoles in pancreatic acinar cells were present in 2/15 X gene knockout mice with a C57BL/6J mouse background, 4/298 ICR(CD-1) mice, 1/110 B6C3F1 mice, and 3/399 CByB6F1-Tg(HRAS)2Jic mice. The vacuoles were usually observed in a unit of the acinus, and the lesions were spread throughout the pancreas. These vacuoles contained weakly basophilic material that was positive for the periodic acid-Schiff reaction. Immunohistochemically, the vacuoles were positive for calreticulin antibody. Electron microscopy revealed globular dilatation of the rough endoplasmic reticulum (rER). According to these findings, vacuolation of pancreatic acinar cells is caused by the accumulation of misfolded proteins and enlargement of the rER.

Binding Characteristics and Analgesic Effects of Mirogabalin, a Novel Ligand for the α2δ Subunit of Voltage-Gated Calcium Channels.

Mirogabalin ([(1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]acetic acid), a novel ligand for the α2δ subunit of voltage-gated calcium channels, is being developed to treat pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. In the present study, we investigated the in vitro binding characteristics and in vivo analgesic effects of mirogabalin compared with those of pregabalin, a standard α2δ ligand. Mirogabalin showed potent and selective binding affinities for the α2δ subunits, while having no effects on 186 off-target proteins. Similar to pregabalin, mirogabalin did not show clear subtype selectivity (α2δ-1 vs. α2δ-2) or species differences (human vs. rat). However, in contrast to pregabalin, mirogabalin showed greater binding affinities for human α2δ-1, human α2δ-2, rat α2δ-1, and rat α2δ-2 subunits; further, it had a slower dissociation rate for the α2δ-1 subunit than the α2δ-2 subunit. Additionally, in experimental neuropathic pain models, partial sciatic nerve ligation rats and streptozotocin-induced diabetic rats, mirogabalin showed more potent and longer lasting analgesic effects. In safety pharmacological evaluations, mirogabalin and pregabalin inhibited rota-rod performance and locomotor activity in rats; however, the safety indices of mirogabalin were superior to those of pregabalin. In conclusion, mirogabalin shows potent and selective binding affinities for the human and rat α2δ subunits, and slower dissociation rates for the α2δ-1 subunit than the α2δ-2 subunit. It shows potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for side effects of the central nervous system. These properties of mirogabalin can be associated with its unique binding characteristics.

DS-7250, a Diacylglycerol Acyltransferase 1 Inhibitor, Enhances Hepatic Steatosis in Zucker Fatty Rats via Upregulation of Fatty Acid Synthesis.

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis. Since Dgat1-/- mice fed a high-fat diet (HFD) are resistant to hepatic steatosis, DGAT1 inhibitors are expected to have antifatty liver effects. To evaluate the hepatic effects of DS-7250, a selective DGAT1 inhibitor, vehicle or 10 mg/kg of DS-7250 was administered orally to male Fisher 344 (F344) and Zucker fatty (ZF) rats fed a standard diet or HFD for 14 or 28 days. ZF rats showed slight hepatic steatosis regardless of feeding conditions. DS-7250 exacerbated hepatic steatosis in ZF rats fed an HFD compared with the vehicle control. Hepatic steatosis did not occur in F344 rats fed an HFD, in which systemic exposures of DS-7250 were comparable to those in ZF rats. There was a higher expression of genes involved in lipid uptake and fatty acid synthesis in ZF rats compared to F344 rats under HFD conditions. DS-7250 upregulated key genes involved in de novo lipogenesis, which causes hepatic steatosis independently of DGAT1, in ZF rats fed an HFD compared with the vehicle control. These data suggest that ZF rats were more susceptible to hepatic steatosis due to their genetic characteristics and DS-7250 exacerbated hepatic steatosis independently of DGAT1.

Minimal change disease of the kidney in a young Sprague Dawley rat.

Minimal change disease (MCD) can be experimentally induced in rats, but spontaneous cases have not been reported. Herein, we present a case of MCD in rats that resembled the phenotypes of human MCD. A 9-week-old male Sprague Dawley rat developed continuous albuminuria for 2 weeks and was sacrificed at 11 weeks of age. Histological testing revealed no glomerular or renal tubular abnormalities on light microscopy. Immunofluorescence revealed absence of immunoglobulin G or immunoglobulin M deposition in the glomerulus. Extensive foot process effacement of glomerular podocytes was observed by electron microscopy, with rearrangement of the actin cytoskeleton at the base of the fused foot processes. The rat did not show desmin-positive podocytes, and the nephrin showed a normal liner pattern of distribution along the glomerular capillary loop throughout the glomeruli. These pathological characteristics corresponded to those of human MCD, and the glomerular lesion was considered a rare case of rat MCD.

UNC569-induced Morphological Changes in Pigment Epithelia and Photoreceptor Cells in the Retina through MerTK Inhibition in Mice.

Mer proto-oncogene tyrosine kinase (MerTK), which is expressed in the retinal pigment epithelium (RPE), regulates phagocytosis of shed photoreceptor outer segments (POS). To investigate the effects of drug-induced MerTK inhibition on the retina, UNC569, a specific MerTK inhibitor, was orally administered to male mice at a concentration of 60, 100, or 150 mg/kg for up to 14 days. Furthermore, MerTK inhibition in the retinal tissue sample was examined using a phosphorylation assay following a single dose of UNC569 at 100 mg/kg. In electron microscopic examination, UNC569 at 100 mg/kg or more increased phagosomes and phagolysosomes in the RPE. In addition, UNC569 at 150 mg/kg increased chromatin-condensed nuclei in the outer nuclear layer, indicating the early phase of apoptosis of photoreceptor cells. MiR-183, miR-96, and miR-124, which are enriched in photoreceptor cells, were elevated in the plasma of mice following treatment of 150-mg/kg UNC569, in conjunction with the photoreceptor lesion. Additionally, 100-mg/kg UNC569 inhibited MerTK phosphorylation in the retina. These results suggest that MerTK inhibition impaired phagocytic function of the retina, leading to accumulation of shed POS within the POS layer and increasing phagosomes and phagolysosomes in the RPE to delay POS renewal, resulting in apoptosis of photoreceptor cells.

Gene expression analysis of Arc mRNA as a neuronal cell activity marker in the hippocampus and amygdala in two-way active avoidance test in rats.

INTRODUCTION: Immediate early genes are widely used as neuronal cell activity markers in neuroscience. The present study investigated the relationship between their expression and abnormality in context fear conditioning. METHODS: The learning test (two-way active avoidance test) was conducted in male rats administered with nonselective muscarinic antagonist scopolamine or selective dopamine D1-like receptor antagonist SCH 23390 at a dose level of 2.0 or 0.1mg/kg, respectively, for 4days. Expression levels of Arc and Fos mRNA in the hippocampus and amygdala were also evaluated on the second day of dosing by fluorescent in situ hybridization (FISH) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). RESULTS: Scopolamine had no effect on avoidance rate, but decreased freezing in the two-way active avoidance test. SCH 23390 decreased avoidance rate and increased freezing. In FISH and RT-qPCR assays, scopolamine decreased Arc mRNA in the hippocampus and amygdala, whereas SCH 23390 increased Arc mRNA in the hippocampus. By contrast, scopolamine and SCH 23390 did not change Fos mRNA expression compared to Arc mRNA expression. DISCUSSION: The results of the learning test indicated that scopolamine or SCH 23390 respectively inhibited fear or context conditioning in rats. Furthermore, alteration of the expression of Arc mRNA but not of Fos mRNA in the hippocampus and amygdala of the brain was suggested to be a sensitive neuronal cell activity marker to detect behavioral abnormality in the two-way active avoidance test.

Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.

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.