Centrilobular Hepatocyte Necrosis Research Articles

Acetaminophen Overdose Reveals Protease-Activated Receptor 4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium.

Hepatic endothelial cell (EC) dysfunction and centrilobular hepatocyte necrosis occur with acetaminophen (APAP) overdose. The protease thrombin, which is acutely generated during APAP overdose, can signal through protease-activated receptors 1 and 4 (PAR1/PAR4). PAR1 is a high-affinity thrombin receptor that is known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generated during APAP overdose to determine (1) if hepatic endothelial PAR4 is a functional receptor, and (2) endothelial-specific functions for PAR1 and PAR4 in a high thrombin setting. We generated mice with conditional deletion(s) of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte congestion/bleeding, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial Translating Ribosome Affinity Purification followed by next-generation sequencing (TRAPseq). We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability but no effect on hepatocyte necrosis. Additionally, mice with loss of endothelial Par1 and Par4 had reduced permeability at an earlier time point after APAP overdose when compared to mice singly deficient in either receptor in ECs. We also found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

Histopathological changes in the Liverof fatal burn victimsin relation to different survival period

: Annually about 2 million people suffer from various modes of burn injuries worldwide of whom more than a lakh die. In India about 60,000 people suffer from burns annually, more than 50,000 are treated in hospitals and about 10,000 succumb to thermal injury. : The primary importance of present study is to have a better understanding of changes occurring in this vital organ in relation to survival period of burn victims, to emphasis that hepatic dysfunction is also an important determinant to survival of burn victims. : Forensic Medicine and Toxicology Department of a tertiary care hospital. A descriptive study was performed on 37 burn deceased of both the sexes of different age groups brought into the mortuary of the Department of Forensic Medicine during the period of July 2009 to December 2010. Liver specimens were collected and the H& E stained slides studied separately by two independent observers. Fatty infiltrations in the liver were present at various stages i.e. microvesicular steatosis to macrovesicular steatosis, followed by fatty cyst in victims with survival periods ranging from 2 days to 30 days. Thus as the survival period increased, the proportion of involvement of the hepatic lobule increased with focal / confluent centrilobular hepatocyte necrosis in 54.5% victims of 8-15 days survival and ultimately ended into massive hepatocyte necrosis leading to complete loss of lobular architecture in victims who survived for 16- 30 days. : The present study indicates that liver damage in the form of hepatic steatosis leading to hepatocyte necrosis and latter complete loss of liver architecture and consecutively hepatic dysfunction occurs with increasing survival period of burn victims therefore, maintenance of Liver integrity and function are crucial for post burn survival.

Preclinical Investigation of a Potent Geranylgeranyl Diphosphate Synthase Inhibitor

The isoprenoid biosynthesis pathway (IBP) is responsible for the synthesis of isoprenoids used for the prenylation of proteins belonging to the Ras small GTPase superfamily. Geranylgeranyl diphosphate synthase (GGDPS) is the enzyme in the IBP pathway that catalyzes the synthesis of the 20-carbon isoprenoid GGDP. The enzymes geranylgeranyl transferase (GGTase) I and II utilize GGDP as the isoprenoid substrate for their geranylgeranylation reactions. Rab proteins mediate vesicle trafficking within the cell and their activity is dependent on geranylgeranylation. Our prior work has demonstrated that agents that disrupt Rab geranylgeranylation disrupt monoclonal protein trafficking in myeloma cells, resulting in induction of the unfolded protein response (UPR) pathway and apoptosis. Because myeloma cells express near maximal levels of UPR-associated proteins to aid in the continuous production of monoclonal proteins, they are particularly sensitive to activation of the pro-apoptotic arm of the UPR. We are therefore pursuing the development of GGDPS inhibitors as a novel therapeutic strategy for myeloma. Our lead compound VSW1198 (Figure) is the most potent GGDPS inhibitor reported to date with an IC50 of 45nM against purified enzyme, high specificity for GGDPS over the related enzyme farnesyl diphosphate synthase, and measurable cellular activity at concentrations as low as 30 nM (15 ng/mL). Due to its potent activity against myeloma cells in vitro, we were interested in evaluating the metabolic stability, pharmacokinetic (PK) profile, distribution pattern, and toxicology profile of VSW1198 in preparation for in vivo efficacy studies. Single dose testing via IV administration in CD-1 mice revealed a maximum tolerated dose of 0.5 mg/kg. Doses > 1mg/kg resulted in liver toxicity that peaked around 6-7 days post-injection. Histopathological examination of the livers demonstrated centrilobular hepatocyte necrosis and apoptosis with invasion of inflammatory cells and panlobular hepatocyte swelling. Analysis of blood samples showed elevated transaminase levels without effects on bilirubin, alkaline phosphatase, lactic dehydrogenase, or albumin, consistent with the observed pattern of hepatic injury. A control compound, RAM3059, also was tested in vivo . This compound has a similar isoprenoid triazole bisphosphonate structure (Figure) but is 380-fold less potent than VSW1198 as a GGDPS inhibitor. Mice injected with RAM3059 (1.6 mg/kg IV) were grossly and histologically normal with no signs of liver toxicity, suggesting liver injury is not due to nonspecific toxicity from the triazole bisphosphonate structure. No effects on kidney function, calcium, creatine kinase, or blood counts were observed. Histopathological examination of the brains and kidneys from the treated mice revealed no abnormalities. A PK and tissue distribution study included 11 blood samples obtained over a period from 5 minutes to 7 days post-injection of VSW1198 (0.5 mg/kg IV, n=5 per time point). Tissue samples (liver, kidney, spleen, lung, heart, brain, and bone marrow) were collected at 2, 8, 24, 48, and 72 hours post-injection (n=5 per time point). VSW1198 was extracted from plasma and tissue samples, derivatized by reaction with diazomethane, and analyzed via LC-MS/MS. The maximum concentration (C0 ) is 611.0 (+ 183.3) ng/mL, the area under curve (AUC0-∞ ) is 2355.9 (+ 160.3) hr*ng/mL, and the plasma elimination half-life (t1/2 ) is 38.0 (± 10.9) hrs. Detectable concentrations of VSW1198 at 72 hours (5.0 + 1.0 ng/mL) indicate prolonged systemic exposure. Analysis of tissues samples is underway and will be reported. The metabolic stability of VSW1198 was assessed using human liver microsomes (HLM) and mouse S9 cells for phase I and phase II metabolism. In both the HLM and mouse S9 studies, VSW1198 showed complete stability while a positive control showed rapid metabolic degradation. In summary, these studies demonstrate the metabolic stability and systemic distribution of the potent GGDPS inhibitor VSW1198. Multi-dose testing and efficacy studies in a myeloma xenograft mouse model are currently underway to assess further the clinical potential of GGDPS inhibitor therapy. [Display omitted] DisclosuresHolstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Bovine Babesiosis Diagnosed in Formalin-Fixed, Paraffin-Embedded Tissues by Using In Situ Hybridization.

Bovine babesiosis, caused by Babesia divergens, is in general a rare disease in Europe. Nonetheless, local outbreaks can cause severe economic damage, and postmortem identification represents a diagnostic challenge. During a recent outbreak in May 2018 in northern Germany, 21 animals of a herd of 150 cattle died within 40 days having had clinical signs of fever and hemoglobinuria. Gross examination of 4 of the 21 deceased animals revealed a tick infestation, jaundice, and dark brown staining of urine and kidneys. Histologically, there were iron-positive deposits, hyperplasia of the red pulp of the spleen, and centrilobular necrosis of hepatocytes. In several locations, small basophilic granules suggestive of intraerythrocytic parasites were visible in hematoxylin-eosin- and Giemsa-stained sections. Peripheral blood smears from a living cow from the herd and polymerase chain reaction (PCR) of feeding ticks revealed B. divergens infection. In situ hybridization (ISH) was applied on formalin-fixed, paraffin-embedded (FFPE) tissue of the necropsied cattle to confirm babesiosis in these animals postmortem. Digoxigenin-labeled DNA probes were generated based on a specific nucleotide sequence for B. divergens, obtained by PCR and sequencing of DNA isolates from infected Ixodes ricinus ticks from deceased cattle. ISH using these probes allowed postmortem diagnosis of B. divergens infection in routinely fixed FFPE tissues.

Chlorogenic acid prevents hepatotoxicity in arsenic-treated mice: role of oxidative stress and apoptosis

Arsenic is a potent and toxic heavy metal found in the environment that causes health problems, including liver disease, in humans and animals. Chlorogenic acid (CA) is the most abundant caffeoylquinic acid isomer present in plants. This study aims to assess how CA protects the liver tissue following sodium arsenite (NaAsO2)-induced toxicity in mice. Male Swiss mice were allocated into 5 groups: Control, intragastrically administered CA (200 mg/kg), intragastrically administered NaAsO2 (5 mg/kg), and two groups administered with CA (100 and 200 mg/kg) and NaAsO2. CA was administered 30 min before NaAsO2 and all the mice were treated daily for 28 days. To investigate the biochemical, histopathological, immunohistochemical, and molecular changes, blood and liver samples were collected. NaAsO2 treatment increased the liver function biomarkers such as alanine transaminase, aspartate transaminase, alkaline phosphatase, and total bilirubin. Lipid and nitric oxide production was elevated. Glutathione content and the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase decreased, indicating a disturbance in redox homeostasis. Histopathological examination revealed a granular degeneration of hepatocytes, infiltration of inflammatory cells, and centrilobular hepatocyte necrosis. Furthermore, tumor necrosis factor-α and interleukin-1β were upregulated upon NaAsO2 treatment, suggesting the induction of inflammation. Moreover, NaAsO2 triggered apoptosis in the liver by upregulating Bax and caspase-3 and downregulating Bcl-2. However, CA abrogated the biochemical, molecular, and histological changes, reflecting its hepatoprotective role in response to NaAsO2 treatment. Our findings demonstrate that CA could be a potential therapeutic to minimize NaAsO2-induced hepatic injury.

Acute Liver Failure Associated with Influenza A Virus Infection: an Autopsy Case Report

An 84-year-old man with chronic renal failure, anemia, and diabetes was admitted for hemodialysis initiation. His vital signs were stable until the eighteenth hospital day, before acquiring an influenza A virus infection. Three days later, he died of septic shock with severe liver impairment. His leukocyte count, prothrombin time (PT-INR), and liver enzyme levels such as aspartate transaminase and alanine aminotransferase, were significantly increased. Hypercytokinemia was also observed. Autopsy revealed bilateral diffuse pneumonia with neutrophil infiltration. The liver showed extensive centrilobular hepatocyte necrosis. Immunohistochemistry for influenza A nucleoprotein revealed positivity in the ciliated columnar epithelium of the bronchi and negativity in the trachea, lungs, and liver. Hypoxic hepatitis is characterized by an abrupt and massive increase in aminotransferase levels (＞ 20 times upper normal limit) due to anoxic centrilobular hepatocyte necrosis. The occurrence of hypoxic hepatitis requires a pre-existing, chronic condition, such as anemia, causing reduced oxygen supply to the liver, followed by an acute decrease in hepatic oxygen supply, such as septic shock. Therefore, this report suggests that hypoxic hepatitis can be an important causative factor for acute liver failure associated with influenza virus infection.

Acute liver failure secondary to severe systemic disease from fatal hemophagocytic lymphohistiocytosis: Case report and systematic literature review.

AIMTo systematically review liver disease associated with hemophagocytic lymphohistiocytosis (HLH), propose reasonable contraindications for liver transplantation for liver failure in HLH, and report an illustrative case.METHODSSystematic review according to PRISMA guidelines of hepatic manifestations of HLH using computerized literature search via PubMed of articles published since 1980 with keywords (“hemophagocytic lymphohistiocytosis” or “HLH”) AND (“liver” or “hepatic”). Two authors independently performed literature search and incorporated articles into this review by consensus. Illustrative case report presented based on review of medical chart, and expert re-review of endoscopic photographs, radiologic images, and pathologic slides.RESULTSA 47-year-old Caucasian male, was hospitalized with high-grade pyrexia, rash, total bilirubin = 45 g/dL, moderately elevated hepatic transaminases, ferritin of 3300 ng/dL, leukopenia, and profound neutropenia (absolute neutrophil count < 100 cells/mm³). Viral serologies for hepatitis A, B, and C were negative. Abdominal computed tomography scan and magnetic resonance imaging revealed no hepatic or biliary abnormalities. Pathologic analysis of liver biopsy revealed relatively well-preserved hepatic parenchyma without lymphocytic infiltrates or macrophage invasion, except for sparse, focal hepatocyte necrosis. Bone marrow biopsy and aspirate revealed foamy macrophages engulfing mature and precursor erythrocytes, consistent with HLH. Interleukin-2 receptor (CD25) was highly elevated, confirming diagnosis of HLH according to Histiocytic Society criteria. Patient initially improved after high-dose prednisone therapy. Patient was judged not to be a liver transplant candidate despite model for end stage liver disease (MELD) score = 33 because liver failure was secondary to severe systemic disease from HLH, including septic shock, focal centrilobular hepatocyte necrosis from hypotension, bone marrow failure, and explosive immune activation from HLH. The patient eventually succumbed to overwhelming sepsis, progressive liver failure, and disseminated intravascular coagulopathy. Systematic review reveals liver injury is very common in HLH, and liver failure can sometimes occur. Data on liver transplantation for patients with HLH are very limited, and so far the results have shown a generally much worse prognosis than for other liver transplant indications. Liver transplantation should not be guided solely by MELD score, but should include liver biopsy results and determination whether liver failure is from intrinsic liver injury vs multisystem (extrahepatic) organ failure from HLH.CONCLUSIONThis case report illustrates that liver transplantation may not be warranted when liver failure associated with HLH is primarily from multisystem failure from HLH. Liver biopsy may be very helpful in determining the severity and pathophysiology of the liver disease.

Aflatoxicosis in African greater cane rats (Thryonomys swinderianus)

Aim:Aflatoxicosis is a widespread problem in captive animals fed on stored food and has been reported in various animals both domestic and wild. This report documents the clinicopathologic, microbial diagnostic findings and therapeutic regime for a study on the presentation, management, and outcome of aflatoxicosis in greater cane rats.Materials and Methods:A total of 65 greater cane rats suspected to be exposed to the toxin were examined clinically along with their environment. Feed samples, recently deceased carcasses and some moribund carcasses were collected for the study. Carcasses were subjected to gross and histopathologic investigations while feed and organs were subjected to microbiological investigations.Results:Gross lesions included hepatic lipidosis with ecchymotic hemorrhages, distended gallbladder, and renomegaly with ecchymosis among others. Histopathology revealed loss of hepatocellular architecture with massive centrilobular hepatocyte necrosis and diffuse steatotic damage characterized by macrovacuoles. Other histologic findings included pulmonary congestion, moderate renal tubular degeneration, and necrosis of epithelial tubular cells. Aspergillus flavus was isolated from the feed and ingesta. Total aflatoxin detected in feed sample was found to be over 400 ppm. Klebsiella species, Staphylococcus species, and Bacillus species were isolated from the liver and intestinal content. Management was attempted using Fungizal® (Avico, Jordan) (which contains Thymol, benzoic acid, sorbic acid, and kaolin) and Orego-Stim® (Saife, USA) (which contains carvacrol and thymol) which were instituted in feed and Superliv® (Ayurvet, India) (polyherbal) liquid was instituted in water for 5 days at manufacturers’ dosage. All clinical signs disappeared, and no more deaths were recorded following management.Conclusion:This report concludes that aflatoxicosis causes severe mortality in greater cane rats and can be prevented and managed successfully.

Circulating liver-specific microRNAs in cynomolgus monkeys.

Circulating microRNAs (miRNAs) can potentially be used as sensitive and specific biomarkers for tissue injury. However, the usefulness of circulating miRNAs as safety biomarkers in nonclinical toxicological studies using nonhuman primates is debatable owing to the limited information on organ-specific miRNAs. Therefore, a systematic investigation was performed to address this point. We identified organ-specific miRNAs from cynomolgus monkeys by next-generation sequencing analysis, which revealed that miR-122 was only abundant in the liver, whereas miR-192 was abundant in the liver, stomach, intestines, and kidney. The sequences of these miRNAs were identical to their human counterparts. Next, the absolute miR-122 and miR-192 levels were qualified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to determine the circulating levels of the miRNAs. No significant differences in the levels of circulating miRNAs between sexes were noted, and there was greater interindividual variation in miR-122 (20-fold variation) than in miR-192 (8-fold variation), based on their dynamic ranges. Finally, we evaluated the fluctuation in circulating liver-specific miRNAs in a monkey model of acetaminophen-induced hepatotoxicity. Acetaminophen with L-buthionine-(S,R)-sulfoximine induced hepatotoxicity in all the animals, which was characterized histopathologically by centrilobular necrosis and vacuolation of hepatocytes. Circulating miR-122 and miR-192 levels increased more than ALT levels after 24 h, indicating that circulating miR-122 and miR-192 may serve as sensitive biomarkers for the detection of hepatotoxicity in cynomolgus monkeys. This review describes the fundamental profiles of circulating liver-specific miRNAs in cynomolgus monkeys and focusses on their organ specificity, circulating levels, and fluctuations in drug-induced hepatotoxicity.

Microcystin toxicosis in nursery pigs

This case report documents a clinical case of blue-green algae toxicosis, caused by microcystin toxins, in 5-week-old pigs. Mortality during the investigation was elevated by approximately 7.5% in three affected groups, with a final mortality of 11.4%, and 50% of the population demonstrating clinical signs of various degrees. Affected pigs grew slowly and had distended abdomens. Histological examination of tissue samples revealed hepatic centrilobular necrosis with chronic-active periacinar individual hepatocyte necrosis and regeneration or centrilobular hepatocyte necrosis with hemorrhage. Additional testing of the feed revealed no toxicity concerns. Algae were present on the surface of a small area of standing water near the pond that had a waterway to the main water supply. There was a small waterway that connected the standing water to the main pond. Water sampled from that small area tested positive for microcystin. On the basis of these findings, it was determined that the toxicity was caused by algae growth in that area. The affected area was removed to prevent further exposure, and no clinical signs have been present since the standing water area was drained. To the knowledge of the authors, this report describes the first documented case of microcystin toxicosis in nursery pigs.

Contributions of caspase-8 and -9 to liver injury from CYP2E1-produced metabolites of halogenated hydrocarbons

1. Drug-induced liver injury is difficult to predict at the pre-clinical stage. This study aimed to clarify the roles of caspase-8 and -9 in CYP2E1 metabolite-induced liver injury in both rats and cell cultures in vitro treated with carbon tetrachloride (CCl4), halothane or sevoflurane. The human hepatocarcinoma functional liver cell line was maintained in 3-dimensional culture alone or in co-culture with human acute monocytic leukemia cells.2. In vivo, laboratory indices of liver dysfunction and histology were normal after administration of sevoflurane. CCl4 treatment increased blood AST/ALT levels, liver caspase-3 and -9 activities and liver malondialdehyde, accompanied by centrilobular hepatocyte necrosis. Halothane increased AST/ALT levels, caspase-3 and -8 activities (but not malondialdehyde) concomitant with widespread hepatotoxicity. In vitro, CCl4 treatment increased caspase-9 activity and decreased both mitochondrial membrane potential (MMP) and cell viability. In co-culture, halothane increased caspase-8 activity and decreased MMP and cellular viability. There were no toxic responses in CYP2E1 knockdown in monoculture and co-culture.3. CYP2E1-inducing compounds play a pivotal role in halogenated hydrocarbon toxicity.4. Changes in hepatocyte caspase-8 and -9 activities could be novel biomarkers of metabolites causing DILI, and in pre-clinical development of new pharmaceuticals can predict nascent DILI in the clinical stage.

A monkey model of acetaminophen-induced hepatotoxicity; phenotypic similarity to human.

Species-specific differences in the hepatotoxicity of acetaminophen (APAP) have been shown. To establish a monkey model of APAP-induced hepatotoxicity, which has not been previously reported, APAP at doses up to 2,000 mg/kg was administered orally to fasting male and female cynomolgus monkeys (n = 3-5/group) pretreated intravenously with or without 300 mg/kg of the glutathione biosynthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO). In all the animals, APAP at 2,000 mg/kg with BSO but not without BSO induced hepatotoxicity, which was characterized histopathologically by centrilobular necrosis and vacuolation of hepatocytes. Plasma levels of APAP and its reactive metabolite N-acethyl-p-benzoquinone imine (NAPQI) increased 4 to 7 hr after the APAP treatment. The mean Cmax level of APAP at 2,000 mg/kg with BSO was approximately 200 µg/mL, which was comparable to high-risk cutoff value of the Rumack-Matthew nomogram. Interestingly, plasma alanine aminotransferase (ALT) did not change until 7 hr and increased 24 hr or later after the APAP treatment, indicating that this phenotypic outcome was similar to that in humans. In addition, circulating liver-specific miR-122 and miR-192 levels also increased 24 hr or later compared with ALT, suggesting that circulating miR-122 and miR-192 may serve as potential biomarkers to detect hepatotoxicity in cynomolgus monkeys. These results suggest that the hepatotoxicity induced by APAP in the monkey model shown here was translatable to humans in terms of toxicokinetics and its toxic nature, and this model would be useful to investigate mechanisms of drug-induced liver injury and also potential translational biomarkers in humans.

Long-term influence of bone marrow-derived mesenchymal stem cells on liver ischemia-reperfusion injury in a rat model.

The aim of this study was to evaluate the long-term usefulness of intraportal injection of the bone marrow-derived mesenchymal stem cells (BM-MSCs) in limitation of experimentally induced ischemia-reperfusion injury (IRI) in a rat model. Twenty Wistar rats were divided into 3 groups: donor group (n=5), study group (n=10), and control group (n=5). IRI was performed using a modified hanging-weight system after left portal triad occlusion in study group animals. Isolated autologous BM-MSCs were labeled with fluorochrome PKH-26 then intraportally injected into the rats in the study group. Control group animals were intraportally injected with 1 ml of PBS. Follow-up was 3 months, after which animals were sacrificed for histopathological examination. Migration of BM-MSCs into different organs was examined. H&E staining of liver tissue sections from "time zero" biopsies did not show many irregularities in structural or histological construction compared to liver sections from the control group. However, a small amount of centrilobular hepatocyte necrosis and coagulative necrosis with neutrophil infiltration areas was observed in liver sections of the study group. The migration assay of BM-MSCs labeled with PKH-26 showed the highest positive BM-MSCs staining (6%) in the spleen, while few positively stained cells were found (2%) in liver sections. No BM-MSCs were detected in brain, kidney, or lung tissues. These results suggest that intraportal bone marrow-derived mesenchymal stem cell injection is safe and cells do not migrate chaotically to other organs after targeted implementation.

Protection afforded by pre- or post-treatment with 4-phenylbutyrate against liver injury induced by acetaminophen overdose in mice

Acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) is a widely used analgesic/antipyretic drug with few adverse effects at therapeutic doses; suicidal or unintentional overdose of APAP frequently induces severe hepatotoxicity. To explore a new and effective antidote for APAP hepatotoxicity, this study examined the effects of sodium 4-phenylbutyrate (4-PBA) on liver injury induced by APAP overdose in mice. Liver injury was induced in C57BL/6 male mice by intraperitoneal injection of APAP (400mg/kg). The effects of 4-PBA (100–200mg/kg) treatment at 1h before the APAP injection were evaluated with serum alanine aminotransferase (ALT) and blood ammonia levels, hepatic pathological changes, including histopathology, DNA damage, nitrotyrosine formation, and mRNA or protein expression involved in the development of hepatotoxicity, such as X-box binding protein-1 (XBP1), c-Jun N-terminal kinase (JNK), C/EBP homologous protein (CHOP) and B-cell lymphoma 2 interacting mediator of cell death (Bim). In addition, glutathione depletion and CYP2E1 protein expression, which are measures of the metabolic conversion of APAP to a toxic metabolite, were examined. Furthermore, we examined the effects of post-treatment with 4-PBA against APAP-induced hepatotoxicity in mice. When administered at 1h before APAP injection, 4-PBA significantly prevented the increase in serum ALT and blood ammonia levels, centrilobular necrosis of hepatocytes, DNA fragmentation, and nitrotyrosine formation induced by APAP in mice. 4-PBA also inhibited hepatic Xbp1 mRNA splicing and JNK phosphorylation induced by APAP, but did not suppress CHOP and Bim mRNA and protein expression. In addition, 4-PBA had little effect on hepatic glutathione depletion and CYP2E1 expression, parameters of toxic APAP metabolite production. Post-treatment with 4-PBA administration at 1 or 2h after APAP injection also attenuated the increase in serum ALT and blood ammonia levels and hepatic pathological changes in APAP-induced hepatotoxicity in mice. Although post-treatment with 4-PBA did not show any effects on hepatic Xbp1 mRNA splicing and JNK phosphorylation, it drastically attenuated the DNA fragmentation induced by APAP. The precise molecular mechanisms of the protection afforded by 4-PBA against APAP hepatotoxicity in mice are unclear, but they seem to involve inhibition of hepatocellular DNA fragmentation. We suggest that 4-PBA is a promising candidate as an antidote against APAP-induced liver injury.

Chronological changes in circulating levels of soluble tumor necrosis factor receptors 1 and 2 in rats with carbon tetrachloride-induced liver injury

Carbon tetrachloride (CCl4) facilitates the generation of hepatotoxins that can result in morphologic abnormalities, and these abnormalities are reasonably characteristic and reproducible for each particular toxin. It is also known that tumor necrosis factor-alpha (TNF-α) may participate in CCl4-induced liver injury (CILI). In this study, we observed the chronological changes in circulating soluble tumor necrosis factor receptors 1 and 2 (sTNF-R1 and -R2) in rats with CILI. Laboratory data; circulating levels of TNF-α, sTNF-R1, and sTNF-R2; and TNF-α levels in liver tissues were measured at various time-points.In the CCl4 group, the plasma aspartate aminotransferase (AST, 7694±3041IU/l)/alanine aminotransferase (ALT, 3241±2159IU/l) levels peaked at 48h after CCl4 administration, but the other laboratory data did not differ significantly from the corresponding data in the controls. Centrilobular hepatocyte necrosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells near the central vein area were observed via hematoxylin eosin (HE) and TUNEL staining, respectively, at 24 and 48h after CCl4 administration. Compared to the control group, the CCl4 group did not show significantly the increased circulating TNF-α levels. But TNF-α levels in the liver tissues first peaked at 1h (5261±2253pg/g liver), and a second peak was observed at 12h (3806±533pg/g liver) after CCl4 administration. Compared to the control group, the CCl4 group showed significantly increased circulating levels of both sTNF-R1 (797±121pg/ml) and sTNF-R2 (5696±626pg/ml) 1h after CCl4 administration. Since the hepatocyte apoptosis may be resulted from binding of TNF-α with TNF-R1 at 24h after administration, and consequently the circulating TNF-R2 level might be approximately 10-fold higher than the circulating TNF-R1 level.In conclusion, increased circulating levels of sTNF-R1 and -R2 potentially contribute to drug-induced liver injury, together with AST/ALT.

Glatiramer Acetate Induced Hepatotoxicity

Glatiramer acetate (Copaxone), a polypeptide has been approved for treating patients with active relapsing-remitting multiple sclerosis. We report the first case of severe acute hepatitis after commencing treatment for multiple sclerosis with glatiramer acetate. A 31-year-old female with multiple sclerosis presented with anorexia, lethargy and jaundice five weeks after commencing glatiramer acetate. She had never received beta-interferon treatment. Investigations revealed a bilirubin of 0.109 mmol/L (0.002-0.02 mmoL/L) and prothrombin time of 21 secs (9-15 secs). Her liver function tests were normal before commencing glatiramer acetate. A liver biopsy performed approximately 6 weeks after commencement of glatiramer acetate showed predominantly centrilobular hepatocyte necrosis with portal-venous bridging, along with mild portal and interface hepatitis. The necrosis was not accompanied by an acute inflammatory or chronic inflammatory infiltrate. The features were not suggestive of autoimmune hepatitis but consistent with drug toxicity. The liver tests returned to normal within 2 months after cessation of glatiramer acetate. Physicians should be aware that glatiramer acetate can be associated with uncommon but yet significantly severe liver toxicity.

Evidence of Oxidative Stress and Associated DNA Damage, Increased Proliferative Drive, and Altered Gene Expression in Rat Liver Produced by the Cholangiocarcinogenic Agent Furan

Furan is a potent cholangiocarcinogen in rat by an as yet undefined mechanism. The risk to man remains unclear. Using a time-course stop study design, we have investigated the potential of furan to induce oxidative stress and DNA damage associated with inflammatory and regenerative responses in rat liver. Furan was administered via oral gavage (30 mg/kg b.w. 5 daily doses per week), and livers were analyzed at time points between eight hr and three months. A one-month recovery group previously treated for three months was also included. There was a marked association between CYP2E1 expression and DNA oxidation (8-oxo-dG) in areas of centrilobular hepatocyte necrosis seen after a single dose. After one-month recovery from three-month treatment, 8-oxo-dG was still observed in areas of furan-induced cholangiofibrosis. Furan-induced changes in the expression of various genes associated with oxidative stress, DNA damage, and cell cycle control were identified during treatment and recovery. We propose that furan-induced cholangiocarcinomas emerge from areas of cholangiofibrosis as a result of a combination of chronic, persistent indirect damage to DNA through oxygen radicals coupled with persistent proliferative signals, including loss of connexin 32, that act to convert this DNA damage to fixed mutations.

Intoxicação espontânea pelas larvas de Perreyia flavipes em bovinos no Estado de Santa Catarina, Brasil

Neste trabalho, é descrito um surto de intoxicação por Perreyia flavipes ocorrido em agosto de 2006, em um rebanho bovino de 280 animais, no município de Sombrio, em Santa Catarina. Ocorreram 17 mortes em um lote de 77 bovinos de raça mista com idades entre um e dois anos mantidos em um piquete (P1) de aproximadamente 90 hectares por cerca de 30 dias. Após as primeiras duas mortes, os animais foram movidos para um segundo piquete (P2), onde ainda ocorreram 15 mortes nos cinco dias após a transferência. Seis bovinos foram necropsiados, e amostras de tecidos foram avaliadas para exame histológico. Os achados de necropsia incluíam hemorragias subcutâneas na mucosa do abomaso e superfície subepicárdica. Ascite e edema do abomaso, duodeno, pâncreas e vesícula biliar foram observados. O fígado apresentava-se amarelado com petéquias subcapsulares e acentuação do padrão lobular. No rúmen de três bovinos, foram encontrados fragmentos de corpos e cabeças de larvas de P. flavipes em meio ao conteúdo ruminal. Histologicamente o fígado apresentava tumefação hepatocelular e necrose centrolobular e mediozonal, difusa e acentuada, com hemorragia e congestão centrolobular. Havia depleção e necrose linfóide no baço, placas de Peyer e linfonodos mesentéricos. O diagnóstico de intoxicação pelas larvas da P. flavipes foi baseado nos dados epidemiológicos, nos achados de necropsia e nas alterações histopatológicas. As lesões hepáticas e em tecidos linfóides, além da grande quantidade de larvas de P. flavipes no primeiro piquete (P1) e no rúmen de animais necropsiados, foram dados importantes para o diagnóstico.

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague–Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3–6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

Avaliação clínica da infecção experimental de bezerros com Salmonella Dublin

The clinical conditions of healthy calves infected with experimental 108CFU of Salmonella Dublin were evaluated and the viability of the experimental model in disease induction in calves was verified. Twelve 10 to 15-day-old male Holstein calves were examined. They were allocated into two groups, control and experimentally infected with 108CFU of Salmonella Dublin. Animals were submitted to clinical examination after inoculation and at every 12 hours, during seven days after the experimental infection. Samples of rectal swabs were collected for Salmonella Dublin isolation. All animals had severe diarrhea, with mucus and bleeding, 12 to 84 hours after the experimental infection with Salmonella Dublin, accompanied by fever, dehydration and respiratory signs. The isolation of Salmonella Dublin from rectal swabs occurred 12 hours after the infection. Two out of the six animals inoculated with Salmonella Dublin died with symptoms of enteritis, fibrinous pneumonia, centrilobular hepatic steatosis, hepatocyte necrosis, spleen congestion, interstitial nephritis, and tubular degeneration. Thus, the oral administration of 108CFU of Salmonella Dublin induced clinical signs of salmonellosis in 10 to 15-day-old calves.