Centrilobular Hepatocytes Research Articles

Hepatic angiotensin-converting enzyme 2 expression in metabolic dysfunction-associated steatotic liver disease and in patients with fatal COVID-19.

Metabolic dysfunction-associated steatotic liver disease (MASLD), characterised by hepatic lipid accumulation, causes inflammation and oxidative stress accompanied by cell damage and fibrosis. Liver injury (LI) is also frequently reported in patients hospitalised with coronavirus disease 2019 (COVID-19), while pre-existing MASLD increases the risk of LI and the development of COVID-19-associated cholangiopathy. Mechanisms of injury at the cellular level remain unclear, but it may be significant that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes COVID-19, uses angiotensin-converting expression enzyme 2 (ACE2), a key regulator of the 'anti-inflammatory' arm of the renin-angiotensin system, for viral attachment and host cell invasion. To determine if hepatic ACE2 levels are altered during progression of MASLD and in patients who died with severe COVID-19. ACE2 protein levels and localisation, and histological fibrosis and lipid droplet accumulation as markers of MASLD were determined in formalin-fixed liver tissue sections across the MASLD pathological spectrum (isolated hepatocellular steatosis, metabolic dysfunction-associated steatohepatitis (MASH) +/- fibrosis, end-stage cirrhosis) and in post-mortem tissues from patients who had died with severe COVID-19, using ACE2 immunohistochemistry and haematoxylin and eosin and picrosirius red staining of total collagen and lipid droplet areas, followed by quantification using machine learning-based image pixel classifiers. ACE2 staining is primarily intracellular and concentrated in the cytoplasm of centrilobular hepatocytes and apical membranes of bile duct cholangiocytes. Strikingly, ACE2 protein levels are elevated in non-fibrotic MASH compared to healthy controls but not in the progression to MASH with fibrosis and in cirrhosis. ACE2 protein levels and histological fibrosis are not associated, but ACE2 and liver lipid droplet content are significantly correlated across the MASLD spectrum. Hepatic ACE2 levels are also increased in COVID-19 patients, especially those showing evidence of LI, but are not correlated with the presence of SARS-CoV-2 virus in the liver. However, there is a clear association between the hepatic lipid droplet content and the presence of the virus, suggesting a possible functional link. Hepatic ACE2 levels were elevated in nonfibrotic MASH and COVID-19 patients with LI, while lipid accumulation may promote intra-hepatic SARS-CoV-2 replication, accelerating MASLD progression and COVID-19-mediated liver damage.

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.

Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50–3000 ppm fluxapyroxad for 7 days and 250–3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250–3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1–100 μM fluxapyroxad or 500 μM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid β-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1–100 μM fluxapyroxad or 500 μM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1–16 μM fluxapyroxad or 500 μM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

Citrus limon (L.) Osbeck Fruit Peel Extract Attenuates Carbon Tetrachloride-Induced Hepatocarcinogenesis in Sprague-Dawley Rats.

Traditional herbal medicine practitioners in the Ashanti region of Ghana use the fruit peels of Citrus limon (L.) Osbeck (C. limon) in preventive and curative treatment of many cancers including liver cancer. This ethnobotanical claim remains to be verified scientifically. Aim of the Study. This study investigated prophylactic hepatoprotective and anti-HCC effects of C. limon peel extract (LPE) in CCl4/olive oil-induced HCC-like rats. After preparation of LPE, it was subjected to phytochemical screening using standard phytochemical methods. A total of 30 healthy adult male Sprague-Dawley rats (weighing 150-200 g) were randomly assigned into six groups of 5 rats each. Rats in the control group received olive oil (5 mL/kg ip) twice weekly for 16 weeks. Rats in the model group received CCl4/olive oil (2 mL/kg, ip) twice weekly for 16 weeks. Rats in capecitabine (10 mg/kg po) and LPE (50, 100, and 200 mg/kg po) groups received CCl4/olive oil (2 mL/kg, i.p) in the morning and their respective treatments in the afternoon twice a week for 16 weeks. Rats in all groups had free access to food and water ad libitum. Body weight and survival rates were monitored. Rats were sacrificed under deep anesthesia, blood was collected, and liver and other organs were isolated. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), prothrombin time, bilirubin, C-reactive protein (CRP), alpha- (α-) fetoprotein (AFP), and liver histology were assessed. Alkaloids, tannins, flavonoids, terpenoids, and saponins were detected in LPE. Model rats demonstrated increased serum levels of AFP, CRP, ALP, GGT, ALT, and AST, prothrombin time, total bilirubin, direct bilirubin, blood lymphocyte, and monocyte counts, but decreased serum albumin and total protein compared to control rats. Unlike the control, model rats demonstrated fat accumulation in periportal and centrilobular hepatocytes and neoplastic transformation. Semiquantitation of periodic acid Schiff- (PAS-) stained liver sections showed decreased glycogen storage in hepatocytes of model rats compared to control rats. Compared to the model, LPE treatment protected against CCl4-induced hepatocarcinogenesis, which was evidenced by decreased AFP, CRP, liver enzymes, total and direct bilirubin, prothrombin time, and blood lymphocyte and monocyte counts; attenuation of fat accumulation; and increased glycogen storage, albumin, and total protein. LPE abates CCl4-induced hepatocarcinogenesis by attenuating liver inflammation and improving metabolic, biosynthetic, and detoxification functions of the liver. The prophylactic hepatoprotective and anti-hepatocarcinogenic effects of LPE are attributable to its phytochemical composition raising hopes of finding potential anticancer bioactive compounds from C. limon fruit peels.

Diagnostic performance of transient elastography in differentiation between porto-sinusoidal vascular liver disease and compensated cirrhosis.

The efficacy of transient elastography (TE) in the differential diagnosis between porto-sinusoidal vascular disease (PSVD) and compensated cirrhosis has not been sufficiently studied. We aimed to investigate the diagnostic performance of TE and identify histological lesions associated with liver stiffness. We conducted a retrospective cohort study including patients with PSVD and cirrhosis (Child-Turcotte-Pugh class A) and healthy subjects. Both the PSVD and cirrhotic patients had at least one sign of PH. The area under the receiver operating characteristic curve (AUROC) was used for differentiation. Ninety-two patients with PSVD (median age: 53 years, 33% male), 100 patients with compensated cirrhosis and 101 healthy subjects were included. The median TE-LSM in the PSVD patients (10.0 [7.0-13.0] kPa) was significantly lower than that in the cirrhotic patients (21.0 [15.0-28.0] kPa, p < .001) but was significantly higher than that in the healthy subjects (5.1 [4.6-6.0] kPa, p < .001). The AUROCs of TE-LSM for the discrimination of PSVD from the cirrhosis and healthy subjects were 0.886 (95% CI: 0.833-0.928) and 0.913 (95% CI: 0.864-0.949), respectively. The sensitivity and specificity to discriminate PSVD from compensated cirrhosis were 78.3% and 82.0%, respectively, at a cut-off of 13.6 kPa. Furthermore, portal fibrosis and aberrant cytokeratin 7 expression of centrilobular hepatocytes were significantly associated with higher TE-LSM (≥10.0 kPa). TE-LSM can be used to differentiate PSVD from compensated cirrhosis. Pathological features in association with increased liver stiffness are identified.

28-day repeated dose toxicity and toxicokinetics study on new melatonergic antidepressant GW117 in beagle dogs.

GW117 is new melatonergic antidepressant being developed to show better antidepressant action than agomelatine. The purpose of this study was to evaluate the toxicity and to determine potential target organs after oral (gavage) administration of the test article GW117 for 28 days and to assess the reversibility after a 4-week recovery phase in beagle dogs. Toxicokinetics was also evaluated. Four groups were designed in this study, including the vehicle control group and the GW117 50, 150 and 500 mg/kg/day groups, with 5 dogs/sex/group. Body weight, hematology, clinical chemistry, gross necropsy, organ weight, histopathology, and other indicators were examined. Results showed that animals dosed at ≥150 mg/kg/day showed gastrointestinal reactions (watery feces and dark green/red brown feces), with a dose-response relationship in the incidence and severity grade. Female dogs at 500 mg/kg/day had an increase in organ weight and ratios of the liver at the end of the dosing phase. Histopathology examination showed that some animals at 500 mg/kg/day, especially female animals, had minimal centrilobular hepatocyte hypertrophy in the liver, which reversed after 28-day recovery. With the exception of the above, no GW117-related abnormality was noted. Meanwhile, there were no sexual differences in drug exposure and accumulation after the first and last dosing. The no observed adverse effect dose level (NOAEL) was 150 mg/kg/day, under which mean Cmax and AUC0 → t were 583.5 and 2767.0 ng/ml*h for females and 663.2 and 4046.3 ng/ml*h for males on Day 28.

Liver Fetuin-A at Initiation of Insulin Resistance

Hepatokines (liver secreted proteins with possible distant action) are emerging potential players in insulin resistance in type 2 diabetic patients. Here, we explored the effect of a high-fat diet on the expression of fetuin-A, one of those candidate liver proteins, and its relationship with liver macrophage activation. Mice were fed a normal diet or a high-fat diet for 3 days, known to initiate steatosis and liver insulin resistance. A preventive liver macrophage depletion was obtained by intravenous injection of clodronate-loaded liposomes. The mRNA and protein expression of fetuin-A was evaluated by qPCR, Western blot and immunofluorescence on different insulin-sensitive tissues (liver, adipose tissue, and muscle). Short-term high-fat diet-induced steatosis, liver macrophage activation, and hepatic insulin resistance together with a significantly increased expression of liver AHSG (α2-HS glycoprotein/fetuin-A) mRNA and serum fetuin-A concentration. On immunofluorescence, fetuin-A was mostly expressed in centrilobular hepatocytes. This increase in fetuin-A under high-fat diet was not evidenced in other peripheral insulin-sensitive tissues (skeletal muscle and adipose tissue). The mRNA expression of α2-HS glycoprotein was 800 times higher within the liver compared with the adipose tissue or the muscle. Liver macrophage depletion that significantly ameliorated insulin sensitivity was associated with a significant decrease in α2-HS glycoprotein mRNA expression. In conclusion, this study demonstrated liver fetuin-A overexpression at the initiation of high-fat diet feeding, concurrent with hepatic steatosis and insulin resistance. Targeting liver macrophages in this setting reduced liver α2-HS glycoprotein expression suggesting that fetuin-A acts as an hepatokine with proinsulin resistance effects.

Livestock fatalities attributed to a massive attack of Psorophora columbiae following Hurricane Laura

The objective of this case report is to characterize the sudden fatality of livestock associated with a massive attack of dark rice field mosquitoes (Psorophora columbiae) after the passage of Hurricane Laura in southwest Louisiana late August 2020. In 3 days, approximately 30 deer, 600 beef cattle, 100 sheep and 30 horses died following a brief history of weakness or, in most cases, no clinical signs prior to death. Several on-site visits were performed by veterinarians to evaluate animals and perform necropsies. Two deer were received for necropsy, and tissue samples from 1 bull and 1 horse were evaluated at the Louisiana Animal Diagnostic Disease Laboratory. In addition, postmortem examination was performed on another 15 animals by veterinarians. Similar gross alterations were observed in all necropsies. Lesions included multifocal petechiae and ecchymoses in the skin and the subcutaneous tissues. Both deer also had pale discoloration and accentuated lobular patterns in their livers. Histologically, the livers of the deer and the bull exhibited centrilobular degeneration and hepatocyte dissociation suggestive of severe anemia. The description of this animal fatality event associated with Psorophora colum- biae attack demonstrates the need to establish prevention and/or intervention protocols in areas prone to hurricanes and tropical depressions.

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&amp; 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.

Phosphatidylcholine (18:0/20:4), a potential biomarker to predict ethionamide-induced hepatic steatosis in rats.

Ethionamide (ETH), a second‐line drug for multidrug‐resistant tuberculosis, is known to cause hepatic steatosis in rats and humans. To investigate predictive biomarkers for ETH‐induced steatosis, we performed lipidomics analysis using plasma and liver samples collected from rats treated orally with ETH at 30 and 100 mg/kg for 14 days. The ETH‐treated rats developed hepatic steatosis with Oil Red O staining‐positive vacuolation in the centrilobular hepatocytes accompanied by increased hepatic contents of triglycerides (TG) and decreased plasma TG and total cholesterol levels. A multivariate analysis for lipid profiles revealed differences in each of the 35 lipid species in the plasma and liver between the control and the ETH‐treated rats. Of those lipids, phosphatidylcholine (PC) (18:0/20:4) decreased dose‐dependently in both the plasma and liver. Moreover, serum TG‐rich very low‐density lipoprotein (VLDL) levels, especially the large particle fraction of VLDL composed of PC containing arachidonic acid (20:4) involved in hepatic secretion of TG, were decreased dose‐dependently. In conclusion, the decreased PC (18:0/20:4) in the liver, possibly leading to suppression of hepatic TG secretion, was considered to be involved in the pathogenesis of the ETH‐induced hepatic steatosis. Therefore, plasma PC (18:0/20:4) levels are proposed as mechanism‐related biomarkers for ETH‐induced hepatic steatosis.

S2792 Marked Elevation in Serum Aminotransferases in Immune-Mediated Necrotizing Myopathy

Introduction: This is an elderly female diagnosed with immune-mediated necrotizing myopathy with marked elevations in serum aminotransaminases. Case Description/Methods: A 71 year old female was admitted for progressive proximal muscle weakness and lower extremity myalgia with an initial creatinine kinase (CK) of 10,520U/L concerning for rhabdomyolysis. Gastroenterology was consulted given an elevation in liver-associated enzymes in a hepatocellular injury pattern (AST 552U/L and ALT 653U/L) thought to be due to muscle breakdown. After a few days of intravenous fluids, the myalgias continued and her CK remained elevated. Rheumatology was consulted and an MRI of the extremities was pursued revealing T2 hyper-intensity and enhancement in multiple muscle segments concerning for an inflammatory myopathy. A muscle biopsy was obtained along the right thigh confirming an inflammatory change with necrotic muscle fibers. Furthermore, an anti-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) returned strongly positive ( >200U). Her atorvastatin 10mg PO qHS was discontinued. Chronic liver disease serologic evaluation resulted in a positive anti-smooth muscle antibody (ASMA) and an immunoglobulin G (IgG) level of 1218mg/dL. Given the concern for a concomitant autoimmune hepatitis, an EUS-guided liver biopsy was pursued which showed centrilobular hepatocyte drop-out with associated moderate mixed inflammation, increased iron deposition (2+ out of 4), with patchy mild fibrous portal expansion with rare periportal fibrosis without features consistent with autoimmune hepatitis and were thought to be due to the underlying immune-mediated necrotizing myopathy (IMNM). She was diagnosed with anti-HMGCR myopathy and was started on a course of prednisone. After 1 month of corticosteroid therapy and one week of azathioprine, her LAEs remained elevated (AST 577U/L and ALT 645U/L) and she continued to be symptomatic with worsening weakness. Rheumatology will be initiating a course of intravenous immunoglobulin G (IVIG) given the failure to respond to corticosteroid and thiopurine combination therapy. Discussion: This is a rare example of markedly elevated liver enzymes in a hepatocellular injury pattern of greater than 20 times the upper limit of normal mimicking autoimmune hepatitis in association with IMNM refractory to corticosteroid and thiopurine combination therapy. Salvage therapy with intravenous immunoglobulin G (IVIG) will be started by rheumatology. Liver enzyme levels are expected to downtrend with improvement in CK.Figure 1.: The top of the image illustrates centrilobular hepatocyte drop-out with associated inflammation including plasma cells and ceroid laden macrophages. Contrast these changes with the normal portal tract in the bottom portion of the image. (400X magnification).

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.

Single-Dose Toxicity Study on ML171, a Selective NOX1 Inhibitor, in Mice.

Background ML171 is a potent nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor with isoform selectivity only for NOX1. This study is aimed at investigating the safety of ML171 after a single intraperitoneal (IP) injection in mice. Methods The toxicity of a single dose of ML171 was evaluated in 6-week-old Institute of Cancer Research (ICR) mice in a good laboratory practice (GLP) laboratory. Twenty-five mice of each sex were assigned to five groups: negative control, vehicle control, and 125, 250, and 500 mg/kg of ML171. All mice were acclimatized for one week before beginning the study. Mice received an IP injection of ML171 or vehicle. The general condition and mortality of the animals were observed. The mice were sacrificed to evaluate histopathology 14 days after the administration of ML171 or vehicle. Results Bodyweights were not significantly different in any group. Three males and one female died due to ML171 administration in the 500 mg/kg dose group. Autopsies of the surviving mice did not reveal any significant abnormalities after the injection of 125 mg/kg of ML171. However, the anterior lobe edge of the liver was thickened and adhesions between the liver and adjacent organs were observed in mice treated with 250 or 500 mg/kg of ML171. In addition, hypertrophy of centrilobular hepatocytes and inflammatory cell infiltration were observed after injection of 250 and 500 mg/kg of ML171. Conclusion Our results indicate that the lethal IP injection dose of ML171 is 500 mg/kg for both males and females. Mortality were not observed for lower doses of ML171. The safe dose of single IP ML171 in ICR mice was 250 mg/kg or less. Further studies are needed to confirm the safety of ML171 in the human body.

Yellow Fever as Cause of Death of Titi Monkeys (Callicebus Spp.)

From 2016 to 2018, an epidemic wave of yellow fever (YF) occurred in Brazil, affecting a large number of Platyrrhini monkeys. Titi monkeys (Callicebus spp.) were severely affected yet pathological characterizations are lacking. This study characterized epizootic YF in 43 titi monkeys (Callicebus spp.) with respect to the microscopic lesions in liver, kidney, spleen, heart, brain, and lung, as well as the distribution of immunolabeling for YF virus antigen, and the flaviviral load in the liver. Of 43 titi monkeys examined, 18 (42%) were positive for yellow fever virus (YFV) by immunohistochemistry or reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Affected livers had consistent marked panlobular necrotizing hepatitis, lipidosis, and mild inflammation, with intense immunolabeling for YFV mainly in centrilobular hepatocytes (zone 1; P = .05). In the spleen, consistent findings were variable lymphoid depletion (10/11), lymphoid necrosis (lymphocytolysis; 4/11), and immunolabeling for YFV in histiocytic cells (3/16). The main finding in the kidney was multifocal acute necrosis of tubular epithelium (5/7) that was occasionally associated with intracytoplasmic immunolabeling for YFV (6/15). These data indicate that titi monkeys are susceptible to YFV infection, developing severe hepatic lesions and high viral loads, comparable to humans and Alouatta spp. Thus, Callicebus spp. may be reliable sentinels for YF surveillance.

Risk Assessment of "Other Substances" – L-Tyrosine

The Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) has, at the request of the Norwegian Food Safety Authority (Mattilsynet; NFSA), assessed the risk of "other substances" in food supplements and energy drinks sold in Norway. VKM has assessed the risk of doses given by NFSA. These risk assessments will provide NFSA with the scientific basis for regulating the addition of "other substances" to food supplements and other foods.&#x0D; "Other substances" are described in the food supplement directive 2002/46/EC as substances other than vitamins or minerals that have a nutritional or physiological effect. It is added mainly to food supplements, but also to energy drinks and other foods. VKM has not in this series of risk assessments of "other substances" evaluated any claimed beneficial effects from these substances, only possible adverse effects.&#x0D; The present report is limited to the use of L-tyrosine in food supplements. Risks related to tyrosine added to food and drinks, protein hydrolysates or high dietary protein intake are outside the scope of the opinion. The report is based on previous risk assessments of tyrosine and scientific papers retrieved from a comprehensive literature search.&#x0D; L-tyrosine, an aromatic amino acid, is considered a conditionally indispensable amino acid because it can be synthesised from L-phenylalanine in the liver. The magnitude of endogenous synthesis of L-tyrosine is not known, but is related to the intake of phenylalanine.&#x0D; L-tyrosine is a precursor of several biologically active substances, including catecholamine neurotransmitters, thyroid hormones and melanin skin pigments. L-tyrosine is available from all protein-containing foods such as meat, eggs, fish, dairy products, grains and pulses. According to information from the NFSA, L-tyrosine is an ingredient in food supplements sold in Norway. The NFSA has requested a risk assessment of the following doses of L-tyrosine food supplements: 1250 mg/day, 1500 mg/day, 1750 mg/day, and 2000 mg/day. Dietary intake of tyrosine in Norway is not known, but data from NHANES III (USA) suggest a mean dietary intake of about 2.8 g per day.&#x0D; In phase 1 seven previous reports that assessed the safety of L-tyrosine supplementation in humans were identified. For the present report, a literature search was performed to retrieve relevant human or animal studies on the safety of L-tyrosine. One relevant animal study assessing the toxicity of tyrosine by feeding rats different doses of tyrosine daily by oral gavage for 13 weeks was identified. No human studies have been identified.&#x0D; No major specific issues related to adverse effects from L-tyrosine used as food supplements were identified in previous reports. However, a lack of studies in healthy adult individuals as well as in children was pointed out, and in particular the absence of long-term studies in healthy individuals. &#x0D; A lowest observed adverse effect level (LOAEL) and a no observed adverse effect level (NOAEL) of 2000 and 600 mg/kg bw per day, respectively, for tyrosine have been identified in a 90-day toxicological study in rats. At 2000 mg/kg bw per day, significant increases, were found in weights of livers and kidneys in addition to increased plasma lipids and hypertrophy of centrilobular hepatocytes in both sexes.&#x0D; The NOAEL at 600 mg/kg per day was used to calculate the margin of exposure (MOE), the ratio of the NOAEL to the specified doses of 1250, 1500, 1750 and 2000 mg/day of Ltyrosine in food supplements. The MOE-values range from 13 for the highest supplement dose in children to 34 for the lowest supplement dose in adults.&#x0D; Given the low MOE-values (range 13-34), and the severity of the adverse effects at the LOAEL (3.3 times the NOAEL), VKM concludes that all the specified doses may represent a risk of adverse effects. No evidence was found to assume specific tolerance levels for L-tyrosine for children or adolescents. Therefore, a similar tolerance as for adults relative to body weight was assumed for these age groups. &#x0D; Based on these data, the Norwegian Scientific Committee for Food Safety (VKM) concludes that:&#x0D; &#x0D; In adults (≥18 years), the specified doses of 1250, 1500, 1750, and 2000 mg/day Ltyrosine in food supplements may represent a risk of adverse health effects.&#x0D; In adolescents (14 to &lt;18 years), the specified doses of 1250, 1500, 1750, and 2000 mg/day L-tyrosine in food supplements may represent a risk of adverse health effects.&#x0D; In children (10 to &lt;14 years), the specified doses of 1250, 1500, 1750, and 2000 mg/day L-tyrosine in food supplements may represent a risk of adverse health effects.&#x0D; &#x0D; Children below 10 years were not included in the terms of reference.

Different dietary fats impact on biochemical and histological parameters and gene expression of lipogenesis-related genes in rats

The effects of different types of dietary fats on some biochemical and histological parameters were investigated and gene expression of lipogenesis-related genes in rats was evaluated. Five groups of Wister rats were randomly selected. Rats in group 1 (control) were given only rodent chow. Rats in groups 2, 3, 4 and 5 were fed with rodent chow supplemented with 10% buffalo butter oil, buffalo butter oil solid fraction (S30), hydrogenated palm kernel oil shortening, and margarine, respectively. As a result of adding shortening (group 4), there was an increase in the rats’ weight and some of the serum biochemical parameters compared to the other experimental groups. Those increments were parallel to an increase in serum acetate of the same group. Histologically, photographs of the livers of rats in group 3 showed slight cytoplasmic vacuolization of centrilobular hepatocytes and asporadic cell necrosis. The livers of rats in group 4, however, showed activation of Kupffer cells, hydropic degeneration of hepatocytes and cytoplasmic vacuolization of centrilobular hepatocytes. The gene expression of sterol regulatory element-binding protein (SREBP) 1c and fatty acid synthase (FAS) showed that feeding rats with S30 and shortening led to an increase in SREBP-1c and FAS mRNA levels, thus indicating the activation of lipogenesis genes in the liver. Supplementing rat diets with margarine showed no changes in SREBP-1c and FAS mRNA levels compared with other treatments. In conclusion, diets with shortening or S30 resulted in an increase in weight gain, histopathological lesions, lipid profile levels changes and a significant up-regulation of SREBP-1c and FAS gene expression levels compared with butter oil and margarine.

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.

Is the hepatocyte ultrastructural zonal heterogeneity changed by overnight (16 h) fasting? Morphometric study

ABSTRACTBackground and objectives: Hepatocyte ultra-structure is influenced by feeding status, circadian rhythm, and zone location. The goal of the present study was to study the effect of overnight fasting on the hepatocyte ultrastructure and the zonal heterogeneity and to discuss the functional correlation.Methods: A total of 14 male albino rats were divided into two groups: negative control group fed ad libitum and overnight fasting rats for 16 hours. The different subcellular structures of both centrilobular and periportal hepatocytes in both control and fasted groups were compared by transmission electron microscopy. Morphometric analysis of the electron micrographs was also done using imageJ software.Results: The lysosomes surface density, mitochondrial volume and surface densities were significantly higher in periportal hepatocytes however surface density of smooth endoplasmic reticulum (SER) and peroxisomes were significantly higher in centrilobular hepatocytes of the control group. Fasting caused a significant decrease in the surface density of rough endoplasmic reticulum and glycogen volume density but with significant increase in SER surface density with more mitochondrial fusion and stronger mitochondrial ER contacts, isolation membranes, and autophagosomes. The zonal differences were maintained after fasting. The organelles appeared normal with no signs of degeneration.Conclusion: The organelles appeared normal with no signs of degeneration and the zonal differences were maintained after fasting. The change in hepatocyte ultrastructure after fasting may be related to autophagy.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7–28 days and 8000 ppm metazachlor for 3–28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3–28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5–50 μM) and NaPB (500 μM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5–25 μM metazachlor or 500 μM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

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.