Investigation of Reactive Oxygen Species production in Human Hepatocytes

1. Aim/Background: Reactive oxygen species (ROS) have been identified as compounds responsible for producing cellular damage. The purpose of this research is to examine if there is production of reactive oxygen species through free radical intermediates within human hepatocytes treated with morphine, bilirubin, or furosemide. The investigation examines the early stages of biotransformation by measuring the levels of reactive oxygen species produced inside of the treated hepatocytes within the first and second hours of treatment. The experiment was designed upon a case of a jaundiced (elevated bilirubin) infant who received morphine and furosemide and later died through unknown mechanisms. The experiment looks to examine if these drug compounds could contribute to cellular damage. This can help to further understand the potential interactions and complications of free radical intermediates produced during the phases of biotransformation. 2. Method: Previously cultured human hepatocytes were washed by centrifugation and re-suspended in 1x supplemental buffer to a concentration of 1x106 cells/mL and seeded in a dark clear bottom 96-well microplate at 100,000 stained cells/well. The cells were treated with either furosemide, morphine, bilirubin, a Tert-Butyl hydro peroxide (TBHP) positive control, or left as a background. Reactive oxygen generated in the presence of these agents were quantified by fluorescence excitation/emission measurement at 495nm/529nm. Fluorescence was measured at one and two hours. ROS generated convert 2',7'-dichlorodihydrofluorescein diacetate to 2',7'-dichlorodihydrofluorescein within the cells, which fluoresces. The fluorescence intensity detected is equivalent to the level of ROS generated. Wells that were untreated were used as blanks and subtracted from background and TBPH. 3. Results: Furosemide and Morphine did not produce statistically significant levels of ROS (p >0.05) above the background in both hours 1 and 2 of biotransformation and ROS measurement (Figure 1). Although Bilirubin did not produce statistically significant (p >0.05) levels of ROS above the background (Figure 2) during the first hour, it did produce statistically significant levels in the second hour of biotransformation. Each compound’s level of ROS was reduced during the second hour, signaling the removal of intermediate ROS metabolites (Figure 2). The production of ROS in each compound signifies that there is biotransformation to an intermediate that produces ROS. 4. Conclusion: The production of ROS above the background by each of the compounds shows there is an intermediate free radical compound that is produced during the biotransformation of each compound [21]. In this study, although furosemide and morphine did not produce statistically significant levels of ROS in both hours of biotransformation, bilirubin did produce significant levels of ROS in the second hour of biotransformation. This finding is in line with previous studies that shows morphine to offer protective effects against ROS production [16, 17]; and bilirubin demonstrating deleterious production of ROS at high doses [18]. Further work must be done to examine the correlation between the levels of ROS and extent of hepatocellular damage.

Post-synaptic scaffold protein TANC2 in psychiatric and somatic disease risk

Understanding the shared genetic aetiology of psychiatric and medical comorbidity in neurodevelopmental disorders (NDDs) could improve patient diagnosis, stratification and treatment options. Rare TANC2 (Tetratricopeptide Repeat, Ankyrin Repeat and Coiled-Coil Containing 2) disrupting variants were disease-causing in NDD patients. This post-synaptic scaffold protein, essential for dendrite formation in synaptic plasticity, plays an unclarified but critical role in development. We here report a novel homozygous-viable Tanc2 disrupted function model where mutant mice were hyperactive and had impaired sensorimotor gating consistent with NDD patient psychiatric endophenotypes. Yet, a multi-systemic analysis revealed the pleiotropic effects of Tanc2 outside the brain such as growth failure and hepatocellular damage. This was associated with aberrant liver function including altered hepatocellular metabolism. Integrative analysis indicates that these disrupted Tanc2 systemic effects relate to interaction with Hippo developmental signalling pathway proteins and will increase the risk for comorbid somatic disease. This highlights how NDD gene pleiotropy can augment medical comorbidity susceptibility underscoring the benefit of holistic NDD patient diagnosis and treatment for which large-scale preclinical functional genomics can provide complementary pleiotropic gene function information.

Hyperenzymemia after vaccination against COVID-19: complex equation with simple variables

The article presents clinical case of the jaundice development and severe hyperenzymemia in GAM-Covid-VAK (Sputnik V) vaccination against COVID-19 in a 69-year-old patient. History — systematic use of non-steroidal anti-inflammatory drugs due to persisting pain after knee arthroplasty in 2018; frequent trips for several years to another region for sanatorium treatment, the use of mineral water. The diseases caused by hepatitis viruses, drug damage and post-vaccination reaction were included in diagnostic search. The markers of hepatitis B and C infection viruses were not detected during the enzyme immunoassay and polymerase chain reaction. The indicator for determining the relationship of a drug with the liver damage development was 6 points (borderline value) and only indicated the likelihood of drug hepatotoxicity. At the same time, it is known from history that repeated administration of the drug did not cause liver dysfunctions. The diagnosis of coronavirus infection was established based on the identification of SARS-CoV-2 in the hospital with repeated laboratory testing and competing diagnosis of hepatitis A has been confirmed on the basis of hepatocellular damage and the presence of serological marker of hepatitis A virus (immunoglobulin M antibodies). The treatment was continued in the infectious hospital, where the diagnosis of co-infection was confirmed. The pneumofibrotic changes in the S5 region of the left lung were revealed according to computed tomography. The normalization of aminotransferase activity and bilirubin was noted during dynamic observation. Apparently HAV infection led to a decrease in the immune response, the formation of an insufficient level of neutralizing antibodies in vaccinated against COVID-19 patient M. and contributed to the development of a new coronavirus infection with minimal manifestations in contact with SARS-CoV-2.

Fifteen-minute update: International normalised ratio as the treatment end point in children with acute paracetamol poisoning

Paracetamol is one of the most frequent reasons for poisonings across the UK with an estimated 90,000 patients and 150 deaths annually. International normalised ratio (INR) may be elevated due to hepatocellular damage and is frequently used to monitor progress on N-acetyl cysteine. N-acetyl cysteine is associated with reduced activity of vitamin K dependent clotting factors leading to a benign elevation of INR. In asymptomatic children with normal aspartate transaminase/alanine transaminase, isolated borderline elevation of INR following paracetamol overdose should be reviewed for possible N-acetyl cysteine induced elevation of INR. Due to these factors, in those with borderline persistent elevation of INR, N-acetyl cysteine can be safety stopped if INR is falling on two or more consecutive tests and is <3.0.

Hepatoprotective Effects of Vernonia amygdalina (Astereaceae) Extract on CCl4-Induced Liver Injury in Broiler Chickens

The aim of this study was to evaluate the effect of Vernonia amygdalina leaf extract (VALE) on the carbon tetrachloride-induced hepatotoxicity (CCl4) in broiler chickens. A total of 360-day-old broilers were divided into 4 treatments of 90 birds each consisting of 6 replicates of 15 birds each. The treatments were birds offered 1 mL/kg BW saline (control group), 100 mg/kg BW VALE, 1 mL/kg BW CCl4 (CCl4-treated group), and 100 mg/kg BW VALE + 1 mL/kg BW CCl4 (VALE + CCl4 group). Blood samples were collected at 42 days of age and analyzed for the liver enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and selected biochemical parameters. The experiment was laid out in a completely randomized design. The results obtained showed that VALE had the potential to mitigate the adverse effects of CCl4 on protein and lipid metabolism as reflected in the low serum malondialdehyde (MDA) levels, which is a marker of lipid peroxidation. The aqueous extract of Vernonia amygdalina (VA) at a dose of 100 mg/kg body weight showed a moderate hepatoprotective effect by reducing serum AST levels (p < 0.05). The levels of serum AST, ALP, ALT, and GGT were significantly increased in CCl4-treated birds compared to the control group, reflecting carbon tetrachloride-induced liver damage. The VALE + CCl4 group showed a significantly higher amount of ALP compared to birds treated with carbon tetrachloride, suggesting a hepatoprotective effect. To conclude, Vernonia amygdalina aqueous extract can be used to confer protection against hepatotoxicity, which can induce severe hepatocellular damage in birds.

Hepatoprotective Effects of Calotropis procera (Ait.) R. Br Root Bark Extracts against Diethylnitrosamine Induced Liver Injury in Rats

Background: Diethylnitrosamine (DEN) is a hepatotoxin whose metabolic activation by liver cytochromes P450 is responsible for the necrosis, mutagenicity and carcinogenicity of liver cells. The purpose of this study was to evaluate the protective effects of Calotropis procera roots bark against DEN induced hepatocellular damage in rats. Material and Methods: Hepatoprotective activity of the ethanolic extract of Calotropis procera root bark were evaluated by induction of liver injury with DEN in Wistar male rats distributed in six groups of six. Serum hepatic markers, alanine amino transferase (ALAT), aspartate amino transferase (ASAT), alkaline phosphatase (ALP), total protein and albumin were evaluated and the enzymes antioxidant activities, superoxide dismutase (SOD) and catalase, as well as the level of malonedialdehyde (MDA) were determined in the liver homogenate. Histological analysis was carried out on sections of rat livers. Phytoconstituents have also been studied. Results: Pretreatment of rats with the extract showed a significant decrease in ALAT, ASAT and ALP while there was an increase in total protein and albumin compared to rats treated only with DEN. It also showed a significant increase in SOD and catalase and a decrease in MDA levels suggesting the hepatoprotective effect of the extract. Observation of liver sections confirmed the results of the biochemical parameters which would attest that the extract is hepatoprotective. Phytoconstituents such as sterols, triterpenes and phenolic compounds have been demonstrated. Conclusion: Ethanolic extract of Calotropis procera roots bark has shown hepatoprotective effects that could be due to its content in sterols and triterpenic and phenolic compounds.

Protective Role of Plasminogen Deficiency in Non-Alcoholic Fatty Liver Disease and Glucose Dysmetabolism

Obesity is global health problem with 40% of the world population being classified as overweight (BMI &gt; 25) and 13% as obese (BMI &gt; 30). Obesity drives chronic metabolic inflammation leading to metabolic syndrome, cardiovascular disease, fatty liver disease, Type II diabetes, and certain cancers. A documented clinical manifestation of obesity is perturbed and dysregulated hemostatic system leading to a procoagulant and anti-fibrinolytic state that ultimately results in an increased risk of thrombosis. Previous work suggested that clotting system components thrombin and fibrin engage in reciprocal mechanisms and contribute to the development of obesity. Specifically, we have shown that fibrin accumulates within obese adipose tissue and liver of obesity patients and mice challenged with a high fat diet (HFD) and colocalizes with macrophages, a key driver of inflammation in obesity. Despite the fact that obesity is known to be linked to an impaired fibrinolytic system, a potential functional contribution of fibrinolytic proteases to the development of obesity and associated downstream diseases has been understudied. Here, we tested the hypothesis that elimination of the fibrinolytic protease plasminogen would increase HFD-driven fibrin deposition and exacerbate macrophage accumulation and subsequent weight gain and obesity-associated pathologies. Contrary to our hypothesis, plasminogen-deficient (Plg-) mice gained as much weight as the Plg+ mice after 20 weeks on HFD. However, whereas the liver mass of HFD-challenged Plg+ mice was significantly higher than that of low fat diet (LFD)-fed mice, the livers of HFD-fed Plg- mice had a mass comparable to LFD-fed mice. HFD-fed Plg- mice had reduced hepatocellular damage, measured by plasma ALT activity, as well as reduced hepatosteatosis, measured by hepatic triglyceride content and liver histology, compared to HFD-fed Plg+ mice. Circulating cholesterol levels in HFD-fed Plg -/- mice were comparable to LFD-fed Plg- mice, while it was significantly elevated in HFD-fed Plg+ mice. While the epididymal white adipose tissue mass was higher in HFD-fed Plg- mice compared to HFD-fed Plg+ mice, the brown adipose tissue mass was comparable. However, there was an upregulation of uncoupling protein-1 (UCP-1) expression BAT of HFD-fed Plg- mice. Glucose clearance was more efficient in HFD-fed Plg- mice compared to HFD-fed Plg+ mice in a glucose tolerance test. Collectively, our data suggest that plasmin(ogen) contributes to HFD-induced fatty liver disease and glucose dysmetabolism. Disclosures No relevant conflicts of interest to declare.

Loss of hepatic Flcn protects against fibrosis and inflammation by activating autophagy pathways

Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease worldwide and can progress to non-alcoholic steatohepatitis (NASH), which is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat these conditions, but it is clear now that modulation of lipid synthesis and autophagy are key biological mechanisms that could help reduce or prevent these liver diseases. The folliculin (FLCN) protein has been recently identified as a central regulatory node governing whole body energy homeostasis, and we hypothesized that FLCN regulates highly metabolic tissues like the liver. We thus generated a liver specific Flcn knockout mouse model to study its role in liver disease progression. Using the methionine- and choline-deficient diet to mimic liver fibrosis, we demonstrate that loss of Flcn reduced triglyceride accumulation, fibrosis, and inflammation in mice. In this aggressive liver disease setting, loss of Flcn led to activation of transcription factors TFEB and TFE3 to promote autophagy, promoting the degradation of intracellular lipid stores, ultimately resulting in reduced hepatocellular damage and inflammation. Hence, the activity of FLCN could be a promising target for small molecule drugs to treat liver fibrosis by specifically activating autophagy. Collectively, these results show an unexpected role for Flcn in fatty liver disease progression and highlight new potential treatment strategies.

Cause-and-effect relationship between thyroid and liver diseases

Thyroid gland (TG) and the liver are in a complex relationship in both physiological and pathological conditions. Thyroid hormones accelerate metabolic processes, intensify the synthesis of proteins and vitamins, play an important role in the development and differentiation of all cells, including hepatocytes. In addition to the central role in the deiodination of thyroid hormones with the formation of their more active and inactivated forms, the liver also carries out their transport. Dysfunction of TG can lead to changes in liver function, and in liver diseases, abnormalities in the metabolism of thyroid hormones can occur. Most often, liver pathology in diseases of TG is manifested by an increase in the serum activity of enzymes of cytolysis and/or cholestasis. Changes in liver function tests are often observed in patients with thyrotoxicosis. They are based on oxidative stress or cholestasis. The increased activity of osteoblasts in hyperthyroidism leads to an increase in the bone fraction of alkaline phosphatase, which must be taken into account in the differential diagnosis. Hepatotoxicity of thyreostatic drugs is relatively common, ranging from minimal hepatocellular damage to fulminant liver failure. In the case of hypothyroidism, the pathophysiological mechanisms are mainly represented by lipid metabolism disorders leading to fatty degeneration. It should be remembered that severe hypothyroidism can be manifested by hyperammonemia and edematous-ascitic syndrome, requiring differential diagnosis with liver failure. Treatment of liver pathology in TG diseases includes normalization of thyroid status, and in cases of drug hepatitis – temporary withdrawal of a potentially hepatotoxic drug. The data on the association of hypothyroidism and non-alcoholic fatty liver disease in the aspect of developing new therapies are very interesting.

Liver histology in short telomere syndrome: A case report and review of the literature

Introduction/Objective Short telomere syndrome (STS) is a genetically inherited syndrome resulting in premature telomere shortening. STS encompasses a spectrum of clinical manifestations, including dyskeratosis congenita, premature hair graying, bone marrow failure, immunodeficiency, pulmonary fibrosis and liver disease. Liver histopathologic features in STS patients have not been well characterized. Methods/Case Report A 46-year-old man presented for dyspnea and cryptogenic cirrhosis. He had a complicated medical history significant for immune thrombocytopenic purpura and splenectomy, recurrent respiratory tract infections, pneumonia, sepsis, primary immunodeficiency, pulmonary mucosa-associated lymphoid tissue lymphoma, and severe hepatopulmonary syndrome. He and his brother had gray hair in their late 20s. He also had a long history of intermittently elevated liver enzymes starting at age 33. A liver biopsy performed 12 years before showed chronic portal inflammation with hepatocellular damage without significant fibrosis. These clinical manifestations prompted an evaluation for a possible telomere biology disorder, which revealed the telomere length was critically short and fell at or below the first percentile for age, supportive of the diagnosis. The most recent liver biopsy showed marked portal lymphocytic inflammation with interface hepatitis, bile ductular reaction and frequent foci of lobular inflammation with focal hepatocyte dropout. Some hepatocytes around the portal tracts were swollen with feathery degeneration and occasional Mallory-Denk bodies. A Rhodanine stain highlighted copper granules in the periportal hepatocytes, suggesting chronic cholestasis. Trichrome and reticulin stains demonstrated portal/periportal/pericellular/perisinusoidal fibrosis and focal bridging fibrosis. Results (if a Case Study enter NA) NA Conclusion Partly due to the rarity of STS and the risk of bleeding associated with biopsies, liver histology was described in only few limited studies with small samples of STS patients, including inflammation, nodular regenerative hyperplasia, steatohepatitis, hemosiderosis, cholestasis, cirrhosis, and large cell change of hepatocytes. Our case and others suggest liver disease associated with STS demonstrates a spectrum of histopathology. Being aware of these histomorphologic features in STS is important for establishing the correct diagnosis.