Hepatocyte Cell Culture Research Articles

Preliminary feasibility study using a solution of synthetic enzymes to replace the natural enzymes in polyhemoglobin-catalase-superoxide dismutase-carbonic anhydrase: effect on warm ischemic hepatocyte cell culture.

This is a study on a simple solution of chemically prepared small chemical molecules of synthetic enzymes: catalase, superoxide dismutase, and carbonic anhydrase (CAT, SOD, and CA). We carried out a study to see if these synthetic enzymes can replace the natural enzymes (CAT, SOD, and CA) and avoid the need for the complicated cross-linking of natural enzymes to PolyHb to form PolyHb-CAT-SOD-CA. We compared the effect a solution of these three synthetic enzymes has on the viability of warm-ischemic hepatocytes that were exposed to nitrogen for 1h at 37°C. PolyHb significantly increased the viability. The three synthetic enzymes themselves also significantly increased the viability. The use of both PolyHb and the three synthetic enzymes resulted in an additive effect in the recovery of viability. Increasing the concentration of the synthetic enzymes resulted in further increase in the effect due to the synthetic enzymes. Implications: In addition to PolyHb, there are a number of other HBOC oxygen carriers. However, only Biopure's HBOC product has received regulatory approval, but only in Russia and South Africa. None of the HBOCs has received regulatory approval by other countries. If regulatory agencies require HBOCs to have antioxidant or CO2 transport properties, all that is needed is to add or inject the solution of synthetic enzymes as a separate component.

In Vitro Evaluation of the Cytotoxic Potential of Thiosemicarbazide Coordinating Compounds in Hepatocyte Cell Culture.

Cancer is a global medical problem and, despite research efforts in the field of tumor treatment, there is currently a shortage of specific anticancer drugs. Most anticancer drugs show significant side effects. The liver is the organ that has central functions in drug metabolism, being a major target of the harmful action of anticancer compounds. In this context, it is essential to evaluate the cytotoxic effects of potential anticancer substances. Therefore, hepatotoxicity and hepatocyte viability were determined in vitro to evaluate the action of seven new local thiosemicarbazide coordination compounds (CCT) on normal liver cells. Doxorubicin was used as a reference substance. The control group consisted of hepatocytes not exposed to CCT action. The cell viability of hepatocytes treated with CCT decreased significantly by 5-12% compared to the control, but was statistically significantly higher by 5-14% compared to doxorubicin, except after CMD-8 and CMT-67 influence, when it does not change. Thus, new local CCT had a selective effect on hepatocytes in vitro and were less hepatotoxic compared to doxorubicin, which may be the basis for further study of its potential in anticancer drugs.

Evaluation of drug carrier hepatotoxicity using primary cell culture models

This study aims to establish a primary rat hepatocyte culture model to evaluate dose-dependent hepatotoxic effects of drug carriers (lipopolymer nanoparticles; LPNs) temporal. Primary rat hepatocyte cell cultures were used to determine half-maximal Inhibition Concentrations (IC50) of the drug-carrier library. Drug-carrier library, at concentrations <50 μg/mL, is benign to primary rat hepatocytes as determined using albumin and urea secretions. Albumin, as a hepatic biomarker, exhibited a more sensitive and faster outcome, compared to urea, for the determination of the IC50 value of LPNs. Temporal measurements of hepatic biomarkers including urea and albumin, and rigorous physicochemical (hydrodynamic diameter, surface charge, etc.) characterization, should be combined to evaluate the hepatotoxicity of drug carrier libraries in screens.

Toxic Effects of Sodium Lauryl Sulfate on Antioxidant Defense System and DNA Damage in Fish Primary Hepatocyte Cultures

Abstract Synthetic detergents which have a major role in environmental pollution accumulate over time and reach levels that harm nature. The surfactants which are abundantly used as cleaning components are discharged into the Van Lake with the sewage water. These chemicals accumulating in the lake may reach a level that could affect the only fish species of the lake, the Van fish. This study aimed to determine the antioxidant levels of Van fish hepatocyte cell culture medium treated with sodium lauryl sulphate (SLS) and to assess the DNA damage. The effect of SLS was assessed by its dose (1x10−5, 1x10-6, 1x10−7 M) and treatment time (24 h, 48 h). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and DNA damage (8-OHdG) were determined in the SLS hepatocyte culture. SOD and GSH-Px were higher on 24 h and 48 h compared to the control group. A significant increase was observed in CAT level in the first 24 h, especially in 1x10−6 and 1x10-5 M concentration. At 48 h, it was observed that the CAT level decreased significantly as the concentration increased. It was determined that MDA and 8-OHdG levels increased depending on concentration and time. In conclusion, different concentrations of SLS affected antioxidant levels in the primary hepatocyte culture of Van Fish and were found to cause an increase in the levels of MDA and 8-OHdG.

Abstract 116: Angptl3 Regulates Hepatic Lipoprotein Production: A New Model For Lipid Lowering?

Background: Angiopoietin-like protein 3 (ANGPTL3 ) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein cholesterol (LDL). Although ANGPTL3 modulates lipolytic pathways in lipoprotein metabolism, its potential intra-hepatocyte role in very low-density lipoprotein (VLDL) assembly and secretion remains unknown. Furthermore, although ANGPTL3 inhibition reduces low-density lipoprotein cholesterol (LDL), humans with complete LDL receptor (LDLR) deficiency exhibit LDLR-independent LDL lowering. Methods: We established hepatocyte cell culture systems, including CRISPR/Cas9-edited HepG2 cells with ANGPTL3 “knocked out” (KO). We also generated hepatocyte-like cells from patient-derived isogenic pluripotent stem cells (iPSCs) with and without ANGPTL3 KO. Radiolabeling was used to quantify apolipoprotein and triglyceride synthesis. Negative stain electron microscopy (NS-EM) was used to quantify the size of secreted lipoproteins. Results: We observed reduction of both apolipoprotein B100 and triglyceride synthesis and secretion in ANGPTL3 KO HepG2 cells. NS-EM demonstrated ANGPTL3-deficient HepG2 cells secrete significantly smaller lipoprotein particles compared to WT control cells. Together, these three findings suggest ANGPTL3 deficiency in hepatocytes results in the production of fewer, smaller, underlipidated lipoprotein particles. Conclusion: Our results point to an important intracellular role for ANGPTL3 within hepatocytes, suggesting that ANGPTL3 modifies the assembly and remodeling, as well as clearance, of hepatically secreted lipoproteins. Further experiments are planned to identify and characterize mechanistic mediators of this process. These studies have potential to identify additional targets for reducing plasma lipids via LDLR-independent pathways.

Prophylactic role of Premna herbacea, a dietary leafy vegetable in managing hepatic steatosis via regulating AMPK/SREBP1/ACC/HMGCR signaling pathway

Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of liver dysfunction ranging from hepatic steatosis (intracellular triglyceride accumulation) to steatohepatitis. This study investigated the beneficial effect of Premna herbacea (a dietary leafy vegetable of North East India) and its active constituent in managing hepatic steatosis using high fat diet (HFD)-fed male Wistar rat and hepatocyte (CC1) cell culture models. Administration with methanolic leaf extract of Premna herbacea (PHME) (250 mg/kg body weight, oral gavage, 20 weeks) reduced the gain in body weight and rise in serum lipid (triglyceride and total cholesterol) levels and enzymes (alkaline phosphatase and aspartate aminotransferase) responsible for liver dysfunction in high fat diet (HFD)-fed rats. PHME administration further upregulated the phosphorylation of AMPK, ACC, and HMGCR, downregulated SREBP1 signaling pathways of lipid metabolism, and prevented hepatic steatosis in liver tissues of HFD-fed rats. Moreover, studies on cultured hepatocytes revealed that treatment with both crude extract and its ethyl acetate fraction (PHEA) significantly reduced intracellular lipid accumulation in palmitate (PA, 0.75 mM)-treated cells. Chemical profiling (HPLC, HRMS, 1H and 13C NMR) of PHEA demonstrated the presence of Verbascoside, a caffeoyl phenylethanoid glycoside as a major phyto-constituent, which prevented cellular lipid accumulation and regulated the alteration in AMPK/SREBP/ACC/HMGCR signaling pathways of lipid metabolism in PA-treated cells. In conclusion, this study showed the prophylactic role of Premna herbacea and its active molecule, Verbascoside as a potential functional food in ameliorating hepatic steatosis by regulating AMPK/SREBP/ACC/HMGCR signaling cascade.

In vitro toxicity of glyphosate in Atlantic salmon evaluated with a 3D hepatocyte-kidney co-culture model

A novel 3D Atlantic salmon co-culture model was developed using primary hepatocytes and kidney epithelial cells isolated from the same fish. Mono and co-cultures of primary hepatocytes and kidney epithelial cells were exposed for 48 h to glyphosate (5, 50 and 500 μM). For comparison, cells were also exposed to chlorpyrifos, benzo(a)pyrene and cadmium. Cell staining, cell viability assessments, RT-qPCR and global metabolomic profiling were used to examine the toxicological effects on liver and renal function and to compare responses in 3D and 2D cultures. The 3D hepatocyte cell culture was considered superior to the 2D culture due to the ATP binding cassette subfamily B member 1 (Abcb1) response and was thus used further in co-culture with kidney cells. Metabolomic analysis of co-cultured cells showed that glyphosate exposure (500 μM) altered lipid metabolism in both hepatocytes and kidney cells. Elevated levels of several types of PUFAs and long-chain fatty acids were observed in exposed hepatocytes, owing to increased uptake and phospholipid remodelling. Glyphosate suppressed the expression of estrogen receptor 1 (Esr1) and vitellogenin (Vtg) and altered histidine metabolism in exposed hepatocytes. Increased levels of cholesterol and downregulation of clusterin (Clu) suggest that glyphosate treatment affected membrane stability in Atlantic salmon kidney cells. This study demonstrates the usefulness of applying 3D co-culture models in risk assessment.

Chinese herbal component, Praeruptorin E, enhances anti-asthma efficacy and prevents toxicity of aminophylline by targeting the NF-κB/PXR/CYP3A4 pathway

BackgroundAminophylline is widely used for the treatment of asthma, but the therapeutic dose is very close to the toxic dose, which makes this drug prone to accumulation poisoning. In the present study, we explored whether the Chinese herbal component, Praeruptorin E (PE), enhances anti-asthma efficacy and prevents the toxicity of aminophylline.MethodsFirst, an ovalbumin (OVA)-induced mouse model of asthma, immunohistochemistry, pathological staining, and bronchoalveolar lavage fluid (BALF) were used to detect the lung condition of asthmatic mice. The content of Th2 cytokines in serum was measured by enzyme-linked immunosorbent assay (ELISA), and the expression of related proteins was detected by Western blotting and immunofluorescence. Concentrations of theophylline and its metabolites in rat serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). siRNA transfection and chromatin immunoprecipitation (ChIP) were used to investigate the mechanism of PE.ResultsPE was found to synergize with aminophylline to reduce the infiltration of inflammatory cells, collagen deposition, and mucus hyperplasia in the lungs of asthmatic mice. It inhibited the expression of Th2 cytokines, interleukin (IL)-4, IL-5, and IL-13; promoted lung tissue repair; and reduced the toxic effect of aminophylline on the heart. Moreover, LC-MS/MS analysis showed that PE reduced the plasma concentration of the parent theophylline and its metabolite 1,3-dimethyluric acid (1,3-DMU). PE facilitated aminophylline’s suppression of nuclear factor-κB (NF-κB), and increased the expression of the xenobiotic nuclear receptor pregnane X receptor (PXR) and its primary target gene, CYP3A11 [this is the mouse homolog of cytochrome P450 3A (CYP3A)] in the asthmatic mouse liver and in the L-02 human fetal hepatocyte cell culture model. In addition, the ChIP assay revealed that PE attenuated the binding of NF-κB to the promoter region of the PXR gene and prevented the suppression of PXR gene expression by NF-κB.ConclusionsPE has a dual function in enhancing the immune regulation and anti-inflammatory effects of theophylline, as well as preventing theophylline toxicity by targeting the NF-κB/PXR/CYP3A4 axis. PE is a promising herbal medicine that will benefit asthmatics taking theophylline.

Mesenchymal stromal cells mitigate liver damage after extended resection in the pig by modulating thrombospondin-1/TGF-\u03b2

Post-surgery liver failure is a serious complication for patients after extended partial hepatectomies (ePHx). Previously, we demonstrated in the pig model that transplantation of mesenchymal stromal cells (MSC) improved circulatory maintenance and supported multi-organ functions after 70% liver resection. Mechanisms behind the beneficial MSC effects remained unknown. Here we performed 70% liver resection in pigs with and without MSC treatment, and animals were monitored for 24 h post surgery. Gene expression profiles were determined in the lung and liver. Bioinformatics analysis predicted organ-independent MSC targets, importantly a role for thrombospondin-1 linked to transforming growth factor-β (TGF-β) and downstream signaling towards providing epithelial plasticity and epithelial-mesenchymal transition (EMT). This prediction was supported histologically and mechanistically, the latter with primary hepatocyte cell cultures. MSC attenuated the surgery-induced increase of tissue damage, of thrombospondin-1 and TGF-β, as well as of epithelial plasticity in both the liver and lung. This suggests that MSC ameliorated surgery-induced hepatocellular stress and EMT, thus supporting epithelial integrity and facilitating regeneration. MSC-derived soluble factor(s) did not directly interfere with intracellular TGF-β signaling, but inhibited thrombospondin-1 secretion from thrombocytes and non-parenchymal liver cells, therewith obviously reducing the availability of active TGF-β.

Interrupting tumor necrosis factor-alpha signaling prevents parenteral nutrition-associated cholestasis in mice.

We have recently reported a mouse model of PN-associated cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid, bilirubin and sterol transporters Abcb11, Abcc2 and Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice. First, recombinant TNFα was administered to mice as well as in hepatocyte cell culture. Second, Tnfr1/2KO or wild-type (WT) mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN). Finally, WT/DSS-PN mice were also infused with infliximab at 10 mg/kg on days 3 and 10 of PN. PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and bilirubin. Intraperitoneal injection of TNFα into WT mice or TNFα treatment of Huh7 hepatocarcinoma cells and primary mouse hepatocytes suppressed messenger RNA (mRNA) transcription of bile (Abcb11, Abcc2]) and sterol transporters (Abcg5/8) and their regulators Nr1h3 and Nr1h4. DSS-PN mice with PNAC had increased hepatic TNFα mRNA expression and significant reduction of mRNA expression of Abcb11, Abcc2, Abcg5/8, Nr1h3, and Nr1h4. In contrast, PNAC development was prevented and mRNA expression normalized in both Tnfr1/2KO /DSS-PN mice and DSS-PN mice treated with infliximab. TNFα is a key mediator in the pathogenesis of PNAC through suppression of hepatocyte Abcb11, Abcc2, and Abcg5/8. Pharmacologic targeting of TNFα as a therapeutic strategy for PNAC thus deserves further investigation.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium.

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

Evidence of Cold Induced Cytoskeletal Arrest in Hepatocytes of the Western Painted Turtle

The western painted turtle (Chrysemys picta belli) overwinters in ice-covered water bodies and can survive without oxygen (anoxia) for months, during which time metabolic rate decreases so that ATP demand does not exceed anoxic ATP supply. In order to characterize the contribution of the cytoskeleton to overwintering metabolic depression in non-excitable turtle cells, we have optimized a hepatocyte cell culture system. Actin and tubulin cycling are large consumers of cellular ATP pools, and we hypothesized that their cycling activity, and consequently capacity for structural rearrangement, would be downregulated by overwintering conditions. Turtles were acclimated to either 22°C or 4°C for at least 20 days and isolated hepatocytes were plated on either fibronectin or polylysine at varying temperatures, or in cyanide containing media, which acted as a chemical proxy for anoxia. Low incubation temperature, but not cyanide, prevented hepatocytes from forming stable attachment to fibronectin, suggesting inhibition of cell spreading and arrest of actin structural rearrangement by low temperature. Attachment to polylysine, a predominately passive and electrostatic phenomenon, was not inhibited by low temperature or cyanide. Hepatocytes isolated from 4°C acclimated turtles were 30% smaller in 2D area than those of 22°C acclimated turtles. Three-hour cyanide exposure of hepatocytes from 22°C acclimated turtles also resulted in a 30% shrinkage of 2D area, indicating that temperature depression and anoxia may have overlapping inhibitory effects on cell spreading and underlying cytoskeletal turnover. Previous research has shown that cyanide treatment causes reduction of hepatocyte mitochondrial membrane potential. Ongoing work is demonstrating that, regardless of whole animal temperature acclimation, low temperature exposure results in decreased mitochondrial membrane potential as well as increased cytosolic calcium, signaling which may contribute to inhibition of energetically costly actin rearrangement.

Intestinal Epithelial Cell-Derived Extracellular Vesicles Modulate Hepatic Injury via the Gut-Liver Axis During Acute Alcohol Injury.

Binge drinking, i.e., heavy episodic drinking in a short time, has recently become an alarming societal problem with negative health impact. However, the harmful effects of acute alcohol injury in the gut-liver axis remain elusive. Hence, we focused on the physiological and pathological changes and the underlying mechanisms of experimental binge drinking in the context of the gut-liver axis. Eight-week-old mice with a C57BL/6 background received a single dose (p.o.) of ethanol (EtOH) [6 g/kg b.w.] as a preclinical model of acute alcohol injury. Controls received a single dose of PBS. Mice were sacrificed 8 h later. In parallel, HepaRGs and Caco-2 cells, human cell lines of differentiated hepatocytes and intestinal epithelial cells intestinal epithelial cells (IECs), respectively, were challenged in the presence or absence of EtOH [0–100 mM]. Extracellular vesicles (EVs) isolated by ultracentrifugation from culture media of IECs were added to hepatocyte cell cultures. Increased intestinal permeability, loss of zonula occludens-1 (ZO-1) and MUCIN-2 expression, and alterations in microbiota—increased Lactobacillus and decreased Lachnospiraceae species—were found in the large intestine of mice exposed to EtOH. Increased TUNEL-positive cells, infiltration of CD11b-positive immune cells, pro-inflammatory cytokines (e.g., tlr4, tnf, il1β), and markers of lipid accumulation (Oil Red O, srbep1) were evident in livers of mice exposed to EtOH, particularly in females. In vitro experiments indicated that EVs released by IECs in response to ethanol exerted a deleterious effect on hepatocyte viability and lipid accumulation. Overall, our data identified a novel mechanism responsible for driving hepatic injury in the gut-liver axis, opening novel avenues for therapy.

Direct and Label-Free Monitoring of Albumin in 2D Fatty Liver Disease Model Using Plasmonic Nanogratings.

Non-alcoholic fatty liver (NAFLD) is a metabolic disorder related to a chronic lipid accumulation within the hepatocytes. This disease is the most common liver disorder worldwide, and it is estimated that it is present in up to 25% of the world’s population. However, the real prevalence of this disease and the associated disorders is unknown mainly because reliable and applicable diagnostic tools are lacking. It is known that the level of albumin, a pleiotropic protein synthesized by hepatocytes, is correlated with the correct function of the liver. The development of a complementary tool that allows direct, sensitive, and label-free monitoring of albumin secretion in hepatocyte cell culture can provide insight into NAFLD’s mechanism and drug action. With this aim, we have developed a simple integrated plasmonic biosensor based on gold nanogratings from periodic nanostructures present in commercial Blu-ray optical discs. This sensor allows the direct and label-free monitoring of albumin in a 2D fatty liver disease model under flow conditions using a highly-specific polyclonal antibody. This technology avoids both the amplification and blocking steps showing a limit of detection within pM range (≈0.26 ng/mL). Thanks to this technology, we identified the optimal fetal bovine serum (FBS) concentration to maximize the cells’ lipid accumulation. Moreover, we discovered that the hepatocytes increased the amount of albumin secreted on the third day from the lipids challenge. These data demonstrate the ability of hepatocytes to respond to the lipid stimulation releasing more albumin. Further investigation is needed to unveil the biological significance of that cell behavior.

Fermented Wheat Germ Extract as a Redox Modulator: Alleviating Endotoxin-Triggered Oxidative Stress in Primary Cultured Rat Hepatocytes.

Bioactive compounds such as benzoquinone derivates presented in fermented wheat germ extract (FWGE) have several positive effects on overall health status of humans and animals alike. Since available data regarding the antioxidant activity of FWGE are limited, the aim of our study was to investigate its effects on the cellular redox homeostasis applying primary hepatocyte cell cultures of rat origin. Cultures were challenged to lipopolysaccharide (LPS) treatment for 2 or 8 hours to trigger inflammatory response. Further, culture media were concomitantly supplemented with or without FWGE (Immunovet®, 0.1% and 1%). In order to monitor the metabolic activity of the cell cultures, CCK-8 test was applied, while reactive oxygen species (ROS) production was measured using Amplex Red method. Malondialdehyde concentration of culture media as a specific marker of lipid peroxidation and the activity of glutathione peroxidase in cell lysates were also determined to monitor the redox status of the cultures. Based on our findings, it can be concluded that FWGE did not show cytotoxic effects in any applied concentration in cell cultures. Furthermore, FWGE efficiently decreased cellular ROS production and lipid peroxidation rate in case of LPS-induced inflammatory response. However, without LPS treatment, higher concentration of FWGE increased the rate of both ROS and malondialdehyde synthesis. This observation may refer to the prooxidant activity of high dose FWGE, which is an important beneficial effect regarding tumor cells. However, in case of noninflamed hepatocytes, considering the results of glutathione peroxidase activity, the application of the product did not result in severe oxidative distress. In accordance with the abovementioned findings, FWGE as a redox modulator, applied in the appropriate concentration, can serve as a promising candidate in the supplementary therapy of patients suffering from various inflammatory diseases, decreasing the free radical generation, thus avoiding the occurrence of cytotoxic effects.

Flow cytometric micronucleus assay and TGx-DDI transcriptomic biomarker analysis of ten genotoxic and non-genotoxic chemicals in human HepaRG\u2122 cells

BackgroundModern testing paradigms seek to apply human-relevant cell culture models and integrate data from multiple test systems to accurately inform potential hazards and modes of action for chemical toxicology. In genetic toxicology, the use of metabolically competent human hepatocyte cell culture models provides clear advantages over other more commonly used cell lines that require the use of external metabolic activation systems, such as rat liver S9. HepaRG™ cells are metabolically competent cells that express Phase I and II metabolic enzymes and differentiate into mature hepatocyte-like cells, making them ideal for toxicity testing. We assessed the performance of the flow cytometry in vitro micronucleus (MN) test and the TGx-DDI transcriptomic biomarker to detect DNA damage-inducing (DDI) chemicals in human HepaRG™ cells after a 3-day repeat exposure. The biomarker, developed for use in human TK6 cells, is a panel of 64 genes that accurately classifies chemicals as DDI or non-DDI. Herein, the TGx-DDI biomarker was analyzed by Ion AmpliSeq whole transcriptome sequencing to assess its classification accuracy using this more modern gene expression technology as a secondary objective.MethodsHepaRG™ cells were exposed to increasing concentrations of 10 test chemicals (six genotoxic chemicals, including one aneugen, and four non-genotoxic chemicals). Cytotoxicity and genotoxicity were measured using the In Vitro MicroFlow® kit, which was run in parallel with the TGx-DDI biomarker.ResultsA concentration-related decrease in relative survival and a concomitant increase in MN frequency were observed for genotoxic chemicals in HepaRG™ cells. All five DDI and five non-DDI agents were correctly classified (as genotoxic/non-genotoxic and DDI/non-DDI) by pairing the test methods. The aneugenic agent (colchicine) yielded the expected positive result in the MN test and negative (non-DDI) result by TGx-DDI.ConclusionsThis next generation genotoxicity testing strategy is aligned with the paradigm shift occurring in the field of genetic toxicology. It provides mechanistic insight in a human-relevant cell-model, paired with measurement of a conventional endpoint, to inform the potential for adverse health effects. This work provides support for combining these assays in an integrated test strategy for accurate, higher throughput genetic toxicology testing in this metabolically competent human progenitor cell line.

Cryogel-Integrated Biochip for Liver Tissue Engineering

Microfluidic systems and polymer hydrogels have been widely developed for tissue engineering. Yet only a few tools combining both approaches, especially for in vitro liver models, are being explored. In this study, an alginate-based cryogel-integrated biochip was engineered for dynamic hepatocyte cell culture in three dimensions (3D). The alginate cryogel was covalently cross-linked in the biochip at subzero temperatures (T < 0 °C) to create a scaffold with high mechanical stability and an interconnected macroporous network. By varying the alginate concentration and the cross-linker ratio, the Young’s modulus of the cryogel can be fine-tuned between 1.5 and 29 kPa, corresponding to the range of stiffness of the different physiological states of the liver. We demonstrated that HepG2/C3A cells can be cultured and maintained viable under dynamic conditions in this device up to 7 days. Albumin synthesis and glucose consumption increased over the cell culture days. Moreover, a 3D cell structure was observed across the entire height of the biochip, which was preserved following alginate lyase treatment to remove the cryogel-based scaffold. In summary, these results suggest a promising cell culture technology with great potential for pharmaceutical industries to engineer healthy and cirrhotic liver models for middle-throughput predictive toxicology and drug screening.

MiR-130b is a potent stimulator of hepatic very-low-density lipoprotein assembly and secretion via marked induction of microsomal triglyceride transfer protein

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase (FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog (PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.

Reduced miR-181d level in obesity and its role in lipid metabolism via regulation of ANGPTL3

Obesity impacts the endocrine and metabolic functions of the adipose tissue. There is increasing interest in the role of epigenetic factors in obesity and its impact on diabetes and dyslipidemia. One such substance, miR-181, reduces plasma triglyceride levels in mice by targeting isocitrate dehydrogenase 1. In the other hand, the adipocyte differentiation and lipid regulating hormone angiopoietin-like 3 (ANGPTL3) is a known regulator of circulating apolipoproteins through its inhibition of the lipoprotein lipase activity. We aimed to study the miR-181d expression in the blood and adipose tissue in a cohort of obese and non-obese people, assessing its possible role in obesity. We also aimed to confirm whether miR-181d can bind and regulate ANGPTL3. miR-181d expression levels were investigated in 144 participants, 82 who were non-obese (body mass index [BMI] < 30) and 62 who were obese (BMI > 30). miR-181d levels in plasma and adipose tissue were measured by RT-PCR. Hepatocyte cell cultures were assessed by overexpression and 3′-UTR-luciferase assays for miR-181d binding to its target protein and its effect on the protein. The plasma levels of ANGPTL3 were also measured by ELISA. The miR-181d levels were significantly lower in obese than in non-obese individuals. In vitro analysis confirmed miR-181 binding to and repression of the ANGPTL3 transcript. Obesity leads to alterations in miR-181d expression. Its downregulation in obese humans was inversely correlated with ANGPTL3, a protein involved in adipocyte differentiation and lipid metabolism. miR-181d can be used as an inhibitor of ANGPTL3 to reduce the TG plasma level.

Bardoxolone methyl as a novel potent antiviral agent against hepatitis B and C viruses in human hepatocyte cell culture systems.

Antiviral drugs against hepatitis B virus (HBV) relieve symptoms experienced by patients with hepatitis; however, these drugs cannot eliminate HBV infection from all patients completely. On the other hand, direct antiviral agents (DAAs) against hepatitis C virus (HCV) can achieve near-complete elimination of HCV infection. However, recent reports have claimed that DAAs pose a risk for HBV reactivation among patients with HBV and HCV co-infection. This suggests that an effective anti-viral strategy for both HBV and HCV would be extremely useful. We hypothesized that an activator of nuclear factor-erythroid factor 2 (Nrf2) could be a candidate, because heme oxygenase-1 (HO-1), a product of the Nrf2-target gene, was shown to be related to suppression of genome replication in both HBV and HCV. In this study, the potential of bardoxolone methyl (BARD), an Nrf2 activator, was examined in cell culture systems against HBV and HCV. We investigated that BARD had a suppressive effect on the production of extracellular HBV DNA in several HBV culture systems. In addition, BARD treatment reduced the levels of intracellular HBV pregenome RNA (pgRNA), a transcript from the HBV genome and a template of HBV genome replication. HCV genome replication was also suppressed in HCV subgenomic replicon-bearing cells by BARD treatment. BARD might be a novel treatment for patients with HBV and HCV co-infection.