Human Liver-derived Cell Line Research Articles

Synergistic toxicity with copper contributes to NAT2-associated isoniazid toxicity

Anti-tuberculosis (AT) medications, including isoniazid (INH), can cause drug-induced liver injury (DILI), but the underlying mechanism remains unclear. In this study, we aimed to identify genetic factors that may increase the susceptibility of individuals to AT-DILI and to examine genetic interactions that may lead to isoniazid (INH)-induced hepatotoxicity. We performed a targeted sequencing analysis of 380 pharmacogenes in a discovery cohort of 112 patients (35 AT-DILI patients and 77 controls) receiving AT treatment for active tuberculosis. Pharmacogenome-wide association analysis was also conducted using 1048 population controls (Korea1K). NAT2 and ATP7B genotypes were analyzed in a replication cohort of 165 patients (37 AT-DILI patients and 128 controls) to validate the effects of both risk genotypes. NAT2 ultraslow acetylators (UAs) were found to have a greater risk of AT-DILI than other genotypes (odds ratio [OR] 5.6 [95% confidence interval; 2.5–13.2], P = 7.2 × 10−6). The presence of ATP7B gene 832R/R homozygosity (rs1061472) was found to co-occur with NAT2 UA in AT-DILI patients (P = 0.017) and to amplify the risk in NAT2 UA (OR 32.5 [4.5–1423], P = 7.5 × 10−6). In vitro experiments using human liver-derived cell lines (HepG2 and SNU387 cells) revealed toxic synergism between INH and Cu, which were strongly augmented in cells with defective NAT2 and ATP7B activity, leading to increased mitochondrial reactive oxygen species generation, mitochondrial dysfunction, DNA damage, and apoptosis. These findings link the co-occurrence of ATP7B and NAT2 genotypes to the risk of INH-induced hepatotoxicity, providing novel mechanistic insight into individual AT-DILI susceptibility.Yoon et al. showed that individuals who carry NAT2 UAs and ATP7B 832R/R genotypes are at increased risk of developing isoniazid hepatotoxicity, primarily due to the increased synergistic toxicity between isoniazid and copper, which exacerbates mitochondrial dysfunction-related apoptosis.

Effect of cell treatment procedures on in vitro genotoxicity assessment.

So far, the majority of in vitro toxicological experiments are conducted after an acute 24h treatment that does not represent a realistic human chemical exposure. Recently, new in vitro approaches have been proposed to study the chemical toxicological effect over several days in order to be more predictive of a representative exposure scenario. In this study, we investigated the genotoxic potential of chemicals (direct or bioactived clastogen, aneugen and apoptotic inducer) with the γH2AX and pH3 biomarkers, in the human liver-derived HepaRP cell line. We used different treatment durations, with or without a three-day recovery stage (release period), before genotoxicity measurement. Data were analysed with the Benchmark Dose approach. We observed that the detection of clastogenic compounds (notably for DNA damaging agents) was more sensitive after three days of repeated treatment compared to one or three treatments over 24h. In contrast, aneugenic chemicals were detected as genotoxic in a similar manner whether after a 24h exposure or a three-day repeated treatment. Globally, the release period decreases the genotoxicity measurement substantially. For DNA damaging agents, after high concentration treatments, γH2AX induction was always observed after a three-day release period. In contrast, for DNA topoisomerase inhibitors, no effect could be observed after the release period. In conclusion, in the HepaRP cell line, there are some important differences between a one-day acute and a three-day repeated treatment protocol, indicating that different cell treatment procedures may differentiate chemical genotoxic mechanisms of action more efficiently.

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies.

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.

Oxidized Low-Density Lipoproteins Trigger Hepatocellular Oxidative Stress with the Formation of Cholesteryl Ester Hydroperoxide-Enriched Lipid Droplets.

Oxidized low-density lipoproteins (oxLDLs) induce oxidative stress in the liver tissue, leading to hepatic steatosis, inflammation, and fibrosis. Precise information on the role of oxLDL in this process is needed to establish strategies for the prevention and management of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Here, we report the effects of native LDL (nLDL) and oxLDL on lipid metabolism, lipid droplet formation, and gene expression in a human liver-derived C3A cell line. The results showed that nLDL induced lipid droplets enriched with cholesteryl ester (CE) and promoted triglyceride hydrolysis and inhibited oxidative degeneration of CE in association with the altered expression of LIPE, FASN, SCD1, ATGL, and CAT genes. In contrast, oxLDL showed a striking increase in lipid droplets enriched with CE hydroperoxides (CE-OOH) in association with the altered expression of SREBP1, FASN, and DGAT1. Phosphatidylcholine (PC)-OOH/PC was increased in oxLDL-supplemented cells as compared with other groups, suggesting that oxidative stress increased hepatocellular damage. Thus, intracellular lipid droplets enriched with CE-OOH appear to play a crucial role in NAFLD and NASH, triggered by oxLDL. We propose oxLDL as a novel therapeutic target and candidate biomarker for NAFLD and NASH.

Polystyrene nanoplastics induce profound metabolic shift in human cells as revealed by integrated proteomic and metabolomic analysis.

Nanoplastics (NPLs) are widespread in our environment. However, their impacts on human health and precise toxicity mechanisms remain poorly understood. Here we studied the internalization, release, and cytotoxicity of polystyrene nanoplastics (PSNPs) using the renal tubular epithelial cell line HKC and human derived liver cell line HL-7702. We also employed an integrated proteomic and metabolomic approach to investigate the potential biological effects of PSNPs on HKC cells. The abundances of 4770 proteins and 100 metabolites were quantified, with 785 proteins and 17 metabolites detected with altered levels in response to PSNPs. Most of the differential proteins and metabolites were enriched in a variety of metabolic pathways, for example, glycolysis, citrate cycle, oxidative phosphorylation, and amino acid metabolism, suggesting the potential effects of NPLs on global cellular metabolism shift in human cells. The altered energy metabolism induced by PSNPs was further confirmed by a Seahorse analysis. Moreover, lysosomal distribution study and western blotting showed that mTORC1 signaling, a central regulator of cellular metabolism, was inhibited upon nanoplastic exposure, likely serving as the link between lysosome dysfunction and metabolic defects. Taken together, our findings systematically mapped the key molecular changes induced by PSNPs in human cells and provide comprehensive biological insights for the risk estimation of NPLs contamination.

In vitro genotoxic and mutagenic effects of water samples from Sapucaia and Esteio streams (Brazil) under the influence of different anthropogenic activities

Pollution of aquatic ecosystems is associated with the discharge of mainly industrial and urban effluents, which may cause damage to public health. This study aims to evaluate the cytotoxic, genotoxic, and mutagenic potential of surface water samples under the influence of different anthropogenic effluents in a human-derived liver cell line (HepG2). Samples were collected in Esteio and Sapucaia streams (Rio Grande do Sul; Brazil), which flow into the Sinos River, a source of water supply for more than one million people. Physicochemical and microbiological analyses were performed as well as an analysis of inorganic elements using the PIXE technique (Particle-Induced X-Ray Emission). The presence of pharmaceutical compounds and caffeine was evaluated by gas chromatography coupled to mass spectrometry. The cytotoxicity, genotoxicity, and mutagenicity of the samples were evaluated in HepG2 cells by cell viability assays, alkaline Comet Assay and Cytokinesis-block micronucleus (CBMN) assay. We verified alterations in the physicochemical and microbiological parameters and detected caffeine, diethyltoluamide, and different inorganic elements that corresponded to elements from domestic and industrial effluents and agricultural runoff. Although the samples in the concentration used were not cytotoxic, water samples from all sites induced DNA damage. However, it is difficult to attribute these damages to a specific substance since the factors are a complex mixture of different compounds. Despite this, it is observed that both urban and industrial contributions had a similar effect in the cells evaluated. Such results demonstrate the need to perform biomonitoring of surface waters under anthropogenic influence, especially those that flow into rivers that are a source of public supply water. We also highlight the need for research into emerging pollutants in these aquatic environments.

Lysophosphatidylethanolamine Affects Lipid Accumulation and Metabolism in a Human Liver-Derived Cell Line.

The physiological functions of lysophosphatidylethanolamine (lysoPE) have not been fully elucidated. In this study, the effects of lysoPE on lipogenesis and lipolysis were investigated in a cultured human liver-derived cell line. The intracellular lipid profile was investigated in detail using liquid chromatography–tandem mass spectrometry (LC-MS/MS) to better understand the underlying mechanism. The expression of genes related to lipid metabolism and catabolism was analyzed using real-time PCR. LysoPE supplementation induced cellular lipid droplet formation and altered triacylglycerol (TAG) profiles. Furthermore, lysoPE downregulated expression of the TAG hydrolyzation regulation factor ATGL, and reduced the expression of fatty acid biosynthesis-related genes SREBP1 and SCD1. LC-MS/MS-based lipidomic profiling revealed that the addition of lysoPE 18:2 increased the PE species containing linoleic acyl, as well as the CE 18:2 species, likely due to the incorporation of linoleic acyl from lysoPE 18:2. Collectively, these findings suggest that lysoPE 18:2 is involved in lipid droplet formation by suppressing lipolysis and fatty acid biosynthesis. Thus, lysoPE might play a pathological role in the induction of fatty liver disease.

Directional Drug Transport through Membrane-Supported Monolayers of Human Liver-Derived Cell Lines.

The aim of this work is to develop a new assay system for screening biliary excretion drugs. When monolayers of human liver-derived cell lines HepG2 and Huh-7 were grown on an insert membrane, the efflux ratio (ER: ratio of the apparent permeability coefficient in the basal-to-apical direction (Papp,B-to-A) to that in the apical to basal direction (Papp,A-to-B)) of sulfobromophthalein (BSP), a model substrate of multidrug resistance-associated protein 2 (MRP2), was greater than 1.0, indicating transport of BSP in the efflux direction. The efflux transport was significantly suppressed by MK-571, an inhibitor of MRPs, in both cell lines. Expression of MRP2 mRNA in HepG2 and Huh-7 was 3.5- and 1.4-fold higher, respectively, than in primary human hepatocytes, while expression of P-glycoprotein and breast cancer resistance protein mRNAs was markedly lower, supporting the idea that MRP2 is the main mediator of directional BSP transport in this assay system. The advantage of our system is the potential to quantitatively evaluate biliary excretion of MRP2 substrates in vitro.

Micronucleus assays with the human derived liver cell line (Huh6): A promising approach to reduce the use of laboratory animals in genetic toxicology

The inadequate representation of enzymes which catalyze the activation/detoxification of xenobiotics in cells that are currently used in genotoxicity testing of chemicals leads to a high number of false positive results and the number of follow up studies with rodents could be reduced by use of more reliable in vitro models. We found earlier that several xenobiotic drug metabolizing enzymes are represented in the human derived liver cell line Huh6 and developed a protocol for micronucleus (MN) experiments which is in agreement with the current OECD guideline. This protocol was used to test 23 genotoxic and non-genotoxic reference chemicals; based on these results and of earlier findings (with 9 chemicals) we calculated the predictive value of the assay for the detection of genotoxic carcinogens. We found a sensitivity of 80% and a specificity of 94% for a total number of 32 chemicals; comparisons with results obtained with other in vitro assays show that the validity of MN tests with Huh6 is higher as that of other experimental models. These results are promising and indicate that the use of Huh6 cells in genetic toxicology may contribute to the reduction of the use of laboratory rodents; further experimental work to confirm this assumption is warranted.

Evaluation of the genetic toxicity of sofosbuvir and simeprevir with and without ribavirin in a human-derived liver cell line.

Direct-acting antivirals have revolutionized the treatment of chronic hepatitis C. Sofosbuvir and simeprevir are prescribed worldwide. However, there is a scarcity of information regarding their genotoxicity. Therefore, the present study assessed the cytotoxic and genotoxic effects of sofosbuvir and simeprevir, alone and combined with ribavirin. HepG2 cells were analyzed using the in vitro cytokinesis-block micronucleus cytome assay. Cells were treated for 24 h with sofosbuvir (0.011-1.511 mM), simeprevir (0.156-5.0 µM), and their combinations with ribavirin (0.250-4.0 mM). No significant differences were observed in the nuclear division cytotoxicity index, reflecting the absence of cytotoxic effects associated to sofosbuvir. However, the highest concentration of simeprevir showed a significant difference for the nuclear division cytotoxicity index. Moreover, significant results were observed for nuclear division cytotoxicity index in two combinations of sofosbuvir plus ribavirin and only in the highest combination of simeprevir plus ribavirin. Additionally, our results showed that sofosbuvir did not increase the frequency of chromosomal damage, but simeprevir significantly increased the frequency of micronuclei at the highest concentrations. The combination index demonstrated that both sofosbuvir and simeprevir produced antagonism to the genotoxic effects of ribavirin. In conclusion, our results showed that simeprevir, but not sofosbuvir, has genotoxic effects in HepG2 cells.

DNA damage of human derived liver cell line HL-7702 induced by organic extracts from surface water in Pearl River Delta, China.

Many organic pollutants attract public health concern due to their genotoxicity. To investigate the genotoxicity of organic matter in surface water of the Pearl River Delta (PRD). Organic substances of 24 samples (dry and wet season) from North River, West River and East River were extracted from 60 L source water by XAD-2 macroporous resin. DNA damage effect of organic extracts was tested in human derived liver cells (HL-7702), using single cell gel electrophoresis (SCGE) assay. The results showed that 100% organic extracts (24/24) could induce DNA damage in HL-7702 cells when the concentration was above 1.0 L surface water/ml culture, no significant difference of DNA damage between dry and wet seasons was observed. The organic substance-induced DNA damage in HL-7702 cells was significantly (P < 0.05) correlated with the contents of Dissolved Organic Carbon in both seasons and Total Suspended Solids in dry season. In conclusion, organic extracts induced genetic damage in HL-7702 cells, indicating potential genotoxicity of organic pollutants of surface water from PRD, South China.

Improvement of DNA Vector Delivery of DOTAP Lipoplexes by Short-Chain Aminolipids.

Cellular delivery of DNA vectors for the expression of therapeutic proteins is a promising approach to treat monogenic disorders or cancer. Significant efforts in a preclinical and clinical setting have been made to develop potent nonviral gene delivery systems based on lipoplexes composed of permanently cationic lipids. However, transfection efficiency and tolerability of such systems are in most cases not satisfactory. Here, we present a one-pot combinatorial method based on double-reductive amination for the synthesis of short-chain aminolipids. These lipids can be used to maximize the DNA vector delivery when combined with the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). We incorporated various aminolipids into such lipoplexes to complex minicircle DNA and screened these systems in a human liver-derived cell line (HuH7) for gene expression and cytotoxicity. The lead aminolipid AL-A12 showed twofold enhanced gene delivery and reduced toxicity compared to the native DOTAP:cholesterol lipoplexes. Moreover, AL-A12-containing lipoplexes enabled enhanced transgene expression in vivo in the zebrafish embryo model.

Cytome micronucleus assays with a metabolically competent human derived liver cell line (Huh6): A promising approach for routine testing of chemicals?

One of the main problems of in vitro genotoxicity tests is the inadequate representation of drug metabolizing enzymes in most indicator cell lines which are currently used. We identified recently a human derived liver cell line (Huh6) which detected induction of DNA damage by representatives of different groups of promutagens without enzyme mix and showed that these cells are more suitable in terms of reproducibility and sensitivity as other currently used liver derived lines. We developed a protocol for micronucleus (MN) cytome assays with these cells and validated the procedure in experiments with representatives of different groups of directly and indirectly acting genotoxic carcinogens (MMS, cisplatin, PhIP, IQ, NDMA, B(a)P, AFB1, etoposide, and H2O2). The optimal cytochalasin B concentration in combination with 48 hr treatment was found to be 1.5 μg/mL and leads to a cytokinesis block proliferation index in the range between 1.7 and 2.0. The morphological characteristics of different nuclear anomalies which reflect DNA damage (MN, nuclear bridges, and buds) and their baseline frequencies in untreated cells were characterized, and the rates which are required to cause significant effects were calculated. All compounds caused dose dependent induction of MN when the cells were treated for 24 hr, longer and shorter exposure times were less effective. Experiments with different serum levels (fetal bovine serum [FBS]) showed that 10% FBS in the medium (instead of 4%) causes a substantial increase of the sensitivity of the cells. Our results indicate that the new protocol is a promising approach for routine testing of chemicals. Environ. Mol. Mutagen. 60: 134–144, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Amido Black 10B a widely used azo dye causes DNA damage in pro- and eukaryotic indicator cells

Acid Black 10B (AB10B) is widely used for the production of textiles, leather and prints. It is a representative of azo dyes and it is well documented that some of these compounds are mutagenic per se, and that cleavage products (in particular aromatic amines) may cause damage of the genetic material and cancer. Since no toxicological data on AB10B have been published, we evaluated its mutagenic activity in Salmonella/microsome assays and studied its acute toxic and genotoxic properties in a human derived liver cell line (HepG2) which retained the activities of drug metabolizing enzymes. The compound did not cause cytotoxicity (MTT assay), but clear genotoxic effects were detected in pro- and eukaryotic indicator cells. Dose dependent induction of his+ revertants was seen in strain TA98 which detects frameshift mutations without metabolic activation; a more pronounced effect was seen in its derivative YG1024 which overexpresses N-acetyltransferase. Induction of single/double strand breaks by Comet assay was detected with concentrations > 0.125 mg/mL in liver derived cells; as well as increased rates for micronucleus (reflecting structural and numeric chromosomal aberrations) and nuclear buds which are a consequence of gene amplifications were seen with a higher dose (2.0 mg/mL) (p < 0.05; Tukey's test). The mutational pattern which was observed in the bacterial tests indicates that the cleavage product p-nitroaniline may cause the genotoxic effects of the dye. Our findings indicate that exposure of humans and the release of the compound into the environment may lead to adverse effects due to its DNA damaging activity.

Hepatotoxicity of fusariotoxins, alone and in combination, towards the HepaRG human hepatocyte cell line

While the reality of mycotoxin co-occurrence in food commodities is now established, their effects in mixtures are not well studied. The present study investigated the individual and combined effects of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA) and moniliformin (MON) fusariotoxins on cell viability and cell death mechanisms in proliferating HepaRG cells, a human derived liver cell line. In addition, DON-ZEA being one of the most widespread mycotoxin mixtures in grains worldwide, its effect on the expression levels of genes encoding for sets of hepatocyte-specific functions was studied. After 48 h, T2 appeared to be the most cytotoxic tested fusariotoxins, followed by NIV, DON and ZEA. Furthermore, at low cytotoxic doses, all tested fusariotoxin mixtures (DON-MON, DON-FB1, DON-ZEA and NIV-T2) acted synergistically on cell death. Interestingly, during the first 18 h of exposure, only FB1 and ZEA alone and in combination with DON seemed to induce cell apoptosis and necrosis. At the gene level, after only 1 h, DON-ZEA combination induced expression of drug-metabolizing enzymes contrary to individual exposures. Thus, the observed synergy of fusariotoxin mixtures suggested that their simultaneous presence in food commodities can induce a toxic risk that should be better taken into consideration.

Genotoxic and chemopreventive assessment of Cynara scolymus L. aqueous extract in a human-derived liver cell line

Cynara scolymus L., popularly known as artichoke, is consumed as food and used as tea infusions for pharmacological purposes to treat liver dysfunctions and other conditions. Scientific data on the safety and protective effect of artichoke in human-derived liver cells is missing. This study investigated the genotoxic and modulatory effect of a liophilized extract suspended in water of C. scolymus L. leaves. Four extract concentrations (0.62, 1.25, 2.5 and 5.0 mg/mL) were evaluated using the comet assay on human hepatocyte cultures, HepG2 cells. Genotoxicity was assessed after two treatment periods, 1 and 24 h. Antigenotoxicity was evaluated against oxidative lesions induced by hydrogen peroxide in pre-, simultaneous and post-treatment protocols. Artichoke leaves aqueous extract induced genotoxic effects in HepG2 cells after 1- and 24-h treatments. In turn, extract concentrations of 0.62, 1.25 and 2.5 mg/mL, exhibited a protective effect in pretreatment, compared to hydrogen peroxide alone. However, in simultaneous and post-treatment protocols, only the lowest concentration reduced the frequency of DNA damage induced by hydrogen peroxide. In addition, in the simultaneous treatment protocol, the highest artichoke extract concentration increased hydrogen peroxide genotoxicity. It can be concluded that artichoke is genotoxic, in vitro, to HepG2 cells, but can also modulate hydrogen peroxide DNA damage.

Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [14C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [14C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.

Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

Hepatic nutrient and hormonal regulation of the PANcreatic-DERived factor (PANDER) promoter

PANcreatic-DERived factor (PANDER, FAM3B) has been shown to regulate glycemic levels via interactions with both pancreatic islets and the liver. Although PANDER is predominantly expressed from the endocrine pancreas, recent work has provided sufficient evidence that the liver may also be an additional tissue source of PANDER production. At physiological levels, PANDER is capable of disrupting insulin signaling and promoting increased hepatic glucose production. As shown in some animal models, strong expression of PANDER, induced by viral delivery within the liver, induces hepatic steatosis. However, no studies to date have explicitly characterized the transcriptional regulation of PANDER from the liver. Therefore, our investigation elucidated the nutrient and hormonal regulation of the hepatic PANDER promoter. Initial RNA-ligated rapid amplification of cDNA ends identified a novel transcription start site (TSS) approximately 26 bp upstream of the PANDER translational start codon not previously revealed in pancreatic β-cell lines. Western evaluation of various murine tissues demonstrated robust expression in the liver and brain. Promoter analysis identified strong tissue-specific activity of the PANDER promoter in both human and murine liver-derived cell lines. The minimal element responsible for maximal promoter activity within hepatic cell lines was located between −293 and −3 of the identified TSS. PANDER promoter activity was inhibited by both insulin and palmitate, whereas glucose strongly increased expression. The minimal element was responsible for maximal glucose-responsive and basal activity. Co-transfection reporter assays, chromatin-immunoprecipitation (ChIP) and site-directed mutagenesis revealed that the carbohydrate-responsive element binding protein (ChREBP) increased PANDER promoter activity and interacted with the PANDER promoter. E-box 3 was shown to be critical for basal and glucose responsive expression. In summary, in-vitro and in-vivo glucose is a potent stimulator of the PANDER promoter within the liver and this response may be facilitated by ChREBP.

Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication

Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive‐strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell‐culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver‐derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7‐Lunet cells supported HAV‐ and HCV‐RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA‐122 and phosphatidylinositol 4‐kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine‐triphosphate–binding cassette transporters and FK506‐binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. Conclusion: We established a cell‐culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. (Hepatology 2015;62:397–408