Human Liver Cell Line HepG2 Research Articles

Iron Regulates Cellular Proliferation by Enhancing the Expression of Glucose Transporter GLUT3 in the Liver.

Iron is often accumulated in the liver during pathological conditions such as cirrhosis and cancer. Elevated expression of glucose transporters GLUT1 and GLUT3 is associated with reduced overall survival in patients with hepatocellular carcinoma. However, it is not known whether iron can regulate glucose transporters and contribute to tumor proliferation. In the present study, we found that treatment of human liver cell line HepG2 with ferric ammonium citrate (FAC) resulted in a significant upregulation of GLUT3 mRNA and protein in a dose-dependent manner. Similarly, iron accumulation in mice fed with high dietary iron as well as in mice injected intraperitoneally with iron dextran enhanced the GLUT3 expression drastically in the liver. We demonstrated that iron-induced hepatic GLUT3 upregulation is mediated by the LKB1/AMPK/CREB1 pathway, and this activation was reversed when treated with iron chelator deferiprone. In addition, inhibition of GLUT3 using siRNA prevented iron-mediated increase in the expression of cell cycle markers and cellular hyperproliferation. Furthermore, exogenous sodium beta-hydroxybutyrate treatment prevented iron-mediated hepatic GLUT3 activation both in vitro and in vivo. Together, these results underscore the importance of iron, AMPK, CREB1 and GLUT3 pathways in cell proliferation and highlight the therapeutic potential of sodium beta-hydroxybutyrate in hepatocellular carcinoma with high GLUT3 expression.

Impact of culture medium on the interpretation of qRT-PCR data in HepG2 incubated with lactobacilli.

Recently, an increasing number of studies have investigated the mechanism of action of lactobacilli in the treatment of non-alcoholic fatty liver disease. Using four computational tools (NormFinder, geNorm, Delta Ct, and BestKeeper), six potential reference genes (RGs) were analyzed in the human liver cell line HepG2 cultivated 24 h in the presence of two strains of heat-killed lactobacilli, Limosilactobacillus reuteri E and Lactiplantibacillus plantarum KG4, respectively, in different cultivation media [Dulbecco´s Modified Eagle´s Medium (DMEM) high glucose or Roswell Park Memorial Institute (RPMI)]. The analysis revealed that the suitability of RG was similar between the two lactobacilli but quite different between the two media. The commonly used RGs, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase were the most unstable in DMEM high glucose. Normalization of the mRNA expression of the target gene encoding sterol regulatory element-binding protein 1c (SREBP-1c) to different RGs resulted in different expression profiles. This demonstrates that validation of candidate RGs under specific experimental conditions is crucial for the correct interpretation of quantitative polymerase chain reaction data. In addition, the choice of media has a profound impact on the effect of lactobacilli on lipogenesis at the gene expression level, as shown by the transcription factor SREBP-1c.

Molecular interactions between metformin and D-limonene inhibit proliferation and promote apoptosis in breast and liver cancer cells

BackgroundCancer is a fatal disease that severely affects humans. Designing new anticancer strategies and understanding the mechanism of action of anticancer agents is imperative.Hypothesis/PurposeIn this study, we evaluated the utility of metformin and D-limonene, alone or in combination, as potential anticancer therapeutics using the human liver and breast cancer cell lines HepG2 and MCF-7.Study designAn integrated systems pharmacology approach is presented for illustrating the molecular interactions between metformin and D-limonene.MethodsWe applied a systems-based analysis to introduce a drug–target–pathway network that clarifies different mechanisms of treatment. The combination treatment of metformin and D-limonene induced apoptosis in both cell lines compared with single drug treatments, as indicated by flow cytometric and gene expression analysis.ResultsThe mRNA expression of Bax and P53 genes were significantly upregulated while Bcl-2, iNOS, and Cox-2 were significantly downregulated in all treatment groups compared with normal cells. The percentages of late apoptotic HepG2 and MCF-7 cells were higher in all treatment groups, particularly in the combination treatment group. Calculations for the combination index (CI) revealed a synergistic effect between both drugs for HepG2 cells (CI = 0.14) and MCF-7 cells (CI = 0.22).ConclusionOur data show that metformin, D-limonene, and their combinations exerted significant antitumor effects on the cancer cell lines by inducing apoptosis and modulating the expression of apoptotic genes.

Cyclodextrin Metal-Organic Frameworks as a Drug Delivery System for Selected Active Pharmaceutical Ingredients.

The cyclodextrin-based metal-organic frameworks (CD MOFs) are a suitable molecular platform for drug delivery systems of various active pharmaceutical ingredients (APIs). The low toxicity and cost-efficient synthesis make CD MOFs an attractive host for the encapsulation of APIs. In this study, we created a model system based on γCD-K MOFs with widely used drugs carmofur (HCFU), 5-fluorouracil (5-FU), and salicylic acid (HBA) to study host-guest encapsulation methods using different crystallization protocols. The host-guest complexes of API:CD MOF in an in-depth study were investigated by liquid chromatography-mass spectrometry (LC-MS), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and 19F- and 13C-detected solid-state NMR spectroscopy (ssNMR). These techniques confirmed the structure and interaction sites within the encapsulation product in the host-guest complex. We also evaluated the toxicity and biocompatibility of the API:CD MOF complex using in vitro and in vivo methods. The cytotoxicity, hepatotoxicity, and neurotoxicity were established with cell lines of fibroblasts (BJ), human liver cell line (HepG2), and human oligodendrocytic cells (MO3.13). Then, Danio rerio was used as an in vivo experimental model of ecotoxicity. The results showed the choice of γCD-K-5 as the most protective and safe option for drug encapsulation to decrease its toxicity level against normal cells.

Differential eigengene network analysis reveals benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin consensus regulatory network in human liver cell line HepG2

Aryl hydrocarbon receptor (AHR) is one of the main mediators of the toxic effects of benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, a vast number of BaP- and TCDD-affected genes may suggest a more complex transcriptional regulatory network driving common adverse effects of these two chemicals. Unlike TCDD, BaP is rapidly metabolized in the liver, yielding products with a questionable ability to bind and activate AHR. In this study, we used transcriptomics data from the BaP- and TCCD-exposed human liver cell line HepG2, and performed differential eigengene network analysis to understand the correlation among genes and to untangle the common regulatory mechanism in the action of BaP and TCDD. The genes were grouped into 11 meta-modules with an overall preservation of 0.72 and were also segregated into three consensus time clusters: 12, 24, and 48 h. The analysis showed that the consensus genes in each time cluster were either directly regulated by the AHR or the AHR-TF interactions. Some TFs form a direct physical interaction with AHR such as ESR1, FOXA1, and E2F1, whereas others, including CTCF, RXRA, FOXO1, CEBPA, CEBPB, and TP53 show an indirect interaction with AHR. The analysis of biological processes (BPs) identified unique and common BPs in BaP and TCDD samples, with DNA damage response detected in all three time points. In summary, we identified a consensus transcriptional regulatory network common for BaP and TCDD consisting of direct AHR targets and AHR-TF targets. This analysis sheds new light on the common mechanism of action of a genotoxic (BaP) and non-genotoxic (TCDD) chemical in liver cells.

Pollution Characteristics of Different Components of PM2.5 in Taiyuan during 2017–2020 Wintertime and Their Toxicity Effects on HepG2 Cells

Fine particulate matter (PM2.5) is a common pollutant, and its health risk has attracted much attention. Studies have shown that PM2.5 exposure is associated with liver disease. The composition of PM2.5 is complex, and its hepatotoxic effects and lipid metabolism process are not well understood. In this study, we detected the concentrations of PM2.5 and its components (metals and polycyclic aromatic hydrocarbon (PAHs)) in the winter in Taiyuan, Shanxi Province, China, from 2017 to 2020 and then assessed the health risks. We also investigated the effects of different components (whole particles (WP), water-soluble particles (WSP), organic particles (OP)) of PM2.5 on the cytotoxicity and lipid metabolism in human liver cell line (HepG2) after 24 h of treatment. The changes in cytotoxicity indexes (LDH, IL-6, reactive oxygen species (ROS)) and lipids (triglyceride (TG), free fatty acid (FFA)) were measured after 24 h. The mRNA expression of lipid metabolism-related factors (SREBP1, CD36, MTTP) was determined by real-time quantitative RT-qPCR. Finally, the correlation between metals and PAHs with higher PM2.5 content in 4 years and biomarkers was analyzed. The results showed that: (1) The PM2.5 pollution was severe in Taiyuan during winter in 2017 and the subsequent four years. The calculation results of the metal enrichment factor (EF) value and PAHs characteristic ratio of PM2.5 showed that PM2.5 pollution sources differed in different years. (2) Exposure to metals and PAHs in PM2.5 did not cause a non-carcinogenic risk. Metals had no cancer risk, while PAHs inhaled in PM2.5 in 2017 and 2018 had a potential cancer risk. The atmospheric PM2.5 pollution in Taiyuan has had a downward trend, but the PAHs in the PM2.5 of 2017–18, when the pollution is relatively serious, have a potential carcinogenic risk. (3) WP, OP and WSP inhibited cell survival rate from 2017 to 2020, and OP had higher cytotoxicity at the same concentration. (4) WP, OP and WSP increased the levels of LDH, IL-6, TNF-α, ROS, MDA, TG and FFA, and inhibited SOD activity in a dose-effect relationship. The organic components in PM2.5 are more toxic to HepG2 cells and affect the expression of lipid metabolism-related factors at the transcriptional level. (5) The mRNA expressions of factors related to lipid synthesis, uptake, oxidation and output were up-regulated after treatment with PM2.5 and its components, suggesting a lipid metabolism disorder. (6) The biomarkers were associated with certain metals (Zn, Pb, Cu and Cr) and PAHs in PM2.5. These suggested that PM2.5- and PM2.5-bound organic matter caused HepG2 cytotoxicity and affected lipid metabolism.

Emerging mycotoxins induce hepatotoxicity in pigs’ precision-cut liver slices and HepG2 cells

Emerging mycotoxins are currently gaining more attention due to their high frequency of contamination in foods and grains. However, most data available in the literature are in vitro, with few in vivo results that prevent establishing their regulation. Beauvericin (BEA), enniatins (ENNs), emodin (EMO), apicidin (API) and aurofusarin (AFN) are emerging mycotoxins frequently found contaminating food and there is growing interest in studying their impact on the liver, a key organ in the metabolization of these components. We used an ex vivo model of precision-cut liver slices (PCLS) to verify morphological and transcriptional changes after acute exposure (4 h) to these mycotoxins. The human liver cell line HepG2 was used for comparison purposes. Most of the emerging mycotoxins were cytotoxic to the cells, except for AFN. In cells, BEA and ENNs were able to increase the expression of genes related to transcription factors, inflammation, and hepatic metabolism. In the explants, only ENN B1 led to significant changes in the morphology and expression of a few genes. Overall, our results demonstrate that BEA, ENNs, and API have the potential to be hepatotoxic.

Synthesis and SARs study of novel spiro-oxindoles as potent antiproliferative agents with CDK-2 inhibitory activities.

A series of 16 novel spirooxindole analogs 8a-p were designed and constructed via cost-effective single-step multicomponent [3+2] cycloaddition reaction of azomethine ylide (AY) generated in situ from substituted isatin (6a-d) with suitable amino acids (7a-c) and ethylene-engrafted pyrazole derivatives (5a,b). The potency of all compounds was assayed against a human breast cancer cell line (MCF-7) and a human liver cell line (HepG2). Spiro compound 8c was the most active member among the synthesized candidates, with exceptional cytotoxicity against the MCF-7 and HepG2 cell lines, with IC50 values of 0.189 ± 0.01 and 1.04 ± 0.21 µM, respectively. The candidate 8c exhibited more potent activity (10.10- and 2.27-fold) than the standard drug roscovitine (IC50 = 1.91 ± 0.17 µM (MCF-7) and 2.36 ± 0.21 µM (HepG2)). Compound 8c was investigated for epidermal growth factor receptor (EGFR) inhibition; it exhibited promising IC50 values of 96.6 nM compared with 67.3 nM for erlotinib. The IC50 value of 8c (34.98 nM) exhibited cyclin-dependent kinase 2 (CDK-2) inhibition, being more active than roscovitine the (IC50 = 140 nM) in targeting the CDK-2 kinase enzyme. Additionally, for apoptosis induction of compound 8c in MCF-7, it upregulated the expression levels of proapoptotic genes for P53, Bax, caspases-3, 8, and 9 at up to 6.18, 4.8, 9.8, 4.6, 11.3 fold-change, respectively, and downregualted the level of the antiapoptotic gene for Bcl-2 by 0.14-fold. Finally, a molecular docking study of the most active compound 8c highlighted a good binding affinity with Lys89 as the key amino acid for CDK-2 inhibition.

Genotoxicity assessment of per- and polyfluoroalkyl substances mixtures in human liver cells (HepG2)

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous, toxic, and persistent environmental chemicals of concern that have been widely detected in all environmental matrices including human biological fluids. Although humans are exposed to complex mixtures of PFAS, it remains uncertain whether the co-exposure to PFAS mixtures could induce genotoxic damage in humans. Hence, this study evaluated the combined genotoxicity of PFAS mixtures in a human cell line system. To assess the possible genotoxic damage caused by human exposure to PFAS and their mixtures, we investigated their potential to induce cytotoxicity (cell viability) and genotoxicity (DNA damage) in a human liver cell line (HepG2). The selected PFAS include perfluorononanoic acid (PFNA), perfluorooctane sulfonic acid (PFOS), perfluorodecanoic acid (PFDA), perfluorooctanoic acid (PFOA), and perfluorohexane sulfonic acid (PFHxS). The interaction toxicities of these PFAS in binary mixtures were also determined using the additive index approach. The results revealed that exposure to PFNA, PFOS, PFDA, PFOA, and PFHxS singly and in binary mixtures induced a concentration-dependent decrease in cell viability. The additive index values indicated that the binary mixtures of PFOS + PFNA, PFOS + PFDA, and PFOS + PFOA displayed synergistic interaction, whereas the binary mixtures of PFOS + PFHxS, PFOA + PFNA, PFOA + PFDA, and PFOA + PFHxS behaved additively. Using the alkaline Comet assay, the potential of PFAS and their mixtures to induce DNA damage was evaluated based on a 1:1 ratio of the concentration of respective compounds required to produce a 1/10th of effective concentrations causing 50 % inhibition in cell viability (EC50). The results revealed that exposure to PFNA, PFOS, PFDA, PFOA, and PFHxS singly and in binary mixtures (PFOS + PFNA, PFOS + PFDA, PFOS + PFOA, PFOS + PFHxS, PFOA + PFNA, PFOA + PFDA, and PFOA + PFHxS) caused a moderate increase in cellular DNA damage, but no dose-response relationship was observed. Overall, this study indicates that the tested PFAS causes a concentration-dependent decrease in cell viability and only a modest increase in cellular DNA damage under these conditions.

Lycopene mitigates acrylamide and glycidamide induced cellular toxicity via oxidative stress modulation in HepG2 cells

Abstract Acrylamide (ACR), a carcinogen and neurotoxin formed in carbohydrate-rich high temperature processed foods, undergo epoxidation in cells to form more toxic metabolite glycidamide (GLY). The present study investigated antioxidant potential of lycopene (LYC) against ACR and GLY induced toxicity in human liver cell line (HepG2). The treatment of HepG2 cells with LYC for 2 h prior to ACR and GLY exposure enhanced cell viability by 10% and 8% and decreased reactive oxygen species (ROS) production in a dose dependent manner. Also, significantly increased antioxidant enzyme activity such as superoxide dismutase (SOD), catalase (CAT) activity and glutathione (GSH) level, with reduction in malondialdehyde (MDA) and 8-oxo-dG level on LYC pre-treatment. Mitochondrial function on LYC pre-treated cells exhibit an increased mitochondrial membrane potential (MMP) level. Data supports the enhancement of antioxidant defence by LYC, against ACR and GLY-induced toxicity.

SIK1 Regulates CRTC2-Mediated Gluconeogenesis Signaling Pathway in Human and Mouse Liver Cells.

The regulation of hepatic gluconeogenesis is of great significance to improve insulin resistance and benefit diabetes therapy. cAMP-Regulated Transcriptional Co-activator 2 (CRTC2) plays a key role in regulating hepatic gluconeogenesis through controlling the expression of gluconeogenic rate-limiting enzymes such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Recently, salt-inducible kinase 1 (SIK1) has been identified to play an important role in glucose metabolism disorders, but whether and how SIK1 regulates the CTRC2 signaling in liver cells under high glucose conditions has rarely been intensively elucidated. Here, we show that high glucose stimulation resulted in time-dependent down-regulated expression of SIK1, phosphorylated SIK1 at T182 site, and phosphorylated CRTC2 at S171 site, as well as upregulated expression of total CRTC2 and its downstream targets G6Pase and PEPCK in the human liver cell line HepG2. The nuclear expression levels of SIK1 and CRTC2 were time-dependently upregulated upon high glucose challenge, which was accompanied by enhanced cytoplasm-to-nucleus translocation of SIK1. Manipulation of SIK1 activity using plasmid-mediated SIK1 over-expression and the use of the SIKs inhibitor HG-9-91-01 confirmed that SIK1 regulated the CRTC2 signaling pathway in HepG2 cells. Furthermore, in mouse primary hepatocytes, high glucose exposure down-regulated SIK1 expression, and promoted SIK1 nuclear accumulation. While HG-9-91-01 treatment suppressed SIK1 expression and released the inhibitory effects of SIK1 on the expressions of key molecules involved in the CRTC2 signaling pathway, additional ectopic expression of SIK1 using adenovirus infection reversed the impacts of HG-9-91-01 on the expressions of these molecules in mouse hepatocytes. Therefore, SIK1 regulates CRTC2-mediated gluconeogenesis signaling pathway under both physiological and high glucose-induced pathological conditions. The modulation of the SIK1-CRTC2 signaling axis could provide an attractive means for treating diabetes.

Consideration of Pannexin 1 channels in COVID-19 pathology and treatment.

Pannexin1 (PANX1) is a ubiquitously expressed, channel-forming protein found in a number of tissues thoughout the body (e.g. lung, vasculature, liver, central nervous system, immune system) that is important in many key physiological and immune responses. PANX1 channels passively flux ATP (predominantly), multiple metabolites, and likely other small anions. PANX1 channels regulate inflammation and host responses to several pathogens, including viruses. While there is currently no evidence suggesting novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and PANX1 directly interact, there is an urgent need for therapeutic strategies, especially those targeting the hyper-inflammation and cytokine storm that occurs in severe cases of COVID-19. Here we argue that PANX1, and drugs known to target PANX1 (including the FDA-approved drug probenecid), should be the focus of further investigation in the context of SARS-CoV-2 infection and its associated pathology in COVID-19 patients.

Identification of Anti-tuberculosis Compounds From Aurone Analogs.

The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains has made tuberculosis (TB) control more difficult. Aurone derivatives have demonstrated promising anti-bacterial activities, but their effects against Mtb have not been thoroughly determined. In this study, we aimed to develop anti-TB compounds from aurone analogs. We used a fluorescent protein tdTomato labeled Mtb CDC1551 strain to screen 146 synthesized aurone derivatives for effective anti-TB compounds. The 9504, 9505, 9501, 9510, AA2A, and AA8 aurones inhibited the growth of Mtb with minimal inhibitory concentrations of 6.25, 12.5, 25, 25, 25, and 50 μM, respectively. We also examined cytotoxicities of the six leads against the human liver cell line HepG2, the primate kidney cell line Vero and human monocyte THP-1 derived macrophages. Three of the aurone leads (9504, 9501, and 9510) showed low cytotoxic effects on all three cell lines and high Mtb inhibitory efficacy (selectivity index > 10). Aurone 9504, 9501, AA2A, or AA8 significantly reduced the Mtb load in the lungs of infected mice after a 12-days treatment. We determined that the aurone leads inhibit Mtb chorismate synthase, an essential enzyme for aromatic acid synthesis. Our studies demonstrate the promise of synthetic aurones as novel anti-TB therapeutics.

Manganese-oxidizing bacteria form multiple cylindrospermopsin transformation products with reduced human liver cell toxicity

Cylindrospermopsin (CYN) is a toxic alkaloid highly persistent in aquatic environments. Biological removal of CYN was described previously. However, no transformation products formed by biological processes could be identified so far. Here, we describe that various manganese-oxidizing bacteria (MOB) transform CYN completely at an initial mean concentration of 7 mg L−1 (17 μM) within 3 to 34 days. Regardless of the strain, and transformation rate, transformation of CYN by MOB led to the same seven transformation products identified by mass spectrometry, which suggests that the removal of CYN by MOB follows a similar mechanism. Oxidation was the main transformation process, and the uracil moiety was the most susceptible part of the CYN molecule. In vitro cytotoxicity tests with the transformation products of CYN formed by one of the tested strains against the two human liver cell lines HepG2 and HepaRG, revealed that the transformation products were substantially less toxic than pure CYN for both cell lines. The results suggest that incubation with MOB might be an option for water treatment to remove CYN and may allow more detailed studies on the fate of CYN in the environment.

Functional analyses of copper transporter genes in the human liver cell line HepG2

Copper (Cu) is an essential element regulated by four genes (hCTR1, hATOX1, hATP7A, and hATP7B in humans and zctr1, zatox1, zatp7a, and zatp7b in zebrafish) in copper uptake, distribution, and transport in animal cells. Zebrafish (Danio rerio) shows a higher endogenous ratio of zatp7a to zatp7b in the liver, is relatively intolerant to copper ions and has a different zatp7a and zatp7b expression patterns in different organs. As high-affinity copper transporters, both zctr1 and hCTR1 increased copper toxicity, whereas hATOX1 and zatox1 slightly reduced copper toxicity in HepG2 cells after copper administration for 24 h. The transfected zatp7b functioned in HepG2 cells as effectively as hATP7B after both 24-h and 96-h copper exposure, but zatp7a failed to function in HepG2 cells as effectively as hATP7A. Our findings suggest that ATP7A dysfunction would increase cytotoxicity in the liver; the reason for zebrafish's copper intolerance could be the bulk dysfunction and abnormal localization of zATP7A.

Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited.We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.

Combined effects and toxicological interactions of perfluoroalkyl and polyfluoroalkyl substances mixtures in human liver cells (HepG2)

The combined effects and toxicological interactions of perfluoroalkyl and polyfluoroalkyl substances (PFAS) mixtures remain largely unknown even though they occur as complex mixtures in the environment. This study investigated the toxicity of individual and combined PFAS to human liver cell line (HepG2). The Combination Index (CI)-isobologram equation method was used to determine the toxicological interactions of PFAS in binary, ternary and multi-component mixtures. The results indicated that the cytotoxicity of individual PFAS to HepG2 cells increased with increasing carbon chain lengths when separated into non-sulfonated and sulfonated groups. The respective cytotoxicity of PFAS is in the order of PFDA > PFNA > PFOA > PFHpA for perfluoroalkyl carboxylic acids and in the order of PFOS > PFHxS for perfluoroalkane sulfonic acids. The toxicological interaction of PFOS and PFOA with other PFAS clearly showed a different pattern of combined toxicity in HepG2 Cells. The binary, ternary, and multi-component combinations of PFOS with PFOA, PFNA, PFDA, PFHxS, and PFHpA displayed synergistic interactions for almost all inhibitory effect levels tested, whereas, either synergistic or antagonistic effect was observed in mixtures with PFOA. Overall, the pattern of interactions of PFAS mixtures is predominated by synergism, especially at low to medium effect levels; the exceptions to this were the antagonistic interactions found in mixture with PFOA, PFHxS, and PFHpA. These cytotoxicity results may have an implication on the health risk assessment of PFAS mixtures.

The Fusarium mycotoxin, 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) induces vacuolization in HepG2 cells

The mycotoxin 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) has been isolated from cultures of the fungus Fusarium avenaceum, one of the most prevalent Fusarium species. AOD is an analogue of sphinganine and 1-deoxysphinganine, important intermediates in the de novo biosynthesis of cellular sphingolipids. Here we studied cellular effects of AOD using the human liver cell line HepG2 as a model system. AOD (10 μM) induced a transient accumulation of vacuoles in the cells. The effect was observed at non-cytotoxic concentrations and was not linked to cell death processes. Proteomic analyses indicated that protein degradation and/or vesicular transport may be a target for AOD. Further studies revealed that AOD had only minor effects on the initiation rate of macropinocytosis and autophagy. However, the AOD-induced vacuoles were lysosomal-associated membrane protein-1 (LAMP-1) positive, suggesting that they most likely originate from lysosomes or late endosomes. Accordingly, both endosomal and autophagic protein degradation were inhibited. Further studies revealed that treatment with concanamycin A or chloroquine completely blocked the AOD-induced vacuolization, suggesting that the vacuolization is dependent of acidic lysosomes. Overall, the results strongly suggest that the increased vacuolization is due to an accumulation of AOD in lysosomes or late endosomes thereby disturbing the later stages of the endolysosomal process.

Promotion of cadmium uptake and cadmium-induced toxicity by the copper transporter CTR1 in HepG2 and ZFL cells

Cadmium (Cd2+) is considered a human carcinogen as it causes oxidative stress and alters DNA repair responses. However, how Cd2+ is taken up by cells remains unclear. We hypothesized that Cd2+ could be transported into cells via a membrane copper (Cu) transporter, CTR1. CTR1 expression was not affected by Cd2+ exposure at the mRNA or protein level. Stable cell lines overexpressing either hCTR1, in the human liver cell line HepG2, or zCTR1, in the zebrafish liver cell line ZFL, were created to study their responses to Cd2+ insult. It was found that both HepG2 and ZFL cells overexpressing CTR1 had higher Cd2+ uptake and thus became sensitive to Cd2+. In contrast, hCTR1 knockdown in HepG2 cells led to a reduced uptake of Cd2+, making the cells relatively resistant to Cd2+. Localization studies revealed that hCTR1 had a clustered pattern after Cd2+ exposure, possibly in an attempt to reduce both Cd2+ uptake and Cd2+-induced toxicity. These in vitro results indicate that CTR1 can transport Cd2+ into the cell, resulting in Cd2+ toxicity.

The Factor VIII Variant X5 Enhances Hemophilia a Gene Therapy Efficiency By Its Improved Secretion

Introduction. Adeno-associated virus (AAV)-based factor VIII (FVIII) gene therapy holds great promise to provide clinical benefit in patients with hemophilia A. However, very high doses are currently needed to achieve therapeutic factor levels and the durability appears to be limited to a couple of years. Vector efficiency could be improved by employing more potent liver-specific promoters, but this might come at the price of overstraining the cellular protein folding capacity, causing FVIII to misfold in the lumen of the Endoplasmic Reticulum (ER). This event would in turn activate the unfolded protein response, cause oxidative stress, and if not resolved may even induce cell death. Aims. The objective of the presented study was to test whether the B-domain deleted (BDD)-FVIII-X5 variant can overcome the secretion challenge of high level FVIII expression in the context of hepatic gene therapy. Methods. The human FVIII variant BDD-FVIII-X5 harboring 5 amino acid exchanges in the A1 domain was previously isolated in a screen aimed at identifying those residues in porcine FVIII that are critical for efficient secretion. BDD-FVIII and BDD-FVIII-X5 were produced in Chinese Hamster Ovary (CHO) cells and purified to apparent homogeneity using standard procedures. The preparations were assayed for total protein by UV absorbance at 280 nm and FVIII activity by a chromogenic assay. Both FVIII variants were vectorized using AAV8 and tested in the human liver cell line HepG2 and FVIII knockout mice (E17) at various doses. Resulting samples were assayed for FVIII chromogenic activity. The potential immunogenic risk was evaluated in three hemophilic mouse strains (E17, human FVIII transgenic, humanized HLA-DRB1*1501). Results. A characterization of purified recombinant Refacto-like BDD-FVIII and the corresponding X5 variant revealed similarity of the two proteins and their specific activities in particular, indicating that introduction of the 5 amino acids from porcine FVIII did not alter functionality of human BDD-FVIII. In vitro expression of BDD-FVIII-X5 in a human liver cell line resulted in substantially increased FVIII activity levels in the supernatant compared with the non-modified BDD-FVIII, commensurate with enhanced secretion of the X5 variant. Intravenous delivery of liver-targeted AAV8 vectors carrying the BDD-FVIII-X5 transgene achieved substantial increases in plasma coagulation activity over BDD-FVIII in FVIII-deficient mice, even when highly efficient codon-optimized F8 nucleotide sequences were employed. Evaluation of the immunogenicity of the BDD-FVIII-X5 variant by an immunological risk assessment did not reveal any increased immunogenic risk compared to BDD-FVIII. Conclusions: The fully active BDD-FVIII-X5 variant demonstrated improved secretion in vitro and in vivo, resulting in substantially higher FVIII levels in a hemophilia A mouse model. No signs of enhanced immunogenicity were noted in a comparative immunogenicity study. The results obtained warrant further exploration of the BDD-FVIII-X5 variant for a next generation hemophilia A gene therapy. Disclosures Horling: Baxalta Innovations GmbH, a Takeda company: Employment. Lengler:Baxalta Innovations GmbH, a Takeda company: Employment. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. De La Rosa:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Hoellriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Reipert:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Xiao:Ivygen: Other: Patent application on FVIII-X5 has been submitted. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.