Human Hepatocellular Cell Line HepG2 Research Articles

1H NMR spectroscopic characterisation of HepG2 cells as a model metabolic system for toxicology studies

The immortalised human hepatocellular HepG2 cell line is commonly used for toxicology studies as an alternative to animal testing due to its characteristic liver-distinctive functions. However, little is known about the baseline metabolic changes within these cells upon toxin exposure. We have applied 1H Nuclear Magnetic Resonance (NMR) spectroscopy to characterise the biochemical composition of HepG2 cells at baseline and post-exposure to hydrogen peroxide (H2O2). Metabolic profiles of live cells, cell extracts, and their spent media supernatants were obtained using 1H high-resolution magic angle spinning (HR-MAS) NMR and 1H NMR spectroscopic techniques. Orthogonal partial least squares discriminant analysis (O-PLS-DA) was used to characterise the metabolites that differed between the baseline and H2O2 treated groups. The results showed that H2O2 caused alterations to 10 metabolites, including acetate, glutamate, lipids, phosphocholine, and creatine in the live cells; 25 metabolites, including acetate, alanine, adenosine diphosphate (ADP), aspartate, citrate, creatine, glucose, glutamine, glutathione, and lactate in the cell extracts, and 22 metabolites, including acetate, alanine, formate, glucose, pyruvate, phenylalanine, threonine, tryptophan, tyrosine, and valine in the cell supernatants. At least 10 biochemical pathways associated with these metabolites were disrupted upon toxin exposure, including those involved in energy, lipid, and amino acid metabolism. Our findings illustrate the ability of NMR-based metabolic profiling of immortalised human cells to detect metabolic effects on central metabolism due to toxin exposure. The established data sets will enable more subtle biochemical changes in the HepG2 model cell system to be identified in future toxicity testing.

Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

Phytochemical Biological and Cytotoxic Activities of Calothamnus quadrifidus Leaves Cultivated in Egypt

The hydro distilled essential oil obtained from Calothamnus quadrifidus leaves was analyzed by gas liquid chromatography-mass spectroscopy. Quantification of the oil constituents was carried out using the gas liquid chromatography-flame ionization detection device where 1,8-cineole was quantified as 525.62 μg/ml. The oil showed potential antioxidant activity when tested by 1,1-diphenyl-2-picrylhydrazine radical scavenging assay where the concentration required to scavenge 1,1-diphenyl-2-picrylhydrazine by 50 %=37.3 μg. The cytotoxic activity of the essential oil was evaluated using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay compared with 1,8-cineole and doxorubicin. Calothamnus quadrifidus leaves essential oil cytotoxic activity was significant against human lung cell line A-549 (2.37 μg), human colon cell line HCT-116 (2.39 μg), human hepato-cellular cell line HepG-2 (2.55 μg) and intestinal epithelium carcinoma Caco-2 (5.80 μg) cell lines, respectively. The oil cytotoxicity exceeded 1,8-cineole except for intestinal epithelium carcinoma Caco-2. Additionally, the antimicrobial activity was evaluated using agar disc diffusion and minimum inhibitory concentration where the oil exhibited the best activity against Penicillium italicum and Geotrichum candidum with minimum inhibitory concentration=0.49 and 0.98 μg, respectively. Structure elucidation of the pure compounds isolated from Calothamnus quadrifidus leaves was done according to their chromatographic behavior, chemical and spectroscopic data. These compounds were isolated for the first time from Calothamnus quadrifidus leaves and identified as gallic acid, gallic acid methyl ester, 7,4`-dimethoxy-apigenin, 6,7-dimethoxy-apigenin, (Bis[3(2-methyl propan-1- one)-2, 6 dihydroxy-4- methoxy phenyl] methane, quercetin 3-O-beta-D-arbinopyranoside and quercetin 3-O-beta-D-4C1-xylopyranoside. In addition to the molecular docking of novel compound 3 was done in peroxiredoxins and the binding mode was found. Conformation 2 was found to be the most stable and may be responsible for the antioxidant effect as it showed the best free binding energy and affinity when compared to conformations.

Cytotoxic and morphological effects of microcystin-LR, cylindrospermopsin, and their combinations on the human hepatic cell line HepG2.

Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are toxins produced by different cyanobacterial species, which are found mainly in freshwater reservoirs. Both of them can induce, separately, toxic effects in humans and wildlife. However, little is known about the toxic effects of the combined exposure, which could likely happen, taking into account the concomitant occurrence of the producers. As both cyanotoxins are well known to induce hepatic damage, the human hepatocellular HepG2 cell line was selected for the present study. Thus, the cytotoxicity of both pure cyanotoxins alone (0-5 μg/mL CYN and 0-120 μg/mL MC-LR) and in combination for 24 and 48 h was assayed, as long as the cytotoxicity of extracts from CYN-producing and nonproducing cyanobacterial species. The potential interaction of the combination was evaluated by the isobologram or Chou-Talalay's method, which provides a combination index as a quantitative measure of the two cyanotoxins interaction's degree. Moreover, a morphological study of the individual pure toxins and their combinations was also performed. Results showed that CYN was the most toxic pure cyanotoxin, being the mean effective concentrations obtained ≈4 and 90 μg/mL for CYN and MC-LR, respectively after 24 h. However, the simultaneous exposure showed an antagonistic effect. Morphologically, autophagy, at low concentrations, and apoptosis, at high concentrations were observed, with affectation of the rough endoplasmic reticulum and mitochondria. These effects were more pronounced with the combination. Therefore, it is important to assess the toxicological profile of cyanotoxins combinations in order to perform more realistic risk evaluations.

Equine Mesenchymal Stromal Cells from Different Sources Efficiently Differentiate into Hepatocyte-Like Cells.

Adult equine hepatocytes have proven challenging to culture long term in vitro as they rapidly lose their morphology and functionality, thus limiting studies on liver function and response to disease. In this study, we describe for the first time the differentiation of equine mesenchymal stromal cells (MSC) from a variety of sources into functional hepatocyte-like cells (HLC). First, we differentiated equine umbilical cord blood (UCB)-derived MSC into HLC and found that these cells exhibited a distinct polygonal morphology, stored glycogen as visualized by periodic acid Schiff's reagent staining, and were positive for albumin and other hepatocyte-specific genes. Second, we demonstrated that UCB-HLC could be revived following cryopreservation and retained their phenotype for at least 10 days. Third, we differentiated three sets of MSC from bone marrow (BM), adipose tissue (AT), and peripheral blood (PB), matched within the same horse. We achieved a 100% differentiation success rate with BM, 0% with AT, and 66% with PB. An additional set of nine PB-MSC samples resulted in an overall success rate of 42% (n = 12), and age or gender did not seem to have an effect on the success of hepatic differentiation from that source. In a final set of experiments, we evaluated the use of these HLC as tools in different fields of biomedical research like virology, to study viral growth, and toxicology, to study chemicals with hepatic toxicity. Equine HLC were found susceptible for infection with the equine herpesviruses type 1 (EHV-1), -2, and -5, and exhibited a more sensitive dose-dependent response to arsenic toxicity than the commonly used human hepatocellular cell line HepG2. Taken together, these data indicate that equine MSC can be efficiently differentiated into HLC and these equine HLC could be a useful tool for in vitro studies.

Strategies to fine-map genetic associations with lipid levels by combining epigenomic annotations and liver-specific transcription profiles

Characterization of the epigenome promises to yield the functional elements buried in the human genome sequence, thus helping to annotate non-coding DNA polymorphisms with regulatory functions. Here, we develop two novel strategies to combine epigenomic data with transcriptomic profiles in humans or mice to prioritize potential candidate SNPs associated with lipid levels by genome-wide association study (GWAS). First, after confirming that lipid-associated loci that are also expression quantitative trait loci (eQTL) in human livers are enriched for ENCODE regulatory marks in the human hepatocellular HepG2 cell line, we prioritize candidate SNPs based on the number of these marks that overlap the variant position. This method recognized the known SORT1 rs12740374 regulatory SNP associated with LDL-cholesterol, and highlighted candidate functional SNPs at 15 additional lipid loci. In the second strategy, we combine ENCODE chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) data and liver expression datasets from knockout mice lacking specific transcription factors. This approach identified SNPs in specific transcription factor binding sites that are located near target genes of these transcription factors. We show that FOXA2 transcription factor binding sites are enriched at lipid-associated loci and experimentally validate that alleles of one such proxy SNP located near the FOXA2 target gene BIRC5 show allelic differences in FOXA2-DNA binding and enhancer activity. These methods can be used to generate testable hypotheses for many non-coding SNPs associated with complex diseases or traits.

Protective effects of alkaloid compounds from Nelumbinis Plumula on tert-butyl hydroperoxide-induced oxidative stress.

This study was conducted to investigate the effect of Nelumbinis Plumula total alkaloid (NPA) and its main alkaloid components on oxidative stress induced by tert-butyl hydroperoxide (t-BHP) in the human hepatocellular HepG2 cell line. According to HPLC analysis, several major alkaloid compounds such as liensinine, isoliensinine and neferine were present in NPA. The cytotoxic effects in 0.55 mM t-BHP-induced HepG2 cells were significantly inhibited by NPA and the major compound in NPA, neferine, showed the strongest activities. The protective effect of neferine against oxidative stress induced by t-BHP may be associated with decreased ROS formation, TBARS generation, LDH release and increased GSH levels, suggesting their involvement of the cytoprotective on oxidative stress. The effects were comparable with quercetin, which was used as positive control. Overall, total alkaloid and alkaloid compounds from Nelumbinis Plumula displayed a significant cytoprotective effect against oxidative stress. Further study is needed to elucidate the relationship between the chemical structures of the components in NPA and their protective effect on oxidative stress.

Cellular assessment of the extract of bambangan (Mangifera pajang) as a potential cytoprotective agent for the human hepatocellular HepG2 cell line

This study was conducted to investigate the potential of bambangan (Mangifera pajang) fruit extracts in the protection against oxidative damage caused by tert-butyl hydroperoxide in the human hepatocellular HepG2 cell line. Proteins which might be involved in the cytoprotective mechanism were investigated using western blotting technique. Quercetin was used as a positive control. The results showed that only the kernel extract of M. pajang and quercetin displayed cytoprotective activity in HepG2 cells, with EC50 values of 1.2 and 5.3μg/ml, respectively. Expression of quinone reductase, glutathione reductase and methionine sulfoxide reductase A proteins were significantly up-regulated by quercetin, suggesting their involvement in the cytoprotective activity of quercetin. However, expressions of only glutathione reductase and methionine sulfoxide reductase A proteins were significantly up-regulated by the kernel extract, again suggesting their involvement in the cytoprotective activity of bambangan kernel extract. Future study is needed to investigate the involvement of other cytoprotective proteins in the cytoprotection mechanism.

The Hepatocarcinoma Cell Line HepG2 Does Not Express a GHS-R1a-Type Ghrelin Receptor

The interaction of ghrelin, a 28-residue acylated peptide, with the growth hormone secretagogue receptor 1a (GHS-R1a) has been studied mostly in cells expressing a recombinant GHS-R1a. As awareness is growing on the importance to study G protein-coupled receptors in a natural environment, we studied the effect of ghrelin in the human hepatocellular HepG2 cell line because it has been described in literature to respond to ghrelin. Despite extensive efforts, we were not able to confirm mRNA expression of GHS-R1a by reverse transcription PCR, radioligand binding, or ghrelin-induced GHS-R1a receptor activation; therefore, we conclude that HepG2 cells do not express GHS-R1a. On the other hand, we confirmed a modest effect of ghrelin on the up-regulation of IRS-1 phosphorylation, which might suggest the existence of an alternative ghrelin receptor in HepG2 cells.

Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes

Unilamellar activated cationic liposomes containing 3β[ N-( N′, N′-dimethylaminopropane)-carbamoyl] cholesterol, dioleoyl phosphatidylethanolamine (DOPE) and the N-hydroxysuccinimide ester of cholesteryl hemisuccinate (4:5:1, molar ratio) have been prepared and their DNA-binding capacity has been assessed in a gel retardation assay. Ternary complexes composed of activated cationic liposomes, carbodiimide–cationized asialoorosomucoid (Me +AOM) and pRSVL plasmid DNA were assembled for receptor-mediated DNA delivery into cells expressing the asialoglycoprotein receptor (ASGP-R). Binding of complexes in which Me +AOM was replaced by fluoresceinated Me +AOM (FMe +AOM) to the human hepatocellular cell line HepG2 at 4 °C was severely reduced by co-incubation with asialoorosomucoid (AOM). Moreover, assemblies containing liposomes, pRSVL DNA and Me +AOM (8:1:4, w/w/w) promoted high levels of luciferase activity in this cell line (1.3×10 7 relative light units/mg soluble cell protein). Assays conducted in the presence of a hundred-fold excess of the ligand AOM afforded considerably lower levels of transfection (2.5×10 5 relative light units/mg soluble cell protein). In contrast, the highest level of luciferase activity achieved with liposome, pRSVL DNA, AOM complexes was only a quarter of the best levels obtained with liposome, pRSVL DNA, Me +AOM assemblies. These findings strongly support the notion that complexes gain entry into hepatocyte-derived cells by ASGP-R mediation and that they are potentially useful gene carriers to liver hepatocytes.

Alpha6beta1 integrin expression in hepatocarcinoma cells: regulation and role in cell adhesion and migration.

Liver carcinogenesis is associated with striking changes in the integrin repertoire of hepatocytes, including the overexpression of the laminin and collagen receptors alpha1beta1 and the de novo induction of the laminin receptor alpha6beta1. Our aim was to analyze the role of pro-inflammatory cytokines, interferons and fibrogenic cytokines TGF-beta and FGF2 in the regulation of the expression of beta1 integrins by neoplastic hepatocytes. The 2 human hepatocellular cell lines HepG2 and Hep3B were used as models. Integrin expression was assessed by qualitative methods (immunocytochemistry, Western blotting) and semi-quantitative techniques (FACS, cellular ELISA), before and after stimulation by TNFalpha, IL1-beta, TGF-beta, FGF2, interferon gamma and interferon alpha-2b. HepG2 and Hep3B constitutively expressed alpha1, alpha2, alpha6 and beta1 chains. A 24 to 48-hr stimulation with pro-inflammatory cytokines, TGF-beta and FGF2 induced a significant increase in the concentrations of all integrin chains. The maximum induction was registered for beta1 chain, which presented increases amounting up to 3, 4 and 7 times the control values in the presence of, respectively, TNF alpha/IL1-beta, TGF-beta and FGF2. Interferons had no direct effect on integrin expression and partially antagonized the effects of TNF alpha and TGF-beta. The increased concentrations of integrin chains were associated with an increased membrane expression of the corresponding dimers and with an increased adhesion of stimulated hepatocytes to laminin, which was antagonized by neutralizing anti-beta1 and anti-alpha6 antibodies. Finally, anti-alpha6 antibody inhibited the migration of HepG2 and Hep3B cells in reconstituted basement membrane. Our results suggest that the stimulation of alpha6beta1 integrin expression in hepatocarcinoma cells is essential for cell adhesion and migration.