HepaRG Cell Cultures Research Articles

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Low prevalence of hepatitis E virus in the liver of Corsican pigs slaughtered after 12 months despite high antibody seroprevalence.

Hepatitis E virus (HEV) infection can be acute and benign or evolve to chronic hepatitis with rapid progression toward cirrhosis or liver failure in humans. Hence, hepatitis E (HE) disease is a major public health concern. In countries where pig populations are highly contaminated with HEV, human cases of HE are mainly foodborne, occurring frequently after consumption of raw or undercooked pork products or liver. Among factors associated to the presence of HEV in pork livers from intensive rearing systems, early slaughter (≤6 months) seems to be major. In Corsica, local pigs are raised in extensive farming systems and slaughtered after 12 months. To evaluate if slaughter of pigs over 12 months reduces the risk of HEV presence in livers, 1197 liver samples were randomly collected in 2 Corsican slaughterhouses. Presence of HEV RNA was detected in liver and HEV seroprevalence was determined in paired serum. The sampling included 1083 livers from animals between 12 and 48 months and 114 livers from animals<12 months. The samples were predominantly from semi-extensive and extensive farms (n=1154). Estimated HEV seroprevalence was high, that is, >88%, and HEV RNA prevalence in adult pig livers (>12 months old) was low, that is, 0.18%. However, in livers from younger animals(<12 months), including piglets below 6 months old, 5.3% (6/114) of the samples were positive for HEV RNA. Sequences recovered from positive livers belonged to HEV genotype 3c and 3f. The presence of infectious HEV was confirmed in two livers by the detection of HEV replication in HepaRG cell cultures. Thus, this study demonstrates the low prevalence of HEV in livers of pigs over 12 months, even in farms with high HEV circulation. This observation may open new perspectives on the preferential use of livers from animals older than 12 months in raw pork liver products.

Connexin-Based Channel Activity Is Not Specifically Altered by Hepatocarcinogenic Chemicals.

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

Cholestasis Differentially Affects Liver Connexins.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.

Robustness testing and optimization of an adverse outcome pathway on cholestatic liver injury.

Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated withnew input information.

The Power of Resolution: Contextualized Understanding of Biological Responses to Liver Injury Chemicals Using High-throughput Transcriptomics and Benchmark Concentration Modeling.

Prediction of human response to chemical exposures is a major challenge in both pharmaceutical and toxicological research. Transcriptomics has been a powerful tool to explore chemical-biological interactions, however, limited throughput, high-costs, and complexity of transcriptomic interpretations have yielded numerous studies lacking sufficient experimental context for predictive application. To address these challenges, we have utilized a novel high-throughput transcriptomics (HTT) platform, TempO-Seq, to apply the interpretive power of concentration-response modeling with exposures to 24 reference compounds in both differentiated and non-differentiated human HepaRG cell cultures. Our goals were to (1) explore transcriptomic characteristics distinguishing liver injury compounds, (2) assess impacts of differentiation state of HepaRG cells on baseline and compound-induced responses (eg, metabolically-activated), and (3) identify and resolve reference biological-response pathways through benchmark concentration (BMC) modeling. Study data revealed the predictive utility of this approach to identify human liver injury compounds by their respective BMCs in relation to human internal exposure plasma concentrations, and effectively distinguished drug analogs with varied associations of human liver injury (eg, withdrawn therapeutics trovafloxacin and troglitazone). Impacts of cellular differentiation state (proliferated vs differentiated) were revealed on baseline drug metabolizing enzyme expression, hepatic receptor signaling, and responsiveness to metabolically-activated toxicants (eg, cyclophosphamide, benzo(a)pyrene, and aflatoxin B1). Finally, concentration-response modeling enabled efficient identification and resolution of plausibly-relevant biological-response pathways through their respective pathway-level BMCs. Taken together, these findings revealed HTT paired with differentiated in vitro liver models as an effective tool to model, explore, and interpret toxicological and pharmacological interactions.

Activation of Polyamine Catabolism by N¹,N11-Diethylnorspermine in Hepatic HepaRG Cells Induces Dedifferentiation and Mesenchymal-Like Phenotype.

Tumorigenesis is accompanied by the metabolic adaptation of cells to support enhanced proliferation rates and to optimize tumor persistence and amplification within the local microenvironment. In particular, cancer cells exhibit elevated levels of biogenic polyamines. Inhibitors of polyamine biosynthesis and inducers of their catabolism have been evaluated as antitumor drugs, however, their efficacy and safety remain controversial. Our goal was to investigate if drug-induced modulation of polyamine metabolism plays a role in dedifferentiation using differentiated human hepatocyte-like HepaRG cell cultures. N1,N11-diethylnorspermine (DENSpm), a potent inducer of polyamine catabolism, triggered an epithelial-mesenchymal transition (EMT)-like dedifferentiation in HepaRG cultures, as shown by down-regulation of mature hepatocytes markers and upregulation of classical EMT markers. Albeit the fact that polyamine catabolism produces H2O2, DENSpm-induced de-differentiation was not affected by antioxidants. Use of a metabolically stable spermidine analogue showed furthermore, that spermidine is a key regulator of hepatocyte differentiation. Comparative transcriptome analyses revealed, that the DENSpm-triggered dedifferentiation of HepaRG cells was accompanied by dramatic metabolic adaptations, exemplified by down-regulation of the genes of various metabolic pathways and up-regulation of the genes involved in signal transduction pathways. These results demonstrate that polyamine metabolism is tightly linked to EMT and differentiation of liver epithelial cells.

Synergistic effect of interleukin-17 and tumour necrosis factor-α on inflammatory response in hepatocytes through interleukin-6-dependent and independent pathways.

The proinflammatory cytokines interleukin (IL)-17 and tumour necrosis factor (TNF)-α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL-6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL-6, IL-17 and/or TNF-α. To determine the contribution of the IL-6 pathway in the IL-17/TNF-α-mediated effect, an anti-IL-6 receptor antibody was used. IL-17 and TNF-α increased in synergy IL-6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL-17/TNF-α synergistic cooperation enhanced the levels of C-reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL-6. IL-17/TNF-α also up-regulated IL-8, monocyte chemoattractant protein (MCP)-1 and chemokine (C-C motif) ligand 20 (CCL20) chemokines in synergy through an IL-6-independent pathway. Interestingly, first exposure to IL-17, but not to TNF-α, was crucial for the initiation of the IL-17/TNF-α synergistic effect on IL-6 and IL-8 production. In HepaRG cells, IL-17 enhanced IL-6 mRNA stability resulting in increased IL-6 protein levels. The IL-17A/TNF-α synergistic effect on IL-6 and IL-8 induction was mediated through the activation of extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase, nuclear factor-κB and/or protein kinase B (Akt)-phosphatidylinositol 3-kinase signalling pathways. Therefore, the IL-17/TNF-α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL-6 for CRP and ASAT induction. Independently of IL-6, the IL-17A/TNF-α combination may also induce immune cell recruitment by chemokine up-regulation. IL-17 and/or TNF-α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction.

Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures.

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.

Effect of the Catechol-O-Methyltransferase Inhibitors Tolcapone and Entacapone on Fatty Acid Metabolism in HepaRG Cells.

Tolcapone and entacapone are catechol-O-methyltransferase inhibitors used in patients with Parkinson's disease. For tolcapone, patients with liver failure have been reported with microvesicular steatosis observed in the liver biopsy of 1 patient. We therefore investigated the impact of tolcapone and entacapone on fatty acid metabolism in HepaRG cells exposed for 24 h and on acutely exposed mouse liver mitochondria. In HepaRG cells, tolcapone induced lipid accumulation starting at 100 µM, whereas entacapone was ineffective up to 200 µM. In HepaRG cells, tolcapone-inhibited palmitate metabolism and activation starting at 100 µM, whereas entacapone did not affect palmitate metabolism. In isolated mouse liver mitochondria, tolcapone inhibited palmitate metabolism starting at 5 µM and entacapone at 50 µM. Inhibition of palmitate activation could be confirmed by the acylcarnitine pattern in the supernatant of HepaRG cell cultures. Tolcapone-reduced mRNA and protein expression of long-chain acyl-CoA synthetase 1 (ACSL1) and protein expression of ACSL5, whereas entacapone did not affect ACSL expression. Tolcapone increased mRNA expression of the fatty acid transporter CD36/FAT, impaired the secretion of ApoB100 by HepaRG cells and reduced the mRNA expression of ApoB100, but did not relevantly affect markers of fatty acid binding, lipid droplet formation and microsomal lipid transfer. In conclusion, tolcapone impaired hepatocellular fatty acid metabolism at lower concentrations than entacapone. Tolcapone increased mRNA expression of fatty acid transporters, inhibited activation of long-chain fatty acids and impaired very low-density lipoprotein secretion, causing hepatocellular triglyceride accumulation. The findings may be relevant in patients with a high tolcapone exposure and preexisting mitochondrial dysfunction.

Short-term treatment with taurolidine is associated with liver injury

BackgroundTaurolidine has been used for peritonitis, oncological and catheter-lock treatment because of its anti-inflammatory properties. It has been suggested that taurolidine has no severe side-effects, but after long-term use morphological and functional changes of the liver were reported. The aim of this study was to investigate the effect of short-term use of taurolidine on the liver.MethodsIn HepaRG cell cultures and on a novel liver biochip dose-dependent effects of taurolidine treatment on hepatocyte adherence and cell viability was investigated. Furthermore, liver enzymes and interleukin- (IL-) 6 were measured in supernatants. Male rats were treated with low- or high-dose taurolidine, respectively, and compared to controls with physiological saline solution administration regarding blood serum parameters and histology.ResultsIn HepaRG cell cultures, hepatocyte adherence was significantly decreased, cell death and cleaved caspase-3 were significantly increased after administration of taurolidine in a dose-dependent manner. High-dose application of taurolidine led to elevated liver enzymes and IL-6 secretion in hepatic organoid. After 24 h a significant increase of serum GLDH and ASAT was observed in rats treated with high-dose taurolidine treatment.ConclusionsOur results suggest that taurolidine caused liver injury after short-term use in in vitro and in vivo models probably due to direct toxic effects on hepatocytes. Therefore, the taurolidine dose should be titrated in further investigations regarding liver injury and inflammation.

Optimisation of in vitro sample preparation for LC-MS metabolomics applications on HepaRG cell cultures

The addition of stabilizers during liquid–liquid extraction improves the precision of untargeted LC-MS metabolomics workflows.

Metabolomics analysis of the toxicity pathways of triphenyl phosphate in HepaRG cells and comparison to oxidative stress mechanisms caused by acetaminophen

Since the publication of REACH guidelines, the need for in vitro tools for toxicity testing has increased. We present here the development of a hepatotoxicity testing tool using human HepaRG cell cultures and metabolomics. HepaRG cells were exposed to either 4mM acetaminophen (APAP) as reference toxicant for oxidative stress or 50μM triphenyl phosphate (TPHP) as toxicant with unknown toxicity pathways (TPs). After 72h exposure, cells were subjected to quenching and liquid–liquid extraction which resulted in a polar and an apolar fraction. Analysis of fractions was performed by ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-QTOF-MS). Significantly up or down regulated metabolites were selected by univariate statistics prior to identification. In order to obtain robust and specific TP biomarkers, the experiment was also repeated using a different culture medium composition to assess which metabolites show consistent changes. Potential biomarkers belonging to different TPs were found for APAP and TPHP. For APAP, the biomarkers were related to a decrease in unsaturated phospholipids, and for TPHP to an accumulation of phosphoglycerolipids and increase of palmitoyl lysophosphatidylcholine. This first proof-of-concept opens new perspectives for the analysis of other (reference) toxicants with different TPs and it can be used to expand the in vitro tool for hepatotoxicity screening of various compounds.

Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure

Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1–2μM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15–20μM. Overall, the mass balance of CPZ decreased in the course of 24h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure.Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data.

Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

In vitro biokinetics of chlorpromazine and the influence of different dose metrics on effect concentrations for cytotoxicity in Balb/c 3T3, Caco-2 and HepaRG cell cultures

The extrapolation of in vitro to in vivo toxicity data is a challenge. Differences in sensitivity between cell systems may be due to intrinsic properties of the cell but also because of differences in exposure. In this study, the cytotoxicity and biokinetics of the antipsychotic chlorpromazine (CPZ) were studied in in vitro assays using different cell types and exposure conditions. Different dose metrics were assessed to express the sensitivity to CPZ.The biokinetics of CPZ were measured in cell cultures of Balb/c 3T3, Caco-2 and HepaRG cells. Cytotoxicity was measured by Alamar Blue and expressed using different dose metrics, including the nominal, measured total and measured free CPZ medium concentrations.CPZ was taken up by the cells; the highest amounts in the cell compartments were found in the Caco-2 and HepaRG cells. CPZ was highly protein-bound in the Caco-2 cell medium containing 10% fetal bovine serum, resulting in lower bioavailable exposure concentrations. Moreover, also uptake into the cells strongly influenced the concentration in the medium. The Balb/c 3T3 cells were the most sensitive to the toxic effect of CPZ. The use of different dose metrics influenced the cytotoxicity results found in the three cell types. The data show that in comparing the sensitivity of the tested cell systems, the freely dissolved concentration is a more appropriate dose metric than total concentration in the medium. The ranking in sensitivity of the three cell types for CPZ was dependent on the dose metric used.

Antiproliferative and iron chelating efficiency of the new bis-8-hydroxyquinoline benzylamine chelator S1 in hepatocyte cultures

If a new generation of iron chelators specifically devoted for cancer chemotherapy emerged these last years, any of them has not yet been approved at this time. Accordingly, there is a need to optimize new chelating molecules for iron chelation therapy and cancer treatment. So, the objective of the present investigation was to characterize the antiproliferative activity and the iron chelating capacity of the iron chelator S1 [bis-N-(8-hydroxyquinoline-5-ylmethyl)benzylamine]. Its effects were compared to O-trensox which binds ferric iron with a very high affinity (pFe3+=29.5). For this purpose, primary rat hepatocyte stimulated by EGF and human hepatoma HepaRG cell cultures were used. In these models, the anti-proliferative effect, the inhibition of DNA synthesis and the iron-chelating efficiency of increasing concentrations of S1 and O-trensox (0 up to 200μM) were investigated. In the two cell culture models, we observed that S1 was about 100 times more efficient than O-trensox and the antiproliferative effect of S1 in HepaRG cells appeared at concentrations as low as 0.1μM without cytotoxicity. Moreover, the stoichiometry of S1 for iron seemed to be in the range S1/Fe3+=1. Using the calcein fluorescence assay, we demonstrated that the affinity of S1 for iron was better than that of O-trensox since it was at least two times more effective to restore the fluorescence of calcein previously quenched by iron. So, the iron chelating efficiency of S1 could explain at least partially its higher anti-proliferative effect compared to O-trensox. Finally, these results suggest that molecules such as S1 may constitute a promising starting point to improve cancer treatment.

An adaptation of the human HepaRG cells to the in vitro micronucleus assay

The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of chemicals because of its simplicity of scoring and wide applicability in different cell types. However, most of the cells currently in use are devoid of the enzyme equipment required for activation of promutagens in the genotoxic metabolites. We postulated that the human HepaRG cell line, which can express xenobiotic metabolising enzymes at levels close to those found in primary human hepatocytes and has retained the indefinite growth capacity of transformed cells, could represent a more suitable model for genotoxicity testing of chemicals requiring metabolic activation. Based on the recommendations of the Organisation for Economic Co-operation and Development test guideline TG 487 for testing of chemicals, HepaRG cell cultures containing >80% mature hepatocytes were treated in situ with various chemicals for 24 h followed by a 3-day mitogenic stimulation with epidermal growth factor without cytokinesis block. In such culture conditions, HepaRG cells underwent >1.5 cell cycle per cell during the mitogenic stimulation. While non-genotoxic compounds (mannitol and staurosporine) did not increase the rate of micronucleated mononucleated cells, all aneugens (colchicine, nocodazole and dichlorodiphenyldichloroethylene) as well as the direct acting clastogen methyl methanesulfonate and clastogens requiring metabolic activation (aflatoxin B1, benzo(a)pyrene and 2-nitrofluorene) induced a statistically significant concentration-related increase in the number of mono-micronucleated cells. The micronucleus test was also performed after 7-day repeat exposure of HepaRG cells to the chemicals. Noticeably, a time-dependent effect was obtained with the three clastogens requiring metabolic activation. In conclusion, our results obtained with HepaRG hepatocytes exposed to various genotoxic compounds requiring or not bioactivation, compared favorably with those reported in various other cell types. They support the view that metabolically competent HepaRG cells have unique potential benefits for testing genotoxic compounds using the in vitro micronucleus assay.

Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes. Part II

Cell cycle progression is dependent on the intracellular iron level and chelators can lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of some new synthetic calix[4]arene podands bearing diamino-tetraesters, diamino-tetraalcohols, diamino-tetraacid and tetraaryloxypentoxy groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the human hepatocarcinoma HepaRG cell cultures using cell nuclei counting after staining with the DNA intercalating fluorescence dye, Hoechst 33342. Their cytotoxicity was evaluated by the extracellular LDH activity. Preliminary results indicated that their antiproliferative effect was mainly due to their cytotoxicity. The efficiency of these compounds, being comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that the new substituted calix[4]arenes could open the way to valuable new approaches for medicinal chemistry scaffolding.

Stable Expression, Activity, and Inducibility of Cytochromes P450 in Differentiated HepaRG Cells

HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.