Long term exposure to environmental concentrations of diesel exhaust particles does not impact the phenotype of human bronchial epithelial cells.

Chronic exposure to diesel engine exhausts is associated with an increased risk of pulmonary diseases including lung cancer. Diesel engine exhausts contain large amounts of diesel exhaust particles (DEP) on which are adsorbed several carcinogenic compounds such as polycyclic aromatic hydrocarbons. Acute toxicity of high concentrations of DEP has been largely demonstrated in various in vitro cellular models. In contrast, the cellular and molecular impacts of low environmental concentrations of DEP on the phenotype of chronically exposed lung epithelial cells remain to be investigated. In the present study, we show that long term exposure (6 months) to 2 µg/ml (0.4 µg/cm2) DEP (standard reference material 1650b) increased cytochrome P4501A mRNA levels in the human bronchial epithelial BEAS-2B cell line. However, chronic exposure to DEP did not change cell morphology, trigger epithelial-mesenchymal transition or increase anchorage-independent cell growth. Moreover, DEP increase neither the levels of reactive oxygen species or those of γ-histone H2AX, nor the expression of interleukin-6 and interleukin-8. Our results thus demonstrate that the chronic exposure to low DEP concentrations could increase cytochrome P501A gene expression in BEAS-2B cells but did not induce molecular effects related to genotoxicity, oxidative stress or inflammation.

Transcriptomic analysis of untreated and drug-treated differentiated HepaRG cells over a 2-week period.

Previous works have shown that differentiated human HepaRG cells can exhibit drug metabolism activities close to those of primary human hepatocytes for several weeks at confluence. The present study was designed to evaluate their long-term functional stability and their response to repeated daily drug treatments over a 14-day period, using a transcriptomic approach. Our data show that less than 1% of the expressed genes were markedly deregulated over this two weeks period and mainly included down-regulation of genes related to the cell cycle and from 3 days, overexpression of genes involved in xenobiotic and lipid metabolism. After daily treatment with the three PPAR agonists, fenofibrate, troglitazone and rosiglitazone qualitative and/or quantitative changes in gene profiling were observed depending on the compound and duration of treatment. The highest increase in the number of deregulated genes as a function of drug treatment was seen with rosiglitazone. The most up-regulated genes common across the three compounds were mainly related to lipid and xenobiotic metabolisms. All the data support the conclusion that human HepaRG cells have an exceptional functional stability at confluence and that they are suitable for investigations on chronic effects of drugs and other chemicals.

Understanding the biokinetics of ibuprofen after single and repeated treatments in rat and human in vitro liver cell systems.

Common in vitro toxicity testing often neglects the fate and intracellular concentration of tested compounds, potentially limiting the predictability of in vitro results for in vivo extrapolation. We used in vitro long-term cultures of primary rat (PRH) and human hepatocytes (PHH) and HepaRG cells to characterise and model the biokinetic profile of ibuprofen (IBU) after single and daily repeated exposure (14 days) to two concentrations. A cross-model comparison was carried out at 100µM, roughly corresponding to the human therapeutic plasma concentration. Our results showed that IBU uptake was rapid and a dynamic equilibrium was reached within 1 or 2 days. All three cell systems efficiently metabolised IBU. In terms of species-differences, our data mirrored known in vivo results. Although no bioaccumulation was observed, IBU intracellular concentration was higher in PRH due to a 10-fold lower metabolic clearance compared to the human-derived cells. In HepaRG cells, IBU metabolism increased over time, but was not related to the treatment. In PHH, a low CYP2C9 activity, the major IBU-metabolising CYP, led to an increased cytotoxicity. A high inter-individual variability was seen in PHH, whereas HepaRG cells and PRH were more reproducible models. Although the concentrations of IBU in PRH over time differed from the concentrations found in human cells under similar exposure conditions.

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 14 days. 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 1-2 µM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20 µM. Overall, the mass balance of CPZ decreased in the course of 24 h, 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.

In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments.

The limited value of in vitro toxicity data for the in vivo extrapolation has been often attributed to the lack of kinetic data. Here the in vitro kinetics of amiodarone (AMI) and its mono-N-desethyl (MDEA) metabolite was determined and modelled in primary human hepatocytes (PHH) and HepaRG cells, after single and repeated administration of clinically relevant concentrations. AMI bioavailability was influenced by adsorption to the plastic and the presence of protein in the medium (e.g. 10% serum protein reduced the uptake by half in HepaRG cells). The cell uptake was quick (within 3h), AMI metabolism was efficient and a dynamic equilibrium was reached in about a week after multiple dosing. In HepaRG cells the metabolic clearance was higher than in PHH and increased over time, as well as CYP3A4. The interindividual variability in MDEA production in PHHs was not proportional to the differences in CYP3A4 activities, suggesting the involvement of other CYPs and/or AMI-related CYP inhibition. After repeated treatment AMI showed a slight potential for bioaccumulation, whereas much higher intracellular MDEA levels accumulated over time, especially in the HepaRG cells, associated with occurrence of phospholipidosis. The knowledge of in vitro biokinetics is important to transform an actual in vitro concentration-effect into an in vivo dose-effect relationship by using appropriate modelling, thus improving the in vitro-to-in vivo extrapolation.

Impact of inflammation on chlorpromazine-induced cytotoxicity and cholestatic features in HepaRG cells.

Several factors are thought to be implicated in the occurrence of idiosyncratic adverse drug reactions. The present work aimed to question as to whether inflammation is a determinant factor in hepatic lesions induced by chlorpromazine (CPZ) using the human HepaRG cell line. An inflammation state was induced by a 24-hour exposure to proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß; then the cells were simultaneously treated with CPZ and/or cytokine for 24 hours or daily for 5 days. The inflammatory response was assessed by induction of C-reactive protein and IL-8 transcripts and proteins as well as inhibition of CPZ metabolism and down-regulation of cytochrome 3A4 (CYP3A4) and CYP1A2 transcripts, two major cytochrome P450 (P450) enzymes involved in its metabolism. Most effects of cotreatments with cytokines and CPZ were amplified or only observed after five daily treatments; they mainly included increased cytotoxicity and overexpression of oxidative stress-related genes, decreased Na(+)-taurocholate cotransporting polypeptide mRNA levels and activity, a key transporter involved in bile acids uptake, and deregulation of several other transporters. However, CPZ-induced inhibition of taurocholic acid efflux and pericanalicular F-actin distribution were not affected. In addition, a time-dependent induction of phospholipidosis was noticed in CPZ-treated cells, without obvious influence of the inflammatory stress. In summary, our results show that an inflammatory state induced by proinflammatory cytokines increased cytotoxicity and enhanced some cholestatic features induced by the idiosyncratic drug CPZ in HepaRG cells. These changes, together with inhibition of P450 activities, could have important consequences if extrapolated to the in vivo situation.

Different dose-dependent mechanisms are involved in early cyclosporine a-induced cholestatic effects in hepaRG cells.

Mechanisms involved in drug-induced cholestasis in humans remain poorly understood. Although cyclosporine A (CsA) and tacrolimus (FK506) share similar immunosuppressive properties, only CsA is known to cause dose-dependent cholestasis. Here, we have investigated the mechanisms implicated in early cholestatic effects of CsA using the differentiated human HepaRG cell line. Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively after addition of 10µM CsA; it peaked at around 2 h and was reversible. These early effects were associated with generation of oxidative stress and deregulation of cPKC pathway. At higher CsA concentrations (≥50µM) alterations of efflux and uptake activities were enhanced and became irreversible, pericanalicular F-actin microfilaments were disorganized and bile canaliculi were constricted. These changes were associated with induction of endoplasmic reticulum stress that preceded generation of oxidative stress. Concentration-dependent changes were observed on total bile acid disposition, which were characterized by an increase and a decrease in culture medium and cells, respectively, after a 24-h treatment with CsA. Accordingly, genes encoding hepatobiliary transporters and bile acid synthesis enzymes were differently deregulated depending on CsA concentration. By contrast, FK506 induced limited effects only at 25-50µM and did not alter bile canaliculi. Our data demonstrate involvement of different concentration-dependent mechanisms in CsA-induced cholestasis and point out a critical role of endoplasmic reticulum stress in the occurrence of the major cholestatic features.

Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.

Impact of isomalathion on malathion cytotoxicity and genotoxicity in human HepaRG cells.

Isomalathion is a major impurity of technical grade malathion, one of the most abundantly applied insecticides; however little is known about its hepatotoxicity. In the present study, cytotoxicity and genotoxicity of malathion and isomalathion either individually or in combination, were assessed using the metabolically competent human liver HepaRG cell line. Isomalathion reduced cell viability starting at a 100 µM concentration after a 24h exposure. It also significantly induced caspase-3 activity in a dose-dependent manner starting at 5 µM. On the contrary, even at concentrations as high as 500 µM malathion affected neither cell viability nor caspase-3 activity. Moreover, co-exposure of both compounds resulted in decreased toxicity of isomalathion. By contrast, malathion and isomalathion either separately or in combination, slightly induced micronuclei formation at low concentrations and had additive genotoxic effects when combined at 25 µM. Individually or combined isomalathion directly inhibited activity of carboxyesterases which are involved in detoxication of malathion. In addition, transcripts of CYP2B6 and CYP3A4, two CYPs responsible for malathion phase I metabolism, were strongly induced by the mixture while isomalathion alone only moderately decreased CYP1A2 and increased CYP2B6 transcripts. However, these CYPs were not altered at the protein or activity levels. Taken altogether, our results showed that isomalathion was much more cytotoxic than malathion while both compounds had comparable genotoxic effects in HepaRG hepatocytes at low concentrations and brought further support to the importance of considering impurities and interactions during evaluation of health risks of pesticides.