Mapping Interindividual Variability of Toxicodynamics Using High-Throughput Transcriptomics and Primary Human Hepatocytes from Fifty Donors.

BACKGROUND: Understanding the variability across the human population with respect to toxicodynamic responses after exposure to chemicals, such as environmental toxicants or drugs, is essential to define safety factors for risk assessment to protect the entire population. Activation of cellular stress response pathways are early adverse outcome pathway (AOP) key events of chemical-induced toxicity and would elucidate the estimation of population variability of toxicodynamic responses. OBJECTIVES: We aimed to map the variability in cellular stress response activation in a large panel of primary human hepatocyte (PHH) donors to aid in the quantification of toxicodynamic interindividual variability to derive safety uncertainty factors. METHODS: High-throughput transcriptomics of over 8,000 samples in total was performed covering a panel of 50 individual PHH donors upon 8 to 24 h exposure to broad concentration ranges of four different toxicological relevant stimuli: tunicamycin for the unfolded protein response (UPR), diethyl maleate for the oxidative stress response (OSR), cisplatin for the DNA damage response (DDR), and tumor necrosis factor alpha (TNFα) for NF-κB signaling. Using a population mixed-effect framework, the distribution of benchmark concentrations (BMCs) and maximum fold change were modeled to evaluate the influence of PHH donor panel size on the correct estimation of interindividual variability for the various stimuli. RESULTS: Transcriptome mapping allowed the investigation of the interindividual variability in concentration-dependent stress response activation, where the average of BMCs had a maximum difference of 864-, 13-, 13-, and 259-fold between different PHHs for UPR, OSR, DDR, and NF-κB signaling-related genes, respectively. Population modeling revealed that small PHH panel sizes systematically underestimated the variance and gave low probabilities in estimating the correct human population variance. Estimated toxicodynamic variability factors of stress response activation in PHHs based on this dataset ranged between 1.6 and 6.3. DISCUSSION: Overall, by combining high-throughput transcriptomics and population modeling, improved understanding of interindividual variability in chemical-induced activation of toxicity relevant stress pathways across the human population using a large panel of plated cryopreserved PHHs was established, thereby contributing toward increasing the confidence of in vitro-based prediction of adverse responses, in particular hepatotoxicity. https://doi.org/10.1289/EHP11891.

Evaluation of human relevance of Nicofluprole-induced rat thyroid disruption.

Nicofluprole is a novel insecticide of the phenylpyrazole class conferring selective antagonistic activity on insect GABA receptors. After repeated daily dietary administration to Wistar rats for 28/90 days, Nicofluprole induced increases in thyroid (and liver) weight, associated with histopathology changes. Nicofluprole did not inhibit thyroid peroxydase nor sodium/iodide symporter, two key players in the biosynthesis of thyroid hormones, indicating the absence of a direct thyroid effect. The results seen in rats suggested a mode of action of Nicofluprole driven by the molecular initiating event of CAR/PXR nuclear receptor activation in livers, with key events of increases in liver weight and hypertrophy, decreasing circulatory thyroid hormones, a compensatory increase in TSH release and follicular cell hypertrophy. To explore the relevance of these changes to humans, well established in vitro rat and human sandwich-cultured hepatocytes were exposed to Nicofluprole up to 7 days. A concentration-dependent CYP3A induction (PXR-activation), an increase in T4-glucuronoconjugation accompanied by UGT1A/2B inductions was observed in rat but not in human hepatocytes. The inductions seen with Nicofluprole in rat (in vivo and in vitro in hepatocytes) that were absent in human hepatocytes represent another example of species-selectivity of nuclear CAR/PXR receptor activators. Importantly, the different pattern observed in rat and human models demonstrate that Nicofluprole-related thyroid effects observed in the rat are with no human relevance.

Primary Human Hepatocyte Spheroids as Tools to Study the Hepatotoxic Potential of Non-Pharmaceutical Chemicals.

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.

Systematic transcriptome-based comparison of cellular adaptive stress response activation networks in hepatic stem cell-derived progeny and primary human hepatocytes.

Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to other in vitro liver models. Here, we systematically compared the transcriptomic profiles upon chemical activation in HLCs, hiPSC, primary human hepatocytes (PHH) and HepG2 liver cancer cells. We used targeted RNA-sequencing to map concentration transcriptional response using benchmark concentration modeling for the various stress responses in the different test systems. We found that HLCs are very sensitive towards oxidative stress and inflammation conditions as corresponding genes were activated at over 3 fold lower concentrations of the corresponding pathway inducing compounds as compared to PHH. PHH were the most sensitive model when studying UPR related effects. Due to the non-proliferative nature of PHH and HLCs, these do not pose a good/sensitive model to pick up DNA damage responses, while hiPSC and HepG2 were more sensitive in these conditions. We envision that this study contributes to a better understanding on how HLCs can contribute to the assessment of cell physiological stress response activation to predict hepatotoxic events.

Utility of Extrapolating Human S1500+ Genes to the Whole Transcriptome: Tunicamycin Case Study.

The TempO-Seq S1500+ platform(s), now available for human, mouse, rat, and zebrafish, measures a discrete number of genes that are representative of biological and pathway co-regulation across the entire genome in a given species. While measurement of these genes alone provides a direct assessment of gene expression activity, extrapolating expression values to the whole transcriptome (~26 000 genes in humans) can estimate measurements of non-measured genes of interest and increases the power of pathway analysis algorithms by using a larger background gene expression space. Here, we use data from primary hepatocytes of 54 donors that were treated with the endoplasmic reticulum (ER) stress inducer tunicamycin and then measured on the human S1500+ platform containing ~3000 representative genes. Measurements for the S1500+ genes were then used to extrapolate expression values for the remaining human transcriptome. As a case study of the improved downstream analysis achieved by extrapolation, the "measured only" and "whole transcriptome" (measured + extrapolated) gene sets were compared. Extrapolation increased the number of significant genes by 49%, bringing to the forefront many that are known to be associated with tunicamycin exposure. The extrapolation procedure also correctly identified established tunicamycin-related functional pathways reflected by coordinated changes in interrelated genes while maintaining the sample variability observed from the "measured only" genes. Extrapolation improved the gene- and pathway-level biological interpretations for a variety of downstream applications, including differential expression analysis, gene set enrichment pathway analysis, DAVID keyword analysis, Ingenuity Pathway Analysis, and NextBio correlated compound analysis. The extrapolated data highlight the role of metabolism/metabolic pathways, the ER, immune response, and the unfolded protein response, each of which are key activities associated with tunicamycin exposure that were unrepresented or underrepresented in one or more of the analyses of the original "measured only" dataset. Furthermore, the inclusion of the extrapolated genes raised "tunicamycin" from third to first upstream regulator in Ingenuity Pathway Analysis and from sixth to second most correlated compound in NextBio analysis. Therefore, our case study suggests an approach to extend and enhance data from the S1500+ platform for improved insight into biological mechanisms and functional outcomes of diseases, drugs, and other perturbations.

Weight of Evidence and Human Relevance Evaluation of the Benfluralin Mode of Action in Rats (Part II): Thyroid carcinogenesis.

Benfluralin is an herbicide of the dinitroaniline class used to control grasses and weeds. In a 2 year dietary study in rats, benfluralin increased incidences of thyroid follicular adenoma and carcinoma at high dietary concentrations (≥2500 ppm). The benfluralin toxicology database suggests the mode of action (MOA) is initiated by induction of liver metabolizing enzymes, particularly thyroid hormone specific UGTs, a major pathway for T4 clearance in rats. As reported with phenobarbital, this effect triggers negative feedback regulation, increasing thyroid stimulating hormone (TSH) release into circulating blood. When sustained over time, this leads to thyroid changes such as follicular hypertrophy, hyperplasia and thyroid follicular tumors with chronic exposures. The described MOA was previously established in rat studies with various chemical activators of xenobiotic receptors in the liver. It is generally considered as non-relevant in humans, due to differences between humans and rats in T4 turnover and susceptibility to this carcinogenic MOA. A structured methodology based on the IPCS/MOA/Human Relevance framework was used in the evaluation of available benfluralin data, and the conclusion was determined that the carcinogenic potential of benfluralin in the thyroid is not relevant in humans.

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis.

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRß), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFß1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.

Transcriptomic Analysis of Cholestatic Compounds In Vitro.

Drug-induced cholestasis is one of the most severe manifestations of drug-induced liver injury. Drug-induced cholestasis is characterized by an accumulation of endogenous metabolites normally excreted in the bile such as bile salts, cholesterol, bilirubin, or drug metabolites. The possibility to determine early in the drug development process whether a compound presents a risk of inducing drug-induced cholestasis is key information. Since preclinical repeated dose toxicity studies have limited predictive value, large efforts in identifying alternative in vitro models with improved prediction are being made. One of the best current models for in vitro human liver is primary human hepatocytes, and we recently reported that primary human hepatocytes can be kept as long-term cultures in 2D-sandwich configuration when regularly renewing the Matrigel overlay, thereby making the model useful for repeat exposure-related toxicities, as well as for the study of adaptive responses. This primary human hepatocyte culture system combined with transcriptomics carries the future promise to identify individual gene expression profiles predictive of increased drug-induced cholestasis risk.This chapter describes the various steps for culturing and exposing primary human hepatocytes to drugs during long-term 2D-sandwich culture, performing RNA extraction, gene chip assay and selecting hepatotoxic signature using the IPA software and highlighting genes involved in bile acid homeostasis.

Three-Dimensional Spheroid Primary Human Hepatocytes in Monoculture and Coculture with Nonparenchymal Cells.

Recent advances in the development of various culture platforms are promising for achieving more physiologically relevant in vitro hepatic models using primary human hepatocytes (PHHs). Previous studies have shown the value of PHHs three-dimensional (3D) spheroid models, cultured in low cell number (1330-2000 cells/3D spheroid), to study long-term liver function as well as pharmacological drug effects and toxicity. In this study, we report that only plateable PHHs aggregate and form compact 3D spheroids with a success rate of 79%, and 96% reproducibility. Out of 3D spheroid forming PHH lots, 65% were considered stable (<50% ATP decrease) over the subsequent 14 days of culture, with reproducibility of a given PHH lot being 82%. We also report successful coculturing of PHHs with human liver nonparenchymal cells (NPCs). Crude P1c-NPC fractions were obtained by low centrifugation of the PHH supernatant fraction followed by a few days of culture before harvesting and cryopreservation. At aggregation of PHHs/P1c-NPCs (2:1 ratio 3D spheroids), liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells were successfully integrated and remained present throughout the subsequent 14-day culture period as revealed by mRNA expression markers and immunostaining. Increased mRNA expression of albumin (ALB), apolipoprotein B (APOB), cytochrome P450 3A4 (CYP3A4), and increased albumin secretion compared to PHH 3D spheroid monocultures highlighted that in a 3D spheroid coculture, configuration with NPCs, PHH functionality is increased. We thus achieved the development of a more integrated coculture model system requiring low cell numbers, of particular interest due to the scarcity of human liver NPCs.

Inter-individual differences in the susceptibility of primary human hepatocytes towards drug-induced cholestasis are compound and time dependent.

Cholestasis represents a major subtype of drug-induced liver injury and novel preclinical models for its prediction are needed. Here we used primary human hepatocytes (PHH) from different donors in 2D-sandwich (2D-sw) and/or 3D-spheroid cultures to study inter-individual differences in the response towards cholestatic hepatotoxins after short-term (48-72 hours) and long-term repeated exposures (14 days). The cholestatic liabilities of drugs were determined by comparing cell viability upon exposure to the highest non-cytotoxic drug concentration in the presence and absence of a non-cytotoxic concentrated bile acid mixture. In 2D-sw culture, cyclosporine A and amiodarone presented clear cholestatic liabilities in all four PHH donors tested, whereas differences in the susceptibility of the various PHH donors towards the cholestatic toxicity of bosentan, chlorpromazine and troglitazone were observed. In PHH from one donor, the cholestatic liabilities of chlorpromazine and troglitazone could only be detected after long-term repeated exposures when maintained in 3D-spheroid culture, but not after short-term exposures in either 2D-sw or 3D-spheroid culture, suggesting that cholestatic hepatotoxicity may require time to develop. In conclusion, inter-individual susceptibility exists towards drug-induced cholestasis, which depends on the compound as well as the exposure time.

Inter-Subject Variability in OCT1 Activity in 27 Batches of Cryopreserved Human Hepatocytes and Association with OCT1 mRNA Expression and Genotype.

PURPOSE: OCT1/3 (Organic Cation Transporter-1 and -3; SLC22A1/3) are transmembrane proteins localized at the basolateral membrane of hepatocytes. They mediate the uptake of cationic endogenous compounds and/or xenobiotics. The present study was set up to verify whether the previously observed variability in OCT activity in hepatocytes may be explained by inter-individual differences in OCT1/3 mRNA levels or OCT1 genotype. METHODS: Twenty-seven batches of cryopreserved human hepatocytes (male and female, age 24-88 y) were characterized for OCT activity, normalized OCT1/3 mRNA expression, and OCT1 genetic mutation. ASP+ (4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide) was used as probe substrate. RESULTS: ASP+ uptake ranged between 75 ± 61 and 2531 ± 202 pmol/(min × million cells). The relative OCT1 and OCT3 mRNA expression ranged between 0.007-0.46 and 0.0002-0.005, respectively. The presence of one or two nonfunctional SLC22A1 alleles was observed in 13 batches and these exhibited significant (p = 0.04) association with OCT1 and OCT3 mRNA expression. However, direct association between genotype and OCT activity could not be established. CONCLUSION: mRNA levels and genotype of OCT only partially explain inter-individual variability in OCT-mediated transport. Our findings illustrate the necessity of in vitro transporter activity profiling for better understanding of inter-individual drug disposition behavior.

Evaluation of transcriptomic signature as a valuable tool to study drug-induced cholestasis in primary human hepatocytes.

Primary human hepatocyte (PHH) sandwich cultures from five different donors were daily exposed to cyclosporine A (CsA), ibuprofen (IBU), chlorpromazine (CPZ), amiodarone (AMI) and paracetamol (APAP) at their respective Cmax (total) for short-term (1-3 days) and long-term treatment (14 days). Whole genome mRNA profiles (34,693 genes in total) were conducted using an Illumina microarray platform. The impact of compound treatments on gene signatures involved in liver differentiation, cholestasis and in bile acid homeostasis was evaluated. Notably, PHH from the five donors showed a highly comparable phenotype of terminally differentiated hepatocytes. As expected, PHH exposed to 100 µM APAP showed no signs of hepatotoxicity both after short- and long-term treatment. CsA at 0.7 µM, IBU at 100 µM, AMI at 2.5 µM and CPZ at 0.1-0.2 µM presented, in line with their cholestatic syndromes reported at therapeutic doses, transcriptomic signatures of cholestasis in PHH cultures; deregulation of genes involved in bile acid homeostasis further confirmed this finding. The strength of the cholestasis signature obtained after treatment with CsA, IBU and AMI could be directly related to the basal expression of the respective drug metabolizing enzymes in the various PHH cultures from different individuals. Our data show that the PHH model system combined with transcriptomics carries the future promise to identify individual gene expression profiles predictive of increased cholestasis risk. As the present work suggests possible correlation between mRNA levels of ADME relevant genes and a transcriptomic signature of cholestasis, particular focus on this research question could be the emphasis of additional data collection.

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

Transcriptomics hit the target: Monitoring of ligand-activated and stress response pathways for chemical testing.

High content omic methods provide a deep insight into cellular events occurring upon chemical exposure of a cell population or tissue. However, this improvement in analytic precision is not yet matched by a thorough understanding of molecular mechanisms that would allow an optimal interpretation of these biological changes. For transcriptomics (TCX), one type of molecular effects that can be assessed already is the modulation of the transcriptional activity of a transcription factor (TF). As more ChIP-seq datasets reporting genes specifically bound by a TF become publicly available for mining, the generation of target gene lists of TFs of toxicological relevance becomes possible, based on actual protein-DNA interaction and modulation of gene expression. In this study, we generated target gene signatures for Nrf2, ATF4, XBP1, p53, HIF1a, AhR and PPAR gamma and tracked TF modulation in a large collection of in vitro TCX datasets from renal and hepatic cell models exposed to clinical nephro- and hepato-toxins. The result is a global monitoring of TF modulation with great promise as a mechanistically based tool for chemical hazard identification.

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.

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.

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.

Transcriptomic hepatotoxicity signature of chlorpromazine after short- and long-term exposure in primary human sandwich cultures.

Drug-induced liver injury is the most frequent reason for market withdrawal of approved drugs, and is difficult to predict in animal models. Here, we analyzed transcriptomic data derived from short- and long-term cultured primary human hepatocytes (PHH) exposed to the well known human hepatotoxin chlorpromazine (CPZ). Samples were collected from five PHH cultures after short-term (1 and 3 days) and long-term (14 days) repeat daily treatment with 0.1 or 0.2 µM CPZ, corresponding to C(max). Two PHH cultures were additionally treated with 1 µM CPZ, and the three others with 0.02 µM CPZ. Differences in the total number of gene changes were seen between donors and throughout treatment. Specific transcriptomic hepatotoxicity signatures were created for CPZ and consisted of inflammation/hepatitis, cholestasis, and liver proliferation in all five donors, as well as fibrosis and steatosis, which were observed in four of five donors. Necrosis was present in three of five donors, and an indicative signature of cirrhosis was observed after long-term 14-day repeat treatment, also in three of five donors. The inter-donor variability in the inflammatory response to CPZ treatment was associated with variability in the strength of the response of the transcriptomic hepatotoxicity signatures, suggesting that features of inflammation could be related to the idiosyncratic hepatotoxic effects of CPZ in humans.

Oncosuppressive suicide gene virotherapy "PVH1-yCD/5-FC" for pancreatic peritoneal carcinomatosis treatment: NFκB and Akt/PI3K involvement.

Peritoneal carcinomatosis is common in advanced pancreatic cancer. Despite current standard treatment, patients with this disease until recently were considered incurable. Cancer gene therapy using oncolytic viruses have generated much interest over the past few years. Here, we investigated a new gene directed enzyme prodrug therapy (GDEPT) approach for an oncosuppressive virotherapy strategy using parvovirus H1 (PV-H1) which preferentially replicates and kills malignant cells. Although, PV-H1 is not potent enough to destroy tumors, it represents an attractive vector for cancer gene therapy. We therefore sought to determine whether the suicide gene/prodrug system, yCD/5-FC could be rationally combined to PV-H1 augmenting its intrinsic oncolytic activity for pancreatic cancer prevention and treatment. We showed that the engineered recombinant parvovirus rPVH1-yCD with 5-FC treatment increased significantly the intrinsic cytotoxic effect and resulted in potent induction of apoptosis and tumor growth inhibition in chemosensitive and chemoresistant cells. Additionally, the suicide gene-expressing PV-H1 infection reduced significantly the constitutive activities of NFκB and Akt/PI3K. Combination of their pharmacological inhibitors (MG132 and LY294002) with rPVH1-yCD/5-FC resulted in substantial increase of antitumor activity. In vivo, high and sustained expression of NS1 and yCD was observed in the disseminated tumor nodules and absent in normal tissues. Treatment of mice bearing intraperitoneal pancreatic carcinomatosis with rPVH1-yCD/5-FC resulted in a drastic inhibition of tumor cell spreading and subsequent increase in long-term survival. Together, the presented data show the improved oncolytic activity of wPV-H1 by yCD/5-FC and thus provides valuable effective and promising virotherapy strategy for prevention of tumor recurrence and treatment. In the light of this study, the suicide gene parvovirotherapy approach represents a new weapon in the war against pancreatic cancer. Moreover, these preliminary accomplishments are opening new field for future development of new combined targeted therapies to have a meaningful impact on advanced cancer.

Plateable cryopreserved human hepatocytes for the assessment of cytochrome P450 inducibility: experimental condition-related variables affecting their response to inducers.

UNLABELLED: 1. RATIONALE: The aim of the present study was to assess the stability of cryopreserved human hepatocytes over 5 years and to explore experimental condition-related variables such as seeding density, culture matrix and medium, start and duration of treatment that could potentially affect the quality of cultures and their response to cytochrome P450 (CYP) inducers. 2. RESULTS: 63/125 batches of cryopreserved human hepatocytes were plateable after thawing. Of those, 17 batches showed reproducible recovery, viability and plateability (less than 5% intra-batch variability) up to 5 years. When cultured in collagen home-coated 48-well plates at a seeding density allowing 70% confluence, cryopreserved human hepatocytes display activities equivalent to fresh counterparts. Their response to CYP inducers is maximal and equivalent to fresh counterpart for an incubation of 72 h starting at Day 2 or Day 3 after plating when cultured in modified Hepatocyte Maintenance Medium (HMM). The number of cryopreserved human hepatocytes can be further reduced by using a cocktail of CYP substrates for the assessment of their inducibility. 3. CONCLUSIONS: Experimental condition-related variables, such as seeding density, culture matrix and medium, start and duration of treatment, affecting the response of plateable thawed cryopreserved human hepatocytes to cytochrome P450 inducers can be reduced by optimizing critical steps of the protocols.