In Vitro Models for Studying Chronic Drug-Induced Liver Injury

[EN] Drug-induced liver injury (DILI) is a major clinical problem in terms of patient morbidity and mortality, cost to healthcare systems and failure of the development of new drugs. The need for consistent safety strategies capable of identifying a potential toxicity risk early in the drug discovery pipeline is key. Human DILI is poorly predicted in animals, probably due to the well-known interspecies differences in drug metabolism, pharmacokinetics, and toxicity targets. For this reason, distinct cellular models from primary human hepatocytes or hepatoma cell lines cultured as 2D monolayers to emerging 3D culture systems or the use of multi-cellular systems have been proposed for hepatotoxicity studies. In order to mimic long-term hepatotoxicity in vitro, cell models, which maintain hepatic phenotype for a suitably long period, should be used. On the other hand, repeated-dose administration is a more relevant scenario for therapeutics, providing information not only about toxicity, but also about cumulative effects and/or delayed responses. In this review, we evaluate the existing cell models for DILI prediction focusing on chronic hepatotoxicity, highlighting how better characterization and mechanistic studies could lead to advance DILI prediction.This work has been supported by the Institute of Health Carlos III (ISCIII, Plan Estatal de I+D+i 2013-2016) and co-financed by the European Regional Development Fund "A way to achieve Europe" (FEDER) through grant PI21/00223, by the Spanish Ministry of Science and Innovation Ministry-Spanish Research Agency through the Project PID2019-106000RB-C22/AEI/10.13039/501100011033, and by the Generalitat Valenciana (PROMETEO/2019/060).Donato, MT.; Gallego-Ferrer, G.; Tolosa, L. (2022). In Vitro Models for Studying Chronic Drug-Induced Liver Injury. International Journal of Molecular Sciences. 23(19):1-30. https://doi.org/10.3390/ijms231911428130231

New microRNA biomarkers for drug-induced steatosis and their potential to predict the contribution of drugs to non-alcoholic fatty liver disease

Background and Aims: Drug-induced steatosis is a major reason for drug failure in clinical trials and post-marketing withdrawal; and therefore, predictive biomarkers are essential. These could be particularly relevant in non-alcoholic fatty liver disease (NAFLD), where most patients show features of the metabolic syndrome and are prescribed with combined chronic therapies, which can contribute to fatty liver. However, specific biomarkers to assess the contribution of drugs to NAFLD are lacking. We aimed to find microRNAs (miRNAs) responsive to steatotic drugs and to investigate if they could become circulating biomarkers for drug-induced steatosis. Methods: Human HepG2 cells were treated with drugs and changes in miRNA levels were measured by microarray and qRT-PCR. Drug-induced fat accumulation in HepG2 was analyzed by high-content screening and enzymatic methods. miRNA biomarkers were also analyzed in the sera of 44 biopsy-proven NAFLD patients and in 10 controls. Results: We found a set of 10 miRNAs [miR-22-5p, -3929, -24-2-5p, -663a, -29a-3p, -21 (5p and 3p), -27a-5p, -1260 and -202-3p] that were induced in human HepG2 cells and secreted to the culture medium upon incubation with model steatotic drugs (valproate, doxycycline, cyclosporin A and tamoxifen). Moreover, cell exposure to 17 common drugs for NAFLD patients showed that some of them (e.g., irbesartan, fenofibrate, and omeprazole) also induced these miRNAs and increased intracellular triglycerides, particularly in combinations. Finally, we found that most of these miRNAs (60%) were detected in human serum, and that NAFLD patients under fibrates showed both induction of these miRNAs and a more severe steatosis grade. Conclusion: Steatotic drugs induce a common set of hepatic miRNAs that could be used in drug screening during preclinical development. Moreover, most of these miRNAs are serum circulating biomarkers that could become useful in the diagnosis of iatrogenic steatosis

Impact of Betamethasone Pretreatment on Engrafment of Cord Blood-Derived Hematopoietic Stem Cells

Hematopoietic stem cell (HSC) transplantation is crucial to cure hematologic malignancies. Umbilical cord blood (UCB) is a source of stem cells, but 90% of UCB units are discarded due to low cellularity. Improving the engraftment capacities of CD34 + stem cells would allow the use of UCB that were so far rejected. Betamethasone induces long-term transcriptomic and epigenomic changes in immune cells through glucocorticoid receptor. We hypothesize that discarded UCB could be used owing to improvements induced by betamethasone. Isolated CD34 + HSC from UCB were exposed to the synthetic glucocorticoids betamethasone and fluticasone for 20 h, and cell phenotype was determined before transplantation. NSG mice were sub-lethally irradiated (1 Gy or 2 Gy) 6 h before intravenously transferring 2-5 × 10 5 CD34 + HSC. The peripheral blood engraftment levels and the leukocyte subsets were followed up for 20 weeks using flow cytometry. At end point, the engraftment and leukocyte subsets were determined in the spleen and bone marrow. We demonstrated that betamethasone has surprising effects in recovering immune system homeostasis. Betamethasone and fluticasone increase CXCR4 and decrease HLA class II and CD54 expression in CD34 + HSCs. Both glucocorticoids-exposed cells showed a similar engraftment in 2 Gy-irradiated NSG mice. Interestingly, betamethasone-exposed cells showed enhanced engraftment in 1 Gy-irradiated NSG mice, with a trend to increase regulatory T cell percentage when compared to control. Betamethasone induces alterations in CD34 + HSCs and improve the engraftment, leading to a faster immune system recovery, which will contribute to engrafted cells survival

Regional overlap of pathologies in Lewy body disorders

Q2Q1Artículo original216-224Paciente adultoLewy body disorders (LBD) are common neurodegenerative diseases characterized by the presence of aggregated a-synuclein in Lewy bodies and Lewy neurites in the central and peripheral nervous systems. The brains of patients with LBD often display other comorbid pathologies, i.e. insoluble tau, b-amyloid aggregates, TAR DNA-binding protein 43 (TDP-43) deposits, and argyrophilic grain disease (AGD). The incidence and physiological relevance of these concurrent pathological findings remain controversial. We performed a semiquantitative detailed mapping of a-synuclein, tau, bamyloid (Ab), TDP-43, and AGD pathologies in 17 areas in 63 LBD cases (44 with Parkinson disease [PD], 28 with dementia, and 19 with dementia with Lewy bodies). APOE and MAPT genetic variants were also investigated. A majority of LBD cases had 2 or 3 concomitant findings, particularly Alzheimer disease-related pathology. Pathological stages of tau, b-amyloid and a-synuclein pathologies were increased in cases with dementia. Ab score was the best correlate of the time to dementia in PD. In addition, b-amyloid deposition correlated with a-synuclein load in all groups. MAPT H1 haplotype did not influence any assessed pathology in PD. These results highlight the common concurrence of pathologies in patients with LBD that may have an impact on the clinical expression of the diseases

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity

Foxa1 Reduces Lipid Accumulation in Human Hepatocytes and Is Down-Regulated in Nonalcoholic Fatty Liver

Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance

Efectes de citocines en la viabilitat de motoneurones i cèl·lules musculars: vies de senyalització implicades

[spa] La esclerosis lateral amiotrófica (ELA) es una enfermedad neurodegenerativa caracterizada por una pérdida selectiva de motoneuronas. Aunque se desconocen las causas exactas de la degeneración, se han propuesto diferentes mecanismos implicados como la excitotoxicidad, la pérdida de soporte trófico y la neuroinflamación. En esta tesis se han investigado los efectos de estos tres mecanismos en la viabilidad de las motoneuronas espinales. Tanto la excitotoxicidad por administración de un inhibidor de la recaptación de glutamato como la privación de suero provocan la pérdida de motoneuronas espinales en cultivo organotípico de médula espinal. Además, el VEGF protege de esta degeneración via la PI3-K/Akt. Por otra parte, hemos demostrado que la citoquina proinflamatoria TNF- potencia la excitotoxicidad glutamatérgica de las motoneuronas espinales mediante, entre otros mecanismos, la inhibición del transportador de glutamato GLT-1. Esta citoquina induce apoptosis en miotubos diferenciados, que es revertida en presencia de IFN- via NF-B.[cat] L’esclerosi lateral amiotròfica (ELA) és una malaltia neurodegenerativa caracteritzada per una pèrdua selectiva de motoneurones. Encara que es desconeixen les causes exactes de la degeneració, s’han proposat diferents mecanismes que hi participen com l’excitotoxicitat, la pèrdua de suport tròfic o la neuroinflamació. En aquesta tesi s’han investigat els efectes d’aquests tres mecanismes en la viabilitat de les motoneurones espinals. Tant l’excitotoxicitat induïda per l’administració d’un inhibidor de la recaptació de glutamat com la privació de sèrum provoquen la degeneració de les motoneurones espinals en cultiu organotípic de medul•la espinal. A més, el VEGF protegeix d’aquesta degeneració via la PI3-K/Akt. D’altra banda, hem demostrat que la citocina proinflamatòria TNF- potencia l’excitotoxicitat glutamatèrgica de les motoneurones espinals via la regulació a la baixa del transportador de glutamat GLT-1, entre d’altres. Aquesta citocina indueix apoptosi en miotúbuls diferenciats, que és revertida en presència d’IFN- via NF-B.[eng] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motoneurons. Although the molecular mechanisms causing the disease remain unknown, excitotoxicity, loss of trophic support and neuroinflammation have been proposed as causes of degeneration. The effects of these mechanisms in the viability of spinal motoneurons have been investigated. Both excitotoxicity induced by the inhibition of glutamate uptake the and serum starvation produce a selective loss of motoneurons in organotípic spinal cord cultures. In addition to this, we have demonstrated that VEGF protects motoneurons from degeneration via PI3-K/Akt. On the other hand, we have shown that proinflammatory cytokine TNF- exacerbates glutamatergic excitotoxicity of spinal motoneurons though the downregulation of glutamate transporter GLT-1. This cytokine induces apoptosis in differentiated myotubes, which is reverted in the presence of IFN- NF-B

High-Content Screening for the Detection of Drug-Induced Oxidative Stress in Liver Cells

Drug-induced liver injury (DILI) remains a major cause of drug development failure, post-marketing warnings and restriction of use. An improved understanding of the mechanisms underlying DILI is required for better drug design and development. Enhanced reactive oxygen species (ROS) levels may cause a wide spectrum of oxidative damage, which has been described as a major mechanism implicated in DILI. Several cell-based assays have been developed as in vitro tools for early safety risk assessments. Among them, high-content screening technology has been used for the identification of modes of action, the determination of the level of injury and the discovery of predictive biomarkers for the safety assessment of compounds. In this paper, we review the value of in vitro high-content screening studies and evaluate how to assess oxidative stress induced by drugs in hepatic cells, demonstrating the detection of pre-lethal mechanisms of DILI as a powerful tool in human toxicology

High-Content Screening for the Detection of Drug-Induced Oxidative Stress in Liver Cells

Drug-induced liver injury (DILI) remains a major cause of drug development failure, post-marketing warnings and restriction of use. An improved understanding of the mechanisms underlying DILI is required for better drug design and development. Enhanced reactive oxygen species (ROS) levels may cause a wide spectrum of oxidative damage, which has been described as a major mechanism implicated in DILI. Several cell-based assays have been developed as in vitro tools for early safety risk assessments. Among them, high-content screening technology has been used for the identification of modes of action, the determination of the level of injury and the discovery of predictive biomarkers for the safety assessment of compounds. In this paper, we review the value of in vitro high-content screening studies and evaluate how to assess oxidative stress induced by drugs in hepatic cells, demonstrating the detection of pre-lethal mechanisms of DILI as a powerful tool in human toxicology

Cell Models and Omics Techniques for the Study of Nonalcoholic Fatty Liver Disease: Focusing on Stem Cell-Derived Cell Models

Nonalcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in western countries. The molecular mechanisms leading to NAFLD are only partially understood, and effective therapeutic interventions are clearly needed. Therefore, preclinical research is required to improve knowledge about NAFLD physiopathology and to identify new therapeutic targets. Primary human hepatocytes, human hepatic cell lines, and human stem cell-derived hepatocyte-like cells exhibit different hepatic phenotypes and have been widely used for studying NAFLD pathogenesis. In this paper, apart from employing the different in vitro cell models for the in vitro assessment of NAFLD, we also reviewed other approaches (metabolomics, transcriptomics, and high-content screening). We aimed to summarize the characteristics of different cell types and methods and to discuss their major advantages and disadvantages for NAFLD modeling