RESUMO
Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.
Assuntos
Rotas de Resultados Adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fígado Gorduroso/induzido quimicamente , Praguicidas/toxicidade , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Células Hep G2 , Humanos , Imidazóis/toxicidade , Fígado/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Receptores Citoplasmáticos e Nucleares , Medição de Risco , Triglicerídeos/metabolismoRESUMO
Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.
Assuntos
Rotas de Resultados Adversos , Fígado Gorduroso/induzido quimicamente , Fungicidas Industriais/toxicidade , Triazóis/toxicidade , Bioensaio , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fígado Gorduroso/metabolismo , Expressão Gênica , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Mitocôndrias Hepáticas/efeitos dos fármacos , Modelos Biológicos , Reação em Cadeia da Polimerase , Receptores Citoplasmáticos e Nucleares/metabolismo , Medição de Risco , Triglicerídeos/metabolismoRESUMO
Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HCS) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Hepatócitos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Acetaminofen/toxicidade , Amodiaquina/toxicidade , Animais , Carbamazepina/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Dietilestilbestrol/toxicidade , Eritromicina/toxicidade , Furosemida/toxicidade , Hepatócitos/metabolismo , Masculino , Vermelho Neutro/metabolismo , Ratos , Testes de Toxicidade , Tretinoína/toxicidadeRESUMO
Pesticides as well as many other environmental pollutants are considered as risk factors for the initiation and the progression of cancer. In order to evaluate the in vitro effects of chemicals present in the diet, we began by combining viability, real-time cellular impedance and high throughput screening data to identify a concentration "zone of interest" for the six xenobiotics selected: endosulfan, dioxin, carbaryl, carbendazim, p'p'DDE and hydroquinone. We identified a single concentration of each pollutant allowing a modulation of the impedance in the absence of vital changes (nuclear integrity, mitochondrial membrane potential, cell death). Based on the number of observed modulations known to be involved in hepatic homeostasis dysfunction that may lead to cancer progression such as cell cycle and apoptosis regulators, EMT biomarkers and signal transduction pathways, we then ranked the pollutants in terms of their toxicity. Endosulfan, was able to strongly modulate all the studied cellular processes in HepG2 cells, followed by dioxin, then carbendazim. While p,p'DDE, carbaryl and hydroquinone seemed to affect fewer functions, their effects nevertheless warrant close scrutiny. Our in vitro data indicate that these xenobiotics may contribute to the evolution and worsening of hepatocarcinoma, whether via the induction of the EMT process and/or via the deregulation of liver key processes such as cell cycle and resistance to apoptosis.
Assuntos
Carcinoma Hepatocelular/induzido quimicamente , Poluentes Ambientais/toxicidade , Ensaios de Triagem em Larga Escala , Neoplasias Hepáticas/induzido quimicamente , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Progressão da Doença , Impedância Elétrica , Transição Epitelial-Mesenquimal , Células Hep G2 , HumanosRESUMO
Atrazine (ATZ) is probably the most widely used herbicide in the world. However there are still many controversies regarding its impacts on human health. Our investigations on the role of pesticides in liver dysfunctions have led us to detect an inhibition of FSP1 expression of 70% at 50µm and around 95% at 500µM of ATZ (p<0.01). This gene encodes the protein S100a4 and is a clinical biomarker of epithelial-mesenchymal transition (EMT), a key step in the metastatic process. Here we investigated the possible effect of ATZ on cell migration and noticed that it prevents the EMT and motility of the HepG2 cells induced by the phorbol ester TPA. ATZ decreases Fak pathway activation but has no effect on the Erk1/2 pathway known to be involved in metastasis in this cell line. These results suggest that ATZ could be involved in cell homeostasis perturbation, potentially through a S100a4-dependant mechanism.
Assuntos
Atrazina/toxicidade , Movimento Celular/efeitos dos fármacos , Herbicidas/toxicidade , Proteínas S100/biossíntese , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Western Blotting , Linhagem Celular , Fibronectinas/biossíntese , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Proteína A4 de Ligação a Cálcio da Família S100 , Transdução de Sinais/efeitos dos fármacosRESUMO
Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used 12-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and ß-catenin that activated the Wnt pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the Wnt/ß-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Antígenos CD , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Hep G2 , Hepatoblastoma/fisiopatologia , Humanos , Neoplasias Hepáticas/fisiopatologia , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail , Proteínas WntRESUMO
Persistent organic pollutants (POPs) are a group of organic or chemicals that adversely affect human health and are persistent in the environment. These highly toxic compounds include industrial chemicals, pesticides such as organochlorines, and unwanted wastes such as dioxins. Although studies have described the general toxicity effects of organochlorine pesticides, the mechanisms underlying its potential carcinogenic effects in the liver are not well understood. In this study, we analyzed the effect of three organochlorine pesticides (dichlorodiphenyltrichloroethane, heptachlore and endosulfan) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the epithelial to mesenchymal transition (EMT) in primary cultured human hepatocytes. We found that these compounds modified the hepatocyte phenotype, inducing cell spread, formation of lamellipodia structures and reorganization of the actin cytoskeleton in stress fibers. These morphological alterations were accompanied by disruption of cell-cell junctions, E-cadherin repression and albumin down-regulation. Interestingly, these characteristic features of dedifferentiating hepatocytes were correlated with the gain of expression of various mesenchymal genes, including vimentin, fibronectin and its receptor ITGA5. These various results show that organochlorines and TCDD accelerate cultured human hepatocyte dedifferentiation and EMT processes. These events could account, at least in part, for the carcionogenic and/or fibrogenic activities of these POPs.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hidrocarbonetos Clorados/toxicidade , Praguicidas/toxicidade , Caderinas/metabolismo , Carcinógenos/toxicidade , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , DDT/toxicidade , Endossulfano/toxicidade , Fibronectinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Heptacloro/toxicidade , Humanos , Dibenzodioxinas Policloradas/toxicidade , Vimentina/genéticaRESUMO
Endosulfan is an organochlorine pesticide commonly used in agriculture yet classified by the Stockholm Convention in 2011 as a persistent organic pollutant (POP). Its potential toxicity makes its continued use a major public health concern. Despite studies in laboratory animals, the molecular mechanisms underlying the carcinogenic effects of endosulfan in human liver remain poorly understood. In this study, we investigated the phenotypical effects of endosulfan on HepG2 liver cells. First, we found that endosulfan disrupted the anoikis process. Indeed, cells exposed to endosulfan were initially sensitized to anoikis and thereafter recovered their resistance to this process. This phenomenon occurred in parallel to the induction of the epithelial to mesenchymal (EMT) process, as demonstrated by: (1) reorganization of the actin cytoskeleton together with activation of the FAK signaling pathway; (2) repression of E-cadherin expression; (3) induction of Snail and Slug; (4) activation of the WNT/ß-catenin pathway; and (5) induction and reorganization of mesenchymal markers (S100a4, vimentin, fibronectin, MMP-7). Secondly, despite the acquisition of mesenchymal characteristics, HepG2 cells exposed to endosulfan failed to migrate. This incapacity to acquire a motile phenotype could be attributed to a disruption of the interaction between the ECM and the cells. Taken together, these results indicate that endosulfan profoundly alters the phenotype of liver cells by inducing cell detachment and partial EMT as well as disrupting the anoikis process. All these events account, at least in part, for the carcinogenic potential of endosulfan in liver.
Assuntos
Anoikis/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Endossulfano/efeitos adversos , Células Hep G2/efeitos dos fármacos , Inseticidas/efeitos adversos , Western Blotting , Caspases/metabolismo , Ensaios de Migração Celular , Citoesqueleto/efeitos dos fármacos , Relação Dose-Resposta a Droga , Imunofluorescência , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/efeitos dos fármacos , Quinases Associadas a rho/efeitos dos fármacosRESUMO
Although many studies of lindane toxicity have been carried out, we still know little about the underlying molecular mechanisms. We used a microarray specifically designed for studies of the hepatotoxic effects of xenobiotics to evaluate the effects of lindane on specific gene expression in primary cultured rat hepatocytes. These genes were assigned to detoxication processes (CYP3A4, Gsta2, CYP4A1), cell signalling pathways and apoptosis (Eif2b3, Eif2b4, PKC). In this study, we demonstrate that lindane up-regulates PKC by increasing oxidative stress. TEMPO (a well known free radical scavenger) and Ro 31-8220 (an inhibitor of classical PKCs) prevented the inhibition of spontaneous and intrinsic apoptosis pathway (characterised by Bcl-xL induction, Bax down-regulation, caspases inhibition) and the induction of necrosis by lindane in rat hepatocytes. Thus, these findings indicate that several dependent key signalling pathways, including detoxification, apoptosis, PKC activity and redox status maintenance, contribute to lindane-induced toxicity in primary cultured rat hepatocytes. This may account more clearly for the acute and chronic effects of lindane in vivo, with the induction of cell death and tumour promotion, respectively.
Assuntos
Hepatócitos/efeitos dos fármacos , Hexaclorocicloexano/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Caspases/genética , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Regulação para Baixo , Hepatócitos/citologia , Hepatócitos/metabolismo , Indóis/farmacologia , Masculino , Necrose/induzido quimicamente , Necrose/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Lindane, a persistent organochlorine pesticide, is recognized as a major public health concern because of its potential toxic effects on human health. Despite observations pointing to the toxicity of lindane, mechanisms underlying its deleterious effects in liver have yet to be understood. In this study, we investigated the effects of lindane on autophagic, apoptotic and necrotic cell death in primary cultured rat hepatocytes. We found that lindane deregulated the autophagic process as demonstrated by (1) the formation of enlarged acidic vesicles labeled with LC3, Rab7 and LAMP1 (specific markers of autophagic vacuole maturation), (2) the conversion of LC3-I (the cytosolic form) into LC3-II (membrane bound), (3) the deregulation of the Beclin 1 protein expression and (4) the enhanced formation of the Bcl-xL/Beclin 1 complex. Lindane induced vacuolization together with the inhibition of spontaneous and intrinsic apoptosis. This disruption of cell suicide was linked to Bcl-xL up-regulation, Bax down-expression, prevention of cytochrome c release, and inhibition of caspase-9 and -3 activities. Lindane-induced disruption of apoptosis and autophagy occurred in parallel with necrosis induction in rat hepatocytes. In consequence, we proposed that lindane toxicity in primary rat hepatocytes could be jointly attributed to the disruption of autophagic process, the inhibition of apoptotic cell death and the induction of necrosis. These events account, at least in part, for the involvement of both cytotoxic and carcinogenic signaling pathways in the action of lindane in the liver.