RESUMEN
Oxidative stress is the consequence of an abnormal increase of reactive oxygen species (ROS). ROS are generated mainly during the metabolism in both normal and pathological conditions as well as from exposure to xenobiotics. Xenobiotics can, on the one hand, disrupt molecular machinery involved in redox processes and, on the other hand, reduce the effectiveness of the antioxidant activity. Such dysregulation may lead to oxidative damage when combined with oxidative stress overpassing the cell capacity to detoxify ROS. In this work, a green fluorescent protein (GFP)-tagged nuclear factor erythroid 2-related factor 2 (NRF2)-regulated sulfiredoxin reporter (Srxn1-GFP) was used to measure the antioxidant response of HepG2 cells to a large series of drug and drug-like compounds (2230 compounds). These compounds were then classified as positive or negative depending on cellular response and distributed among different modeling groups to establish structure-activity relationship (SAR) models. A selection of models was used to prospectively predict oxidative stress induced by a new set of compounds subsequently experimentally tested to validate the model predictions. Altogether, this exercise exemplifies the different challenges of developing SAR models of a phenotypic cellular readout, model combination, chemical space selection, and results interpretation.
Asunto(s)
Estrés Oxidativo , Xenobióticos , Humanos , Especies Reactivas de Oxígeno , Células Hep G2 , Estudios Prospectivos , Relación Estructura-ActividadRESUMEN
A comprehensive understanding of the dynamic activation and crosstalk between different cellular stress response pathways that drive cell adversity is crucial in chemical safety assessment. Various chemicals have electrophilic properties that drive cell injury responses in particular oxidative stress signaling and inflammatory signaling. Here we used bacterial artificial chromosome-based GFP cellular stress reporters with live cell confocal imaging, to systematically monitor the differential modulation of the dynamics of stress pathway activation by six different soft electrophiles: sulforaphane, andrographolide, diethyl maleate, CDDO-Me, ethacrynic acid and tert-butyl hydroquinone. The various soft electrophiles showed differential potency and dynamics of Nrf2 activation and nuclear translocation. These differences in Nrf2 dynamics correlated with distinct activation pattern of Nrf2 downstream targets SRNX1 and HMOX1. All soft electrophiles caused a strong dose dependent suppression of a cytokine-induced NFĸB response represented by suppression of NFĸB nuclear oscillation and inhibition of the downstream target gene activation A20 and ICAM1, which followed the potency of Nrf2 modulation but occurred at higher concentration close to saturation of Nrf2 activation. RNAi-based depletion of RelA resulted in a prolonged presence of Nrf2 in the nucleus after soft electrophile treatment; depletion of Nrf2 caused the induction of NFĸB signaling and activation of its downstream targets A20 and ICAM1. A systematic transcriptome analysis confirmed these effects by soft electrophiles on Nrf2 and NFκB signaling crosstalk in human induced-pluripotent stem cell-derived hepatocyte-like cells. Altogether our data indicate that modulation of Nrf2 by soft electrophiles may have consequences for efficient inflammatory signaling.
Asunto(s)
Antioxidantes , Factor 2 Relacionado con NF-E2 , Antioxidantes/farmacología , Hepatocitos , Humanos , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Ácido Oleanólico/análogos & derivados , Estrés OxidativoRESUMEN
Cells are exposed to oxidative stress and reactive metabolites every day. The Nrf2 signaling pathway responds to oxidative stress by upregulation of antioxidants like glutathione (GSH) to compensate the stress insult and re-establish homeostasis. Although mechanisms describing the interaction between the key pathway constituents Nrf2, Keap1 and p62 are widely reviewed and discussed in literature, quantitative dynamic models bringing together these mechanisms with time-resolved data are limited. Here, we present an ordinary differential equation (ODE) based dynamic model to describe the dynamic response of Nrf2, Keap1, Srxn1 and GSH to oxidative stress caused by the soft-electrophile diethyl maleate (DEM). The time-resolved data obtained by single-cell confocal microscopy of green fluorescent protein (GFP) reporters and qPCR of the Nrf2 pathway components complemented with siRNA knock down experiments, is accurately described by the calibrated mathematical model. We show that the quantitative model can describe the activation of the Nrf2 pathway by compounds with a different mechanism of activation, including drugs which are known for their ability to cause drug induced liver-injury (DILI) i.e., diclofenac (DCF) and omeprazole (OMZ). Finally, we show that our model can reveal differences in the processes leading to altered activation dynamics amongst DILI inducing drugs.
Asunto(s)
Hepatocitos , Factor 2 Relacionado con NF-E2 , Humanos , Glutatión/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés OxidativoRESUMEN
Nuclear factor erythroid-2 related factor 2 (NRF2) encoded by the NFE2L2 gene is a transcription factor critical for protecting cells from chemically-induced oxidative stress. We developed computational procedures to identify chemical modulators of NRF2 in a large database of human microarray data. A gene expression biomarker was built from statistically-filtered gene lists derived from microarray experiments in primary human hepatocytes and cancer cell lines exposed to NRF2-activating chemicals (oltipraz, sulforaphane, CDDO-Im) or in which the NRF2 suppressor Keap1 was knocked down by siRNA. Directionally consistent biomarker genes were further filtered for those dependent on NRF2 using a microarray dataset from cells after NFE2L2 siRNA knockdown. The resulting 143-gene biomarker was evaluated as a predictive tool using the correlation-based Running Fisher algorithm. Using 59 gene expression comparisons from chemically-treated cells with known NRF2 activating potential, the biomarker gave a balanced accuracy of 93%. The biomarker was comprised of many well-known NRF2 target genes (AKR1B10, AKR1C1, NQO1, TXNRD1, SRXN1, GCLC, GCLM), 69% of which were found to be bound directly by NRF2 using ChIP-Seq. NRF2 activity was assessed across ~9840 microarray comparisons from ~1460 studies examining the effects of ~2260 chemicals in human cell lines. A total of 260 and 43 chemicals were found to activate or suppress NRF2, respectively, most of which have not been previously reported to modulate NRF2 activity. Using a NRF2-responsive reporter gene in HepG2 cells, we confirmed the activity of a set of chemicals predicted using the biomarker. The biomarker will be useful for future gene expression screening studies of environmentally-relevant chemicals.
Asunto(s)
Minería de Datos , Bases de Datos Genéticas , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transcriptoma , Biomarcadores/metabolismo , Células Hep G2 , HumanosRESUMEN
Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hepatocitos/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Diferenciación Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Daño del ADN/efectos de los fármacos , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Células Hep G2 , Hepatocitos/patología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Microscopía Confocal/métodos , Esferoides Celulares/patología , Estrés Fisiológico/efectos de los fármacosRESUMEN
Formation of the reactive amodiaquine quinoneimine (AQ-QI) and N-desethylamodiaquine quinoneimine (DEAQ-QI) plays an important role in the toxicity of the anti-malaria drug amodiaquine (AQ). Glutathione conjugation protects against AQ-induced toxicity and GSTP1 is able to conjugate its quinoneimine metabolites AQ-QI and DEA-QI with glutathione. In this study, HepG2 cells transiently transfected with the human GSTP1 construct were utilized to investigate the protective effect of GSTP1 in a cellular context. HepG2 cells were exposed to synthesized QIs, which bypasses the need for intracellular bioactivation of AQ or DEAQ. Exposure was accompanied by decreased cell viability, increased caspase 3 activity, and decreased intracellular GSH levels. Using high-content imaging-based BAC-GFP reporters, it was shown that AQ-QI and DEAQ-QI specifically activated the endoplasmic reticulum (ER) stress response. In contrast, oxidative stress, DNA damage, or inflammatory stress responses were not activated. Overexpression of GSTP1 resulted in a two-fold increase in GSH-conjugation of the QIs, attenuated QI-induced cytotoxicity especially under GSH-depletion condition, abolished QIs-induced apoptosis but did not significantly inhibit the activation of the ER stress response. In conclusion, these results indicate a protective role of GSTP1 by increasing enzymatic detoxification of AQ-QI and DEAQ-QI and suggest a second protective mechanism by interfering with ER stress induced apoptosis.
RESUMEN
Drug-induced liver injury remains a concern during drug treatment and development. There is an urgent need for improved mechanistic understanding and prediction of DILI liabilities using in vitro approaches. We have established and characterized a panel of liver cell models containing mechanism-based fluorescent protein toxicity pathway reporters to quantitatively assess the dynamics of cellular stress response pathway activation at the single cell level using automated live cell imaging. We have systematically evaluated the application of four key adaptive stress pathway reporters for the prediction of DILI liability: SRXN1-GFP (oxidative stress), CHOP-GFP (ER stress/UPR response), p21 (p53-mediated DNA damage-related response) and ICAM1 (NF-κB-mediated inflammatory signaling). 118 FDA-labeled drugs in five human exposure relevant concentrations were evaluated for reporter activation using live cell confocal imaging. Quantitative data analysis revealed activation of single or multiple reporters by most drugs in a concentration and time dependent manner. Hierarchical clustering of time course dynamics and refined single cell analysis allowed the allusion of key events in DILI liability. Concentration response modeling was performed to calculate benchmark concentrations (BMCs). Extracted temporal dynamic parameters and BMCs were used to assess the predictive power of sub-lethal adaptive stress pathway activation. Although cellular adaptive responses were activated by non-DILI and severe-DILI compounds alike, dynamic behavior and lower BMCs of pathway activation were sufficiently distinct between these compound classes. The high-level detailed temporal- and concentration-dependent evaluation of the dynamics of adaptive stress pathway activation adds to the overall understanding and prediction of drug-induced liver liabilities.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Modelos Biológicos , Estrés Fisiológico/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Hep G2 , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Análisis de la Célula Individual , Estrés Fisiológico/efectos de los fármacos , Máquina de Vectores de Soporte , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis and generally activated during toxicity in a chemical-specific manner. Here, we established a platform containing a panel of distinct adaptive stress response reporter cell lines based on BAC-transgenomics GFP tagging in HepG2 cells. Our current panel of eleven BAC-GFP HepG2 reporters together contains (1) upstream sensors, (2) downstream transcription factors and (3) their respective target genes, representing the oxidative stress response pathway (Keap1/Nrf2/Srxn1), the unfolded protein response in the endoplasmic reticulum (Xbp1/Atf4/BiP/Chop) and the DNA damage response (53bp1/p53/p21). Using automated confocal imaging and quantitative single-cell image analysis, we established that all reporters allowed the time-resolved, sensitive and mode-of-action-specific activation of the individual BAC-GFP reporter cell lines as defined by a panel of pathway-specific training compounds. Implementing the temporal pathway activity information increased the discrimination of training compounds. For a set of >30 hepatotoxicants, the induction of Srxn1, BiP, Chop and p21 BAC-GFP reporters correlated strongly with the transcriptional responses observed in cryopreserved primary human hepatocytes. Together, our data indicate that a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses, thus assisting in the future mechanism-based safety assessment of chemicals.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Cromosomas Artificiales Bacterianos , Proteínas Fluorescentes Verdes/análisis , Imagen Molecular/métodos , Pruebas de Toxicidad/métodos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Hep G2/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
A quantitative dynamics pathway map of the Nrf2-mediated oxidative stress response and p53-related DNA damage response pathways as well as the cross-talk between these pathways has not systematically been defined. To allow the dynamic single cell evaluation of these pathways, we have used BAC-GFP recombineering to tag for each pathway's three key components: for the oxidative stress response, Keap1-GFP, Nrf2-GFP, and Srxn1-GFP; for the DNA damage response, 53bp1-GFP, p53-GFP, and p21-GFP. The dynamic activation of these individual components was assessed using quantitative high throughput confocal microscopy after treatment with a broad concentration range of diethyl maleate (DEM; to induce oxidative stress) and etoposide (to induce DNA damage). DEM caused a rapid activation of Nrf2, which returned to baseline levels at low concentrations but remained sustained at high concentrations. Srxn1-GFP induction and Keap1-GFP translocation to autophagosomes followed later, with upper boundaries reached at high concentrations, close to the onset of cell death. Etoposide caused rapid accumulation of 53bp1-GFP in DNA damage foci, which was later followed by the concentration dependent nuclear accumulation of p53-GFP and subsequent induction of p21-GFP. While etoposide caused activation of Srxn1-GFP, a modest activation of DNA damage reporters was observed for DEM at high concentrations. Interestingly, Nrf2 knockdown caused an inhibition of the DNA damage response at high concentrations of etoposide, while Keap1 knockdown caused an enhancement of the DNA damage response already at low concentrations of etoposide. Knockdown of p53 did not affect the oxidative stress response. Altogether, the current stress response landscapes provide insight in the time course responses of and cross-talk between oxidative stress and DNA-damage and defines the tipping points where cell injury may switch from adaptation to injury.
Asunto(s)
Daño del ADN/efectos de los fármacos , Etopósido/toxicidad , Maleatos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Genes Reporteros , Células Hep G2 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Over the past decade, major leaps forward have been made on the mechanistic understanding and identification of adaptive stress response landscapes underlying toxic insult using transcriptomics approaches. However, for predictive purposes of adverse outcome several major limitations in these approaches exist. First, the limited number of samples that can be analyzed reduces the in depth analysis of concentration-time course relationships for toxic stress responses. Second these transcriptomics analysis have been based on the whole cell population, thereby inevitably preventing single cell analysis. Third, transcriptomics is based on the transcript level, totally ignoring (post)translational regulation. We believe these limitations are circumvented with the application of high content analysis of relevant toxicant-induced adaptive stress signaling pathways using bacterial artificial chromosome (BAC) green fluorescent protein (GFP) reporter cell-based assays. The goal is to establish a platform that incorporates all adaptive stress pathways that are relevant for toxicity, with a focus on drug-induced liver injury. In addition, cellular stress responses typically follow cell perturbations at the subcellular organelle level. Therefore, we complement our reporter line panel with reporters for specific organelle morphometry and function. Here, we review the approaches of high content imaging of cellular adaptive stress responses to chemicals and the application in the mechanistic understanding and prediction of chemical toxicity at a systems toxicology level.
Asunto(s)
Adaptación Biológica/efectos de los fármacos , Compuestos Orgánicos/toxicidad , Estrés Fisiológico/efectos de los fármacos , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales Bacterianos/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Genes Reporteros , Humanos , Compuestos Orgánicos/química , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
BACKGROUND: DAYSLEEPER is a domesticated transposase that is essential for development in Arabidopsis thaliana [Nature, 436:282-284, 2005]. It is derived from a hAT-superfamily transposon and contains many of the features found in the coding sequence of these elements [Nature, 436:282-284, 2005, Genetics, 158:949-957, 2001]. This work sheds light on the expression of this gene and localization of its product in protoplasts and in planta. Using deletion constructs, important domains in the protein were identified. RESULTS: DAYSLEEPER is predominantly expressed in meristems, developing flowers and siliques. The protein is mainly localized in the nucleus, but can also be seen in discrete foci in the cytoplasm. Using several vesicular markers, we found that these foci belong to vesicular structures of the trans-golgi network, multivesicular bodies (MVB's) and late endosomes. The central region as well as both the N- and the C-terminus are essential to DAYSLEEPER function, since versions of DAYSLEEPER deleted for these regions are not able to complement the daysleeper phenotype. Like hAT-transposases, we show that DAYSLEEPER has a functionally conserved dimerization domain [J Biol Chem, 282:7563-7575, 2007]. CONCLUSIONS: DAYSLEEPER has retained the global structure of hAT transposases and it seems that most of these conserved features are essential to DAYSLEEPER's cellular function. Although structurally similar, DAYSLEEPER seems to have broadened its range of action beyond the nucleus in comparison to transposases.