RESUMO
In an intercropping system, the interplay between cereals and legumes, which is strongly driven by the complementarity of below-ground structures and their interactions with the soil microbiome, raises a fundamental query: Can different genotypes alter the configuration of the rhizosphere microbial communities? To address this issue, we conducted a field study, probing the effects of intercropping and diverse maize (Zea mays L.) and bean (Phaseolus vulgaris L., Phaseolus coccineus L.) genotype combinations. Through amplicon sequencing of bacterial 16S rRNA genes from rhizosphere samples, our results unveil that the intercropping condition alters the rhizosphere bacterial communities, but that the degree of this impact is substantially affected by specific genotype combinations. Overall, intercropping allows the recruitment of exclusive bacterial species and enhances community complexity. Nevertheless, combinations of maize and bean genotypes determine two distinct groups characterized by higher or lower bacterial community diversity and complexity, which are influenced by the specific bean line associated. Moreover, intercropped maize lines exhibit varying propensities in recruiting bacterial members with more responsive lines showing preferential interactions with specific microorganisms. Our study conclusively shows that genotype has an impact on the rhizosphere microbiome and that a careful selection of genotype combinations for both species involved is essential to achieve compatibility optimization in intercropping.
Assuntos
Agricultura , Fabaceae , Agricultura/métodos , Zea mays/genética , Raízes de Plantas , Rizosfera , RNA Ribossômico 16S/genética , Fabaceae/genética , Solo , Bactérias/genética , Genótipo , Microbiologia do SoloRESUMO
Transcriptomic analysis is a powerful method in the utilization of New Approach Methods (NAMs) for identifying mechanisms of toxicity and application to hazard characterization. With this regard, mapping toxicological events to time of exposure would be helpful to characterize early events. Here, we investigated time-dependent changes in gene expression levels in iPSC-derived renal proximal tubular-like cells (PTL) treated with five diverse compounds using TempO-Seq transcriptomics with the aims to evaluate the application of PTL for toxicity prediction and to report on temporal effects for the activation of cellular stress response pathways. PTL were treated with either 50 µM amiodarone, 10 µM sodium arsenate, 5 nM rotenone, or 300 nM tunicamycin over a temporal time course between 1 and 24 h. The TGFß-type I receptor kinase inhibitor GW788388 (1 µM) was used as a negative control. Pathway analysis revealed the induction of key stress-response pathways, including Nrf2 oxidative stress response, unfolding protein response, and metal stress response. Early response genes per pathway were identified much earlier than 24 h and included HMOX1, ATF3, DDIT3, and several MT1 isotypes. GW788388 did not induce any genes within the stress response pathways above, but showed deregulation of genes involved in TGFß inhibition, including downregulation of CYP24A1 and SERPINE1 and upregulation of WT1. This study highlights the application of iPSC-derived renal cells for prediction of cellular toxicity and sheds new light on the temporal and early effects of key genes that are involved in cellular stress response pathways.
Assuntos
Células-Tronco Pluripotentes Induzidas , Transcriptoma , Perfilação da Expressão Gênica , RimRESUMO
Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies.
Assuntos
Células Epiteliais , Rim , Humanos , Transporte de Elétrons/genética , Estudos Prospectivos , Rim/metabolismo , Linhagem Celular , Células Epiteliais/metabolismoRESUMO
Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Tricloroetileno , Humanos , Cisteína/toxicidade , Cisteína/metabolismo , Tricloroetileno/toxicidade , Tricloroetileno/metabolismo , Transcriptoma , Fator 2 Relacionado a NF-E2/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Glutationa/metabolismo , FenótipoRESUMO
Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.
Assuntos
Complexo I de Transporte de Elétrons , Mitocôndrias , Transporte de Elétrons , Células Hep G2 , Hepatócitos , HumanosRESUMO
Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Transporte de Elétrons/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Praguicidas/toxicidade , Biomarcadores , Linhagem Celular , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Proteostase/efeitos dos fármacos , Medição de Risco , TranscriptomaRESUMO
Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.
Assuntos
Agroquímicos/toxicidade , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Desacopladores/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/patologia , Consumo de Oxigênio/efeitos dos fármacosRESUMO
In the original publication of the article.
RESUMO
Temporal and spatial characterization of gene expression is a prerequisite for the understanding of cell-, tissue-, and organ-differentiation. In a multifaceted approach to investigate gene expression in the tail plate of the free-living marine flatworm Macrostomum lignano, we performed a posterior-region-specific in situ hybridization screen, RNA sequencing (RNA-seq) of regenerating animals, and functional analyses of selected tail-specific genes. The in situ screen revealed transcripts expressed in the antrum, cement glands, adhesive organs, prostate glands, rhabdite glands, and other tissues. Next we used RNA-seq to characterize temporal expression in the regenerating tail plate revealing a time restricted onset of both adhesive organs and copulatory apparatus regeneration. In addition, we identified three novel previously unannotated genes solely expressed in the regenerating stylet. RNA interference showed that these genes are required for the formation of not only the stylet but the whole male copulatory apparatus. RNAi treated animals lacked the stylet, vesicula granulorum, seminal vesicle, false seminal vesicle, and prostate glands, while the other tissues of the tail plate, such as adhesive organs regenerated normally. In summary, our findings provide a large resource of expression data during homeostasis and regeneration of the morphologically complex tail regeneration and pave the way for a better understanding of organogenesis in M. lignano.
Assuntos
Regulação da Expressão Gênica , Genes de Helmintos , Proteínas de Helminto/genética , Platelmintos/fisiologia , Regeneração/genética , Cauda/fisiologia , Animais , Proteínas de Helminto/biossíntese , Organismos Hermafroditas , Hibridização In Situ , Microvilosidades , Especificidade de Órgãos , Platelmintos/genética , Interferência de RNA , RNA de Helmintos/biossíntese , RNA de Helmintos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Regeneração/fisiologia , Transcriptoma , Cicatrização/genéticaRESUMO
Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose â galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.
Assuntos
Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/induzido quimicamente , Células-Tronco Neurais/efeitos dos fármacos , Síndromes Neurotóxicas/diagnóstico , Testes de Toxicidade/métodos , Metabolismo dos Carboidratos , Meios de Cultura , Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Galactose/metabolismo , Galactose/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Células-Tronco Neurais/ultraestrutura , Neuritos/efeitos dos fármacos , Desacopladores/toxicidadeRESUMO
The utilisation of genome-wide transcriptomics has played a pivotal role in advancing the field of toxicology, allowing the mapping of transcriptional signatures to chemical exposures. These activities have uncovered several transcriptionally regulated pathways that can be utilised for assessing the perturbation impact of a chemical and also the identification of toxic mode of action. However, current transcriptomic platforms are not very amenable to high-throughput workflows due to, high cost, complexities in sample preparation and relatively complex bioinformatic analysis. Thus, transcriptomic investigations are usually limited in dose and time dimensions and are, therefore, not optimal for implementation in risk assessment workflows. In this study, we investigated a new cost-effective, transcriptomic assay, TempO-Seq, which alleviates the aforementioned limitations. This technique was evaluated in a 6-compound screen, utilising differentiated kidney (RPTEC/TERT1) and liver (HepaRG) cells and compared to non-transcriptomic label-free sensitive endpoints of chemical-induced disturbances, namely phase contrast morphology, xCELLigence and glycolysis. Non-proliferating cell monolayers were exposed to six sub-lethal concentrations of each compound for 24 h. The results show that utilising a 2839 gene panel, it is possible to discriminate basal tissue-specific signatures, generate dose-response relationships and to discriminate compound-specific and cell type-specific responses. This study also reiterates previous findings that chemical-induced transcriptomic alterations occur prior to cytotoxicity and that transcriptomics provides in depth mechanistic information of the effects of chemicals on cellular transcriptional responses. TempO-Seq is a robust transcriptomic platform that is well suited for in vitro toxicity experiments.
Assuntos
Perfilação da Expressão Gênica/métodos , Rim/citologia , Fígado/citologia , Testes de Toxicidade/métodos , Transcriptoma/efeitos dos fármacos , Bromatos/toxicidade , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ciclosporina/toxicidade , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ocratoxinas/toxicidade , Ácido Valproico/toxicidadeRESUMO
Hazard assessment (HA) requires toxicity tests to allow deriving protective points of departure (PoDs) for risk assessment irrespective of a compound's mode of action (MoA). The scope of in vitro test batteries (ivTB) thereby necessitated for systemic toxicity is still unclear. We explored the protectiveness regarding systemic toxicity of an ivTB with a scope, which was guided by previous findings from rodent studies, where examining six main targets, including liver and kidney, was sufficient to predict the guideline scope-based PoD with high probability. The ivTB comprises human in vitro models representing liver, kidney, lung and the neuronal system covering transcriptome, mitochondrial dysfunction and neuronal outgrowth. Additionally, 32 CALUX®- and 10 HepG2 BAC-GFP reporters cover a broad range of disturbance mechanisms. Eight compounds were chosen for causing adverse effects such as immunotoxicity or anemia in vivo, i.e., effects not directly covered by assays in the ivTB. PoDs derived from the ivTB and from oral repeated dose studies in rodents were extrapolated to maximum unbound plasma concentrations for comparison. The ivTB-based PoDs were one to five orders of magnitude lower than in vivo PoDs for six of eight compounds, implying that they were protective. The extent of in vitro response varied across test compounds. Especially for hematotoxic substances, the ivTB showed either no response or only cytotoxicity. Assays better capturing this type of hazard would be needed to complement the ivTB. This study highlights the potentially broad applicability of ivTBs for deriving protective PoDs of compounds with unknown MoA.
Animal tests are used to determine which amount of a chemical is toxic ('threshold of toxicity') and which organs are affected. In principle, the threshold can also be derived solely from tests with cultured cells. However, only a limited number of cell types can practically be tested, so one challenge is to determine how many and which types shall be tested. In animal studies, only few organs including liver and kidney are regularly among those most sensitively affected. We explored whether a cell-based test battery representing these sensitive organs and covering important mechanisms of toxicity can be used to derive protective human thresholds. To challenge this approach, eight chemicals were tested that primarily cause effects in organs not directly represented in our test battery. Results provided protective thresholds for most of the investigated compounds and gave indications how to further improve the approach towards a full-fledged replacement for animal tests.
Assuntos
Testes de Toxicidade , Transcriptoma , Humanos , Medição de RiscoRESUMO
Adverse Outcome Pathways (AOPs) are increasingly used to support the integration of in vitro data in hazard assessment for chemicals. Quantitative AOPs (qAOPs) use mathematical models to describe the relationship between key events (KEs). In this paper, data obtained in three cell lines, LHUMES, HepG2 and RPTEC/TERT1, using similar experimental protocols, was used to calibrate a qAOP of mitochondrial toxicity for two chemicals, rotenone and deguelin. The objectives were to determine whether the same qAOP could be used for the three cell types, and to test chemical-independence by cross-validation with a dataset obtained on eight other chemicals in LHUMES cells. Repeating the calibration approach for both chemicals in three cell lines highlighted various practical difficulties. Even when the same readouts of KEs are measured, the mathematical functions used to describe the key event relationships may not be the same. Cross-validation in LHUMES cells was attempted by estimating chemical-specific potency at the molecular initiating events and using the rest of the calibrated qAOP to predict downstream KEs: toxicity of azoxystrobin, carboxine, mepronil and thifluzamide was underestimated. Selection of most relevant readouts and accurate characterization of the molecular initiating event for cross-validation are critical when designing in vitro experiments targeted at calibrating qAOPs.
Assuntos
Rotas de Resultados Adversos , Linhagem Celular , Modelos Teóricos , Medição de Risco , Testes de ToxicidadeRESUMO
The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Túbulos Renais Proximais/citologia , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Análise de Sequência de RNA/métodosRESUMO
Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides rotenoids and strobilurins each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts including transcriptomics in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.
Assuntos
Síndromes Neurotóxicas , Praguicidas , Animais , Simulação por Computador , Humanos , Síndromes Neurotóxicas/etiologia , Medição de Risco , IncertezaRESUMO
Human induced pluripotent stem cells (iPSC) have the potential to radically reduce the number of animals used in both toxicological science and disease elucidation. One initial obstacle culturing iPSC is that they require daily medium exchange. This study attempts to clarify why and propose some practical solutions. Two iPSC lineages were fed at different intervals in a full growth area (FGA) or a restricted growth area (RGA). The FGA consisted of a well coated with Matrigel™ and the RGA consisted of a coated coverslip placed in a well. Glucose, lactate, extracellular pH and cell cycle phases were quantified. Without daily feeding, FGA cultured iPSC had significantly reduced growth rates by day 2 and began to die by day 3. In contrast, RGA cultured cells grew to confluence over 3days. Surprisingly, glucose was not exhausted under any condition. However, extracellular pH reached 6.8 after 72h in FGA cultures. Artificially reducing medium pH to 6.8 also inhibited glycolysis and initiated an increase in G0/G1 phase of the cell cycle, while adding an additional 10mM bicarbonate to the medium increased glycolysis rates. This study demonstrates that iPSC are highly sensitive to extracellular acidification, a likely limiting factor in maintenance of proliferative and pluripotent status. Culturing iPSC in RGA prevents rapid extracellular acidification, while still maintaining pluripotency and allowing longer feeding cycles.
Assuntos
Técnicas de Cultura de Células , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas , Ácidos , Ciclo Celular , Morte Celular , Diferenciação Celular , Células Cultivadas , Colágeno , Combinação de Medicamentos , Corpos Embrioides , Glucose/metabolismo , Glicólise , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Laminina , ProteoglicanasRESUMO
The kidney is a major target for drug-induced injury, primarily due the fact that it transports a wide variety of chemical entities into and out of the tubular lumen. Here, we investigated the expression of the main xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1 at an mRNA and/or protein level. RPTEC/TERT1 cells expressed OCT2, OCT3, OCTN2, MATE1, MATE2, OAT1, OAT3 and OAT4. The functionality of the OCTs was demonstrated by directional transport of the fluorescent dye 4-Di-1-ASP. In addition, P-glycoprotein activity in RPTEC/TERT1 cells was verified by fluorescent dye retention in presence of various P-glycoprotein inhibitors. In comparison to proliferating cells, contact inhibited RPTEC/TERT1 cells expressed increased mRNA levels of several ABC transporter family members and were less sensitive to cyclosporine A. We conclude that differentiated RPTEC/TERT1 cells are well suited for utilisation in xenobiotic transport and pharmacokinetic studies.