RESUMO
Per- and poly-fluoroalkyl substances (PFAS) have a wide range of elimination half-lives (days to years) in humans, thought to be in part due to variation in proximal tubule reabsorption. While human biomonitoring studies provide important data for some PFAS, renal clearance (CLrenal) predictions for hundreds of PFAS in commerce requires experimental studies with in vitro models and physiologically-based in vitro-to-in vivo extrapolation (IVIVE). Options for studying renal proximal tubule pharmacokinetics include cultures of renal proximal tubule epithelial cells (RPTECs) and/or microphysiological systems. This study aimed to compare CLrenal predictions for PFAS using in vitro models of varying complexity (96-well plates, static 24-well Transwells and a fluidic microphysiological model, all using human telomerase reverse transcriptase-immortalized and OAT1-overexpressing RPTECs combined with in silico physiologically-based IVIVE. Three PFAS were tested: one with a long half-life (PFOS) and two with shorter half-lives (PFHxA and PFBS). PFAS were added either individually (5 µM) or as a mixture (2 µM of each substance) for 48 h. Bayesian methods were used to fit concentrations measured in media and cells to a three-compartmental model to obtain the in vitro permeability rates, which were then used as inputs for a physiologically-based IVIVE model to estimate in vivo CLrenal. Our predictions for human CLrenal of PFAS were highly concordant with available values from in vivo human studies. The relative values of CLrenal between slow- and faster-clearance PFAS were most highly concordant between predictions from 2D culture and corresponding in vivo values. However, the predictions from the more complex model (with or without flow) exhibited greater concordance with absolute CLrenal. Overall, we conclude that a combined in vitro-in silico workflow can predict absolute CLrenal values, and effectively distinguish between PFAS with slow and faster clearance, thereby allowing prioritization of PFAS with a greater potential for bioaccumulation in humans.
Assuntos
Simulação por Computador , Fluorocarbonos , Túbulos Renais Proximais , Modelos Biológicos , Humanos , Fluorocarbonos/farmacocinética , Túbulos Renais Proximais/metabolismo , Meia-Vida , Taxa de Depuração Metabólica , Fluxo de Trabalho , Eliminação Renal , Poluentes Ambientais/farmacocinética , Poluentes Ambientais/metabolismo , Células Epiteliais/metabolismoRESUMO
In next generation risk assessment (NGRA), the Dietary Comparator Ratio (DCR) can be used to assess the safety of chemical exposures to humans in a 3R compliant approach. The DCR compares the Exposure Activity Ratio (EAR) for exposure to a compound of interest (EARtest) to the EAR for an established safe exposure level to a comparator compound (EARcomparator), acting by the same mode of action. It can be concluded that the exposure to a test compound is safe at a corresponding DCR ≤ 1. In this study, genistein (GEN) was selected as a comparator compound by comparison of reported safe internal exposures to GEN to its BMCL05, as no effect level, the latter determined in the in vitro estrogenic MCF7/Bos proliferation, T47D ER-CALUX, and U2OS ERα-CALUX assay. The EARcomparator was defined using the BMCL05 and EC50 values from the 3 in vitro assays and subsequently used to calculate the DCRs for exposures to 14 test compounds, predicting the (absence of) estrogenicity. The predictions were evaluated by comparison to reported in vivo estrogenicity in humans for these exposures. The results obtained support in the DCR approach as an important animal-free new approach methodology (NAM) in NGRA and show how in vitro assays can be used to define DCR values.
Assuntos
Estrogênios , Receptores de Estrogênio , Humanos , Estrogênios/toxicidade , Linhagem Celular Tumoral , Genisteína/toxicidade , Medição de RiscoRESUMO
Protein phosphorylation is the most common type of post-translational modification where serine, threonine or tyrosine are reversibly bound to the phosphate group of ATP in a reaction catalyzed by protein kinases. Phosphorylation plays an important role in regulation of cell homeostasis, including but not limited to signal perception and transduction, gene expression and function of proteins. Protein phosphorylation happens on a fast time scale and represents an energy-efficient way for the cell to adapt to exposure to chemical stressors. To understand the cascade of cellular signaling induced by exposure to chemicals, we have exposed HepG2 cells to three chemicals with different modes of action, namely, caffeine, coumarin, and quercetin in a concentration and time response manner. Significantly upregulated and downregulated phosphosites were screened to analyze the activation/deactivation of signaling pathways by protein kinases. In total, 69, 44 and 12 signaling pathways were found enriched in caffeine, coumarin and quercetin treated cells, respectively, of which 9 pathways were co-enriched with 11 jointly responded kinases. Among identified co-responded kinases, CDK1, MAPK1 and MAPK3 play important roles in cell cycle and insulin signaling pathways. Quantitative phosphoproteomics can sensitively distinguish the effects of different chemicals on cells, allowing the assessment of chemical safety through changes in substrates and metabolic pathways at the cellular level, which is important for the development of non-animal approaches for chemical safety assessment.
Assuntos
Cafeína , Cumarínicos , Quercetina , Cafeína/farmacologia , Cumarínicos/farmacologia , Células Hep G2 , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Proteômica , Quercetina/farmacologiaRESUMO
Organ-on-chip (OoC) technology is full of engineering and biological challenges, but it has the potential to revolutionize the Next-Generation Risk Assessment of novel ingredients for consumer products and chemicals. A successful incorporation of OoC technology into the Next-Generation Risk Assessment toolbox depends on the robustness of the microfluidic devices and the organ tissue models used. Recent advances in standardized device manufacturing, organ tissue cultivation and growth protocols offer the ability to bridge the gaps towards the implementation of organ-on-chip technology. Next-Generation Risk Assessment is an exposure-led and hypothesis-driven tiered approach to risk assessment using detailed human exposure information and the application of appropriate new (non-animal) toxicological testing approaches. Organ-on-chip presents a promising in vitro approach by combining human cell culturing with dynamic microfluidics to improve physiological emulation. Here, we critically review commercial organ-on-chip devices, as well as recent tissue culture model studies of the skin, intestinal barrier and liver as the main metabolic organ to be used on-chip for Next-Generation Risk Assessment. Finally, microfluidically linked tissue combinations such as skin-liver and intestine-liver in organ-on-chip devices are reviewed as they form a relevant aspect for advancing toxicokinetic and toxicodynamic studies. We point to recent achievements and challenges to overcome, to advance non-animal, human-relevant safety studies.
Assuntos
Dispositivos Lab-On-A-Chip , Medição de Risco/métodos , Toxicologia/métodos , Alternativas aos Testes com Animais/métodos , Alternativas aos Testes com Animais/tendências , Humanos , Intestinos/metabolismo , Fígado/metabolismo , Medição de Risco/tendências , Pele/metabolismo , Técnicas de Cultura de Tecidos , Toxicologia/tendênciasRESUMO
Selection of appropriate fit-for-purpose in vitro and in silico models is critical for non-animal safety assessment of chemical-induced hepatoxicity. The present study evaluated the feasibility of integrating in vitro data from three-dimensionally (3D)-cultured HepaRG cells and physiologically based pharmacokinetic (PBPK) modeling to predict chemical-induced liver toxicity. A 3D organoid culture system was established using an ultralow attachment method. HepaRG cells cultured in a two-dimensional (2D) monolayer and under 3D conditions were exposed to acetaminophen (APAP) at concentrations of 0.16-20 mM. The results showed that the viability of both 3D- and 2D cultured cells was significantly decreased by APAP in a concentration-dependent manner. Furthermore, 3D cultures were more sensitive to APAP-induced mitochondrial damage than 2D cultures were, based on measurements of mitochondrial superoxide accumulation and mitochondrial membrane potential loss. PBPK simulations using nominal in vitro concentrations showed that the APAP concentration eliciting mitochondrial damage was closer to the predicted peak liver concentration in humans in 3D cultures than it was in 2D cultures. In summary, our results suggest that combining in vitro data from 3D HepaRG cultures and PBPK modeling provides a promising tool for assessment of liver injury.
Assuntos
Acetaminofen/farmacocinética , Analgésicos não Narcóticos/farmacocinética , Técnicas de Cultura de Células , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/efeitos dos fármacos , Modelos Biológicos , Células Cultivadas , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , HumanosRESUMO
The Threshold of Toxicological Concern (TTC) is an important risk assessment tool which establishes acceptable low-level exposure values to be applied to chemicals with limited toxicological data. One of the logical next steps in the continued evolution of TTC is to develop this concept further so that it is representative of internal exposures (TTC based on plasma concentration). An internal TTC (iTTC) would provide threshold values that could be utilized in exposure-based safety assessments. As part of a Cosmetics Europe (CosEu) research program, CosEu has initiated a project that is working towards the development of iTTCs that can be used for the human safety assessment. Knowing that the development of an iTTC is an ambitious and broad-spanning topic, CosEu organized a Working Group comprised a balance of multiple stakeholders (cosmetics and chemical industries, the EPA and JRC and academia) with relevant experience and expertise and workshop to critically evaluate the requirements to establish an iTTC. Outcomes from the workshop included an evaluation on the current state of the science for iTTC, the overall iTTC strategy, selection of chemical databases, capture and curation of chemical information, ADME and repeat dose data, expected challenges, as well as next steps and ongoing work.
Assuntos
Cosméticos/toxicidade , Animais , Cosméticos/efeitos adversos , Cosméticos/metabolismo , Europa (Continente) , Humanos , Medição de RiscoRESUMO
Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2-ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As2O3), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2-ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As2O3-challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2-ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As2O3-induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As2O3-induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2-ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents.
Assuntos
Antineoplásicos/toxicidade , Elementos de Resposta Antioxidante/efeitos dos fármacos , Leucemia Monocítica Aguda/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Células 3T3 , Animais , Antineoplásicos/uso terapêutico , Elementos de Resposta Antioxidante/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Etionamida/uso terapêutico , Etionamida/toxicidade , Células Hep G2 , Humanos , Isoniazida/uso terapêutico , Isoniazida/toxicidade , Leucemia Monocítica Aguda/tratamento farmacológico , Camundongos , Células U937RESUMO
The field of toxicity testing for non-pharmaceutical chemicals is in flux with multiple initiatives in North America and the EU to move away from animal testing to mode-of-action based in vitro assays. In this arena, there are still obstacles to overcome, such as developing appropriate cellular assays, creating pathway-based dose-response models and refining in vitro-in vivo extrapolation (IVIVE) tools. Overall, it is necessary to provide assurances that these new approaches are adequately protective of human and ecological health. Another major challenge for individual scientists and regulatory agencies is developing a cultural willingness to shed old biases developed around animal tests and become more comfortable with mode-of-action based assays in human cells. At present, most initiatives focus on developing in vitro alternatives and assessing how well these alternative methods reproduce past results related to predicting organism level toxicity in intact animals. The path forward requires looking beyond benchmarking against high dose animal studies. We need to develop targeted cellular assays, new cell biology-based extrapolation models for assessing regions of safety for chemical exposures in human populations, and mode-of-action-based approaches which are constructed on an understanding of human biology. Furthermore, it is essential that assay developers have the flexibility to 'validate' against the most appropriate mode-of-action data rather than against apical endpoints in high dose animal studies. This chapter demonstrates the principles of fit-for-purpose assay development using pathway-targeted case studies. The projects include p53-mdm2-mediated DNA-repair, estrogen receptor-mediated cell proliferation and PPARα receptor-mediated liver responses.
Assuntos
Testes de Toxicidade/métodos , Toxicologia , Alternativas aos Testes com Animais , Animais , Dano ao DNA , Ensaios de Triagem em Larga Escala , Humanos , Técnicas In Vitro , PPAR alfa/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The development and validation of scientific alternatives to animal testing is important not only from an ethical perspective (implementation of 3Rs), but also to improve safety assessment decision making with the use of mechanistic information of higher relevance to humans. To be effective in these efforts, it is however imperative that validation centres, industry, regulatory bodies, academia and other interested parties ensure a strong international cooperation, cross-sector collaboration and intense communication in the design, execution, and peer review of validation studies. Such an approach is critical to achieve harmonized and more transparent approaches to method validation, peer-review and recommendation, which will ultimately expedite the international acceptance of valid alternative methods or strategies by regulatory authorities and their implementation and use by stakeholders. It also allows achieving greater efficiency and effectiveness by avoiding duplication of effort and leveraging limited resources. In view of achieving these goals, the International Cooperation on Alternative Test Methods (ICATM) was established in 2009 by validation centres from Europe, USA, Canada and Japan. ICATM was later joined by Korea in 2011 and currently also counts with Brazil and China as observers. This chapter describes the existing differences across world regions and major efforts carried out for achieving consistent international cooperation and harmonization in the validation and adoption of alternative approaches to animal testing.
Assuntos
Alternativas aos Testes com Animais/métodos , Cooperação Internacional , Estudos de Validação como Assunto , Animais , Humanos , Toxicologia/métodosRESUMO
The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher's linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.
Assuntos
Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Transformação Celular Neoplásica , Espectroscopia de Infravermelho com Transformada de Fourier , Animais , Carcinógenos/classificação , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/patologia , Mesocricetus , Análise Multivariada , Análise de Componente PrincipalRESUMO
SUMMARY: IRootLab is a free and open-source MATLAB toolbox for vibrational biospectroscopy (VBS) data analysis. It offers an object-oriented programming class library, graphical user interfaces (GUIs) and automatic MATLAB code generation. The class library contains a large number of methods, concepts and visualizations for VBS data analysis, some of which are introduced in the toolbox. The GUIs provide an interface to the class library, including a module to merge several spectral files into a dataset. Automatic code allows developers to quickly write VBS data analysis scripts and is a unique resource among tools for VBS. Documentation includes a manual, tutorials, Doxygen-generated reference and a demonstration showcase. IRootLab can handle some of the most popular file formats used in VBS. License: GNU-LGPL. AVAILABILITY: Official website: http://irootlab.googlecode.com/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Software , Espectrofotometria Infravermelho/métodos , Análise Espectral Raman/métodos , Interface Usuário-ComputadorRESUMO
Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.
Assuntos
Técnicas de Cocultura , Hepatócitos , Ensaios de Triagem em Larga Escala , Fígado , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Testes de Toxicidade/métodos , Linhagem Celular , Biomarcadores/metabolismo , Xenobióticos/toxicidadeRESUMO
Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.
Assuntos
Túbulos Renais Proximais , Sistemas Microfisiológicos , Humanos , Reprodutibilidade dos Testes , Estudos Prospectivos , RimRESUMO
Omic-based technologies are of particular interest and importance for hazard identification and health risk characterization of chemicals. Their application in the new approach methodologies (NAMs) anchored on cellular toxicity pathways is based on the premise that any apical health endpoint change must be underpinned by some alterations at the omic levels. In the present study we examined the cellular responses to two chemicals, caffeine and coumarin, by generating and integrating multi-omic data from multi-dose and multi-time point transcriptomic, proteomic and phosphoproteomic experiments. We showed that the methodology presented here was able to capture the complete chain of events from the first chemical-induced changes at the phosphoproteome level, to changes in gene expression, and lastly to changes in protein abundance, each with vastly different points of departure (PODs). In HepG2 cells we found that the metabolism of lipids and general cellular stress response to be the dominant biological processes in response to caffeine and coumarin exposure, respectively. The phosphoproteomic changes were detected early in time, at very low doses and provided a fast, adaptive cellular response to chemical exposure with 7-37-fold lower points of departure comparing to the transcriptomics. Changes in protein abundance were found much less frequently than transcriptomic changes. While challenges remain, our study provides strong and novel evidence supporting the notion that these three omic technologies can be used in an integrated manner to facilitate a more complete understanding of pathway perturbations and POD determinations for risk assessment of chemical exposures.
Assuntos
Segurança Química , Proteômica , Transcriptoma , Cafeína/toxicidade , Perfilação da Expressão Gênica/métodos , Medição de RiscoRESUMO
Next-generation risk assessment (NGRA) for environmental chemicals involves a weight of evidence (WoE) framework integrating a suite of new approach methodologies (NAMs) based on points of departure (PoD) obtained from in vitro assays. Among existing NAMs, the omic-based technologies are of particular importance based on the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omics level, such as transcriptome, proteome, metabolome, epigenome and genome. Transcriptomic assay plays a leading role in providing relatively conservative PoDs compared with apical endpoints. However, it is unclear whether and how parameters measured with other omics techniques predict the cellular response to chemical perturbations, especially at exposure levels below the transcriptomically defined PoD. Multi-omics coverage may provide additional sensitive or confirmative biomarkers to complement and reduce the uncertainty in safety decisions made using targeted and transcriptomics assays. In the present study, we conducted multi-omics studies of transcriptomics, proteomics and phosphoproteomics on two prototype compounds, coumarin and 2,4-dichlorophenoxyacetic acid (2,4-D), with multiple chemical concentrations and time points, to understand the sensitivity of the three omics techniques in response to chemically-induced changes in HepG2. We demonstrated that, phosphoproteomics alterations occur not only earlier in time, but also more sensitive to lower concentrations than proteomics and transcriptomics when the HepG2 cells were exposed to various chemical treatments. The phosphoproteomics changes appear to approach maximum when the transcriptomics alterations begin to initiate. Therefore, it is proximal to the very early effects induced by chemical exposure. We concluded that phosphoproteomics can be utilized to provide a more complete coverage of chemical-induced cellular alteration and supplement transcriptomics-based health safety decision making.
Assuntos
Socorristas , Proteômica , Humanos , Proteômica/métodos , Transcriptoma , Proteoma , Perfilação da Expressão GênicaRESUMO
Using morphological transformation as an endpoint, the Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) is an in vitro system with a high sensitivity and specificity for testing the carcinogenic potential of test agents. Advantages of the assay are that SHE cells are metabolically competent, genetically stable and acquire spontaneous transformation with a low frequency; additionally, it detects both genotoxic and non-genotoxic carcinogens. However, in comparison with other short-term mammalian cell assays, it is time consuming, laborious and, most importantly, the visual scoring of morphological transformation might be subjective. In this review, we examine the background to the test and why it has the potential for use in safety risk assessment. Additionally, we propose a novel approach to objectively interrogate and classify SHE colonies using vibrational spectroscopy coupled to a mathematical framework for high-throughput screening. It is our view that this alternative approach has the potential to improve the sensitivity and specificity of the in vitro SHE assay.
Assuntos
Carcinógenos/farmacologia , Transformação Celular Neoplásica , Embrião de Mamíferos/citologia , Animais , Proliferação de Células , Forma Celular , Células Cultivadas/efeitos dos fármacos , Cricetinae , Mesocricetus , Transplante de Neoplasias , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has utility in the assessment of potential chemical carcinogenicity (both genotoxic and non-genotoxic mechanisms of action). The assay uses morphological transformation as an end point and has a reported sensitivity of 87%, specificity of 83% and overall concordance of 85% with in vivo rodent bioassay data. However, the scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with benzo[a]pyrene, 3-methylcholanthrene, anthracene, N-nitroso-N-methylnitroguanidine, ortho-toluidine HCl, 2,4-diaminotoluene or D-mannitol for 7 days before fixation with methanol. Identified colonies were interrogated by acquiring a minimum of five infrared (IR) spectra per colony using attenuated total reflection Fourier-transform IR spectroscopy. Individual IR spectra were acquired over a spatial area of approximately 250 × 250 µm. Resultant data were analysed using Fisher's linear discriminant analysis and feature histogram algorithms to extract classifying biomarkers of test agent-specific effects or transformation in SHE cells. Clustering of spectral points suggested co-segregation or discrimination of test agent categories based on mechanism of action. Towards transformation, unifying alterations were associated with alterations in the Amide I and Amide II peaks; these were consistently major classifying biomarkers for transformed versus non-transformed SHE cells. Our approach highlights a novel method towards objectively screening and classifying SHE cells, be it to ascertain test agent treatment based on mechanism of action or transformation.
Assuntos
Carcinógenos/classificação , Transformação Celular Neoplásica , Mutagênicos/classificação , Animais , Biomarcadores/metabolismo , Carcinógenos/toxicidade , Células Cultivadas , Cricetinae , Interpretação Estatística de Dados , Embrião de Mamíferos/citologia , Concentração de Íons de Hidrogênio , Modelos Lineares , Mesocricetus , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.
Assuntos
Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Animais , Biomarcadores/análise , Linhagem Celular , Transformação Celular Neoplásica , Congressos como Assunto , Cosméticos/toxicidade , Humanos , Estudos de Validação como AssuntoRESUMO
Applying Fourier-transform infrared (FTIR) spectroscopy (or related technologies such as Raman spectroscopy) to biological questions (defined as biospectroscopy) is relatively novel. Potential fields of application include cytological, histological and microbial studies. This potentially provides a rapid and non-destructive approach to clinical diagnosis. Its increase in application is primarily a consequence of developing instrumentation along with computational techniques. In the coming decades, biospectroscopy is likely to become a common tool in the screening or diagnostic laboratory, or even in the general practitioner's clinic. Despite many advances in the biological application of FTIR spectroscopy, there remain challenges in sample preparation, instrumentation and data handling. We focus on the latter, where we identify in the reviewed literature, the existence of four main study goals: Pattern Finding; Biomarker Identification; Imaging; and, Diagnosis. These can be grouped into two frameworks: Exploratory; and, Diagnostic. Existing techniques in Quality Control, Pre-processing, Feature Extraction, Clustering, and Classification are critically reviewed. An aspect that is often visited is that of method choice. Based on the state-of-art, we claim that in the near future research should be focused on the challenges of dataset standardization; building information systems; development and validation of data analysis tools; and, technology transfer. A diagnostic case study using a real-world dataset is presented as an illustration. Many of the methods presented in this review are Machine Learning and Statistical techniques that are extendable to other forms of computer-based biomedical analysis, including mass spectrometry and magnetic resonance.
Assuntos
Biologia/métodos , Armazenamento e Recuperação da Informação/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Estatística como Assunto/métodos , Bases de Dados Factuais , Humanos , Terminologia como AssuntoRESUMO
The bacterial reverse mutation assay (Ames test) is a biological assay used to assess the mutagenic potential of chemical compounds. In this paper approaches for the development of an in silico mutagenicity screening tool are described. Three individual in silico models, which cover both structure activity relationship methods (SARs) and quantitative structure activity relationship methods (QSARs), were built using three different modelling techniques: (1) an in-house alert model: which uses SAR approach where alerts are generated based on experts judgements; (2) a kNN approach (k-Nearest Neighbours), which is a QSAR model where a prediction is given based on outcomes of its k chemical neighbours; (3) a naive Bayesian model (NB), which is another QSAR model, where a prediction is derived using a Bayesian formula through preselected identified informative chemical features (e.g., physico-chemical, structural descriptors). These in silico models, were compared against two well-known alert models (DEREK and ToxTree) and also against three different consensus approaches (Categorical Bayesian Integration Approach (CBI), Partial Least Squares Discriminate Analysis (PLS-DA) and simple majority vote approach). By applying these integration methods on the validation sets it was shown that both integration models (PLS-DA and CBI) achieved better performance than any of the individual models or consensus obtained by simple majority rule. In conclusion, the recommendation of this paper is that when obtaining consensus predictions for Ames mutagenicity, approaches like PLS-DA or CBI should be the first choice for the integration as compared to a simple majority vote approach.