Toxicological Risk Assessment of Emerging Nanomaterials: Cytotoxicity, Cellular Uptake, Effects on Biogenesis and Cell Organelle Activity, Acute Toxicity and Biodistribution of Oxide Nanoparticles

The lack of toxicological data on nanomaterials makes it difficult to assess the risk related to their exposure, and as a result further investigation is required. This chapter presents the synthesis of controlled oxide nanoparticles followed by the evaluation of their safety profile or toxicity (iron, titanium and zinc oxides). The controlled surface chemistry, dispersion in several media, morphology and surface charge of these nanoparticles are presented (transmission electron microscopy, dynamic light scattering, zeta potential, X-ray photoelectron spectroscopy). Classical cytotoxic and cellular uptake studies on different cancer cell lines from liver, prostate, heart, brain and spinal cord are discussed. The incidence of nanoparticles on biogenesis and activity of cell organelles is also highlighted, as well as their biodistribution in animal models. The acute toxicity on zebrafish embryo model is also presented. Finally, the stress is put on the influence and the necessity of controlling the protein corona, a layer of plasma proteins physically adsorbed at the surface of such nanoparticles as a result of their presence in the bloodstream (or relevant biological fluids)

Systems approach to the study of stretch and arrhythmias in right ventricular failure induced in rats by monocrotaline.

We demonstrate the synergistic benefits of using multiple technologies to investigate complex multi-scale biological responses. The combination of reductionist and integrative methodologies can reveal novel insights into mechanisms of action by tracking changes of in vivo phenomena to alterations in protein activity (or vice versa). We have applied this approach to electrical and mechanical remodelling in right ventricular failure caused by monocrotaline-induced pulmonary artery hypertension in rats. We show arrhythmogenic T-wave alternans in the ECG of conscious heart failure animals. Optical mapping of isolated hearts revealed discordant action potential duration (APD) alternans. Potential causes of the arrhythmic substrate; structural remodelling and/or steep APD restitution and dispersion were observed, with specific remodelling of the Right Ventricular Outflow Tract. At the myocyte level, [Ca(2+)]i transient alternans were observed together with decreased activity, gene and protein expression of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA). Computer simulations of the electrical and structural remodelling suggest both contribute to a less stable substrate. Echocardiography was used to estimate increased wall stress in failure, in vivo. Stretch of intact and skinned single myocytes revealed no effect on the Frank-Starling mechanism in failing myocytes. In isolated hearts acute stretch-induced arrhythmias occurred in all preparations. Significant shortening of the early APD was seen in control but not failing hearts. These observations may be linked to changes in the gene expression of candidate mechanosensitive ion channels (MSCs) TREK-1 and TRPC1/6. Computer simulations incorporating MSCs and changes in ion channels with failure, based on altered gene expression, largely reproduced experimental observations

Développement de modèles de cancer par xénotransplantation chez le poisson zèbre

L'utilisation de modèles animaux a permis la découverte de mécanismes importants du développement tumoral et la mise au point de nouveaux traitements. Le poisson zèbre (Danio rerio), est de plus en plus utilisé dans le cadre de ces recherches du fait de ses nombreux avantages comme la transparence de ses larves. Des approches par transplantation de cellules tumorales de mammifères se sont développées récemment chez ce téléostéen. Elles permettent d étudier le devenir des cellules injectées et leurs réponses aux traitements en tenant compte des interactions entre cellules cancéreuses et leur environnement. Dans ce contexte, nous avons mis au point des modèles de leucémies par transplantation de cellules de lignées humaines et validé la pertinence de ces modèles dans des études pharmacologiques. Une seconde approche, basée sur la transplantation de cellules leucémiques primaires de patients a permis une caractérisation des cellules susceptibles de coloniser les larves de poisson. Le monoxyde d azote (NO) semble jouer un rôle ambivalent dans le développement tumoral. Nous avons développé un nouveau modèle in vivo afin de mieux comprendre l origine et les conséquences de la production de NO dans l environnement tumoral. L utilisation de diaminofluorophores dans un modèle de xénotransplantation de gliomes de rat nous a permis de mettre en évidence une production de NO in situ dans l environnement tumoral. Enfin, la possibilité de suivre le développement de cellules cancéreuses in vivo d étudier des protéines potentiellement impliquées dans la progression tumorale. Nous avons caractérisé une famille de protéines susceptibles d intervenir dans ce processus : les nonaspanines.New cancer model development allowed new approach to understand important mechanisms involved in tumour progression and to develop new treatments. Due to its advantages, such as embryos transparency, zebrafish (Danio rerio) is more and more used in biological experimentation, and recently in cancer research. By transplanting mammalian tumour cells in zebrafish larvae, several models have been obtained. Such models allowed cancer cells development and response to several treatment , considering cancer cells and tumour interactions. We developed leukaemia models based on human cell lines transplantations. We also validated our models in pharmacological assays. Another model was obtain by injecting human leukaemia primary cells extract from patient blood and allowed us to define leukaemia cell fraction that can colonize zebrafish larvae. Nitric oxide (NO) involvement during tumour progression is ambivalent and still discussed. We developed a new in vivo model to analyse nitric oxide production and roles in tumour environment by using diaminofluorophores in a rat glioma xenograft model. By following tumour cell in vivo, we are able to study proteins that may be involved in tumour progression. We characterized a protein family called nonaspanins.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Developmental defects in zebrafish for classification of EGF pathway inhibitors.

AbstractOne of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway.In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems

Detection of nitric oxide by diaminofluorescein visualizes the skeleton in living zebrafish.

Several in vivo stainings, such as Calcein, Alizarin Red and Quercetin are commonly used to visualize ossification in living teleost specimen. These staining techniques represent important tools for bone research in fish, but do not visualize cartilage. In the present study, we show that nitric oxide (NO) labelling by DAF-FM DA visualizes both bone and cartilage in vivo during zebrafish skeletogenesis. NO detection performed in Tg(osterix:mCherry) or in combination with Alizarin Red in wild-type zebrafish reveals that intense staining through NO labelling colocalizes with the appearance of osteoblasts and characterizes ossified structures. Cartilage structures are clearly distinguished in the living larvae, although the labelling is less intensive when compared to ossified structures. This method is the first and easy to handle alternative to cartilage and bone double stainings on fixed samples. In contrast to most live skeletal stainings, which only stain the mineralized bone structures, this protocol in addition allows in vivo visualization of cartilage

Effects of some controversial food additives on zebrafish embryonic development

Background information: There are rising concerns about potential hazardous properties of food additives, forcing legislator to tighten management policy and requiring extensive, yet animal- minimized, testing strategies. The zebrafish embryo is an emerging model system for chemical testing with many advantages that made it amenable to high-throughput assays at the in vivo level. In this study, we applied a panel of tests to evaluate toxicity, particularly neurobehavioral effects, of seven substances including standard compounds and controversial food additives. Methods: Zebrafish wildtype and transgenic fluorescent embryos were exposed to different concentrations of four food additives: Sodium benzoate (SB), Monosodium glutamate (MSG), Tartrazine (TTZ), and Quinoline yellow (QY). Method validation was carried out using three other substances: Ethanol (EtOH), Dimethyl sulfoxide (DMSO), 3,4-Dichloroaniline (DCA). Morphological and lethal effects were recorded and the data were analysed to determine median lethal concentration (LC50), median effective concentration (EC50), effective concentration 10% (EC10), and teratogenic index (TI) values as well as concentration-response equations. Delayed effects of substances on larval locomotion were inspected using the light/dark challenge. Gene expression analysis was carried out using transgenic fluorescent lines. Results: LC50 values of three standard compounds (EtOH, DMSO, and DCA) reveal a high correlation with previously validated data, proving the reliability of our method. Effects of each substance on zebrafish embryonic morphology and lethality were determined as well as the corresponding concentration-response curves. Calculated toxicological indexes revealed that SB belongs to Cat.3 aquatic toxicity class, while QY is the most teratogenic substance. At EC10, all additives exhibited a delayed effect on zebrafish larval locomotion in compound-specific patterns. Observation of transgenic fluorescent embryos and locomotion analysis of hatched larvae reveal that SB could decrease the zebrafish motoneuron differentiation rate, while TTZ exhibited anti-angiogenic effects. Conclusion: Our results demonstrate that our test panel is reliable as a means to assess and categorise chemical toxicity. Also, our data suggest the need to reconsider the safety of food additives SB, TTZ, and QY as well as other controversial food additives in further studies

Data from: Phenotype classification of zebrafish embryos by supervised learning

Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100% agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification

Classification phénotypique d'embryons de poissons zèbres par apprentissage supervisé

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Monitoring cutting- tool wear using signals from the control system

Varnost in zanesljivost delovanja industrijskih obdelovalnih postopkov je pomemben pogoj za gospodarsko donosnost. Motnje v postopku, kakor so kolizija,preobremenitev, izpad in obraba orodja, niso popolnoma razumljive in povzročajo napake proizvodnega sistema. Da bi preprečili vpliv različnih motenj obdelovalnega postopka, npr. obrabo in lom orodja, posvečajo moderni tehnoloski sistemi posebno pozornost napovedovanju stanja rezalnega orodja. Številne teorije o spremljanju skušajo klasificirati in pojasniti obrabo orodja, vendar se nobena ni dala zadovoljivih rezultatov, ki bi hkrati zagotovila prilagodljivo in preprosto obvladovanje postopka za sprejemljivo ceno. Brezzančna struktura modernega ali digitalnega krmiljenja odpira nove možnosti in perspektive v tem pogledu. V mnogih primerih kombinacija signalov digitalne opreme in internih podatkov krmilnega sistema stroja, skupaj z izpolnjenimi metodami analize signalov, lahko nadomesti zunanje sisteme za spremljanje. Vgradnje programskega modula za nadzor postopka v krmilni sistem stroja omogoča hitre reakcije, če se pojavijo motnje postopka, in sicer brez dodatnega povečanja računalniške opreme.Ta prispevek preučuje občutljivost signalov v nadzornem sistemu na postopke obrabe rezalnega orodja pri čelnem struženju.The safety and reliability of operation of industrial manufacturing processes is a very important prerequisite for economic production. Process disturbancessuch as collision, overload, breakdown and tool wear are not yet fully understood, and cause production-system failures. In order to prevent the effects of excess wear or eventual tool breakdown, modern technological systems pay particular attention to predicting the condition of tool. Numeroustheories of monitoring have tried to classify and explain tool wear, but none have given completely satisfactory results as yet, while at the same time ensuring flexible, simple and cost-effective process control. The open structure of modern digital control opens up new possibilities: in many cases the combination of digital plant signals and the internal data of the machine control system, along with advanced methods of signal analysis, can replace external control systems. The integration of a process-control software moduleinto the machine control system allows fast reactions, should there be any process disturbances, without any additional hardware expansion. This paper studies the sensitivity of signals contained in the control system to the cutting-tool wear processes in face turning