NuclEAtEd ERYthROcYtEs - A NEW EXPERIMENtAl CEll MOdEl
Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele VieţiiVol. 21, supp. 1, 2011, pp. 23-34 2011 Vasile Goldis University Press (www.studiauniversitatis.ro)NuclEAtEd ERYthROcYtEs - A NEW EXPERIMENtAl cEllMOdEl fOR AssEssINg IN VITRO tOXIcItY, EcOtOXIcItY ANdtO dEtERMINE thE sAfEtY Of fREsh fIsh PROducts.A REVIEWDaniela BRATOSIN1,2*, Alexandrina RUGINA1, Ana-Maria GHEORGHE1, Iulian STANA2,Violeta TURCUS2, Eugenia FAGADAR3, Aurel ARDELEAN21National Institute of Biological Science Research & Development (INCDSB), Bucharest, Romania2”Vasile Goldis” Western University of Arad, Faculty of Natural Sciences, Arad, Romania3Romanian Academy Chemistry Institute from Timisoara, RomaniaABSTRACT. The human activities have a negative impact to the environment, consisting in the water contaminationwith toxic products, heavy metals or with xenobiotic substances. Manufactured nanomaterials (nanoparticles,nanotubes, nanosheets and nanowires) have recent applications in drug delivery, medical devices, cosmetics,chemical catalysts, optoelectronics, electronics and magnetics. Some nanomaterials have been found to be toxicto humans and other organisms either upon contact or after persistent environmental exposure. In present, themeasurements of the pollution degree are made with two methods: phisyco-chemical methods and ecotoxicologicaltest (bioassay or environmental biosensors).Our results indicate that flow cytometric analysis of nucleated red blood cells viability using calceinAM and cell death discrimination could provide a rapid and accurate experimental cellular model for effectivelyscreening and evaluating biological responses for in vitro nanotoxicology and can be used in ecotoxicology asbioassays for the ecological monitoring of aquatic environment. In the some time, our results indicate that theuse of nucleated erythocytes could be potentially useful for the development of rapid and low cost safety tests toassess fisheries product quality.Key words: nucleated erythrocytes, toxicity, ecotoxicology, pollutants, nanomaterials, apoptosis, flow cytometryINTRODUCTIONEnvironmental pollution is one of the ever surgingproblems receiving careful attention in our country as wellas in the world. The human activities have a negative impactto the environment, consisting in the water contaminationwith toxic products, heavy metals or with xenobioticsubstances. The process of impurification of the surfaceand underground waters due to the human activities hashigh dimensions, like the permanent diversification ofthese toxic substances, was determined by the evolutionof the industrial processes. In recent years, some studieshave indicated that living organisms are affected fromelements present in the environment and the aquaticenvironment represents the largest sink for accumulationof xenobiotics. In the last decade, eutrophication causedby global industrialisation and anthropogenic impactson ecosystem can lead to biological damage. Heavymetal analysis demonstrated the presence of nickel, zinc,aluminium and manganese, as a clear demonstration ofwater quality deterioration. Copper, zinc and iron aretrace essential metals for different physiological functions(various enzymes and other cellular proteins), even throughtheir excess can lead to biological damage by excessiveintracellular accumulation. Increasing or decreasing levelsof these elements in living tissues cause important effectson metabolism.The revolution in nanotechnology brings advantagesin diverse areas of our lives such as engineering,information technology and medicine, etc. (Gross M.,1999; Kim D. et al., 2005; Akerman M.A. et al., 2002).Improvements in nanoscale materials synthesis andcharacterization have given scientists great control overthe fabrication of materials measuring between 1 and 100nm, unlocking many unique size-dependent propertiesand, thus, promising many new and/or improvedtechnologies (Oberdörster G. et al., 2005; ASTM E 245606, 2006). Recent years have found the integration of suchmaterials into commercial goods and a current estimatesuggests there are over 800 nanoparticle-containingconsumer products. The production of nanoparticles willincrease from 2300 tons produced today to 58000 tonsby 2020 (Maynard, 2006). Manufactured nanomaterials(nanoparticles, nanotubes, nanosheetsand nanowires)have recent applications in drug delivery, medicaldevices, cosmetics, chemical catalysts, optoelectronics,electronics and magnetics. Some nanomaterials havebeen found to be toxic to humans and other organismseither upon contact or after persistent environmentalexposure (Oberdörster G., 2004; Zhu S. et al., 2006;Griffitt R.J. et al., 2007; Usenko C.Y. et al., 2007).Despite this increase in the prevalence of engineerednanomaterials, little is known about their potential impact
Bratosin D., Rugina A., Gheorghe A.M, Stana I., Turcus V., Fagadar E., Ardelean A.on environmental health and safety (Moore M.N., 2006;Crosera M. et al., 2009). The field of nanotoxicologyhas formed in response to this lack of informations andresulted in a flurry of research studies. Nanotoxicologyis an emerging discipline (Oberdörster E. et al., 2005),a gap between the nanomaterials safety evaluation andthe nanotechnology development that produces newnanomaterials, new applications and new products.Nanotoxicology relies on many analytical methods forthe characterization of nanomaterials as well as on theirimpact on in vitro and in vivo functions (Lewinski N. etal., 2008, Hassellöv M. et al., 2008).Ecotoxicology has been established in the last twodecades as an environmental natural science, evolving onone hand from toxicology, and on the other hand fromapplied ecology or environmental chemistry.Ecotoxicology deals with the interactions betweenenvironmental chemicals and biota, thereby focusingon adverse effects at different levels of biologicalorganisation, from the molecular, cellular, tissue, organand organism level, up to populations and ecosystems.Ecotoxicological research on selected pollutantsrequires an interdisciplinary effort, consideringphysicochemical, molecular, toxicological, physiologicaland ecological processes. Only an integrated approachconsidering environmental chemical, toxicological andecological concepts may be suitable for understandingecotoxicological effects in contaminated ecosystemsEcotoxicological research is aimed at anunderstanding of toxicological phenomena in a variety ofbiota, populations and ecosystems, and diverse aspectssuch as mechanisms of toxic action and ecologicalprocesses in contaminated systems are considered.Another more prospective approach is based oninvestigating potential toxicological effects in laboratoryassays that may be used for extrapolation to the field.In present, the measurements of the pollution degreeare made with two methods: phisyco-chemical methodsand ecotoxicological test (bioassay or environmentalbiosensors). The main limits of the analytical methodsare the increased expenses of the equipments and thelack of the toxicological informations about bio risk.The biosensor, like a general definition, represents anysystem which detects the presence of the substratum,by utilization of the biological component which givesa signal, which can be quantified. Cellular biosensorsare systems which combine analytical devices and cellsto obtain biological signals like recognizing elements.Biosensors have two intriguing characteristics: (1) theyhave a naturally evolved selectivity to biological orbiologically active analytes; and (2) biosensors have thecapacity to respond to analytes in physiologically relevantmanner. Due their characteristics, these biosensors candetect the variations of the environment and can beused in the ecotoxicology tests and in monitoring of theenvironment, where pollution source and the nature ofthe toxic substances cannot be predicted. That’s why, the164majority cellular biosensors will be used in monitoring ofthe environmental toxicity.Bioassays play a role in this process; however,more comprehensive studies on contaminated systemsand ecological and toxicological processes are needed inaddition. Often bioassays do not consider the processesin the ecosystem, and neglect environmental factors thatinfluence toxicity. However, they are valuable tools inthe characterisation of the toxic action of chemicals, andin the understanding of associated toxicity.In ecotoxicological research, cellular effect studiesare as important as studies in laboratory species becausethe primary interaction between chemicals and biotaoccurs at the surface of or in cells. Whether chemicalinduced alterations in cell structure and physiologywill develop into an adverse toxic effect depends onmany parameters, including adaptive responses. Therelation between cellular toxicological responses totoxicity at higher biological levels is a key question inecotoxicology. Hence, cellular toxicology provides anessential concept in understanding ecotoxicologicalprocesses, since it plays a key role in elucidating toxicmodes of action, and diagnoses toxicological effects athigher biological levels.In the present is not known exactly the limits of thepollution for human security, of the major ecosystemsand of the ecosphere because it is not known the capacityof support of the ecosystems. The pollutions can be muchdiversified: chemical substances (organically substances,metals, oils, gases); physical factors (heat, noise,radiations, etc) or biological (pathogenic embryo) andthey can activate each other, sometimes the establishmentof limits concentrations approved is not efficient, evendangerous. In present is trying to find some molecularbiomarkers able to measure the risk brought by thewater pollution, to the pisciculture food, especially. Inecotoxicology the biomarkers is an obvious change or/and measurable on the molecular, biochemical, cellular,physiological or behavioral level, which shows theactual or the last exposition of an individual on a leastone chemical polluting substance. A biomarker measuredon the individual level, didn’t find the ecotoxicologicalsignification only it is describes, explains and predictsthe pollution effects on the populations and communitieswhich develops in their natural environment. They arethree kinds of biomarkers: biomarkers of expositionon the xenobiotic, shows that the polluting substancespresent in the environment penetrate the organism , theybeen the results of the interaction between pollutantswith biological molecules in the tissue and/ or the liquidsof the body; biomarkers of the exposure effect allowthe demonstration of the fact that a xenobiotic get to theorganism and has a toxic effect or notion a critical target(stress biomarkers of sensibility on the effect made byexposure receive the phenomen of variation of geneticorigin of the response to the contamination with pollutionsand it is translated by a variation of the sensibility (theStudia Universitatis “Vasile Goldiş”, Seria Ştiinţele VieţiiVol. 21, supp. 1, 2011, pp. 23-34 2011 Vasile Goldis University Press (www.studiauniversitatis.ro)
Nucleated Erythrocytes - A New Experimental Cell Model For AssessingIn Vitro Toxicity, Ecotoxicity And To Determine The Safety Of Fresh Fish Products.A Reviewincreasing of the glutation S transpherase quantity or thedecreasing of the sensibility acetilcholinsterase, whichcan be biomarkers).The human society, in present, is deal with problemswhich aim to the quality of life and the safety of thepeoples: environmental pollution and the food safety,which are in directly depends.In this way, the evaluation of the pollution ofnatural aquatic ecosystems and those from the fishfarms especially, and the estimating the risk degreewhich has on the food, are very important task. Inpresent, the researchers are trying new biological testsfor identification of new, sensitive biomarkers fordetermination of immediate and later effects of differentsubstances on the aquatic environment, in general andon the human health especially. Their identification canlead to the imagination of the cellular biosensors ableto monitoring the aquatic ecosystems, according to theactual laws.To assess aquatic pollution degree or for assessingcytotoxicity or ecotoxicity of nanoparticles, wedeveloped a new experimental cell system based on theuse of nucleated RBCs from fishes and batrachians whichare directly exposed to pollutants or to nanoparticlesabsorbed by different ways.Despite their structural simplicity, the erythrocytesof lower vertebrates preserve nucleus and mitochondria,both the sensors of the PCD machinery. As well as playinga central role in the physiology of respiration, these cellscan represented an outstanding model to study xenobioticinduced damage to different cellular compartments. Littleis known about the effect of environmental toxicantson apoptosis induction. The two modes of cell death(apoptosis and necrosis) differ fundamentally in theirmorphology, biochemistry and biological relevance. Weand others have recently shown that programmed celldeath (PCD) of nucleated erythrocytes is related to anapoptotic mechanism (Bratosin D. et al. 2004).In our study, to evaluate cell-nanomaterialsinteractions, nucleated RBCs were exposed to differentconcentrations of pollutants or nanocomposites andanalyzed by flow cytometry, after 24h incubationendpoints for morphological changes (FSC/SSC),apoptosis/necrosis analysis (FITC-annexin-V labeling/PI) and viability (calcein-AM method) or measurementof reactive oxigen species (ROS).The toxicological analysis were performedcomparatively on the porphyrin base or metalloporphyrinand for each porphyrin bare derivative on the correspondentporphyrin-silica-hybrid nanomaterials obtained by solgel synthesis in one step acid catalysis or by two stepsacid-base catalysis, using tetraethylorthosilicate (TEOS)as silica precursor (Bratosin D. et al., 2011) or , on heavymetal analysis (nickel, zinc, aluminium and manganese).Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele VieţiiVol. 21, supp. 1, 2011, pp. 23-34 2011 Vasile Goldis University Press (www.studiauniversitatis.ro)DETECTION OF ALTERED MORPHOLOGY BY LIGHTSCATTERING FLOW CYTOMETRY AND MICROSCOPY.Multiparametric flow cytometric analysis whichdiscriminates and quantifies viable, apoptotic and necroticcells via measurement of forward and side light scatter(proportional to cell diameter and internal granularity,respectively) is a very rapid and sensible method.As shown in Figure 1, flow cytometric analysisannounce significant morphological changes of nucleatedRBCs incubated for 24 h in saline supernatants ofdifferent nanomaterials (P1-P8) compared to nucleatedRBCs incubated only in saline isotonic solution (T24h).In fact, the XGeo Mean values (cell side scatter)vary from 168 (P6) to 268 for P3 as compared to thestatistical value of normal RBCs, i.e. 182 6. In the sameway, the YGeo Mean values (cell density scatter) varyfrom 190 for (P1) to 461 (P2) or 588 for P3 as comparedto the statistical value of normal RBCs, i.e. 237 17.Optical microscopy entirely confirmed these dataand showed that morphological changes of nucleatederythrocytes were associated with cell shrinkage(decreased forward scatter and increased side scatter),one of characteristic features of apoptosis. Images ofmicroscopic analyses of nucleated erythrocytes incubatedin supernatants obtained by preincubation of nanomaterialsin saline solutions show that highlights the morphologicalchanges are not uniform for all samples, neither theintensity nor that the manner of expression, showing thatthey accurately reflect the toxicity of different samples.Change of discoid morphology to rounded forms, brings tomind an apoptosis phenomenon. They are very numerousin samples P2 and P3, and when they are accompanied by atransparent appearance, providing that these cells are dead.Samples P4 and P7 induce an unexpected morphologicalaspect, comparable to a “bicycle wheel”. Very interesting,in the sample P1 and P5, the nanomaterials produce evenmore bizarre forms, a sort of “mega pores” or “holes.” Thesame phenomenon is also observed in P8 sample, but lessobvious. These morphological changes were confirmedby scanning electron microscopy (Fig 2).FLOW CYTOMETRIC MEASUREMENT OF ROSPRODUCTION.In order to test how aluminium concentrationsinfluences ROS generation, normal nucleatederythrocytes incubated at 20ºC for 24h (control normalsample) was compared with erythrocytes stimulated by2mM H2O2, as positive sample (Fig.1A). The calculatedaverage of the MFI values for erythrocytes exposedto various concentration of aluminium showed asignificantly higher ability of aluminium to generateROS as compared to the normal sample (Fig. 3). TheMFI for unstimulated normal RBC sample was 34compared to 48 for normal H2O2-stimulated sample. Theresults showed that the presence of aluminium caused anincrease in fluorescence, between MFI 58 to MFI 79,depending of aluminium concentrations.165
Bratosin D., Rugina A., Gheorghe A.M, Stana I., Turcus V., Fagadar E., Ardelean A.ABFig. 1. Comparative morphological shape changes analyses by flow cytometry (A) and optical microscopy (B) of normalnucleated erythrocytes (To and T24h) and exposed to nanomaterials (P1 to P8) at 0,008 g/ml. Dot-plot analysis FSC/SSC of cells shape changes. Abscissae: forward scatter (cell size); ordinates: side scatter (cell density, granularity andrefractiveness).Data are representative of three analysis giving similar results. Number of counted cells: 10,000. Resultspresented are from one representative experiment of three performed.Black arrows: erythrocytes with “mega pores” or “holes” White arrows: “bicycle wheel” erythrocyte shape.Results presented are from one representative experiment of three performed. Cells were visualized using an invertedmicroscope MCX 1600 for bright field (Micros Autrich)166Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele VieţiiVol. 21, supp. 1, 2011, pp. 23-34 2011 Vasile Goldis University Press (www.studiauniversitatis.ro)
Nucleated Erythrocytes - A New Experimental Cell Model For AssessingIn Vitro Toxicity, Ecotoxicity And To Determine The Safety Of Fresh Fish Products.A ReviewFig. 2. Scanning electron microscopic analysis of normal nucleated erythrocytes (a and b) of Rana sp., exposed to theaction of sample P1 (meso-tetra-tolyl-porphyrin) at 0.008 g/ ml (c-f) and to the sample P3 (Zn (II)-meso-tetra-piridilporphyrin) at 0.008 g / ml (g, h). the results presented are representative experiments.Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele VieţiiVol. 21, supp. 1, 2011, pp. 23-34 2011 Vasile Goldis University Press (www.studiauniversitatis.ro)167
Bratosin D., Rugina A., Gheorghe A.M, Stana I., Turcus V., Fagadar E., Ardelean A.Fig. 3. Comparative histogram of the reactive oxigen species (ROS) produced in red blood cells of Rana esculentaunder the action of aluminum measured by flow cytometry. 1: red blood cells incubated at 20 C for 24hours, 8: positivecontrol with red blood cells stimulated with2 mM H2O2, 1-7: fluorescence of histograms for erythrocytes incubatedwith increasing concentrations of aluminum. MFI: mean fluorescence intensity of 2’, 7’-dichlorofluorescein (DCF).INFLUENCE OF PORPHYRINS ON CELL VIABILITYMEASURED WITH CALCEIN-AM ASSAYWe recently devised a new flow cytometric assayfor the measurement of cells viability using calceinAM (Bratosin D. et al., 2005). The assay is based onthe use of acetoxymethyl ester of calcein (calceinAM), a fluorescein derivative and nonfluorescent vitaldye that passively crosses the cell membrane of viablecells and is converted by cytosolic esterases into greenfluorescent calcein which is retained by cells with intactmembranes. In this regard, it is important to mentionthat we have previously demonstrated that the loss ofesterase activity was an early event that occurred beforephosphatidylserine exposure (Bratosin D. et al., 2005).Application of this assay for analysing the effect ofnanoma
NuclEAtEd ERYthROcYtEs - A NEW EXPERIMENtAl cEll MOdEl fOR AssEssINg IN VITRO tOXIcItY, EcOtOXIcItY ANd tO dEtERMINE thE sAfEtY Of fREsh fIsh PROducts. A REVIEW Daniela BRATOSIN1,2*, Alexandrina RUGINA1, Ana-Maria GHEORGHE1, Iulian STANA2, Violeta TURCUS 2, Eugenia FAGADAR3, Aurel ARDELEAN 1
cell anaemia have been investigated with respect to their nature, catalytic activity and location. It has previously been suggested that membrane-associated iron catalyses processes leading to oxidative membrane damage in these erythrocytes. Additionally, the potential contribution of . 2.3 Quantification and Characterisation of Iron .
Keywords: Power analysis, minimum detectable effect size, multilevel experimental, quasi-experimental designs Experimental and quasi-experimental designs are widely applied to evaluate the effects of policy and programs. It is important that such studies be designed to have adequate statistical power to detect meaningful size impacts, if they .
Experimental and quasi - experimental desi gns for generalized causal inference: Wadsworth Cengage learning. Chapter 1 & 14 Campbell, D. T., & Stanley, J. C. (1966). Experimental and quasi -experimental designs for research. Boston: Hougton mifflin Company. Chapter 5. 3 & 4 Experimental Design Basics READINGS
experimental or quasi-experimental designs. The eval . Principles in Experimental Designs (New York, McGraw Hill, 1962). the details of experimental design, attentionisfocused on
Quasi experimental designs are similar to true experimental designs but in quasi experiments, the experimenter lacks the degree of control over the conditions that is possible in a true experiment Some research studies may necessitate the use of quasi designs rather than true experimental designs Faulty experimental design on the .
6. Stages in experimental design and scientific methodology 7.Development of experimental design disciplines and their implementation 4. Can provide examples of experimental designs in real cases. 5. Can explain several concepts of treatment design types, environment, and measurement (response). 6. Can explain the stages of experimental design and
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