Dissertationen zum Thema „Toxicity testing In vitro“

This study describes the development of a novel method of three dimensional perfused cell culture for in vitro toxicity testing. Multiple parallel perfused microbioreactors (TissueFlex^TM) were adopted to provide a well-controlled cell culture environment. Alginate and collagen type I, commonly used as hydrogel scaffolds to support cell culture, were tested as the scaffolding materials for this application. Alginate supports cell proliferation, but does not support cell attachment. Collagen gel (type I), good for cell attachment but with poor mechanical strength, could be used at the high concentration of 5mg/ml to prevent the degradation of the gel. Improvement of collagen biomechanical property by a purpose-designed compressor to physically induce cross-linking showed promising results and merits further study. The suitability of alamarBlue® assay, a common non-toxic non-destructive viability assay method, was confirmed for this study and the protocol was optimised. To demonstrate the effectiveness of three dimensional perfused cell culture, human mesenchymal stem cells (MSC) seeded in collagen type I were employed to test the cell inhibition of two antibiotics, trimethoprim and pyrimethamine. The results displayed the perfusion system has greater advantage and sensitivity than the static system, as does these of 3D scaffolds, compared with 2D. Such differences are related to the continuous supply of fresh culture medium to keep cells at a stable pH, temperature, oxygen, and a more physiological like environment. The cytotoxicity of two stereoisomer compounds, obtained confidentially from Pfizer. Ltd., was assessed using the developed method and compared to conventional 2D static and perfused culture by using rat adipose mesenchymal stem cells. The results successfully distinguished toxic and non-toxic compounds and also demonstrated that the 3D perfused system improved the prediction of drug toxicity over 2D culture. 3D perfused bioreactors were applied to hepatotoxicity study using freshly isolated rat hepatocytes. Only algimatrix^TM supported hepatocyte spheroid formation among those tested including collagen type I, alginate beads, poly lactic acid fibres, and Algimatrix^TM. A new variation of TissueFlex^TM bioreactor with micro-patterned surface, designed specifically for hepatocyte self-assembly culture without use of any scaffold, was tested. The results demonstrated that, compared with the standard sandwich culture, the self-assembly culture in the micro-patterned bioreactors showed high cell viability, biomarkers expression, as well as more physiological immunocytochemistry. Moreover, the differential gene expression indicated that self-assembly culture could provide more relevant information regarding metabolising processes than the 2D sandwich culture, which would potentially improve hepatotoxicity prediction. In conclusion, 3D perfused cell culture for in vitro toxicity testing improved the predictivity, reliability and physiological relevance of drug toxicity compared to traditional 2D culture.

A large range of polymers are used in building and mass transport interiors which released more toxic products during combustion. This work explores the cytotoxicity of selected chemicals and smoke derived from materials combustion. A selection of polymers and fiberglass reinforced polymer (FRP) composites used in building and railway carriage interiors including: polyethylene (PE), polypropylene (PP), polycarbonate (PC), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), melamine plywood, and two FRPs were studied. A small scale laboratory fire test using a vertical tube furnace was designed for the generation of combustion products. The volatile organic compounds were identified using ATD-GCMS (Automatic Thermal Desorption-Gas Chromatography Mass Spectrometry). The in vitro techniques were developed for human cells exposure to fire effluents including the indirect (impinger) and direct (air/liquid interface using Harvard Navicyte Chamber) exposure. Cytotoxic effects were assessed based on cell viability using a range of in vitro assays. Human skin tissue was also used as preliminary study to assess the toxic effects at the tissue level. A minor change in the cellular function of the skin from the exposure of PMMA combustion products was observed. The combustion study was conducted under different burning stage of fire: non-flaming and flaming combustion. Results suggested that PVC was the most toxic material for both non-flaming (IC50 1.24 mg/L) and flaming combustion (IC50 1.99 mg/L). The degree of toxicity generated depends on the fire stage: non-flaming or flaming combustion. Some materials revealed to be more toxic under flaming combustion (PP, PC, FRPs), whilst others (PVC, PMMA, PE, and melamine plywood) appear to be more toxic under non-flaming combustion. A strong correlation was shown between the change in toxicity as measured by IC50 and TLC and the change in concentration of volatile organic compounds (VOCs) and particulates. A comparison between in vitro data versus published in vivo combustion data indicated the in vitro results to be more sensitive than animal toxicity data. The outcome of this study has the potential for an alternative method to current fire toxicity standard, whilst providing more accurate toxicity information for fire safety professionals, materials manufacturer, building designers and consumer safety data.

Exposure to air contaminants is significantly associated with both short-term and long-term health effects. However, the precise mechanisms that derive such effects are not always understood. While an extensive background database from in vivo toxicological studies have been developed, most toxicity data is from oral and dermal chemical exposures rather than inhalation exposure. There is a need to explore new alternative approaches to provide toxicity information particularly on this technically demanding area. This research explores the potential of in vitro methods for toxicity assessment of workplace air contaminants. A tiered approach for in vitro toxicity testing of workplace contaminants was designed in which appropriate air sampling and exposure techniques were developed. A diversified battery of in vitro assays including the MTS (tetrazolium salt, Promega), NRU (neutral red uptake, Sigma) and ATP (adenosine triphosphate, Promega) and a multiple human cell system including: A549- lung derived cells; HepG2-liver derived cells, and skin fibroblasts were used. Primarily the application and merits of in vitro methods for prediction of toxicity of selected workplace contaminants including Ammonium hydroxide, Cadmium chloride, Cobalt chloride, Formaldehyde, Glutaraldehyde, Manganese chloride, Mercuric chloride, Sodium dichromate, Sulphureous acid and Zinc chloride was confirmed. To study the toxicity of airborne contaminants an indirect exposure method was established using air sampling techniques followed by static and dynamic direct exposure methods by culturing cells on porous membranes to reveal representative data relating to human airborne exposures. The static method enabled the measurement of an airborne IC50 (50% inhibitory concentration) value for selected volatile organic compounds (VOCs) including: Xylene (IC50 = 5,350-8,200 ppm) and Toluene (IC50 = 10,500- 16,600 ppm) after 1 hr exposure. By implementing the dynamic method, airborne IC50 values were calculated for gaseous contaminants including: NO2 (IC50 = 11 ?? 3.54 ppm; NRU), SO2 (IC50 = 48 ?? 2.83 ppm; ATP) and NH3 (IC50 = 199 ?? 1.41 ppm; MTS). A higher sensitivity of in vitro methods was observed compared to in vivo published data. A range of in vitro bioassays in conjunction with exposure techniques developed in this thesis may provide an advanced technology for a comprehensive risk assessment of workplace air contaminants.

L'objectif de cette thèse est d'améliorer la compréhension de la toxicité de diverses nanoparticules de synthèse (ENPs) pour l'homme et l'écosystème. Les travaux réalisés s’appuient sur la combinaison de données toxicologiques et d’un modèle environnemental - le modèle USEtox. En tant qu'élément important de l'évaluation de l'impact du cycle de vie, le facteur de caractérisation (CF) a été utilisé, dans ce travail, comme indicateur de toxicité pour l'homme et l'écosystème. Pour avoir accès aux courbes dose-réponse et à différentes données toxicologiques, des expériences in vitro ont été réalisées en exposant des neutrophiles porcins fraîchement isolés à trois types de nanoparticules de synthèse. Les modifications morphologiques, les taux de mortalité et la chimioluminescence des neutrophiles ont été évaluées. De plus, pour estimer le temps de persistance des nanoparticules de synthèse dans l'écosystème eau douce, un modèle basé sur la science des colloïdes a été développé. Il prend en compte les comportements spécifiques des nanoparticules de synthèse et inclut des recommandations sur le choix des paramètres hydrologiques régionaux. Enfin, une enquête documentaire exhaustive a été réalisée pour recueillir les données écotoxicologiques de diverses nanoparticules de synthèse. Dans le cadre du modèle USEtox, le CF toxicologique non cancérogène pour cuivre NPs et les CF écotoxicologiques pour 14 ENPs sont recommandés. Ces valeurs des CF pourraient être utiles à l'avenir pour évaluer les impacts environnementaux des produits contenant des ENPs
The objective of this thesis is to improve understandings of toxicity of various engineered nanoparticles (ENPs) to human and ecosystem. It is realized via coordinating toxicological data and a scientific consensus environmental model -- the USEtox model. As an important element in life cycle impact assessment, the characterization factor (CF) is employed as a toxicity indicator for human and ecosystem in this work. To obtain the firsthand dose-response phenomena and human toxicological data, in vitro experiments have been conducted by exposing freshly isolated porcine neutrophils to three kinds of ENPs (i.e. copper, nickel and aluminum oxide nanoparticles). The morphologies, mortality rates, and chemiluminescence, of neutrophils are observed or monitored. Additionally, to estimate the persistence time of ENPs in freshwater ecosystem, a fate model on the basis of colloid science is developed. It takes nano-specific behaviors of ENPs into account and includes recommendations of regionalized hydrological parameters. Finally, a comprehensive literature survey is accomplished to collect the ecotoxicological data of various ENPs. Under the framework of USEtox model, the non-carcinogenic human toxicological CFs for Copper NPs and the ecotoxicological CFs for 14 ENPs are recommended. These CF values could be useful in the future when evaluating the environmental impacts of products containing ENPs

Botulism is a foodborne intoxication caused by ingestion of Clostridium botulinum neurotoxin (BoNT). Preliminary studies focussed on the production of polyclonal antisera against BoNT/E by immunizing a rabbit with botulinal toxoid type E. The antiserum was subsequently used to detect BoNT/E using the slot blot immunoassay where samples were applied to a slot blot filtration manifold and drawn by vacuum through a membrane. The membrane was then immunologically processed before chemiluminescent detection. However, the antisera lacked specificity and cross-reacted with closely related clostridia strains.
The specificity of the antisera was increased by adsorbing cross-reactive antibodies from whole antisera with affinity columns made with total proteins from culture supernatants of closely related clostridia. Alternatively, specific antibodies were isolated with an affinity column prepared with C. botulinum type E toxoid.
Different methods of concentrating BoNT/E in each sample prior to testing them were evaluated to increase the sensitivity of the assay.
The slot blot immunoassay was then evaluated for detection of BoNT/E in mixed cultures and in food samples. (Abstract shortened by UMI.)

This project developed methodology for in vitro toxicity assessment of contaminated sites using the Promega?? MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay performed on human cells (HepG2 and Skin fibroblasts). The project included the development of a method for extracting contaminants from soil based on leaching and centrifugation. A number of solvents and surfactants were assessed for their suitability as extracting agents. The Zwitterionic surfactant CHAPS ({3[(3-Cholamidopropyl) dimethylammonio] propanesulphonic acid}), which is an irritant in vivo, was found suitable for in vitro toxicity assessment applications. CHAPS was found to be the least toxic surfactant in vitro when tested on skin fibroblasts (NOEC: 1800??577 ppm, IC50: 4000??577 ppm) and HepG2 cells (NOEC: 833??289 ppm, IC50: 5300??287 ppm). The chosen surfactant was used in three different methods for extraction of Toluene and Xylene spiked in 2 g and 10g soil. The combination comprising of 0.1% (s/w) CHAPS and cosolvent 1% (w/w) Isopropanol, at their respective NOEC (No Observed Effective Concentration) toxicity values, showed good recovery of the nonpolar organic compounds in comparison to the recovery by 0.1% CHAPS and 0.5% CHAPS. The study found additive interactions to be the most common form of toxicity for 16 concentration combinations of Formaldehyde (polar), Toluene and Xylene (nonpolar) when compared to predicted toxicity (R2=0.943, P<0.0001). When assessing the in vitro toxicity of unknown (blind) contaminated soil samples, the Hazard Index (HI) predicted from the chemical analyses results showed a relatively good correlation (R2>0.7062, n=26) when compared to the experimental toxicity results on HepG2 cells. Furthermore, the comparison of Australian Health Investigation Levels (HIL) with in vitro toxicity testing gave similar correlation (R2>0.6882, n=26) on HepG2 cells. The overall project suggests the potential application of the zwitterionic surfactant (CHAPS) in sampling contaminants from soils in an in vitro toxicity assessment. This study demonstrates the application of in vitro toxicity assessment using human cells for the prediction of toxic risk as a sentinel to human toxicity from a contaminated site.

Orientadores: Nelci Fenalti Hoerhr, Roberto Rittner Neto
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-06T21:30:04Z (GMT). No. of bitstreams: 1 Sega_EstelaMunhoz_M.pdf: 1403353 bytes, checksum: 0aec5b888298032d359cc1b3cf0905b6 (MD5) Previous issue date: 2006
Resumo: Esse estudo analisou as propriedades toxicológicas de novos compostos organofosforados. Foram realizados experimentos para avaliar a atividade anticolinesterásica desses organofosforados, in vitro, no sangue total através do método de Ellman modificado. Para determinar a sua citotoxicidade foram utilizadas células PC 12, com as quais avaliamos a viabilidade celular após contato com os organofosforados e determinamos a IC 50, encontrando valores muito diferentes para os diversos organofosforados estudados. Estudos de toxicidade aguda in vivo foram realizados com camundongos, através da metodologia recomendada pela OECD nos quais determinamos a DL50 para três dos organofosforados estudados, sendo que um apresentou toxidade moderada. Foram analisados os efeitos dos solventes nas constantes de acoplamento JHH, JHH, JNC e 2Jnc em espectros de RMN de LH e 13C do cloreto de acetilcolina. Os valores das constantes de acoplamento em solventes de diferentes constantes dielétricas (s) não sofreram variações, indicando uma ausência de efeitos de solvente no equilíbrio conformacional do cloreto de acetilcolina (ACh). As constantes de acoplamento mostram que o sistema OCH2CH2N+ tem uma conformação gaúche (sinclinal). O Jnh e Jkc são observados na maioria dos solventes, mas não em solventes clorados e não são dependentes da viscosidade do solvente, esse comportamento foi explicado usando dados de medidas de Ti. Os valores dos coeficientes de difusão de RMN mostraram que a ACh tem uma grande tendência de se agregar quando dissolvida em solventes clorados, fato que pode explicar as diferenças observadas em valores de T1 para o 14N
Abstract: This study analyzed the properties of the news organophosphorus. Experiments had been carried through to evaluate the inhibition of acetylcholinesterase of these organophosphorus, in vitro, through the modified EUman's method. In order to determine its cytotoxicity cells PC 12 had been used, with which we evaluate the cellular viability after contact with the organophosphorus and determined the IC50, different values were found for the diverse organophosphorus. Studies of acute toxicity had been carried through with mice, following the methodology recommended by the OECD in which determine the DL50 for three of the organophosphorus studied, being that one presented moderate toxicity. Coupling constants values ( Jhh and Jnc) obtained from the 'H and 13 C NMR spectra of acetylcholine chloride (ACh) in several solvents with a wide range of dielectric constants (e) are remarkably invariant, indicating an absence of solvent effects in the conformational equilibrium of this compound. Those values show that the OCH2CH2N+ system occurs in a synclinal conformation. The Jnh and Jnc are observable in most solvents, but not in chlorine-containing solvents and are not dependent on solvent viscosity. This behavior was explained using data from Ti measurements. The measurement of NMR diffusion coefficients show that ACh has a greater tendency to aggregate when dissolved in chlorinated solvents, a fact that could explain the observed differences in 14N T1
Mestrado
Patologia Clinica
Mestre em Ciências Médicas

Preclinical testing is an important part of cancer drug development. The aim of this thesis was to establish and evaluate preclinical in vitro methods useful in the development of new anticancer drugs. In paper I, the development of non-clonogenic assays (FMCA-GM) using CD34+ stem cells for assessment of haematological toxicity was described. A high correlation was seen when comparing the 50% inhibitory concentrations (IC50) from FMCA-GM with the IC50 from the established clonogenic assay (CFU-GM). In paper II, FMCA-GM was complemented with additional cell models, establishing a normal cell panel. In vitro toxicity towards the five normal cell types was compared with known clinical adverse event profiles. The normal cell panel roughly reflected the tissue specific toxicities but was most useful in the prediction of therapeutic index. In paper III the use of peripheral blood lymphocytes from human, dog, rat and mouse to detect species differences in cellular drug sensitivity was described. Good agreement between our method and the established CFU-GM assay was observed. In paper II the benefit of using primary tumour cells from patients to predict cancer diagnosis-specific activity was studied. The in vitro activity of fourteen anticancer drugs was tested in tumour samples of both haematological and solid tumour origin. In general, clinical activity was well reflected. In paper IV, the efficacy and toxicity models were applied for experimental follow-up of a novel inhibitor of the ubiquitin-proteasome system, CB3 (Phosphoric acid, 2,3-dihydro-1,1-dioxido-3-thienyl diphenyl ester). In the preliminary characterization of CB3, antitumour activity and a favourable toxicity profile were displayed, although the exact mechanism of action remains to be elucidated. CB3 will therefore be further investigated. In conclusion, the work presented here contributes to different parts of the preclinical drug development and the methods may aid in the characterization of anticancer compounds

Relative to traditional methods, air-liquid interface (ALI) exposures constitute a superior in vitro model for assessing the toxicological activity of complex aerosols. By removing the medium barrier, aerosols can be delivered to the cells at their apical surface. This project investigated the utility of the commercially available VITROCELL® exposure system for comparative toxicological assessment of complex aerosols (freshly-generated diluted diesel exhaust and simulated urban smog). The system setup was modified to improve control of aerosol properties (temperature and humidity) and cellular responses (dynamic range). Following optimization, cytotoxicity (WST-1 and LDH assays) and expression of selected genes involved in proinflammatory signalling and oxidative stress responses (via quantitative RT-PCR) were quantified following 1 hour aerosol exposures. The results showed only limited, variable responses following exposures to high concentrations of diesel exhaust. Lack of consistent and robust responses are likely due to poor deposition of particulate matter from the test aerosols.

Drug metabolism is a specialised subset of xenobiotic metabolism, pertaining to the breakdown and elimination of pharmaceutical drugs. The enzymes involved in these pathways are the cytochrome P450 family of isozymes. Metabolism is an important factor in determining the pharmacological effects of drugs. The main aim of this study was to develop a system whereby the major metabolites of drugs can be produced in vitro. An in vitro system was developed and optimised using commercially prepared microsomes from rat liver and coumarin (by monitoring its conversion to 7-hydroxycoumarin) as a model. The optimum running conditions for the incubations were 50 μM coumarin, 50 μg protein/ml microsomes, 1 mM NADP⁺, 5 mM G6P and 1U/ml G6PDH incubated for 30 minutes at 38℃. The HPLC method for the detection of coumarin and 7-hydroxycoumarin was also validated with respect to linearity, reproducibility, precision, accuracy and lower limits of detection and quantification. The system developed was then tested using microsomes prepared from fresh bovine liver on these ten drugs of interest in doping control in horse racing: diazepam, nordiazepam, oxazepam, promazine, acepromazine, chlorpromazine, morphine, codeine, etoricoxib and lumiracoxib. The bovine liver microsomes were prepared using differential centrifugation and had activity on a par with the commercial preparations. This in vitro system metabolised the drugs and produced both phase I and II metabolites, similar to those observed in humans and horses in vivo. For example, the major metabolites of the benzodiazepine drug, diazepam, nordiazepam, temazepam and oxazepam as well as the glucuronidated phase II products were all found after incubations with the bovine liver microsomes. The metabolism of the drugs was also investigated in silico using the computational procedure, MetaSite. MetaSite was able to successfully predict known metabolites for most of the drugs studied. Differences were observed from the in vitro incubations and this is most likely due to MetaSite using only human cytochrome P450s for analysis.

This study examined the effects of low to moderate doses of manganese (0, 250, or 750 _g per day from PD 1-21) on a comprehensive battery of behaviors in rats during the neonatal period, preweanling period, and in adulthood.

Exposure to methyl mercury (MeHg) is a global concern. Increased chronic exposure to MeHg among fish and marine mammal consuming populations will increase the risk of prenatal exposure and as a result, the risk of infant brain damage and neurotoxcity. It is therefore important to understand the role of environmental factors, such as nutrition, in determining susceptibility to MeHg toxicity. Three nutrients (selenium (Se), vitamin C and vitamin E) were selected for examination of their interactions with the mechanisms of McHg cytotoxicity in vitro. Two hybrid neural cell lines (M03.13 and NSC-34) were evaluated for their usefulness in the study of MeHg cytotoxicity. Sixteen toxic endpoints were selected for investigation of growth, viability, structure and biochemistry. Both cell lines responded to MeHg exposure in a dose dependent manner for the majority of endpoints suggesting that both MO3.13 and NSC-34 cells undergo structural and biochemical changes during exposure to McHg, but that MO3.13 cells are more sensitive to DNA, mitochondria) membrane damage and glutathione (GSH) depletion and that NSC-34 cells are more sensitive to protein damage and apoptosis. Se exposure lessened the MeHg-induced decrease in DNA and GSH concentrations in both cell lines. In NSC-34 cells, Se also increased F-actin concentrations and prevented an increase in caspase-3 activity. Se may alter the mechanism of cell death by preventing McHg disruption of DNA replication thus maintaining the production and function of peptides (GSH) and protein (polymerized actin) that aid in MeHg detoxification and neural function. In NSC-34 cells, vitamin C prevented the induction of caspase-3 activity and lessened DNA damage and GSH depletion. Vitamin E lessened GSH depletion and lessened G-actin depletion. Both vitamin C and E improved GSH status, but vitamin C also delayed McHg damage of DNA and prevented early signs of apoptosis suggesting these two vitamins interfere with MeHg metabolism by diffe

O presente estudo in vitro objetivou avaliar a longo prazo o potencial antimicrobiano residual e a citotoxicidade de soluções químicas de limpeza de prótese quando incorporadas à resina acrílica termopolimerizável após sucessivos ciclos de imersão noturna diária. Discos (10mm x 1mm) de resina acrílica termopolimerizável para base de prótese (Lucitone 550) foram submetidos a três ciclos diários de desinfecção (8h/cada) em hipoclorito de sódio a 1% (NaClO), digluconato de clorexidina a 2% (CLX) ou água destilada (controle) durante 91 (T91) ou 183 dias (T183), simulando o período de 9 meses ou 1,5 ano de imersão noturna diária realizada pelo paciente. Inicialmente, foi utilizado o método de concentração inibitória mínima em caldo para determinar o possível efeito residual (incorporação) das soluções à resina acrílica. Metade dos discos imersos em cada agente de limpeza em um dos tempos de imersão (n=5) foi inoculada (1x107cels/mL) com um dos patógenos associados à estomatite protética: Candida albicans (Ca) e Staphylococcus aureus (Sa). Os discos foram incubados a 37oC para análise em espectrofotômetro após 24h, 7 e 14 dias. Os valores de absorbância foram convertidos em porcentagens de inibição microbiana. Confirmada a ação antimicrobiana residual dos agentes de limpeza incorporados à resina acrílica, foi então analisada sua citotoxicidade in vitro sobre fibroblastos gengivais humanos (L929). Os efeitos citotóxicos foram avaliados por meio do ensaio colorimétrico MTT [brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5-difeniltetrazólio] para a determinação da viabilidade celular, após as células serem expostas por 24h às amostras de cada condição experimentais (n=18) previamente imersas em uma das soluções por um dos períodos avaliados (T91 ou T183). A citotoxicidade foi determinada com base na atividade mitocondrial em relação a corpos de prova não submetidos à imersão nas soluções testadas. Os resultados do ensaio do MTT foram analisados estatisticamente por ANOVA-1 fator seguida pelo teste post-hoc de Tukey HSD (a=0,05). Para os períodos T91 e T183, não houve inibição microbiana com a imersão em água (controle) em até 14 dias de incubação. A CLX inibiu progressivamente o crescimento microbiano ao longo dos 14 dias para ambos os períodos de imersão (Ca: 19 a 73,58%; Sa: 0 a 87,08%), sendo observada maior ação antimicrobiana em T183. O NaClO apresentou discreta inibição microbiana apenas no período de 14 dias tanto em T91 (Ca: 0%; Sa: 2,70%) quanto em T183 (Ca: 8,50%; Sa: 15,08%). De acordo com os resultados do teste de MTT, as soluções químicas de limpeza testadas apresentaram uma redução significativa da viabilidade celular quando comparado às células controles propagadas apenas em meio de cultura (p<0,002). A CLX resultou na menor viabilidade celular em ambos os períodos de imersão (p<0,018). As amostras de resina acrílica imersas em água ou NaClO em T91 e T183 apresentaram viabilidade celular estatisticamente similar às amostras não imersas (p>0,05). Pode-se concluir que a CLX incorporada à resina acrílica para base de prótese apresentou um efeito antimicrobiano residual em ambos os períodos de imersão noturna, o que não foi observado com o NaClO. Por outro lado, a CLX residual resultou em efeito intensamente citotóxico aos fibroblastos gengivais humanos quando comparada ao NaClO e à agua destilada, que se apresentaram discretamente citotóxicos. Esses resultados sugerem precaução na seleção de agentes de limpeza para prótese como meio de prevenção e tratamento adjunto da estomatite protética, pois mesmo em concentrações baixas, recomendadas para imersão noturna, podem apresentar algum grau de toxicidade às mucosas de suporte protético.
This in vitro study aimed to evaluate the long-term residual antimicrobial activity and cytotoxicity of chemical denture cleansers incorporated into a heat-polymerized acrylic resin after successive cycles of daily overnight soaking. Discs (10mm x 1mm) were prepared from heat-polymerized acrylic resin (Lucitone 550) and submitted to three daily immersion (8h/each) in 1% sodium hypochlorite (NaClO), chlorhexidine digluconate 2% (CHX) or distilled water (control) for 91 days (T91) or 183 days (T183), simulating the period of 9 months or 1.5 year of nocturnal immersion performed by the patient. Initially, the method of minimum inhibitory concentration in broth was used to determine the possible residual effect (incorporation) of the acrylic resin solution. Half of the disks immersed in each cleaning agent for one of the period immersion (n=5) was inoculated (1x107cells/mL) with pathogens associated with denture stomatitis: Candida albicans (Ca) or Staphylococcys aureus (Sa). The disks were incubated at 37oC for analysis in a spectrophotometer after 24h, 7 and 14 days. The absorbance values were expressed as percentages of microbial inhibition. Confirmed the residual antimicrobial action of cleaning agents incorporated into the acrylic resin, its cytotoxicity was analyzed in vitro on human gingival fibroblasts (L929). Cytotoxic effects were evaluated by the colorimetric assay MTT [3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] to determine cellular viability after the cells were exposed for 24h to the samples of each experimental condition (n=18) previously immersed in one of the solutions for the evaluation periods (T91 or T183). Cytotoxicity was determined based on mitochondrial activity compared to the specimens not subjected to immersion in the solutions. The MTT assay results were 1-way ANOVA followed by Tukey\'s HSD post-hoc test (a=0.05). For the periods T91 and T183, no microbial inhibition was observed with immersion in water (control) for up to 14 days of incubation. The CHX progressively inhibited microbial growth over the 14 days for both immersion times (Ca: 19 to 73.58%, Sa: 0 to 87.08%); with greater antimicrobial activity in T183. The NaClO showed a slight microbial inhibition only in the 14-day period in both T91 (Ca: 0%; Sa: 2.70%) and T183 (Ca: 8.50%; Sa: 15.08%). According to the results of the MTT assay, the chemical cleaning solutions tested showed a significant reduction in cell viability when compared to the control cells propagated in normal culture medium (p<0.002). The CHX resulted in the lowest cell viability in both immersion periods (p<0.018). The acrylic samples immersed in water or NaClO in T91 and T183 showed cell viability statistically similar to nonimmersed samples (p>0.05). CHX incorporated into the acrylic resin denture base had a residual antimicrobial effect on both immersion periods, which was not observed with NaClO. On the other hand, the residual CHX were severely cytotoxic to human gingival fibroblasts compared to NaClO and distilled water which were slightly cytotoxic. These results suggest caution in selecting denture cleaning agents as a method of prevention and adjunct treatment of denture stomatitis because even at low concentrations recommended for overnight immersion, they may exhibit some degree of toxicity to the denture bearing mucosa.

NPs are toxic to a wide range of organisms across trophic levels; gram-positive and gram-negative bacteria (Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and Escherichia coli), algae (Pseudokirchneriella subcapitata), crustaceans (Daphnia magna and Thamnocephalus platyurus), fish (rainbow trout, zebrafish, trout) and plants (Lactuca sativa L. and Raphanus sativus L). Due to their lack of target specificity, NPs may pose an environmental risk. The antibacterial properties of Ag and Cu nanoparticles (NP) are enhanced by their large reactive surface area, compared to bulk counterparts. Toxicity of NPs is attributed to their solubility and subsequent release of ions. However, the cytotoxic effects of NPs cannot always be attributed to the free ion fraction. The underpinning objective of this study was to link the response of microbial biosensors to detailed chemical analysis of NP dissolution products. NPs were suspended in Millipore water and in the presence of the steric stabiliser Na citrate and the resulting NP solubility characterised. Using chemical analysis this study quantified the flux of total dissolved metal (total [M]) and free metal ions [M+] from Ag and Cu NPs (Chapter 3). Two bioluminescent biosensors were used to assess the bioavailable metal fraction ([M]bio) of NP dissolution (Chapters 5 and 6). E. coli HB101 pUCD607 (bacterial) and M. citricolor (fungal) were chosen to represent NP toxicity across trophic levels using the same response mechanism. Additionally, the metal-induced bioreporter, P. fluorescens DF57-Cu15, was used to quantify the Cu bioavailability of Cu NP dissolution. By combining chemical and biological analysis this study inferred NP toxicity is not mass dependent, toxicity is dissolution dependent. Dissolution of Ag and Cu NPs in Millipore water was mostly in the [M+] form. This remained the case for Ag NPs in the presence of Na citrate. However, dissolution of Cu NPs in Na citrate was mostly as total [Cu]. This was due to Cu ions complexing readily with citrate. Toxicity of Ag NP dissolution in Millipore water was concentration dependent. Total [Ag] correlated with E. coli HB101 toxicity response. The addition of Na citrate reduced Ag NP dissolution and therefore reduced toxicity to E. coli HB101. M. citricolor was less sensitive than E. coli HB101 to the dissolution products of Ag NPs in Millipore water. However, the sensor was more sensitive to the dissolution of Ag NPs in Na citrate than E. coli HB101. Cu NPs were chemically stable in Millipore water. The bioreporter P. fluorescens DF57-Cu15 was not induced by Millipore suspensions and E. coli HB101 was not inhibited. However, M. citricolor responded to [Cu]bio of Millipore suspensions with a maximum 54% inhibition of bioluminescence. P. fluorescens DF57-Cu15 was induced by the dissolution products of Cu NPs with the addition of Na citrate, only at high NP concentrations (> 500 mg/L). [Cu]bio of the Na citrate suspensions was toxic to E. coli HB101. However, toxicity was greater for M. citricolor with a maximum biosensor inhibition of 83%. There was no correlation between total [Cu], [Cu2+] or [Cu]bio with the response of either biosensors nor the bioreporter. Interpretation of Ag and Cu NP toxicity was made possible by the combining of chemical and biological toxicity assessment. Dissolution of Ag NPs suspended in Millipore water could be attributed as the main factor in toxicity to E. coli HB101 because of the knowledge gained by chemical analysis. It also allowed the conclusion that NP dissolution was a key factor to toxicity in all cases but biological assessment attributed NP assimilation as a contributing factor. Biological assessment is vital as no chemical analysis can quantify [M]bio, especially when [M]bio was perceived differently by biosensors of different trophic levels and modes of action. Combining chemical and biological assessment in this study was essential for interpreting NP toxicity.

To meet the demands of political, ethical and scientific pressures on animal testing, research into possible alternatives is required. Data obtained with animal models often cannot be related to humans. Testing with current cell-based assays, microdosing and pharmacokinetic models contribute to reducing animal testing and improving the drug development process. Micro-fabrication and rapid prototyping techniques offer potential solutions to reduce the need for animal toxicity testing. The aim of this research was to develop biological platforms for in vitro toxicity testing to provide physiologically relevant, high-throughput solutions to reduce animal testing. This was achieved by investigating and integrating microfabrication methods of microfluidics, dielectrophoresis and additive manufacturing. Three approaches were taken: (i) micro-pattern protein arrays for primary hepatocyte cell culture enclosed within microfluidics devices for high-throughput toxicity testing. It was observed that hepatocytes attached to the micro-pattern within microfluidics and maintained viability, however liver specific functions observed by florescence assays, the P450 enzymes, were observed to be reduced compared to Petri dish conditions. (ii) A biomimetic dielectrophoretic cell patterning technique to form liver lobule-like tissue structures within agar on a paper substrate was developed for toxicity testing. Observation of these biomimetic micro liver structures showed high viability (80-90%) and an increase in liver specific function marker albumin protein (20%) compared to control samples after 48 hours. (iii) Rapid prototyping methods were explored with regard to fabrication of microfluidic chips for the automated trapping, imaging and analysis of zebrafish embryos. Monolithic microfluidic chips for zebrafish were developed to be suitable for optical based toxicity assays. The biocompatibility of 3D printed materials was investigated. A method to render the photopolymer Dreve Fototec 7150 compatible with zebrafish culture was observed to provide 100% viability. Future development of this research will aim to (i) develop the liver lobule-like system to use layers of multiple cell types to form complex micro-liver models using additive manufactured microfluidic systems for toxicity testing. (ii) Automation of zebrafish handing using additive manufactured microfluidic devices for in-situ analysis of dechorionated zebrafish for high-throughput toxicity studies.

Current regulatory risk assessment strategies have several limitations, such as linking subcellular changes to higher-level biological effects, and an improved knowledge-based approach is needed. Ecotoxicogenomic techniques have been proposed as having the potential to overcome the current limitations, providing greater mechanistic information for ecotoxicological testing. In this thesis, metabolomics is explored as a novel method for toxicity testing using Daphnia magna. Initially I evaluated the potential application of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) based metabolomics for use in regulatory toxicity testing. Subsequently, I aimed to use this approach to discriminate between toxicant modes of action (MOA) and to link toxicant induced metabolic effects to reduced reproductive output in D. magna. FT-ICR MS metabolomics was determined to be a feasible approach for toxicity testing of both whole-organism homogenates and haemolymph of D. magna. It is capable of discriminating between life-stages of D. magna as well as determining toxicant-induced metabolic effects. Highly predictive multivariate classification models were capable of significantly discriminating between four different toxicant MOAs; achievable in both haemolymph and whole-organism extracts, with the latter being the more information-rich sample type. Multivariate regression models were predictive of reduced reproductive output in D. magna following toxicant exposure, and determined that a metabolic biomarker signature was significantly able to predict the reproductive output of D. magna. Ultimately this research has concluded that an FT-ICR MS metabolomics approach for use in regulatory toxicity testing using Daphnia magna is both viable and can provide valuable information.

Introduction aux problemes de la peroxydation radicalaire des lipides, notamment au travers de la toxicite de l'oxygene. Effets des composes organonitres sur cette toxicite (etudes in vivo, in vitro, etudes de la mutagenicite)

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaCO-2 cell model, there was no significant change in the transport of [3H] l-proJine by the cell after co-incubation with either dapsone or cyclophosphamide (50!!M) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50!!M) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (1 OO!!M ) . With the test involving the NG11 S-401l-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after co-incubation with either phenytoin or cyclophosphamide (50!!M) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (SO!!M) (0.44 + 0.04 mM), sulphamethoxazole (SOIlM) (0.43 + 0.08mM) and carbamazepine (SO!!M) (0.47 + 0.034 mM), when coincubated with the rat microsomal system, compared to the control (0.52 + 0.07mM). There was no significant depletion in glutathione when the erythrocytes were coincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50~lM) (0.953 + 011 OmM) when co-incubated the rat microsomal system, compared to the control (1.124 + 0.032mM). Differences were considered statistically significant for p