Poly Brominated Diphenyl Ethers (PBDEs) are known flame retardants with highly persistent and lipophilic in nature. The continued usage of PBDE in various products amplifies the human burden of PBDEs. It is therefore, important to study the potential toxicological and/or biological effects of PBDE exposure in human. In this study we investigated the mode of action of PBDE induced toxicity in human liver by exposing human hepatocarcinoma cells in a time (24-72h) and dose (0-100μM) dependent manner. The highest test dose caused an inhibition in cell viability up to 50% after 72h, whereas lower doses (<50μM) showed slight increase in cell viability. Likewise, higher doses caused significant accumulation of intracellular ROS over time. Further, increase in caspase-3 enzyme levels and DNA fragmentation showed that, lower brominated PBDEs induce liver toxicity through accumulation of toxic metabolites and reactive oxygen species over time leading to caspase-mediated apoptotic cell death.

Les cellules endothéliales (CE), monocouche de cellules hexagonales jointives situées à la face interne de la cornée, assurent la transparence de ce tissu essentiel à la vision. Peu avant la naissance, ces CE perdent leur capacité proliférative en restant bloquées en phase G1du cycle cellulaire. Le non remplacement des cellules mortes est responsable de certaines pathologies cécitantes dont la cornea gutatta et les dystrophies bulleuses du pseudophaque sont les prototypes et comptent parmi les premières indications de greffe de cornée. Les mécanismes moléculaires impliqués dans l’arrêt de la prolifération de ces CE restent très partiellement expliqués. La première partie de cette thèse a pour objectif d’identifier si des changements d’expression transcriptionnelle des gènes régulateurs du cycle cellulaire interviennent au cours de l’organoculture (OC) et de la culture in vitro, afin de définir au mieux les cibles potentielles à inhiber ou celles à surexprimer pour re-déclencher une prolifération cellulaire contrôlée. Pour la première fois, nous avons mis en évidence des profils transcriptionnels variables en fonction de l’environnement des CE, avec une activation globale de l’expression des gènes en OC de routine et en culture primaire et l’expression accrue de gènes impliqués dans l’arrêt du cycle cellulaire en différents points comme DIRAS3, GADD45A, p15 p16, p18 et p19 ou impliqués dans la régulation du cycle des CE comme le complexe ubiquitine/protéasome (culines, APC...), laissant supposer que les freins antiprolifératifs sont encore plus complexes. Dans la seconde partie, nous avons développé une méthode d’analyse de la viabilité de l’endothélium pan-cornéenne afin d’évaluer au mieux la viabilité des cellules endothéliales sur les greffons. Nous avons développé un outil innovant de mesure combinant un triple marquage Hoechst/Ethidium/Calcéine AM à l’analyse pan-endothéliale permettant d’évaluer de définir la notion originale de densité en cellules viables d’une cornée. Cette technique peut être appliquée à l’analyse de n’importe quel procédé chirurgical ou non, susceptible d’altérer directement ou indirectement l’endothélium cornéen / Corneal endothelial cells (EC) form a monolayer of hexagonal contiguous cells located at the inner surface of the corne and are responsible for maintenance of its transparency, and therefore essential for vision. Just before birth, they lose the proliferative capacity and remain blocked in the G1 phase of the cell cycle. The absence of cell replacement by mitosis induces is responsible for certain blinding diseases such as cornea gutatta and pseudophakic bullous keratopathy, two prototype endothelial diseases that are among the first indications of corneal graft. The molecular mechanisms involved is the non-proliferation of EC remain only partially explained. The first part of this thesis aimed to identify whether changes in transcriptional expression of cell cycle regulators genes occurred during organ culture (OC) and during in vitro culture, in order to better define the potential targets to inhibit or to overexpress necessary to trigger a controlled cell proliferation. Forthe first time we have highlighted variable transcriptional profiles depending on endothelial environment, with a glolal activation of gene expression in routine OC and in primary cultures, especially with an increased expression of gene involved in cell cycle arrest at different points like DIRAS3, GADD45, p15 p16, p18 and p19 or involved in cycle regulation as the ubiquitin/proteasome complex (Culines, APC ...), suggesting that the antiproliferative brakes are even more complex than previously reported. In a second part we developed an original method of pan-corneal endothelial viability assessment. This innovative measurement tool combines a triple Hoechst/Ethidium/Calcein-AM labeling with a part endothelial image analysis allowing to define the new concept of viable endothelial cell density, that represent the reaviable cell pool of a cornea. This technique can be applied to the analysis of any procedure (surgical or not), likely to directly or indirectly alter the corneal endothelium

Magister Scientiae - MSc / Basella alba (BA) and Hibiscus macranthus (HM) are used by traditional healers in Cameroon to treat male sexual fertility problems. Previous studies showed that in vivo administration of the leaf extracts of both plants caused a significant increase in rat seminal vesicle weight and spermatozoa numbers was accompanied by a significant increase in serum testosterone. The aim of this study was to establish the effects of BA and HM extracts on Sertoli cell functions. TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells (Mather, 1982). Sertoli cell play a key role in spermatogenesis by regulating and supporting germ cell development. Therefore, any alterations in Sertoli cell physiology or structure may lead to impaired spermatogenesis, germ cell loss and male infertility. Developing germ cells in the seminiferous tubule require a constant supply of lactate and pyruvate (Jutte et al, 1981; 1982) and toxicant induced alterations in these nutrients have been shown to induce germ cell necrosis (Monsees et al., 2000). TM4 Sertoli cells were cultured in DMEM/Ham F-12 (M) for one day and exposed to 0.01, 0.1, 1, 10, 100 μg/ml of BA and HM extracts, respectively, for four further days. The extracts were dissolved in 0.5 % DMSO in M, while 0.5 % and 2% DMSO in M were used as negative or positive controls, respectively, and 100mM ethanol as positive control where indicated. Results obtained from the Sertoli cells exposed to BA extracts, showed that the plant extract had no significant effect on the cell viability but induced a significant concentration-dependent increase in lactate (19-67%) and pyruvate levels (39-102%) and a concentration-dependent decrease in the protein content (9-42%). The H&E histological study confirmed that the BA extract had no cytotoxic effect, as there were no changes in the morphology of the cell. Likewise, apoptotic study using DAPI showed no alteration in the nucleus when compared to the negative control. The HM plant extract significantly enhanced mitochondrial dehydrogenase activity (7fold) in the Sertoli cells but caused only slight alterations in the lactate and pyruvate levels. There was no effect seen in the protein content of the Sertoli cells. H&E and DAPI staining revealed that there were neither changes in the morphology of the cells nor any alteration regarding the mitotic and apoptotic indices. Thus, the HM extract did not have a cytotoxic effect on the cells. This study demonstrated that the Basella alba methanol extract may enhance spermatogenesis as it stimulated the source of energy required for the development of germ cells without exerting a cytotoxic effect. The Hibiscus macranthus extract stimulated mitochondrial dehydrogenase activities and may thus trigger changes in Sertoli cell physiology. In summary, both plant extracts enhanced certain Sertoli cell functions and thus might explain the positive in vivo effects of the combined plant extracts on rat spermatogenesis observed by Moundipa et al. (1999). / South Africa

Ivy geranium (Pelargonium peltatum) is a heat susceptible species with its heat tolerance varying among varieties. Reactive oxygen species (ROS) and in-vivo defense systems are related to plant heat damage and heat tolerance. Application of chelated-iron has also been reported to enhance ivy geranium heat tolerance; however, the correlation of ROS, relative enzyme stability, and iron content to differences in heat tolerance in ivy geraniums is unknown. Here we show that the H2O2 content and ROS scavenging enzyme stability in ivy geranium varies with varieties and active iron is not related to heat tolerance in ivy geranium. H2O2 content in mature leaves in both heat tolerant 'Beach' and sensitive 'Butterfly' increased under heat stress, but 'Butterfly' had a relatively greater increase of this toxic compound. Catalase (CAT) activities in young leaves in both varieties decreased. In young leaves of 'Butterfly', CAT activities decreased to a level significantly lower than that in old leaves while this did not occur in 'Beach'. Superoxide dismutase (SOD) activities in 'Butterfly' young leaves were also decreased. All these phenomenon coincided with the heat tolerance differences of the two varieties. Active iron content only changed with leaf age and did not vary between varieties or treatments. Our results demonstrated that ROS scavenging ability and relative enzyme stability may indicate heat tolerance in ivy geranium and that iron deficiency was not the cause of heat damage. Cell Membrane Themostability (CMT) and Triphenyl Tetrazolium Chloride (TTC) cell viability tests are alternative, laboratory-based screening methods for screening for heat-tolerance. Both CMT and TTC tests can represent the variance in heat tolerance observed in ivy geraniums. The results of both CMT and TTC tests correlated well with plant width and growth indexes although their correlations to plant chlorosis were low. Unlike TTC, CMT strongly correlated with plant width. CMT and TTC tests are complementary laboratory-based methods that can be applied to cultivar screening for heat tolerance in ivy geraniums.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Studentka: Lucie Vašková Školitel: RNDr. Jana Maixnerová, Ph.D. Název diplomové práce: Study on cytotoxicity of compounds in vitro The subject of this diploma thesis was to assess the effect of newly synthesized antimycobacterial substances on the viability of human hepatocellular carcinoma (HepG2) cells. The tested substances were esters (HE-nMe, HE-4PHOPH, HE-KARVA, HE-2NAFT, HE-METRO, HE-CH2PY, HE-8CHIN) and thioesters (HES-4H, HES- nETH) of antituberculotic isoniazid. Experiments performed with these substances have shown, that like isoniazid, the substances inhibit InhA enzyme in mycobacteria and therefore interfere with cell wall biosynthesis. Isoniazid is a drug standardly used in the first line of TB treatment. Together with other first-line antituberculotics, some hepatotoxic potential has been reported during treatment. To assess the possible cytotoxic effect of the tested isoniazid derivatives, the standard human hepatocyte cell line HepG2 was chosen as the cell model. Cell viability was assessed by a colorimetric method that measures the metabolic activity of cells based on the reduction of the tetrazolium compound MTS. Obtained values were quantitatively compared using the toxicological...

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Solos ácidos cobrem cerca de 30% das áreas agricultáveis do mundo. Nestes solos, geralmente ocorrem baixas concentrações de cátions como cálcio e magnésio, enquanto a acidez (pH <5,5) promove a solubilização de Al3+. A exposição de raízes a pH ácido e/ou ao Al3+ inibe o crescimento radicular, reduz a viabilidade de células do ápice, promove estresse oxidativo, e pode causar desarranjos na parede celular. Na parede, é provável que tanto o H+ como o Al3+ atuem sobre a pectina, comprometendo a sua estrutura e funcionalidade. Por outro lado, peroxidases classe III (GPOX) parecem desempenhar um papel central nas modificações da parede celular, são induzidas por H+ e Al3+ e algumas isoformas são associadas à pectina. O objetivo deste trabalho foi caracterizar as respostas radiculares da cultivar Micro-Tom de tomateiro (Solanum lycopersicum L.) à toxidez por H+ e Al3+ e avaliar a capacidade do cálcio e do boro em aliviar esta toxidez com relação à inibição do crescimento radicular, queda na viabilidade celular e alterações na atividade de GPOX. Em raízes expostas a pH 4,0, após 30 min já foi possível observar a redução na viabilidade de células do ápice, avaliada através da absorção de azul de Evans. Observou-se elevação significativa na atividade de GPOX após 2 h de tratamento a pH 4,0. Apesar da defasagem na sua indução em relação à queda na viabilidade celular, a atividade de GPOX parece ser um melhor indicador da ação de H+. O uso do inibidor da GPOX (SHAM; ácido salicilhidroxâmico) indicou que estas enzimas desempenham um papel no sentido de impedir maiores danos às células por pH baixo. O cálcio (10 mM) aliviou totalmente a toxidez por H+ (pH 4,0 e 4,5) e Al3+ (10 ?M) em relação a crescimento radicular e viabilidade celular. O boro (30 ?M) aliviou totalmente a toxidez por H+ e Al3+ em relação a viabilidade celular, mas aliviou apenas parcialmente ou em nada a inibição do crescimento radicular por H+ ou Al3+, respectivamente. Tanto o cálcio quanto o boro suprimiram totalmente a indução da atividade de GPOX. O cálcio e o boro apresentaram interação positiva sobre o crescimento radicular a pH 5,8 e 4,5, mas não na presença de Al3+ e nem em relação à viabilidade celular ou atividade de GPOX. De modo geral, encontrou-se boa correlação entre viabilidade celular e atividade de GPOX. Tomado no seu conjunto, os dados evidenciam um papel importante de GPOX na toxidez por H+ e Al3+ e sugerem que, apesar de distintos, a toxidez por estes íons possivelmente apresentam alguns aspectos e mecanismos em comum. Também corroboram outros trabalhos que evidenciam a ação destes íons sobre a parede celular, em particular a matriz péctica. Estudos futuros deverão examinar se a GPOX que é induzida por H+ e Al3+ está associada à pectina da parede. / Acid soils cover about 30% of the arable land in the world. These soils usually have low concentrations of cations, such as calcium and magnesium, whereas the low pH (pH <5.5) increases the solubility of Al3+. Exposure of roots to low pH and/or Al3+ inhibits root growth, reduces cell viability of the root apex, promotes oxidative stress, and can cause derrangements in the cell wall. It is likely that both H+ and Al3+ act on pectin of the cell wall, affecting its structure and functionality. On the other hand, class III peroxidases (GPOX) appear to play a role in modifications of the cell wall, are induced by H+ and Al3+ and some isoforms are associated with pectin. The aim of this study was to characterize the responses of roots of the Micro-Tom cultivar of tomato (Solanum lycopersicum L.) to the toxicity of H+ and Al3+ and evaluate the ability of calcium and boron to alleviate this toxicity with respect to inhibition of root growth, decrease in cell viability and changes in the activity of GPOX. In roots exposed to pH 4.0, after 30 min it was already possible to observe a decrease in cell viability at the apex, as assessed by the uptake of Evans blue. We observed a significant increase in GPOX activity after 2 h of treatment at pH 4.0. Despite the lag in their induction in relation to the decrease in cell viability, GPOX activity seems to be a better indicator of the action of H+. The use of inhibitors of GPOX activity indicates that these enzymes play a role in preventing further damage to cells by low pH. Calcium (10 mM) completely alleviated H+ (pH 4.0 or 4.5) and Al3+ (10 mM) toxicity with respect to root growth and cell viability. Boron (30 mM) completely alleviated H+ and Al3+ toxicity with respect to cell viability, but only partly alleviated or had no effect on the inhibition of root growth by H+ or Al3+, respectively. Both calcium and boron completely suppressed the induction of GPOX activity. Calcium and boron displayed a positive interactive effect on root growth at pH 5.8 and 4.5, but not in the presence of Al3+ and not in relation to cell viability or GPOX activity. In general, good correlations were found between cell viability and GPOX activity. Taken together, the data indicate a significant role of GPOX activity in the toxicity of H+ and Al3+ and suggest that although different, the toxicity of these ions possibly share some common aspects and mechanisms. The data also corroborates other studies that indicate the cell wall as a target of toxicity, particularly the pectic matrix. Further studies should examine whether GPOX activity, which is induced by H+ and Al3+ is associated with pectin in the wall.

Durante a fase de obturação dos canais radiculares, pode ocorrer o contato direto do material obturador com os tecidos periapicais, por tempo indeterminado, o que pode retardar, dificultar ou impedir a ocorrência do processo de reparo, por isso, para o êxito do tratamento endodôntico, a seleção de um material de obturação do canal radicular adequado é tão essencial como a técnica operatória. Esse contato pode ocorrer por extravasamento na forma de puff ou mesmo sem extravasamento, pois componentes derivados desses materiais podem entrar em contato direto com os tecidos, através de numerosas conexões, como, por exemplo, os túbulos dentinários, canais acessórios, canais laterais e o forame apical, causando irritação e possível desconforto pós-operatório. Outra forma pela qual os materiais obturadores de canais radiculares podem entrar em contato com os tecidos periapicais é através do processo realização de tratamento endodôntico em dentes decíduos, devido ao processo de rizólise ou tratamento endodôntico em dentes imaturos (ápice aberto). Por esses motivos, a biocompatibilidade dos materiais obturadores de canais radiculares é de extrema importância, diante disso objetivo deste trabalho foi avaliar a citotoxicidade, por meio do Ensaio do MTT, de seis materiais endodônticos usados em dentes decíduos e permanentes (AH Plus, GuttaFlow 2, Endomethasone N, Vitapex®, Calen® espessada e MTA ProRoot) recém espatulados, em cultura primária de linfócitos do sangue periférico de humanos, por 24 horas. Os resultados foram submetidos a análise estatística pelo teste one-way ANOVA e pós-teste de Tukey, com nível de significância de 5%. Observou-se que o Endomethasone N foi o mais citotóxico sobre linfócitos, O AH Plus, e a Calen® espessada foram citotóxicas a partir de 25 mg/mL, enquanto o MTA ProRoot, GuttaFlow 2 e o Vitapex® foram os menos citotóxicos sobre linfócitos humanos, podendo-se concluir que o Vitapex® (usados em dentes decíduos) e o GuttaFlow 2 e MTA ProRoot (usados em dentes permanentes) foram os materiais obturadores menos citotóxicos sobre linfócitos humanos. / During the filling phase of root canals, there may be direct contact of the filling material with the periapical tissues, for an (indefinite) unknown period, which may delay, hinder or prevent the occurrence of the repair process. The objective of this study is to evaluate the cytotoxicity by MTT assay of six endodontic materials used in primary and permanent teeth (AH Plus , GuttaFlow 2, Endomethasone N, Vitapex®, Calen® thickened and MTA ProRoot) newly spatulate in primary cultures of peripheral human blood lymphocytes for 24 hours. The results were statistically analyzed by one-way ANOVA and Tukey\'s test at 5% significance level. It was observed that the Endomethasone N was the most cytotoxic material for the lymphocytes, the AH Plus, and Calen® thickened were cytotoxic from 25 mg/mL, while the MTA ProRoot, GuttaFlow 2 and Vitapex® were less cytotoxic for human lymphocytes, allowing to conclude that the Vitapex® (used in deciduous teeth) and GuttaFlow 2 and MTA ProRoot (used in permanent teeth) were less cytotoxic sealers on human lymphocytes.

Materials based on novel Off-Stoichiometry Thiol-Ene polymers, abbreviated OSTE, show promising properties as materials forlow cost and scalable manufacturing of micro- and nanosystems such as lab-on-chip devices. The OSTE materials have tunablemechanical properties, offer possibility for low temperature bonding to many surfaces via tunable surface chemistry, and can beused in soft lithography. Unlike the commonly used elastomer poly(dimethylsiloxane), PDMS, the OSTE materials have lowpermeability for gasses, are resistant to common solvents and can be more permanently surface modified.In this master’s thesis project, the OSTE materials have been evaluated with focus on compatibility with cells, possibility fornanostructuring using soft lithography and the use of OSTE as a flexible support for conducting polymers.Results from cell seeding studies with HEP G2 cells suggest that cells can proliferate on a low thiol off-stoichiometry OSTEmaterial for at least five days. The biocompatibility for this type of OSTE material may be similar to poly(styrene). However, highlevels of free thiol monomers in the material decrease cell viability considerably.By using soft lithography techniques it is possible to fabricate OSTE nanochannels with at least the dimensions of 400 nm x 15nm. Combined with the advantages of using the OSTE materials, such as low temperature bonding and possibility for stablesurface modifications, a candidate construction material for future development of systems for DNA analysis is at hand.OSTE can serve as a flexible support for an adsorbed film of a conducting polymer with the possibility for future applicationssuch as electronic interfaces in microsystems. In this project, a film of PEDOT:PSS with the electrical resistance of ~5 kΩ wascreated by adsorption to an flexible OSTE material. Furthermore, results suggest that it is possible to further optimize theconductivity and water resistance of PEDOT:PSS films on OSTE.

OBJECTIVE: Low level light therapy (LLLT) is being widely used in wound healing and pain relief, and its use is expected to be expanded rapidly to treatment of other disorders as well in a foreseeable future. However, before its expansion, the fear that LLLT could initiate or promote metastasis must be addressed thoroughly. As an initial effort towards this end, the current study evaluates the safety of LLLT in vitro using two different human cancer cell lines (Michigan Cancer Foundation-7 (MCF-7) and Jurkat E6-1) by determining the viability of cells after low level light (LLL) application while treatment under anti-cancer chemotherapeutic drugs (5-fluorouracil (5-FU) and cisplatin) separately on each cell line. METHODS: Two human cancer cell lines (MCF-7 and Jurkat E6-1) were cultured throughout the experiments. Two different anti-cancer chemotherapeutic drugs (5-FU and cisplatin) were used to treat both cell lines. The half maximal inhibitory concentration (IC50) of each drug on each cell line was determined by treating each cell line with varying concentrations of each drug. A total of 3 or 4 trials were done for each cell line with each drug, and the range of concentration was narrowed closer to the IC50 value at each successive trial. Once the IC50 concentrations were determined, each cell line was treated with 808 nm LLL at varying energy densities in a single dose using a light emitting diode (LED) source both in the absence and the presence of each drug at one IC50. A total of 3 or 5 trials were done for each cell line with each drug, and for each trial, six different energy densities ranging from 0 J/cm2 (control) to 10 J/cm2 were applied. The energy densities were varied for each trial with control always being used. After application of LLL, the viability of cells was determined, and three different 1-way ANOVA (Analysis of Variance) analyses were done to compare the viability of cells at each energy density to that of control. RESULTS: The IC50 of 5-FU in MCF-7 and Jurkat E6-1 cells was determined as 70 µM and 20 µM respectively. The IC50 of cisplatin in MCF-7 and Jurkat E6-1 cells was determined as 17 µM and 7 µM respectively. No significant difference (P > 0.05) in the viability of MCF-7 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of 5-FU at IC50 (70 µM). No significant difference (P > 0.05) in the viability of MCF-7 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of cisplatin at IC50 (17 µM). No significant difference (P > 0.05) in the viability of Jurkat E6-1 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of 5-FU at IC50 (20 µM). However, a significant increase (0.01 < P < 0.05) in the viability of cells was observed when treating Jurkat E6-1 cells with 10 J/cm2 of LLL in the presence of cisplatin at IC50 (7 µM) compared to control group (0 J/cm2). Except for the comparison mentioned previously, no significant difference in the viability of Jurkat E6-1 cells was observed between each group treated with different energy density of LLL and control group (0 J/cm2) both in the absence and the presence of cisplatin at IC50 (7 µM). No definite trend in the viability of cells was observed with increasing energy density of LLL for each cell line either in the absence of the presence of each drug at IC50. CONCLUSIONS: The application of LLL at 808 nm with energy densities ranging from 0.1 J/cm2 to 10 J/cm2 under an LED source did not induce cell proliferation or death compared to control (0 J/cm2) for each cell line in the absence or the presence of each drug, and no definite trend was observed with increasing energy density. The study suggests that LLLT at these parameters may be safe to use on cancer patients, but further studies on different cancer cell lines and animal models with different parameters (wavelength, energy density, dosage) of LLL are warranted.