O fator alimentar pode ser considerado um promotor tumorigênico responsável pela etiologia do câncer gástrico no estado do Pará. A mandioca (Manihot esculenta Crantz) é uma das muitas espécies da Amazônia consumida de modo indiscriminado pelos habitantes da região norte. O cianeto, seu principal componente tóxico, pode ser liberado durante o processamento da mandioca por meio da hidrólise do glicosídeo cianogênico linamarina. No organismo, o cianeto bloqueia a cadeia de transporte de elétrons inibindo a respiração celular e, provocando, entre outras coisas, a produção de radicais livres que podem agir no DNA, através da formação de adutos exocíclicos. O objetivo deste trabalho foi a avaliação da atividade citotóxica, genotóxica e mutagênica de folhas e tucupi, crus e cozidos, de mandioca mansa e brava, usando os ensaios do MTT, cometa e citoma em células HepG2. Os resultados obtidos demonstraram que a viabilidade celular decai à medida que a concentração aumenta na maioria dos grupos de tratamento. No ensaio do cometa, a análise visual demonstrou que o cianeto de potássio, usado como padrão, foi genotóxico em todas as concentrações testadas (5,0; 15,0 e 25,0 ?g/mL). As amostras de folhas de mandioca brava foram genotóxicas somente nas concentrações 15,0 e 25,0 ?g/mL (cruas) e 5,0; 15,0 e 25,0 ?g/mL (cozidas). As amostras de mandioca mansa foram genotóxicas apenas quando cozidas (5,0 e 25,0 ?g/mL). Já as amostras de tucupi, tanto cruas quanto cozidas, demonstraram dano ao DNA de células HepG2 em todas as concentrações testadas (20,0; 40,0 e 60.0 ?g/mL). Utilizando análise de fragmentação de DNA observamos que o cianeto de potássio foi genotóxico apenas na avaliação da porcentagem de DNA na cauda. Já amostras de mandioca brava e mansa, de maneira geral, demonstraram valores de porcentagem de DNA na cauda, tail moment e olive moment maiores em relação ao grupo controle negativo, mas, somente as folhas de mandioca, em algumas concentrações, demonstraram valores estatisticamente significativos. Observando o ensaio do citoma, as concentrações de folhas e tucupi, crus e cozidos, tanto de mandioca brava quanto de mandioca mansa mostraram um discreto aumento no número de micronúcleos em células HepG2 binucleadas, estatisticamente não significativo, em relação ao grupo controle negativo. Já o número de pontes nucleoplasmáticas e brotos nucleares foi menor que no grupo controle. Os danos aqui observados pelo ensaio do cometa podem estar transitoriamente presentes ii como intermediários formados durante o reparo de lesões no DNA. A ausência de brotos nucleares, pontes nucleoplasmáticas e resultados estatísticos significativos em relação ao grupo controle negativo, não nos permitem afirmar presença de mutagenicidade em células HepG2 tratadas com mandioca tanto brava quanto mansa. De maneira geral, o cozimento das amostras não foi um fator determinante na diminuição do dano observado em células HepG2. Nossos resultados confirmam apenas citotoxicidade e genotoxicidade das variedades da mandioca nas concentrações e sistema celular utilizados. Os mecanismos moleculares que envolvem a genotoxicidade da mandioca requerem estudos futuros. Para o consumo da mandioca devem ser levados em consideração tipo de processamento realizado para extração de compostos cianogênicos, níveis de glicosídeos cianogênicos nos produtos consumidos, quantidade de mandioca consumida e estado nutricional do consumidor / The food factor can be considered a tumor causing agent reponsasible for the etiology of gastric cancer in the state of Pará Cassava ( Manihot esculenta Crants is one of the many food species of the Amazon indiscriminately consumed by the inhabitants of the northern region Cyanide, its main toxic component, can be released during cassava processing through the hydrolysis of the cyanogenic glycoside linamarin. In the body, cyanide blocks the electron transport chain by inhibiting cell respiration which brings about, among other things, the production of free radicals which can act upon DNA through the formation of exocyclical adducts. The main goal of this study was the assessment of the citotoxic, genotoxic and mutagenic role of the leaves and tucupi juice of wild and sweet cassava, both raw and cooked, using the MTT, comet and cytome assays in HepG2 cells. The results gathered have shown that cell viability decreases as the concentration increases in most treatment groups. In the comet assay, a visual analysis has shown that potassium cyanide, used as a standard, was genotoxic in all concentrations tested (5.0, 15.0 and 25.0 ?g/mL). Samples of wild cassava leaves were genotoxic only in concentrations of 15.0 and 25.0 ?g/mL for raw leaves, and 5.0, 15.0 and 25.0 ?g/mL for cooked leaves. Samples of sweet cassava leaves were genotoxic only when cooked (5.0 and 25.0 ?g/mL). Yet, samples of tucupi juice, both raw and cooked, have shown damage to DNA in HepG2 cells at all concentrations tested (20.0, 40.0 and 60.0 ?g/mL). In performing DNA fragmentation analysis, it was observed that potassium cyanide was genotoxic only in the assessment of percentage DNA in tail. Whereas the wild and sweet cassava samples, in a general fashion, have shown greater values of percentage DNA in tail, tail moment and olive moment than the negative control group, but only the cassava leaves revealed statistically significant values, in some concentrations. For the cytome assay, concentrations of leaves and of tucupi juice, raw and cooked, of both wild and sweet cassava, have shown a slight increase in the number of micronuclei in binucleated HepG2 cells, not statistically significant as compared to the negative control group. On the other hand, the number of nucleoplasmic bridges and nuclear buds in binucleated cells was lower than the value found in the negative control group. The damages verified herein by the comet assay may be transiently present as intermediates formed during repair of DNA lesions. The absence of iv nucleoplasmic bridges, nuclear buds and of statistically significant results vis-à-vis the negative control group do not warrant the assertion for the presence of mutagenicity in HepG2 cells treated with either wild or sweet cassava. In general, the cooking of the samples was not determining factor of the decrease in damage observed in HepG2 cells. Our results only confirm cytotoxicity and genotoxicity of wild and sweet cassava species in the concentrations and cell system used. The molecular mechanisms involving genotoxicity of cassava require further studies. For the consumption of cassava, the way of processing performed for extracting cyanogen contents, the levels of cyanogenic glycosides in the products consumed, amount of cassava consumed as well as the nutritional condition of the consumer must be taken into account.

The current dietary recommendations for optimal health are designed to increase our intake of two bioactive omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), abundant naturally in fatty fish such as salmon. Health authorities recommend that the general population consume two to three fatty fish meals per week (1) for optimal health and for the prevention of cardiovascular disease. However, some modern Western societies consume only modest amounts of fish and seafood (2;3). Land based vegetable oils may provide an alternative to meet these needs. Linseed and canola oils are rich in alpha-linolenic acid (ALA, 18:3n-3) (4). ALA can be converted endogenously to EPA and DHA and suggests that increasing the dietary intake of ALA may increase the conversion and accumulation of DHA in tissues and plasma. However, elevated dietary intakes of ALA in animals and humans results in an increased level of EPA in tissues yet there is little or no change in the level of DHA (5-7). The current consensus is that the synthesis of DHA from ALA in humans is limited yet the mechanisms involved in regulating the accumulation of DHA in tissues are poorly understood. The reputed rate-limiting enzyme in the conversion of fatty acids is delta 6 desaturase (D6D). ALA is a substrate for D6D and undergoes a series of desaturation and elongation reactions to yield n-3 long chain polyunsaturated fatty acids (LCPUFA). The final step in the synthesis of DHA from ALA involves translocation of its immediate fatty acid precursor, 24:6n-3 from the endoplasmic reticulum to the peroxisome to be partially beta-oxidised to yield DHA. The involvement of multiple enzymes in the desaturation-elongation pathway, and the integration of other pathways, such as phospholipid biosynthesis, suggests there are various steps that may regulate the accumulation of DHA in cell membranes. This thesis aimed to examine the possible regulatory steps in the conversion of fatty acids to LCPUFA, particularly in the synthesis of DHA from n-3 fatty acid precursors. The human hepatoma cell line, HepG2, was used as an in vitro cell system to examine the accumulation of individual fatty acids and their metabolites in isolation from other competing fatty acid substrates. The accumulation of linoleic acid (LA, 18:2n-6) and ALA in HepG2 cell phospholipids following supplementation with increasing concentrations of each respective fatty acid correlated with that described in vivo, as was the accumulation of their conversion products. The accumulation of DHA in cells supplemented with ALA reached a plateau at concentrations above 5 micro g/ml and paralleled the accumulation of 24:6n-3 in cell phospholipids, suggesting that the delta 6 desaturation of 24:6n-3 was prevented by increasing concentrations of ALA, thereby limiting the accumulation of DHA. The accumulation of DHA in cells supplemented with eicosapentaenoic acid (EPA, 20:5n-3) or docosapentaenoic acid (DPA, 22:5n-3) was significantly greater than the level of DHA that accumulated in cells supplemented with ALA. However, regardless of substrate, the level of DHA in cell membranes reached a plateau at substrate concentrations above 5 micro g/ml. This thesis further aimed to examine the effect of fatty acid supplementation on the mRNA expression of D6D in HepG2 cells. The expression and activity of D6D mRNA is subject to nutritional and hormonal regulation. The mRNA expression of D6D in HepG2 cells following supplementation with oleic acid (OA, 18:1n-9), LA, ALA, arachidonic acid (AA, 20:4n-6) or EPA was examined by real time RT PCR. The expression of D6D mRNA was reduced by up to 50% in cells supplemented with OA, LA, ALA , AA or EPA compared with control cells and suggests that fatty acids modulate the expression of the key enzyme involved in the conversion of fatty acids. The effect of fatty acid co-supplementation on the fatty acid composition of HepG2 cell phospholipids was also examined in an attempt to gain insights into the role of D6D and the enzymes involved in peroxisomal beta-oxidation on the accumulation of DHA from n-3 fatty acid precursors. The reduction in the accumulation of DHA in cells co-supplemented with DPA and docosatetraenoic acid (DTA, 22:4n-6) was greater than in cells co-supplemented with DPA and LA, suggesting that peroxisomal beta-oxidation may have a greater role in determining the accumulation of DHA from DPA than the activity of D6D. Further investigation should be directed towards understanding the role that peroxisomal beta-oxidation may play in the synthesis of DHA from precursor fatty acids. The fatty acid composition of cell membranes in vivo is a result of several physiological processes including dietary intake, phospholipids biosynthesis and fatty acid conversion as well as catabolic processes. This thesis demonstrates that a greater understanding of the regulation of the conversion of fatty acids will help to define dietary approaches that enhance the synthesis of n-3 LCPUFA from n-3 fatty acid precursors to lead to improved outcomes for health.

O fator alimentar pode ser considerado um promotor tumorigênico responsável pela etiologia do câncer gástrico no estado do Pará. A mandioca (Manihot esculenta Crantz) é uma das muitas espécies da Amazônia consumida de modo indiscriminado pelos habitantes da região norte. O cianeto, seu principal componente tóxico, pode ser liberado durante o processamento da mandioca por meio da hidrólise do glicosídeo cianogênico linamarina. No organismo, o cianeto bloqueia a cadeia de transporte de elétrons inibindo a respiração celular e, provocando, entre outras coisas, a produção de radicais livres que podem agir no DNA, através da formação de adutos exocíclicos. O objetivo deste trabalho foi a avaliação da atividade citotóxica, genotóxica e mutagênica de folhas e tucupi, crus e cozidos, de mandioca mansa e brava, usando os ensaios do MTT, cometa e citoma em células HepG2. Os resultados obtidos demonstraram que a viabilidade celular decai à medida que a concentração aumenta na maioria dos grupos de tratamento. No ensaio do cometa, a análise visual demonstrou que o cianeto de potássio, usado como padrão, foi genotóxico em todas as concentrações testadas (5,0; 15,0 e 25,0 ?g/mL). As amostras de folhas de mandioca brava foram genotóxicas somente nas concentrações 15,0 e 25,0 ?g/mL (cruas) e 5,0; 15,0 e 25,0 ?g/mL (cozidas). As amostras de mandioca mansa foram genotóxicas apenas quando cozidas (5,0 e 25,0 ?g/mL). Já as amostras de tucupi, tanto cruas quanto cozidas, demonstraram dano ao DNA de células HepG2 em todas as concentrações testadas (20,0; 40,0 e 60.0 ?g/mL). Utilizando análise de fragmentação de DNA observamos que o cianeto de potássio foi genotóxico apenas na avaliação da porcentagem de DNA na cauda. Já amostras de mandioca brava e mansa, de maneira geral, demonstraram valores de porcentagem de DNA na cauda, tail moment e olive moment maiores em relação ao grupo controle negativo, mas, somente as folhas de mandioca, em algumas concentrações, demonstraram valores estatisticamente significativos. Observando o ensaio do citoma, as concentrações de folhas e tucupi, crus e cozidos, tanto de mandioca brava quanto de mandioca mansa mostraram um discreto aumento no número de micronúcleos em células HepG2 binucleadas, estatisticamente não significativo, em relação ao grupo controle negativo. Já o número de pontes nucleoplasmáticas e brotos nucleares foi menor que no grupo controle. Os danos aqui observados pelo ensaio do cometa podem estar transitoriamente presentes ii como intermediários formados durante o reparo de lesões no DNA. A ausência de brotos nucleares, pontes nucleoplasmáticas e resultados estatísticos significativos em relação ao grupo controle negativo, não nos permitem afirmar presença de mutagenicidade em células HepG2 tratadas com mandioca tanto brava quanto mansa. De maneira geral, o cozimento das amostras não foi um fator determinante na diminuição do dano observado em células HepG2. Nossos resultados confirmam apenas citotoxicidade e genotoxicidade das variedades da mandioca nas concentrações e sistema celular utilizados. Os mecanismos moleculares que envolvem a genotoxicidade da mandioca requerem estudos futuros. Para o consumo da mandioca devem ser levados em consideração tipo de processamento realizado para extração de compostos cianogênicos, níveis de glicosídeos cianogênicos nos produtos consumidos, quantidade de mandioca consumida e estado nutricional do consumidor / The food factor can be considered a tumor causing agent reponsasible for the etiology of gastric cancer in the state of Pará Cassava ( Manihot esculenta Crants is one of the many food species of the Amazon indiscriminately consumed by the inhabitants of the northern region Cyanide, its main toxic component, can be released during cassava processing through the hydrolysis of the cyanogenic glycoside linamarin. In the body, cyanide blocks the electron transport chain by inhibiting cell respiration which brings about, among other things, the production of free radicals which can act upon DNA through the formation of exocyclical adducts. The main goal of this study was the assessment of the citotoxic, genotoxic and mutagenic role of the leaves and tucupi juice of wild and sweet cassava, both raw and cooked, using the MTT, comet and cytome assays in HepG2 cells. The results gathered have shown that cell viability decreases as the concentration increases in most treatment groups. In the comet assay, a visual analysis has shown that potassium cyanide, used as a standard, was genotoxic in all concentrations tested (5.0, 15.0 and 25.0 ?g/mL). Samples of wild cassava leaves were genotoxic only in concentrations of 15.0 and 25.0 ?g/mL for raw leaves, and 5.0, 15.0 and 25.0 ?g/mL for cooked leaves. Samples of sweet cassava leaves were genotoxic only when cooked (5.0 and 25.0 ?g/mL). Yet, samples of tucupi juice, both raw and cooked, have shown damage to DNA in HepG2 cells at all concentrations tested (20.0, 40.0 and 60.0 ?g/mL). In performing DNA fragmentation analysis, it was observed that potassium cyanide was genotoxic only in the assessment of percentage DNA in tail. Whereas the wild and sweet cassava samples, in a general fashion, have shown greater values of percentage DNA in tail, tail moment and olive moment than the negative control group, but only the cassava leaves revealed statistically significant values, in some concentrations. For the cytome assay, concentrations of leaves and of tucupi juice, raw and cooked, of both wild and sweet cassava, have shown a slight increase in the number of micronuclei in binucleated HepG2 cells, not statistically significant as compared to the negative control group. On the other hand, the number of nucleoplasmic bridges and nuclear buds in binucleated cells was lower than the value found in the negative control group. The damages verified herein by the comet assay may be transiently present as intermediates formed during repair of DNA lesions. The absence of iv nucleoplasmic bridges, nuclear buds and of statistically significant results vis-à-vis the negative control group do not warrant the assertion for the presence of mutagenicity in HepG2 cells treated with either wild or sweet cassava. In general, the cooking of the samples was not determining factor of the decrease in damage observed in HepG2 cells. Our results only confirm cytotoxicity and genotoxicity of wild and sweet cassava species in the concentrations and cell system used. The molecular mechanisms involving genotoxicity of cassava require further studies. For the consumption of cassava, the way of processing performed for extracting cyanogen contents, the levels of cyanogenic glycosides in the products consumed, amount of cassava consumed as well as the nutritional condition of the consumer must be taken into account.

Realizou-se um estudo estrutura-atividade sobre os efeitos citoprotetor/citotóxico de 5 flavonóides envolvendo processos mitocondriais, com ênfase na apoptose. Nossos resultados mostram que a dupla ligação na posição 2-3 / grupos 3-OH em conjugação com a função 4-oxo no anel C da estrutura dos flavonóides parece favorecer a interação destes compostos com a membrana mitocondrial, diminuindo a sua fluidez, e tanto inibindo a cadeia respiratória das mitocôndrias, quanto causando desacoplamento. Por outro lado, a estrutura o-di-OH no anel B parece favorecer a inibição da cadeia respiratória, sendo que a ausência desta estrutura parece favorecer a atividade desacopladora. Os flavonóides que não afetaram a respiração mitocondrial, induziram a transição de permeabilidade mitocondrial. A capacidade dos flavonóides em liberar o Ca2+ acumulado pelas mitocôndrias correlaciona-se com a sua capacidade de afetar a respiração mitocondrial e sua inabilidade em induzir a transição de permeabilidade mitocondrial. Já os dados referentes aos estudos da atividade protetora contra a formação de radicais livres demonstraram que a quercetina, luteolina e a galangina foram substancialmente mais potentes que a taxifolina e a catequina em conferir proteção contra a lipoperoxidação, embora somente a quercetina tenha sido um efetivo seqüestrador tanto de DPPH?, quanto de O2?-. Esses resultados sugerem que a dupla ligação na posição 2-3 em conjugação com a função 4-oxo na estrutura dos flavonóides são os fatores mais importantes na atividade antioxidante dos flavonóides sobre as mitocôndrias. Ainda, a presença da estrutura o-di-OH no anel B, conforme observado na quercetina, favorece essa atividade via seqüestro de O2?-, enquanto que a ausência dessa característica estrutural na galangina, a favorece via diminuição da fluidez de membrana e/ou desacoplamento mitocondrial. Os ensaios realizados para avaliar o efeito dos flavonóides sobre as células HepG2 mostraram que galangina, luteolina e quercetina são capazes de induzir morte celular. Esse efeito parece estar associado à dissipação do potencial de membrana mitocondrial e à diminuição na capacidade energética celular, mais pronunciada no caso dos dois primeiros; porém, como essa diminuição na concentração de ATP não é drástica, a morte celular pode ocorrer por apoptose. Os experimentos realizados para avaliar essa possibilidade sugerem uma ativação das caspases via mitocôndria, observada pelo aumento das atividades de caspase -9 e -3 e também pela exposição de fosfatidil serina. A taxifolina que não apresentou capacidade de produzir dano celular, apresentou, por outro lado, capacidade de prevenir parcialmente a diminuição de viabilidade das células HepG2 induzida pelo pró-oxidante t-butilhidroperóxido, aparentemente por meio de sua atividade antioxidante que inibiu, também de forma parcial, o acúmulo de espécies reativas de oxigênio. / We carried out a structure-activity study addressing the protective/toxic effects of 5 flavonoids (quercetin, taxifolin, luteolin, catechin and galangin) upon mitochondrial aspects with emphasis on the mechanisms potentially involved in cell apoptosis. The major findings were: The 2,3 double bond/3-OH group in conjugation with the 4-oxo function on the C-ring in the flavonoid structure seems favour the interaction of these compounds with the mitochondrial membrane, decreasing its fluidity either inhibiting the respiratory chain of mitochondria or causing uncoupling; while the o-di-OH on the B-ring seems favour the respiratory chain inhibition, the absence of this structure seems favour the uncoupling activity. The flavonoids not affecting the respiration of mitochondria induced MPT. The ability of flavonoids to induce the release of mitochondria-accumulated Ca2+ correlated well with their ability to affect mitochondrial respiration on the one hand, and their inability to induce MPT, on the other. The data concerning the protective activity against the free radical formation showed that quercetin, luteolin and galangin were far more potent than taxifolin and catechin in affording protection against lipid peroxidation, although only quercetin was an effective scavenger of both DPPH? and O2?-. These results suggest that the 2,3-double bound in conjugation with the 4-oxo function in the flavonoid structure are major determinants of the antioxidant activity of flavonoids on mitochondria, the presence of an o-di-OH structure on the B-ring, as occurring in quercetin, favouring this activity via O2?- scavenging, while the absence of this structural feature in galangin, favouring it via decrease in membrane fluidity and/or mitochondrial uncoupling. The assays addressing the flavonoids effects on HepG2 cells showed that galangin, luteolin and quercetin are able to induce cell death. This effect appears to be linked to the mitochondrial membrane potential dissipation and consequent decrease in the cellular energy charge, more pronounced for the galangin or luteolin treatment. However, since this decrease in ATP content is not drastic, the apoptosis process can occur. The set of experiments performed in order to evaluate this possibility demonstrated caspases-9 and -3 activation and also phosphatidylserine exposure on the cell membrane. On the other hand, taxifolin, that did not present ability to induce injury to the cell, was able to partially inhibit a viability decrease of HepG2 cell exposed to the pro-oxidant t-butylhydroperoxide, probably on account of its capacity to partially decrease ROS formation.

Realizou-se um estudo estrutura-atividade sobre os efeitos citoprotetor/citotóxico de 5 flavonóides envolvendo processos mitocondriais, com ênfase na apoptose. Nossos resultados mostram que a dupla ligação na posição 2-3 / grupos 3-OH em conjugação com a função 4-oxo no anel C da estrutura dos flavonóides parece favorecer a interação destes compostos com a membrana mitocondrial, diminuindo a sua fluidez, e tanto inibindo a cadeia respiratória das mitocôndrias, quanto causando desacoplamento. Por outro lado, a estrutura o-di-OH no anel B parece favorecer a inibição da cadeia respiratória, sendo que a ausência desta estrutura parece favorecer a atividade desacopladora. Os flavonóides que não afetaram a respiração mitocondrial, induziram a transição de permeabilidade mitocondrial. A capacidade dos flavonóides em liberar o Ca2+ acumulado pelas mitocôndrias correlaciona-se com a sua capacidade de afetar a respiração mitocondrial e sua inabilidade em induzir a transição de permeabilidade mitocondrial. Já os dados referentes aos estudos da atividade protetora contra a formação de radicais livres demonstraram que a quercetina, luteolina e a galangina foram substancialmente mais potentes que a taxifolina e a catequina em conferir proteção contra a lipoperoxidação, embora somente a quercetina tenha sido um efetivo seqüestrador tanto de DPPH?, quanto de O2?-. Esses resultados sugerem que a dupla ligação na posição 2-3 em conjugação com a função 4-oxo na estrutura dos flavonóides são os fatores mais importantes na atividade antioxidante dos flavonóides sobre as mitocôndrias. Ainda, a presença da estrutura o-di-OH no anel B, conforme observado na quercetina, favorece essa atividade via seqüestro de O2?-, enquanto que a ausência dessa característica estrutural na galangina, a favorece via diminuição da fluidez de membrana e/ou desacoplamento mitocondrial. Os ensaios realizados para avaliar o efeito dos flavonóides sobre as células HepG2 mostraram que galangina, luteolina e quercetina são capazes de induzir morte celular. Esse efeito parece estar associado à dissipação do potencial de membrana mitocondrial e à diminuição na capacidade energética celular, mais pronunciada no caso dos dois primeiros; porém, como essa diminuição na concentração de ATP não é drástica, a morte celular pode ocorrer por apoptose. Os experimentos realizados para avaliar essa possibilidade sugerem uma ativação das caspases via mitocôndria, observada pelo aumento das atividades de caspase -9 e -3 e também pela exposição de fosfatidil serina. A taxifolina que não apresentou capacidade de produzir dano celular, apresentou, por outro lado, capacidade de prevenir parcialmente a diminuição de viabilidade das células HepG2 induzida pelo pró-oxidante t-butilhidroperóxido, aparentemente por meio de sua atividade antioxidante que inibiu, também de forma parcial, o acúmulo de espécies reativas de oxigênio. / We carried out a structure-activity study addressing the protective/toxic effects of 5 flavonoids (quercetin, taxifolin, luteolin, catechin and galangin) upon mitochondrial aspects with emphasis on the mechanisms potentially involved in cell apoptosis. The major findings were: The 2,3 double bond/3-OH group in conjugation with the 4-oxo function on the C-ring in the flavonoid structure seems favour the interaction of these compounds with the mitochondrial membrane, decreasing its fluidity either inhibiting the respiratory chain of mitochondria or causing uncoupling; while the o-di-OH on the B-ring seems favour the respiratory chain inhibition, the absence of this structure seems favour the uncoupling activity. The flavonoids not affecting the respiration of mitochondria induced MPT. The ability of flavonoids to induce the release of mitochondria-accumulated Ca2+ correlated well with their ability to affect mitochondrial respiration on the one hand, and their inability to induce MPT, on the other. The data concerning the protective activity against the free radical formation showed that quercetin, luteolin and galangin were far more potent than taxifolin and catechin in affording protection against lipid peroxidation, although only quercetin was an effective scavenger of both DPPH? and O2?-. These results suggest that the 2,3-double bound in conjugation with the 4-oxo function in the flavonoid structure are major determinants of the antioxidant activity of flavonoids on mitochondria, the presence of an o-di-OH structure on the B-ring, as occurring in quercetin, favouring this activity via O2?- scavenging, while the absence of this structural feature in galangin, favouring it via decrease in membrane fluidity and/or mitochondrial uncoupling. The assays addressing the flavonoids effects on HepG2 cells showed that galangin, luteolin and quercetin are able to induce cell death. This effect appears to be linked to the mitochondrial membrane potential dissipation and consequent decrease in the cellular energy charge, more pronounced for the galangin or luteolin treatment. However, since this decrease in ATP content is not drastic, the apoptosis process can occur. The set of experiments performed in order to evaluate this possibility demonstrated caspases-9 and -3 activation and also phosphatidylserine exposure on the cell membrane. On the other hand, taxifolin, that did not present ability to induce injury to the cell, was able to partially inhibit a viability decrease of HepG2 cell exposed to the pro-oxidant t-butylhydroperoxide, probably on account of its capacity to partially decrease ROS formation.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Simona Hvolková Supervisor: PharmDr. Jana Ramos Mandíková, Ph.D. Title of diploma thesis: Cytotoxicity testing on 2D and 3D model of human liver cells. An inherent part of drug development are in vitro assays, which might be helpful in prediction of drug toxicity. Nowadays, the majority of assays use simple 2D structures for cell growth, but 3D structures with similar conditions to in vivo are becoming more popular. The goal of the study was to assess the cytotoxicity of selected xenobiotics in vitro by both 2D and 3D cell models. The research subjects were drugs from the group of antimycotics (amphotericin B, ketoconazole), NSAIDs (diclofenac, ibuprofen), antipyretics (paracetamol, fenacetine), sodium azide, tamoxifen, para-aminosalicylic acid, methanol and ethanol. For determination of cytotoxicity, the standard colorimetric method (CellTiter 96® ) based on reductive assessment of metabolic active cells was used. For drug testing it was used human standard line of liver cells HepG2. The cells were cultivated in monolayer or in 3D form with the Alvetex® Scaffold technology using high porous networked polystyrene. The parameter of inhibition concentration IC50 was chosen for toxicity assessment of...

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Non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in the liver, is the most common cause of liver diseases in Western countries. NAFLD is a major risk factor for developing hepatocellular carcinoma (HCC); however, in vitro evaluation of hepatic cancerogenesis fails due to a lack of liver models displaying a proliferation of hepatocytes. Originally designed to overcome primary human hepatocyte (PHH) shortages, upcyte hepatocytes were engineered to obtain continuous proliferation and, therefore, could be a suitable tool for HCC research. We generated upcyte hepatocytes, termed HepaFH3 cells, and compared their metabolic characteristics to HepG2 hepatoma cells and PHHs isolated from resected livers. For displaying NAFLD-related HCCs, we induced steatosis in all liver models. Lipid accumulation, lipotoxicity and energy metabolism were characterized using biochemical assays and Western blot analysis. We showed that proliferating HepaFH3 cells resemble HepG2, both showing a higher glucose uptake rate, lactate levels and metabolic rate compared to PHHs. Confluent HepaFH3 cells displayed some similarities to PHHs, including higher levels of the transaminases AST and ALT compared to proliferating HepaFH3 cells. We recommend proliferating HepaFH3 cells as a pre-malignant cellular model for HCC research, while confluent HepaFH3 cells could serve as PHH surrogates for energy metabolism studies.

Bastões plásticos quimioluminescentes, chamados light sticks, são usados por companhias pesqueiras e descartados nas praias. Moradores locais utilizam seus conteúdos como repelentes, óleo bronzeador e medicamento para dores nas articulações. Nós investigamos a reatividade das soluções de bastões de light sticks coletados em praias brasileiras e a toxicidade celular de seus conteúdos e também de soluções de light stick de bastões novos. Produtos da reação dos conteúdos de light stick descartado com 2\'-desoxiguanosina (dGuo) foram analisados por HPLC/UV/ESI-MS/MS. Um aduto foi purificado e caracterizado em espectrômetro de massas por Dissociação Induzida por Colisão (CID) e através de experimentos de 1H RMN. A estrutura do aduto revelou que o produto de degradação de bis(triclorofenil)oxalato é reativo para nucleófilos in vitro. O mesmo aduto foi detectado em DNA de timo de bezerro incubado in vitro com a solução do light stick descartado pelo uso HPLC/ESI-MS/MS. Além do DNA, albumina também foi modificada pelos conteúdos de light stick descartado. Células HepG2 foram incubadas por 16 h com 0.0125% - 0.12% (v/v) das soluções de light sticks: (i) coletadas na praia, (ii) obtidas imediatamente após a reação quimioluminescente no laboratório, e (iii) previamente a reação, contendo ou n-butil-ftalato, difenilantraceno, e bis(triclorofenil)oxalato (solução 1), ou dimetil-ftalato, H2O2, e salicilato de sódio (solução 2). A sobrevivência celular foi avaliada pelo teste do XTT, corante cristal violeta e lactato desidrogenase realizados em placas de 96 poços. Com as concentrações testadas foram obtidas significativamente a morte celular. O dano oxidativo ao DNA celular foi avaliado pela análise da 8-oxo-2\'-desoxiguanosina através do equipamento HPLC/ESI-MS/MS, que revelou aumento de alterações nas células tratadas com 0.006% das soluções de light stick de bastões descartados e novos. Nossos dados apontam genotoxicidade e citotoxicidade das soluções de light sticks e podem contribuir como alerta a autoridades públicas para proibição do uso incontrolado. / Chemiluminescent plastic rods, called light sticks, are used by fishery companies and littered on the shores. Local inhabitants use their contents as repellents, tanning oil, and medicine for joint pain. We have investigated the reactivity of spent light stick solutions collected on brazilian beaches and the cellular toxicity of their contents as well as that of brand-new light stick solutions. Products of the reaction of the spent light stick contents with 2\'-deoxyguanosine (dGuo) were analyzed by HPLC/UV/ESI-MS/MS. A dGuo adduct was purified and characterized in a Collision Induced Dissociation (CID) mass spectrometer and through 1H NMR experiments. The adduct structure revealed that a degradation product of bis(trichlorophenyl)oxalate is reactive towards nucleophiles in vitro. The same adduct was detected in calf thymus DNA incubated in vitro with spent light stick solution, by using HPLC/ESI-MS/MS. Besides DNA, albumin was also modified by spent light stick contents. HepG2 cells were incubated for 16 h with 0.0125% 0.12% (v/v) of light stick solutions: (i) collected on the beaches, (ii) obtained immediately after the chemiluminescent reaction in the laboratory, and (iii) previously the reaction, containing either n-butyl-phthalate, diphenylanthracene, and bis(trichlorophenyl)oxalate (solution 1), or dimethyl-phthalate, H2O2, and sodium salicylate (solution 2). Cell survival was evaluated by the XTT, crystal violet dye, and lactate dehydrogenase assays performed in 96 well plates. The concentrations tested were found to significantly kill cells. Oxidative damage to cellular DNA was assessed by 8-oxo-2\'-deoxyguanosine analysis via an HPLC/ESI-MS/MS equipment, which revealed increased changes in cells treated with 0.006% of spent and brand-new light stick solutions. Our data point to important genotoxicity and cytotoxicity of the light stick solutions and may contribute to alert public policies to ban their uncontrolled use.

Atualmente, a utilização de azo corantes pelas indústrias de tingimento constitui um problema ambiental e de saúde, considerando o lançamento de quantidades elevadas para o meio ambiente e a falta de dados toxicológicos dos corantes disponíveis para as indústrias. Vários estudos têm demonstrado o potencial genotóxico de diversos corantes azóicos, porém para os corantes Disperse Red 1, Disperse Orange 1 e o Disperse Red 13, não foram encontrados dados na literatura relativos à sua capacidade de dano ao material genético. Considerando que esses corantes são empregados em processos de tingimento no Brasil, esse trabalho teve como objetivo a avaliação de sua atividade mutagênica, utilizando o teste de micronúcleo (MN) em linfócitos humanos e em células HepG2. Os resultados obtidos no teste com linfócitos, demonstram que na menor concentração testada (0,2 µg/mL), o número de micronúcleos presentes foi semelhante ao controle negativo, mas esse número aumenta à medida que eleva-se a concentração. No entanto, a partir da concentração de 1,0 µg/mL, este valor começa a decair. Isso provavelmente se deve à citotoxidade dos corantes, levando à morte celular ou redução da divisão celular e, conseqüentemente, não há a formação de micronúcleo. Embora o perfil de mutagenicidade dos três corantes seja semelhante, o corante Disperse Red 13 parece ter maior potencial de dano sobre os linfócitos em relação aos demais, seguido pelo Disperse Red 1 e Disperse Orange 1, respectivamente. Os resultados obtidos para o teste de MN em células HepG2 foram semelhantes aos obtidos no teste feito em linfócitos. O aumento do número de micronúcleos em relação ao aumento da concentração dos corantes, ocorreu até o limite de 2,0 µg/mL em células HepG2, excetuando-se o corante Disperse Red 13, para o qual o limite foi de 1,0 µg/mL. E a partir desses pontos, considerados como limites, ocorreu uma redução no número de MN. Para este sistema celular, os três corantes parecem ter potencial mutagênico bastante semelhante. Portanto, a análise dos resultados mostrou que os corantes Disperse Red 13, Disperse Red 1 e Disperse Orange 1 são mutagênicos para sistemas celulares diferentes. Foi também avaliado Índice de Proliferação do Bloqueio de Citocinese (IPBC), que permite a avaliação de toxicidade celular ou atraso no ciclo celular por meio da determinação da proliferação celular nas culturas. Porém, neste estudo não foram observadas diferenças estatísticas entre o controle negativo e as concentrações testadas. Nossos resultados confirmam que os azo corantes constituem uma importante classe de contaminantes ambientais e devem ser avaliados e utilizados de forma cautelosa. / Currently, the use of azo dyes for the textile industries can causes direct and/or indirect effects on human health and on the environment, considering the discharge of industrial effluents that contain toxic dyes and the lack of reports in the literature about the toxic effects of these compounds. Several studies have been demonstrated the genotoxic effect of diverse azo dyes, however for the dyes Disperse Red 1, Disperse Orange 1 and Disperse Red 13 no information about their capacity to cause DNA damage was found in the literature. Considering that these dyes are used for dying processes in Brazil, the main of this work was the evaluation of the mutagenic activity of Disperse Red 1, Disperse Orange 1 and Disperse Red 13, using the micronucleus assay (MN) in human lymphocytes and HepG2 cells. For the lymphocytes assay, we observed that the number of micronucleus induced by the lowest concentration of each dye (0,2 µg/mL) was similar to the negative control. For the other concentrations we observed a dose response micronucleus formation, until 1,0 µg/mL. Above this concentration, the number of micronucleus has decreased, probably because of the cytotoxic effects of the dyes, which leads to cellular death or reduction of cellular division and, consequently, does not have the micronucleus formation. Although the mutagenicity profile of the three dyes is similar, Disperse Red 13 seems to be the strongest for the lymphocytes, followed by Disperse Red 1 and Disperse Orange 1, respectively. For the HepG2 cells the results were similar to the lymphocytes. For the three dyes we noted a dose dependent increase in the frequency of micronuclei. However, for the HepG2 the threshold for this increase was 2,0 µg/mL, except for Disperse Red 13, which the limit was at 1 µg/ml, after this point a reduction in the MN number occurred. For this cellular system, the three dyes seem to have similar mutagenic potential. Therefore, our results suggest that the dyes Disperse Red 13, Disperse Red 1 and Disperse Orange 1 are potentially mutagenic for different cellular systems. Besides, cytokinesis-block proliferation index (CBPI) was calculated, in order to evaluate cellular toxicity or delay in the cellular cycle through of the determination of the cellular proliferation in the cultures. No statistical difference was detected between the tested concentrations and the negative control. Our results confirmed that azo dyes constitute an important class of environmental contamination and they should be evaluated and used carefully.