Nitrate is a ubiquitous drinking water contaminant with potential adverse effects on human health. However, little is known about nitrate toxicity at the cellular and molecular level. The purpose of this study was to examine the effects of environmentally relevant concentrations of nitrate on cytotoxicity and protein expression in human cell lines. To determine if tissue-specific responses occurred, a human hepatoma cell line (HepG2) and a human embryonic kidney cell line (HEK293) were used. Both potassium and ammonium salts of nitrate were used to determine salt-specific toxicity. Test concentrations of nitrate varied from 1 μg/L to 5000 mg/L. Cells were exposed to a nitrate salt for 24, 48, or 72 hours and then examined for effects on viability (using the Neutral Red assay) or proliferation (using the BrdU ELISA assay). To determine the effects of nitrate on protein expression, levels of PCNA, Hsp70, Hsc70, and VEGF protein were monitored using Western blotting in HepG2 and HEK293 cells exposed to KNO3 or NH4NO3 for 24 hours.<p>Nitrate was cytotoxic to both cell types at high concentrations, with EC50 values between 1557 mg/L (approximately) 5852mg/L for viability, and ~2.5 mg/L 3631 mg/L for proliferation. Several EC50 values were not calculable based on the available data, but appeared to be far greater than 5000 mg/L. Ammonium nitrate was generally more toxic than potassium nitrate, and increasing exposure time generally resulted in greater toxicity. The HepG2 and HEK293 cells displayed similar responses for most assays, except the 24 hour KNO3 Neutral Red assay. Here, HEK293 viability increased with increasing KNO3 concentrations, while HepG2 viability decreased. The reason for this finding is unknown, but may involve cell-specific homeostatic mechanisms. A hormetic-like effect was observed in both cell types in several of the proliferation assays; the biological significance of this effect remains unknown.<p>No significant changes in protein expression were observed under these experimental conditions. Some subtle trends were present, such as a slight increase in Hsp70 expression with increasing nitrate concentration in both cell types. In HepG2 cells, PCNA expression increased slightly with increasing nitrate concentrations; however, the opposite effect was observed in HEK293 cells. This may be due to transcriptional or translational regulation.<p>In summary, environmentally relevant concentrations of nitrate did not appear to evoke significant cytotoxicity or changes in protein expression. Cell viability and proliferation effects were observed at higher concentrations of nitrate. Private water supplies may contain nitrate concentrations above the EC50 values in these experiments. More research is required to determine if this poses a direct threat to human health.

Nitrate is a ubiquitous drinking water contaminant with potential adverse effects on human health. However, little is known about nitrate toxicity at the cellular and molecular level. The purpose of this study was to examine the effects of environmentally relevant concentrations of nitrate on cytotoxicity and protein expression in human cell lines. To determine if tissue-specific responses occurred, a human hepatoma cell line (HepG2) and a human embryonic kidney cell line (HEK293) were used. Both potassium and ammonium salts of nitrate were used to determine salt-specific toxicity. Test concentrations of nitrate varied from 1 μg/L to 5000 mg/L. Cells were exposed to a nitrate salt for 24, 48, or 72 hours and then examined for effects on viability (using the Neutral Red assay) or proliferation (using the BrdU ELISA assay). To determine the effects of nitrate on protein expression, levels of PCNA, Hsp70, Hsc70, and VEGF protein were monitored using Western blotting in HepG2 and HEK293 cells exposed to KNO3 or NH4NO3 for 24 hours.<p>Nitrate was cytotoxic to both cell types at high concentrations, with EC50 values between 1557 mg/L (approximately) 5852mg/L for viability, and ~2.5 mg/L 3631 mg/L for proliferation. Several EC50 values were not calculable based on the available data, but appeared to be far greater than 5000 mg/L. Ammonium nitrate was generally more toxic than potassium nitrate, and increasing exposure time generally resulted in greater toxicity. The HepG2 and HEK293 cells displayed similar responses for most assays, except the 24 hour KNO3 Neutral Red assay. Here, HEK293 viability increased with increasing KNO3 concentrations, while HepG2 viability decreased. The reason for this finding is unknown, but may involve cell-specific homeostatic mechanisms. A hormetic-like effect was observed in both cell types in several of the proliferation assays; the biological significance of this effect remains unknown.<p>No significant changes in protein expression were observed under these experimental conditions. Some subtle trends were present, such as a slight increase in Hsp70 expression with increasing nitrate concentration in both cell types. In HepG2 cells, PCNA expression increased slightly with increasing nitrate concentrations; however, the opposite effect was observed in HEK293 cells. This may be due to transcriptional or translational regulation.<p>In summary, environmentally relevant concentrations of nitrate did not appear to evoke significant cytotoxicity or changes in protein expression. Cell viability and proliferation effects were observed at higher concentrations of nitrate. Private water supplies may contain nitrate concentrations above the EC50 values in these experiments. More research is required to determine if this poses a direct threat to human health.

The cytotoxic effects of exposure to low concentrations of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) that are typically found in groundwater were investigated, in vitro. Most 2,4-D toxicology studies use high concentrations of the herbicide that are above those typically found in groundwater and measure overt biological endpoints. In contrast, this thesis examines the effects of low concentrations of 2,4-D and measures more subtle and sensitive endpoints such as gene expression and the generation of reactive oxygen species. This work derives from recent cDNA microarray analysis conducted in our laboratory that revealed significant alterations in the expression of 238 genes in cells exposed to nanomolar (nM) concentrations of a commercial formulation of 2,4-D. These findings are extended in this thesis to include the in vitro cytotoxic effects of low concentrations of both technical and commercial 2,4-D on two cell lines. Cells derived from liver (HepG2) and kidney (HEK293) respectively, were chosen, since liver and kidney are known to metabolize 2,4-D in vivo. Cell viability was measured using the Resazurin assay, reactive oxygen species (ROS) were measured with 2,7-dichlorofluorescin diacetate (2,7-DCFH-DA), and real timepolymerase chain reaction (RT-PCR) was used to assess changes in mRNA expression while protein expression was examined by Western blot.<p>Cell viability studies revealed that low environmental concentrations (0.1 to 100 nM) of 2,4-D induced small, but statistically significant decreases in cell viability. No concentration or time-dependent decreases in cell viability were observed in cells exposed to either forms of low environmental 2,4-D concentrations. HEK293 cells were more susceptible than HepG2 cells to the toxic effects of both forms of 2,4-D, having statistically significant lower viability at all exposure concentrations and durations. Both forms of 2,4-D reduced cell viability in both cell lines, suggesting that cytotoxicity was induced directly by 2,4-D, and not by the inert ingredients in the commercial formulation.<p>The ROS assays illustrated that 2,4-D induced statistically significant ROS production in HepG2 and HEK293 cell cultures at concentrations greater than 10 µM and 100 nM respectively. This was both a concentration and time-dependent effect in both cell lines. Although HEK293 cells were more susceptible to 2,4-D, they had 50 to 70% less ROS production than HepG2 cells, at all exposure concentrations and times.<p>The RT-PCR and Western blot analyses showed that exposure of HepG2 and HEK293 cells to low 2,4-D concentrations induced (< 2 fold) alterations in mRNA and protein levels of FTL, FTH1 and PCNA however these changes did not consistently vary with concentration.<p>Taken together, cell viability, ROS and gene expression studies show that low environmental 2,4-D concentrations induced subtle in vitro cytotoxic effects. However we have no evidence that these subtle changes pose a serious health threat to exposed humans.

This investigation assesses the role of poly(ADP-ribose) polymerase in ethanol-mediated hepatotoxicity using the untransfected HepG2 hepatocellular carcinoma line, an established, well-characterized toxicological model. HepG2 cells were treated with ethanol at concentrations between 100 mM and 800 mM, and assessed for markers of cytotoxicity. PARP-1 activity in total cell protein lysates was quantified as a proxy of apoptotic induction at six hours. Our results demonstrated a 1.43-fold AST activity increase in culture medium isolates of cells exposed to 800 mM without significant effect on cellular viability. PARP-1 activity varied greatly and results for enzyme activity remained inconclusive. The results suggest a high degree of insensitivity to ethanol toxicity and nuclear enzyme activity, demonstrating the metabolic irrelevance of untransfected HepG2 in ethanol toxicosis. There is a need to characterize phase 1 metabolic enzyme expression profiles relevant to ethanol for CYP2E1 and ADH pathways to facilitate comparisons across toxicological models using transfected, as well as the untransfected HepG2 model.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2015-11-10T11:06:55Z No. of bitstreams: 1 texto completo.pdf: 14494167 bytes, checksum: fab8d8ee99ed6b81f44c5a418b81401f (MD5) / Made available in DSpace on 2015-11-10T11:06:55Z (GMT). No. of bitstreams: 1 texto completo.pdf: 14494167 bytes, checksum: fab8d8ee99ed6b81f44c5a418b81401f (MD5) Previous issue date: 2014-07-02 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / O carcinoma hepatocelular é o mais frequente câncer primário do fígado e já se tornou a quinta neoplasia mais comum no mundo e a segunda causa de óbitos relatados por câncer. Estima-se que 2/3 das neoplasias humanas poderiam ser prevenidos pela modificação do estilo de vida, incluindo a dieta. Estudos farmacológicos intensificaram-se nos últimos anos com plantas da família Annonaceae, cujo interesse científico está nas acetogeninas, uma classe de compostos derivados de ácidos graxos com forte atividade antitumoral. Os experimentos foram conduzidos no Laboratório de Infectologia Molecular Animal, do Departamento de Bioquímica e Biologia Molecular, na Universidade Federal de Viçosa, para avaliar in vitro as propriedades antitumorais de extratos foliares de 11 Anonáceas (9 espécies de 4 gêneros). Para isso, células tumorais hepáticas humanas da linhagem HepG2 (ATCC HB-8065 ) foram expostas a três concentrações dos extratos foliares de Anonáceas e do quimioterápico 5- Fluoruracil (5-FLU), em três períodos de tempo (24, 48 e 72 h), com análise espectrofotométrica da viabilidade celular pela metodologia do corante vermelho neutro. Foram conduzidos ensaios para avaliar o efeito dos extratos vegetais e do quimioterápico sobre o fluxo químico celular no meio de cultura durante os cultivos através da análise por Espectrometria de Emissão Atômica de diferentes especies químicas, e sobre o metabolismo e integridade celular, através da análise de marcadores tumorais e intermediários metabólicos. Todos os ensaios induziram à morte celular, com resultados distintos entre plantas da mesma espécie, com até 100% de citotoxicidade, sendo que nove Anonáceas exibiram efeito citotóxico com até 1/8 da concentração daquela utilizada para o quimioterápico 5-FLU. Nenhum dos ensaios com extratos foliares exibiu retomada da proliferação celular após 48 ou 72 h, o que foi verificado para os cultivos com o quimioterápico, clara evidência de quimiorresistência tumoral. O mecanismo de morte celular por apoptose foi confirmado através da citometria de fluxo para dois dos extratos escolhidos, incluindo 5-FLU. Não houve um padrão no efeito dos extratos sobre o fluxo de íons ou dos marcadores tumorais durante os cultivos das células hepatocarcinogênicas, que se mostrou variável dentro da espécie ou entre elas, ou dentro de um mesmo gênero ou entre eles, com interferência na absorção de alguns íons especialmente, Ca, Na, P, S e depleção intracelular total de outros (Se, Zn) para alguns dos tratamentos. Marcadores bioquímicos como aspartato aminotransferase (AST), alanina aminotransferase (ALT) e lactato desidrogenase (LDH) tiveram seus níveis elevados para alguns dos extratos utilizados, incluindo 5-FLU, resultado coincidente com a redução na absorção da glicose pelos hepatócitos tumorais, indícios da alteração no metabolismo e integridade celular. / The hepatocellular carcinoma is the most common primary liver cancer and has become the fifth most common cancer worldwide and the second leading cause of cancer deaths reported. It is estimated that two thirds of human cancers could be prevented by changes in lifestyle, including diet. Pharmacological studies have intensified in recent years with the Annonaceae plant family, whose scientific interest is in the acetogenins, a class of compounds derived from fatty acids with strong antitumor activity. The experiments were conducted at the Laboratory of Molecular Animal Infectious Diseases, Department of Biochemistry and Molecular Biology, Federal University of Viçosa, to evaluate the in vitro antitumor properties of leaf extracts of 11 annonaceae (9 species of 4 genera). For this, human liver cancer cells HepG2 line (ATCC HB-8065 ) were exposed to three different concentrations of leaf extracts of Annonaceae and of the chemotherapeutic 5-fluorouracil (5-flu) in three time periods (24, 48 and 72 h), with spectrophotometric analysis of cell viability by the neutral red dye method. Assays were conducted to evaluate the effect of plant extracts and chemotherapeutic chemical flow cell on the culture medium during the cultivation by atomic emission spectrometry analysis of different chemical species, and the cellular integrity and metabolism, by analysis of tumor markers and metabolic intermediates. All assays induced cell death, with results between different plants of the same species, with up to 100% cytotoxicity, nine annonacea exhibited cytotoxic effect up to one eighth of the concentration of that used for the chemotherapeutic 5-flu. None of the trials with leaf extracts exhibited recovery of cell proliferation after 48 or 72 h, which was observed in cultures with chemotherapy, clear evidence of tumor chemoresistance. The mechanism of cell death by apoptosis was confirmed by flow cytometry for two extracts selected, including 5-flu. There was no pattern in the effect of the extracts on the flow of ions or of tumor markers during the cultivation of hepatic ocarcinogenic cells, which was variable within species or between or within the same genus or between them, interfering with the absorption of especially some ions, Ca, Na, P, S, and total depletion of other intracellular (Se, Zn) for some treatments. Biochemical markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) had their elevated levels for some of the extracts used, including 5-flu coincident result with a reduction in glucose uptake by tumor hepatocytes, evidence of changes in metabolism and cellular integrity.

Ingeniero Civil en Biotecnología / Ingeniero Civil Químico / Debido a que el hígado posee funciones únicas, es de gran interés desarrollar herramientas que permitan conseguir un mejor entendimiento global del comportamiento de las células hepáticas bajo cambios de las condiciones de crecimiento causado por agentes externos, tales como cambios en la suplementación de la fuente de carbono o la utilización de drogas en el medio de cultivo. El objetivo de este de esta memoria es establecer una metodología que permita realizar estudios metabólicos en células de origen hepático, con herramientas in silico que complemente los trabajos de investigaciones in vitro. Para ello, se utilizará la línea celular HepG2, ya que presenta ventajas frente a otras células de origen hepático, como su alta capacidad proliferativa y su fenotipo estable. Junto a esto se diseñó un modelo hepático para describir el metabolismo intracelular, que considera 37 metabolitos y 32 reacciones, abarcando las principales vías metabólicas de una célula hepática. En un primer estudio experimental se analizó como afecta en el metabolismo de las células la aplicación de agente nocivo acetaminofén (APAP), y si el daño es contrarrestable utilizando N-acetilcisteína (NAC) como agente protector. Como resultado se obtuvo que la concentración utilizada de APAP en el medio (1 [mM]) es altamente tóxica ya que frena de inmediato el crecimiento celular, entrando a una fase de muerte celular. Del análisis de flujo metabólico aplicado a este caso se obtuvo que existe una alta tasa específica de producción de lactato, y por ende, los flujos son redireccionados para que aumenten los niveles de piruvato intracelular a partir de diversa fuentes de carbono además de la glucosa (triptófano y triglicéridos), y sea utilizado en la síntesis de lactato. Con respecto a la protección, sólo fue posible obtener una curva de crecimiento comparable al control cuando se utilizó NAC (5 [mM]) 3 horas después que se administrara APAP. La aplicación previa tuvo el mismo resultado que el caso en que sólo se utiliza APAP. El análisis de flujo metabólico del único caso en que hubo una protección efectiva está sujeto a errores de cálculos de tasas específicas, al igual que el caso control. Grandes desbalances de carbono y nitrógeno, junto con una distribución de flujos internos con comportamientos no esperados, no permiten comparar los resultados entre los casos. Sin embargo, este hecho refleja lo importante que es tener mediciones precisas y un buen diseño experimental para calcular las tasas específicas de consumo/producción, para así obtener un correcto funcionamiento del modelo hepático planteado. En el segundo estudio, se analizaron los cambios metabólicos que se generan al utilizar diferentes suplementaciones del carbohidrato en el medio. Los casos fueron una suplementación pura de glucosa, una pura de fructosa y una mezcla de ellas (en una razón 1:1). Al realizar las curvas de crecimiento, se obtuvieron parámetros de crecimiento similares, siendo el caso de fructosa pura en donde las células crecen a menor velocidad, dado que están acostumbradas a crecer en un medio con glucosa. Este hecho se ratifica al realizar el análisis de flujo metabólico, donde el caso de mayor consumo de hexosa, producción de lactato y síntesis de triglicéridos fue el caso en que se suplementó exclusivamente con glucosa. En los casos en que existía fructosa en el medio, las células presentaron una tasa menor de consumo de azúcar, y por consiguiente una menor producción de triglicéridos. Además en estos dos casos, los flujos hacia síntesis de lactato y síntesis de triglicéridos son más bajo, pero con un flujo permanente en el ciclo TCA cercano al doble que el caso de suplementación con glucosa pura. El modelo hepático fue capaz representar el metabolismo de la línea celular HepG2 en ambos estudios. Sin embargo, debido a los resultados del primero, es necesario realizar más ensayos de toxicidad de sustancias, para validar el modelo y la metodología empleada en este trabajo, con el fin de caracterizar y mejorar funciones hepáticas en sistemas de cultivo in vitro.

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.

Der Scavenger Receptor BI (SR-BI) vermittelt den selective lipid transfer von Cholesterol und Vitamin E aus HDL in die Leber. Die zelluläre Aufnahme verschiedener Lipide aus HDL über den selben Mechanismus, vermittelt durch den selben Rezeptor wirft die Frage auf, ob diese Aufnahmeprozesse einander beeinflussen. Aktuelle Forschungsergebnisse zeigen, daß die Aufnahme von neutralen Lipiden (Cholesterolester, Triacylglycerol) aus HDL in die Zelle von der Lipidzusammensetzung der Donorpartikel abhängen könnte. Wir untersuchten, ob der Vitamin-E-Gehalt von HDL die Aufnahme und den Efflux von Cholesterol in und aus HepG2-Zellen beeinflußt. Die Inkubation von HepG2-Zellen mit [3H]Cholesterol-markiertem HDL mit ansteigendem Vitamin-E-Gehalt ergab eine steigende Aufnahme von Vitamin E, während sich die Cholesterolaufnahme nicht veränderte. Der erhöhte zelluläre Gehalt an Vitamin E bewirkte eine Reduktion der PKC-Alpha- und SR-BI-Expression in Verbindung mit einem erniedrigten Cholesterolefflux aus HepG2-Zellen zu nativem HDL als Akzeptorpartikel. Die Verarmung der Zellen an Cholesterol führte zu einer erniedrigten PKC-Alpha- und SR-BI-Expression. Hingegen veränderte die Erhöhung des zellulären Cholesterolgehalts von HepG2-Zellen die PKC-Alpha- und SR-BI-Expression nicht, während der Cholesterolefflux aus HepG2-Zellen zu HDL im Vergleich zur Kontrolle gesteigert war. Wir schließen aus diesen Ergebnissen, daß Vitamin E und Cholesterol die SR-BI-Expression modulieren können und daß SR-BI den Efflux und möglicherweise auch den Influx von Cholesterol in HepG2-Zellen vermittelt. / The scavenger receptor BI (SR-BI) mediates selective lipid transfer of cholesterol and vitamin E from HDL to the liver. Cellular uptake of different lipids from HDL by the same mechanism, mediated by the same receptor rise the question, whether these uptake processes affect each other. Recent results show that the cellular uptake of neutral lipids (cholesterol ester, triacylglycerol) from HDL may depend on the lipid composition of the donor particles. We investigated whether the vitamin E-content of HDL affects cholesterol uptake and efflux by HepG2 cells. Incubation of HepG2 cells with [3H]cholesterol-labeled HDL, which contained increasing vitamin E-concentrations resulted in an increased uptake of vitamin E, whereas the cholesterol uptake did not change. The increased cellular content of vitamin E caused a decreased PKCalpha and SR-BI-expression combined with a decreased cholesterol-efflux from HepG2 cells to native HDL as acceptor. Depletion of cellular cholesterol decreased PKC-alpha and SR-BI-expression in HepG2 cells. Increase of cellular cholesterol of HepG2 cells, however, did not change PKCalpha and SR-BI-expression, whereas cholesterol-efflux from HepG2 cells to HDL increased. We conclude that vitamin E and cholesterol can modulate the SR-BI-expression and that SR-BI mediates the efflux and possibly also the influx of cholesterol by HepG2 cells.

A doença hepática gordurosa não-alcoólica (NAFLD, do inglês) é a manifestação clínica hepática da síndrome metabólica, cuja incidência aumenta consideravelmente em todo o mundo. A NAFLD pode progredir para um estado de esteatohepatite não-alcoólica (NASH, do inglês), caracterizado por inflamação hepatocelular, com ou sem fibrose. Dados na literatura mostram que o coativador-1 alfa do receptor ativado por proliferadores de peroxissoma gama (PGC-1alfa), além de estar envolvido em diversos processos metabólicos, representa uma estratégia terapêutica promissora na modulação da inflamação. Neste projeto investigamos as alterações inflamatórias no fígado induzida por dieta hiperlipídica e o papel do PGC-1alfa nesse processo. Camundongos C57black/6 receberam dieta hiperlipídica contendo 30% de gordura por 10 semanas. O peso dos animais foi avaliado semanalmente. Após a eutanásia, o tecido adiposo intra-abdominal (retroperitoneal e periepididimal) foi coletado e pesado. Analisamos o perfil glicêmico e lipídico sérico e expressão de genes envolvidos no metabolismo glicêmico e lipídico. Avaliou-se também o aspecto histológico e a inflamação do tecido hepático por quantificação das citocinas IL-6, TNF-alfa e IL-1beta. A dieta rica em gordura conduziu a um aumento dos depósitos de gordura intra-abdominal, hiperglicemia e hiperlipidemia. Os animais também apresentavam esteatohepatite, com aumento de citocinas pró-inflamatórias e diminuição na expressão de PGC-1alfa no tecido hepático. O envolvimento do PGC-1alfa na produção de mediadores inflamatórios por hepatócitos foi avaliado em células HepG2 utilizando RNA de interferência (RNAi). O knockdown da expressão de PGC-1alfa causou aumento na expressão e liberação de IL-6 em hepatócitos via aumento na fosforilação de IkBalfa e consequente ativação do NFkB. Portanto, nossos dados mostram que o PGC-1alfa inibe a produção de mediadores inflamatórios (IL-6) em hepatócitos, e fornecem novas evidências das conexões existentes entre as vias metabólicas e imunes / Nonalcoholic fatty liver disease (NAFLD) is the liver clinical manifestation of the metabolic syndrome, whose incidence increases considerably around the world. NAFLD may progress to a state of non-alcoholic steatohepatitis, characterized by hepatocellular inflammation, with or without fibrosis. Data in the literature show that the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alfa), a protein involved in various metabolic processes, represents a promising therapeutic strategy in the modulation of inflammation. In this project, we investigate the inflammatory changes in the liver induced by high fat diet and the role of PGC-1alfa in this process. C57black/6 mice were fed a high fat diet containing 30% fat for 10 weeks. The weight of the animals was measured weekly. After euthanasia, the intra-abdominal adipose tissue (retroperitoneal and periepididymal) was collected and weighed. We have analyzed the glycemic and serum lipid profile and expression of genes involved in glucose and lipid metabolism. We also assessed liver morphology by HE staining and inflammation by quantification of the cytokines IL-6, TNF-alfa and IL-1beta. The high fat diet led to an increase in intra-abdominal fat deposits, hyperglycemia and hyperlipidemia. Animals also presented steatohepatitis, an increased proinflammatory cytokines and decreased PGC-1alfa expression in the hepatic tissue. The involvement of PGC-1alfa on inflammatory mediators production by hepatocytes was evaluated in HepG2 cells using RNA interference (RNAi). The knockdown of PGC-1alfa expression caused an increase in IL6 expression and release by hepatocytes via the increase in the IkBalfa phosphorylation and subsequent activation of NFkB. Therefore, our data show that PGC-1alfa inhibits the production of inflammatory mediators (IL-6) in hepatocytes and provide further evidence of the connections between the metabolic and immune pathways

A doença hepática gordurosa não-alcoólica (NAFLD, do inglês) é a manifestação clínica hepática da síndrome metabólica, cuja incidência aumenta consideravelmente em todo o mundo. A NAFLD pode progredir para um estado de esteatohepatite não-alcoólica (NASH, do inglês), caracterizado por inflamação hepatocelular, com ou sem fibrose. Dados na literatura mostram que o coativador-1 alfa do receptor ativado por proliferadores de peroxissoma gama (PGC-1alfa), além de estar envolvido em diversos processos metabólicos, representa uma estratégia terapêutica promissora na modulação da inflamação. Neste projeto investigamos as alterações inflamatórias no fígado induzida por dieta hiperlipídica e o papel do PGC-1alfa nesse processo. Camundongos C57black/6 receberam dieta hiperlipídica contendo 30% de gordura por 10 semanas. O peso dos animais foi avaliado semanalmente. Após a eutanásia, o tecido adiposo intra-abdominal (retroperitoneal e periepididimal) foi coletado e pesado. Analisamos o perfil glicêmico e lipídico sérico e expressão de genes envolvidos no metabolismo glicêmico e lipídico. Avaliou-se também o aspecto histológico e a inflamação do tecido hepático por quantificação das citocinas IL-6, TNF-alfa e IL-1beta. A dieta rica em gordura conduziu a um aumento dos depósitos de gordura intra-abdominal, hiperglicemia e hiperlipidemia. Os animais também apresentavam esteatohepatite, com aumento de citocinas pró-inflamatórias e diminuição na expressão de PGC-1alfa no tecido hepático. O envolvimento do PGC-1alfa na produção de mediadores inflamatórios por hepatócitos foi avaliado em células HepG2 utilizando RNA de interferência (RNAi). O knockdown da expressão de PGC-1alfa causou aumento na expressão e liberação de IL-6 em hepatócitos via aumento na fosforilação de IkBalfa e consequente ativação do NFkB. Portanto, nossos dados mostram que o PGC-1alfa inibe a produção de mediadores inflamatórios (IL-6) em hepatócitos, e fornecem novas evidências das conexões existentes entre as vias metabólicas e imunes / Nonalcoholic fatty liver disease (NAFLD) is the liver clinical manifestation of the metabolic syndrome, whose incidence increases considerably around the world. NAFLD may progress to a state of non-alcoholic steatohepatitis, characterized by hepatocellular inflammation, with or without fibrosis. Data in the literature show that the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alfa), a protein involved in various metabolic processes, represents a promising therapeutic strategy in the modulation of inflammation. In this project, we investigate the inflammatory changes in the liver induced by high fat diet and the role of PGC-1alfa in this process. C57black/6 mice were fed a high fat diet containing 30% fat for 10 weeks. The weight of the animals was measured weekly. After euthanasia, the intra-abdominal adipose tissue (retroperitoneal and periepididymal) was collected and weighed. We have analyzed the glycemic and serum lipid profile and expression of genes involved in glucose and lipid metabolism. We also assessed liver morphology by HE staining and inflammation by quantification of the cytokines IL-6, TNF-alfa and IL-1beta. The high fat diet led to an increase in intra-abdominal fat deposits, hyperglycemia and hyperlipidemia. Animals also presented steatohepatitis, an increased proinflammatory cytokines and decreased PGC-1alfa expression in the hepatic tissue. The involvement of PGC-1alfa on inflammatory mediators production by hepatocytes was evaluated in HepG2 cells using RNA interference (RNAi). The knockdown of PGC-1alfa expression caused an increase in IL6 expression and release by hepatocytes via the increase in the IkBalfa phosphorylation and subsequent activation of NFkB. Therefore, our data show that PGC-1alfa inhibits the production of inflammatory mediators (IL-6) in hepatocytes and provide further evidence of the connections between the metabolic and immune pathways