<p> Per- and polyfluoroalkyl substances (PFAS) are a class of persistent environmental contaminants that have become ubiquitous, resulting in widespread exposure among humans and wildlife. Amphibians are regularly exposed in the field, making them susceptible to sublethal effects of PFAS exposure. In amphibians exposed to PFAS, deleterious effects have been observed, including reduction in body condition measured using the scaled mass index (SMI) and degraded hepatic condition, among others. PFAS may dysregulate lipid metabolism by altering signaling cascades regulated by peroxisome proliferator activated receptors (PPAR), but whether changes in energy stores can explain changes in amphibian SMI and/or hepatic condition remain underexplored. Since lipids are a critical energy reserve for anurans, understanding whether lipid metabolism is being perturbed is critical. The central objective of this thesis was to investigate the effect of PFAS on lipid homeostasis in <em>Xenopus laevis </em>tadpoles within the context of a PPAR mechanism of action (MOA), considering apical, molecular, and lipidomic endpoints. I conducted three studies: (a) a study to characterize SMI and the relative expression of the hepatic xPPARα/β/γ during metamorphosis, (b) a pharmaceutical exposure to assess the <em>in vivo</em> effects of xPPARα/β/γ agonism on hepatic gene expression for select downstream targets (<em>apoa5, fabp1, acox1,​ pck1</em>), and (c) a chronic PFAS exposure to investigate the effects of environmentally relevant concentrations (PFOS, PFHxS, PFOA, PFHxA at 0.5 ppb; binary mixture of PFOS:PFHxS at 1 ppb) on lipid homeostasis through apical endpoints (mass, snout vent length, SMI, hepatic condition), relative hepatic gene expression, and Multiple Reaction Monitoring (MRM) profiling of the hepatic lipidome for changes in relative class abundance. In study (a), I identified SMI and hepatic expression of <em>xPPARα/β/γ</em> is dynamic during late metamorphosis, indicating the potential for heightened susceptibility. However, in study (b), pharmaceutical agonists had no effect on <em>X. laevis</em> at high doses. For study (c), I did not observe effects on a majority of apical endpoints, including SMI, but detected a significant sex-specific reduction in hepatic condition for male<em> X. laevis</em> tadpoles exposed to single-chemical perfluorosulfonic acid (PFSA) treatments. For gene expression, I observed a transient downregulation for apolipoprotein-V (<em>apoa5</em>) at Nieuwkoop and Faber (NF) stage 62 for <em>X. laevis</em> tadpoles exposed to single-chemical perfluorocarboxylic acid (PFCA) treatments. Lipid profiling detected transient dysregulation of predominantly membrane lipids in-response to short-chain PFAS treatments at NF 58. Overall, our findings indicate PFAS may exert toxicity during anuran metamorphosis through multiple mechanisms of action (MOA) with sex-specific and developmental-stage specific outcomes.</p>

Background and aims: Treatment of acute fulminant liver damage arising as a result of various origins (ischemia-reperfusion injury, toxic shock, an infectious cause or cholestasis) still remains a major clinical problem. We currently do not have available clinically proven, pharmacologically effective and universal compound for the treatment of acute liver injury. The main aim of my research work was, therefore, to test the potential hepatoprotective effect of selected cytoprotective drugs and try to find out or suggest their mechanism of action, which we have examined in the systems for the intracellular gaseous signaling molecules NO and CO, where the key enzymes for their formation are NOS / HO respectively. My PhD study had two main directions: 1) Experimental study of the relationship between HO / CO and NOS / NO systems in the environment of hepatotoxic substances on isolated primary rat hepatocytes and in rat model, 2) Evaluation of ameliorative effect of selected substances in the hepatotoxicity models and to test the relationship of this effect on changes in some parameters of cytotoxicity / cytoprotection, antioxidant parameters, gene expression of mRNA for selected genes and histological changes in the state of cells / tissues / organs. Methods: We measured urea, bilirubin and liver...

Made available in DSpace on 2014-07-29T16:11:52Z (GMT). No. of bitstreams: 1 dissertacao guilherme.pdf: 2090110 bytes, checksum: a1c991816c551ebd8ad069c1df1f9f72 (MD5) Previous issue date: 2008-09-25 / The araticum (Annona coriacea Mart.) is a typical fruit of the brazilian cerrado used popularly to overcome inflammatory processes. The family Annonaceae presents as the main constituents acetogenins, a class of substances with great cytotoxic and genotoxic potential, and cited as responsible for a disease similar to parkinson in a Caribbean population that used the fruit as much as food and for medicinal purposes. The aim of this study was to evaluate the putative activity of crude ethanolic extract of seeds of A. coriacea on the cerebral cortex of mice exposed at doses of 12.5, 25, 50 and 100 mg / kg, and evaluate its activity on different areas of the brain, the liver and kidneys. We used 30 adult male Swiss mice divided into groups control, solvent and treated (12.5, 25, 50 and 100 mg / kg). The extract was administered orally for four days. The target organs were extirpated, fixed in 70% ethanol (v / v) and processed for histological method - hematoxylin and eosin. The analysis of the slides was performed by image processing system for counting cells and other morphometric analysis. The morphological studies showed no significant changes to the brain in different areas, just as no changes were detected in the kidneys. On the other hand, was found a reduction on the frequency of cells per area of the liver, like as an reduction on the consumption of food, water and production of excreta. We conclude with this work a possible hepatotoxic activity induced by exposure to crude ethanol extract of seeds of A. Coriacea Mart., observed by the decrease in frequency of cells per area of the liver, correlated with reductions in consumption of food / water and production of excreta by animals. / O Araticum (Annona coriacea Mart.) é um fruto típico do cerrado brasileiro utilizado popularmente para remediar processos inflamatórios. A família Annonaceae apresenta como principais constituintes as acetogeninas, uma classe de substâncias com grande potencial citotóxico, genotóxico e ainda citado como o responsável por uma doença similar ao parkinsonismo em uma população caribenha que utilizava o fruto tanto como alimento quanto para fins medicinais. O objetivo deste estudo foi avaliar a putativa atividade do extrato bruto etanólico das sementes da A. coriacea sobre o córtex cerebral de camundongos expostos nas doses de 12,5; 25; 50 e 100 mg/kg, além de avaliar sua atividade sobre diferentes áreas do encéfalo, sobre o fígado e rins. Foram utilizados 30 camundongos Swiss machos adultos divididos em grupos controle, solvente e tratados (12,5; 25; 50 e 100 mg/kg). O extrato foi administrado por via oral durante quatro dias. Os órgãos-alvo foram extirpados, fixados em etanol 70% (v/v) e processados para método histológico - hematoxilina e eosina. A análise das lâminas foi executada via sistema de processamento de imagens para a contagem das células e demais análises morfométricas. Os estudos morfológicos não demonstraram alterações significativas para o cérebro em suas diferentes áreas, da mesma forma que não foram detectadas alterações nos rins. Por outro lado, foi detectado uma redução da freqüência de células por área do fígado, assim como verificamos uma diminiução no consumo de ração, água e produção de excreta. Concluimos com este trabalho uma possível atividade hepatotóxica induzida pela exposição ao extrato bruto etanólico das sementes da A. Coriacea Mart., observada pela diminuição da freqüência de células por área do fígado, correlacionado com reduções de consumo de ração/água e produção de excretas pelos animais.

Abstract The hepatic inducible Cytochrome P450s (CYPs) generally prime xenobiotics for elimination. Murine CYP2A5 and human CYP2A6 share similar xenobiotic substrates and some regulatory features. Recently, they were shown to oxidize bilirubin, a byproduct of heme catabolism and a dose-dependent anti- or pro-oxidant, to biliverdin. In this study, the putative role of the redox-sensitive, cytoprotective transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the regulation of hepatic Cyp2a5 expression and induction under diverse hepatotoxic conditions and altered heme homeostasis was characterized. The coordination of Cyp2a5 and the Nrf2 target gene Heme oxygenase-1 (Hmox1), which determines bilirubin formation from heme, responses to heavy metals and modulators of heme homeostasis, was studied in cultured wildtype and Nrf2(-/-) mouse primary hepatocytes. Nrf2 was essential for the basal hepatic expression of CYP2A5 in the endoplasmic reticulum (ER) and mitochondria, as well as for its induction by cadmium, lead, methyl mercury and phenethyl isothiocyanate. A functional Nrf2 binding antioxidant response element (ARE) about -2.4 kilobases upstream of the Cyp2a5 transcriptional start site was identified. In contrast to Hmox1, a target of BTB and CNC homology 1 (Bach)-mediated repression via AREs, the regulation of Cyp2a5 did not clearly involve Bach1. Excessive heme induced mainly ER-localized CYP2A5 via Nrf2, which was limited by the Nrf2-independent HMOX1 induction. In heme synthesis blockades, CYP2A5 was enhanced via Nrf2 and additional factors, such as the peroxisome proliferator-activated receptor &#947; coactivator-1&#945; (PGC-1&#945;). The typical CYP2A5 inducers phenobarbital, dibutyryl-cyclic adenosine monophosphate (db-cAMP) and PGC-1&#945; enhance heme synthesis; CYP2A5 was induced via Nrf2 in acute but not chronic phenobarbital exposure without a clear connection to heme, while the responses to db-cAMP and PGC-1&#945; were sensitized in the absence of Nrf2. This suggests novel crosstalk between Nrf2 and PGC-1&#945;. In this study, Cyp2a5 was identified as a sensitive indicator of hepatic Nrf2 pathway activation that could be used, e.g. for in vitro screening of drug candidate hepatotoxicity. The similar subcellular localization and coordination of CYP2A5 and HMOX1 expression in altered heme metabolism support the postulated role for CYP2A5 in bilirubin homeostasis. / Tiivistelmä Vierasaineet stimuloivat maksan Sytokromi P450 (CYP)-entsyymejä, mikä yleensä lisää niiden eliminaatiota. Hiiren CYP2A5 ja ihmisen CYP2A6 ovat lähisukua katalyyttisten ja osin säätelyllisten yhteneväisyyksiensä puolesta. Vastikään niiden osoitettiin katalysoivan hemin hajoamistuotteen, bilirubiinin hapettumista biliverdiiniksi, mikä saattaisi säädellä sen annosriippuvaisia vaikutuksia antioksidanttina ja oksidanttina. Työssä tutkittiin solustressiä aistivan, suojaavan transkriptiotekijän Nrf2 osuutta Cyp2a5-geenin aktivaatiossa maksatoksisissa olosuhteissa ja hemimetabolian muutoksissa. Cyp2a5:n ja bilirubiinin tuotosta vastaavan, Nrf2-säädellyn Hemioksigenaasi-1 (Hmox1):n vasteita verrattiin viljellyissä villityypin ja poistogeenisen Nrf2(-/-) hiiren primaarimaksasoluissa. Tulokset osoittavat, että Nrf2 ylläpitää CYP2A5:n ilmentymistä endoplasmisella kalvostolla (ER) ja mitokondrioissa sekä välittää sen stimulaation altisteilla kadmium, lyijy, metyylielohopea ja fenetyyli-isotiosyanaatti. Toimiva Nrf2-vasteinen antioksidanttivaste-elementti (ARE) tunnistettiin n. -2,4 kiloemäsparia Cyp2a5-geenin luennan aloituskohdasta ylävirtaan. BTB ja CNC homologia 1 (Bach1)-tekijä, joka on tärkeä Hmox1-säätelijä ja ARE-välitteinen transkription estäjä, ei selkeästi osallistu Cyp2a5:n säätelyyn. Hemin ylimäärä stimuloi CYP2A5:n määrää ER-kalvostolla, Nrf2-riippumattomasti stimuloituvan HMOX1 rajoittaessa Nrf2-reitin aktivaatiota. Hemisynteesin estyessä Nrf2 aktivoi Cyp2a5-geeniä muiden mekanismien kuten peroksisomiproliferaattori-aktivoituva reseptori gamman koaktivaattori-1&#945; (PGC-1&#945;) kanssa. Fenobarbitaali (PB), dibutyryyli-syklinen adenosiinimonofosfaatti (db-cAMP) ja PGC-1&#945; lisäävät tunnetusti hemisynteesiä. Nrf2 havaittiin Cyp2a5:n aktivaatiolle välttämättömäksi akuutissa mutta ei kroonisessa PB-altistuksessa ilman selkeästi havaittua hemin osuutta. Cyp2a5-geenin db-cAMP- ja PGC-1&#945;-vasteinen stimulaatio voimistui merkittävästi toimivan Nrf2-reitin puuttuessa, mikä osoittaa vuoropuhelua Nrf2 ja PGC-1&#945; välillä. Väitöskirjatyössä Cyp2a5 tunnistettiin herkäksi Nrf2-reitin aktivaation maksamarkkeriksi, jota voitaisiin hyödyntää esim. lääkeainekandidaattien maksatoksisuuden seulonnassa soluviljelyssä. CYP2A5:n ja HMOX1:n solunsisäinen kohdentuminen ja ekspressio koordinoituvat hemimetabolian muutoksissa, mikä tukee teoriaa CYP2A5:n roolista bilirubiinin metaboliassa maksassa.

Les valeurs toxicologiques de référence pour les contaminants de l’environnement sont généralement basées sur les résultats d’études animales dont la pertinence pour l’humain est remise en question. L’objectif de mon projet était d’évaluer une approche combinant les études in vitro sur des cellules humaines et la modélisation pharmacocinétique pour établir des valeurs toxicologiques de référence. Nous avons mené une étude de cas sur l’hépatotoxicité de l’acide perfluorooctanoïque (APFO). Un point de départ (POD) a été déterminé sur la base des concentrations sans effet néfaste observable (NOAEL) d’études in vitro. Ce POD a été traduit en équivalents de surveillance biologique, qui ont été comparés aux concentrations mesurées dans les études épidémiologiques sur l’exposition à l’APFO et l’hépatotoxicité. Des apports quotidiens tolérables (AQT) ont été estimés par dosimétrie inverse à l’aide d’un modèle pharmacocinétique. Nous avons utilisé un POD de 1 μM des études in vitro, qui a été traduit en une concentration plasmatique de 414 ng/mL. Pour atteindre cette concentration chez l’enfant exposé prénatalement et par l’allaitement, nous avons estimé que la concentration plasmatique chez la mère au début de la grossesse doit être de 155 ng/mL. Ces concentrations ont été traduites en équivalents de biosurveillance de 4,14 ng/mL (adulte) et 1,55 ng/mL (femme enceinte) (facteurs d’incertitude combinés : 100 à partir du POD), des valeurs se situant dans l’intervalle des concentrations médianes dans les études épidémiologiques (0,49 à 16 ng/mL). Des AQT de 0,121 (hépatoxicité chez l’adulte) et 0,047 ng/kg/jour (hépatotoxicité développementale) ont également été obtenus (facteurs d’incertitude combinés : 300) à partir du même POD. En conclusion, les résultats laissent croire que l’approche évaluée offre une alternative adéquate et prudente pour l’établissement de valeurs toxicologiques de référence. / Acceptable exposure levels for environmental contaminants are generally calculated based on animal studies, but the scientific community is currently questioning their relevance to humans. The objective of this project was to evaluate a new approach combining in vitro studies on human cells and pharmacokinetic modeling to derive acceptable exposure levels. We conducted a case study on the hepatotoxicity of perfluorooctanoic acid (PFOA). We determined a point of departure (POD) based on no observable adverse effect levels (NOAELs) from in vitro studies using human cells. We then translated this POD into a biomonitoring equivalent and compared it to concentrations measured in epidemiological studies on PFOA exposure and hepatotoxicity. A Tolerable Daily Intake (TDI) was estimated using a pharmacokinetic model. We used a POD of 1 μM from in vitro studies and translated it into a 414 ng/mL plasma concentration. To reach this concentration in children exposed prenatally and by breastfeeding, we estimated that the plasma concentration in the mother during pregnancy must be 155 ng/mL. We translated these concentrations into biomonitoring equivalents of 4.14 ng/mL (adult) and 1.55 ng/mL (pregnant women) when using a composite uncertainty factor of 100. These biomonitoring equivalents are within the range of median concentrations in epidemiologic studies (0.49 to 16 ng/mL). TDIs of 0.121 and 0.047 ng/kg/day for adult hepatoxicity and developmental hepatotoxicity, respectively, were estimated using a composite uncertainty factor of 300. In conclusion, the results suggest that the evaluated approach offers an adequate and conservative alternative to derive acceptable exposure levels.

Background & Aims: Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. The aim of this work is to find early signal markers of drug-induced injury of the liver by focusing on select antioxidant/oxidant and apoptotic genes. As well, to address the relationship between conventional liver dysfunction markers and the measured mRNA and protein expressions in the D-galactosamine/lipopolysaccharide and tert-butylhydroperoxide hepatotoxicity models. Furthermore, potential hepatoprotective capabilities of antioxidant polyphenols quercetin and curcumin were evaluated in relation to its modulation of the oxidative stress and apoptotic parameters in the given xenobiotic hepatotoxicity models. Methods: Biochemical markers testing the hepatic function included aminotransferases (ALT, AST) and bilirubin. Measurements of TBARS and conjugated dienes were used to assess lipoperoxidation. Plasma levels of catalase and reduced glutathione were used as indicators of the oxidative status of the cell. Real time PCR was used to analyse the mRNA expressions of the inducible nitric oxide synthase (NOS-2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD-1), glutathione peroxidase (Gpx-1), caspase 3 (Casp3), BH3 interacting domain death agonist (Bid) and Bcl-2...