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Hepatotoxicity is a serious adverse health effect caused by drugs and other chemical toxins generally detected in the later stages of drug development or in whole animal studies. Thus, development of screening approaches available for earlier identification of hepatotoxic molecules is necessary. A novel in vitro- in silico test system for the evaluation of the molecular mechanisms of xenobiotic toxicity in primary hepatocyte systems is presented here. It is well established that hepatocytes in vitro are most representative of hepatotoxicity in vivo, and are most useful for the determination of xenobiotic hepatotoxicity mechanisms at the molecular and cellular level. There is an on-going interest in Quantitative Structure-Activity Relationships (QSAR) in toxicology, as it can identify correlations between chemical structure and biological activity. QSAR can be used to evaluate the effects of metabolism and toxicity as many physicochemical descriptors reflect simple molecular properties that can provide insight into the physicochemical nature of the activity under consideration. QSARs were determined for hepatotoxicity of halobenzenes, p-benzoquinones, α,β-unsaturated carbonyl compounds and nitroaromatics towards isolated hepatocytes. A molecular link was established for their proposed toxicity pathways. For example oxidative activation was linked to EHOMO (energy of the highest occupied molecular orbital) values and hydrophobicity (log P) of the chemicals, while reductive activation was linked with ELUMO (energy of the lowest molecular orbital) values and log P. Such relationships may thus be useful for predicting toxicity of other chemicals of the same mechanism of toxicity. Due to the complexity involved in the phenomena of hepatotoxicity, unravelling of structure-hepatotoxicity relationships is a complicated task. A conceptual framework for QSAR modeling is proposed that involves recognition of molecular initiating events as potential endpoints to improve the prediction potential of QSAR models. Acute toxicity of reactive chemicals could be based on an initial reaction with biomolecules, thus the theory of covalent binding reactivity was used to test this concept. Reactivity assays with thiol and amine surrogate nucleophiles were used to determine susceptibility to toxicity. The derived QSAR expressions suggested that covalent binding reactivity is a good correlate to hepatotoxicity, however only if electrophilicity was the main mechanism of toxicity.

Hepatotoxicity is a serious adverse health effect caused by drugs and other chemical toxins generally detected in the later stages of drug development or in whole animal studies. Thus, development of screening approaches available for earlier identification of hepatotoxic molecules is necessary. A novel in vitro- in silico test system for the evaluation of the molecular mechanisms of xenobiotic toxicity in primary hepatocyte systems is presented here. It is well established that hepatocytes in vitro are most representative of hepatotoxicity in vivo, and are most useful for the determination of xenobiotic hepatotoxicity mechanisms at the molecular and cellular level. There is an on-going interest in Quantitative Structure-Activity Relationships (QSAR) in toxicology, as it can identify correlations between chemical structure and biological activity. QSAR can be used to evaluate the effects of metabolism and toxicity as many physicochemical descriptors reflect simple molecular properties that can provide insight into the physicochemical nature of the activity under consideration. QSARs were determined for hepatotoxicity of halobenzenes, p-benzoquinones, α,β-unsaturated carbonyl compounds and nitroaromatics towards isolated hepatocytes. A molecular link was established for their proposed toxicity pathways. For example oxidative activation was linked to EHOMO (energy of the highest occupied molecular orbital) values and hydrophobicity (log P) of the chemicals, while reductive activation was linked with ELUMO (energy of the lowest molecular orbital) values and log P. Such relationships may thus be useful for predicting toxicity of other chemicals of the same mechanism of toxicity. Due to the complexity involved in the phenomena of hepatotoxicity, unravelling of structure-hepatotoxicity relationships is a complicated task. A conceptual framework for QSAR modeling is proposed that involves recognition of molecular initiating events as potential endpoints to improve the prediction potential of QSAR models. Acute toxicity of reactive chemicals could be based on an initial reaction with biomolecules, thus the theory of covalent binding reactivity was used to test this concept. Reactivity assays with thiol and amine surrogate nucleophiles were used to determine susceptibility to toxicity. The derived QSAR expressions suggested that covalent binding reactivity is a good correlate to hepatotoxicity, however only if electrophilicity was the main mechanism of toxicity.

In this thesis I examined molecular mechanisms involved in acute and chronic liver injury, and also studied basic pathways mediating tumour promotion. Acute hepatic failure secondary to paracetamol poisoning is associated with high mortality. C-jun (NH2) terminal kinase (JNK) is a member of the mitogen activated protein kinase family and is a key intracellular signaling molecule involved in the control of cell fate. Paracetamol induced hepatic JNK activation in both human and murine paracetamol hepatotoxicity, and in a murine model preceded the onset of hepatocyte death. JNK inhibition in vivo (using two JNK inhibitors with different mechanisms of action) markedly reduced mortality in murine paracetamol hepatotoxicity. In addition, delayed administration of JNK inhibitor was more effective than N-acetylcysteine following paracetamol poisoning in mice. JNK inhibition was not protective in acute carbon tetrachloride or anti-Fas antibody mediated hepatic injury, suggesting specificity for the role of JNK in paracetamol hepatotoxicity. Furthermore, disruption of the JNK1 or JNK2 genes did not protect against paracetamol-induced hepatic damage. Pharmacological JNK inhibition had no effect on paracetamol metabolism, but markedly inhibited hepatic TNF-alpha production following paracetamol poisoning. These data demonstrate a central role for JNK in the pathogenesis of paracetamol induced liver failure, thereby identifying JNK as an important therapeutic target in the treatment of paracetamol hepatotoxicity. Liver fibrosis with loss of tissue architecture and subsequent hepatic failure represents a massive healthcare burden worldwide. Expression of Galectin-3 (a beta-galactoside binding animal lectin) is upregulated in established human fibrotic liver disease, during the development of experimental liver fibrosis and is temporally and spatially related to the induction and resolution of experimental hepatic fibrosis. Disruption of the gene encoding Galectin-3 blocks transdifferentiation of precursors to myofibroblasts in vitro and in vivo, markedly attenuating hepatic scarring in a murine model of liver fibrosis. Inhibition of Galectin-3 expression by siRNA in primary murine and human hepatic stellate cells significantly reduced myofibroblast activation and procollagen(I) expression. The reduction in hepatic fibrosis observed in the Galectin-3-/- mouse occurred despite equivalent liver injury and inflammation, and similar tissue expression of TGF-beta. TGF-beta failed to transactivate Galectin-3-/- hepatic stellate cells, in contrast with wild type hepatic stellate cells. However TGF-beta stimulated signaling via Smad-2 and 3 was equivalent in both Galectin-3-/- and wild type hepatic stellate cells indicating that Galectin-3 is required for TGF-beta mediated myofibroblast activation and matrix production. This supports a novel and important mechanistic role for Galectin-3 in the regulation of myofibroblast activation and consequent liver fibrosis. Finally, in vivo siRNA knockdown of Galectin-3 inhibited myofibroblast activation following hepatic injury and may therefore provide a novel therapeutic approach to the prevention and treatment of liver fibrosis. CD98hc (a ligand for Galectin-3) constitutively and specifically associates with beta1 integrins and is highly expressed on the surface of human tumour cells irrespective of the tissue of origin. CD98hc promotes both anchorage- and serum-independent growth. Using chimeras of CD98hc and the type II membrane protein CD69 demonstrated that the transmembrane domain of CD98hc is necessary and sufficient for integrin association in cells. Furthermore, CD98hc/β1 integrin association is required for focal adhesion kinase-dependent phosphoinositol 3-hydroxykinase activation and cellular transformation. Amino acids 82-87 in the putative cytoplasmic/transmembrane region appear to be critical for the oncogenic potential of CD98hc and provide a novel mechanism for tumour promotion by integrins.

Made available in DSpace on 2014-06-11T19:33:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-03Bitstream added on 2014-06-13T21:06:00Z : No. of bitstreams: 1 furtado_ks_dr_botfm.pdf: 1065360 bytes, checksum: 96620ea9848aeb92bccef945922a61fc (MD5) / O café e a cafeína são dois potenciais agentes preventivos contra o desenvolvimento ou avanço dos processos de fibrose/cirrose e carcinogênese hepática em humanos, entretanto suas ações são controversas e muitas vezes inconclusivas. Devido a isto, o objetivo deste trabalho foi verificar a ação do café ou da cafeína isolada no fígado de ratos Wistar tratados com tioacetamida (TAA) ou tetracloreto de carbono (CCl4). Para tanto, os dados experimentais foram distribuídos em dois artigos. No primeiro artigo, foram avaliados os efeitos do café convencional, descafeinado e da cafeína isolada na hepatotoxicidade induzida pela TAA em ratos Wistar. Para tanto, 60 os animais foram divididos em 5 grupos experimentais: G1 (controle negativo), G2 (controle positivo tratado com TAA 200 mg/Kg i.p.), G3 (TAA + café convencional), G4 (TAA + café descafeinado) e G5 (TAA + cafeína a 0,1%). Ao final de 8 semanas de tratamento os ratos foram eutanasiados para coleta do sangue (análises séricas) e do fígado (análises histológicas, histoquímicas e moleculares). De maneira geral os animais tratados com café/cafeína (G3-G5) apresentaram níveis da enzima alanina aminotransferase (ALT), área ocupada por colágenos I e III e expressão da proteína TGF-β1 menores que o grupo controle positivo (G2). Adicionalmente, os grupos G3 e G5 apresentaram menor número de núcleos PCNA positivos em fase S do que o grupo G2. O grupo G3 também apresentou menor número de focos GST-P positivos que o grupo G2. Ademais, os grupos G4 e G5 apresentaram as maiores atividades de MMP-2 ativa. Em conclusão, tanto o café convencional como o descafeinado como a cafeína a 0,1% apresentaram efeitos benéficos, mostrando que os outros componentes do café, mesmo sem a cafeína, ou que somente a cafeína são capazes de reduzir a hepatotoxicidade no fígado de ratos Wistar tratados com TAA. No segundo artigo... / Consumption of coffee beverages reduces the incidence of liver disease. However, whether these beneficial effects on human health are due to caffeine or other specific components in the beverage remains controversial. There, the present study aimed to study evaluated the protective effects of coffee beverages or caffeine on liver toxicity induced by repeated administration of the hepatotoxicant thioacetamide (TAA) in male Wistar rats. Animals were randomized into five groups: untreated controls (G1) TAA only (G2, 200 mg/Kg b.w. twice a week for 8 weeks, i.p.), TAA+conventional coffee (G3), TAA+decaffeinated coffee (G4) and TAA+caffeine (G5, 0.1% in the drinking water). At the end of 8 weeks, the animals were euthanized and blood and liver samples were collected. Serum ALT levels were lower in animals that received coffee and caffeine (p < 0.001). In addition, liver oxidized glutathione (p < 0.05), fibrosis/inflammation score (p < 0.001) and TGF-β expression (p ≤ 0.001) was reduced in these groups when compared to TAA-only rats. Moreover, conventional coffee and caffeine reduced PCNA S-phase index (p < 0.001) but only conventional coffee reduced cleaved caspase-3 index (p < 0.001) and active metalloproteinase 2 (p ≤ 0.004) in the liver from TAA-treated animals. In conclusion, consumption of conventional and decaffeinated coffee and caffeine has beneficial effects against TAA-induced liver injury in Wistar rats

Secoisolariciresinol (SECO) is the major lignan present in flaxseed, but unlike the structurally related lignan nordihydroguaiaretic acid, it is not associated with toxicity. The major phase I metabolite of SECO is lariciresinol, likely formed as a result of para-quinone methide (p-QM) formation followed by an intramolecular cyclization, thereby minimizing any toxicity associated with the p-QM. Four analogues of SECO were used to investigate substituent effects on lignan metabolism and formation of reactive quinones. HPLC methods were developed for analysis of SECO analogues and their metabolites. The stability of SECO analogues (1 mM) in a 50 mM Na2HPO4 buffer at pH 6.0 and 7.4 were quantified. Enzymatic oxidation experiments using mushroom tyrosinase and microsomes harvested from male Sprague-Dawley rats were performed with and without a GSH trapping system. Mass spectrometry and LC-MS were used to identify metabolites. Life Technologies was contracted to perform IC50 inhibition assays on SECO and the SECO analogues against CYP3A4, CYP3A5, CYP2C9 and CYP2C19 cytochrome P450 isoforms. All SECO analogues were stable at pH 6.0. SECO-2 was stable at pH 7.4 but SECO-1, -3 and -4 were unstable at pH 7.4. Autoxidation of SECO -1, -3 and -4 were 1st order reactions with t1/2 of 9.0 h, 1.7 h and 7.0 h respectively. Mushroom tyrosinase oxidations were performed to generate ortho-quinone standards. SECO-1 -3 and -4 were oxidized by mushroom tyrosinase but SECO-2 was not. Trapping with GSH produces aromatic ring conjugates for SECO-1, -3, -4. Results from microsomal oxidations for SECO-1, -3 and -4 are consistent with these standards. SECO-2 was metabolized by a microsomal system to produce a benzyl GSH adduct. Dealkylation products were also observed. All SECO analogues formed quinones but interestingly, GSH conjugation was competitive with intramolecular cyclization. All cytochrome P450 isoforms were inhibited by every analogue tested to varying degrees, a potential cause of toxicity concerns. Quinones are known to cause toxicity in vivo, including cytotoxicity, immunotoxicity, and carcinogenesis. Our results suggest that since the phenol and catechol lignans form GSH adducts in addition to intramolecular cyclization products, this class of lignans have the potential to cause toxicity.

A cisplatina ainda é um dos agentes quimioterápicos mais efetivos. No entanto, em elevadas doses pode ocorrer hepatotoxicidade. Alguns antioxidantes têm sido mostrado amenizar a hepatotoxicidade induzida pela cisplatina mas o mecanismo molecular envolvido não está bem esclarecido.No presente estudo nós investigamos moleculares subjacente ao efeito protetor da dimetiltiuouréia (DMTU), um conhecido eqüestrador de radical hidroxil, contra a lesão oxidativamitocondrial hepática induzida pela cisplatina em ratos. Ratos Wistar machos adultos ( 200 a 220g) foram divididos entre 4 grupos de 8 animais cada. O grupo controle foi tratado apenas com uma injeção intraperitoneal (i.p.) de solução salina (1 ml/ 100g de peso). Ao grupo DMTU foi administrado apenas DMTU (500 mg/kg de peso, i.p., seguido de 125 mg/kg, i.p., duas vezes ao dia até o sacrifício). Ao grupo cisplatina foi administrado uma injeção única de cisplatina (10 mg/kg de peso, i.p.). Ao grupo DMTU + cisplatina foi administrado DMTU (500mg/kg de peso, i.p.), pouco antes da injeção da cisplatina (10 mg/kg de peso, i.p.), seguido por injeções de DMTU (125 mg/kg de peso, i.p.) duas vezes ao dia até o sacrifício ( 72 horas após o tratamento). A hepatotoxicidade foi evidenciada no grupo cisplatina pelo aumento dos níveis séricos de alanina (ALT) e aspartato (AST)aminotransferases. O mecanismo de hepatotoxicidade induzido pela cisplatina mostrou-se envolvido na rigidez de membrana; na redução da razão glutationa reduzida em relação a glutationa oxidada (GSH/GSSG); na redução dos níveis de ATP, GSH e NADPH; na lipoperoxidação; na lesão oxidativa da cardiolipina e de proteínas com grupos fidrílicos. Mais ainda, a morte celular por apoptose foi também demonstrada e os achados fortemente sugerem a participação do xi mecanismo sinalizador mitocondrial neste processo; o DMTU não apresentou nenhum efeito direto sobre a mitocôndria e inibiu substancialmente a lesão mitocondrial induzida pela cisplatina, prevenindo a hepatotoxicidade. Todos os seguintes efeitos induzidos pela cisplatina foram previnidos pelo DMTU: (a) elevação dos níveis séricos de AST e ALT; (b) redução dos níveis de ATP hepático;(c)peroxidação lipídica;(d)oxidação da cardiolipina; (e)oxidação de proteínas sulfidrílicas; (f) rigidez da membrana mitocondrial; (g) oxidação de GSH; (h)oxidação de NADPH e (i) morte celular por apoptose. Os resultados mostraram o papel principal da mitocôndria e dos radicais hidroxilas na proteção do fígado saudável contra a lesão hepática induzida pela cisplatina, delineando um número de etapas que podem ser consideradas no desenvolvimento de futuros agentes citoprotetores / Cisplatin is still one of the most effective chemotherapeutic agents. However, at higher doses hepatotoxicity may occur. Some antioxidants have been shown to ameliorate cisplatin-induced hepatotoxicity but the involved molecular mechanism has not been clarified. In the present study we investigated the molecular mechanism underlying the protective effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, against liver mitochondrial oxidative damage induced by cisplatin in rats.Adult male Wistar rats (200 to 220g) were divided into 4 groups of 8 animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1ml/100g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p, followed by 125 mg/Kg, i.p., twice a day until sacrifice). The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU+cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 hours after the treatment). epatotoxicity was evidenced in the cisplatin group by the increased serum levels of alanine (ALT) and aspartate (AST) aminotransferases. The mechanism of cisplatininduced hepatotoxicity was found to involve membrane rigidification; decreased GSH/GSSG ratio, ATP, GSH and NADPH levels; lipid peroxidation; oxidative damage of cardiolipin and protein sulfhydryl groups. Moreover, cell death by apoptosis was also demonstrated and the findings strongly suggest the participation of the mitochondrial signaling pathway in this process; DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial injury, therefore preventing the hepatotoxicity. All the following cisplatin-induced xiv effects were prevented by DMTU: (a) elevation of AST and ALT serum levels; (b) decreased hepatic ATP levels; (c) lipid peroxidation; (d)cardiolipin oxidation; (e) sulfhydryl protein oxidation; (f) mitochondrial membrane rigidification; (g) GSH oxidation; (h) NADPH oxidation and (h) apoptotic cell death. Results show the central role of mitochondria and hydroxyl radicals in the protection of healthy liver against cisplatin-induced injury, highlighting a number of steps that might be considered in the development of novel cytoprotective agents.