RESUMO
Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), chemicals present in a multitude of consumer products, are persistent organic pollutants. Both compounds induce hepatotoxic effects in rodents, including steatosis, hepatomegaly and liver cancer. The mechanisms of PFOA- and PFOS-induced hepatic dysfunction are not completely understood. We present evidence that PFOA and PFOS induce their hepatic effects via targeting hepatocyte nuclear factor 4-alpha (HNF4α). Human hepatocytes treated with PFOA and PFOS at a concentration relevant to occupational exposure caused a decrease in HNF4α protein without affecting HNF4α mRNA or causing cell death. RNA sequencing analysis combined with Ingenuity Pathway Analysis of global gene expression changes in human hepatocytes treated with PFOA or PFOS indicated alterations in the expression of genes involved in lipid metabolism and tumorigenesis, several of which are regulated by HNF4α. Further investigation of specific HNF4α target gene expression revealed that PFOA and PFOS could promote cellular dedifferentiation and increase cell proliferation by down regulating positive targets (differentiation genes such as CYP7A1) and inducing negative targets of HNF4α (pro-mitogenic genes such as CCND1). Furthermore, in silico docking simulations indicated that PFOA and PFOS could directly interact with HNF4α in a similar manner to endogenous fatty acids. Collectively, these results highlight HNF4α degradation as novel mechanism of PFOA and PFOS-mediated steatosis and tumorigenesis in human livers.
Assuntos
Ácidos Alcanossulfônicos/toxicidade , Caprilatos/toxicidade , Fluorocarbonos/toxicidade , Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/biossíntese , Hepatócitos/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Fator 4 Nuclear de Hepatócito/efeitos dos fármacos , Humanos , Masculino , Camundongos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Regulação para Cima/efeitos dos fármacosRESUMO
Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress.
Assuntos
Antibacterianos/toxicidade , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Replicação do DNA/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fluoroquinolonas/toxicidade , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Naftiridinas/toxicidade , Fator de Necrose Tumoral alfa/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Ativação Enzimática , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Fígado/enzimologia , Fígado/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Trovafloxacin (TVX) is a drug that has caused idiosyncratic, drug-induced liver injury (IDILI) in humans. In a murine model of IDILI, otherwise nontoxic doses of TVX and the inflammagen lipopolysaccharide (LPS) interacted to produce pronounced hepatocellular injury. The liver injury depended on a TVX-induced, small but significant prolongation of tumor necrosis factor-α (TNF) appearance in the plasma. The enhancement of TNF expression by TVX was reproduced in vitro in RAW 264.7 murine macrophages (RAW cells) stimulated with LPS. The current study was designed to identify the molecular target of TVX responsible for this response in RAW cells. An in silico analysis suggested a favorable binding profile of TVX to eukaryotic topoisomerase II-α (TopIIα), and a cell-free assay revealed that TVX inhibited eukaryotic TopIIα activity. Topoisomerase inhibition is known to lead to DNA damage, and TVX increased the DNA damage marker phosphorylated histone 2A.X in RAW cells. Moreover, TVX induced activation of the DNA damage sensor kinases, ataxia telangiectasia mutated (ATM) and Rad3-related (ATR). The ATR inhibitor NU6027 [6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine] prevented the TVX-mediated increases in LPS-induced TNF mRNA and protein release, whereas a selective ATM inhibitor [2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933)] was without effect. TVX prolonged TNF mRNA stability, and this effect was largely attenuated by NU6027. These results suggest that TVX can inhibit eukaryotic topoisomerase, leading to activation of ATR and potentiation of TNF release by macrophages, at least in part through increased mRNA stability. This off-target effect might contribute to the ability of TVX to precipitate IDILI in humans.
Assuntos
Antígenos de Neoplasias/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fluoroquinolonas/toxicidade , Macrófagos/metabolismo , Naftiridinas/toxicidade , Animais , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Fluoroquinolonas/antagonistas & inibidores , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Naftiridinas/antagonistas & inibidores , Compostos Nitrosos/farmacologia , Pirimidinas/farmacologia , Pironas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Idiosyncratic drug-induced liver injury (IDILI) continues to be a significant human health problem. IDILI is characterized as occurring in a minority of individuals exposed to a drug, yet it accounts for as much as 17% of all cases of acute liver failure. Despite these concerns, the mechanisms underlying IDILI remain unknown. Trovafloxacin (TVX), which causes IDILI in humans, also causes hepatocellular death in vitro when combined with tumor necrosis factor-alpha (TNF) treatment. However, the molecular mechanisms involved in this toxicity are not fully characterized. The purpose of this study was to identify mechanisms by which TVX and TNF interact to cause hepatocellular death, with a focus on a human hepatocyte cell line. TVX and TNF interacted to cause cytotoxicity in HepG2 cells at drug concentrations similar to those in people undergoing TVX therapy. TVX/TNF treatment caused apoptosis and DNA damage in HepG2 cells that depended on caspase activation. Prolonged activation of JNK occurred in TVX/TNF-induced cytotoxicity, and treatment with the JNK selective inhibitor SP600125 attenuated cytotoxicity. TVX/TNF cotreatment also caused cytotoxicity in isolated primary murine hepatocytes that was dependent on caspase activation. These results increase understanding of molecular signaling pathways involved in hepatocellular death caused by a drug with idiosyncratic liability in the presence of TNF.
Assuntos
Antibacterianos/toxicidade , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fluoroquinolonas/toxicidade , Hepatócitos/efeitos dos fármacos , Naftiridinas/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Inibidores de Proteínas Quinases , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. In previous studies, cotreatment of rats with SLD and a modestly inflammatory dose of lipopolysaccharide (LPS) led to liver injury, whereas neither SLD nor LPS alone caused liver damage. In studies presented here, further investigation of this animal model revealed that the concentration of tumor necrosis factor-alpha (TNF-alpha) in plasma was significantly increased by LPS at 1 h, and SLD enhanced this response. Etanercept, a soluble TNF-alpha receptor, reduced SLD/LPS-induced liver injury, suggesting a role for TNF-alpha. SLD metabolites in plasma and liver were determined by LC/MS/MS. Cotreatment with LPS did not increase the concentrations of SLD or its metabolites, excluding the possibility that LPS contributed to liver injury through enhanced exposure to SLD or its metabolites. The cytotoxicities of SLD and its sulfide and sulfone metabolites were compared in primary rat hepatocytes and HepG2 cells; SLD sulfide was more toxic in both types of cells than SLD or SLD sulfone. TNF-alpha augmented the cytotoxicity of SLD sulfide in primary hepatocytes and HepG2 cells. These results suggest that TNF-alpha can enhance SLD sulfide-induced hepatotoxicity, thereby contributing to liver injury in SLD/LPS-cotreated rats.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/sangue , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Sulindaco/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sinergismo Farmacológico , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/toxicidade , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Ratos , Ratos Sprague-Dawley , Sulindaco/toxicidade , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangueRESUMO
Therapy employing the fluoroquinolone antibiotic, trovafloxacin (TVX) was curtailed due to idiosyncratic hepatotoxicity. Previous studies in mice showed that a nonhepatotoxic inflammatory stress induced by tumor necrosis factor alpha (TNF) synergized with a nonhepatotoxic dose of TVX to cause liver injury. The purpose of this study was to explore mechanisms by which TVX interacts with TNF to cause liver injury. TVX pretreatment prolonged the peak of plasma TNF after its administration. This prolongation of TNF by TVX was critical to the development of hepatotoxicity. The prolongation of TNF concentration in plasma was primarily due to reduced clearance when compared with secondary biosynthesis. TNF is cleared from plasma by binding to soluble TNF receptors (TNFRs) which are eliminated by the kidney; however, the plasma concentrations of soluble TNFRs were not reduced, and biomarkers of renal dysfunction were not elevated in TVX/TNF-treated mice. Two injections of TNF mimicked the prolongation of the TNF peak by TVX and caused liver injury, but injury was less severe than after TVX/TNF coexposure. TVX enhanced the induction of proinflammatory cytokines by TNF. Additionally, TVX sensitized Hepa1c1c7 cells to TNF-induced killing in a concentration-dependent manner and increased both potency and efficacy of TNF to activate effector caspases that were critically involved in cell death from TVX/TNF coexposure. In summary, TVX reduced the clearance of TNF independent of either receptor shedding or kidney dysfunction. Additionally, TVX interacted with TNF to enhance inflammation and sensitize hepatocytes to TNF-induced cell death.
Assuntos
Anti-Infecciosos/toxicidade , Morte Celular/efeitos dos fármacos , Fluoroquinolonas/toxicidade , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Naftiridinas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Humanos , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/fisiologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Ratos , Fator de Necrose Tumoral alfa/sangueRESUMO
1. Our goal was to investigate the body distribution of serotonin (5-hydroxytryptamine; 5-HT) in rats infused with 5-HT (25 microg/kg per min) for 7 days and the contribution of the 5-HT transporter (SERT) for 5-HT uptake into the tissues. 2. Mini-osmotic pumps containing 5-HT or vehicle were implanted in rats knocked out for SERT (SERT-KO) or in wild-type (WT) rats. On the 8th day, tissues were harvested for measurements of 5-HT by high-performance liquid chromatography (HPLC). The 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA) was also measured by HPLC, because an increase in 5-HIAA in tissues from rats receiving 5-HT reflects 5-HT uptake followed by metabolism. 3. In WT rats infused with 5-HT, an increase in 5-HT or 5-HIAA was observed in the heart, pancreas, thyroid, adrenal gland, kidney, seminal vesicle, bladder, prostate, liver, oesophagus, stomach, femur, trachea, lung and spleen compared with vehicle-infused rats. An increase in 5-HT and 5-HIAA was not observed in aorta, vena cava and jejunum. In tissues from SERT-KO rats infused with 5-HT, the content of 5-HT or 5-HIAA was decreased in most of the tissues studied compared with 5-HT-infused WT rats. Although 5-HT uptake in the kidney, seminal vesicle, prostate, jejunum and trachea is SERT dependent, it is SERT independent in the pancreas. The remaining tissues display SERT-dependent and -independent mechanisms for 5-HT uptake. 4. Altogether, tissues from different systems, such as the cardiovascular, endocrine, genitourinary and gastrointestinal, accumulate 5-HT mainly via SERT and, thus, these systems are potential targets for drugs that interfere with 5-HT homeostasis.