RESUMEN
Recent environmental sampling around a landfill site in the UK demonstrated that unidentified xenoestrogens were present at higher levels than control sites; that these xenoestrogens were capable of super-activating (resisting ligand-dependent antagonism) the murine variant 2â¯ERß and that the ionic liquid 1-octyl-3-methylimidazolium chloride (M8OI) was present in some samples. To determine whether M8OI was a contributor to the xenoestrogen pool in the soils, activation of human estrogen receptors by M8OI was examined. M8OI activated the human ERα in MCF7 cells in a dose-response manner. These effects were inhibited by the ER antagonist ICI182780; occurred in the absence of any metabolism of M8OI and were confirmed on examination of ER-dependent induction of trefoil factor 1 mRNA in MCF7 cells. M8OI also super-activated the murine variant 2â¯ERß in a murine hepatopancreatobiliary cell line. The human ERß was not activated by M8OI when expressed in HEK293â¯cells. These data demonstrate that M8OI is a xenoestrogen capable of activating the human ERα and super-activating the murine variant 2â¯ERß.
Asunto(s)
Receptor alfa de Estrógeno/agonistas , Imidazoles/farmacología , Líquidos Iónicos/farmacología , Animales , Células Cultivadas , Receptor alfa de Estrógeno/metabolismo , Células HEK293 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Imidazoles/química , Imidazoles/metabolismo , Líquidos Iónicos/química , Líquidos Iónicos/metabolismo , Células MCF-7 , Ratones , Estructura MolecularRESUMEN
BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is an autoimmune-associated chronic liver disease triggered by environmental factors, such as exposure to xenobiotics, which leads to a loss of tolerance to the lipoic acid-conjugated regions of the mitochondrial pyruvate dehydrogenase complex, typically to the E2 component. We aimed to identify xenobiotics that might be involved in the environmental triggering of PBC. METHODS: Urban landfill and control soil samples from a region with high PBC incidence were screened for xenobiotic activities using analytical, cell-based xenobiotic receptor activation assays and toxicity screens. RESULTS: A variety of potential xenobiotic classes were ubiquitously present, as identified by their interaction with xenobiotic receptors - aryl hydrocarbon receptor, androgen receptor and peroxisome proliferator activated receptor alpha - in cell-based screens. In contrast, xenoestrogens were present at higher levels in soil extracts from around an urban landfill. Furthermore, two landfill sampling sites contained a chemical(s) that inhibited mitochondrial oxidative phosphorylation and induced the apoptosis of a hepatic progenitor cell. The mitochondrial effect was also demonstrated in human liver cholangiocytes from three separate donors. The chemical was identified as the ionic liquid [3-methyl-1-octyl-1H-imidazol-3-ium]+ (M8OI) and the toxic effects were recapitulated using authentic pure chemical. A carboxylate-containing human hepatocyte metabolite of M8OI, bearing structural similarity to lipoic acid, was also enzymatically incorporated into the E2 component of the pyruvate dehydrogenase complex via the exogenous lipoylation pathway in vitro. CONCLUSIONS: These results identify, for the first time, a xenobiotic in the environment that may be related to and/or be a component of an environmental trigger for PBC. Therefore, further study in experimental animal models is warranted, to determine the risk of exposure to these ionic liquids. LAY SUMMARY: Primary biliary cholangitis is a liver disease in which most patients have antibodies to mitochondrial proteins containing lipoic acid binding site(s). This paper identified a man-made chemical present in soils around a waste site. It was then shown that this chemical was metabolized into a product with structural similarity to lipoic acid, which was capable of replacing lipoic acid in mitochondrial proteins.
Asunto(s)
Colangitis/inducido químicamente , Imidazoles/toxicidad , Contaminantes del Suelo/toxicidad , Xenobióticos/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Receptor alfa de Estrógeno/efectos de los fármacos , Células Hep G2 , Humanos , Imidazoles/aislamiento & purificación , Hígado/efectos de los fármacos , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Plaguicidas/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Ratas , Contaminantes del Suelo/análisis , Xenobióticos/aislamiento & purificaciónRESUMEN
The pancreas and liver are closely related developmentally and trans-differentiation of cells from one tissue into the cells of the other has been documented to occur after injury or exposure to selected growth factors or glucocorticoid hormones. To generate a readily-expandable source of human hepatocyte-like (H-13) cells, the human pancreatic adenocarcinoma cell (HPAC) line was stably transfected with a construct encoding the variant 2 hepatocyte nuclear factor 4 α (HNF4α) using a piggyBac vector and transient expression of a transposase. Through induction of transgene HNF4α regulated via an upstream glucocorticoid response element in combination with existing modulating effects of glucocorticoid, H-13â¯cells were converted into quantitatively similar hepatocyte-like (H-13/H) cells based on expression of a variety of hepatocyte proteins. H-13/H cells also demonstrated the ability to store glycogen and lipids. These data provide proof of concept that regulated expression of genes associated with hepatocyte phenotype could be used to generate quantitatively functional human hepatocyte-like cells using a readily expandable cell source and simple culture protocol. This approach would have utility in Toxicology and Hepatology research.
Asunto(s)
Glucocorticoides/farmacología , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Páncreas/citología , Páncreas/efectos de los fármacos , Perfilación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Humanos , Páncreas/metabolismo , Células Tumorales CultivadasRESUMEN
The rodent pancreatic AR42J-B13 (B-13) cell line differentiates into non-replicative hepatocyte-like cells in response to glucocorticoid mediated via the glucocorticoid receptor (GR). The aims of this study were to identify a human cell line that responds similarly and investigate the mechanisms underpinning any alteration in differentiation. Exposing the human pancreatic adenocarcinoma (HPAC) cell line to 1-10⯵M concentrations of dexamethasone (DEX) resulted an inhibition of proliferation, suppressed carcinoembryonic antigen expression, limited expression of pancreatic acinar and hepatic gene expression and significant induction of the constitutively-expressed hepatic CYP3A5 mRNA transcript. These changes were associated with a pulse of genomic DNA methylation and suppressed notch signalling activity. HPAC cells expressed high levels of GR transcript in contrast to other nuclear receptors - such as the glucocorticoid-activated pregnane X receptor (PXR) - and GR transcriptional function was activated by DEX in HPAC cells. Expression of selected hepatocyte transcripts in response to DEX was blocked by co-treatment with the GR antagonist RU486. These data indicate that the HPAC response to glucocorticoid exposure includes an inhibition in proliferation, alterations in notch signalling and a limited change in the expression of genes associated with an acinar and hepatic phenotype. This is the first demonstration of a human cell responding to similarly to the rodent B-13 cell regarding formation of hepatocyte-like cells in response to glucocorticoid. Identifying and modulating the ablating factor(s) may enhance the hepatocyte-like forming capacity of HPAC cells after exposure to glucocorticoid and generate an unlimited in vitro supply of human hepatocytes for toxicology studies and a variety of clinical applications.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Glucocorticoides/farmacología , Hepatocitos/efectos de los fármacos , Páncreas/citología , Línea Celular , Dexametasona/farmacología , Hepatocitos/citología , Humanos , Hígado/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores de Glucocorticoides/efectos de los fármacos , Neoplasias PancreáticasRESUMEN
Tartrazine is a food colour that activates the transcriptional function of the human oestrogen receptor alpha in an in vitro cell model. Since oestrogens are cholestatic, we hypothesised tartrazine will cause periportal injury to the liver in vivo. To test this hypothesis, tartrazine was initially administered systemically to mice resulting in a periportal recruitment of inflammatory cells, increased serum alkaline phosphatase activity and mild periportal fibrosis. To determine whether an oestrogenic effect may be a key event in this response, tartrazine, sulphonated metabolites and a food additive contaminant were screened for their ability to interact with murine oestrogen receptors. In all cases, there were no interactions as agonists or antagonists and further, no oestrogenicity was observed with tartrazine in an in vivo uterine growth assay. To examine the relevance of the hepatic effects of tartrazine to its use as a food additive, tartrazine was orally administered to transgenic NF-κB-Luc mice. Pre- and concurrent oral treatment with alcohol was incorporated given its potential to promote gut permeability and hepatic inflammation. Tartrazine alone induced NF- κB activities in the colon and liver but there was no periportal recruitment of inflammatory cells or fibrosis. Tartrazine, its sulphonated metabolites and the contaminant inhibited sulphotransferase activities in murine hepatic S9 extracts. Given the role of sulfotransferases in bile acid excretion, the initiating event giving rise to periportal inflammation and subsequent hepatic pathology through systemic tartrazine exposure is therefore potentially associated an inhibition of bile acid sulphation and excretion and not on oestrogen receptor-mediated transcriptional function. However, these effects were restricted to systemic exposures to tartrazine and did not occur to any significant effect after oral exposure.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Colorantes de Alimentos/toxicidad , Hígado/efectos de los fármacos , Tartrazina/toxicidad , Administración Oral , Animales , Línea Celular , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Etanol/toxicidad , Femenino , Inyecciones Intraperitoneales , Hígado/metabolismo , Pruebas de Función Hepática , Luciferasas de Luciérnaga/genética , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/genéticaRESUMEN
Tissue homeostasis requires an effective, limited wound-healing response to injury. In chronic disease, failure to regenerate parenchymal tissue leads to the replacement of lost cellular mass with a fibrotic matrix. The mechanisms that dictate the balance of cell regeneration and fibrogenesis are not well understood. Here we report that fibrogenic hepatic stellate cells (HSCs) in the liver are negative regulators of hepatocyte regeneration. This negative regulatory function requires stimulation of the 5-hydroxytryptamine 2B receptor (5-HT(2B)) on HSCs by serotonin, which activates expression of transforming growth factor ß1 (TGF-ß1), a powerful suppressor of hepatocyte proliferation, through signaling by mitogen-activated protein kinase 1 (ERK) and the transcription factor JunD. Selective antagonism of 5-HT(2B) enhanced hepatocyte growth in models of acute and chronic liver injury. We also observed similar effects in mice lacking 5-HT(2B) or JunD or upon selective depletion of HSCs in wild-type mice. Antagonism of 5-HT(2B) attenuated fibrogenesis and improved liver function in disease models in which fibrosis was pre-established and progressive. Pharmacological targeting of 5-HT(2B) is clinically safe in humans and may be therapeutic in chronic liver disease.