Methylimidazolium ionic liquids - a new class of forever chemicals with endocrine disrupting potential.

A class of chemical with a potentially important perceived future contribution to the net zero carbon goal (as "green" solvents) is the methylimidazolium ionic liquids (MILs). These solvents are used in industrial processes such as biofuel production yet little is known about their environmental stability or toxicity in man although one MIL - 1-octyl-3-methylimidazolium (M8OI) - has been shown to activate the human estrogen receptor alpha (ERα). The stabilities of the chloride unsubstituted methylimidazolium (MI) and MILs possessing increasing alkyl chain lengths (2C, 1-ethyl-3-methylimidazolium (EMI); 4C, 1-butyl-3-methylimidazolium (BMI); 6C; 1-hexyl-3-methylimidazolium (HMI), 8C, M8OI; 10C, 1-decyl-3-methylimidazolium (DMI)) were examined in river water and a human liver model system. The MILs were also screened for their abilities to activate the human ERα in vitro and induce uterine growth in pre-pubertal rats in vivo. Short chain MILs (EMI, BMI and HMI) underwent negligible metabolism and mineralisation in river water; were not metabolised in a model of human liver metabolism; activated the human ERα in vitro and were estrogenic in vivo in rats. A structure-based computational approach predicted short chain MIL binding to both the estrogen binding site and an additional site on the human estrogen receptor alpha. Longer chain MILs (M8OI and DMI) were metabolised in river water and partially mineralised. Based on structure-activity considerations, some of these environmentally-derived metabolites may however, remain a hazard to the population. MILs therefore have the potential to become forever chemicals with adverse effects to both man, other animals and the environment in general.

The methylimidazolium ionic liquid M8OI is a substrate for OCT1 and p-glycoprotein-1 in rat.

The methylimidazolium ionic liquid M8OI was recently found to be present in both the environment and man. In this study, M8OI disposition and toxicity were examined in an established rat progenitor-hepatocyte model. The progenitor B-13 cell was approx. 13 fold more sensitive to the toxic effects of M8OI than the hepatocyte B-13/H cell. However, this difference in sensitivity was not associated with a difference in metabolic capacities. M8OI toxicity was significantly decreased in a dose-dependent manner by co-addition of the OCT1 (SLC22A1) inhibitor clonidine, but not by OCT2 or OCT3 inhibitors in B-13 cells. M8OI toxicity was also dose-dependently increased by the co-addition of p-glycoprotein-1 (ABCB1B, multi drug resistant protein 1 (MDR1)) substrates/inhibitors. Excretion of B-13-loaded fluorophore Hoechst 33342 was also inhibited by the p-glycoproteins substrate cyclosporin A and by M8OI in a dose-dependent manner. Comparing levels of OCT and p-glycoprotein transcripts and proteins in B-13 and B-13/H cells suggest that the lower sensitivity to M8OI in B-13/H cells is predominantly associated with their higher expression of p-glycoprotein-1. These data together therefore suggest that a determinant in M8OI toxicity in rats is the expression and activity of the p-glycoprotein-1 transporter.

The methylimidazolium ionic liquid M8OI is detectable in human sera and is subject to biliary excretion in perfused human liver.

A methylimidizolium ionic liquid (M8OI) was recently found to be contaminating the environment and to be related to and/or potentially a component of an environmental trigger for the autoimmune liver disease primary biliary cholangitis (PBC). The aims of this study were to investigate human exposure to M8OI, hepatic metabolism and excretion. PBC patient and control sera were screened for the presence of M8OI. Human livers were perfused with 50µM M8OI in a closed circuit and its hepatic disposition examined. Metabolism was examined in cultured human hepatocytes and differentiated HepaRG cells by the addition of M8OI and metabolites in the range 10-100 µM. M8OI was detected in the sera from 5/20 PBC patients and 1/10 controls. In perfused livers, M8OI was cleared from the plasma with its appearance - primarily in the form of its hydroxylated (HO8IM) and carboxylated (COOH7IM) products - in the bile. Metabolism was reflected in cultured hepatocytes with HO8IM production inhibited by the cytochrome P450 inhibitor ketoconazole. Further oxidation of HO8IM to COOH7IM was sequentially inhibited by the alcohol and acetaldehyde dehydrogenase inhibitors 4-methyl pyrazole and disulfiram respectively. Hepatocytes from 1 donor failed to metabolise M8OI to COOH7IM over a 24 h period. These results demonstrate exposure to M8OI in the human population, monooxygenation by cytochromes P450 followed by alcohol and acetaldehyde dehydrogenase oxidation to a carboxylic acid that are excreted, in part, via the bile in human liver.

Emerging risk from "environmentally-friendly" solvents: Interaction of methylimidazolium ionic liquids with the mitochondrial electron transport chain is a key initiation event in their mammalian toxicity.

Recent studies have identified the 8C alkyl chain methylimidazolium ionic liquid 1-octyl-3-methylimidazolium in the environment and its potential to trigger the auto-immune liver disease primary biliary cholangitis. The toxicity of a range of methylimidazolium ionic liquids were therefore examined. Oxygen consumption was rapidly inhibited, with potency increasing with alkyl chain length. This preceded caspase 3/7 induction and DNA fragmentation. Time- and dose-dependent loss of dye reduction capacities reflected these effects, with a >700 fold difference in potency between 2C and 10C alkyl chain liquids. None of the ionic liquids directly inhibited mitochondrial complexes I-IV or complex V (F0F1-ATPase). However, dithionite reduction and ESR spectroscopy studies indicate a one electron reduction of oxygen in the presence of a methylimidazolium ionic liquid, suggesting methylimidazolium ionic liquids function as mitochondrial electron acceptors. However, only longer chain ionic liquids form a non-aqueous phase or micelle under aqueous physiological conditions and lead to increases in reactive oxygen species in intact cells. These data therefore suggest that the longer chain methylimidazolium liquids are toxic in sensitive liver progenitor cells because they both readily integrate within the inner mitochondrial membrane and accept electrons from the electron chain, leading to oxidative stress.

Renal injury and hepatic effects from the methylimidazolium ionic liquid M8OI in mouse.

The ionic liquid 1-octyl-3-methylimidazolium (M8OI) has been found in the environment and identified as a hazard for triggering the liver disease primary biliary cholangitis (PBC). Given limited toxicity data for M8OI and other structurally-related ionic liquids, target organs for M8OI toxicity were examined. Adult male C57Bl6 mice were acutely exposed to 0-10 mg/kg body weight M8OI via 2 intraperitoneal injections (time zero and 18 h) and effects examined at 24 h. At termination, tissue histopathology, serum and urinary endpoints were examined. No overt pathological changes were observed in the heart and brain. In contrast, focal and mild to multifocal and moderate degeneration with a general trend for an increase in severity with increased dose was observed in the kidney. These changes were accompanied by a dose-dependent increased expression of Kim1 in kidney tissue, marked elevations in urinary Kim1 protein and a dose-dependent increase in serum creatinine. Hepatic changes were limited to a significant dose-dependent loss of hepatic glycogen and a mild but significant increase in portal tract inflammatory recruitment and/or fibroblastic proliferation accompanied by a focal fibrotic change. Cultured mouse tissue slices reflected these in vivo effects in that dose-dependent injury was observed in kidney slices but not in the liver. Kidney slices accumulated higher levels of M8OI than liver slices (e.g. at 10 µM, greater than 4 fold) and liver slices where markedly more active in the metabolism of M8OI. These data indicate that the kidney is a target organ for the toxic effects of M8OI accompanied by mild cholangiopathic changes in the liver after intraperitoneal administration.

Changes in the gut microbiota of mice orally exposed to methylimidazolium ionic liquids.

Ionic liquids are salts used in a variety of industrial processes, and being relatively non-volatile, are proposed as environmentally-friendly replacements for existing volatile liquids. Methylimidazolium ionic liquids resist complete degradation in the environment, likely because the imidazolium moiety does not exist naturally in biological systems. However, there is limited data available regarding their mammalian effects in vivo. This study aimed to examine the effects of exposing mice separately to 2 different methylimidazolium ionic liquids (BMI and M8OI) through their addition to drinking water. Potential effects on key target organs-the liver and kidney-were examined, as well as the gut microbiome. Adult male mice were exposed to drinking water containing ionic liquids at a concentration of 440 mg/L for 18 weeks prior to examination of tissues, serum, urine and the gut microbiome. Histopathology was performed on tissues and clinical chemistry on serum for biomarkers of hepatic and renal injury. Bacterial DNA was isolated from the gut contents and subjected to targeted 16S rRNA sequencing. Mild hepatic and renal effects were limited to glycogen depletion and mild degenerative changes respectively. No hepatic or renal adverse effects were observed. In contrast, ionic liquid exposure altered gut microbial composition but not overall alpha diversity. Proportional abundance of Lachnospiraceae, Clostridia and Coriobacteriaceae spp. were significantly greater in ionic liquid-exposed mice, as were predicted KEGG functional pathways associated with xenobiotic and amino acid metabolism. Exposure to ionic liquids via drinking water therefore resulted in marked changes in the gut microbiome in mice prior to any overt pathological effects in target organs. Ionic liquids may be an emerging risk to health through their potential effects on the gut microbiome, which is implicated in the causes and/or severity of an array of chronic disease in humans.

The toxicity of the methylimidazolium ionic liquids, with a focus on M8OI and hepatic effects.

Ionic liquids are a diverse range of charged chemicals with low volatility and often liquids at ambient temperatures. This characteristic has in part lead to them being considered environmentally-friendly replacements for existing volatile solvents. However, methylimidazolium ionic liquids are slow to break down in the environment and a recent study at Newcastle detected 1 octyl 3 methylimidazolium (M8OI) - an 8 carbon variant methylimidazolium ionic liquid - in soils in close proximity to a landfill site. The current M8OI toxicity database in cultured mammalian cells, in experimental animal studies and in model indicators of environmental impact are reviewed. Selected analytical data from the Newcastle study suggest the soils in close proximity to the landfill site, an urban soil lacking overt contamination, had variable levels of M8OI. The potential for M8OI - or a structurally related ionic liquid - to trigger primary biliary cholangitis (PBC), an autoimmune liver disease thought to be triggered by an unknown agent(s) in the environment, is reviewed.

Expression of serine/threonine protein kinase SGK1F promotes an hepatoblast state in stem cells directed to differentiate into hepatocytes.

The rat pancreatic AR42J-B13 (B-13) cell line differentiates into non-replicative hepatocyte-like (B-13/H) cells in response to glucocorticoid. Since this response is dependent on an induction of serine/threonine protein kinase 1 (SGK1), this may suggest that a general pivotal role for SGK1 in hepatocyte maturation. To test this hypothesis, the effects of expressing adenoviral-encoded flag tagged human SGK1F (AdV-SGK1F) was examined at 3 stages of human induced pluripotent stem cell (iPSC) differentiation to hepatocytes. B-13 cells infected with AdV-SGK1F in the absence of glucocorticoid resulted in expression of flag tagged SGK1F protein; increases in ß-catenin phosphorylation; decreases in Tcf/Lef transcriptional activity; expression of hepatocyte marker genes and conversion of B-13 cells to a cell phenotype near-similar to B-13/H cells. Given this demonstration of functionality, iPSCs directed to differentiate towards hepatocyte-like cells using a standard protocol of chemical inhibitors and mixtures of growth factors were additionally infected with AdV-SGK1F, either at an early time point during differentiation to endoderm; during endoderm differentiation to anterior definitive endoderm and hepatoblasts and once converted to hepatocyte-like cells. SGK1F expression had no effect on differentiation to endoderm, likely due to low levels of expression. However, expression of SGK1F in both iPSCs-derived endoderm and hepatocyte-like cells both resulted in promotion of cells to an hepatoblast phenotype. These data demonstrate that SGK1 expression promotes an hepatoblast phenotype rather than maturation of human iPSC towards a mature hepatocyte phenotype and suggest a transient role for Sgk1 in promoting an hepatoblast state in B-13 trans-differentiation to B-13/H cells.

The ionic liquid 1-octyl-3-methylimidazolium (M8OI) is an activator of the human estrogen receptor alpha.

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 2 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 2 ERß in a murine hepatopancreatobiliary cell line. The human ERß was not activated by M8OI when expressed in HEK293 cells. These data demonstrate that M8OI is a xenoestrogen capable of activating the human ERα and super-activating the murine variant 2 ERß.

Identification of a xenobiotic as a potential environmental trigger in primary biliary cholangitis.

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.

HNF4alpha expression amplifies the glucocorticoid-induced conversion of a human pancreatic cell line to an hepatocyte-like cell.

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-13 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.

Glucocorticoid-induced pancreatic-hepatic trans-differentiation in a human cell line in vitro.

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 µM 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.

B-13 progenitor-derived hepatocytes (B-13/H cells) model lipid dysregulation in response to drugs and chemicals.

Lipid dysregulation is a common hepatic adverse outcome after exposure to toxic drugs and chemicals. A donor-free rat hepatocyte-like (B-13/H) cell was therefore examined as an in vitro model for investigating mechanisms. The B-13/H cell irreversibly accumulated triglycerides (steatosis) in a time- and dose-dependent manner when exposed to fatty acids, an effect that was potentiated by the combined addition of hyperglycaemic levels of glucose and insulin. B-13/H cells also expressed the LXR nuclear receptors and exposure to their activators - T0901317 or GW3965 - induced luciferase expression from a transfected LXR-regulated reporter gene construct and steatosis in a dose-dependent manner with T0901317. Exposing B-13/H cells to a variety of cationic amphiphilic drugs - but not other hepatotoxins - also resulted in a time- and dose-dependent accumulation of phospholipids (phospholipidosis), an effect that was reduced by over-expression of lysosomal phospholipase A2. Through application of this model, hepatotoxin methapyrilene exposure was shown to induce phospholipidosis in both B-13 and B-13/H cells in a time- and dose-dependent manner. However, methapyrilene was only toxic to B-13/H cells and inhibitors of hepatotoxicity enhanced phospholipidosis, suggesting phospholipidosis is not a pathway in toxicity for this withdrawn drug. In contrast, pre-existing steatosis had minimal effect on methapyrilene hepatotoxicity in B-13/H cells. These data demonstrate that the donor free B-13 cell system for generating hepatocyte-like cells may be employed in studies of fatty acid- and LXR activator-induced steatosis and phospholipidosis and in the dissection of pathways leading to adverse outcomes such as hepatotoxicity.

Hepatic effects of tartrazine (E 102) after systemic exposure are independent of oestrogen receptor interactions in the mouse.

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.