Impaired Function of Solute Carrier Family 19 Leads to Low Folate Levels and Lipid Droplet Accumulation in Hepatocytes.

Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/-1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.

Abundance of Cytochromes in Hepatic Extracellular Vesicles Is Altered by Drugs Related With Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a serious worldwide health problem that accounts for more than 50% of acute liver failure. There is a great interest in clinical diagnosis and pharmaceutical industry to elucidate underlying molecular mechanisms and find noninvasive biomarkers for this pathology. Cell-secreted extracellular vesicles (EVs) have provided a new biological source to identify low disease invasive markers. Despite the intense research developed on these vesicles, there is currently a gap on their patho-physiological effects. Here, we study EVs secreted by primary rat hepatocytes challenged with galactatosamine (GalN), acetaminophen, or diclofenac as DILI in vitromodels. Proteomics analysis of these EVs revealed an increase in enzymes already associated with liver damage, such as catecholamine-methyl transferase and arginase 1. An increase in translation-related proteins and a decrease in regulators of apoptosis were also observed. In addition, we show the presence of enzymatic activity of P450 cytochrome 2d1 in EVs. The activity specifically is decreased in EVs secreted by hepatocytes after acetaminophen treatment and increased in EVs derived from GalN-treated hepatocytes. By using in vivo preclinical models, we demonstrate the presence of this cytochrome activity in circulation under normal conditions and an increased activity after GalN-induced injury. Conclusion: Hepatocyte-secreted EVs carry active xenobiotic-metabolizing enzymes that might be relevant in extracellular metabolism of drugs and be associated with DILI. (Hepatology Communications 2018;0:00-00).

Combining mouse embryonic stem cells and zebrafish embryos to evaluate developmental toxicity of chemical exposure.

The assays in this study utilize mouse embryonic stem cells (mESCs) and zebrafish embryos to evaluate the potential developmental toxicity of industrial and pharmaceutical chemicals. A set of eleven chemicals of known mammalian in vivo teratogenicity were tested in the assays and correlations to mammalian data. Using mESCs, proliferation, differentiation, and cytotoxicity of the chemicals were measured. In zebrafish embryos, lethality and the lowest effect level concentrations for morphological malformations were determined. Clustering of the assays based on frequency of affected assays resulted in a ranking of the test compounds that correlated to in vivo rodent data (R = 0.88, P < 0.001). We conclude that the combination of ESC- and zebrafish-based assays provides a valuable platform for the prioritization of pharmaceutical and industrial chemicals for further testing of developmental toxicity in rodents.

Identification of vascular disruptor compounds by analysis in zebrafish embryos and mouse embryonic endothelial cells.

To identify vascular disruptor compounds (VDCs), this study utilized an in vivo zebrafish embryo vascular model in conjunction with a mouse endothelial cell model to screen a subset of the U.S. Environmental Protection Agency (EPA) ToxCast Phase I chemical inventory. In zebrafish, 161 compounds were screened and 34 were identified by visual inspection as VDCs, of which 28 were confirmed as VDCs by quantitative image analysis. Testing of the zebrafish VDCs for their capacity to inhibit endothelial tube formation in the murine yolk-sac-derived endothelial cell line C166 identified 22 compounds that both disrupted zebrafish vascular development and murine endothelial in vitro tubulogenesis. Putative molecular targets for the VDCs were predicted using EPA's Toxicological Prioritization Index tool and a VDC signature based on a proposed adverse outcome pathway for developmental vascular toxicity. In conclusion, our screening approach identified 22 novel VDCs, some of which were active at nanomolar concentrations.

Quantitative proteomic analysis of hepatocyte-secreted extracellular vesicles reveals candidate markers for liver toxicity.

Extracellular vesicles have created great interest as possible source of biomarkers for different biological processes and diseases. Although the biological function of these vesicles is not fully understood, it is clear that they participate in the removal of unnecessary cellular material and act as carriers of various macromolecules and signals between the cells. In this report, we analyzed the proteome of extracellular vesicles secreted by primary hepatocytes. We used one- and two-dimensional liquid chromatography combined with data-independent mass spectrometry. Employing label-free quantitative proteomics, we detected significant changes in vesicle protein expression levels in this in vitro model after exposure to well-known liver toxins (galactosamine and Escherichia coli-derived lipopolysaccharide). The results allowed us to identify candidate markers for liver injury. We validated a number of these markers in vivo, providing the basis for the development of novel methods to evaluate drug toxicity. This report strongly supports the application of proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems. Analysis of such systems should help to identify specific markers for various biological processes and pathological conditions. BIOLOGICAL SIGNIFICANCE: Identification of low invasive candidate marker for hepatotoxicity. Support to apply proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems to identify low invasive markers for diseases.

Transcriptome of extracellular vesicles released by hepatocytes.

The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

Serum UPLC-MS/MS metabolic profiling in an experimental model for acute-liver injury reveals potential biomarkers for hepatotoxicity.

A key interest in clinical diagnosis and pharmaceutical industry is to have a repertoire of noninvasive biomarkers to-individually or in combination-be able to infer or predict the degree of liver injury caused by pathological conditions or drugs. Metabolomics-a comprehensive study of global metabolites-has become a highly sensitive and powerful tool for biomarker discovery thanks to recent technological advances. An ultra-performance liquid chromatography/time-of-flight tandem mass spectrometry (UPLC/TOF MS/MS)-based metabolomics approach was employed to investigate sera from galactosamine-treated rats to find potential biomarkers for acute liver injury. Hepatic damage was quantified by determining serum transaminase activity and in situ liver histological lesions. Principal component analysis in combination with coefficient of correlation analysis was used for biomarker selection and identification. According to the data, serum levels of several metabolites including glucose, amino acids, and membrane lipids were significantly modified, some of them showing a high correlation with the degree of liver damage determined by histological examination of the livers. In conclusion, this study supports that UPLC-MS/MS based serum metabolomics in experimental animal models could be a powerful approach to search for biomarkers for drug- or disease-induced liver injury.

Isolation of urinary exosomes from animal models to unravel noninvasive disease biomarkers.

In the last years, disease biomarker discovery has highly evolved thanks to the application of high--throughput technologies such as proteomics. However, due to the elevated complexity and abundance of some of the proteins in the samples the analysis of subcellular compartments has been revealed to be fundamental in order to identify underrepresented clinically relevant proteins. In this sense, extracellular microvesicles including exosomes that are present in different body fluids constitute a suitable and convenient subcellular compartment to identify disease biomarkers. On the other hand, animal models offer numerous advantages over human samples in order to accelerate the identification of candidate biomarkers. In this chapter we provide a detailed methodology to purify and analyze urinary exosomes that can be applied to the study of different diseases that have good animal models.

Proteomic analysis of microvesicles from plasma of healthy donors reveals high individual variability.

Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications.

Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples.

PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of hepatic disease in order to inform the diagnosis, prognosis and treatment. EXPERIMENTAL DESIGN: We have investigated the proteome of urinary vesicles present in urine samples obtained from experimental models for the study of liver injury, as an approach for identifying potential biomarkers for hepatic disease. RESULTS: The biochemical and proteomic characterization of highly purified exosome-like urinary vesicles has identified 28 proteins previously unreported in these vesicles, and many that have been previously associated with diseases, such as the prion-related protein. Furthermore, in urine samples from D-galactosamine-treated rats, a well-characterized experimental model for acute liver injury, we have detected a severe reduction in some proteins that normally are clearly detected in urinary vesicles. Finally, differential protein content on urinary vesicles from a mouse model for chronic liver injury has been also identified. CONCLUSIONS AND CLINICAL RELEVANCE: Our results argue positively that urinary vesicles could be a source for identifying non-invasive biomarkers of liver injury. We proposed some proteins such as Cd26, Cd81, Slc3A1 and Cd10 that have been found to be differentially expressed in urinary vesicles from some of the analyzed models as potential biomarkers for liver injury.

Overview of extracellular microvesicles in drug metabolism.

IMPORTANCE OF THE FIELD: Liver is the major body reservoir for enzymes involved in the metabolism of endogenous and xenobiotic compounds. Recently, it has been shown that hepatocytes release exosome-like vesicles to the extracellular medium, and the proteomic characterization of these hepatocyte-secreted exosomes has revealed the presence of several of these enzymes on them. AREAS COVERED IN THIS REVIEW: A systematic bibliographic search focused on two related aspects: i) xenobiotic-metabolizing enzymes that have been detected in microvesicles (MVs); and ii) MVs that are in the blood stream or secreted by cell types with clear interactions with this fluid. WHAT THE READER WILL GAIN: A discussion of these hepatocyte-secreted vesicles along with other MVs as enzymatic carriers in the context of extrahepatic drug-metabolizing systems. TAKE HOME MESSAGE: The contribution of many tissues including the liver to the MV plasma population is supported by several reports. On the other hand, many enzymes involved in the metabolism of drugs have been detected in MVs. Together, these observations support a role of hepatic-MVs in spreading the liver metabolizing activities through the body contributing in this manner to extrahepatic drug metabolism systems what could be relevant for body homeostasis and pharmaceutical interests.

Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes.

Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study, we described and characterized for the first time exosome secretion in nontumoral hepatocytes, and with the use of a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we demonstrated that exosomal membrane proteins can constitute an interesting tool to express nonexosomal proteins into exosomes with therapeutic purposes.