Cultures Of Hepatocytes Research Articles

Determination of cytotoxic, genotoxic, and oxidative damage from deltamethrin on primary hepatocyte culture of Lake Van fish, Alburnus tarichi

ABSTRACT Deltamethrin is a widely used pyrethroits worldwide. Although the chemical is used to combat insects, it has effects on other non-target organisms. Deltamethrin is extensively used in agriculture, animal husbandry, and domestic areas in the Lake Van basin. The aim of this study was to determine the cytotoxic, genotoxic, and oxidative damage from deltamethrin on the primary hepatocyte culture of Lake Van fish (Alburnus tarichi). In this study, the toxic effects of different concentrations (0.01, 0.1, 1, and 10 µM) of deltamethrin in the primary hepatocyte culture of Lake Van fish were investigated via liver enzymes aspartase aminotransferase (AST) and alanine aminotransferase (ALT), and the total antioxidant status (TAS), total oxidant status (TOS), lipid peroxidation (MDA), and DNA damage (8-OHdG). Deltamethrin caused an increase in the AST and ALT levels dependent on the dosage and time. The TAS and TOS levels increased at the end of 24 h and there was no difference at the end of 48 h. Deltamethrin did not affect the MDA level, but increased the 8-OHdG (P < 0.05). In conclusion, it can be said that high doses of deltamethrin (1 and 10 µM) have a toxic effect on the primary hepatocyte culture of Lake Van fish.

Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ has been implicated in the pathogenesis of various human diseases including fatty liver. Although nuclear translocation of PPARγ plays an important role in PPARγ signaling, details of the translocation mechanisms have not been elucidated. Here we demonstrate that PPARγ2 translocates to the nucleus and activates signal transduction through H2O2-dependent formation of a PPARγ2 and transportin (Tnpo)1 complex via redox-sensitive disulfide bonds between cysteine (Cys)176 and Cys180 of the former and Cys512 of the latter. Using hepatocyte cultures and mouse models, we show that cytosolic H2O2/Tnpo1-dependent nuclear translocation enhances the amount of DNA-bound PPARγ and downstream signaling, leading to triglyceride accumulation in hepatocytes and liver. These findings expand our understanding of the mechanism underlying the nuclear translocation of PPARγ, and suggest that the PPARγ and Tnpo1 complex and surrounding redox environment are potential therapeutic targets in the treatment of PPARγ-related diseases.

3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber.

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.

Plasma membrane vesicles of human umbilical cord mesenchymal stem cells ameliorate acetaminophen-induced damage in HepG2 cells: a novel stem cell therapy

BackgroundAcetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury.MethodsPMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated.ResultsThe hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that the redox state and activities of aminotransferases were restored in APAP-treated HepG2 cells.ConclusionsThe results suggest that PMVs from hUCMSCs could be used as a novel stem cell therapy for the treatment of APAP-induced liver injury.

Evaluation of Erythromycin as a Tool to Assess CYP3A Contribution of Low Clearance Compounds in a Long-Term Hepatocyte Culture

Long-term hepatocyte culture systems such as HepatoPac are well suited to evaluate the metabolic turnover of low clearance (CL) drugs because of their sustained metabolic capacity and longer-term viability. Erythromycin (ERY), a moderate, mechanism-based inhibitor of CYP3A, was evaluated as a tool in the HepatoPac model to assess contribution of CYP3A to the clearance of drug candidates. ERY inhibited CYP3A activity by 58% and 80% at 3 and 10 μM, respectively, for up to 72 hours. At 30 µM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CLint was inhibited 58% by 3 μM of ERY, 75% by 15 μM of ERY, 89% by 30 μM of ERY, and 94% by 60 μM of ERY. ERY (30 μM) did not markedly affect CLint of substrates for several other major cytochrome P450 isoforms evaluated and did not markedly inhibit uridine diphosphoglucuronosyl transferase (UGT) isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, or 2B15 as assessed using recombinant UGTs. ERY only mildly increased CYP3A4 gene expression by 2.1-fold (14% of rifampicin induction) at 120 µM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 µM), arylhydrocarbon receptor, constitutive androstane receptor, and pregnane-X-receptor activation are not likely to markedly increase levels of other drug-metabolizing enzymes or transporters. ERY at concentrations up to 60 µM was not toxic for up to 6 days of incubation. Use of ERY to selectively inhibit CYP3A in high-functioning, long-term hepatocyte models such as HepatoPac can be a valuable strategy to evaluate the contribution of CYP3A metabolism to the overall clearance of slowly metabolized drug candidates. SIGNIFICANCE STATEMENT: This work describes the use of erythromycin as a selective inhibitor of CYP3A to assess the contribution of CYP3A in the metabolism of compounds using long-term hepatocyte cultures.

Visualization and isolation of zone-specific murine hepatocytes that maintain distinct cytochrome P450 oxidase expression in primary culture

Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/β-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.

Synthesis and evaluation of new 4-peptidamido-2-fluorobenzyl phosphoramide mustard conjugates as prodrugs activated by prostate-specific antigen

In our continued efforts to develop targeted prodrugs activated by prostate-specific antigen (PSA), we designed and synthesized novel phosphoramide mustard peptide conjugates using previously optimized PSA substrates. Initial Nu/Nu mouse PK studies indicated that prodrug I (glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) exhibits high clearance with significant extrahepatic metabolism in vivo. Substrate optimization studies were thus carried out to further improve PSA specificity and enable the design of prodrugs with reduced in vivo clearance and enhanced tumor selectivity. To assess the utility of the newly optimized sequences as promoieties, they were coupled to phosphoramide mustard using a 4-amino-2-fluorobenzyl alcohol linker akin to prodrug I. In the presence of human PSA, prodrug I was rapidly cleaved with a half-life (t1/2) of 35 min. Prodrugs II (glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) and III (GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) were hydrolyzed at slower rates with t1/2 values of 80 and 107 min, respectively. These results we observed here are different from our previously reported data but may be explained by the fact that PSA-activated release of phosphoramide mustard and reactive quinonimine methides resulted in mechanism-based inhibition of PSA, thereby preventing further hydrolysis of prodrugs I–III. Prodrug I was cytotoxic to PSA-producing LNCaP cells with an IC50 value of 7.3 μM and demonstrated 14-fold selectivity over the non-PSA-producing DU145. Despite its poor in vitro antiproliferative activity (IC50 = 30 µM), prodrug III was found to be more stable against non-PSA-mediated hydrolysis compared with prodrug I as revealed by metabolite profiling studies, which was in agreement with its improved stability in human hepatocyte cultures. These results suggested that a combination of the peptide sequence GABA ← mGly-Ala-Ser-Chg-Gln with optimal linkers and/or other cytotoxic agents can help achieve an adequate balance between PSA cleavage rate and enhanced resistance to non-PSA-mediated hydrolysis.

1921-P: Nonalcoholic Steatohepatitis in Mice with Hepatocyte-Specific Deletion of Mitochondrial Fission Factor

Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred as mitochondrial dynamics (MtD), to adapt to nutritional demand. Evidence suggested that impaired MtD leads to metabolic abnormalities such as nonalcoholic steatohepatitis (NASH)-like phenotypes, which are characterized by hepatic steatosis, inflammation and fibrosis. However, how MtD is involved in the development of NASH is poorly understood. In this study, we found mitochondrial fission factor (Mff), a critical mediator of mitochondrial fission, is upregulated in the liver from mice fed high-fat diet (HFD). To explore the role of Mff in the development of NASH, we generated liver-specific deletion of Mff (MffLiKO) mice. Histological analysis revealed that mitochondria are enlarged in the liver from MffLiKO mice fed normal chow diet (NCD), which is followed by hepatocyte apoptosis, slightly increased infiltration of inflammatory macrophages, and fibrosis. However, no lipid accumulation was observed in the liver from MffLiKO mice fed NCD. That is, only the impairment of Mff-regulated mitochondrial fission did not develop NASH-like phenotypes. On the other hand, the liver of MffLiKO mice fed HFD developed NASH-like phenotype with severer steatosis. Expression of mRNAs related to endoplasmic reticulum (ER) stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic triglyceride secretion was downregulated, with reduced hepatic triglyceride secretion in MffLiKO mice in vivo and in primary cultures of Mff-deficient hepatocytes in vitro. Interestingly, thapsigargin-induced ER stress suppressed triglyceride secretion in primary cultured hepatocytes in vitro. This study demonstrates that MffLiKO develop NASH-like-phenotypes in response to HFD and suggests that impaired MtD induces ER stress and thus reduces hepatic triglyceride secretion, which may play a role in the pathogenesis of NASH. Disclosure Y. Takeichi: None. T. Miyazawa: None. S. Sakamoto: None. R. Sakamoto: None. M. Nomura: None. Y. Ogawa: Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker’s Bureau; Self; Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation.

Human Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells.

The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver’s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a growing interest in the use of tissue-specific extracellular matrix (ECM) to provide cells with an in vitro environment that more closely resembles that of the native tissue. In the present study, we have developed a method that allows for the isolation and downstream application of the human liver’s main cell types from cryopreserved material. We also isolated and solubilized human liver ECM (HL-ECM), analyzed its peptidomic and proteomic composition by mass spectrometry and evaluated its interest for the culture of distinct primary human liver cells. Our analysis of the HL-ECM revealed proteomic diversity, type 1 collagen abundance and partial loss of integrity following solubilization. Solubilized HL-ECM was evaluated either as a coating or as a medium supplement for the culture of human primary hepatocytes, hepatic stellate cells and liver sinusoidal endothelial cells. Whereas the solubilized HL-ECM was suitable for cell culture, its impact on the phenotype and/or functionality of the human liver cells was limited. Our study provides a first detailed characterization of solubilized HL-ECM and a first report of its influence on the culture of distinct human primary liver cells.

Lipid droplets disrupt mechanosensing in human hepatocytes.

Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer death in the world. Although most cases occur in stiff, cirrhotic livers, and stiffness is a significant risk factor, HCC can also arise in noncirrhotic livers in the setting of nonalcoholic fatty liver disease (NAFLD). We hypothesized that lipid droplets in NAFLD might apply mechanical forces to the nucleus, functioning as mechanical stressors akin to stiffness. We investigated the effect of lipid droplets on cellular mechanosensing and found that primary human hepatocytes loaded with the fatty acids oleate and linoleate exhibited decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers. The presence of large lipid droplets in hepatocytes resulted in increased nuclear localization of the mechano-sensor Yes-associated protein (YAP). In cirrhotic livers from patients with NAFLD, hepatocytes filled with large lipid droplets showed significantly higher nuclear localization of YAP as compared with cells with small lipid droplets. This work suggests that lipid droplets induce a mechanical signal that disrupts the ability of the hepatocyte to sense its underlying matrix stiffness and that the presence of lipid droplets can induce intracellular mechanical stresses.NEW & NOTEWORTHY This work examines the impact of lipid loading on mechanosensing by human hepatocytes. In cirrhotic livers, the presence of large (although not small) lipid droplets increased nuclear localization of the mechanotransducer YAP. In primary hepatocytes in culture, lipid droplets led to decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers; the presence of large lipid droplets resulted in increased YAP nuclear localization. Collectively, the data suggest that lipid droplets induce intracellular mechanical stress.

Cytotoxic dimeric half‐sandwich Ru(II), Os(II) and Ir(III) complexes containing the 4,4′‐biphenyl‐based bridging ligands

A series of dinuclear half‐sandwich Ru(II), Os(II) and Ir(III) complexes [Ru2(μ‐Ln)(η6‐pcym)2Cl2](PF6)2 (1, 4), [Os2(μ‐Ln)(η6‐pcym)2Cl2](PF6)2 (2, 5) and [Ir2(μ‐Ln)(η5‐Cp*)2Cl2](PF6)2 (3, 6), based on 4,4′‐biphenyl‐based bridging Schiff base ligands N,N′‐(biphenyl‐4,4′‐diyldimethylidyne)bis‐2‐(pyridin‐2‐yl)methanamine (L1; for 1–3) and N,N′‐(biphenyl‐4,4′‐diyldimethylidyne)bis‐2‐(pyridin‐2‐yl)ethanamine (L2; for 4–6) is reported; pcym = 1‐methyl‐4‐(propan‐2‐yl)benzene, Cp* = pentamethylcyclopentadienyl. The complexes were characterized by relevant analytical techniques (i.e. elemental analysis, FT‐IR, NMR, ESI‐MS), and their in vitro cytotoxicity was assessed at six cancerous and two non‐cancerous (healthy) human cell lines. Overall, complexes 4–6, containing the L2 bridging ligand, revealed higher cytotoxicity as compared with 1–3 and, thus, they were studied in greater detail. The best‐performing complex 6 exceeded at least twice the in vitro cytotoxicity of cisplatin and showed high selectivity towards the cancer cells over the normal ones, including the primary culture of human hepatocytes. In contrast to cisplatin, complexes 4–6 did not induce the cell cycle modification of the treated A2780 human ovarian carcinoma cells (studied by flow cytometry and Western blot analysis). High levels of superoxide anion were induced by complexes 4–6 at the A2780 cells. The levels of activated forms of Caspase‐3 and Caspase‐8 at the A2780 cells treated by Ru(II) complex 4 were comparable with cisplatin, while complexes 5 and 6 had only a minor effect on activation of these caspases.

Glucagon-Dependent Suppression of mTORC1 is Associated with Upregulation of Hepatic FGF21 mRNA Translation.

Fibroblast growth factor 21 (FGF21) is a peptide hormone that acts to enhance insulin sensitivity and reverse many of the metabolic defects associated with consumption of a high-fat diet. Recent studies show that the liver is the primary source of FGF21 in the blood, and that hepatic FGF21 expression is upregulated by glucagon. Interestingly, glucagon acts to upregulate FGF21 production by primary cultures of rat hepatocytes and H4IIE and HepG2 hepatocarcinoma cells independent of changes in FGF21 mRNA abundance, suggesting that FGF21 protein expression is regulated post-transcriptionally. Based on these observations, the goal of the present study was to assess whether or not FGF21 mRNA is translationally regulated. The results show that FGF21 mRNA translation and secretion of the hormone are significantly upregulated in H4IIE cells exposed to 25 nM glucagon, independent of changes in FGF21 mRNA abundance. Furthermore, the glucagon-induced upregulation of FGF21 mRNA translation is associated with suppressed activity of the mechanistic target of rapamycin in complex 1 (mTORC1). Similarly, the results show that rapamycin-induced suppression of mTORC1 leads to upregulation of FGF21 mRNA translation with no change in FGF21 mRNA abundance. In contrast, activation of mTORC1 by refreshing the culture medium leads to downregulation of FGF21 mRNA translation. Notably, re-feeding fasted rats also leads to downregulation of FGF21 mRNA translation concomitantly with activation of mTORC1 in the liver. Overall, the findings support a model in which glucagon acts to upregulate FGF21 production by hepatocytes through suppression of mTORC1 and subsequent upregulation of FGF21 mRNA translation.

Excess DHA Induces Cell Cycle Arrest by Activating the P53/Cycling Pathway in Blunt Snout Bream (Megalobrama amblycephala)

Pervious study showed that diets with high levels of docosahexaenoic acid (DHA) cause growth retardation in Megalobrama amblycephala. In order to explore the mechanisms, a feeding trial and a primary hepatocyte culture experiment were designed. In vivo, fish (average weight 26.40±0.11 g) were randomly divided into groups and fed three levels of DHA (0%, 0.2% and 1.6% diets, respectively.) for 8 weeks. The results showed that the final body weight and weight gain of fish fed 1.6% DHA were significantly lower than that of fish fed 0.2% DHA (P < 0.05). P53 gene and protein expression levels were significantly increased (P < 0.05), while expression of downstream Cyclin D1 and Cyclin E1 was significantly inhibited in the 1.6% DHA group when compared with the 0% DHA and 0.2% DHA groups (P < 0.05). In vitro, primary hepatocytes isolated from Megalobrama amblycephala incubated with 500 μM DHA showed significantly increased cell cycle arrest and apoptosis (P < 0.05), significantly increased P53 gene and protein levels (P < 0.05), and significantly decreased Cyclin D1 and Cyclin E1 levels (P < 0.05), when compared with other groups. When primary hepatocytes were incubated with DHA and a P53 inhibitor (pifithrin-α), P53 expression and P53-mediated signaling were inhibited, cell cycle progression recovered and apoptosis was reduced. In summary, high levels of DHA activated the P53/Cyclin pathway to induce cell cycle arrest. Inhibition of P53 activity may be a potential way of reducing the side effects of DHA on the growth of Megalobrama amblycephala.

SUN-555 Vitamin E Sequestration by Liver Fat in Vitro and in Women with Hepato-Steatosis

BACKGROUND: The global obesity epidemic has sobering consequences to human health. Especially concerning is obesity-associated hepato-steatosis (HS), a common cause of chronic liver disease in the Americas and Western Europe that precedes non-alcoholic steatohepatitis (NASH). Maintenance of normal body weight is the only current means to prevent HS and NASH. We hypothesized that excess liver fat in obesity-associated HS could act as a pathophysiologic chemical depot for fat-soluble vitamins and alter normal physiology. Because clinical trials with Vitamin E (α-T) have shown that NASH partially responds to this supplement, we selected α-T as a model vitamin to test the sequestration hypothesis. INTERVENTIONS: Under an IND and IRB-approved protocol, two deuterium-labeled α-tocopherols (d3-α-T and d6-α-T) were administered orally and intravenously, respectively, to 10 healthy women and 6 women with HS. Serial blood samples obtained over 72 h were analyzed by LC-MS/MS. In parallel, we performed studies in hepatocytes in cell culture and mouse model. RESULTS: In healthy women who received oral d3- and intravenous d6-α-T, 85% of the initial plasma peak d6-α-T disappeared within 20 minute and reappeared in the plasma peaking between 6-8 h. Compared to healthy subjects, subjects with HS had similar d6-α-T entry rates into liver, but reduced release rates into plasma (p<0.001). Similarly, pharmacokinetics parameters (AUC and Maximum Concentration [Cmax]), were reduced (AUC0-8,p<0.01;Cmax p<0.02) in HS subjects, indicating reduced hepatic d6-α-T output. Consistently, livers of mice fed with a high fat diet (42% fat) had more vitamin E compared to controls diet (5% fat), with both diets having the same α-T content CONCLUSION: These findings suggest the unique role of the liver in vitamin E physiology which is dysregulated by excess liver fat (measured by magnetic resonance spectroscopy). Considered together, the findings imply that obesity-associated HS may produce unrecognized hepatic α-T sequestration, which might subsequently drive liver disease. The data here raise the intriguing possibility that timely α-T supplementation might attenuate progression of HS to NASH, perhaps by correcting an unrecognized fat-induced, localized, hepatic vitamin E deficiency prior to onset of inflammation, hepatitis, and fibrosis. Additionally, our findings raise the possibility that HS may similarly alter hepatic physiology of other fat-soluble vitamins.

Systemic modified messenger RNA for replacement therapy in alpha 1-antitrypsin deficiency

Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.

Discovery of novel hepatocyte eQTLs in African Americans.

African Americans (AAs) are disproportionately affected by metabolic diseases and adverse drug events, with limited publicly available genomic and transcriptomic data to advance the knowledge of the molecular underpinnings or genetic associations to these diseases or drug response phenotypes. To fill this gap, we obtained 60 primary hepatocyte cultures from AA liver donors for genome-wide mapping of expression quantitative trait loci (eQTL) using LAMatrix. We identified 277 eGenes and 19,770 eQTLs, of which 67 eGenes and 7,415 eQTLs are not observed in the Genotype-Tissue Expression Project (GTEx) liver eQTL analysis. Of the eGenes found in GTEx only 25 share the same lead eQTL. These AA-specific eQTLs are less correlated to GTEx eQTLs. in effect sizes and have larger Fst values compared to eQTLs found in both cohorts (overlapping eQTLs). We assessed the overlap between GWAS variants and their tagging variants with AA hepatocyte eQTLs and demonstrated that AA hepatocyte eQTLs can decrease the number of potential causal variants at GWAS loci. Additionally, we identified 75,002 exon QTLs of which 48.8% are not eQTLs in AA hepatocytes. Our analysis provides the first comprehensive characterization of AA hepatocyte eQTLs and highlights the unique discoveries that are made possible due to the increased genetic diversity within the African ancestry genome.

Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury.

Although CEACAM1 (CC1) glycoprotein resides at the interface of immune liver injury and metabolic homeostasis, its role in orthotopic liver transplantation (OLT) remains elusive. We aimed to determine whether/how CEACAM1 signaling may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. In the mouse, donor liver CC1 null mutation augmented IRI-OLT (CC1-KO→WT) by enhancing ROS expression and HMGB1 translocation during cold storage, data supported by in vitro studies where hepatic flush from CC1-deficient livers enhanced macrophage activation in bone marrow-derived macrophage cultures. Although hepatic CC1 deficiency augmented cold stress-triggered ASK1/p-p38 upregulation, adjunctive ASK1 inhibition alleviated IRI and improved OLT survival by suppressing p-p38 upregulation, ROS induction, and HMGB1 translocation (CC1-KO→WT), whereas ASK1 silencing (siRNA) promoted cytoprotection in cold-stressed and damage-prone CC1-deficient hepatocyte cultures. Consistent with mouse data, CEACAM1 expression in 60 human donor liver biopsies correlated negatively with activation of the ASK1/p-p38 axis, whereas low CC1 levels associated with increased ROS and HMGB1 translocation, enhanced innate and adaptive immune responses, and inferior early OLT function. Notably, reduced donor liver CEACAM1 expression was identified as one of the independent predictors for early allograft dysfunction (EAD) in human OLT patients. Thus, as a checkpoint regulator of IR stress and sterile inflammation, CEACAM1 may be considered as a denominator of donor hepatic tissue quality, and a target for therapeutic modulation in OLT recipients.

Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor That Induces CYP1A1 in Hepatic and Intestinal Cells.

The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.

The effect of graphene oxide on signalling of xenobiotic receptors involved in biotransformation

Graphene oxide (GO) is an engineered nanomaterial which was demonstrated to have outstanding capacity for adsorption of organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), the ligands and activators of the aryl hydrocarbon receptor (AhR). Due to the partially overlapping ligand capacity of AhR and pregnane X receptor (PXR), we tested the impact of GO particles on their signalling. While reporter gene assay revealed potentiating effect of GO on ligand-activated AhR-dependent luciferase activity, there was no effect for PXR. However, inducible target genes for AhR (CYP1A1) or PXR (ABCB1) were decreased at mRNA as well as protein levels by the presence of GO in HepG2 (for AhR), LS180 (for PXR) or primary human hepatocytes (both receptors). Moreover, the presence of GO diminished PXR and AhR protein levels in primary cultures of human hepatocytes. This was partially reversed by proteasome inhibitor MG132 for AhR but not for PXR. In conclusion, GO decreases ligand-stimulated activities of AhR and PXR in human cells.

Role of autophagy in Dynamic Remodeling of Cultured Mouse Hepatocytes

Primary culture of hepatocytes is a valuable tool to study mechanisms involved in liver metabolic, endocrine and secretory functions. However, freshly isolated hepatocytes dedifferentiate and lose many features of hepatocyte including the transporters and metabolic enzymes in the course of primary culture. Here, we investigated the role and mechanisms of autophagy in the dynamic remolding of long‐term cultured mouse hepatocytes. Mouse hepatocytes were freshly isolated from wild type mice and liver‐specific Atg5 as well as Drp1 knockout mice and cultured for different time points from 2 hours to 7 days. Autophagy and mitophagy flux were determined using the tandem GFP‐mCherry‐LC3 and Cox8‐GFP‐mCherry. We found that hepatocytes gradually lost their typical cuboidal morphology and displayed fibroblast like morphology, which was accompanied by increased stress fibers and highly fused mitochondrial network formation in the course of culture. These mitochondrial dynamic changes were associated with decreased major mitochondrial fusion proteins Mfn1/2 and fission protein Drp1. The levels of hepatic cytochrome P450 enzymes (such as 2E1 and 2B10) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) markedly decreased, suggesting loss of hepatic metabolic features of drug and glucose metabolism in cultured hepatocytes. GFP‐mCherry‐LC3 puncta analysis and Cox8‐GFP‐mCherry analysis revealed that basal autophagic flux and mitophagy increased as early as day 2 and continue to increase in dedifferentiated hepatocytes during the culture. This mitophagy was independent of Parkin, as the levels of Parkin were dramatically decreased after 12 hours culture in hepatocytes. Mitophagy was markedly blunted in cultured Drp1 knockout hepatocytes, suggesting that elongated mitochondrial may be protective against autophagic degradation. Importantly, the levels of P450 enzymes, Parkin and GADPH remained higher in Atg5 knockout hepatocytes compared with wild type hepatocytes during the culture, suggesting the autophagy may be involved in the degradation of these metabolic enzymes. In conclusion, these findings indicate that autophagy is important for the remodeling of cultured hepatocytes and manipulation of autophagy process may be helpful to maintain some of the hepatocyte features during the culture.