Analysis of host factor networks during hepatitis B virus infection in primary human hepatocytes.

BACKGROUND: Chronic hepatitis B virus (HBV) infection affects around 250 million people worldwide, causing approximately 887,000 deaths annually, primarily owing to cirrhosis and hepatocellular carcinoma (HCC). The current approved treatments for chronic HBV infection, such as interferon and nucleos(t)ide analogs, have certain limitations as they cannot completely eradicate covalently closed circular DNA (cccDNA). Considering that HBV replication relies on host transcription factors, focusing on host factors in the HBV genome may provide insights into new therapeutic targets against HBV. Therefore, understanding the mechanisms underlying viral persistence and hepatocyte pathogenesis, along with the associated host factors, is crucial. In this study, we investigated novel therapeutic targets for HBV infection by identifying gene and pathway networks involved in HBV replication in primary human hepatocytes (PHHs). Importantly, our study utilized cultured primary hepatocytes, allowing transcriptomic profiling in a biologically relevant context and enabling the investigation of early HBV-mediated effects. METHODS: PHHs were infected with HBV virion particles derived from HepAD38 cells at 80 HBV genome equivalents per cell (Geq/cell). For transcriptomic sequencing, PHHs were harvested 1, 2-, 3-, 5-, and 7 days post-infection (dpi). After preparing the libraries, clustering and sequencing were conducted to generate RNA-sequencing data. This data was processed using Bioinformatics tools and software to analyze DEGs and obtain statistically significant results. Furthermore, qRT-PCR was performed to validate the RNA-sequencing results, ensuring consistent findings. RESULTS: We observed significant alterations in the expression patterns of 149 genes from days 1 to 7 following HBV infection (R2 > 0.7, q < 0.05). Functional analysis of these genes identified RNA-binding proteins involved in mRNA metabolism and the regulation of alternative splicing during HBV infection. Results from qRT-PCR experiments and the analysis of two validation datasets suggest that RBM14 and RPL28 may serve as potential biomarkers for HBV-associated HCC. CONCLUSIONS: Transcriptome analysis of gene expression changes during HBV infection in PHHs provided valuable insights into chronic HBV infection. Additionally, understanding the functional involvement of host factor networks in the molecular mechanisms of HBV replication and transcription may facilitate the development of novel strategies for HBV treatment.

Sex hormones differently regulate lipid metabolism genes in primary human hepatocytes.

BACKGROUND: Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is higher in men than in women. Hormonal and genetic causes may account for the sex differences in MASLD. Current human in vitro liver models do not sufficiently take the influence of biological sex and sex hormones into consideration. METHODS: Primary human hepatocytes (PHHs) were isolated from liver specimen of female and male donors and cultured with sex hormones (17ß-estradiol, testosterone and progesterone) for up to 72 h. mRNA expression levels of 8 hepatic lipid metabolism genes were analyzed by RT-qPCR. Sex hormones and their metabolites were determined in cell culture supernatants by LC-MS analyses. RESULTS: A sex-specific expression was observed for LDLR (low density lipoprotein receptor) with higher mRNA levels in male than female PHHs. All three sex hormones were metabolized by PHHs and the effects of hormones on gene expression levels varied depending on hepatocyte sex. Only in female PHHs, 17ß-estradiol treatment affected expression levels of PPARA (peroxisome proliferator-activated receptor alpha), LIPC (hepatic lipase) and APOL2 (apolipoprotein L2). Further changes in mRNA levels of female PHHs were observed for ABCA1 (ATP-binding cassette, sub-family A, member 1) after testosterone and for ABCA1, APOA5 (apolipoprotein A-V) and PPARA after progesterone treatment. Only the male PHHs showed changing mRNA levels for LDLR after 17ß-estradiol and for APOA5 after testosterone treatment. CONCLUSIONS: Male and female PHHs showed differences in their expression levels of hepatic lipid metabolism genes and their responsiveness towards sex hormones. Thus, cellular sex should be considered, especially when investigating the pathophysiological mechanisms of MASLD.

The effect of pregnancy-related hormones on hepatic transporters: studies with premenopausal human hepatocytes.

Introduction: Pregnancy results in significant changes in drug pharmacokinetics (PK). While previous studies have elucidated the impact of pregnancy-related hormones (PRH) on mRNA or protein expression and activity of major hepatic metabolizing enzymes, their effect on hepatic drug transporters remains largely unexplored. Therefore, we investigated the effect of a cocktail of PRH on the mRNA expression and activity of hepatic transporters. Methods: Plated human hepatocytes (PHH) from 3 premenopausal donors were incubated, in triplicate, for 72 h, with vehicle (DMSO < 0.01%), rifampin (10 µM; positive control) or a cocktail of PRH consisting of estrone, estradiol, estriol, estetrol, progesterone, cortisol, testosterone, oxytocin, and placental growth hormone. The PRH concentrations replicated 0.1×, 1×, or 10× of the plasma concentrations of these hormones observed during each of the three trimesters of pregnancy. After treatment, mRNA expression (quantified by qPCR) of hepatic influx and efflux transporters as well as the activity of influx transporters was quantified (uptake of a selective substrate ± corresponding transporter inhibitor). The data were expressed relative to that in the control (vehicle) group. Significance was evaluated by ANOVA (followed by Dunn's multiple comparisons) or unpaired t-test when the within-lot data were analyzed, or repeated measures ANOVA (followed by Dunn's multiple comparisons) or paired t-test when data from all 3 lots were analyzed (p < 0.05). Results and Discussion: In general, a) PRH cocktails significantly induced transporter mRNA expression in the following order OAT2 ≈ NTCP ≈ OCT1 > OATP2B1 and repressed mRNA expression in the following order OATP1B3 > OATP1B1; b) these changes translated into significant induction of OAT2 (T1-T3) and NTCP (T2-T3, in only two lots) activity at the 1× PRH concentration. Compared with the influx transporters, the induction of mRNA expression of efflux transporters was modest, with mRNA expression of MRP2 and BSEP being induced the most. Conclusion: Once these data are verified through in vivo probe drug PK studies in pregnancy, they can be populated into physiologically based pharmacokinetic (PBPK) models to predict, for all trimesters of pregnancy, transporter-mediated clearance of any drug that is a substrate of the affected transporters.

MicroRNAs modulate SARS-CoV-2 infection of primary human hepatocytes by regulating the entry factors ACE2 and TMPRSS2.

BACKGROUND AND AIMS: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) preferentially infects the respiratory tract; however, several studies have implicated a multi-organ involvement. Hepatic dysfunctions caused by SARS-CoV-2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non-coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS-CoV-2 and to evaluate the potential of miRNAs for modulating viral infection. METHODS: We analysed liver autopsies from a coronavirus disease 19 (COVID-19)-positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS-CoV-2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT-PCRs, western blots and luciferase reporter assays were performed. RESULTS: We could detect SARS-CoV-2 RNA in COVID-19-positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS-CoV-2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS-CoV-2 receptor expression and SARS-CoV-2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR-141-3p to the SARS-CoV-2 genome. CONCLUSION: We confirm that PHH are susceptible to SARS-CoV-2 infection and demonstrate selected miRNAs targeting SARS-CoV-2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS-CoV-2 and associated dysfunctions in COVID-19.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus.

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

Induction of steatosis in primary human hepatocytes recapitulates key pathophysiological aspects of metabolic dysfunction-associated steatotic liver disease.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease. Its limited treatment options warrant novel pre-clinical models for target selection and drug validation. We have established and extensively characterized a primary human steatotic hepatocyte in vitro model system that could guide treatment strategies for MASLD. METHODS: Cryopreserved primary human hepatocytes from five donors varying in sex and ethnicity were cultured with free fatty acids (FFA) in 3D collagen sandwich for 7 days and the development of MASLD was followed by assessing classical hepatocellular functions. As proof of concept, the effects of the drug Firsocostat (GS-0976) on in vitro MASLD phenotypes were evaluated. RESULTS: Incubation with FFA induced steatosis, insulin resistance, mitochondrial dysfunction, inflammation, and alterations in prominent human gene signatures similar to patients with MASLD, indicating the recapitulation of human MASLD in this system. As the application of Firsocostat rescued clinically observed fatty liver disease pathologies, it highlights the ability of the in vitro system to test drug efficacy and potentially characterize their mode of action. CONCLUSIONS: Altogether, our human MASLD in vitro model system could guide the development and validation of novel targets and drugs for the treatment of MASLD. IMPACT AND IMPLICATIONS: Due to low drug efficacy and high toxicity, a clinical treatment option for MASLD is limited. To facilitate earlier stop-go decisions in drug development, we have established a primary human steatotic hepatocyte in vitro model. As the model recapitulates clinically relevant MASLD characteristics at high phenotypic resolution, it can serve as a pre-screening platform and guide target identification and validation in MASLD therapy.

Tankyrase1/2 inhibitor XAV-939 reverts EMT and suggests that PARylation partially regulates aerobic activities in human hepatocytes and HepG2 cells.

The maintenance of a highly functional metabolic epithelium in vitro is challenging. Metabolic impairments in primary human hepatocytes (PHHs) over time is primarily due to epithelial-to-mesenchymal transitioning (EMT). The immature hepatoma cell line HepG2 was used as an in vitro model to explore strategies for enhancing the hepatic phenotype. The phenotypic characterization includes measuring the urea cycle, lipid storage, tricarboxylic acid-related metabolites, reactive oxygen species, endoplasmic reticulum calcium efflux, mitochondrial membrane potentials, oxygen consumptions rate, and CYP450 biotransformation capacity. Expression studies were performed with transcriptomics, co-immunoprecipitation and proteomics. CRISPR/Cas9 was also employed to genetically engineer HepG2 cells. After confirming that PHHs develop an EMT phenotype, expression of tankyrase1/2 was found to increase over time. EMT was reverted when blocking tankyrases1/2-dependent poly-ADP-ribosylation (PARylation) activity, by biochemical and genetic perturbation. Wnt/ß-catenin inhibitor XAV-939 blocks tankyrase1/2 and treatment elevated several oxygen-consuming reactions (electron-transport chain, OXHPOS, CYP450 mono-oxidase activity, phase I/II xenobiotic biotransformation, and prandial turnover), suggesting that cell metabolism was enhanced. Glutathione-dependent redox homeostasis was also significantly improved in the XAV-939 condition. Oxygen consumption rate and proteomics experiments in tankyrase1/2 double knockout HepG2 cells then uncovered PARylation as master regulator of aerobic-dependent cell respiration. Furthermore, novel tankyrase1/2-dependent PARylation targets, including mitochondrial DLST, and OGDH, were revealed. This work exposed a new mechanistic framework by linking PARylation to respiration and metabolism, thereby broadening the current understanding that underlies these vital processes. XAV-939 poses an immediate and straightforward strategy to improve aerobic activities, and metabolism, in (immature) cell cultures.

Deciphering the phospho-signature induced by hepatitis B virus in primary human hepatocytes.

Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

Examining the hepatotoxic potential of cannabidiol, cannabidiol-containing hemp extract, and cannabinol at consumer-relevant exposure concentrations in primary human hepatocytes.

Hemp extracts and consumer products containing cannabidiol (CBD) and/or other phytocannabinoids derived from hemp have entered the marketplace in recent years. CBD is an approved drug in the United States for the treatment of certain seizure disorders. While effects of CBD in the liver have been well characterized, data on the effects of other cannabinoids and hemp extracts in the liver and methods for studying these effects in vitro are limited. This study examined the hepatotoxic potential of CBD, CBD concentration-matched hemp extract, and cannabinol (CBN), at consumer-relevant concentrations determined by in silico modeling, in vitro using primary human hepatocytes. Primary human hepatocytes exposed to between 10-nM and 25-µM CBD, CBN, or hemp extract for 24 and 48 h were evaluated by measuring lactate dehydrogenase release, apoptosis, albumin secretion, urea secretion, and mitochondrial membrane potential. Cell viability was not significantly affected by CBD, CBN, or the hemp extract at any of the concentrations tested. Exposure to hemp extract induced a modest but statistically significant decrease in albumin secretion, urea secretion, and mitochondrial membrane potential at the highest concentration tested whereas CBD only induced a modest but statistically significant decrease in albumin secretion compared with vehicle control. Although this study addresses data gaps in the understanding of cannabinoid hepatoxicity in vitro, additional studies will be needed to determine how these results correlate with relevant consumer exposure and the biological effects of cannabinoids in human liver.

Perfluoropolyether-Based Gut-Liver-on-a-Chip for the Evaluation of First-Pass Metabolism and Oral Bioavailability of Drugs.

In the evolving field of drug discovery and development, multiorgans-on-a-chip and microphysiological systems are gaining popularity owing to their ability to emulate in vivo biological environments. Among the various gut-liver-on-a-chip systems for studying oral drug absorption, the chip developed in this study stands out with two distinct features: incorporation of perfluoropolyether (PFPE) to effectively mitigate drug sorption and a unique enterohepatic single-passage system, which simplifies the analysis of first-pass metabolism and oral bioavailability. By introducing a bolus drug injection into the liver compartment, hepatic extraction alone could be evaluated, further enhancing our estimation of intestinal availability. In a study on midazolam (MDZ), PFPE-based chips showed more than 20-times the appearance of intact MDZ in the liver compartment effluent compared to PDMS-based counterparts. Notably, saturation of hepatic metabolism at higher concentrations was confirmed by observations when the dose was reduced from 200 µM to 10 µM. This result was further emphasized when the metabolism was significantly inhibited by the coadministration of ketoconazole. Our chip, which is designed to minimize the dead volume between the gut and liver compartments, is adept at sensitively observing the saturation of metabolism and the effect of inhibitors. Using genome-edited CYP3A4/UGT1A1-expressing Caco-2 cells, the estimates for intestinal and hepatic availabilities were 0.96 and 0.82, respectively; these values are higher than the known human in vivo values. Although the metabolic activity in each compartment can be further improved, this gut-liver-on-a-chip can not only be used to evaluate oral bioavailability but also to carry out individual assessment of both intestinal and hepatic availability.

Heterocyclic Amines Disrupt Lipid Homeostasis in Cryopreserved Human Hepatocytes.

Metabolic dysfunction associated-steatotic liver disease (MASLD)/metabolic dysfunction-associated steatohepatitis (MASH) is the liver manifestation of metabolic syndrome, which is characterized by insulin resistance, hyperglycemia, hypertension, dyslipidemia, and/or obesity. Environmental pollutant exposure has been recently identified as a risk factor for developing MASH. Heterocyclic amines (HCAs) are mutagens generated when cooking meat at high temperatures or until well-done. Recent epidemiological studies reported that dietary HCA exposure may be linked to insulin resistance and type II diabetes, and we recently reported that HCAs induce insulin resistance and glucose production in human hepatocytes. However, no previous studies have examined the effects of HCAs on hepatic lipid homeostasis. In the present study, we assessed the effects of two common HCAs, MeIQx (2-amino-3, 8-dimethylimidazo [4, 5-f] quinoxaline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4, 5-b] pyridine), on lipid homeostasis in cryopreserved human hepatocytes. Exposure to a single concentration of 25 µM MeIQx or PhIP in human hepatocytes led to dysregulation of lipid homeostasis, typified by significant increases in lipid droplets and triglycerides. PhIP significantly increased expression of lipid droplet-associated genes, PNPLA3 and HSD17B13, and both HCAs significantly increased PLIN2. Exposure to MeIQx or PhIP also significantly increased expression of several key genes involved in lipid synthesis, transport and metabolism, including FASN, DGAT2, CPT1A, SCD, and CD36. Furthermore, both MeIQx and PhIP significantly increased intracellular cholesterol and decreased expression of PON1 which is involved in cholesterol efflux. Taken together, these results suggest that HCAs dysregulate lipid production, metabolism, and storage. The current study demonstrates, for the first time, that HCA exposure may lead to fat accumulation in hepatocytes, which may contribute to hepatic insulin resistance and MASH.

Metabolism of highly potent synthetic opioid nitazene analogs: N-ethyl-N-(1-glucuronyloxyethyl) metabolite formation and degradation to N-desethyl metabolites during enzymatic hydrolysis.

The metabolism of the highly potent synthetic opioids metonitazene, etonitazene, and protonitazene was investigated in fresh human hepatocytes. In the hydrolyzed culture medium, N-desethyl-, N,N-di-desethyl-, O-desalkyl-, N-desethyl-O-desalkyl-, N,N-di-desethyl-O-desalkyl-, and N-oxidated metabolites were detected as phase I metabolites, whereas in the unhydrolyzed culture medium, O-glucuronides of phase I metabolites with O-dealkylation were detected as phase II metabolites. The detected phase I metabolites were identified by comparing their analytical data with those of synthesized authentic standards. In contrast, phase II metabolites were identified by comparing their analytical data with those of the glucuronidated products formed by the incubation of the corresponding substrates with human liver microsomes in the presence of uridine diphosphate glucuronic acid. In addition to the aforementioned metabolites, some putative N-ethyl-N-(1-glucuronyloxyethyl) metabolites were detected in the unhydrolyzed culture medium. Purification and hydrolysis experiments revealed that N-ethyl-N-(1-glucuronyloxyethyl) metabolites formed the corresponding N-desethyl metabolites via unstable N-ethyl-N-(1-hydroxyethyl) metabolites during enzymatic hydrolysis.

Induction of human hepatic cytochrome P-450 3A4 expression by antifungal succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

Human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene.

Following isotonitazene scheduling in 2019, the availability of alternative 2-benzylbenzimidazole opioids (nitazenes) on the global drug market increased, resulting in many fatalities worldwide. Nitazenes are potent µ-opioid receptor agonists with strong narcotic/analgesic effects, and their concentrations in biological matrices are low, making the detection of metabolite biomarkers of consumption crucial to document use in clinical and forensic settings. However, there is little to no data on the metabolism of the most recently available nitazenes, especially desnitro-analogues. The aim of the research was to assess isotonitazene, metonitazene, etodesnitazene, and metodesnitazene human metabolism and identify specific metabolite biomarkers of consumption. The four analogues were incubated with 10-donor-pooled human hepatocytes, and the incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry and data mining with Compound Discoverer (Thermo Scientific); the analysis was supported by in silico metabolite predictions with GLORYx open-access software. Metabolites were identified in postmortem blood and/or urine samples from two metonitazene-positive and three etodesnitazene-positive cases following the same workflow, with and without glucuronide hydrolysis in urine, to confirm in vitro results. Twelve, nine, twenty-two, and ten metabolites were identified for isotonitazene, metonitazene, etodesnitazene, and metodesnitazene, respectively. The main transformations were N-deethylation at the N,N-diethylethanamine side chain, O-dealkylation, and further O-glucuronidation. In vitro and autopsy results were consistent, demonstrating the efficacy of the 10-donor-pooled human hepatocyte model to predict human metabolism. We suggest the parent and the corresponding O-dealkyl- and N-deethyl-O-dealkyl metabolites as biomarkers of exposure in urine after glucuronide hydrolysis, and the corresponding N-deethyl metabolite as additional biomarker in blood.

Preclinical efficacy and safety of encapsulated proliferating human hepatocyte organoids in treating liver failure.

Alginate-encapsulated hepatocyte transplantation is a promising strategy to treat liver failure. However, its clinical application was impeded by the lack of primary human hepatocytes and difficulty in controlling their quality. We previously reported proliferating human hepatocytes (ProliHHs). Here, quality-controlled ProliHHs were produced in mass and engineered as liver organoids to improve their maturity. Encapsulated ProliHHs liver organoids (eLO) were intraperitoneally transplanted to treat liver failure animals. Notably, eLO treatment increased the survival of mice with post-hepatectomy liver failure (PHLF) and ameliorated hyperammonemia and hypoglycemia by providing liver functions. Additionally, eLO treatment protected the gut from PHLF-augmented permeability and normalized the increased serum endotoxin and inflammatory response, which facilitated liver regeneration. The therapeutic effect of eLO was additionally proved in acetaminophen-induced liver failure. Furthermore, we performed assessments of toxicity and biodistribution, demonstrating that eLO had no adverse effects on animals and remained non-tumorigenic.

Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models.

Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction-associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.

The differences in drug disposition gene induction by rifampicin and rifabutin are unlikely due to different effects on important pregnane X receptor (NR1I2) splice variants.

Rifampicin and rifabutin can activate the pregnane X receptor (PXR, NR1I2), thereby inducing pharmacokinetically important genes/proteins and reducing exposure to co-administered drugs. Because induction effects vary considerably between these antibiotics, differences could be due to unequal rifamycin-induced activation or tissue expression of the three major NR1I2 splice variants, PXR.1 (NM_003889), PXR.2 (NM_022002), and PXR.3 (NM_033013). Consequently, PXR activation (PXR reporter gene assays) and mRNA expression levels of total NR1I2, PXR.1, PXR.2, and PXR.3 were investigated by polymerase chain reaction in colon and liver samples from eleven surgical patients, in LS180 cells, and primary human hepatocytes. Compared to the colon, total NR1I2 mRNA expression was higher in the liver. Both tissues showed similar expression levels of PXR.1 and PXR.3, respectively. PXR.2 was not quantifiable in the colon samples. Rifampicin and rifabutin similarly enhanced PXR.1 and PXR.2 activity when transfected into LS180 cells, while PXR.3 could not be activated. In LS180 cells, rifampicin (10 µM) reduced total NR1I2 and PXR.3 expression 2-fold after 24 h, while rifabutin (10 µM) increased total NR1I2, PXR.1, PXR.2, and PXR.3 mRNA by approx. 50% after 96-h exposure. In primary human hepatocytes, rifampicin (10 µM) suppressed total NR1I2, PXR.1, and PXR.3 after 48-h exposure, and rifabutin (10 µM) had no significant impact on total NR1I2 or any of the splice variants studied. In conclusion, both antibiotics activated the studied PXR splice variants similarly but modified their expression differently. While rifampicin can suppress mRNA of PXR forms, rifabutin rather increases their expression levels.

Comparison of in vitro thyroxine (T4) metabolism between Wistar rat and human hepatocyte cultures.

In vitro assays remain relatively new in exploring human relevance of liver, in particular nuclear receptor-mediated perturbations of the hypothalamus-pituitary-thyroid axis seen in rodents, mainly in the rat. Consistent with in vivo data, we confirm that thyroid hormone thyroxine metabolism was 9 times higher in primary rat hepatocytes (PRH) than in primary human hepatocytes (PHH) cultured in a 2D sandwich (2Dsw) configuration. In addition, thyroxine glucuronide (T4-G) was by far the major metabolite formed in both species (99.1% in PRH and 69.7% in PHH) followed by thyroxine sulfate (T4-S, 0.7% in PRH and 18.1% in PHH) and triiodothyronine/reverse triiodothyronine (T3/rT3, 0.2% in PRH and 12.2% in PHH). After a 7-day daily exposure to orphan receptor-mediated liver inducers, T4 metabolism was strongly increased in PRH, almost exclusively through increased T4-G formation. These results were consistent with the inductions of glucuronosyltransferase Ugt2b1 and canalicular transporter Mrp2. PHH also responded to activation of the three nuclear receptors, with mainly induction of glucuronosyltransferase UGT1A1 and canalicular transporter MRP2. Despite this, T4 disappearance rate and secreted T4 metabolites were only slightly increased in PHH. Overall, our data highlight that cryopreserved hepatocytes in 2Dsw culture allowing long-term exposure and species comparison are of major interest in improving liver-mediated human safety assessment.

SPOP regulates the expression profiles and alternative splicing events in human hepatocytes.

Speckle type BTB/POZ protein (SPOP) may have cancer promoting or inhibiting effects. At present, the role of SPOP in hepatocellular carcinoma (HCC) has rarely been studied. In this study, to investigate the effects of SPOP in HCC and elucidate the underlying molecular mechanisms of its relationship with genes, differentially expressed genes (DEGs) were classified through RNA sequencing. The gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes functional pathway analysis were used to further predict the function of DEGs after the overexpression of SPOP. The biological function of SPOP-regulated alternative splicing events in cells is comprehensively assessed. The Cancer Genome Atlas database and Gene Expression Omnibus dataset were performed to evaluate the correlation between SPOP and HCC progression. Due to SPOP overexpression, 56 DEGs in the HCC related pathway were further identified. The results showed that SPOP overexpression facilitated the cell proliferation and changed the gene expression profiles of human normal hepatocytes. SPOP-regulated alternative splicing events were involved in pathways associated with cellular processes, metabolism, environmental information procession, organismal systems, and so on. In conclusion, SPOP may potentially exhibit tumor-promoting effects, necessitating further investigations to unveil its molecular mechanisms comprehensively.

In vitro testing of host-targeting small molecule antiviral matriptase/TMPRSS2 inhibitors in 2D and 3D cell-based assays.

The outbreak of coronavirus disease 2019 (COVID-19) pandemic strongly stimulated the development of small molecule antivirals selectively targeting type II transmembrane serine proteases (TTSP), required for the host-cell entry of numerous viruses. A set of 3-amidinophenylalanine derivatives (MI-21, MI-472, MI-477, MI-485, MI-1903 and MI-1904), which inhibit the cleavage of certain viral glycoproteins was characterized in 2D and 3D primary human hepatocyte models on collagen- and Matrigel-coating using a CCK-8 assay to evaluate their cytotoxicity, a resorufin-based method to detect redox imbalances, fluorescence and ultrafiltration experiments to evaluate their interactions with human serum albumin (HSA) and α-acidic glycoprotein (AGP), and luminescence measurement to assess CYP3A4 modulation. For elucidation of selectivity of the applied compounds towards matriptase, transmembrane serine protease 2 (TMPRRS2), thrombin and factor Xa (FXa) Ki values were determined. It was proven that cell viability was only deteriorated by inhibitor MI-1903, and redox status was not influenced by administration of the selected inhibitors at 50 µM for 24 h. MI-472 and MI-477 formed relatively stable complexes with AGP. CYP3A4 inhibition was found to be strong in PHHs exposed to all inhibitors with the exception of MI-21, which seems to be a promising drug candidate also due to its better selectivity towards matriptase and TMPRSS2 over the blood clotting proteases thrombin and FXa. Our in vitro pharmacokinetic screening with these inhibitors helps to select the compounds with the best selectivity and safety profile suitable for a further preclinical characterization without animal sacrifice.