Primary Human Hepatocyte Spheroids as Tools to Study the Hepatotoxic Potential of Non-Pharmaceutical Chemicals

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.

Collagen Fibers-based Self-assembled 3D Spheroids Alleviate Thioacetamide-induced Liver Cirrhosis in Rats

Cirrhosis refers to irreversible liver damage where healthy tissue is replaced by scar tissue which impairs liver function. There is no cure and current treatments only prevent further liver damage; thus novel therapeutic options are urgently needed. Here, we report a new approach that enables the formation of the self-assembled 3D spheroids of adipose-derived stem cells (ADSCs) and murine hepatocytes (AML12) via reconstituted collagen fibers. Compared with the spheroids formed in the commercially available EZSHERE dish, the collagen fiber-based ADSC/hepatocyte spheroids offer a notable benefit in structure formation and paracrine factor secretion. To test regenerative capability of the collagen fiber-based 3D ADSC/hepatocyte spheroids, a rat model of thioacetamide (TAA)-induced liver cirrhosis was employed. The transplantation of the collagen fiber-based 3D ADSC/hepatocyte spheroids show an improvement in liver function and ameliorates pathological liver cirrhosis in TAA-treated rats. In summary, our data show collagen fiber-based self-assembled 3D ADSC/hepatocyte spheroids to possess excellent regenerative capacity in response to TAA-induced liver injury, promising an alternative therapeutic strategy for liver cirrhosis.

Hepatotoxicity of copper sulfide nanoparticles towards hepatocyte spheroids using a novel multi-concave agarose chip method

Aim: To explore the hepatotoxicity of copper sulfide nanoparticles (CuSNPs) toward hepatocyte spheroids. Materials & methods: Other than the traditional agarose method to generate hepatocyte spheroids, we developed a multi-concave agarose chip (MCAC) method to investigate changes in hepatocyte viability, morphology, mitochondrial membrane potential, reactive oxygen species and hepatobiliary transporter by CuSNPs. Results: The MCAC method allowed a large number of spheroids to be obtained per sample. CuSNPs showed hepatotoxicity in vitro through a decrease in spheroid viability, albumin/urea production and glycogen deposition. CuSNPs also introduced hepatocyte spheroid injury through alteration of mitochondrial membrane potential and reactive oxygen species, that could be reversed by N-acetyl-l-cysteine. CuSNPs significantly decreased the activity of BSEP transporter by downregulating its mRNA and protein levels. Activity of the MRP2 transporter remained unchanged. Conclusion: We observed the hepatotoxicity of CuSNPs in vitro with associated mechanisms in an advanced 3D culture system.

Expression of Cytochrome P450 Enzymes Is Induced by Flaxseed Enterolignans

Objectives The major flaxseed lignan, secoisolariciresinol diglucoside (SDG), is metabolized to bioactive enterolignans in the intestine and liver. Once formed, enterolignans are subject to hepatic conjugation, similar to cytochrome P450 (CYP)-mediated conjugation of many drugs. CYPs also metabolize food-derived phytochemicals; therefore, adding flaxseed to the diet may promote drug-nutrient interactions affecting the potency of medications. Clinical trials have shown blood pressure-lowering effects of flaxseed in hypertensive patients; however, it is unknown if this stems from direct actions of flaxseed bioactives or indirectly from increased circulating levels of anti-hypertensive (AH) medications resulting from competition for CYP-mediated conjugation and inactivation. Our objective was to determine if enterolignans produced from flaxseed SDG affect expression or activity of CYP enzymes that metabolize metoprolol (MET), a commonly-prescribed AH medication. Methods 3D human hepatocyte spheroids were treated with 25 μM MET or 1, 10, or 100 μM enterodiol (END) for 48 hours, after which RNA was extracted for qPCR analysis of expression of CYP2C9 and CYP3A4, two CYP isozymes involved in MET metabolism. Spheroids were also treated with the same concentrations of MET and END for 1 and 48 hours to measure MET and END metabolites using HPLC/LC-QTOF-MS analysis. Differences were tested with 1-way ANOVA with Tukey's post-hoc test. Results MET, 1 μM END, and 10 μM END all induced an approximate 2-fold increase in CYP2C9 expression, while 100 μM END induced a 4-fold increase (P < 0.05). CYP3A4 expression was induced 5-fold by MET, while 1 μM and 10 μM END produced only a 2-fold increase in CYP3A4 expression (P < 0.05). 100 μM END led to an almost 14-fold increase in CYP3A4 expression (P < 0.05). Compared to 1 hour of co-treatment with 25 μM MET + END, O-demethylmetoprolol in media was significantly lower after 48 hours (P < 0.05), suggesting competition between END and MET for CYP metabolism. Conclusions Our initial experiments in 3D human hepatic spheroids are the first to show that metabolites of flaxseed lignans induce expression of CYP2C9 and CYP3A4. The fact that flaxseed enterolignans induce the same CYPs as MET supports the hypothesis of shared metabolism and potential metabolic competition between enterolignans and AH medications. Funding Sources NSERC Discovery Grant.

High-throughput transcriptomic analysis of human primary hepatocyte spheroids exposed to per- and polyfluoroalkyl substances (PFAS) as a platform for relative potency characterization

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focussed on four model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at four time-points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS; (2) identify similarities in biological responses; (3) compare PFAS potency through benchmark concentration analysis; and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (i.e., was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

The Efficacy of the Hepatocyte Spheroids for Hepatocyte Transplantation

The safety and short-term efficacy of hepatocyte transplantation (HCTx) have been widely proven. However, issues such as reduced viability and/or function of hepatocytes, insufficient engraftment, and lack of a long-term effect have to be overcome for widespread application of HCTx. In this study, we evaluated hepatocyte spheroids (HSs), formed by self-aggregation of hepatocytes, as an alternative to hepatocytes in single-cell suspension. Hepatocytes were isolated from C57BL/6 J mice liver using a three-step collagenase perfusion technique and HSs were formed by the hanging drop method. After the spheroids formation, the HSs showed significantly higher mRNA expression of albumin, ornithine transcarbamylase, glucose-6-phosphate, alpha-1-antitrypsin, low density lipoprotein receptor, coagulation factors, and apolipoprotein E (ApoE) than 2 dimensional (2D)-cultured hepatocytes ( p < 0.05). Albumin production by HSs was significantly higher than that by 2D-cultured hepatocytes (9.5 ± 2.5 vs 3.5 ± 1.8 μg/dL, p < 0.05). The HSs, but not single hepatocytes, maintained viability and albumin mRNA expression in suspension (92.0 ± 2.8% and 1.03 ± 0.09 at 6 h). HSs (3.6 × 106 cells) or isolated hepatocytes (fSH, 3.6 × 106 cells) were transplanted into the liver of ApoE knockout (KO-/-) mice via the portal vein. Following transplantation, serum ApoE concentration (ng/mL) of HS-transplanted mice (1w: 63.1 ± 56.7, 4w: 17.0 ± 10.9) was higher than that of fSH-transplanted mice (1 w: 33.4 ± 13.0, 4w: 13.7 ± 9.6). In both groups, the mRNA levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, MCP-1, and MIP-1β) were upregulated in the liver following transplantation; however, no significant differences were observed. Pathologically, transplanted HSs were observed as flat cell clusters in contact with the portal vein wall on day 7. Additionally, ApoE positive cells were observed in the liver parenchyma distant from the portal vein on day 28. Our results indicate that HS is a promising alternative to single hepatocytes and can be applied for HCTx.