Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.
Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.
- Drug metabolism and disposition: the biological fate of chemicals
- Published almost 5 years ago
Carfilzomib, an irreversible proteasome inhibitor, has a favorable safety profile and significant antitumor activity in patients with relapsed and refractory multiple myeloma (MM). Here we summarize the clinical pharmacokinetics (PK), metabolism, and drug-drug interaction (DDI) profile of carfilzomib. The PK of carfilzomib, infused over 2-10 minutes, was evaluated in patients with solid tumors or MM. Metabolites of carfilzomib were characterized in patient plasma and urine samples. In vitro drug metabolism and DDI studies were conducted in human liver microsomes and hepatocytes. A clinical DDI study was conducted in patients with solid tumors to evaluate the effect of carfilzomib on CYP3A activity. Plasma concentrations of carfilzomib declined rapidly and in a biphasic manner after intravenous administration. The systemic half-life was short and the systemic clearance rate was higher than hepatic blood flow. Carfilzomib was cleared largely extrahepatically via peptidase cleavage and epoxide hydrolysis. Cytochrome P450-mediated metabolism played a minor role, suggesting that coadministration of P450 inhibitors or inducers is unlikely to change its PK profile. Carfilzomib showed direct and time-dependent inhibition of CYP3A in human liver microsome preparations and exposure to carfilzomib resulted in reductions in CYP3A and 1A2 gene expression in cultured human hepatocytes. However, administration of carfilzomib did not affect the PK of midazolam in patients with solid tumors, and there were no safety signals indicative of potential drug interactions. We conclude that the rapid systemic clearance and short half-life of carfilzomib limit clinically significant DDI.
- Drug metabolism and disposition: the biological fate of chemicals
- Published over 4 years ago
Organic anion-transporting polypeptides (OATPs) are multispecific transporters mediating the uptake of endogenous compounds and xenobiotics in tissues that are important for drug absorption and elimination, including the intestine and liver. Silymarin is a popular herbal supplement often used by patients with chronic liver disease; higher oral doses than those customarily used (140 mg three times/day) are being evaluated clinically. The present study examined the effect of silymarin flavonolignans on OATP1B1-, OATP1B3-, and OATP2B1-mediated transport in cell lines stably expressing these transporters, and in human hepatocytes. In overexpressing cell lines, OATP1B1- and OATP1B3-mediated estradiol-17β-glucuronide uptake and OATP2B1-mediated estrone-3-sulfate uptake were inhibited by most of the silymarin flavonolignans investigated. OATP1B1-, OATP1B3-, and OATP2B1-mediated substrate transport was inhibited efficiently by silymarin (IC(50) values of 1.3, 2.2 and 0.3 μM, respectively), silybin A (IC(50) values of 9.7, 2.7 and 4.5 μM, respectively), silybin B (IC(50) values of 8.5, 5.0 and 0.8 μM, respectively), and silychristin (IC(50) values of 9.0, 36.4 and 3.6 μM, respectively). Furthermore, silymarin, silybin A and silybin B (100 μM) significantly inhibited OATP-mediated estradiol-17β-glucuronide and rosuvastatin uptake into human hepatocytes. Calculation of the maximal unbound portal vein concentrations/IC(50) values indicated a low risk for silymarin-drug interactions in hepatic uptake with a customary silymarin dose. The extent of silymarin-drug interactions depends on OATP isoform specificity and concentrations of flavonolignans at the site of drug transport. Clinical investigations that achieve higher concentrations with either increased doses of silymarin or formulations with improved bioavailability may enhance the potential risk of DDIs with OATP substrates.
Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.
It has been known that Arak, Salvadora persica, has a number of medicinal properties. We tried to investigate in vitro scolicidal effect of root extracts of this plant against protoscolices from hydatid cysts of Echinococcus granulosus. Protoscolices were aseptically collected from sheep livers containing hydatid cysts. S. persica root extract was used in 10, 30, and 50 mg/ml concentration for 10, 20, and 30 min. The viability of protoscolices was ascertained by 0.1% eosin staining. Scolicidal activity of S. persica extract at a concentration of 10 mg/ml was 36.3%, 50.3%, and 70.8% after 10, 20, and 30 min of exposure, respectively. The scolicidal effect of this extract at a concentration of 30 mg/ml was 52.9%, 86.7%, and 100% after 10, 20, and 30 min of exposure, respectively. S. persica extract at a concentration of 50 mg/ml, meanwhile, killed 81.4%, 100%, and 100% of protoscolices after 10, 20, and 30 min, respectively. Also, the cytotoxic potential of S. persica was assessed on human liver cells (HepG2) using trypan blue exclusion test. No cytotoxic effect was observed on HepG2 cell line. The present study confirmed for the first time that the ethanolic extract of S. persica has high scolicidal power in vitro. However, in vivo effect of this material remains to be studied for treatment of echinococcosis in humans and herbivorous animals.
- Toxicology in vitro : an international journal published in association with BIBRA
- Published over 5 years ago
The HepaRG cell line is the first human cell line able to differentiate in vitro into mature hepatocyte-like cells. Our main objective within the framework of the EEC-LIINTOP project was to optimize the use of this cell line for drug metabolism and toxicity studies, especially after repeat treatments. The main results showed that differentiated HepaRG cells: (i) retained their drug metabolism capacity (major CYPs, phase 2 enzymes, transporters and nuclear receptors) and responsiveness to prototypical inducers at relatively stable levels for several weeks at confluence. The levels of several functions, including some CYPs such as CYP3A4, were dependent on the addition of dimethyl sulfoxide in the culture medium; (ii) sustained the different types of chemical-induced hepatotoxicity, including steatosis, phospholipidosis and cholestasis, after acute and/or repeat treatment with reference drugs. In particular, drug-induced vesicular steatosis was demonstrated in vitro for the first time. In conclusion, our results from the LIINTOP project, together with other studies reported concomitantly or more recently in the literature, support the conclusion that the metabolically competent human HepaRG cells represent a surrogate to primary human hepatocytes for investigating drug metabolism parameters and both acute and chronic effects of xenobiotics in human liver.
Platelets are the smallest blood constitutes which contain three types of granules; alpha granules, dense granules, and lysosomal granules. Each granule contains various biophysiological substances such as growth factors, cytokines, etc. Platelets have been conventionally viewed as a trigger of inflammatory responses and injury in the liver. Some studies revealed that platelets have strong effects on promoting liver regeneration. This review presents experimental evidence of platelets in accelerating liver regeneration and describes three different mechanisms involved; (1) the direct effect on hepatocytes, where platelets translocate to the space of Disse and release growth factors through direct contact with hepatocytes, (2) the cooperative effect with liver sinusoidal endothelial cells, where the dense concentration of sphingosine-1-phosphate in platelets induces excretion of interleukin-6 from liver sinusoidal endothelial cells, and (3) the collaborative effect with Kupffer cells, where the functions of Kupffer cells are enhanced by platelets.
The repeated dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect genotoxic hepatocarcinogens that can be integrated into a general toxicity study. The assay methods were thoroughly validated by 19 Japanese facilities. Methapyrilene hydrochloride (MP), known to be a non-genotoxic hepatocarcinogen, was examined in the present study. MP was dosed orally at 10, 30 and 100 mg/kg/day to 6-week-old male Crl:CD (SD) rats daily for 14 days. Treatment with MP resulted in an increase in micronucleated hepatocytes (MNHEPs) with a dosage of only 100 mg/kg/day. At this dose level, cytotoxicity followed by regenerative cell growth was noted in the liver. These findings suggest that MP may induce clastogenic effects indirectly on the liver or hepatotoxicity of MP followed by regeneration may cause increase in spontaneous incidence of MNHEPs.
The complexity of liver tumorigenesis is underscored by the recently observed anti-oncogenic effects of oncoproteins, although the mechanisms are unclear. Shp2/Ptpn11 is a proto-oncogene in hematopoietic cells and antagonizes the effect of tumor suppressor Pten in leukemogenesis. In contrast, we show here cooperative functions of Shp2 and Pten in suppressing hepatocarcinogenesis. Ablating both Shp2 and Pten in hepatocytes induced early-onset non-alcoholic steatohepatitis (NASH) and promoted genesis of liver tumor-initiating cells likely due to augmented cJun expression/activation and elevated ROS and inflammation in the hepatic microenvironment. Inhibiting cJun partially suppressed NASH-driven liver tumorigenesis without improving NASH. SHP2 and PTEN deficiencies were detected in liver cancer patients with poor prognosis. These data depict a mechanism of hepato-oncogenesis and suggest a potential therapeutic strategy.