Hepatic HepG2 Research Articles

Calcium hydroxide nanoparticles induce cell death, genomic instability, oxidative stress and apoptotic gene dysregulation on human HepG2 cells

Calcium hydroxide nanoparticles (Ca(OH)2NPs) possess potent antimicrobial activities and unique physical and chemical properties, making them valuable across various fields. However, limited information exists regarding their effects on genomic DNA integrity and their potential to induce apoptosis in normal and cancerous human cell lines. This study thus aimed to evaluate the impact of Ca(OH)2NPs on cell viability, genomic DNA integrity, and oxidative stress induction in human normal skin fibroblasts (HSF) and cancerous hepatic (HepG2) cells. Cell viability and genomic DNA stability were assessed using the Sulforhodamine B (SRB) assay and alkaline comet assay, respectively. Reactive oxygen species (ROS) levels were measured using 2,7-dichlorofluorescein diacetate, while the expression level of apoptosis-related genes (p53, Bax, and Bcl-2) were quantified using real-time PCR (qRT-PCR). The SRB cytotoxicity assay revealed that a 48-hour exposure to Ca(OH)2NPs caused concentration-dependent cell death and proliferation inhibition in both HSF and HepG2 cells, with IC50 values of 271.93 µg/mL for HSF and 291.8 µg/mL for HepG2 cells. Treatment with the IC50 concentration of Ca(OH)2NPs selectively induced significant DNA damage, excessive ROS generation, and marked dysregulation of apoptotic (p53 and Bax) and anti-apoptotic (Bcl-2) gene expression in HepG2 cells, triggering apoptosis. In contrast, exposure of HSF cells to the IC50 concentration of Ca(OH)2NPs caused no significant changes in genomic DNA integrity, ROS generation, or apoptotic gene expression. These findings indicate that Ca(OH)2NPs exhibit concentration-dependent cytotoxicity in both normal HSF and cancerous HepG2 cells. However, exposure to the IC50 concentration was non-genotoxic to normal HSF cells while selectively inducing genotoxicity and apoptosis in HepG2 cancer cells through DNA breaks and ROS-mediated mechanisms. Further studies are required to explore the biological and toxicological properties and therapeutic potential of Ca(OH)2NPs in hepatic cancer treatment.

Application of a dynamic colonic gastrointestinal digestion model to red wines: a study of flavanol metabolism by the gut microbiota and the cardioprotective activity of microbial metabolites.

Over the last decade, research has emphasized the role of the microbiome in regulating cardiovascular physiology and disease progression. Understanding the interplay between wine polyphenols, the gut microbiota, and cardiovascular health could provide valuable insights for uncovering novel therapeutic strategies aimed at preventing and managing cardiovascular disease. In this study, two commercial red wines were subjected to in vitro dynamic gastrointestinal digestion (GIS) to monitor the flavanol-microbiota interaction by evaluating the resulting microbial metabolites. Furthermore, the cardiovascular protective activity of wine flavanol microbial metabolites was investigated, integrating their effects on antihypertensive activity, cholesterol metabolism and insulin resistance into human endothelial (EA.hy926) and hepatic (HepG2) cell lines. A significant production of microbial flavanol metabolites, with a prevalence of phenylpropionic and phenylacetic acids, valerolactones and short chain fatty acids such as butyric acid, was observed, particularly in the transverse and descending colon sections. Incubating HAECs and HepG2 cells with the colon improved cardioprotective parameters. Specifically, an increase in the vasodilator NO, an improvement in the LDL receptors and the HMGCoA enzyme, with positive effects on cholesterol metabolism, and the reduction of glycogen levels improving insulin resistance were observed.

Interaction of Phyllanthus amarus extract and its lignans with human xenobiotic receptors, drug metabolizing enzymes and drug transporters

Ethnopharmacological relevancePhyllanthus amarus is ethnomedicinally used to treat gallbladder stones, kidney stones and chronic liver diseases. P. amarus is gaining popularity as an ingredient in many botanical dietary supplements. Aim of the studyTo evaluate the interaction of P. amarus extract and its lignans with human xenobiotic sensing receptors (PXR and AhR) and their downstream genes. Materials and methodsActivation of PXR and AhR was measured by reporter gene assays. Gene expression analysis was performed in hepatic (HepG2) and intestinal (LS174T) cells by RT-PCR. CYP inhibition assays were carried out in baculosomes. The inhibitory effect on the ABC transporters (P-gp and BCRP) was investigated via rhodamine-123 and Hoechst 33342 uptake assays in Caco-2 and MDR-MDCK cells. Effect on CYP3A4 and CYP1A2 enzyme activity was measured in primary human hepatocytes. ResultsP. amarus extract and its lignans activated AhR and PXR in respective reporter cells. Tested extract and lignans significantly increased CYP3A4 mRNA but inhibited CYP3A4 enzyme activity when tested in primary human hepatocytes and CYP3A4-specific baculosomes. In contrast, increased CYP1A2 mRNA was associated with increased CYP1A2 enzyme activity in hepatocytes. No inhibition of CYP1A2 activity was detected in baculosomes. A weak inhibitory effect on ABC-transporters was observed. ConclusionsResults suggest that overconsumption of P. amarus or P. amarus-containing botanical supplements may change CYP homeostasis which could alter the pharmacokinetics of substrate drugs, thereby elevating the risk of herb-drug interactions (HDIs) when taken concomitantly with conventional medications. Further studies are warranted to strengthen the clinical relevance of these findings.

Mitochondrial dysfunction induced in human hepatic HepG2 cells exposed to the fungicide kresoxim-methyl and to a mixture kresoxim-methyl/boscalid

ABSTRACT The fungicides strobilurins and succinate dehydrogenase inhibitors (SDHIs) are blockers of the electron transport chain (ETC) in fungi. Here, we show that the exposure for 24 h to kresoxym-methyl, a fungicide from the class of strobilurins, alters the mitochondrial respiration in human HepG2 hepatocytes. In addition, we demonstrate an increase in production of mitochondrial superoxide radical anion, a reduction in ATP level, a decrease in the ratio reduced/oxidized glutathione and a decrease in cell viability (assessed by the LDH assay, Presto Blue assay, and Crystal Violet assay). As kresoxym-methyl is associated to boscalid (SDHI) in commercial formulations, we analyzed a potential exacerbation of the induced mitochondrial dysfunction for this combination. For the highest dose at which kresoxym-methyl (5 µM) and boscalid (0.5 µM) did not induce changes in mitochondrial function when used separately, in contrast, when both fungicides were used in combination at the same concentration, we observed a significant alteration of the mitochondrial function of hepatocytes: there was a decrease in oxygen consumption rate, in the ATP level. In addition, the level of mitochondrial superoxide radical anion was increased leading to a decrease in the ratio reduced/oxidized glutathione, and an increase in viability.

In Silico and In Vitro Investigation of Cytotoxicity and Apoptosis of Acridine/Sulfonamide Hybrids Targeting Topoisomerases I and II.

Sulfonamide acridine derivatives have garnered significant attention from medicinal chemists due to their diverse range of biological activities. In this study, eleven compounds were synthesized according to the literature, and their impact on cell growth inhibition, induction of apoptosis, and cell cycle distribution were assessed in three different cell lines. Their inhibitory effects on the topoisomerase (Topo) I and II were investigated in vitro. Molecular docking studies were conducted to predict the binding affinities of these compounds for crystallized downloaded topoisomerases. The compounds were examined in vitro for their anticancer activity against human hepatic (HepG2) colon (HCT-8) and breast (MCF-7) carcinoma cell lines. Compound 8b was the most active against HepG2, HCT-116, and MCF-7 with IC50 14.51, 9.39, and 8.83 µM, respectively, compared to Doxorubicin as reference. In addition, it demonstrated the highest potency among the tested compounds against Topo-I, with an IC50 value of 3.41 µg/mL compared to the control camptothecin (IC50 of 1.46 μM). Compound 7c displayed a significant inhibitory effect on Topo-II, with an IC50 of 7.33 μM, compared to an IC50 value of 6.49 μM via Doxorubicin, the control. Compounds 7c and 8b were assessed against topoisomerases showing induction of apoptosis and a reduction in the S phase of the cell cycle. Molecular docking demonstrated interaction with the active site as with those exhibited by the co-crystallized ligands of the crystallized proteins in both topoisomerases. Compounds 7c and 8b hold promise as potential anticancer drugs due to their anti-proliferative and proapoptotic effects, which are mediated by their action on the topoisomerase enzyme, particularly Topo II.

Oxidative Stress Associated With Increased Reactive Nitrogen Species Generation in the Liver and Kidney Caused by a Major Metabolite Accumulating in Tyrosinemia Type 1.

Tyrosinemia type 1 (TT1) is caused by fumarylacetoacetate hydrolase activity deficiency, resulting in tissue accumulation of upstream metabolites, including succinylacetone (SA), the pathognomonic compound of this disease. Since the pathogenesis of liver and kidney damage observed in the TT1-affected patients is practically unknown, this study assessed the effects of SA on important biomarkers of redox homeostasis in the liver and kidney of adolescent rats, as well as in hepatic (HepG2) and renal (HEK-293) cultured cells. SA significantly increased nitrate and nitrite levels and decreased the concentrations of reduced glutathione (GSH) in the liver and kidney, indicating induction of reactive nitrogen species (RNS) generation and disruption of antioxidant defenses. Additionally, SA decreased the GSH levels and the activities of glutathione peroxidase, glutathione S-transferase, glutathione reductase, and superoxide dismutase in hepatic and renal cells. Noteworthy, melatonin prevented the SA-induced increase of nitrate and nitrite levels in the liver. Therefore, SA-induced increase of RNS generation and impairment of enzymatic and nonenzymatic antioxidant defenses may contribute to hepatopathy and renal disease in TT1.

Cultured Bacteria Isolated from Primary Sclerosing Cholangitis Patient Bile Induce Inflammation and Cell Death.

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by inflammation and progressive fibrosis of the biliary tree. The pathogenesis of PSC remains poorly understood, and there are no effective therapeutic options. Previous studies have observed associations between changes in the colonic and biliary microbiome and PSC. We aimed to determine whether bacterial isolates cultured from PSC patient bile induced disease-associated phenotypes in cells. Bile was collected from PSC patients (n=10) by endoscopic retrograde cholangiography and from non-PSC controls (n=3) undergoing cholecystectomies. Biliary bacteria were cultured anaerobically, and 50 colonies per sample were identified by 16S rRNA sequencing. The effects of supernatants from seven PSC-associated bacterial strains on cellular phenotypes were characterized using human colonic (Caco-2), hepatic (HepG2), and biliary (EGI-1) cells. No bacteria were isolated from non-PSC controls, while bacteria were cultured from most PSC patients. The PSC bile microbiomes exhibited reduced diversity compared to the gut or oral cavity, with one or two bacterial strains predominating. Overall, PSC-associated bacteria produced factors that were cytotoxic to hepatic and biliary cells. Enterococcus faecalis , and to a lesser extent Veillonella parvula , induced epithelial permeability, while Escherichia coli, Fusobacterium necrophorum , and Klebsiella pneumoniae induced inflammatory cytokines in biliary cells. Our data suggest that bacteria cultured from PSC bile induce cellular changes that may contribute to PSC disease pathogenesis. Enterococcus may promote intestinal permeability, facilitating bacterial migration to the biliary tree. Once there, Escherichia, Fusobacterium and Klebsiella , may cause inflammation and damage in biliary and liver cells.

Myricetin inhibits CYP3A4, GST, and MRP1 in hepatic cancer cells

AbstractHerbal and nutritional supplements are widely used to prevent and treat many diseases, including cancer. Tumor cells modify metabolic enzyme systems like CYP3A4 and GST. They also overexpress MRP1, an ATP-binding cassette transporter subfamily G (ABCG2) member. Drug efflux may increase, reducing tumor cell drug accumulation and developing drug resistance that leads to significant obstacles in cancer care. Natural products' ability to overcome cancer's multidrug resistance is interesting. Their ability to affect several targets makes them valuable in addressing drug resistance from diverse approaches. The potential of natural flavonoid; Myricetin (MYR) to modulate CYP3A4, GST, and MRP1 activity and expression in hepatic cancer cells was evaluated to prove its targeting and preventing these pathways of multidrug resistance. The cell proliferation of MYR was determined using an MTT assay. Specific enzyme assays, efflux assay, and gene expression using RT-PCR were used to evaluate MYR effect in hepatic cell lines HepG-2 and Huh-7. MYR has a noteworthy cytotoxic effect compared to doxorubicin (DOX) with IC50 > 100 μM in HepG-2 and Huh-7 cells. MYR showed potent inhibition of CYP3A4 and GST enzyme activity and MRP1 efflux function and downregulated their gene expression in a dose-dependent manner in both cells. MYR100 dose was the most significant effective dose. MRY100 decreased CYP3A4 activity by 67.5% (P < 0.05) and 55% (P < 0.01) and downregulated the gene by 0.2-fold (P < 0.001) and 0.3-fold (P < 0.001) in HepG-2 and Hub-7 cells, respectively. After treatment with MRY100, GST activity decreased significantly in both cells, reaching 47.6% (P < 0.001) and 33.2% and GST gene downregulation was 0.12 and 0.21-fold (P < 0.001). MRY100 inhibited MRP1 efflux pump 2.3 times (P < 0.001) and 1.9 times (P < 0.001) more effectively than PC, resulting in a 0.23-fold and 0.12-fold downregulation of MRP1 genes in HepG-2 and Hub-7 cells. The result will validate the use of MYR to interact with the metabolism phases and could be used as adjuvant therapy in cancer prevention and treatment approaches.

Lacticaseibacillus paracsei HY7207 Alleviates Hepatic Steatosis, Inflammation, and Liver Fibrosis in Mice with Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty acid disease (NAFLD) is caused by a build-up of fat in the liver, inducing local inflammation and fibrosis. We evaluated the effects of probiotic lactic acid-generating bacteria (LAB) derived from a traditional fermented beverage in a mouse model of NAFLD. The LAB isolated from this traditional Korean beverage were screened using the human hepatic cell line HepG2, and Lactocaseibacillus paracasei HY7207 (HY7207), which was the most effective inhibitor of fat accumulation, was selected for further study. HY7207 showed stable productivity in industrial-scale culture. Whole-genome sequencing of HY7207 revealed that the genome was 2.88 Mbp long, with 46.43% GC contents and 2778 predicted protein-coding DNA sequences (CDSs). HY7207 reduced the expression of lipogenesis and hepatic apoptosis-related genes in HepG2 cells treated with palmitic acid. Furthermore, the administration of 109 CFU/kg/day of HY7207 for 8 weeks to mice fed an NAFLD-inducing diet improved their physiologic and serum biochemical parameters and ameliorated their hepatic steatosis. In addition, HY7207 reduced the hepatic expression of genes important for lipogenesis (Srebp1c, Fasn, C/ebpa, Pparg, and Acaca), inflammation (Tnf, Il1b, and Ccl2), and fibrosis (Col1a1, Tgfb1, and Timp1). Finally, HY7207 affected the expression of the apoptosis-related genes Bax (encoding Bcl2 associated X, an apoptosis regulator) and Bcl2 (encoding B-cell lymphoma protein 2) in the liver. These data suggest that HY7207 consumption ameliorates NAFLD in mice through effects on liver steatosis, inflammation, fibrosis, and hepatic apoptosis. Thus, L. paracasei HY7207 may be suitable for use as a functional food supplement for patients with NAFLD.

Synthesis of Novel Acrylamide Graft Copolymer of Acacia nilotica Gum for the Stabilization of Melatonin Nanoparticles for Improved Therapeutic Effect: Optimization Using (3)2 Factorial Design.

The objective of the present study was to optimize the microwave-assisted synthesis of the acrylamide graft copolymer of Acacia nilotica gum (AM-co-ANG). Furthermore, graft copolymer was used for the formulation of a nanoparticulate system using a novel top to bottom solvent antisolvent technique for the delivery of melatonin. Grafting of ANG was optimized by using 32 factorial design, where concentrations of polymer and monomer (acrylamide) were used as independent variables and swelling index in acidic (0.1 N HCl) and basic (1 N NaOH) pH. Grafted polymers were further used to develop and optimize nanoparticulate system using concentration of the graft copolymer and concentration of drug as independent variables. The size of the nanoformulation and entrapment efficiency were selected as dependent variables. Difference in infrared spectrum and absorbance maxima in the ultraviolet region confirm that grafting has taken place. Porous structure and a higher contact angle confirmed hydrophobic nature of AM-co-ANG as compared with the native polymer. Acrylamide graft copolymers show more swelling in 1 N NaOH as compared with 0.1 N HCl. In vitro toxicity studies in hepatic (HepG2 cell line), brain (SHSY5Y cell line), and skin (HaCaT cell line) cells easily predict that synthesized polymer have no cytotoxicity. The entrapment efficiency ranged from 55.24 ± 1.35% to 73.21 ± 1.83%. A nonlinear correlation was observed between independent and dependent variables, as confirmed by multivariate analysis of variance, surface regression, and the correlation report. The prepared formulations were able to release drug up to 12 h. The regression coefficient easily predicted that most of the formulations followed Baker-Lonsdale drug release kinetics.

Design of experiment (DoE) of mucoadhesive valproic acid-loaded nanostructured lipid carriers (NLC) for potential nose-to-brain application

Epilepsy is a highly prevalent neurological disease and valproic acid (VPA) is used as a first-line chronic treatment. However, this drug has poor oral bioavailability, which requires the administration of high doses, resulting in adverse effects. Alternative routes of VPA administration have therefore been investigated, such as the nose-to-brain route, which allows the drug to be transported directly from the nasal cavity to the brain. Here, the use of nanostructured lipid carriers (NLC) to encapsulate drugs administered in the nasal cavity has proved advantageous. The aim of this work was to optimise a mucoadhesive formulation of VPA-loaded NLC for intranasal administration to improve the treatment of epilepsy. The Design of Experiment (DoE) was used to optimise the formulation, starting with component optimisation using Mixture Design (MD), followed by optimisation of the manufacturing process parameters using Central Composite Design (CCD).The optimised VPA-loaded NLC had a particle size of 76.1 ± 2.8 nm, a polydispersity index of 0.190 ± 0.027, a zeta potential of 28.1 ± 2.0 mV and an encapsulation efficiency of 85.4 ± 0.8%. The in vitro release study showed VPA release from the NLC of 50 % after 6 h and 100 % after 24 h. The in vitro biocompatibility experiments in various cell lines have shown that the optimised VPA-loaded NLC formulation is safe up to 75 µg/mL, in neuronal (SH-SY5Y), nasal (RPMI 2650) and hepatic (HepG2) cells. Finally, the interaction of the optimised VPA-loaded NLC formulation with nasal mucus was investigated and mucoadhesive properties were observed. The results of this study suggest that the use of intranasal VPA-loaded NLC may be a promising alternative to promote VPA targeting to the brain, thereby improving bioavailability and minimising adverse effects.

Air-ventilated normothermic mechanical perfusion improves susceptibility to donation after circulatory death and cold preservation-induced cholestatic liver injury through PPAR-γ/UGT1A1 axis.

End-ischemic normothermic mechanical perfusion (NMP) could provide a curative treatment to reduce cholestatic liver injury from donation after circulatory death (DCD) in donors. However, the underlying mechanism remains elusive. Our previous study demonstrated that air-ventilated NMP could improve functional recovery of DCD in a preclinical NMP rat model. Here, metabolomics analysis revealed that air-ventilated NMP alleviated DCD- and cold preservation-induced cholestatic liver injury, as shown by the elevated release of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and γ-glutamyl transferase (GGT) in the perfusate (p < .05) and the reduction in the levels of bile acid metabolites, including ω-muricholic acid, glycohyodeoxycholic acid, glycocholic acid, and glycochenodeoxycholate (GCDC) in the perfused livers (p < .05). In addition, the expression of the key bile acid metabolism enzyme UDP-glucuronosyltransferase 1A1 (UGT1A1), which is predominantly expressed in hepatocytes, was substantially elevated in the DCD rat liver, followed by air-ventilated NMP (p < .05), and invitro, this increase was induced by decreased GCDC and hypoxia-reoxygenation in the hepatic cells HepG2 and L02 (p < .05). Knockdown of UGT1A1 in hepatic cells by siRNA aggravated hepatic injury caused by GCDC and hypoxia-reoxygenation, as indicated by the ALT and AST levels in the supernatant. Mechanistically, UGT1A1 is transcriptionally regulated by peroxisome proliferator-activator receptor-γ (PPAR-γ) under hypoxia-physoxia. Taken together, our data revealed that air-ventilated NMP could alleviate DCD- and cold preservation-induced cholestatic liver injury through PPAR-γ/UGT1A1 axis. Based on the results from this study, air-ventilated NMP confers a promising approach for predicting and alleviating cholestatic liver injury through PPAR-γ/UGT1A1 axis.

Abstract Mo089: Fontan-Associated Liver Disease On A Chip

Introduction: The Fontan procedure is performed in children with single ventricle physiology, and has led to excellent short-term palliation and survival rates but at the cost of long-term complications. Fontan-associated liver disease (FALD) results from hemodynamic changes following the Fontan procedure, leading to elevated central venous pressures, chronic alterations in hepatic perfusion, and resultant hepatic fibrosis. Improved understanding of FALD pathophysiology, which may provide insights for prevention and treatment is challenged by the lack of experimental models. Current in vitro liver models, mostly 2D or suspension hepatic cell cultures, lack biomimicry. This study presents a 3D bioprinted perfusable human liver model for in vitro study of hepatic fibrosis in response to altered flow conditions in FALD patients. Methods: Vascular 3D model of hepatic sinusoid was biofabricated using hybrid bioinks in two distinct peripheral layers and a central lumen. The outer layer was cast with hepatic (HepG2) cells encapsulated in a mixture of gelatin methacrylate (GelMA) and collagen type I. The inner layer consisted of hepatic stellate cells (HSCs) in GelMA. Human endothelial cells (ECs) were seeded onto the central lumen. 3D cellular constructs were cultured in vitro for three weeks in static or dynamic conditions, by circulating the culture media at varying flow conditions to simulate sinusoid pressure characteristic of healthy vs. disease state. Using Volcano system in cath lab, we invasively measured the pressure in mid-channel. Cell viability, proliferation, maturation into functional liver tissue, and tissue stiffness were evaluated via microindentation, flow hemodynamics assays, metabolomic assays, and immunohistochemistry analyses. Results: Bioengineered liver constructs cultured under static and dynamic conditions demonstrated high cell viability and growth and full endothelialization of the sinusoid channel. HepG2 cells exhibited long-term function and maturation, forming clusters. Under FALD flow conditions, there were notable changes in EC-HepG2 cell morphology compared to dynamic flow in the healthy range, accompanied by reduced levels of functional markers. Conclusion: This proof-of-principle study introduces an innovative and highly biomimetic 3D model of hepatic sinusoid, which can serve as a robust platform for in vitro investigations of complex cell-microenvironment interactions in the context of FALD as well as other hepatic disorders.

Abstract Tu149: FDA-Approved Antidepressant Trazodone Potentially Increases the Risk of Dyslipidemia

Cardiovascular disease is the leading cause of death. Many cardiovascular health problems, such as atherosclerosis, are caused by dyslipidemia. One xenobiotic nuclear receptor, Pregnane X Receptor (PXR), plays a significant role in atherosclerosis and dyslipidemia, and is activated by various environmental chemicals, including endocrine-disrupting chemicals (EDCs). EDCs are found in common household items such as plastics, medications, and food. Trazodone is a clinically used medication to treat depression by aiding in restoring the balance of serotonin in the brain. But it is unclear if Trazodone has possible impacts on cardiovascular risk factors such as dyslipidemia. Our preliminary data suggested that Trazodone activated human PXR in both intestinal (LS180) and hepatic (HepG2) cells. We hypothesize that Trazodone could regulate the cholesterol uptake mediated by PXR signaling. In this study we use cell-based transfection assay to evaluate the underlying mechanisms by which Trazodone activates PXR. We found that Trazodone was a more potent agonist of human PXR than mouse PXR. Trazodone could activate PXR more intensely in human liver cells compared with human intestinal cells. Our data suggested that Trazodone was a selective PXR agonist and promoted the dissociation between PXR and its nuclear corepressors. Next, we are to identify the key amino acid residues within PXR ligand binding pocket that interact with Trazodone by using computational docking study along with site-mutagenesis assay. Furthermore, we plan to estimate if Trazodone altered cholesterol uptake by human intestinal cells using fluorescence-labeled cholesterol. In conclusion, we explore the potential molecular mechanisms of how FDA-approved antidepressant Trazodone activates human PXR and increases the possible risk of dyslipidemia, which provides potential evidence on future cardiovascular disease risk assessment for Trazodone as well as other antidepressant drugs.

Nano-emulsion based on Santolina chamaecyparissus essential oil potentiates the cytotoxic and apoptotic effects of Doxorubicin: an in vitro study

Aim This study was aimed at investigating the cytotoxic effect of a novel combination of doxorubicin (DOX) and nano-formulation of Santolina chamaecyparissus L. essential oil (SCEO-NANO) on hepatic (HepG2) and colon (HT29) cancer cell lines. Methods A nano-emulsion was prepared by high-pressure homogenisation, then analysed by zetasizer and Fourier transform infrared spectroscopy. HepG2 and HT29 cells were used in in vitro tests for apoptosis detection. Results Formulated droplet size increased in DOX@SCEO-NANO/DOX to 11.54 ± 0.02 with uniform distribution (PDI = 0.13 ± 0.01), when compared with SCEO-NANO (size: 8.91 ± 0.02 nm; PDI = 0.1 ± 0.02). In both cells, DOX@SCEO-NANO/DOX led to a considerable reduction in colony formation. Compared to DOX, apoprotein proteins were overexpressed in HepG2 cells, showing increases of 8.66-fold for caspase-3 and 4.24-fold for the Bax/Bcl-2 ratio. In HT29 cells, ROS-dependent necrosis and apoptosis were seen. Comparing DOX@SCEO-NANO/DOX versus DOX, greater levels of caspase-3 and the Bax/Bcl-2 ratio were observed. Conclusion The DOX@SCEO-NANO/DOX formulation showed potential for targeted eradication of colon adenocarcinoma and hepatocellular carcinoma cells.

Mitigating Effects of Tanacetum balsamita L. on Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD).

The metabolic syndrome and its associated co-morbidities have been recognized as predisposing risk factors for the development of metabolic-associated fatty liver disease (MAFLD). The present study reports on the beneficial effect of the Tanacetum balsamita methanol-aqueous extract (ETB) at 150 and 300 mg/kg bw on biochemical parameters related to oxidative stress, metabolic syndrome, and liver function in rat animal models with induced MAFLD. ETB was found to be non-toxic with LD50 > 3000 mg/kg and did not affect cell viability of hepatic HEP-G2 cells in a concentration up to 800 μg/mL. The pathology was established by a high-calorie diet and streptozotocin. Acarbose and atorvastatin were used as positive controls. At the higher dose, ETB reduced significantly (p < 0.05) the blood glucose levels by about 20%, decreased lipase activity by 52%, total cholesterol and triglycerides by 50% and 57%, respectively, and restored the amylase activity and leukocytes compared to the MAFLD group. ETB ameliorated oxidative stress biomarkers reduced glutathione and malondialdehyde in a dose-dependent manner. At 300 mg/kg, the beneficial effect of the extract on antioxidant enzymes was evidenced by the elevated catalase, glutathione peroxidase, and superoxide dismutase activity by 70%, 29%, and 44%, accordingly, compared to the MAFLD rats. ETB prevents the histopathological changes related to MAFLD. ETB, rich in 3,5-dicafeoylquinic, chlorogenic, and rosmarinic acids together with the isorhamnetin- and luteolin-glucoside provides a prominent amelioration of MAFLD.

Evaluation of the Potential Cytoprotective Effect of Melatonin in Comparison with Vitamin E and Trolox against Cd2+-Induced Toxicity in SH-SY5Y, HCT 116, and HepG2 Cell Lines.

Cadmium (Cd) toxicity poses a significant threat to cellular health, leading to oxidative stress and cell damage. Antioxidant agents, particularly those of natural origin, have been studied as a potential alternative for mitigating heavy metal toxicity. This study aimed to evaluate the cytoprotective effects of the antioxidant melatonin (MLT) in comparison with Vitamin E (VitE) and Trolox against Cd2+-induced cellular toxicity. The MTT assay was employed to assess cell viability in neuronal SH-SY5Y, colorectal HCT 116, and hepatic HepG2 cell lines. The results showed that all three antioxidants offered some level of protection against Cd toxicity, with Vitamin E proving to be the most effective. MLT also demonstrated a substantial cytoprotective effect, especially at the highest Cd concentration of 30 µM. These findings suggest that MLT, alongside Vit E and Trolox, could be valuable in mitigating the detrimental effects of Cd exposure by reducing the oxidative stress in these cellular models.

Investigation of cannabidiol-induced cytotoxicity in human hepatic cells

Cannabidiol (CBD) is one of the primary cannabinoids present in extracts of the plant Cannabis sativa L. A CBD-based drug, Epidiolex, has been approved by the U.S. FDA for the treatment of seizures in childhood-onset epileptic disorders. Although CBD-associated liver toxicity has been reported in clinical studies, the underlying mechanisms remain unclear. In this study, we demonstrated that CBD causes cytotoxicity in primary human hepatocytes and hepatic HepG2 cells. A 24-h CBD treatment induced cell cycle disturbances, cellular apoptosis, and endoplasmic reticulum (ER) stress in HepG2 cells. A potent ER stress inhibitor, 4-phenylbutyrate, markedly attenuated CBD-induced apoptosis and cell death. Additionally, we investigated the role of cytochrome P450 (CYP)-mediated metabolism in CBD-induced cytotoxicity using HepG2 cell lines engineered to express 14 individual CYPs. We identified CYP2C9, 2C19, 2D6, 2C18, and 3A5 as participants in CBD metabolism. Notably, cells overexpressing CYP2C9, 2C19, and 2C18 produced 7-hydroxy-CBD, while cells overexpressing CYP2C9, 2C19, 2D6, and 2C18 generated 7-carboxy-CBD. Furthermore, CBD-induced cytotoxicity was significantly attenuated in the cells expressing CYP2D6. Taken together, these data suggest that cell cycle disturbances, apoptosis, and ER stress are associated with CBD-induced cytotoxicity, and CYP2D6-mediated metabolism plays a critical role in decreasing the cytotoxicity of CBD.

Synthesis of Nitrostyrylthiazolidine-2,4-dione Derivatives Displaying Antileishmanial Potential.

A series of 61 thiazolidine-2,4-diones bearing a styryl group at position 5 was synthesized in 2-5 steps and their structure was proved by elemental and spectral analyses. The compounds obtained were evaluated in vitro against the promastigote stage of the kinetoplastid parasite Leishmania infantum and the human HepG2 cell line, to determine selectivity indices and to compare their activities with those of antileishmanial reference drugs. The study of structure-activity relationships indicated the potential of some derivatives bearing a nitro group on the phenyl ring, especially when located at the meta position. Thus, among the tested series, compound 14c appeared as a hit compound with good antileishmanial activity (EC50 = 7 µM) and low cytotoxicity against both the hepatic HepG2 and macrophage THP-1 human cell lines (CC50 = 101 and 121 µM, respectively), leading to good selectivity indices (respectively, 14 and 17), in comparison with the reference antileishmanial drug compound miltefosine (EC50 = 3.3 µM, CC50 = 85 and 30 µM, SI = 26 and 9). Regarding its mechanism of action, among several possibilities, it was demonstrated that compound 14c is a prodrug bioactivated, predominantly by L. donovani nitroreductase 1, likely leading to the formation of cytotoxic metabolites that form covalent adducts in the parasite. Finally, compound 14c is lipophilic (measured CHI LogD7.7 = 2.85) but remains soluble in water (measured PBS solubility at pH7.4 = 16 µM), highlighting the antileishmanial potential of the nitrostyrylthiazolidine-2,4-dione scaffold.

Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells.

Hepatic ischemia and hypoxia are accompanied by reduced bile flow, biliary sludge and cholestasis. Hepatobiliary transport systems, nuclear receptors and aquaporins were studied after hypoxia and reoxygenation in human hepatic cells. Expression of Aquaporin 8 (AQP8), Aquaporin 9 (AQP9), Pregnane X receptor (PXR), Farnesoid X receptor (FXR), Organic anion transporting polypeptide 1 (OATP1), and the Multidrug resistance-associated protein 4 (MRP4) were investigated in induced pluripotent stem cells (iPSCs) derived hepatic cells and the immortalized hepatic line HepG2. HepG2 was subjected to combined oxygen and glucose deprivation for 4 h followed by reoxygenation. Expression of AQP8 and AQP9 increased during differentiation in iPSC-derived hepatic cells. Hypoxia did not alter mRNA levels of AQP8, but reoxygenation caused a marked increase in AQP8 mRNA expression. While expression of OATP1 had a transient increase during reoxygenation, MRP4 showed a delayed downregulation. Knock-down of FXR did not alter the expression of AQP8, AQP9, MRP4, or OATP1. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation compared to normoxic controls. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Expression patterns differed between hepatobiliary transport systems during hypoxia and reoxygenation. Expression of AQP8 and AQP9 increased during differentiation in induced pluripotent stem cells. Expression of hepatobiliary transporters varies during hypoxia and reoxygenation. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Hypoxia and reoxygenation may affect aquaporins in hepatic cells and potentially affect bile composition.