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Isolation and use of liver
Abstract
This chapter describes the isolation and use of liver cells. The technique is based on liver perfusion with collagenase after removal of Ca 2+ by preperfusion with a chelator. Moderately high yields of viable, single hepatocytes with a smooth, spherical appearance and essentially free of nonparenchymal cells are obtained. Early attempts to isolate hepatocytes employed mechanical force and, subsequently, perfusion of the liver with Ca 2+- or K+-chelators but were unsuccessful in obtaining viable cells in high yields. This was not achieved until the isolation of rat hepatocytes by the use of the digestive enzymes collagenase and hyaluronidase was introduced by Howard and collaborators. Although most of the techniques recently employed to obtain isolated hepatocytes involve perfusion of the liver with digestive enzymes, attempts are also made to avoid the perfusion step and achieve hepatocyte isolation by incubating cut pieces of the liver in enzyme-containing solutions. The presently available results suggest that isolated hepatocytes—and cell suspensions isolated from certain other target tissues—may be a useful model for drug toxicity studies in the future.
... Group IV and Group V animals were pretreated with methanol extract of Bougainvillea glabra at 250mg/kg and 500mg/kg BW by orally for 30 days respectively. Animals from Group II to Group V were administered with isoproterenol (ISO) 85mg/kg, sub cutaneous on 29 th and 30 th day at an interval of 24 hours [16]. All the animals were sacrificed by decapitation on 31 st day of the experiment. ...
... The values were expressed as mg/dl. TGL 5 Plasma LDH Plasma LDH was estimated by the method of Moldeus et al. (1978) [16]. Cuvettes (1 ml) in potassium phosphate buffer (pH 7.0) using 20 µl sample. ...
... The values were expressed as mg/dl. TGL 5 Plasma LDH Plasma LDH was estimated by the method of Moldeus et al. (1978) [16]. Cuvettes (1 ml) in potassium phosphate buffer (pH 7.0) using 20 µl sample. ...
p>The present study was executed to evaluate the myocardial protective effect of methanol extract of Bougainvillea glabra against isoproterenol induced myocardial necrosis in rats. Myocardial necrosis was induced by subcutaneous injection of isoproterenol (85mg/kg body weight) on 29th and 30th day at an interval of 24 hours. Myocardial necrosis was evident from the changes of marker enzymes in serum, plasma and heart tissue. The activities of serum cardiac marker enzymes such as lactate dehydrogenase (LDH), creatine kinase myoglobin (CK-MB), serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), triglycerides (TG), total cholesterol (TC), high density lipoproteins (HDL), low density lipoproteins (LDL), very low density lipoproteins (VLDL) and total protein (TP) were estimated. In addition, plasma TBARS and plasma LDH levels were also recorded. Antioxidant parameters viz catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx) and malondialdehyde (MDA) levels were performed in heart tissue homogenate. The outcome of the study indicated that, pretreatment with methanol extract of Bougainvillea glabra to isoproterenol induced rats significantly prevented the altered serum cardiac marker enzymes, plasma levels and antioxidant parameters to near normal status. The cardioprotective effect was compared with propranolol (10 mg/kg, oral) which was used as the standard. Histopathological findings exposed a reduced degree of necrosis and inflammation succeeding pretreatment with Bougainvillea glabra. Based on these results, it was suggested that methanol extract of Bougainvillea glabra prevents myocardial necrosis and oxidative stress induced by isoproterenol.</p
... Primary hepatocytes were isolated from T2-ASO and NTC-ASO-treated 12-week-old male mice at the University of California, San Francisco Liver Center using the standard collagenase method. 22 Immunofluorescence Staining and Confocal Microscopy siRNA transfected cells and primary hepatocytes were seeded onto cover glass slides. After 12 hours, cells were incubated with 10 mmol/L NH 4 Cl (09718, Sigma-Aldrich) or sham buffer at 37°C for 24 hours. ...
... Cells were permeabilized in 0.25% Triton-100 in PBS for 10 minutes at room temperature. After washing with PBS, cells were treated with 1% bovine serum albumin, 22 [4,5]P 2 ) or control IgG diluted in 0.1% PBST. After 3 washes with PBS, cells were incubated for 1 hour at room temperature with secondary antibodies diluted in 0.1% PBST and subsequently washed 3× with PBS. ...
Objective:
TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of apoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr
-/-
mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.
... Collagenase perfusion was performed on the liver to obtain all hepatocytes; viability was determined by plasma membrane disruption analyzed with trypan blue (0.2 w/v) exclusion test (Moldeus et al. 1978). Cells suspended at a density of 106 cells/ml in Krebs-Henseleit buffer (pH 7.4) were then combined in 12.5 mM HEPE in round-bottomed flasks. ...
... Intactness of plasma membrane, determined by trypan blue (0.2% w/v) exclusion test, was assessed as an index of viability of isolated hepatocytes (Moldeus et al. 1978). Aliquots of the incubated hepatocytes were taken at different time points during the 3-h incubation period. ...
Perfluorooctanesulfonate (PFOS), an anthropogenic fluorosurfactant, is one of the most common global pollutants. PFOS is used in various consumer products to provide soil, oil, and water resistance to materials used in clothing, upholstery, and food packaging. PFOS is persistent, bioaccumulative, and toxic to mammalian species. In this study, the cellular mechanisms involved in PFOS hepatotoxicity were evaluated. For this purpose, we determined oxidative stress markers including cell lysis, ROS generation, lipid peroxidation, glutathione depletion, mitochondrial membrane potential decrease, lysosomal membrane leakiness, and cellular proteolysis. Our results demonstrated that PFOS liver cytotoxicity was associated with reactive oxygen species (ROS) formation and lipid peroxidation in isolated rat hepatocytes. Incubation of hepatocytes with PFOS caused rapid depletion of hepatocyte glutathione (GSH), an important marker of cellular oxidative stress. Most of the PFOS-induced GSH depletion could be attributed to the expulsion of glutathione disulfide (GSSG). PFOS hepatotoxicity was inhibited by antioxidants and ROS scavengers, mitochondrial permeability transition (MPT) pore sealing agents, and endocytosis inhibitors. Our results suggest that PFOS hepatotoxicity might be the result of oxidative stress-induced lysosomal membrane leakiness and cellular proteolysis in rat hepatocytes.
... Short term cytotoxic activity of A. catechu sequential extracts were assayed by determining the percentage viability of the DLA cells using trypan blue exclusion methods [11] . The cells were aspirated from the peritoneal cavity of tumour bearing mice. ...
... Wistar rats (average weight: 250-300 gr) were used in this study, being kept under optimum temperature with free access to food and water. Liver perfusion by collagenase was done to isolate rat hepatocytes (Moldéus et al. 1978) (Figure 1). Briefly, rats were anesthetized using intraperitoneal (i.p.) injection of xylazine (15 mg/kg) and ketamine (75 mg/kg), then intravenous (i.v.) administration of 0.25 ml heparin (5000 U/ml) into the inferior vena cava was done through a U-shaped abdominal incision, in order to prevent blood coagulation in liver. ...
... In the present study, wistar rats with an average weight of 250-300 gr were used, which were kept under optimum temperature with free access to food and water. For the purpose of hepatocyte isolation, liver perfusion using collagenase enzyme was done (Moldéus et al. 1978) (Figure 1A). For this aim, rats were anesthetized using intraperitoneal (i.p.) injection of ketamine (75 mg/kg) and xylazine (15 mg/kg), a U-shaped incision was done in the abdominal skin, then 0.25 ml heparin (5000 U/ml) was injected intravenously (i.v.) into the inferior vena cava to prevent hepatic blood coagulation. ...
Acrylamide is a chemical that is produced from cooking the high carbohydrate food at the temperature of higher than 120 degree celsus and has axidative stress and couse cancer.
Aurapten is an antioxidant substance which is found in citrus has anti-inflammatory and anti-cancer effect.
For this study we investigated the effect of aurapten on rat liver with and without acrylamide toxicity.
... Zabol University of Medical Sciences Ethics Committee of Animal Experimentation, approved the study. Hepatocytes were isolated using collagenase liver perfusion as described previously (Moldeus et al., 1978) and using trypan blue, cell viability was determined; cell isolation efficiency was always >90% (Hashemzaei et al., 2015). After isolation, hepatocytes at 10 6 cells/ml (10 ml) were suspended in Krebs-Henseleit buffer (pH 7.4) including 12.5 mM HEPES, in flasks with 95% O2 and 5% CO2 at 37 • C (Shahraki et al., 2013a). ...
Objective
Arsenic (As) poisoning is a worldwide public health problem. Arsenic can cause cancer, diabetes, hepatic problems, etc. Hence, we investigated possible hepatoprotective properties of curcumin against As³⁺-induced liver damages in freshly isolated rat hepatocytes.
Materials and Methods
Isolation of hepatocytes was done by the two-step liver perfusion method using collagenase. The EC50 concentration of As³⁺ was used in toxicity assessments and curcumin (2, 5, and 10 µM) was added 15 min before As³⁺ addition to isolated hepatocytes. Curcumin impact was assessed in terms of cytotoxicity, lipid peroxidation induction, reactive oxygen species (ROS) levels, and mitochondrial membrane potential.
Results
As³⁺ significantly increased cytotoxicity, malondialdehyde and ROS levels and induced mitochondrial membrane damage and hepatocyte membrane lysis after 3 hr incubation. Curcumin 2 µM significantly prevented lipid peroxidation induction, ROS formation, and mitochondrial membrane damage; while curcumin 5 µM had no apparent effect on these parameters, curcumin 10 µM potentiated them.
Conclusion
Curcumin only at low doses could ameliorate oxidative stress injury induced by As³⁺ in isolated rat hepatocytes.
... Human liver (HepG2) cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Primary rat hepatocytes were isolated from collagenase perfusion technique as described by Moldeus and co-workers (Moldéus et al., 1978). Cells were cultured in Dulbecco's modified eagle's medium (DMEM) (Invitrogen, Carisbad, CA, USA) with the supplementation of streptomycin (100 lg/ml)-penicillin (100 U/ml) (Invitrogen) and 10% fetal bovine serum (FBS, Invitrogen). ...
Current attention has been given on health effects of combined exposure of nanoscale materials and organic pollutants. Nickel (II) oxide nanoparticles (NiO NPs) displays exceptional properties and is being used in various areas such as batteries, diesel–fuel additives, and biomedicals. Benzo[a]pyrene (BaP) is a ubiquitous pollutant. Cigarette smoke, diesel exhaust, and grilled foods are main sources of BaP exposure. Therefore, combined exposure of NiO NPs and BaP to humans is unavoidable. There is a dearth of knowledge on combined effects of NiO NPs and BaP in humans. This study was aimed to investigate co-exposure effects of NiO NPs and BaP in human liver cells (HepG2) and primary rat hepatocytes. We observed that individual and co-exposure of NiO NPs and BaP induced cytotoxicity, lactate dehydrogenase leakage, lipid peroxidation, depletion of mitochondrial membrane potential, and activation of caspases (-3 and -9) in both types of cells. Individual and co-exposure of NiO NPs and BaP further accelerated the generation of free oxygen radicals (reactive oxygen species and hydrogen peroxide) and depletion of antioxidants (glutathione and various antioxidant enzymes). Remarkably, NiO NPs and BaP exerted synergistic toxicity to both HepG2 cells and primary rat hepatocytes. Moreover, combined toxicity of NiO NPs and BaP in both cells was mediated through free oxygen radicals induced oxidative stress. This work warrants further research on risk assessment of co-exposure effects NiO NPs and BaP in an appropriate in vivo model.
... The viability of cells was evaluated by trypan blue staining (Moldéus et al. 1978). To measure the dry weight (DW), the mass of harvested cells was dried at 50 °C in an oven until constant weight, then weighed with a balance (Yan et al. 2006). ...
The biological approach for synthesizing nanoparticles (NPs) using plant extracts is an efficient alternative to conventional physicochemical methods. Galegine, isolated from Galega (Galega officinalis L.), has anti-diabetic properties. In the present study, silver nanoparticles (AgNPs) loaded onto urea-based periodic mesoporous organosilica (AgNPs/Ur-PMO) were bio-synthesized using G. officinalis leaf extract. The synthesized NPs were characterized and confirmed via analysis methods. Different concentrations of biosynthesized AgNPs/Ur-PMO nanoparticles (0, 1, 5, 10, and 20 mg L⁻¹) were used as elicitors in cell suspension culture (CSC) of G. officinalis. The callus cells from hypocotyl explants were treated at their logarithmic growth phase (8th d) and were collected at time intervals of 24, 72, 120, and 168 h. The viability and growth of cells were reduced (by 17% and 35%, respectively) at higher concentrations and longer treatments of AgNPs/Ur-PMO; however, the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were increased (1.23 and 3.01 fold, respectively in comparison with the control average). The highest total phenolic (2.43 mg g⁻¹ dry weight) and flavonoid (2.22 mg g⁻¹ dry weight) contents were obtained 168 h after treatment with 10 mg L⁻¹ AgNPs/Ur-PMO. An increasing tendency in the antioxidant enzyme activities was also observed in all the elicitor concentrations. Treatment with AgNPs/Ur-PMO (in particular 5 mg L⁻¹ for 120 h) significantly enhanced the galegine content (up to 17.42 mg g⁻¹) about 1.80 fold compared with the control. The results suggest that AgNPs/Ur-PMO can be used as an effective elicitor for enhancing galegine production in the CSC of G. officinalis.
Key points
• The green biosynthesis of AgNPs/Ur-PMO was done using G. officinalis leaf extract
• Its toxicity as an elicitor increased with increasing concentration and treatment time
• AgNPs/Ur-PMO significantly increased the antioxidant capacity and galegine content
Graphical Abstract
... Primary rat hepatocytes were isolated by collagenase perfusion methods as described earlier. 41 42 with some modifications. 43 The MTT assay is based on the principle of the ability of mitochondria of living cells to reduce MTT salt into blue formazan crystals. ...
Bismuth (III) oxide nanoparticles (Bi2O3 NPs) have shown great potential for biomedical applications because of their tunable physicochemical properties. In this work, pure and Zn-doped (1 and 3 mol %) Bi2O3 NPs were synthesized by a facile chemical route and their cytotoxicity was examined in cancer cells and normal cells. The X-ray diffraction results show that the tetragonal phase of β-Bi2O3 remains unchanged after Zn-doping. Transmission electron microscopy and scanning electron microscopy images depicted that prepared particles were spherical with smooth surfaces and the homogeneous distribution of Zn in Bi2O3 with high-quality lattice fringes without distortion. Photoluminescence spectra revealed that intensity of Bi2O3 NPs decreases with increasing level of Zn-doping. Biological data showed that Zn-doped Bi2O3 NPs induce higher cytotoxicity to human lung (A549) and liver (HepG2) cancer cells as compared to pure Bi2O3 NPs, and cytotoxic intensity increases with increasing concentration of Zn-doping. Mechanistic data indicated that Zn-doped Bi2O3 NPs induce cytotoxicity in both types of cancer cells through the generation of reactive oxygen species and caspase-3 activation. On the other hand, biocompatibility of Zn-doped Bi2O3 NPs in normal cells (primary rat hepatocytes) was greater than that of pure Bi2O3 NPs and biocompatibility improves with increasing level of Zn-doping. Altogether, this is the first report highlighting the role of Zn-doping in the anticancer activity of Bi2O3 NPs. This study warrants further research on the antitumor activity of Zn-doped Bi2O3 NPs in suitable in vivo models.
... Primary rat hepatocytes were isolated by collagenase perfusion methods as described earlier. 41 42 with some modifications. 43 The MTT assay is based on the principle of the ability of mitochondria of living cells to reduce MTT salt into blue formazan crystals. ...
Drug resistance and inability to distinguish between cancerous and non-cancerous cells are important obstacles in the treatment of cancer. Zinc oxide nanoparticles (ZnO NPs) is now emerging as a crucial material to challenge this global issue due to its tunable properties. Developing an effective, inexpensive, and eco-friendly method in order to tailor the properties of ZnO NPs with enhanced anticancer efficacy is still challenging. For the first time, we reported a facile, inexpensive, and eco-friendly approach for green synthesis of ZnO-reduced graphene oxide nanocomposites (ZnO-RGO NCs) using garlic clove extract. Garlic has been playing one of the most important dietary and medicinal roles for humans since centuries. We aimed to minimize the use of toxic chemicals and enhance the anticancer potential of ZnO-RGO NCs with minimum side effects to normal cells. Aqueous extract of garlic clove was used as reducing and stabilizing agent for green synthesis of ZnO-RGO NCs from the zinc nitrate and graphene oxide (GO) precursors. A potential mechanism of ZnO-RGO NCs synthesis with garlic clove extract was also proposed. Preparation of pure ZnO NPs and ZnO-RGO NCs was confirmed by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and dynamic light scattering (DLS). The in vitro study showed that ZnO-RGO NCs induce two-fold higher cytotoxicity in human breast cancer (MCF7) and human colorectal cancer (HCT116) cells as compared to pure ZnO NPs. Besides, biocompatibility of ZnO-RGO NCs in non-cancerous human normal breast (MCF10A) and normal colon epithelial (NCM460) cells was higher than those of pure ZnO NPs. This work highlighted a facile and inexpensive green approach for the preparation of ZnO-RGO NCs with enhanced anticancer activity and improved biocompatibility.
... Hepatocytes were prepared by the collagenase perfusion method of Moldeus et al., (1978). ...
Isolated hepatocytes in suspension and in primary monolayer culture were used for study of the metabolism and toxicity of the hepatotoxin, hydrazine. Dye uptake and the leakage of the cytosolic enzyme LDH were used as measures of cytotoxicity in vitro. Only 16mM and higher concentrations were cytotoxic to isolated hepatocytes in suspension. Hydrazine caused a dose-dependent depletion of ATP both in isolated hepatocytes and in rat liver in vivo. The depletion in isolated hepatocytes occured at a non-cytotoxic concentration, and the depletion in liver occured without lethality suggesting that ATP depletion may underlie the hepatotoxicity of hydrazine. The ATP depletion may be related to fatty liver caused by hydrazine. In isolated hepatocytes in suspension non-cytotoxic concentrations of hydrazine effected other biochemical parameters such as GSH, GSSG and NADPH. Hydrazine increased cellular triglycerides in hepatocytes in suspension and caused a parallel decrease in the medium. Therefore, inhibition of triglyceride secretion from the liver may be responsible for hydrazine-induced fatty liver. Hydrazine appeared to be more cytotoxic to hepatocytes in suspension than in primary culture, as measured by LDH leakage and ATP levels. This difference could be due to the loss of metabolising enzymes such as cytochrome P450 in primary culture. However, hydrazine inhibited protein synthesis in cultured hepatocytes at a concentration far below that which had measurable effects on cell viability and ATP levels. The inhibition of protein synthesis is probably involved in the toxicity of hydrazine (reduced lipoprotein synthesis may be responsible for fatty accumulation in hepatocytes). Using 1H and 15N NMR in hepatocytes, various metabolites of hydrazine including some acetylated species were detected. Metabolic inhibitors or low temperatures (4°C), reduced the levels of hydrazine inside the cells, suggesting that hydrazine entry into cells may be energy-dependent. Hydrazine levels in cultured hepatocytes were higher than those in suspension, but in both systems the concentration of hydrazine inside the cells was lower than that outside. Both metabolic inhibitors KCN and DNP increased hydrazine-induced cytotoxicity and ATP depletion. Cytochrome P450 has been implicated in the metabolism of hydrazine. The effects of single and repeated administration of hydrazine on the activity of different forms of this enzyme showed that hydrazine when administered repeatedly increased some of these activities.
... The standard two-phase collagenase perfusion (Moldeus et al. 1978) with minor modifications (Farghali et al. 1986) was used for hepatocytes isolation. Bürker chamber was used for the counting hepatocytes and the cell viability was determined via Trypan blue exclusion method (Strober 2001). ...
Dihydromyricetin (DHM) is a natural flavonoid showing several health promoting effects such as protective activity during severe alcohol intoxication. The mechanism underlying the effects of DHM on alcohol metabolism is virtually unknown. The present paper is focused on clarifying the role of DHM in the liver alcohol elimination at its molecular level. First, impact of DHM on alcohol dehydrogenase (ADH) activity in vitro and the enzyme induction in vivo was examined. Neither the ADH activity nor the enzyme expression were influenced by DHM. Next, the effect of DHM during alcohol intoxication were studied on primary hepatocytes isolated from EtOH-premedicated and untreated rats. The viability of cells exposed to alcohol, estimated based on the released enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), was slightly affected by DHM. Although the expected hepatoprotective effect of DHM was not fully achieved, DHM (in a concentration manner) proved to reduce the level of ROS/RNS in hepatocytes. However, no change in the rate of alcohol metabolism in vivo was found when rats were administered with a single or repeated dose of ethanol supplemented with DHM. In conclusion, the proposed positive effect of DHM during alcohol intoxication has not been proven. Moreover, there is no effect of DHM on the alcohol metabolism. The “hoped-for” DHM hepatoprotective activity can be attributed to the reduction of ROS/RNS levels in cells.
... Hepatocytes were isolated by perfusion with collagenase I as described [52] [53]. Hepatocytes were plated on collagen I (0.3 mg/mL) and let to attach in DMEM containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 0.08 U/mL insulin (culture medium) for 24 hours before proceeding with experiments. ...
ADAM10 and ADAM17 are proteases that affect multiple signalling pathways by releasing molecules from the cell surface. As their substrate specificities partially overlaps, we investigated their concurrent role in liver regeneration and fibrosis, using three liver-specific deficient mouse lines: ADAM10- and ADAM17-deficient lines, and a line deficient for both proteases. In the model of partial hepatectomy, double deficient mice exhibited decreased AKT phosphorylation, decreased release of EGFR activating factors and lower shedding of HGF receptor c-Met. Thus, simultaneous ablation of ADAM10 and ADAM17 resulted in impaired EGFR signalling, while HGF/c-Met signalling pathway was enhanced. In contrast, antagonistic effects of ADAM10 and ADAM17 were observed in the model of chronic CCl 4 intoxication. While ADAM10-deficient mice develop more severe fibrosis manifested by high ALT, AST, ALP and higher collagen deposition, combined deficiency of ADAM10 and ADAM17 surprisingly results in comparable degree of liver damage as in control littermates. Therefore, ADAM17 deficiency is not protective in fibrosis development per se , but can ameliorate the damaging effect of ADAM10 deficiency on liver fibrosis development and results in decreased shedding of TNF RI, while ADAM10 deficiency leads to increased levels of soluble TNF RI in serum. In conclusion, hepatocyte-derived ADAM10 and ADAM17 are important regulators of growth receptor signalling and TNF RI release, and pathological roles of these proteases are dependent on the cellular context.
... The isolation of hepatocytes was performed by the two step collagenase perfusion method. This method is based on the pre-perfusion of the liver with Ca^^ free buffer followed by enzymatic digestion of the liver with collagenase (Moldeus et al. 1978). ...
All prokaryotic and eukaryotic cells in response to stressful insults, such as chemical exposure or hyperthermic treatment upregulate a well conserved set of genes. These genes encode a family of proteins known as stress proteins whose induction is an adaptive response which serves to protect the cells by interacting with denatured or damaged proteins. The aim of the current thesis was to determine the effect of induction of stress proteins on the subsequent toxicity of chemicals in isolated hepatocytes. A further aim was to determine whether the induction of stress proteins following chemical exposure could be used as a sensitive marker of chemical exposure. These studies were performed in two hepatocyte models, hepatocyte monolayers and liver spheroids, and the hepatic effect was also determined in vivo. Hyperthermic treatment of hepatocytes had a bi-phasic effect on the toxicity of subsequent chemical exposure. Exposure of cells to chemicals immediately following hyperthermic treatment resulted in a sensitisation of the cells to the toxin. If the cells were allowed a recovery period prior to chemical exposure the hepatocytes became tolerant to the toxic actions of the chemicals. The reasons for this are thought to be alterations in stress protein and GSH levels. The use of stress proteins as markers was problematic as hepatocyte monolayers had elevated basal stress protein levels resulting in poor induction following chemical exposure. However, liver spheroids had low basal stress protein levels which were significantly induced following chemical exposure to a greater extent than the response observed in vivo. However, in all situations only certain stress proteins were induced following exposure to specific chemicals. Differences in the activation of the transcription factor responsible for stress protein induction (HSF-1) were thought to be responsible for these differences. Therefore, measurement of these components of stress response may not be an all encompassing marker of toxicity but may be of use in determining the toxicity of certain chemicals. However, it does not rule out the possibility that other components of the stress response may be of use as such a marker.
... Tissue viability was assessed by estimating lactate dehydrogenase (LDH) leakage. This enzyme activity was measured both in the culture medium and in explants at each incubation time in a spectrophotometer (T80+UV/Visible Spectrometer; PG Instruments Limited, Lutterworth, England), following the method described by Moldeus et al. (19). Percentages of LDH leakage were calculated as follows: ...
The combination of synthetic anthelmintics and bioactive phytochemicals may be a pharmacological tool for improving nematode control in livestock. Carvone (R-CNE) has shown in vitro activity against gastrointestinal nematodes; however, the anthelmintic effect of bioactive phytochemicals either alone or combined with synthetic drugs has been little explored in vivo. Here, the pharmacological interaction of abamectin (ABM) and R-CNE was assessed in vitro and in vivo. The efficacy of this combination was evaluated in lambs naturally infected with resistant gastrointestinal nematodes. Additionally, the ligand and molecular docking of both molecules to P-glycoprotein (P-gp) was studied in silico. The presence of R-CNE produced a significant (p < 0.05) increase of Rho123 and ABM accumulation in the intestinal explants. After 60 min of incubation, Rho123 incubated with R-CNE had a 67 ± 21% higher concentration (p < 0.01) than when it was incubated alone. In the case of ABM, a significant increase in the intestinal concentrations was observed at 15 and 30 min after incubation with R-CNE. In the in vivo assay, no undesirable effects were observed after the oral administration of R-CNE. The coadministration of the natural compound prolonged ABM absorption in lambs. ABM T½ absorption was 1.57-fold longer (p < 0.05) in the coadministered group. Concentrations of R-CNE between 420 and 2,593 ng/mL were detected in the bloodstream between 1 and 48 h posttreatment. The in vivo efficacy of ABM against gastrointestinal nematodes increased from 94.9 to 99.8% in the presence of R-CNE, with the lower confidence interval limit being >90%. In vitro/in vivo pharmacoparasitological studies are relevant for the knowledge of the interactions and the efficacy of bioactive natural products combined with synthetic anthelmintics. While ADMET (absorption, distribution, metabolism, excretion, and toxicity) predictions and the molecular docking study showed a good interaction between ABM and P-gp, R-CNE does not appear to modulate this efflux protein. Therefore, the pharmacokinetic–pharmacodynamic effect of R-CNE on ABM should be attributed to its effect on membrane permeability. The development of pharmacology-based information is critical for the design of successful strategies for the parasite control.
... Rat hepatocytes were prepared by the two-step collagenase perfusion method [19] from male Wistar rats (6-weeks old) purchased from Japan SLC Inc., (Shizuoka, Japan). The hepatocytes were seeded into 24-well culture plates coated with collagen (BioCoat Collagen I 24-well plate, Corning Inc., Corning, NY, USA) at a cell density of 2 × 10 5 cells/cm 2 in the Lanford medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and allowed to attach to the culture plate during pre-incubation for 3 h at 37 • C with 5% CO 2 . ...
Effects of 4-methyl-2-mercaptobenzimidazole (4-MeMBI) and 5-methyl-2- mercaptobenzimidazole (5-MeMBI) on cytochrome P450 (CYP) activity were examined in primary cultured rat hepatocytes. Hepatocytes from male Wistar rats were cultured in the presence of 4-MeMBI or 5-MeMBI (0-400 μM), and the activity of CYPs 3A2/4 (48 and 96 h) and 1A1/2 (48 h) was determined by measuring the activity of testosterone 6β-hydroxylation and 7-ethoxyresorufin O-deethylation, respectively. As a result, 4-MeMBI and 5-MeMBI (≥12.5 μM) inhibited CYP3A2 activity. On the other hand, 4-MeMBI (≥25 μM) and 5-MeMBI (≥100 μM) induced CYP1A1/2 activity, being consistent with the previous in vivo results. In a comparative metabolism study using primary cultured human hepatocytes from two Caucasian donors, 4-MeMBI and 5-MeMBI induced the activity of CYPs 3A4 and 1A1/2 with individual variability. It was concluded from these results that 4-MeMBI, 5-MeMBI and MBI caused inhibition of CYP3A2 activity in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes were considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms.
... Primary hepatocytes were prepared from male Sprague-Dawley rats (150-200 g) as previously described (31). Briefly, rat liver was perfused with liver perfusion medium (GIBCO-BRL, Gaithersburg, MD) at 37 Њ C for 15 min at 20 mL/min. ...
Introduction:
Tocilizumab is an anti-interleukin-6 antibody that has been used for the treatment of severe coronavirus disease 2019 (COVID-19). However, the concrete evidence of its benefit in reducing the mortality in severe COVID-19 is lacking. Therefore, we performed a systematic review and meta-analysis of relevant studies that compared the efficacy of Tocilizumab in severe COVID-19 vs. standard of care alone.
Methods: Literature search for studies that compared Tocilizumab and Standard of care in the treatment of COVID-19 was done using major online databases from December 2019 to June 14th, 2020. Search words Tocilizumab, anti-interleukin-6 antibody, and COVID-19 or coronavirus 2019 in various combinations were used. Articles in the form of abstracts, letters without original data, case reports, and reviews were excluded. Data was gathered on an excel sheet, and statistical analysis was performed using Review Manager 5.3.
Results:
Five studies were eligible from 693 initial studies, including 3,641 patients (>2283 males). There were thirteen retrospective studies and three prospective studies. There were 2,488 patients in the standard of care group and 1,153 patients in the Tocilizumab group. The death rate in the tocilizumab group, 22.4% (258/1153), was lower than the standard of care group, 26.21% (652/2,488) (Pooled odds ratio 0.57 [95% CI 0.36-0.92] p=0.02). There was a significant heterogeneity (Inconsistency index= 80%) among the included studies.
Conclusion:
The addition of Tocilizumab to the standard of care reduces mortality in severe COVID-19. Larger randomized clinical trials are needed to validate these findings.
... Cell counts were performed. In addition to cell counts and ^H-thymidine incorporation, potential cytotoxic effects of CCBs were also assessed by using the trypan blue exclusion method and by measuring the release of lactate dehydrogenase (LDH) into the supernatant (Moldeus et al. 1978). ...
The involvement of abnormal lipid metabolism in the progression of renal disease and the pathogenesis of chronic graft dysfunction is generally accepted. The dysregulation of lipoprotein homeostasis is involved in the incidence of accelerated cardiovascular disease in this population. Chronic renal dysfunction is also an inflammatory condition. Therefore, the present study was undertaken to investigate various mechanisms involved in the receptor-mediated regulation of intracellular lipoprotein transport and its interactions with inflammatory cytokines. Using human mesangial cell line (HMCL) culture, we demonstrated that HMCL express native LDL receptors. Phorbol 12-myristate 13-acetate (PMA), Angiotensin II (Ang II), TNF-α, and IL-1β induced acetylated-LDL internalisation, scavenger receptor mRNA expression and promoter activity. Both AP-1 and ets motifs were specific response elements to PMA-induced scavenger receptor expression. Conventionally, LDL receptor pathway is not thought to be involved in foam cells formation due to the tight metabolic control through a feedback regulation. However, our studies demonstrated that TNF-α, TGF-β, PDGF, or IL-1β increased LDL binding, LDL receptor mRNA expression and LDL receptor promoter activity. Both TNF-α, and IL-1β overrode the suppression of LDL receptor activity caused by a high concentration of native LDL and caused foam cell formation in HMCL. TNF-α, and IL-1β also increased the expression of a cleavage activating protein (SCAP) for sterol regulatory element binding proteins (SREBP). Diltiazem and verapamil, not nifedipine increased LDL binding, LDL receptor mRNA expression and LDL receptor promoter activity, but they were not able to override sterol-induced inhibition. Our results suggest that inflammatory cytokines may contribute to lipid deposition and foam cell formation in HMC through following pathways: 1) inducing scavenger receptor expression; 2) disregulating LDL receptor gene expression by increasing sterol-independent and mitogenesis-independent gene transcription. The implications of these findings are that inflammatory cytokines are important risk factors for glomerular atherosclerosis. Therefore, future strategies for controlling progression of renal and cardiovascular diseases should include anti-oxidants, lipid-lowering, and anti-inflammatory drugs.
... The effects of different samples on viability of the human normal cell line, human amnion (WISH) were assessed (Moldeus et al., 1978). Dulbecco's modified Eagle's medium (DMEM) with 4.5g/L glucose, and with supplements of 10% (v/v) inactivated fetal calf serum, 1% nonessential amino acids, 1% glutamine, 100U/mL penicillin, and 10mg/mL streptomycin at pH value of 7.4, was utilized to grow cells. ...
AN AGRICULTURAL soil was used to isolate Alternaria alternata. It was molecularly identified and was given an accession number MN645469 at the National Center for Biotechnology Information (NCBI) and a strain identifier to be Alternaria alternata strain NMK1. Four fractions were collected from the acetic acid extract of its filtrate. Fraction no. 2 showed highest antioxidant activity (71 and 64%, at 200 and 150µg/mL) followed by the crude extract (60 % at 200µg/mL). Safety of using the extracted fractions was tested against the normal human amnion (WISH) cell line. Lowest cytotoxicity values were recorded for fraction no. 2 (19%) followed by the crude extract (28%) at 50µg/mL. Aspergillus flavus, Aspergillus niger and Penicillium griseofulvum exhibited highest relative density percentages (28.4, 30.8 and 24.3%) on their respective isolation seeds; wheat, broad bean and kidney bean. Interestingly, fraction no. 2 caused 100% inhibition of these isolated fungal species at 100μg/ mL. The minimum fungicidal concentration (MFC) was estimated to be 50μg/mL against A. flavus and 40μg/mL for A. niger and P. griseofulvum. Generally, subjecting each of the tested isolates to fraction no. 2 led to damage in their DNA as shown by the comet assay. Gas chromatography-mass spectroscopy (GC-MS) profiling of fraction no. 2 showed 12 major compounds with diverse possible biological activities being antiinflammatory, insecticide, anticancer, antineoplastic antifungal, and antimicrobial.
... Hepatocytes were isolated by collagenase perfusion of the rat liver. 18 Viabilities of >85% for hepatocytes (determined with trypan blue exclusion assay) were accepted. Cells were suspended (10 6 cells/ mL) in round bottom flasks rotating in the Krebs-Henseleit buffer (pH 7.4), supplemented with 12.5 mM HEPES under an atmosphere of carbogen gas (95% O 2 and 5% CO 2 ) in a 37 °C water bath; 10 mL of hepatocyte suspension in each flask was preincubated for 30 min prior to addition of As and AsNPs. ...
Background:
Arsenic, an environmental pollutant, is a carcinogenic metalloid and also an anticancer agent.
Objective:
To evaluate the toxicity of arsenic nanoparticles in rat hepatocytes.
Methods:
Freshly isolated rat hepatocytes were exposed to 0, 20, 40, and 100 μM of arsenic nanoparticles and its bulk counterpart. Their viability, reactive oxygen species level, glutathione depletion, mitochondrial and lysosomal damage, and apoptosis were evaluated.
Results:
By all concentrations, lysosomal damage and apoptosis were clearly evident in hepatocytes exposed to arsenic nanoparticles. Evaluation of mitochondria and lysosomes revealed that lysosomes were highly damaged.
Conclusion:
Exposure to arsenic nanoparticles causes apoptosis and organelle impairment. The nanoparticles have potentially higher toxicity than the bulk arsenic. Lysosomes are highly affected. It seems that, instead of mitochondria, lysosomes are the first target organelles involved in the toxicity induced by arsenic nanoparticles.
... Primary hepatocytes were isolated from the rat by a collagenase perfusion technique as described (Moldeus et al. 1979). Hepatocytes from liver were obtained by collagenase digestion of sliced liver. ...
Naturally occurring antioxidants prevent or delay the harmful effect of free radical formation and radioprotection. The present study aimed to investigate the radioprotective effect of dieckol, a naturally occurring marine bioactive phenolic compound on lipid peroxidation and antioxidant status, DNA damage, and inflammation in gamma-radiation-induced rat primary hepatocytes. Isolated hepatocyte cells exposed to gamma-radiation showed an increased level of lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) accompanied with the decrease in the activities of enzymatic (SOD, CAT, and GPx) and non-enzymatic (vitamin C, vitamin E, and GSH) antioxidants associated with increased DNA damage coupled with upregulation of inflammatory proteins (NF-κB and COX-2) compared to control. Treatment of dieckol (5, 10, 20 μM) reduces the γ-radiation-induced toxicity and the associated pro-oxidant and antioxidant imbalance as well as decreasing the DNA damage (tail length, tail moment, %DNA in a tail and olive tail moment) and inflammation in hepatocyte cells. These findings indicate that treatment of dieckol offers protection against γ-radiation-induced cellular damage in the liver cells.
... Short-term cytotoxic activity of B. phoenicea extract was assayed by determining the percentage of viability of the DLA cells using Trypan blue exclusion methods [13]. ...
Objective: Bauhinia phoenicea Wight and Arn. is a medicinal plant endemic to Southern Western Ghats. In the traditional systems of medicine, it is using against various ailments including some oxidative disorders. Detailed studies on the pharmacological activities of this plant are not yet reported. Hence, this paper aimed to prove the efficacy of this plant as a natural antioxidant source.Methods: The sequential extracts of the dried leaf powder were assayed for an antioxidant property using 2,2 diphenyl 1-picrylhydrazyl free radical scavenging assay, in vitro cytotoxicity of the extracts was screened using Trypan blue exclusion method. The antitumor activity of the selected fractions was studied using ascites tumor affected mice and noted the percentage of increase in lifespan.Results: The free radical scavenging activity of all extracts was increasing with increasing concentration of the drug, the least IC50 value was showed by ethanol fraction (41 μg/ml). The plant drug was not toxic to the normal cells and was highly toxic to tumor cell lines. Maximum in vitro cytotoxicity was observed in chloroform fraction (98% cell death at 100 mg/ml) and the least IC50 value was exhibited by the aqueous fraction (34 mg/ml). Both the aqueous and chloroform fractions increased the lifespan of ascites tumor bearing mice, aqueous fraction in 100 mg/ml concentration shows 71.9% increase in lifespan which is near to the result showed by the commercial anticancer drug cyclophosphamide (72.5%).Conclusion: According to our results, it is concluded that leaf of B. phoenicea has significant antioxidant, cytotoxic, and antitumor properties supporting the folk medicinal use of this species. The further procedures of identification of pharmacological active principles are in progress.
... After incubation at 37°C for 10 min, the culture medium were collected and centrifuged at 250×g for 5 min at 4°C. LDH activity of both cell-free supernatants was measured by the method of Moldéus et al. 9) Preparations of Cell-Lysate and Cell-Extract Cells were trypsinized and harvested from a 35-mm-diameter culture dish. After washing with 1 mL of ice-cold phosphate-buffered saline (PBS) twice, the cells were harvested again with centrifugation at 250×g for 5 min at 4°C and suspended in 50 µL of ice-cold NP-40 lysis buffer consisting of 0.1% Nonidet ® P-40, 1 mM ethylenediaminetetraacetic acid (EDTA), 250 mM NaCl, 10% glycerol, 50 mM Tris-HCl buffer (pH 8.0), and 1% Protease Inhibitor Cocktail for Use with Mammalian Cells and Tissue Extract (Nacalai Tesque, Inc., Kyoto, Japan) to make a cell-lysate. ...
2,3-Dimethoxy-5-methyl-p-benzoquinone is a common chemical structure of coenzyme Q (CoQ) that conjugates different lengths of an isoprenoid side chain at the 6-position of the p-benzoquinone ring. In a series of studies to explore the cytotoxic mechanism of CoQ homologues with a short isoprenoid side chain, we found that a CoQ analogue without an isoprenoid side chain, CoQ0, showed marked toxicity against HeLa cells in comparison with cytotoxic homologues. Therefore, we examined the cytotoxic mechanism of CoQ0. Different from the cytotoxic CoQ homologues that induced apoptosis, 100 µM CoQ0 induced necrosis of HeLa cells. The CoQ0-induced cell death was accompanied by a decrease in endogenous non-protein and protein-associated sulfhydryl (SH)-groups, but this improved with the concomitant addition of compounds with SH-groups but not antioxidants without SH-groups. In addition, UV-spectrum analysis suggested that CoQ0 could rapidly form S-conjugated adducts with compounds with SH-groups by Michael addition. On the other hand, enzyme activities of both glyceraldehyde-3-phosphate dehydrogenase, which has a Cys residue in the active site, and α-ketoglutarate dehydrogenase complex, which requires cofactors with SH-groups, CoA and protein-bound α-lipoic acid, and CoA and ATP contents in the cells were significantly decreased by the addition of CoQ0 but not CoQ1. Furthermore, the decrease of an endogenous antioxidant, glutathione (GSH), by CoQ0 treatment was much greater than the predicted increase of endogenous GSH disulfide. These results suggest that CoQ0 rapidly forms S-conjugate adducts with these endogenous non-protein and protein-associated SH-groups of HeLa cells, which disrupts carbohydrate metabolism followed by intracellular ATP depletion and necrotic cell death.
Graphical Abstract Fullsize Image
... Short term cytotoxicity was determined by Trypan blue dye exclusion method [13]. Approximately 1 × 10 7 DLA, EAC or spleen cells were suspended in 1 mL PBS; pH 7.4. ...
Docosahexaenoic acid (DHA) is long chain omega-3 fatty acid with known health benefits and clinical significance. However, 4-hydroxy hexenal (HHE), an enzymatic oxidation product of DHA has recently been reported to have health-damaging effects. This conflict raises major concern on the long-term clinical use of these fatty acids. Even though the enzymatic and non-enzymatic conversion of HHE to nontoxic acid molecules is possible by the aldehyde detoxification systems, it has not yet studied. To address this, primary oxidation products of DHA in lipoxidase system were subjected to non-enzymatic conversion at a physiological temperature over a period of 1 week. The reaction was monitored using HPLC, IR spectroscopy and biochemically (based on the loss of conjugated dienes, lipid peroxides aldehydes). Short term and long term cytotoxicity of the compounds generated at various time points were analyzed. IR and HPLC spectra revealed that the level of aldehydes in the primary oxidation products reduced over time, generating acids and acid derivatives within a week period. In short term and long term cytotoxicity analysis, initial decomposition products were more toxic than the 1-week decomposition products. Further, when primary oxidation products subjected to aldehyde dehydrogenase mediated oxidation; it generated products that are also found to be less toxic. The study suggests the possible non-enzymatic conversion of primary oxidation products of DHA to less cytotoxic acid molecules. Exploration of the physiological roles of these acid molecules may explain the biological potential of omega-3 fatty acids.
... The effects of different treatments on primary rat hepatocytes viability were assessed in cultured cells (Moldeus et al., 1978). Cells were isolated from rat liver by collagenase perfusion through portal vein. ...
... The HepG2, A549, MCF-7 & IMR-90 cell lines were bought from American Type Culture Collection (ATCC) (Manassas, VA). Primary hepatocytes were isolated from rat using collagenase perfusion method 47 . ...
We investigated the anticancer potential of Ag-doped (0.5-5%) anatase TiO2 NPs. Characterization study showed that dopant Ag was well-distributed on the surface of host TiO2 NPs. Size (15 nm to 9 nm) and band gap energy (3.32 eV to 3.15 eV) of TiO2 NPs were decreases with increasing the concentration of Ag dopant. Biological studies demonstrated that Ag-doped TiO2 NP-induced cytotoxicity and apoptosis in human liver cancer (HepG2) cells. The toxic intensity of TiO2 NPs was increases with increasing the amount of Ag-doping. The Ag-doped TiO2 NPs further found to provoke reactive oxygen species (ROS) generation and antioxidants depletion. Toxicity induced by Ag-doped TiO2 NPs in HepG2 cells was efficiently abrogated by antioxidant N-acetyl-cysteine (ROS scavenger). We also found that Ag-doped TiO2 NPs induced cytotoxicity and oxidative stress in human lung (A549) and breast (MCF-7) cancer cells. Interestingly, Ag-doped TiO2 NPs did not cause much toxicity to normal cells such as primary rat hepatocytes and human lung fibroblasts. Overall, we found that Ag-doped TiO2 NPs have potential to selectively kill cancer cells while sparing normal cells. This study warranted further research on anticancer potential of Ag-doped TiO2 NPs in various types of cancer cells and in vivo models.
... The tumour growth on the mice of each group was arbitrated by estimating the diameter of tumour volume in two perpendicular planes using a digital vernier calliper, starting from 7 th day of tumour growth up to 31 th day. The volume of tumour development was calculated using the equation, V=4/3πr1 2 r2, where r1 is the minor diameter and r2 is the major diameter 25 . ...
... Preparation of primary rat hepatocytes was documented two-step collagenase perfusion method [16]. Briefly, 500 ml of HEPES buffer (20 mM) was injected into hepatic portal vein to remove the residual blood and weaken the intercellular junctions. ...
Unlabelled:
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. It is important for HCC cells to resist to apoptosis caused by adverse energy pressure in microenvironment during the HCC tumorigenesis. HRP-3, a member of hepatoma-derived growth factor (HDGF)-related proteins (HRP) family, was shown to be highly up-regulated in HCC tissues and play an important role in HCC pathogenesis based on our previous research. The aim of the study was to investigate the HRP-3's role in HCC cells endurance against energy pressure.
Method:
The HRP-3 expression level in primary rat hepatocytes and human HCC cell lines were examined when changing the extracellular glucose concentration. To assess biological function of HRP-3 during glucose deprivation, HRP-3 stable knockdown and control clones of SMMC-7721 and SK-hep1 were constructed for further analysis including cellular morphology observation, apoptotic sub G1 peak analysis and the mTOR-mediated phosphorylation of S6K1 detection in the absence of glucose.
Results:
Expression level of HRP-3 protein was highly up-regulated both in primary rat hepatocytes and HCC cells as prolonging the stimulation of glucose deprivation. Both morphology and sub-G1 phase analyses indicated that stable knockdown of HRP-3 sensitized HCC cells to glucose deprivation-induced cell apoptosis. Furthermore, silence of HRP-3 prevented the de-phosphorylation of S6K1 induced by glucose deprivation, which was an essential molecular event for HCC cell survival in energy pressure.
Conclusions:
We propose that glucose deprivation-induced HRP-3 up-regulation potentially plays a major role in protecting HCC cells against apoptosis caused by energy pressure.
Lipophagy is a form of selective autophagy that targets the lipid droplets for lysosomal decay and has been implicated in the onset and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). Factors that augment lipophagy have been identified as targets for MASLD therapeutic development. TMEM55B is a key regulator of lysosomal positioning which is critical for the lysosome fusion with the autophagosome, but less studied. Here, we demonstrate that inhibition of TMEM55B in murine models accelerates MASLD onset and progression. In cellular models, TMEM55B deficiency enhances lipophagy, leading to increased fatty acid release from lysosomes to mitochondria but simultaneously impairs mitophagy, causing an accumulation of dysfunctional mitochondria. This imbalance leads to increased lipid accumulation and oxidative stress, worsening MASLD. These findings underscore the importance of lysosomal positioning in lipid metabolism and suggest that augmenting lipophagy may exacerbate disease in the context of mitochondrial dysfunction.
Garcinia talbotii is an evergreen tree whose wild edible fruits are used in culinary preparations and folk medicine. Garcinia talbotii fruits were subjected to proximate analysis and the Garcinia talbotii fruit extract (GFE) was subjected to phenolic profiling, antioxidative effects on HEK-293 cells and antiproliferative effects on Pancreatic Cancer (PC) cells. The fruit showed higher amounts of Vitamin C (188.51 ± 20.38 mg/100 g) with macro and micronutrients. HR-LCMS analysis revealed Chlorogenic acid, Catechin and Protocatechuic acid in higher concentrations. GFE exhibited significant antioxidant effects on HEK-293 cells under H2O2 induced oxidative stress (IC50: 10.5 ± 0.08 μg/ml). Antiproliferative effects of GFE studied against PANC-1 and MIA PaCa-2 cells resulted in reduced cell proliferation and clonogenicity. GFE treatment on PC cells enhanced ROS, triggered mitochondrial depolarization, apoptosis via DNA damage; expression of proteins in DNA Damage Response (DDR) pathway was studied by immunoblotting. To conclude nutrients and bioactive compounds present in GFE contributed to nutraceutical potential of fruit. The study on Garcinia talbotii fruit extract highlights its exceptional nutraceutical value and potent anticancer properties against pancreatic cancer cells through ROS-induced DNA damage and apoptosis. Further exploration of its bioactive compounds holds promise for novel cancer treatments.
Due to exceptional properties, copper oxide nanoparticles (CuO NPs) are now being applied in diverse fields such as agriculture, cosmetics, food industry, and medicine. There is enough scientific evidence that CuO NPs are toxic to living organisms. However, no effective method is available to mitigate the health hazard of the CuO NPs. This study aimed to investigate the possible cytoprotective mechanism of protocatechuic acid (PCA) from the toxicity of CuO NPs in human liver HepG2 cells and primary rat hepatocytes. CuO NPs were synthesized by a facile hydrothermal method. X-ray diffraction and electron microscopy characterization data confirmed the formation of spherical and crystalline CuO NPs with smooth surface morphology. On the basis of preliminary results, cells are divided into four groups; (i) control group, (ii) PCA group (10 µg/mL, a non-cytotoxic dose), (iii) CuO NPs group (IC50), and (iv) co-exposure group (PCA + CuO NPs). Results show that pre-treatment with PCA significantly abrogates the CuO NPs-induced cytotoxicity and lactate dehydrogenase leakage in both types of cells. CuO NPs-induced reactive oxygen species, hydrogen peroxide, and malondialdehyde levels are remarkably mitigated by the PCA pre-treatment. The depletion in glutathione and several antioxidant enzymes (e.g. glutathione peroxidase, superoxide dismutase, and catalase) because of the CuO NPs exposure is effectively restored by PCA. Caspase-3 gene up-regulation and mitochondrial membrane potential depletion due to CuO NPs exposure are also alleviated by the pre-treatment using PCA. Therefore, this first study reports the mitigating potential of PCA against CuO NPs toxicity in liver cells by restoring the antioxidant defense system and quashing the apoptosis.
• TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor, is reportedly excreted predominantly through urinary excretion in an unchanged form in humans, with partial biliary excretion also possible. However, the clearance mechanisms remain unclear. The aim of this study was to investigate the clearance mechanisms in humans and to assess species differences in the excretion routes.
• TP0463518 was not metabolized in rat, dog, or human hepatocytes. TP0463518 is a substrate for human BCRP, OATP1B1, OATP1B3, and OAT3, suggesting that renal uptake by OAT3 is probably the predominant clearance route, with hepatic uptake by OATP1B1 and OATP1B3 contributing partially to clearance in humans.
• A species difference in excretion routes was observed. The unchanged urinary excretion rates in humans, male rats, female rats, dogs, and monkeys were 80.7%, 0.1%, 40.9%, 15.2%, and 72.6%, respectively. Urinary excretion was predominant in humans and monkeys, while only biliary excretion was observed in male rats. Uptake studies using hepatocytes showed that the hepatic uptake clearance in rats was 13.6-fold higher than that in humans. Therefore, not only reabsorption via renal tubules, but also hepatic uptake seems to be involved in the species differences in excretion routes between rats and humans.
Excessive alcohol use often results in alcoholic liver disease (ALD). An early change in the liver due to excessive drinking is hepatic steatosis, which may ultimately progress to hepatitis, liver fibrosis, cirrhosis, and liver cancer. Among these debilitating processes, hepatic steatosis is reversible with the appropriate treatment. Therefore, it is important to find treatments and foods that reverse hepatic steatosis. Black carrot has antioxidant and anti-inflammatory effects. In this study, we examined the effectiveness of black carrot extract (BCE) on hepatic steatosis in in vivo and in vitro ethanol-induced liver injury models. For the in vivo experiments, serum aminotransferase activities enhanced by ethanol- and carbon tetrachloride were significantly suppressed by the BCE diet. Furthermore, morphological changes in the liver hepatic steatosis and fibrosis were observed in the in vivo ethanol-induced liver injury model, however, BCE feeding resulted in the recovery to an almost normal liver morphology. In the in vitro experiments, ethanol treatment induced reactive oxygen species (ROS) levels in hepatocytes at 9 h. Conversely, ROS production was suppressed to control levels and hepatic steatosis was suppressed when hepatocyte culture with ethanol were treated with BCE. Furthermore, we investigated enzyme activities, enzyme protein levels, and messenger RNA levels of alcohol dehydrogenase (ADH), cytochrome p450 2E1 (CYP2E1), and aldehyde dehydrogenase (ALDH) using enzyme assays, western blot, and quantitative reverse transcription-polymerase chain reaction analyses. We found that the activities of ADH, CYP2E1, and ALDH were regulated through the cAMP-PKA pathway at different levels, namely, translational, posttranslational, and transcriptional levels, respectively. The most interesting finding of this study is that BCE increases cAMP levels by suppressing the Pde4b mRNA and PDE4b protein levels in ethanol-treated hepatocytes, suggesting that BCE may prevent ALD.
Carbohydrate polymers were widely used in pharmaceuticals and drug delivery systems due to their biodegradability and biocompatibility. Among them, chitosan (Cs) has been considered in many new drug delivery systems. Poly(ethylene glycol) as a hydrophilic polymer can increase the solubility and stealth functions of nanocarriers. The Fe3O4 nanoparticles functionalized with polymers act as non-toxic drug vehicles for tumor targeting under external magnetic fields. In present study, the Fe3O4/SiO2-NH2 nanoparticles were prepared and then functionalized with methoxy-PEGylated chitosan (Cs-g-mPEG2000) and the hydroxyurea (HU) was loaded on this nanoparticles. The structure, crystallinity, and morphology of HU/Fe3O4/SiO2/Cs-g-mPEG2000 were determined using spectroscopic and electron microscopy analysis. Encapsulation efficiency of HU and the percentage of loading and release rate at different pH values at 37 °C were examined. Maximum drug release was observed at pH = 7.4. According to TEM results, the nanoparticle sizes were between 18 and 157 nm. The cytotoxicity effect of HU-loaded nanoparticles against MCF-7 human breast cancer cell was evaluated using MTT assay and cell cycle arrest analysis. The inhibitory concentration (IC50) values were 249 and 85 μg/mL on the MCF-7 cell line compared to the control group in 24 h and 96 h, respectively. In addition, the expression of p53 and lincRNA-P21 genes in treated cells and control group was assessed using real-time PCR, and the results showed that the ratio of p53 expression to lincRNA-P21 in MCF-7 cells was significantly increased (P < 0.05). The cell cycle arrested in the S-phase and the population of cells increased 1.3-fold compared to the control group.
Edifenphos (EDF) is an organophosphorus pesticide with antifungal and anti-insecticidal properties. However, EDF accumulates in various agricultural products and causes potential hazards to human health. Although numerous reports have indicated EDF accumulation in agricultural products, toxic effects on cellular system is poorly understood. In the present study, we investigated the cytotoxicity and genotoxicity of EDF in rat hepatocytes and its amelioration by apigenin (a dietary flavonoid). Results showed that EDF inhibited the cell viability, induced oxidative stress, DNA damage, loss of mitochondrial membrane potential (ΔΨm) and caspase-9/-3 activation in rat hepatocytes. Incubation of hepatocytes with N-acetyl cysteine (ROS scavenger) significantly abrogated the ROS generation and apoptosis caused by EDF. In addition, this study also showed that apigenin significantly suppressed the toxic effects of EDF by quenching ROS production thereby abrogating the caspase-9/-3 and apoptosis activation in hepatocytes. Taken together, the findings of this study demonstrate that EDF induces cytotoxicity and DNA damage in hepatocytes, and apigenin can be considered as an effective dietary anti-oxidant regimen against EDF- induced toxicity in cellular system.
Communicated by Ramaswamy H. Sarma
Premna serratifolia L. (Lamiaceae) is a medicinal plant, widely distributed in the tropical and subtropical regions and commonly used in traditional medicine. The current study was focused to evaluate the cytotoxic potential of aqueous extract of root of P. serratifolia (AEPS) against human hepatoblastoma cancer cell line (Hep G2).The yield of the dried extract was 5.8% and used for further studies.Cytotoxic potential of AEPS was analyzed by MTT assay, which exhibits IC50 value 1000 µg/mL after 48 h incubation. Hoechst and AO/EtBr staining, ROS measurement, mitochondrial membrane potential, clonogenic and wound healing assays also confirmed the cytotoxic efficacy of AEPS in dose and time-dependent manner. UPLC–Q-TOF–MS/MS analysis of AEPS confirmed the presence of 12polyphenolic compounds, namely 4-hydroxy-3-methoxycinnamic acid, linarin, peonidin-3,5-O-di-beta-glucopyranoside, diosmin, trans-cinnamic acid, daidzein, saponarin, homoorietin, acacetin, sarsasapogenin, phytol and sissotrin. The cytotoxic potential of AEPS might due to presence of biologically active polyphenolic compounds.
The effects of metyrapone on qualitative changes in cytochrome P-450-dependent drug metabolizing activities in primary cultures of rat hepatocytes were investigated. Metyrapone apparently increased benzo(a)pyrene hydroxylation and maintained both ethoxycoumarin-O-deethylation and propoxycoumarin-O-depro-pylation. whereas it had little effect on methoxycoumarin-O-demethylation Furthermore, P-450d (high spin type of P-448) as well as P-450c (low spin type of P-448) were induced by metyrapone. while P-450b and P-450e were not. In conclusion, metyrapone act as a 3-methylcholanthrene-like inducer in the primary cultures of rat hepatocytes
Resveratrol (RES) (trans-3, 5,-4'-trihydroxystilebene) is a multi-biofunctional compound found in a variety of plants such as grapes and mulberries. Studies of nanoencapsulated resveratrol have indicated that this compound can inhibit the growth of cancer cells and free radicals. The aim of this study was to isolate resveratrol from Vitis vinifera, develop and evaluate resveratrol nanostructured lipid carriers (NLCs) and/or resveratrol encapsulated chitosan-coated nanostructured lipid carriers (CSNLCs) using low-viscous chitosan for anticancer therapy. In addition, our study was carried out to examine the prophylactic potential of RES, NLC, and CSNLC on paraquat-induced injury in rat hepatocytes. In this study we isolated resveratrol and encapsulated NLCs in phosphate-buffered saline solution using a phase inversion method. In addition, CSNLCs were prepared by ionic gelation method of NLCs using chitosan. NLCs and CSNLCs were then characterized for their particle size, zeta potential, morphology, and entrapment efficiency. Furthermore, NLCs and CSNLCs were evaluated for their cytotoxic effect on Hep-G2, human HCT-116 (colorectal cancer cell line), lymphoblastic leukemia (1301), and human MCF-7 (Michigan Cancer Foundation-7) cells as well as their effect on caspase-3 and death receptor (DR-4). In addition, incubation of hepatocytes with paraquat resulted in increased formation of TBARS (thiobarbituric acid reactive substances) with a parallel increase in lactate dehydrogenase (LDH) leakage at 1 h after incubation. Time-dependent depletion of cellular glutathione (GSH) was observed starting 2 h after incubation with paraquat. The mean particle size of NLC and CSNLC were 67.0 and 98.41 nm, zeta potential were (-) 24.8 and (+) 31.6 mV, entrapment efficiency were 74.15% and 85.46%, respectively, with the observed shapes of nanoparticle being spherical. The treatment of Hep-G2, human HCT-116, lymphoblastic leukemia (1301), and human MCF-7 cells with NLC led to high inhibition in the cell proliferation as concluded by the low IC50 values 27.7, 17.43, 35.39, and 47.66 μg/mL, respectively, whereas CSNLC had high cytotoxic effect on Hep-G2, human HCT-116, lymphoblastic leukemia (1301), and human MCF-7 cells with low IC50 values 13.29, 10.56, 16.79 and 22.60 μg/mL, respectively. Both NLC and CSNLC possess apoptotic properties through activation of the caspase-3 and death receptor (DR-4). In addition, incubation of hepatocytes with RES, NLC, and CSNLC markedly protected against paraquat-induced formation of TBARS, increase in LDH leakage, and prevented GSH depletion. The most effective doses for ethyl acetate, ethanolic, and aqueous extracts were 7.5, 10, and 12.5 μg, respectively. The results presented here may suggest that nanoencapsulated resveratrol isolated from the stems of V. vinifera to obtain NLC and CSNLC possess anticancer and apoptotic effects on cell proliferation, and therefore, can be used as new approach of pharmaceutical drugs. In addition, the results clearly suggest that the RES, NLC, and CSNLC exerted protective effect against cytotoxicity induced by paraquat. On the contrary, the effect decreased in order of CSNLC, NLC, and RES.
Focal segmental glomerulosclerosis is the final result of a number of interrelated events leading to permanent glomerular injury. It is a feature of many chronic progressive renal diseases. Glomerulosclerosis (GS) and atherosclerosis are thought to occur by similar pathogenic mechanisms. Central to this analogy between the atherosclerosis and GS processes is the origin and functional properties of the glomerular mesangial cell (MC). There are similarities between contractile glomerular MC and vascular smooth muscle cells. Hyperlipidaemia is believed to play a role in atherogenesis and the progression of renal disease. Much research in the past decade has focused on post-secretory modification of lipoproteins such as oxidation and their potential role in promoting atherosclerosis. This study investigated whether low density lipoproteins (LDL) are oxidatively modified by MC and if interaction of oxidatively modified LDL with MC may cause glomerular injury and potentially influence glomerulosclerosis. Our results demonstrate firstly that rat and human mesangial cells have the ability to oxidise LDL in vitro. The oxidative mechanism involves superoxide anions but other free radicals may also be involved. Experiments designed to compare LDL oxidation by MC with other cell types present in the kidney, such as endothelial cells, proximal tubular cells and monocyte/macrophages, demonstrated that endothelial cells oxidatively modified LDL to the greatest extent. Mesangial cells oxidised LDL to a similar extent to that of macrophages. Secondly, mesangial cells express scavenger type receptors that are capable of binding and internalising modified lipoproteins which may result in unregulated uptake of lipoproteins. Finally, exposure of oxidised LDL to mesangial cells caused a decrease in cell proliferation at low concentrations and cytotoxicity at higher concentrations. These results support the hypothesis that that the interaction of modified LDL with mesangial cells contributes to glomerular injury and may play a role the pathogenesis of glomerulosclerosis.
Thioxanthone and its analogues, 2- or 4-isopropylthioxanthone, 2-chlorothioxanthone, 2,4-diethylthioxanthone (DETX) and xanthone, are used as photoinitiators of ultraviolet (UV) light-initiated curable inks. As these photoinitiators were found in numerous food/beverage products packaged in cartons printed with UV-cured inks, the cytotoxic effects and mechanisms of these compounds were studied in freshly isolated rat hepatocytes. The toxicity of DETX was greater than that of other compounds. DETX elicited not only concentration (0-2.0 mm)- and time (0-3 hours)-dependent cell death accompanied by the depletion of cellular adenosine triphosphate (ATP), and reduced glutathione (GSH) and protein thiol levels, but also the accumulation of GSH disulfide and malondialdehyde. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or N-acetyl-l-cysteine (NAC) at a concentration of 5.0 mm ameliorated DETX (1 mm)-induced cytotoxicity. Further, the exposure of hepatocytes to DETX resulted in the induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential, both of which were partially prevented by the addition of NAC. These results indicate that: (1) DETX-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were, at least in part, ameliorated by the addition of fructose; and (3) GSH loss and/or ROS formation was prevented by NAC. Taken collectively, these results suggest that the onset of toxic effects caused by DETX may be partially attributable to cellular energy stress as well as oxidative stress.
The novel psychoactive compounds derived from amphetamine have been illegally abused as recreational drugs, some of which are known to be hepatotoxic in humans and experimental animals. The cytotoxic effects and mechanisms of 5‐(2‐aminopropyl)benzofuran (5‐APB) and N‐methyl‐5‐(2‐aminopropyl)benzofuran (5‐MAPB), both of which are benzofuran analogues of amphetamine, and 3,4‐methylenedioxy‐N‐methamphetamine (MDMA) were studied in freshly isolated rat hepatocytes. 5‐MAPB caused not only concentration‐dependent (0–4.0 mm) and time‐dependent (0–3 h) cell death accompanied by the depletion of cellular ATP and reduced glutathione and protein thiol levels, but also accumulation of oxidized glutathione. Of the other analogues examined at a concentration of 4 mm, 5‐MAPB/5‐APB‐induced cytotoxicity with the production of reactive oxygen species and loss of mitochondrial membrane potential was greater than that induced by MDMA. In isolated rat liver mitochondria, the benzofurans resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA, with a decrease in the rate of state 3 oxygen consumption. Furthermore, the benzofurans caused more of a rapid mitochondrial swelling dependent on the mitochondrial permeability transition than MDMA. 5‐MAPB at a weakly toxic level (1 mm) was metabolized slowly: levels of 5‐MAPB and 5‐APB were approximately 0.9 mm and 50 μm, respectively, after 3 h incubation. Taken collectively, these results indicate that mitochondria are target organelles for the benzofuran analogues and MDMA, which elicit cytotoxicity through mitochondrial failure, and the onset of cytotoxicity may depend on the initial and/or residual concentrations of 5‐MAPB rather than on those of its metabolite 5‐APB. Copyright © 2016 John Wiley & Sons, Ltd.
Objectives: To measure the metabolism and toxicity of 7-chloro-4- (cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-1) and 4-cyclohexylmethyl-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2), two new benzodiazepine analogues found to be effective against Leishmania amastigotes in vitro. Methods: The metabolism of BNZ-1 and -2 was investigated in isolated rat hepatocytes and rat liver microsomes. The toxicity of the compounds was assessed in a murine macrophage cell line by determining cell viability and reduced glutathione (GSH) content. The metabolism and toxicity of flurazepam was assessed for comparison. Key findings BNZ-1 and BNZ-2 underwent similar metabolic transformations by the liver systems, forming N-demethylated and hydroxylated metabolites, with subsequent O-glucuronidation. Flurazepam and both analogue compounds depleted macrophage GSH levels without affecting cell viability at the concentrations used (up to 100 μM), but only flurazepam inhibited glutathione reductase activity, indicating that it is acting by a different mechanism. Conclusions: The exact mechanism responsible for GSH depletion is unknown at present. Further experiments are needed to fully understand the effects of BNZs on the parasite GSH analogue, trypanothione, which may be a direct or indirect target for these agents. Pharmacokinetic evaluation of these compounds is required to further progress their development as potential new treatments for leishmaniasis.
Crocin, the main constituent of saffron (Crocus sativus L.), is a natural carotenoid which is known for its antioxidant activity. Liver as the organ that metabolizes many chemicals is one of the first position that is at risk of environmental pollutants. It is clear that compounds that exhibit antioxidant properties, scavenging of free radicals and inhibition of lipid peroxidation are expected to show hepatoprotective effects. Previous studies have proven the protective effect of crocin on the liver. The aim of this study is to find out the exact hepatoprotective mechanisms of this compound. In the present study, the protective effects of various concentrations of crocin (5, 10, 25, 50 and 100 μg/mL) were examined against oxidative stress toxicity induced by cumene hydroperoxide (CHP) on isolated rat hepatocytes. To find out the exact protective activity of crocin, we evaluated cell lysis, lipid peroxidation, reactive oxygen species (ROS) generation, GSH/GSSG, collapse of mitochondrial membrane potential, lysosomal membrane damage, the release of cytochrome c, and cellular proteolysis. Crocin (50 and 100 µg/mL) reduces cell lysis, lipid peroxidation, ROS generation, collapse of mitochondrial membrane potential, lysosomal membrane damage, cytochrome c release, and cellular proteolysis. It also increase GSH/GSSG. Crocin (50 and 100 µg/mL) reduced liver toxicity not only as an antioxidant but also by protecting the mitochondria and lysosome. Our data demonstrated that crocin is a promising candidate for preventing liver injury associated with oxidative stress. These findings pave the way to further studies evaluating the clinical protective effect of crocin. © 2018, Iranian Journal of Pharmaceutical Research. All rights reserved.
Psychoactive compounds, N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB) and 3,4-methylenedioxy-N-methamphetamine (MDMA), are known to be hepatotoxic in humans and/or experimental animals. As previous studies suggested that these compounds elicited cytotoxicity via mitochondrial dysfunction and/or oxidative stress in rat hepatocytes, the protective effects of fructose and N-acetyl-l-cysteine (NAC) on 5-MAPB- and MDMA-induced toxicity were studied in rat hepatocytes. These drugs caused not only concentration-dependent (0–4 mm) and time-dependent (0–3 hours) cell death accompanied by the depletion of cellular levels of adenosine triphosphate (ATP) and glutathione (reduced form; GSH) but also an increase in the oxidized form of GSH. The toxic effects of 5-MAPB were greater than those of MDMA. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or NAC at a concentration of 2.5 mm prevented 5-MAPB−/MDMA-induced cytotoxicity. In addition, the exposure of hepatocytes to 5-MAPB/MDMA caused the loss of mitochondrial membrane potential, although the preventive effect of fructose was weaker than that of NAC. These results suggest that: (1) 5-MAPB−/MDMA-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were ameliorated, at least in part, by the addition of fructose; and (3) GSH loss via oxidative stress was prevented by NAC. Taken collectively, these results indicate that the onset of toxic effects caused by 5-MAPB/MDMA may be partially attributable to cellular energy stress as well as oxidative stress.
Umbelliferone (UMB) has widespread pharmacological activity, comprising anti-inflammatory, anti-oxidant, anti-genotoxic and anti-immunomodulatory but the anticancer activity remains unknown in human oral carcinoma (HOC) KB cells. MTT assay determinations was revealed that treatment of KB cells with UMB, prevent and reduce the cell proliferation with the IC50 − 200 μM as well as induces loss of cell viability, morphology change and internucleosomal DNA fragmentation in a concentration dependent manner. Acridine orange and ethidium bromide dual staining assay established that UMB induced apoptosis in KB cells in a dose dependent manner. Alkaline comet assay determination revealed UMB has the potential to increase oxidative DNA damage in KB cells through DNA tail formation significantly (p < 0.05). Furthermore, UMB brought a dose-dependent elevation of reactive oxygen species (ROS), which is evidenced by the DCF fluorescence, altered the mitochondrial membrane potential in KB cells. Similarly, we observed increased DNA damage stimulated apoptotic morphological changes in UMB treated cells. Taken together, the present study suggests that UMB exhibits anticancer effect on KB cell line with the increased generation of intracellular ROS, triggered oxidative stress mediated depolarization of mitochondria, which contributes cell death via DNA damage as well as cell cycle arrest at G0/G1 phase. The results have also provided us insight in the pharmacological backgrounds for the potential use of UMB, to target divergent pathways of cell survival and cell death. To conclude UMB could develop as a novel candidate for cancer chemoprevention and therapy, which is our future focus and to develop a connectivity map between in vivo and in vitro activity.
Docetaxel (DTX) is one of the most widely used drugs in oncology due to its high efficacy against several cancers. Though, its routine clinical administration, formulated in tween 80, causes serious side effects. Polylactide-co-glycolide (PLGA), biodegradable polyester synthesized and approved for human use, is employed to overcome these problems.
In this investigation, we compare the cytotoxic mechanisms of DTX and PLGA-DTX in isolated rat hepatocytes. Cytotoxicity of DTX and PLGA-DTX were associated with reactive oxygen species formation, activation of caspases cascade, collapse of mitochondrial membrane potential (MMP), lysosomal membrane leakiness and ATP depletion. Our results also showed that CYP2E1 is involved in the oxidative stress cytotoxicity mechanism and both drugs are detoxified via phase II metabolic methylation. Furthermore, we concluded that PLGA-DTX is bioactivated by GSH. It could also potentiate hepatocyte toxicity through a mitochondrial/lysosomal toxic cross-talk. In addition to these observed differences, it is likely that mode of hepatocyte membrane penetration is different between these compounds.
Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid. Rat liver parenchymal cells transported alpha-aminoisobutyric acid by an energy-dependent Na+-requiring system which displayed Michaelis-Menten kinetics. Addition of insulin to cultured rat liver parenchymal cells resulted in an increased influx of alpha-aminoisobutyric acid which was reflected in a higher initial rate of alpha-aminoisobutyric acid transport as well as an increased accumulation of alpha-aminoisobutyric acid at later time points. Cycloheximide effectively blocked the increase while results with actinomycin D were equivocal. Insulin at concentrations as low as 50 pM was effective in stimulating alpha-aminoisobutyric acid transport while the maximal response was observed at 80 nM.
Suspensions of isolated parenchymal cells were prepared from rat liver by incubation with collagenase and hyaluronidase followed by mechanical treatment. Utilization of 0.15% collagenase together with 0.15% hyaluronidase yielded adequate numbers of cells for experimental purposes. As shown by light and electron microscopy, approximately 75% of the isolated cells retain their structural integrity. The cell suspensions are capable of maintaining endogenous respiration in the presence of 1% albumin for periods of time up to 8 hr. These cell preparations consist almost entirely of parenchymal cells and offer a unique tissue preparation for the study of hepatic metabolism.
Previous studies have implicated the reticuloendothelial cells of the liver in certain aspects of steroid metabolism. The similarity in the metabolism of steroids and polycyclic hydrocarbons suggested that the nonparenchymal cells possibly play a role in these areas. The present study presents evidence that at least one of the microsomal NADPH-requirig enzymes, benzpyrene hydroxylase, is present in nonparenchymal cells and, furthermore, is "inducible." In adult rats treated with 3-methylcholanthrene or beta-naphthoflavone, the nonparenchymal cells exhibited increases in benzpyrene hydroxylase activity of 17-fold and five-fold, respectively. Treatment with phenobarbital resulted in only a slight increase in enzyme activity. Enzyme activity in parenchymal cells under similar conditions was increased sixfold and fivefold by 3-methylcholanthrene and beta-naphthoflavone, respectively, but not by phenobarbital.
A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.
This chapter discusses the physiological implications of drug oxidations to the cell. The conversion of various lipophilic drugs to hydrophilic and excretable products occurs predominantly in the liver as a multistep metabolic process that most often involves cytochrome P-450-linked monooxygenation and subsequent conjugation reactions. During this process, the hepatocyte is exposed not only to the possible toxic effects of the unmetabolized drug but also to those of its metabolites that may sometimes be even more deleterious. Moreover, drugs may compete with endogenous substrates for common enzymes, cofactors and oxygen and may thus also indirectly affect cellular intermediary and energy metabolism. The chapter describes a number of possible physiological implications of drug oxidations to the cells, such as protection against drug toxicity by binding and metabolism through the cytochrome P-450 system; competition between drugs and endogenous substrates for common enzymes, oxygen, and cofactors; and the formation of reactive and toxic drug metabolites. Most of these phenomena are, however, only partly elucidated, and little is known about their physiological importance.
Isolated rat liver cells were found to catalyze the cytochrome P-450 dependent, oxidative metabolism of alprenolol [1-(2-allylphenoxy)-3-isopropylaminopropanol] at a rate similar to that obtained with the isolated microsomal fraction in the presence of a NADPH-generating system. Alprenolol was rapidly taken up into the liver cells and the apparent Michaelis constant was similar to that obtained with liver microsomes. Further, inhibitors of drug metabolism such as SKF 525-A and metyrapone showed similar inhibitory patterns in the cellular and microsomal systems. With liver cells isolated from control rats, NADPH generation from endogenous substrates was sufficient to support optimal alprenolol metabolism, whereas the addition of glucose or lactate stimulated this rate in liver cells isolated from starved, phenobarbital-treated rats. Inhibitors of mitochondrial respiration such as rotenone, antimycin A and KCN and the uncoupler carbonyl cyanide-p-trifluoromethoxy-phenyl hydrazone inhibited cellular alprenolol metabolism, presumably by lowering the ATP/ADP ratio thereby interfering with NADPH generation via the malic enzyme system; this effect was more pronounced in liver cells from starved rats. Furthermore, in analogy with previous findings, the rate of gluconeogenesis from lactate was inhibited during alprenolol metabolism; this effect in turn was most pronounced in liver cells from phenobarbital-treated rats. The findings indicate that under certain conditions, the level of cytoplasmic NADPH may become rate-limiting for drug metabolism linked to cytochrome P-450 and support the previous assumption that in fed rats, generation of cytoplasmic NADPH is mainly via the pentose phosphate shunt whereas the malic enzyme system becomes more important for this purpose during starvation.
Isolated perfused rat livers were dispersed by a two-step procedure of Ca2+ removal (probably including the removal of a Ca2+-dependent adhesion factor) followed by enzymatic treatment. Collagenase, crude or purified, converted all of the parenchymal tissue to a suspension of cells which were 95% intact. Other enzymes such as hyaluronidase, lysozyme and trypsin were ineffective. Since hypoxic conditions during enzymatic treatment did not affect cellular viability, the oxygenation apparatus can be omitted to facilitate sterile preparation of cells.Purification of the parenchymal cells by differential centrifugation reduced contamination by non-parenchymal cells from 10–20% initially to 1–2% finally, with 40% of the parenchymal cells recovered and no loss in viability. The purified cells were 20% binucleated and had the same biochemical composition as intact liver tissue. The cells were active in the synthesis of RNA, protein, glycogen and various metabolites, and showed sensitivity towards steroid and polypeptide hormones. Many of the freshly isolated cells had marked constriction furrows which disappeared upon incubation.
Hepatocytes of adult rats were isolated by infusion of a hyaluronidase collagenase mixture. High yields of cells excluding trypan blue were obtained. These cells, in Hank's buffer containing rat serum and 0.1% glucose, ethylmorphine. The formaldehyde initially formed is further metabolized to tritiated water. Fifteen per cent of the original metabolic activity was observed after 21 hr at 37° in 5% CO2-air, and cumulative metabolism is linear for up to 90 min under these conditions. The Km for the N-demethylation of [3H-CH3-N]ethylmorphine is 50 μM, 20 per cent of the value observed for this reaction by musomal preparations. An active transport of the substrate into the cell is postulated to account for this difference.
Various methods for the preparation of rat hepatocyte suspensions have been compared with regard to cell yield and viability index. A modified method of collagenase/hyaluronidase digestion using a sequential removal and replacement of Ca2+ and the omission of all perfusion steps has been evolved, thereby making the technique potentially viable for use with pieces of liver such as isolated human liver biopsy material. These suspensions contain large numbers of viable cells, and the viability index is also high (>70%). A number of metabolic functions can be readily detected in these cell suspensions, indicating the intactness of the isolated cells. A systematic survey of the enzymic components required for liver digestion is also reported.
Normal adult rat liver cells have been successfully cultured as monolayers without subjecting donor animals to a partial hepatectomy before cell isolation. Coating plastic tissue culture dishes with acid soluble calf skin collagen increases the efficiency of cell attachment. Hepatocytes form a monolayer in 24 hr in serum-free L-15 medium although 10% fetal calf serum for 24 hr increases efficiency of attachment. In serum-free medium the mono-layer remains viable for at least one week without added insulin. Cultured cells maintain tyrosine transaminase and four of the five urea cycle enzymes at levels above or equal to freshly isolated cells for 72 hr. Adenylyl cyclase activities are maintained for at least 72 hr, and are stimulated by epinephrine, glucagon and fluoride. Tyrosine transaminase activity is increased in cultured cells by glucagon and dexamethasone, but urea cycle enzymes are not.
Although considerable knowledge has been gathered on the functional aspects of microsomal monooxygenation, comparatively little has so far been known about the intracellular regulation of this process. For such studies, we have found the isolated rat liver cell system to be a very useful model, combining the convenience of an in vitro system with the access to the complex mechanisms of the intact in vivo system. This model has the advantage over the perfused liver that it readily lends itself to the study of rapid reaction sequences and makes quantitation of short-term drug metabolic reactions easier. It is also superior to liver slices which often show considerable leakage of adenine and pyridine nucleotides and where substrate penetration and oxygen diffusion may present problems depending on the relative thickness of the slice.
To obtain high levels of glutathione in isolated hepatocytes an isolation procedure shorter than 16 min was used. This procedure gave a moderately high yield of viable cells (200--300 X 10(6) cells/10 g liver) with 44 +/- 3 nmol of glutathione/10(6) cells. Incubation in Krebs-Henseleit solution containing 2% albumin resulted in a continuous loss of reduced glutathione from the cells, while incubation in a medium containing amino acids and horse serum resulted in increased levels, suggesting active synthesis for 5 h. A short and apparently harmless depletion of reduced glutathione was induced by diethylmaleate or cumene hydroperoxide. A depletion of reduced glutathione lasting more than 1 h was accompanied by an increased cellular leakage. The depletion was induced by either diethylmaleate plus paracetamol or diethylmaleate alone in higher concentrations. A common mechanism for these toxic responses is suggested.
The metabolism of diphenylhydantoin (DPH) was studied using isolated rat hepatocytes. The Vmax of DPH metabolism when calculated from the disappearance of DPH in the medium was 0.39 to 0.48 μmole/g cell/hr which is comparable to that in microsomal preparations. Disappearance rate of DPH was concentration-dependent but not of a simple Michaelis-Menten type. The formation of a DPH metabolite (glucuronide of hydroxylated DPH, HPPH-G) has an apparent Vmax of 0.29 to 0.32 μmole/g cell/hr. DPH binds extensively to hepatocytes and its distribution between the hepatocytes and the suspension medium was independent of DPH concentration.
Isolated rat liver cells catalyze the metabolism of benzo(alpha)pyrene (BP) with the resulting formation of phenols, dihydrodiols, and conjugates. The rate of the primary oxidative step in the process was similar to that catalyzed by isolated rat liver microsomes in the presence of a reduced nicotinamide adenine dinucleotide phosphate-generating system and responded similarly to various inhibitors, including 2-diethylaminoethyl-2,2-diphenylvalerate, metyrapone, alpha-naphthoflavone, and hexobarbital. The level of cytoplasmic, reduced nicotinamide adenine dinucleotide phosphate was not rate limiting in liver cells isolated from either fed or fasted animals. The conjugates and dihydrodiols formed were readily excreted, whereas low concentrations of phenols accumulated intracellularly. The pattern of metabolites of BP was the same in isolated rat liver cells and in the isolated perfused rat liver. 3-Methylcholanthrene treatment of the rats caused a marked increase in cellular BP metabolism as well as in cytochrome P-450 concentration. The induced hemoprotein revealed characteristics similar to those previously established with isolated liver microsomes, i.e., increase in high-spin form, enhanced affinity for BP as revealed by a lower Michaelis constant, and sensitivity to the inhibitory action of alpha-naphthoflavone. After 3-methylcholanthrene treatment, phenols and dehydrodiols constituted a larger percentage of the total metabolites, indicating a more pronounced stimulation of the oxidative than of the conjugative step of BP metabolism by induction, and the dihydrodiols now tended to accumulate intracellularly.
Cell death and necrosis are important reactions of liver cells to injury that play a role in a wide variety of human liver diseases. A review is given of the important facets known about the biochemical basis of toxic liver cell death. Liver cells can withstand a great many specific biochemical and morphologic changes without loss of viability. Disturbances in RNA and protein synthesis, mitochondrial function, or release of lysosomal enzymes do not play a primary causative role in cell death. Many previous studies have tended to implicate the plasma membrane and its presumed role in maintaining the proper Ca2+ balance as the primary site of the development of irreversible hepatocyte damage. These studies have generally faced a major difficulty in determining if the observed changes are the cause or an effect of cell death. Galactosamine-induced liver cell injury seems to offer a potentially analyzable model for the experimental analysis of liver cell necrosis. Our studies on the role of plasma membrane injury and associated increases in total cellular calcium are reviewed, and a tentative working hypothesis for the pathogenesis of galactosamine-induced liver cell necrosis is presented.
1. Sodium tetraphenylboron (TPB), a specific agent for complexing K+ has been found to dissociate adult mouse liver in vitro into a suspension of single cells. 2. Evidence is presented that this is the result of the removal of K+, which is the major cation involved in aggregation of cells in this tissue. 3. A coordination mechanism for aggregation is proposed in which the negatively charged surfaces in cell-cell aggregates or in cell-matrix-cell aggregates are neutralized by monovalent cations. 4. Two variables of coordination mechanisms, i.e., cation coordinated and the coordination number are used in a model advanced to explain ordered movement and aggregation of cells in tissues.
Isolated hepatocytes are being increasingly used in drug metabolism studies since they possess many of the attributes of both in vitro and in vivo systems. This paper reviews recent work on this use of isolated hepatocytes, with particular emphasis on their value in the study of (a) species and organ differences in xenobiotic metabolism, (b) the relation of metabolism to toxicity and (c) the balance of Phase I/Phase II metabolism.
The affinity and metabolism of alprenolol, a β-receptor antagonist, in rat liver microsomes, perfused liver and in the conscious rat were studied. Alprenolol elicited a type I difference spectrum with a spectral dissociation constant, Ks, of 0.34 μM. The apparent Michaelis-Menten constant, Km in rat liver microsomes was 25 μM and in the 9000xg supernatant fraction 17 μM. In re-circulating perfusions of rat liver in situ alprenolol at an initial concentration of 4.3 μg/ml was rapidly eliminated from the perfusate with a clearance close to the flow rate of the perfusate. The concentration in the perfusate of total metabolites increased rapidly to a high level. In conscious rat with permanent cannulas the blood concentration of alprenolol was determined after intravenous and oral administration of 2.0 mg/kg. The bioavailability of orally given alprenolol was 4 per cent of the given dose. The elimination after intravenous administration was bi-exponential with a half-life time of the β-phase of about 40 minutes.
The possibility that glutathione may protect against acetaminophen induced hepatic necrosis was examined. Pretreatment of mice with diethyl maleate, which depletes hepatic glutathione, potentiated acetaminophen induced hepatic damage. Administration of acetaminophen caused a dose dependent depletion of hepatic glutathione. Glutathione depletion by acetaminophen was enhanced by treatments that potentiate the hepatic necrosis and covalent binding produced by the toxic metabolite of acetaminophen. Conversely, glutathione depletion was inhibited by treatments that protect against these toxic effects. Moreover, covalent binding of this metabolite to hepatic macromolecules did not occur until the availability of glutathione was exhausted through conjugation with the metabolite. Similarly, alteration of glutathione availability by pretreatment with diethyl maleate or cysteine, respectively, increased or decreased covalent binding of the toxic metabolite. It is proposed that a fundamental role of glutathione in the body may be to protect tissues against electrophilic attack by drug metabolites and other alkylating agents.
Various drugs including hexobarbital, lidocaine and nortriptyline were added to suspensions of liver cells isolated from untreated and phenobarbital-treated male rats. Upon drug addition, there was a fast binding to cytochrome P-450, as revealed by the appearance of a rapidly growing type I spectral change in the difference spectrum. When this had reached optimal magnitude, an absorption peak at 437 nm could often be seen to appear and quickly disappear, followed by yet another increase in absorption at about 446 nm; the latter and the type I spectral change then rapidly disappeared. These spectral changes were most pronounced with liver cells from phenobarbital-treated rats which contained markedly increased levels of cytochrome P-450. Also the rate of hexobarbital binding to cytochrome P-450 seemed to be increased after phenobarbital pretreatment. Finally, evidence was obtained that the major part of cytochrome P-450 in the isolated liver cells is present in an oxidized, non-substrate-bound form.
The rate of formation of the type I spectral change upon drug addition to a suspension of isolated rat liver cells was used to study factors that influence drug uptake by the hepatocytes. Although considerably slower than in liver homogenates and microsomes, drug combination with cellular cytochrome P-450 was still rapid and occurred within a few seconds. The effects of varying temperature and concentration and lipid solubility of the drugs studied as well as the lack of effect of preincubation of the cells with rotenone on the rate of formation of the type I spectral change, lead us to suggest that drug uptake into the hepatocytes occurs by a non-energy requiring diffusion process.
Enzymatic activities for the N-demethylation of aminopyrine, the p-hydroxylation of aniline and the UDP-glucuronidation of p-nitrophenol in isolated rat hepatic cells (intact and disrupted by ultrasonication) have been compared on the basis of DNA content to those in homogenates from fresh liver tissue and the crude microsomal fractions prepared from these homogenates. The influence of different preparative procedures on the enzymatic activities are discussed. The inhibitor of drug metabolism SKF 525 A blocks the UDP-glucuronidation only in the intact isolated cells and not in the homogenates. In contrast, N-demethylation is inhibited by SKF 525 A both in the intact cells and in the disrupted cells and in 9000 g supernatants from fresh liver homogenates. This indicates that for the N-demethylation a true enzyme inhibition is involved while for the UDP-glucuronidation a non-specific change in the properties of the membranes leading to a reduced permeability for substrate or cosubstrate is more probable. The suspension of isolated hepatocytes appeared to be suitable to study various aspects of drug metabolism, especially their modifications due to changes in the cellular integrity and changes in the permeability of the membranes.
1.1. A new method, in which a rubber pestle is used to disperse the tissue, has been described for preparation of rat liver cell suspensions.2.2. An evaluation of the advantages and disadvantages of methods used in the past for preparing liver cell suspensions is presented.3.3. The advantages of the new method, which is believed to be superior to all others previously described, are stated. The method yields a suspension which is free of blood cells, other cellular contaminants and cell debris, and in which the hepatic cord cells of the liver tissue are recovered in an almost quantitative yield. The method does not employ an enzymatic or a drastic physical treatment of the tissue at any stage, and takes only 15 min from the time the animal is sacrificed.