Increase In Hepatocytes Research Articles

THU135 Maternal Obesogenic Diet Exposure Affects Post-natal And Pathologic Hepatocyte Proliferation In Offspring

Abstract Disclosure: N. Ramavath: None. H. Hinrichs: None. M. Young: None. M. Thompson: None. Background: Obesity is a complex metabolic syndrome associated with a wide range of abnormalities in liver known as non-alcoholic fatty liver diseases (NAFLD). Evidence supports that maternal obesity/obesogenic diet exposure programs increased NAFLD offspring. The liver responds to lipotoxic hepatocyte injury through hepatocyte proliferation. It is not clear whether maternal obesogenic diet exposure affects the capacity of offspring hepatocytes to proliferate. We hypothesized that early impact of maternal/ obesogenic diet exposure in early life or utero exposure enhances hepatocyte proliferation capacity in offspring. Methods: Female mice were fed chow or high fat-fructose-cholesterol (HFFC) diet for 6 weeks and bred with lean males Liver and serum samples were collected at different postnatal time periods 5, 10, 15, 20, 25, and 30 days to analyze histological and molecular markers for liver proliferation. In addition, a small group of offspring from each group were weaned to the HFFC diet for 7 weeks to analyze progression of NAFLD (non -alcoholic fatty liver diseases). Results: No significant difference between neonatal body weight and liver weight was observed; however, the liver weight/body weight was increased at day 20 in HFFC offspring compared to chow offspring. The number of ki-67 positive hepatocytes peaked in both groups at day 15. An increase in ki-67 positive hepatocytes was observed at 15, 20, and 30 days in HFFC offspring compared to chow offspring. After 7 weeks of HFFC diet feeding, the number of Ki-67 positive hepatocytes was higher in HFFC offspring in comparison with Chow offspring. Conclusion: Maternal obesogenic diet exposure affected post-natal and NAFLD-associated hepatocyte proliferation in offspring with an increase in hepatocyte proliferative capacity. This could have implications for the ability of maternal obesity exposed offspring to respond to hepatic injury and potential for development of hepatic cancer. Presentation: Thursday, June 15, 2023

Titanium dioxide nanoparticles promote histopathological and genotoxic effects in Danio rerio after acute and chronic exposures

Titanium dioxide nanoparticles (TiO2-NPs) are among the most used nanomaterials worldwide, but studies evaluating its genotoxicity and histopathological effects are scarce, dealing with short exposure times and low concentrations for human use. The aim was to evaluate TiO2-NPs genotoxicity and histological alterations in the intestine and liver of zebrafish after exposure to human consumption compatible concentrations. Fishes were acutely (96 hours) and chronically (30 days) exposed to 5.0, 20 and 40 mg L-1 of TiO2-NPs and later euthanized for organ and blood analysis through histological procedures and the micronucleus test, respectively. An increase in the thickness of intestinal villi was observed after acute and chronic exposure in the higher concentrations. The liver showed an increase in vacuolated hepatocytes after both exposures, besides an increase in hepatocytes with peripheral nucleus. Genotoxicity was only observed after chronic exposure, demonstrated by the increase in micronucleus and cell buddings. These findings indicate that TiO2-NPs cause histopathological damage even in acute exposures, as the intestine serves as a barrier for NPs and the liver is an organ that accumulates Ti. Genotoxicity was possibly mediated by reactive oxygen species through chronic inflammation, leading to tissue damage and carcinogenesis in longer exposures that represents human exposure time.

Total glucosides of paeony decreases apoptosis of hepatocytes and inhibits maturation of dendritic cells in autoimmune hepatitis

Total glucosides of paeony (TGP), an active mixture extracted from paeony root, has anti-inflammatory and immunoregulatory effects and is widely used for the treatment of autoimmune diseases such as rheumatoid arthritis. However, the role of TGP in autoimmune hepatitis (AIH) is still unknown. In this study, we aimed to investigate the effect of TGP in autoimmune liver disease (AILD) patients and in concanavalin A (Con A)-induced experimental autoimmune hepatitis (EAH). Changes in biochemical parameters of AILD patients showed that treatment with TGP exerts significant protective effects on liver function, as reflected by decreased levels of serum alanine transaminase, aspartate transaminase, γ-glutamyl transpeptidase and total bilirubin. In EAH mice, we found that pretreatment with TGP reduced the levels of serum liver enzyme levels, histopathological damage and hepatocyte apoptosis. Importantly, flow cytometry analysis showed that pretreatment with TGP reduced the infiltration of mature dendritic cells in the liver. In vitro, TGP pretreatment ameliorated the Con A-induced mitochondrial membrane potential decline, reactive oxygen species increase, and apoptosis increase in hepatocytes. In addition, the levels of Bax, Cleaved Caspase-3 and cytoplasmic Cytochrome C decreased during this process, whereas those of Bcl-2 and mitochondrial Cytochrome C increased. Therefore, TGP might decrease hepatocyte apoptosis through the mitochondrial apoptotic pathway. Moreover, the maturation of bone marrow dendritic cells was also inhibited by TGP treatment. In conclusion, TGP treatment ameliorates AIH by regulating hepatocyte apoptosis and DC maturation. TGP is a potential compound for AIH treatment.

Interleukin 6-dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis.

Our findings indicate that genomic instability derived during the IL6-mediated liver regenerative response within a milieu of chronic inflammation links partial hepatectomy to accelerated hepatocarcinogenesis; this suggests a new therapeutic approach through the usage of an anti-IL6 treatment to extend the tumor-free survival of patients undergoing surgical resection. (Hepatology 2017;65:1600-1611).

Diacylglycerol Accumulation Impairs Short-Term Activation of Phospholipase D by Thyroxine in the Liver Cells

Thyroid hormones (TG) are known modulators of signal transduction. Phospholipase D (PLD) is one of the targets of TG in the stimulated cells. Response of cells to the short-term TG action significantly reduces at old age. Taking into account that diacylglycerol (DAG) accumulation induces the resistance of cells to some of regulatory factors in the target cells the aim of the present study was to determine if DAG content increase in hepatocytes impairs the L-thyroxine (L-T4) short-term action. The experiments were performed in either the [14C]palmitic acid- labeled hepatocytes or [14C]oleic acid-pre-labeled liver cells of 3- and 24-month-old rats. To study the short-term L-T4 action on cells the PLD activation was determined. The DAG production and content in hepatocytes significantly increased at old age and in the young cells pre-treated with palmitic acid. The reduction of DAG level in cells by means of DAG-kinase activator, alfa-tocoferol acetate, or long-term L-T4 treatment improved the short-term hormone action. The above data have indicated that DAG play important role in the L-T4 PLD regulation. The cross-talk between classic and non-genomic pathways of TG regulation of lipid metabolism has been determined.

Biomarkers of Flutamide-Bioactivation and Oxidative Stress In Vitro and In Vivo.

The nonsteroidal androgen-receptor antagonist flutamide is associated with hepatic injury. Oxidative stress and reactive metabolite formation are considered contributing factors to liver toxicity. Here we have used flutamide as a model drug to study the generation of reactive drug metabolites that undergo redox cycling to induce oxidative stress (OS) in vitro and in vivo. Lipid peroxidation (LPO) markers, as well as genes regulated by the redox-sensitive Nrf2 pathway, have been identified as surrogates for the characterization of OS. These markers and metabolism biomarkers for drug bioactivation have been investigated to characterize drug-induced hepatic damage. Rat hepatocytes and in vivo studies showed that several LPO markers, namely the isoprostanes 15R-PD2, dihydro keto PE2, and iPF(2α)-VI, as well as hydroxynonenal mercapturic acid metabolites, had increased significantly by 24 hours after flutamide treatment from 4.9 to 15.3-fold in hepatocytes and from 2.6 to 31.0-fold in rat plasma. Induction of mRNA expression levels for Nrf2-regulated genes was evident as well, with heme oxygenase 1, glutathione-S-transferase π1 and NAD(P)H dehydrogenase showing a 3.6-, 4.1-, and 1.9-fold increase in hepatocytes and 5.6-, 7.5-, and 94.1-fold in rat liver. All effects were observed at drug concentrations that did not show overt liver toxicity. Addition of an in situ hydrogen peroxide-generating system to in vitro experiments demonstrated the formation of a reactive di-imine intermediate as the responsible metabolic pathway for the generation of OS. The dataset suggests that hepatic oxidative stress conditions can be mediated via metabolic activation and can be monitored with suitable biomarkers preceding the terminal damage.

DIACYLGLYCEROL ACCUMULATION IMPAIRS SHORT-TERM ACTIVATION OF PHOSPHOLIPASE D BY THYROXINE IN THE LIVER CELLS

Thyroid hormones (TG) are known modulators of signal transduction. Phospholipase D (PLD) is one of the targets of TG in the stimulated cells. Response of cells to the short-term TG action significantly reduces at old age. Taking into account that diacylglycerol (DAG) accumulation induces the resistance of cells to some of regulatory factors in the target cells the aim of the present study was to determine if DAG content increase in hepatocytes impairs the L-thyroxine (L-T4) short-term action. The experiments were performed in either the [14C]palmitic acid- labeled hepatocytes or [14C]oleic acid-pre-labeled liver cells of 3- and 24-month-old rats. To study the short-term L-T4 action on cells the PLD activation was determined. The DAG production and content in hepatocytes significantly increased at old age and in the young cells pre-treated with palmitic acid. The reduction of DAG level in cells by means of DAG-kinase activator, alfa-tocoferol acetate, or long-term L-T4 treatment improved the short-term hormone action. The above data have indicated that DAG play important role in the L-T4 PLD regulation. The cross-talk between classic and non-genomic pathways of TG regulation of lipid metabolism has been determined.

Effectiveness of xenotransplantation of human fetal hepatocytes in spleen of rats with acute liver failure induced by CCL4

Human’s fetal hepatocytes (HFH) were intrasplenic transplanted white non-pedigree rats with acute liver failure (ALF) challenged by single per oral administration of hepatotropic toxin diluted in oil ССl4 at a dose 10 ml/kg (volumetric correlation 1:1) (10 mL/kg body weight as a 1:1 mixture of CCl4 and mineral oil). Transplantation had positive effect on all biochemical blood parameters of the studying animals. Morphologic study showed that reparative-restorative processes were arising in hepatic parenchyma after administration of HFH into splenic pulp of rats with model of ALF on days 14-21. Substantial and main factor in restoration of parenchyma was restoration of micro topographic interrelations in acinus as well as polyploidy of hepatic cells expressed in increase of hepatocytes’ nuclei sizes and hypertrophy of cells themselves. It is an indirect confirmation of engraftment of HFH in liver of rats with model of ALF. ABDUKHAKIM KHADJIBAEV, MAISA URAZMETOVA, AYBEK MADAMINOV, RAVSHAN MAVLYAN-HODJAEV, RAISA AHMEDOVA, FERUZA YUSUPOVA

Differential Regulation of Bone Marrow-Derived Endothelial Progenitor Cells and Endothelial Outgrowth Cells by the Notch Signaling Pathway

Endothelial progenitor cells (EPCs) are heterogeneous populations of cells that participate in vasculogenesis and promote tissue regeneration. However the different roles of EPC populations in vasculogenesis and tissue regeneration, as well as their regulation and mechanisms remain elusive. In the present study, we cultured bone marrow (BM)-derived early EPCs (EEPCs) and endothelial outgrowth cells (EOCs), and investigated their roles in liver regeneration and their regulation by the Notch signaling pathway. We found that Notch signaling exhibited different effects on the proliferation and migration of EEPCs and EOCs. Our results also showed that while EEPCs failed to form vessel-like structures in a three dimensional sprouting model in vitro, EOCs could sprout and form endothelial cords, and this was regulated by the Notch signaling. We further showed that, by using a conditional knockout model of RBP-J (the critical transcription factor mediating Notch signaling), Notch signaling differentially regulates EEPCs and EOCs. In a partial hepatectomy (PHx) model, EEPCs Notch-dependently benefitted liver regeneration with respect to liver function and hepatocyte proliferation and apoptosis. In contrast, EOCs appeared not directly involved in the recovery of liver function and the increase of hepatocytes. These data suggested that the RBP-J-mediated Notch signaling differentially regulated the two types of EPCs, which showed different roles in liver regeneration.

Fructose and NAFLD: metabolic implications and models of induction in rats

The increase in fructose consumption is paralleled by a higher incidence of obesity worldwide. This monosaccharide is linked to metabolic syndrome, being associated with hypertriglyceridemia, hypertension, insulin resistance and diabetes mellitus. It is metabolized principally in the liver, where it can be converted into fatty acids, which are stored in the form of triglycerides leading to NAFLD. Several models of NAFLD use diets high in simple carbohydrates. Thus, this study aimed to describe the major metabolic changes caused by excessive consumption of fructose in humans and animals and to present liver abnormalities resulting from high intakes of fructose in different periods of consumption and experimental designs in Wistar rats. Two groups of rats were fasted for 48 hours and refed for 24 or 48 hours with a diet containing 63% fructose. Another group of rats was fed an diet with 63% fructose for 90 days. Refeeding for 24 hours caused accumulation of large amounts of fat, compromising 100% of the hepatocytes. The amount of liver fat in animals refed for 48 hours decreased, remaining mostly in zone 2 (medium-zonal). In liver plates of Wistar rats fed 63% fructose for 45, 60 and 90 days it's possible to see that there is an increase in hepatocytes with fat accumulation according to the increased time; hepatic steatosis, however, is mild, compromising about 20% of the hepatocytes. Fructose is highly lipogenic, however the induction of chronic models in NAFLD requires long periods of treatment. The acute supply for 24 or 48 hours, fasted rats can cause big changes, liver steatosis with macrovesicular in all lobular zones.

Significance of morphological alteration by portal vein branch ligation in endotoxin‐induced liver injury after partial hepatectomy

Regenerating liver after partial hepatectomy (PH) is susceptible to endotoxin. This study was conducted to investigate how morphological alteration by preoperative portal vein branch ligation (PVL) affects endotoxin-induced liver injury after PH. Male Sprague-Dawley rats were divided into a PVL group undergoing left PVL and into a non-PVL group receiving a sham operation. Seven days later, animals in both groups were subjected to PH (the left lateral, median and caudate lobes). Lipopolysaccharide (LPS) was intravenously administered to both groups 2 days after PH. A significant increase in hepatocyte and sinusoidal endothelial cell proliferation assessed by Ki-67 immunostaining reached a peak at day 2 and 3 after PVL, respectively, in accordance with the changes in plasma interleukin-6 concentrations after PVL. The proliferation response of these cells after PH was observed in both groups, showing a significantly weaker response in the PVL group. The sinusoidal width after PH was significantly reduced in the non-PVL group when compared with that in the PVL group. LPS administration induced a marked elevation of plasma tumour necrosis factor-alpha levels in the non-PVL group compared with the PVL group. PVL before PH significantly attenuated endotoxin-induced functional and structural liver damage with greater hepatic polymorphonuclear leucocyte infiltration and microcirculatory derangement, resulting in an improvement in the 7-day survival rate. Morphological alteration by PVL is of great advantage in preventing the development of endotoxin-induced liver injury in the regeneration process after PH.

Hepatic pharmacokinetics of taurocholate in the normal and cholestatic rat liver

The disposition kinetics of [3H]taurocholate ([3H]TC) in perfused normal and cholestatic rat livers were studied using the multiple indicator dilution technique and several physiologically based pharmacokinetic models. The serum biochemistry levels, the outflow profiles and biliary recovery of [3H]TC were measured in three experimental groups: (i) control; (ii) 17 alpha-ethynylestradiol (EE)-treated (low dose); and (iii) EE-treated (high dose) rats. EE treatment caused cholestasis in a dose-dependent manner. A hepatobiliary TC transport model, which recognizes capillary mixing, active cellular uptake, and active efflux into bile and plasma described the disposition of [3H]TC in the normal and cholestatic livers better than the other pharmacokinetic models. An estimated five- and 18-fold decrease in biliary elimination rate constant, 1.7- and 2.7-fold increase in hepatocyte to plasma efflux rate constant, and 1.8- and 2.8-fold decrease in [3H]TC biliary recovery ratio was found in moderate and severe cholestasis, respectively, relative to normal. There were good correlations between the predicted and observed pharmacokinetic parameters of [3H]TC based on liver pathophysiology (e.g. serum bilirubin level and biliary excretion of [3H]TC). In conclusion, these results show that altered hepatic TC pharmacokinetics in cholestatic rat livers can be correlated with the relevant changes in liver pathophysiology in cholestasis.

Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

Ultra-wideband (UWB) technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR) could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM) cell. Cells were exposed to UWBR for 2 h at a temperature of 23 degrees C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05) dose-dependent response in cell viability in both serum-treated and serum free medium (SFM) -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

Ezetimibe-induced hyperlipidaemia

Ezetimibe is intestinally active cholesterol absorption inhibitor used to reduce low-density lipoprotein-cholesterol levels. This case report describes a novel side effect with this agent: ezetimibe-induced hyperlipidaemia in a patient with statin intolerance and familial combined hyperlipidaemia. Ezetimibe therapy induced an asymptomatic 770% increase in triglycerides (TGs) (3.51-27.1 mmol/l) and a 190% increase in total cholesterol (9.8-18.5 mmol/ 1) secondary to an increase (4.6-25.9 micromol/l; 560%) in hepatic cholesterol (lathosterol) synthesis. This lipid profile resolved 9 months after cessation of ezetimibe therapy. This report shows that ezetimibe may have long-lasting effects in man far exceeding its plasma half-life and that ezetimibe monotherapy can induce a large increase in hepatocyte very-low-density lipoprotein synthesis in rare individuals with a consequent mixed hyperlipidaemia or possibly hypercholesterolaemia depending on the metabolism and clearance of TG-rich lipoproteins.

Increased susceptibility to liver injury after hemorrhagic shock in rats chronically fed ethanol: role of nuclear factor-kappa B, interleukin-6, and granulocyte colony-stimulating factor.

Chronic ethanol use preceding severe trauma and hemorrhagic shock (HS) is associated with an increased incidence of multiorgan failure (MOF) and death; however, the molecular basis for this increased susceptibility is unknown. We previously demonstrated that production of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), mediated by nuclear factor-kappa B (NF-kappa B), each make essential contributions to organ injury and inflammation in a rodent model of controlled HS, and we proposed in this study to examine the hypothesis that the increased susceptibility to MOF after shock/trauma in the setting of chronic ethanol use is due to an exaggerated activation of NF-kappa B and production of these proinflammatory cytokines. We observed increased HS-induced liver injury 4 h after resuscitation in rats fed the ethanol-containing Lieber-DeCarli liquid diet for 8 weeks compared with rats fed the control liquid diet (3-fold increase in serum alanine aminotransferase [ALT], P = 0.008, and 2-fold increase in focal liver necrosis, P = 0.005). The increased liver injury in the ethanol-fed HS rats was accompanied by a 70% increase in liver NF-kappa B activation (P < 0.05), a 3- to 5-fold increase in hepatocyte and Kupffer cell production of IL-6 and G-CSF (P < 0.05 for each), and a 2-fold increase in neutrophil infiltration (P < 0.005) compared with the control diet-fed HS rats. Thus, increased susceptibility to HS-induced liver injury in the setting of chronic ethanol use may be mediated, at least in part, by increased NF-kappa B activation resulting in increased local production of IL-6 and G-CSF and increased infiltration of neutrophils, which can damage liver cells directly and contribute to impaired sinusoidal blood flow.

Mechanism for Peroxisome Proliferator-activated Receptor-α Activator-induced Up-regulation of UCP2 mRNA in Rodent Hepatocytes

Peroxisome proliferator-activated receptor-alpha (PPARalpha)activators, fish oil feeding, or fibrate administration up-regulated mitochondrial uncoupling protein (UCP2) mRNA expression in mouse liver by 5-9-fold, whereas tumor necrosis factor-alpha (TNFalpha) also up-regulated UCP2 in liver. In this study, the mechanisms for PPARalpha activators-induced up-regulation of UCP2 mRNA, related to TNFalpha and reactive oxygen species (ROS), were investigated. PPARalpha activators-induced UCP2 up-regulation in mouse/rat liver tissues was due to their increases in hepatocytes but not in non-parenchymal cells. Addition of PPARalpha activators, WY14,643 or fenofibrate, to cultured hepatocytes up-regulated UCP2 mRNA by 5-10-fold. PPARalpha activators-induced up-regulation of UCP2 mRNA was not due to increased mRNA stability and required cycloheximide-sensitive short term turnover protein(s). However, expression of PPARalpha/retinoid X receptor-alpha and PGC-1 was not rate-limiting for WY14,643-induced UCP2 up-regulation. In primary hepatocytes, an exogenous oxidant, tert-butyl-hydroperoxide (TBHP), which increased ROS production, up-regulated UCP2 mRNA, whereas WY14,643 treatment did not produce detectable ROS under the condition that fibrate markedly up-regulated UCP2. In in vivo studies, PPARalpha activators moderately up-regulated TNFalpha mRNA expression in mouse liver. An anti-oxidant pyrrolidine dithiocarbamate ammonium salt injection completely prevented their TNFalpha mRNA increases but did not prevent most of their UCP2 mRNA increases. These data indicate that PPARalpha activators up-regulate UCP2 expression in hepatocytes through unknown proteins by increased transcription, and neither ROS nor TNFalpha production are the major causes for PPARalpha activators-induced UCP2 up-regulation.

Inhibitory effect of tetrahydroswertianolin on tumor necrosis factor-α-dependent hepatic apoptosis in mice

We investigated the effect of tetrahydroswertianolin (THS), a hepatoprotective agent from Swertia japonica, on tumor necrosis factor-α (TNF-α)-dependent hepatic apoptosis induced by d-galactosamine ( d-GalN) (700 mg/kg, i.p.) and lipopolysaccharide (LPS) (10 μg/kg, i.p.) in mice. Apoptotic symptoms were observed at the initial stage of liver damage. By 5 hr after intoxication, hepatic DNA fragmentation had risen to 2123%, with the value in untreated mice set at 100%, without a significant elevation of serum alanine transaminase (ALT) activity. There was a parallel increase in hepatocytes undergoing chromatin condensation and apoptotic body formation. By 8 hr after intoxication, serum ALT activity had risen to 3707 U/L. Pretreatment with THS (50 mg/kg, p.o.) at 18 and 2 hr before intoxication significantly reduced DNA fragmentation to 821% of that in untreated mice and prevented the emergence of chromatin condensation and apoptotic body formation. A significant and dose-dependent reduction in serum ALT activity at 8 hr also was observed with THS pretreatment. These effects of THS were different from those observed from pretreatment with glycyrrhizin (GCR), which is a clinically used hepatoprotective agent with membrane-stabilizing activity. GCR pretreatment (100 mg/kg, p.o.) did not inhibit hepatic DNA fragmentation (1588% of untreated mice), although this compound significantly protected against serum ALT elevation (1463 U/L). These data suggest that an inhibitory effect on the progression of hepatic apoptosis prior to liver injury may be involved in the hepatoprotective mechanisms of THS, whereas it appears that GCR affects the processes after apoptosis. In a separate experiment, we found that the concentration of serum TNF-α rose to 2016 pg/mL at 1 hr after intoxication of mice with d-GalN and LPS, but this increase was suppressed by THS pretreatment (10, 50, or 200 mg/kg, p.o.) to 716, 454, or 406 pg/mL, respectively. Further study with a reverse transcriptase-polymerase chain reaction method showed that THS blocked TNF-α production at the transcriptional level. Because TNF-α is a critical mediator to elicit apoptosis in this model, the property of suppressing TNF-α production may be of prime importance for THS inhibition of hepatic apoptosis.

The effect of aging and an oxidative stress on peroxide levels and the mitochondrial membrane potential in isolated rat hepatocytes

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on the mitochondrial membrane potential. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor age. The hydrogen peroxide is significantly increased in both aged and adult rat hepatocytes, however, due to the high basal level in the aged cells, it is higher in aged rat cells not subjected to oxidative stress than that elicited by 50 μM adriamycin in young rat hepatocytes. The results suggest that a hydrogen peroxide increase in hepatocytes of aged rats is unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that the effects of the drug may be due to the concomitant high level of both superoxide and hydrogen peroxide.

Hypocalcemia decreases the early and late responses to epidermal growth factor in rat hepatocytes

Extreme variations in extracellular Ca 2+ concentrations ([Ca 2+] e) modify the signaling generated by many hormones and growth factors. However, the influence of physiological changes in [Ca 2+] e on the response to hepatic mitogens remains largely unknown. To study the influence of [Ca 2+] e on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca 2+] e similar to those observed in normocalcemia or hypocalcemia. To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca 2+] e prevailing in vivo. Intracellular Ca 2+ concentrations ([Ca 2+] i) and DNA synthesis were evaluated after increasing doses of EGF. [Ca 2+] e strongly influenced the [Ca 2+] i response to EGF with significantly smaller [Ca 2+] i increases in hepatocytes of normal rats kept in low [Ca 2+] e compared with those kept in normal [Ca 2+] e. In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca 2+] e. In normal [Ca 2+] e, the EGF-induced increases in [Ca 2+] i were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca 2+] e significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca 2+] e. Furthermore, the blunted responses in [Ca 2+] i mobilization and DNA synthesis associated with hypocalcemia could not be over-come by increasing concentrations of EGF nor by normalization of [Ca 2+] e in vitro. These data demonstrate that [Ca 2+] e within the physiological concentration range can strongly influence the hepatocyte response to EGF. At [Ca 2+] e comparable to that of hypocalcemia in vivo, our data point to the appearance of a phenomenon of hepatocellular resistance to the early (increases in [Ca 2+] i) and late (DNA synthesis) cellular responses to EGF.

Metabolic alterations in hepatocytes promoted by the herbicides paraquat, dinoseb and 2,4-D

The cytotoxic effects of the herbicides paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride), dinoseb (2-sec-butyl-4,6-dinitrophenol) and 2,4-D (2,4-dichlorophenoxyacetic acid) on freshly isolated rat hepatocytes were investigated. Paraquat and 2,4-D (1-10 mM) caused a dose and time dependent cell death accompanied by depletion of intracellular glutathione (GSH) and mirroring increase of oxidized glutathione (GSSG). Dinoseb, the most effective cytotoxic compound under study (used in concentrations 1000 fold lower than paraquat and 2,4-D), exhibited moderate effects upon the level of GSH and GSSG. These limited effects are at variance with significant effects upon the adenine and pyridine nucleotide contents. ATP and NADH levels are rapidly depleted by herbicide metabolism. This depletion is observed in the millimolar range for paraquat and 2,4-D and in the micromolar range for dinoseb. 2,4-D completely depletes cellular ATP, with subsequent cell death, as detected by LDH leakage. Paraquat rapidly depletes NADH, according to the redox cycling of the herbicide metabolism. The most effective compound is dinoseb since it exerts similar effects as described for paraquat and 2,4-D at concentrations 1000 fold lower. Simultaneously with NADH and ATP depletion, the levels of ADP, AMP and NAD+ increase in hepatocytes incubated in the presence of the herbicides. In contrast to NADH, the time course and extent of ATP depletion and fall in energy charge correlate reasonably with the time of onset and rate of cell death. It is concluded that the herbicides, paraquat and 2,4-D are hepatotoxic and initiate the process of cell death by decreasing cellular GSH.(ABSTRACT TRUNCATED AT 250 WORDS)