Rate Of Hepatocytes Research Articles

Perilipin 5 protects the mitochondrial oxidative functions and improves the alcoholic liver injury in mice.

Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important protective factor against hepatic lipotoxicity induced by excessive lipolysis, but its role and molecular mechanism in alcoholic liver disease (ALD) are not fully elucidated. The optimized National Institute on Alcohol Abuse and Alcoholism model was used to construct ALD model mice. Automatic biochemical analyser was used for Biochemical Parameters. The primary hepatocytes and Plin5-overexpressed HepG2 cells (including full-length Plin5 and Plin5 deleting 444-464 aa) were used for invitro experiment. Haematoxylin and Eosin staining, Oil Red O staining, Bodipy 493/503 staining, Periodic Acid-Schiff staining, immunohistochemistry and JC-1 staining were used to evaluate cell morphology, lipids, glycogen, inflammation and membrane potential. Commercially kits are used to detect glycolipid metabolites, such as triglycerides, glycogen, glucose, reactive oxygen species, lactic acids, ketone bodies. Fluorescently labelled deoxyglucose, NBDG, was used for glucose intake. An XF96 extracellular flux analyser was used to determinate oxygen consumption rate in hepatocytes. The morphological and structural damage of mitochondria was evaluated by electron microscopy. Classical ultracentrifugation is used to separate the subcellular organelles of tissues and cells. Immunoblotting and qPCR were used to detect changes in mRNA and protein levels of related genes. Our results showed that the expression of Plin5 in mouse livers was enhanced by alcohol intake, and Plin5 deficiency aggravated the alcohol-induced liver injury. To clarify the mechanism, we found that Plin5 deficiency significantly elevated the hepatic NADH levels and ketone body production in the alcohol-treated mice. As NADH elevation could promote the reduction of pyruvate into lactate and then inhibit the gluconeogenesis, alcohol-treated Plin5-deficient mice exhibited more lactate production and severer hypoglycemia. These results implied that Plin5 deficiency impaired the mitochondrial oxidative functions in the presence of alcohol. In addition, we demonstrated that Plin5 could be recruited onto mitochondria by alcohol, while Plin5 without mitochondrial targeting sequences lost its mitochondrial protection functions. Collectively, this study demonstrated that the mitochondrial Plin5 could protect the alcohol-induced mitochondrial injury, which provides an important new insight on the roles of Plin5 in highly oxidative tissues.

Multi-walled carbon nanotube induced liver injuries possibly by promoting endoplasmic reticulum stress in Cyprinus carpio

The mass production and discharge of carbon nanotubes (CNTs) to the water environment are of great concern since they threaten the health of organisms in the aquatic ecosystem. CNTs induce multi-organ injuries in fish, but limited literature is available regarding the mechanisms involved. In the present study, juvenile common carp (Cyprinus carpio) were exposed to multi-walled carbon nanotubes (MWCNTs) (0.25 mg L−1 and 2.5 mg L−1) for four weeks. MWCNTs caused dose-dependent alterations in the pathological morphology of liver tissues. Ultrastructural changes manifested as nuclear deformation, chromatin condensation, endoplasmic reticulum (ER) disorderly arrangement, mitochondria vacuolation, and mitochondrial membrane destruction. TUNEL analysis indicated that the apoptosis rate in hepatocytes markedly increased upon exposure to MWCNTs. Moreover, the apoptosis was confirmed by significant upregulation of mRNA levels of apoptosis-related genes (Bcl-2, XBP1, Bax, and caspase3) in MWCNTs-exposure groups, except for Bcl-2 expression which was not significantly changed in HSC groups (2.5 mg L−1 MWCNTs). Furthermore, real-time PCR assay indicated the increased expression of ER stress (ERS) marker genes (GRP78, PERK, and eIF2α) in the exposure groups compared to the control groups, suggesting that the PERK/eIF2α signaling pathway involved in the injuries of the liver tissue. Overall, the results above indicate that MWCNTs induce ERS by activating the PERK/eIF2α pathway in the liver of common carp, and resulted in the initiation of apoptosis procedure.

CAN THYROID PROFILE PREDICT THE IMPENDING DANGER OF DECOMPENSATION IN LIVER CIRRHOSIS?

Background: Chronic liver disease is one of the world's leading causes of mortality and morbidity. Thyroid and liver are interdependent in their functioning, Thyroid gland regulates basic metabolic rate in hepatocytes and liver being the site of thyroid hormone metabolism, modulates hormone levels. Assessment of thyroid levels in cirrhosis may prove useful to identify severity of disease and probability of complication rates. In this context we conducted a study to signify the association between thyroid hormones and severity of hepatic disorder in cirrhosis through usage of Child Pugh score. 100 patients a Method: ged 18 years and above, with liver cirrhosis were evaluated in our institute. Thyroid prole and other relevant investigations were carried out to assess severity of liver cirrhosis, correlated with Child Pugh scoring. Results: 74% of the patients in the study were in euthyroid state. Total and free T3, T4 levels were signicantly low and inversely correlated with severity of liver cirrhosis. Most of the patients had normal T4 and TSH levels. Among all, free T3 was found to be the most sensitive marker to assess severity of cirrhosis. Thyroid dysfunction is oft Conclusion: en observed in liver cirrhosis which calls for thyroid prole tests to be conducted with a potential role as prognostic marker.

Vitamin E Can Ameliorate Oxidative Damage of Ovine Hepatocytes In Vitro by Regulating Genes Expression Associated with Apoptosis and Pyroptosis, but Not Ferroptosis.

(1) Background: the current research was conducted to investigate the potential non-antioxidant roles of vitamin E in the protection of hepatocysts from oxidative damage. (2) Methods: primary sheep hepatocytes were cultured and exposed to 200, 400, 600, or 800 μmol/L hydrogen peroxide, while their viability was assessed using a CCK-8 kit. Then, cells were treated with 400 μmol/L hydrogen peroxide following a pretreatment with 50, 100, 200, 400, and 800 μmol/L vitamin E and their intracellular ROS levels were determined by means of the DCF-DA assay. RNA-seq, verified by qRT-PCR, was conducted thereafter: non-treated control (C1); cells treated with 400 μmol/L hydrogen peroxide (C2); and C2 plus a pretreatment with 100 μmol/L vitamin E (T1). (3) Results: the 200–800 μmol/L hydrogen peroxide caused significant cell death, while 50, 100, and 200 μmol/L vitamin E pretreatment significantly improved the survival rate of hepatocytes. ROS content in the cells pretreated with vitamin E was significantly lower than that in the control group and hydrogen-peroxide-treated group, especially in those pretreated with 100 μmol/L vitamin E. The differentially expressed genes (DEGs) concerning cell death involved in apoptosis (RIPK1, TLR7, CASP8, and CASP8AP2), pyroptosis (NLRP3, IL-1β, and IRAK2), and ferroptosis (TFRC and PTGS2). The abundances of IL-1β, IRAK2, NLRP3, CASP8, CASP8AP2, RIPK1, and TLR7 were significantly increased in the C1 group and decreased in T1 group, while TFRC and PTGS2 were increased in T1 group. (4) Conclusions: oxidative stress induced by hydrogen peroxide caused cellular damage and death in sheep hepatocytes. Pretreatment with vitamin E effectively reduced intracellular ROS levels and protected the hepatocytes from cell death by regulating gene expression associated with apoptosis (RIPK1, TLR7, CASP8, and CASP8AP2) and pyroptosis (NLRP3, IL-1β, and IRAK2), but not ferroptosis (TFRC and PTGS2).

13C-METHACETIN BREATHE TEST IN EARLY DIAGNOSTICS OF NON-ALCOHOLIC FATTY LIVER DISEASE

The aim: Of the study was to evaluate the early utility changes of the 13C methacetin breath test parameters in patients with NAFLD. Materials and methods: There were included 50 subjects in the study, among them 35 patients had steatosis and 15 patients had steatohepatitis, including 35 (70.0%) male subjects and 15 (30.0%) female subjects. The control group included 17 apparently healthy volunteers, among them 10 (58.8%) subjects were male and 7 (41.2%) subjects were female. Results: It was determined that metabolism kinetics in case of liver steatosis was significant decreased more than 30% compared to the control group (p = 0.0001) and in case of steatohepatitis that decrease was more than 65% (p = 0.00001) compared with normal values. It resulted in less cumulative dose accumulation in steatosis (p = 0.00001) and steatohepatitis (p = 0.00001). Among the reasons for the decrease in the kinetics of metabolism in steatosis, there were insufficient response of hepatocytes on 10 minutes (<10 dose/h,%) in 40% of cases and reduction of metabolism rate amplitude at 20-40 minutes following methacetin administration. Conclusions: The results of 13C-methacetin breath test demonstrate that in patients with NAFLD there is a gradual slowing of metabolism rate in hepatocytes, which leads to a decrease in cumulative dose.

A combination of ultrasound-targeted microbubble destruction with transplantation of bone marrow mesenchymal stem cells promotes recovery of acute liver injury

BackgroundBone marrow mesenchymal stem cells (BMSCs) can provide an additional source of therapeutic stem cells for regeneration of liver cells during acute liver injury (ALI). However, the insufficient hepatic homing by the transplanted BMSCs limits their applications. Ultrasound-targeted microbubble destruction (UTMD) has been reported to promote the homing of transplanted stem cells into the ischemic myocardium. In this study, we investigated whether UTMD promotes the hepatic homing of BMSCs in ALI rats and evaluated the therapeutic effect.MethodsBMSCs were isolated from the femurs and tibias of Sprague-Dawley (SD) rats. The isolated BMSCs were stably transfected with a lentivirus expressing enhanced green fluorescent protein (EGFP) that can be visualized and quantified in vivo after transplantation. Both tumor necrosis factor α (TNF-α) and stromal cell-derived factor 1 (SDF-1) were used to verify the appropriate ultrasound parameters. The ALI rats were divided into four groups: control, BMSCs, UTMD, and UTMD + BMSCs. The protein and mRNA expression levels of SDF-1, intercellular cell adhesion molecule (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), hepatocyte growth factor (HGF), and monocyte chemotactic protein 1 (MCP-1) in the exposed livers were analyzed at 48 h after treatment. ALI recovery was determined by serum biochemical parameters and histology.ResultsThe isolated rat BMSCs demonstrated a good proliferation potential that was both osteogenic and adipogenic in differentiation and expressed cluster of differentiation (CD) 29 and CD90, but not CD45 or CD11b/c. After BMSC and/or UTMD treatment, the number of GFP-labeled BMSCs in the UTMD + BMSCs group was significantly higher than that of the BMSCs group (9.8 ± 2.3 vs. 5.2 ± 1.1/per high-power field). Furthermore, the expression of GFP mRNA was performed for evaluation of the homing rate of BMSCs in injury sites as well. In addition, the expression levels of SDF-1, ICAM-1, VCAM-1, HGF, and MCP-1 were higher (p < 0.01) in UTMD+BMSCs group. The serum levels of biomarkers were significantly lower in the UTMD + BMSCs group, and the apoptotic rate of hepatocytes in the UTMD + BMSCs group was markedly lower than that of the BMSCs group (all p < 0.05). The hepatic pathology was significantly alleviated in the UTMD + BMSCs group.ConclusionsUTMD treatment efficiently induced a favorable microenvironment for cell engraftment, resulting in improvement of hepatic homing of BMSCs, which was probably mediated through upregulation of the expression of adhesion molecules and cytokines. UTMD treatment appeared to be an effective and noninvasive approach to achieve better efficacy of BMSC-based therapy for repairing a severely injured liver.

Hepatic ATF6 Increases Fatty Acid Oxidation to Attenuate Hepatic Steatosis in Mice Through Peroxisome Proliferator-Activated Receptor α.

The endoplasmic reticulum quality control protein activating transcription factor 6 (ATF6) has emerged as a novel metabolic regulator. Here, we show that adenovirus-mediated overexpression of the dominant-negative form of ATF6 (dnATF6) increases susceptibility to develop hepatic steatosis in diet-induced insulin-resistant mice and fasted mice. Overexpression of dnATF6 or small interfering RNA-mediated knockdown of ATF6 decreases the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα)/retinoid X receptor complex, and inhibits oxygen consumption rates in hepatocytes, possibly through inhibition of the binding of PPARα to the promoter of its target gene. Intriguingly, ATF6 physically interacts with PPARα, enhances the transcriptional activity of PPARα, and triggers activation of PPARα downstream targets, such as CPT1α and MCAD, in hepatocytes. Furthermore, hepatic overexpression of the active form of ATF6 promotes hepatic fatty acid oxidation and protects against hepatic steatosis in diet-induced insulin-resistant mice. These data delineate the mechanism by which ATF6 controls the activity of PPARα and hepatic mitochondria fatty acid oxidation. Therefore, strategies to activate ATF6 could be used as an alternative avenue to improve liver function and treat hepatic steatosis in obesity.

Towards an optimized inhibition of liver stage development assay (ILSDA) for Plasmodium falciparum

BackgroundExperimental vaccines targeting Plasmodium falciparum have had some success in recent years. These vaccines use attenuated parasites, recombinant sporozoite proteins, or DNA and virus combinations to induce cell-mediated immune responses and/or antibodies targeting sporozoite surface proteins. To capitalize on the success of these vaccines and understand the mechanisms by which these vaccines function, it is important to develop assays that measure correlates of protection in volunteers. The inhibition of liver stage development assay (ILSDA) tests antibodies for the ability to block sporozoite development in hepatocytes. As such the ILSDA is an excellent candidate assay to identify correlates of humoral protection, particularly against the liver stage of malaria infection. In addition, the ILSDA can be used as a tool to evaluate novel sporozoite antigens for future vaccine development. Historically the ILSDA has suffered from low sporozoite infection rates, absence of standardized reagents, and the subjectivity associated with the traditional primary outcome measures, which depend on microscopy of stained hepatocyte cultures. This study worked to significantly improve sporozoite infection rates in hepatocytes, modify key steps in the assay protocol to reduce experimental variability, and demonstrate the utility of the ILSDA in testing antibodies targeting the circumsporozoite protein.MethodsCryopreserved primary human hepatocytes, Plasmodium falciparum sporozoites, and circumsporozoite antibodies were used to optimize the ILSDA.ResultsInoculation of cryopreserved primary human hepatocytes with Plasmodium falciparum sporozoites improved liver stage development in the ILSDA compared to HCO4 cells. In the ILSDA, circumsporozoite antibodies suppressed liver stage development in cryopreserved primary human hepatocytes in a concentration-dependent manner. Antibody-mediated suppression of parasite development in the ILSDA at a 96-hour endpoint was more robust than the 24-hour endpoint.ConclusionsILSDA performance is improved by the use of cryopreserved primary human hepatocytes, expediting interactions between sporozoites and hepatocytes, and extending the assay endpoint.

Effects of Aflatoxin on Liver and Protective Effectiveness of Esterified Glucomannan in Merino Rams

The effects of total aflatoxin (AF) given orally on liver in Merino rams were studied. In addition, this study was conducted in order to evaluate the efficacy of an esterified glucomannan (EG) for protection against aflatoxicosis. One-year-old 32 Merino rams were divided into four equal groups. The control group (C) was fed with the commercial feed. The AF group was fed with commercial feed plus 250 μg/day of total AF. The EG group was fed with commercial feed plus 2 g/day of EG. The AF + EG group was fed with commercial feed plus 250 μg/day of total AF and 2 g/day of EG. After feeding period, tissue samples were taken from the liver in order to perform histological analyses. Vacuolar degeneration with small and large droplets and hydropic degeneration in hepatocytes were observed in the AF group. The ceroid pigmentation was observed in macrophages in groups or one by one. It was observed that the fat rate in hepatocytes was 2.6% in the C group, 35.5% in the AF group, 2.9% in the EG group, and 9.6% in the AF + EG group. In conclusion, the adverse effects caused by aflatoxicosis on the liver could be ameliorated by adding EG to the ration.

An updated method for the isolation and culture of primary calf hepatocytes

Primary hepatocytes are commonly used during in vitro studies, but care must be taken with isolation and culture of the cells to ensure their viability. In this study, hepatocytes were isolated from the liver (caudate process) of a newborn calf by the collagenase perfusion and digestion method. The trypan blue exclusion method was used to determine total cell number and the survival rate of hepatocytes, while hepatocyte function was assessed by measuring lactate dehydrogenase, albumin and urea in culture medium supernatants at 24, 48, 72, 96, 120, 144 and 168 h. Results showed that the number of viable cells/g of liver (wet weight) averaged 1.12×10(7) cells/g, with an average hepatocyte viability of 85.7% (range 83-92%). After 48 h of culture, the hepatocytes solidly adhered to the well culture plate and were spread in an epithelioid shape, with clear cell boundaries between the cells and biliary ductule-like structures formed which persisted for up to 10 days. Hepatocyte function was optimal at 72 h after isolation and culture. This simple and economical procedure for the isolation and culture of viable cells may be useful for in vitro bovine hepatocyte studies.

Effect of Apolipoprotein A-I Complex with Tetrahydrocortisone on Protein Biosynthesis and Glucose Absorption by Rat Hepatocytes

We studied the effect of apolipoprotein A-I-tetrahydrocortisone complex on (14)C glucose absorption and lactate accumulation and on the rate of protein biosynthesis in isolated rat hepatocytes. The presence of apolipoprotein A-I-tetrahydrocortisone complex in the incubation medium increased absorption of labeled glucose by hepatocytes by 52%, while lactate content in the conditioning medium increased 4-fold. The rate of protein biosynthesis increased by 80% in comparison with control cells. It is hypothesized that the increase in protein biosynthesis rate in hepatocytes under the effect of apolipoprotein A-I-tetrahydrocortisone complex is due to stimulation of energy metabolism, specifically, of its glycolytic component.

Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q

Plasma lipoprotein(a) (Lp[a]) level is an independent risk factor of cardiovascular disease that is under strong genetic control. We conducted a genome-wide association study of plasma Lp(a) in 386 members of a founder population that adheres to a communal lifestyle, proscribes cigarette smoking, and prepares and eats meals communally. We identified associations with 77 single nucleotide polymorphisms (SNPs) spanning 12.5 Mb on chromosome 6q26-q27 that met criteria for genome-wide significance (P <or= 1.3 x 10(-7)) and were within or flanking nine genes, including LPA. We show that variation in at least six genes in addition to LPA are significantly associated with Lp(a) levels independent of each other and of the kringle IV repeat polymorphism in the LPA gene. One novel SNP in intron 37 of the LPA gene was also associated with Lp(a) levels and carotid artery disease number in unrelated Caucasians (P = 7.3 x 10(-12) and 0.024, respectively), also independent of kringle IV number. This study suggests a complex genetic architecture of Lp(a) levels that may involve multiple loci on chromosome 6q26-q27.

Regulating effect of apoptotic rate of hepatocyte on course of hepatic regeneration

Objective To investigate the role of different apoptotic rate induced by TGF-β1 in different stages of hepatic regeneration after partial hepatectomy(PH).Methods Rat model of 70% PH was established.Primary hepatocytes were isolated by in situ perfusion with collagenase solution from normal rats and those 1 d,3 d,5 d,7 d after PH.Flow cytometry was used to detect apoptotic rate in each group.The apoptosis of primary hepatocytes induced by TGF-β1(5 μg/L)was determined by staining with fluorescent dyes of DNA-binding Hoechst33258 and observed under fluorescent microscope.The effect of TGF-β1 on Bcl-2 expression in hepatoeytes was detected with immunocytochemistry.Results The viability of primary hepatocytes was 92%.The apoptotic rate of normal hepatocyte was 18.89%±3.14%,the apoptotic rate in 1 d was 11.85%±2.51%.After that it began to decrease,it was the lowest on the 3rd d.Then it began to increase gradually,it was 28.82%±5.53% on day 7 after PH.The apoptotic rate of normal hepatocytes induced by TGF-β1 was 58.13%±6.63%,which was higher than that in control groups(17.81%±3.19%,P＜0.05).However,the apoptotic rate of isolated primary hepatocytes had no difference between the groups induced and not induced by TGF-β1 1,3,5 d after PH.Bcl-2 expression in hepatocytes isolated in the early stage after PH was gradually increased and higher than that of normal group.The Bcl-2 expression was decreased significantly after being treated with TGF-β1.Conclusion TGF-β1 induced apoptotic rate has a marked difference in different stages of hepatic regeneration and it possesses an important regulating role for initiation and termination of hepatic regeneration. Key words: Liver regeneration; Apoptosis; TGF-β1

Protective effects of a hibernation-inducer on hepatocyte injury induced by hypothermic preservation

For hepatocyte-based cell therapy to be realistic, the method chosen for cryopreservation or hypothermic preservation is critical. The aim of the present study was to clarify whether D-Ala2-Leu5-enkephalin (DADLE), a hibernation inducer, has protective effects on hepatocytes with regard to hypothermic preservation injury. A suspension of rat hepatocytes was stored at 4 degrees C for 24 h with or without DADLE. Their viability was measured by the trypan blue dye exclusion method, and alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels in the preservation solution were measured. After 24 h of cold storage, viable hepatocytes were cultured at 37 degrees C for another 24 h. Then albumin production and lidocaine clearance were measured. DADLE significantly improved the survival rate of hepatocytes. The levels of ALT and LDH in the preservation solution with DADLE were significantly lower than those in the preservation solution without DADLE. The treated viable hepatocytes maintained both albumin synthesis and lidocaine clearance. DADLE appears to have protective effects on hepatocytes with regard to hypothermic preservation injury in vitro. This hibernation-inducer is useful in prolonged hypothermic preservation for hepatocyte-based therapy.

Effects of total replacement of fish oil by vegetable oils on n-3 and n-6 polyunsaturated fatty acid desaturation and elongation in sharpsnout seabream (Diplodus puntazzo) hepatocytes and enterocytes

The main aim of this work was to determine the impact of total dietary fish oil replacement by vegetable oils on the fatty acid metabolism of sharpsnout seabream hepatocytes and digestive tract enterocytes. Three isonitrogenous (48% crude protein) and isoenergetic (23 MJ/kg) experimental diets were formulated using three different lipid sources: fish oil (FO), rich in n-3 HUFAs; soybean oil (SO), rich in linoleic acid (LA), and linseed oil (LO), especially rich in linolenic acid (LNA). These diets were fed, three times a day to apparent satiation, to triplicate groups of 30 sharpsnout seabream (with an initial average weight of 14.9 g) for nine months at 23.5±1.2 °C. Inclusion of vegetable oils in sharpsnout seabream diet did not have any quantitative nutritional effects on desaturation/elongation of [1-14C] LNA and [1-14C] LA in isolated hepatocytes and total digestive tract enterocytes. Most of the radioactivity found in tissue lipid extracts (94% and 86% for hepatocytes and enterocytes, respectively) using silver nitrate thin-layer chromatography was recovered as [1-14C] C18 PUFA, clearly showing the lack of any significant desaturase/elongase activity in these cells. Only the high levels of HUFA in fish tissues pointed to the existence of some kind of regulatory mechanism, presumably based on HUFA bioaccumulation and C18 PUFA oxidation. Moreover, direct measurements of β-oxidation rates yielded very low values in all cases, the only significant difference being a higher oxidation rate in hepatocytes from fish fed LO versus FO diet.

State of liver mitochondrial respiratory chain in rats with experimental toxic hepatitis

Polarographical determination of oxygen concentration has shown that in rats with experimental hepatitis induced by combined ethanol and CCl4 administration for 4 weeks, the functioning of the hepatocyte mitochondrial respiratory chain is impaired. Development of liver pathology was accompanied by adipose dystrophy, fibrosis, and an increase of triglycerides and lipid peroxidation products in the liver tissue. The endogenous respiration rate in hepatocytes isolated from the pathologically altered liver was 34% higher than in the control. Cell respiration was not stimulated by the addition of the substrates malate and pyruvate with digitonine. An uncoupler of oxidation and phosphorylation, 2,4-dinitrophenol, increased the hepatocyte oxygen consumption rate by 37%, while addition of the inhibitor of the I complex, rotenone, decreased cell respiration in pathologically altered hepatocytes by 27%. The states 3 (V3) and 4 (V4) of mitochondrial respiration with malate + glutamate as substrates were found to be higher by 70% and 56%, respectively, as compared with the control level. When using malate + glutamate or succinate as substrates, V3 and Vd (dinitrophenol respiration) in the toxic hepatitis hepatocyte mitochondria did not differ from the control, which indicates no uncoupling occurred of the oxidation and phosphorylation processes. Cytochrome c oxidase activity was elevated (+80%) as compared with the control. Administration of the hypolipidemic agent symvastatin simultaneously with ethanol and CCl4 resulted in a reduction of the degree of liver adipose dystrophy, prevented activation of lipid peroxidation, and decreased the hepatocyte endogenous respiration rate. Addition of malate + pyruvate, dinitrophenol or rotenone produced oxygen consumption changes similar to those in the control. However, in mitochondria isolated from the pathologically altered liver, symvastatin induced an uncoupling effect on the respiratory chain in the presence of the substrates malate + glutamate, but did not change the cytochrome c oxidase activity. We suggest that functioning of the NCCR complex in the hepatocyte mitochondria of animals with experimental toxic hepatitis is impaired, which leads to an intensive superoxide anion production at the level of this complex. Under these conditions, the defect of the NADH-coenzyme Q-oxidoreductase is compensated by functioning of other complexes of the respiratory chain (SCCR, coenzyme Q-cytochrome c-reductase, cytochrome c oxidase, and ATP-synthase activities).

Comparison of Intrinsic Clearance in Liver Microsomes and Hepatocytes from Rats and Humans: Evaluation of Free Fraction and Uptake in Hepatocytes

Apparent intrinsic clearance (CL(int,app)) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL(int,app) values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL(int,app). This could partly be attributed to the differences in the free fraction (fu). The fu in hepatocyte incubations (fu,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (fu,buffer) but also by the rate constants of uptake (k(up)) and metabolism (k(met)). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [k(up)/(k(met) + k(up))]/[1 + (C(hep)/C(buffer)) x (V(hep)/V(buffer))], where the C(hep), C(buffer), V(hep), and V(buffer) represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the fu,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.

A promoter polymorphism in cholesterol 7α-hydroxylase interacts with apolipoprotein E genotype in the LDL-lowering response to atorvastatin

Bile-acid biosynthesis is a key determinant of intracellular cholesterol and, in turn, cholesterol synthesis rate in hepatocytes. This suggests that variation in the cholesterol 7α-hydroxylase gene ( CYP7A1), a key enzyme in bile-acid biosynthesis, may influence the statin response. To test this hypothesis, a promoter polymorphism (A–204C) in CYP7A1 was examined in 324 hypercholesterolemic patients treated with atorvastatin 10 mg. The variant C allele was significantly and independently associated with poor LDL cholesterol reductions; −39% in wild type allele homozygotes, −37% in variant allele heterozygotes, and −34% in variant allele homozygotes ( p < 0.0001 for trend). Differences were more striking in men, and were enhanced by the coexistence of common variants of apolipoprotein E gene ( APOE), ɛ2 or ɛ4. In subjects having wild type alleles at both loci, the mean reduction in LDL cholesterol was −40%, while the value in subjects having two CYP7A1 variant alleles and at least one variant APOE allele was −31% ( p < 0.0001). Combination analysis of these two loci more accurately predicted the achievement of goal LDL cholesterol, than did both single locus analysis. We concluded that the CYP7A1 A–204C promoter variant was associated with poor response to atorvastatin, which were additively enhanced by common variants in another locus, APOE.

Cadmium uptake kinetics in rat hepatocytes: correction for albumin binding.

The relationship between cytotoxicity and kinetics of cadmium uptake was investigated in primary rat hepatocyte cultures. Primary rat hepatocytes were exposed to cadmium concentrations ranging from 1.0 to 80 micro M in albumin-free buffer or 32 to 8,000 microM in buffer containing physiological concentrations of bovine serum albumin (600 micro M) for 1 h, and cellular toxicity was observed at 23 h postexposure. Hepatocytes exposed to cadmium in the presence of albumin appeared less sensitive to cadmium toxicity when compared to cells exposed in the absence of albumin. The experimentally derived 23-h postexposure EC(50)s for hepatocytes exposed to cadmium in both presence and absence of albumin was 65.5 +/- 2.4 and 14.3 +/- 3.9 microM, respectively. A Scatchard plot of cadmium binding to albumin suggested two high-affinity binding sites. The observed uptake of cadmium by hepatocytes in the absence and presence of albumin consisted of a composite fast uptake rate and cell membrane association (Component I), and a slow, sustained uptake rate (Component II). Cadmium uptake rates in hepatocytes, based on total medium cadmium concentrations, indicated that Component II uptake rates were four times faster under albumin-free exposure conditions. However, when uptake rates were evaluated, based on the calculated equilibrium concentration of free cadmium in the exposure buffer, uptake rates in hepatocytes exposed in the presence of albumin were two times as fast. This faster cadmium uptake in the presence of albumin may result from diffusion-limited, nonequilibrium conditions occurring at the cell surface.

Lack of association between CYP2A5 induction and apoptosis in mouse primary hepatocytes

Upregulation of mouse hepatic cytochrome P450 2A5 (CYP2A5) is a process closely associated with hepatocellular damage and formation of liver tumours. 2-Aminopurine, a protein kinase inhibitor modulating cell cycle control, was recently shown to strongly induce CYP2A5 in mouse hepatocytes. The objective of this study was to determine the association between CYP2A5 induction and apoptosis in mouse primary hepatocytes. Five well-characterised CYP2A5 inducers were tested for their ability to affect apoptosis rate, determined by immunohistochemical in situ 3′-end-labelling technique, in a primary mouse hepatocyte model. Transforming growth factor beta (TGFβ) was used as a positive (proapoptotic) control. Phenobarbital, pyrazole and the mitogen-activated protein kinase inhibitor PD98059 did not significantly affect apoptosis rate in hepatocytes. Norcocaine induced apoptosis at 6 hr (1.8-fold) and 2-aminopurine 12 hr (1.4-fold) after treatment, which is considerably earlier than peaks in the amount of CYP2A5 mRNA. TGFβ reduced CYP2A5 marker activity, coumarin 7-hydroxylase by 74%. These results indicate that in a primary hepatocyte model (a) there is no systematic correlation between apoptosis and CYP2A5 induction; (b) phenobarbital does not significantly affect the rate of apoptosis; and (c) the induction of apoptosis caused by the chemicals tested occurs considerable earlier than elevation of CYP2A5 expression. Thus, no causal link appears to exist between induction of CYP2A5 and apoptotic rate.