Effect of hemosorption on the ultrastructure of hepatocytes in toxic liver damage

Extracorporeal perfusion of toxic blood via carbonic sorbents is an effective method for correcting severe disturbances of hemostasis. Ultrastructural alterations in hepatic cells were studied in experimental toxic liver injury before and after hemosorption. It was established that after hemosorption the processes of intracellular regeneration were significantly activated in the liver parenchyma. The number of crysts in the mitochondria increased as did the electronic density of the matrix. At the same time the number of lysosomes rose as well. However, in persistent unresolved cholestasis, destructive alterations in the hepatic tissue progressed despite the performance of hemosorption.     

Stabilizing action of alpha-tocopherol in postischemic damage to the hydroxylating system of the endoplasmic reticulum membranes of the rat liver

Administration of alpha-tocopherol (50 mg/kg) as an oily emulsion into male rat stomach every 12 hours 2 days before and 3 days after local disturbance in liver circulation prevents postischemia (72 hours after resumption of circulation) repression of amidopyrine-N-demethylation, aniline-hydroxylation, NADP H-neotetrazolium reductase, NADP H oxidase, a decrease in the cytochrome P-450 level, as well as intensification of ascorbate-dependent lipid peroxidation of hepatocyte endoplasmic reticulum membranes. The protective effect of alpha-tocopherol is suggested to be due to its antioxidative and membrane-stabilizing properties.     

Changes in the ultrastructure of rat hepatocytes at different periods after hepatic ischemia

The ultrastructure of rat hepatocytes was investigated at once at 30 min liver ischemia and at different periods after it. In 24 h of recirculation the processes of the recovery of hepatocyte ultrastructure dominated in the liver parenchyma, but even in 14 days of recirculation no complete reconstruction of hepatocyte ultrastructure was observed.     

Changes in the ultrastructure of human hepatocytes in acute phlegmonous cholecystitis

At an acute phlegmonous cholecystitis in the human being, certain changes in ultrastructure of organelles of hepatocytes and slight rearrangements in their surface are revealed. Destruction of microfilaments, hypertrophy and fragmentation are observed in Golgi complex (GC) and in the granular endoplasmic reticulum. The structure of the GC and of the endoplasmic reticulum demonstrates their active functional state, that is evidently manifested as a defensive reaction of the organism to the lesion.     

Necrosis and apoptosis: sequence of liver damage following reperfusion after 60 min ischemia in rats

This study evaluated the time-dependent modes of cell death that occur during the course of reperfusion after 60 min ischemia. The serum ALT level increased immediately after reperfusion, peaked at 6 h and then declined gradually thereafter. This was supported by the H&E staining of the liver tissues taken at 2 h reperfusion, which revealed massive peri-portal necrosis. The succinate driven mitochondrial-swelling rate, release of cytochrome c into the cytoplasm, increase in caspase-3 activity and TUNEL stained tissue were measured to determine the changes in the biochemical markers of apoptosis. The biochemical markers of apoptosis increased by 2 h of reperfusion, peaked at 6 h and remained elevated throughout the 24 h reperfusion period. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), inhibited MPT opening, the release of cytochrome c and caspase-3 activation. This indicates that necrotic death occurs particularly in the peri-portal region in the initial period of reperfusion, and delayed apoptotic death occurs primarily in the peri-central region in the liver tissues undergoing I/R.     

Prolonged lidocaine metabolizing activity of primary hepatocytes with spheroid culture using polyurethane foam as a culture substratum

Primary rat hepatocytes formed spheroids in the pores of polyurethane foam (PUF) used as a culture substratum. The hepatocytes in monolayer and spheroid stationary culture converted lidocaine to monoethylglycinexylidide (MEGX) which was N-deethylation of lidocaine. The metabolic activity of the hepatocytes/spheroid stationary culture system was 1.5∼2.0-fold higher than that of monolayer culture for 10 days. The activity of albumin production and cell survival of hepatocytes in monolayer and spheroid cultures decrease due to lidocaine treatment dependend on the lidocaine concentration, but the activity and cell survival in PUF/spheroid stationary culture were maintained at a higher level than that in monolayer culture under the lidocaine treatment. We developed a device for an in vitro liver model, drug metabolism simulator (DMS), using a PUF/spheroid packed-bed module including 4.00 ± 0.68 × 10⁷ hepatocytes and analyzed pharmacokinetics of lidocaine in a one-compartment model. Lidocaine clearance and extraction ratio of hepatocytes in the DMS corresponded to 1.354 ± 0.318 ml/min/g-liver and 0.677 ± 0.0159/g-liver, respectively (N=4). These values were comparable with in vivo values, 1.930 ml/min g-liver and 0.965/g-liver reported by Nyberg (1977). Consequently, PUF/spheroid culture maintained high lidocaine metabolizing activity over a long term and seems to provide a promising culture system as a drug metabolism simulator which will be used for drug screening, cytotoxicity tests and prediction of pharmacokinetics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modification of DNA damage in rat hepatocytes following whole-body roentgen radiation using cysteamine and 8-bromocaffeine

Using the elastoviscosimetric method it was shown that the level of residual DNA double-strand breaks in rat hepatocytes depended on conditions of total-body X-irradiation: it increased in the presence of a sensitizer, 8-bromocaffeine and decreased in the presence of a protector, cysteamine. This correlation confirms a major role of fixed DNA double-strand breaks in the formation of a lethal effect in a cell.     

Use of antioxidants to prevent damage during acute ischemia and reperfusion of the kidneys

Prophylactic injection of the natural antioxidant alpha-tocopherol and synthetic antioxidants ionol, diludin and 6-mercurascan prevented the development of lesions during acute renal ischemia and subsequent reperfusion. Ionol proved more effective on intraperitoneal injection than on intragastric route of administration. It has been demonstrated that antiischemic effect of ionol is related to its ability to exhibit the process of lipid peroxidation.     

Prevention of arrhythmia in acute ischemia in conscious animals using a serotonin analog

The state of the serotonergic system was studied in adaptation of rats to short-term non-damaging stress actions along with the possibility of protecting the heart of conscious animals against arrhythmias in acute ischemia with the serotonin analogue 4-nitro-5-methoxytryptamine. It was shown that the adaptation resulted in a significant increase in rat midbrain serotonin by 70%. Preliminary administration of the serotonin analogue 3 fold reduced the total duration of arrhythmias and approximately 5 fold--the heart fibrillation rate and the death rate of animals in acute ischemia. The data obtained are in agreement with the idea on the role of stress-limiting systems in prevention of stress-induced and ischemic damages of the organism. They show that protective effects of metabolites of these systems can be successfully reproduced with their synthetic analogues or activators.     

The role of the parasympathetic nervous system in the regulation of microsomal monooxygenase activity in the liver of rats

The effect of vegetative nervous system activation or depression (subdiaphragmatic vagotomy, atropine, proserine and acetylcholine treatments) on the hepatic microsomal enzymes activities has been studied on Wistar male rats. It is found, that hepatic denervation and atropine treatment decreased cytochrome P450 content and aniline hydroxylase activity. Proserine and acetylcholine induced an opposite effect. It is considered that these different changes in the microsomal enzyme activities with variations in the vegetative nervous system state have proved the nervous control of these processes.     

A comparative study of the effects of procaine, lidocaine, tetracaine and dibucaine on the functions and ultrastructure of isolated rat liver mitochondria

The effects of procaine, lidocaine, tetracaine and dibucaine (10(-5) - 10(-2) M) were tested on isolated rat liver mitochondria by measurements of the respiratory rates and of the membrane potential and by electron microscopy. A general concentration-dependent stimulation of the basal state (respiration before ADP addition) was observed for all local anesthetics studied. Up to the concentration of 10(-3) M, the order of stimulation was: procaine less than lidocaine less than dibucaine less than tetracaine. However, with the exception of dibucaine, which inhibited state-3 respiration (ADP present) in a strictly concentration-dependent manner, the other drugs had a biphasic effect: slight stimulation of state 3 at low and moderate concentrations (less than or equal to 10(-3) M) and inhibition at higher concentrations. Nevertheless, due to a stronger stimulation of the basal state, the acceptor control ratio decreases progressively (uncoupling effect) as the concentration of the drugs increases. The only exception to this observation is procaine in the range of 10(-5) - 10(-4) M, where the stimulation of the two respiration states (although small) is approximately equal and thus the uncoupling effect is absent or negligible. Membrane potential recordings suggested that membrane integrity and phosphorylation capacity were negatively affected at high drug concentrations (greater than 10(-3) M), especially in the case of tetracaine and dibucaine, when 5 x 10(-3) M even produced the collapse of the membrane potential and complete loss of the phosphorylation ability. Electron microscopy confirmed these effects, showing an abundance of either swollen or supercondensed mitochondria, with many membrane ruptures. The action mechanisms of the tertiary amines studied are discussed in terms of interaction of drug with the lipid bilayer and with the membrane proteins. It is concluded that both the inhibitory and the uncoupling effects are dependent, in the first place, on the degree of hydrophobicity of each local anesthetic.     

Functioning of the monooxygenase system of the liver in experimental myocardial infarct

The experiments on rats have shown that coronary artery ligation reduces the content of microsomal cytochromes P-450 and b5 and causes amidopyrine-N-demethylation and aniline-p-hydroxylation disturbances that persist throughout a 3-week period of myocardial infarction. The investigation of spontaneous lipid peroxidation of microsomal membranes in myocardial infarction has shown that concentration of malonic dialdehyde in microsomal fraction significantly increased by the 7th day after coronary artery ligation, as compared to sham-operated rats.     

Reconstruction of a membrane monooxygenase cytochrome P 450-containing system in the liver using detergents in solution

Emulgen 913, Triton N-101 and sodium cholate were compared for their reconstituting action on the dimethylaniline N-demethylation system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The comparison showed that emulgen 913 is the most efficient detergent. The optimum molar ratio of the proteins and emulgen appeared to be equal to 1:1:600. Study on the mechanism of emulgen reconstituting action showed that this effect is due to the mixed complex formation between the cytochrome and reductase, the complexes containing five molecules of the flavoprotein and five molecules of cytochrome P-450. No formation of mixed protein aggregates or reconstitution was observed in the absence of the detergent or at its concentrations exceeding the optimum level.     

Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures.

The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 M N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-EDTA buffer at pH 8.7 and at a methimazole concentration of 2 mM. Data are also presented which show that no interferences caused by either cytochrome P450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavin-containing monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.     

Prevention of adrenaline-induced heart damage using the antioxidant ionol

Experiments on an isolated papillary muscle of the rat heart left ventricle have demonstrated that pretreatment with the antioxidant ionol prevents the disturbances of myocardial contractility caused by administration of a large dose of adrenaline. The data obtained are in agreement with the concept that the prophylactic action of antioxidants during stress is determined by the fact that they prevent activation of lipid peroxidation in the heart under the action of excess catecholamines.