Mechanism of mitochondrial dysfunction and cytotoxicity induced by tropolones in isolated rat hepatocytes

The hair cell orientation patterns present on the saccules of fishes may be important for encoding the direction of a sound source. This study was conducted to determine whether primary afferent projections to the medulla are organized by the best directions for the hair cells they innervate. The toadfish saccule has hair cells oriented primarily in the vertical plane: both the rostral and the caudal saccule have hair cell orientations sweeping from 0 degrees to 45 degrees, and the middle saccule has hair cells oriented at 90 degrees. Fluorescent dextran amines were used singly and in combination to label regions of the saccular nerve innervating rostral, middle, and caudal saccule. The projections of those afferents were examined in detail in the anterior and descending octaval nuclei, which are auditory nuclei in this species. There was no evidence of topographic projections based on location along the length of the saccule or based on hair cell orientation. There was some evidence that parallel inputs are present from each region of the saccule examined, which may be based on the 180 degrees opposition of hair cells found throughout the saccule; however, afferents from the rostral, middle, and caudal saccule appear to have overlapping projections to the anterior and descending octaval nuclei. These data suggest that in toadfish, calculations of the direction of the sound source may begin in either of these primary auditory nuclei by comparing afferent input from along the saccule.     

Identification of factors from rat neutrophils responsible for cytotoxicity to isolated hepatocytes

Activated polymorphonuclear neutrophils (PMNs) have been shown to be cytotoxic to rat hepatic parenchymal cells in vitro. This cytotoxicity could be observed without direct cell-cell contact, since the conditioned medium from PMNs activated with formyl-Met-Leu-Phe (fMLP) was effective in hepatocyte killing. To identify the toxic factor(s) released by PMNs, degranulation was induced by fMLP in PMNs pretreated with cytochalasin B. The contents released from the phagocytes were subjected to gel filtration on a Sephadex G-100 column. Resulting fractions were tested for cytotoxicity to isolated hepatocytes by using release of alanine aminotransferase as a marker for hepatocyte injury. Cytotoxicity was associated with fractions containing cathepsin G and elastase and not with other fractions, including those containing myeloperoxidase. The former two enzymes were purified to homogeneity with a carboxymethyl cellulose column. Each of these enzymes demonstrated concentration-dependent cytotoxicity to hepatocytes at concentrations > 2 microgram/mL. Moreover, they exhibited an additive cytotoxic effect. Effective concentrations for the combined cathepsin G and elastase in the incubation mixture were similar to the concentrations of these enzymes in PMN-conditioned medium that produced cytotoxicity to hepatocytes. Cytotoxicity of either purified enzyme or of conditioned medium could be prevented by plasma alpha-1-antitrypsin or soybean trypsin-chymotrypsin inhibitor, which were also potent inhibitors of enzymic activity of both cathepsin G and elastase. By contrast, the serine protease inhibitors, aprotinin and 4-(2-aminoethyl)-benzene-sulfonyl fluoride, were less effective in inhibiting cathepsin G and elastase activities as well as cytotoxicity caused by the purified proteases or PMN-conditioned medium. These results support the hypothesis that cathepsin G and elastase are important mediators of hepatic parenchymal cell killing produced by activated PMNs in vitro.     

Involvement of polyamines in the protection of taurine against the cytotoxicity of hydrazine or carbon tetrachloride in isolated rat hepatocytes

Taurine (2-aminoethanesulfonic acid) is known to protect hepatocyte injury induced by hydrazine or carbon tetrachloride. We investigated whether cellular polyamines are involved in the protective mechanism of taurine in the hepatocyte injury caused by hydrazine or carbon tetrachloride. The agents decreased cellular polyamine concentrations, but the treatment with taurine prevented this decrease. The protection of taurine against hepatic injury was not observed in hepatocytes treated with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase which is a key enzyme in polyamine biosynthesis. The protection of taurine was recovered by the addition of polyamines to DFMO-treated hepatocytes. These results suggest that cellular polyamines play an important role in the protection of taurine in hydrazine or carbon tetrachloride-induced hepatocyte injury.     

Adenosine-mediated inhibition of glutathione synthesis in rat isolated hepatocytes

The modulatory effect of adenosine on hepatic glutathione (GSH) synthesis has been investigated in an experimental model in which GSH synthesis from methionine was monitored in rat isolated hepatocytes previously depleted of GSH. Adenosine inhibited GSH synthesis in this system, with complete inhibition occurring at 1 mM. Studies with adenosine receptor agonists and antagonists and adenosine analogues devoid of agonist activity have indicated that this effect of adenosine is not receptor-mediated nor is it mediated through changes in ATP level or redox balance. Rather, the data are consistent with an inhibitory effect of adenosine on the methionine-->cysteine transsulphuration pathway, probably at the level of the enzyme S-adenosylhomocysteine hydrolase. Submaximal inhibitory concentrations of adenosine produced an effect on GSH synthesis additive to that obtained with a maximal inhibitory concentration of adrenaline, which inhibits GSH synthesis at the level of gamma-glutamylcysteinyl synthetase. Furthermore, exposure of hepatocytes to adenosine subsequent to brief treatment with the toxicant menadione precipitated cytotoxicity. These results suggest that adenosine-mediated inhibition of hepatic GSH synthesis may have a role in various pathological or toxicological states.     

Uptake and intracellular trafficking of asialoglycoprotein-polylysine-DNA complexes in isolated rat hepatocytes

Receptor-mediated gene delivery has been reported for a number of different receptor systems although the intracellular fate of such systems has not been systematically investigated. In this study, we have determined the fate of a commonly used asialoglycoprotein (ASGP)-dependent DNA delivery system in isolated rat hepatocytes. ASPG-polylysine (PLL296) was ionically complexed with pSV-CAT DNA at a molar ratio of 10:1. The resulting complex inhibited 125I-ASGP binding to rat hepatocytes but ASGP only partially inhibited the binding of complex. The ASGP-independent binding was due to the interaction of the PLL component of the complex with plasma membranes and could be minimised by replacing PLL296 with PLL19. Following internalisation, ASGP was cleaved from the complex and translocated to the lysosomes where it was degraded. The DNA, however, remained in an intracellular compartment that cosedimented with plasma membranes in Percoll density gradients. This study shows first that hepatocytes do not process DNA internalised as ASGP complexes in a manner similar to ASGP itself, and second that the differential sorting of the two cleaved molecules leads to a rapid intracellular compartmentalisation of the DNA. Controlled release from this compartment may be a means for prolonged gene expression in gene therapy protocols.     

Incorporation of radioactive amino acids into protein in isolated rat hepatocytes

The incorporation of radioactivity from a 14C-labelled amino acid mixture (algal protein hydrolysate) into protein in isolated rat hepatocytes has been studied. The incorporation rate declined with increasing cell concentration, an effect which could be explained partly by isotope consumption, partly (and largely) by isotope dilution due to the formation of non-labelled amino acids by the cells. At a high extracellular amino acid concentration, the rate of incorporation into protein became independent of cell concentration, because the isotope dilution effect was now quantitatively insignificant. The time course of protein labelling at various cells concentrations correlated better with the intracellular than with the extracellular amino acid specific activity, suggesting that amino acids for protein synthesis were taken from an intracellular pool. With increasing extracellular amino acid concentrations both the intracellular amino acid concentration, the intracellular radioactivity and the rate of incorporation into protein increased. Protein labelling exhibited a distinct time lag at high amino acid concentrations, presumably reflecting the time-dependent expansion of the intracellular amino acid pool. The gradual increase in the rate of protein labelling could be due either to an increased intracellular specific activity, or to a real stimulation of protein synthesis by amino acids, depending on whether the total intracellular amino acid pool or just the expandable compartment is the precursor pool for protein synthesis.     

Nuclear accumulation of G-actin in isolated rat hepatocytes by adenine nucleotides

Distinct inositol and phosphatidylinositol polyphosphates 5-phosphatases have recently been cloned. Primers have been designed coding for highly conserved amino acid regions that are shared between sequences of 5-phosphatases. One of the PCR fragment referred to as 51 C, shows 99% identity to a previously reported sequence (INPPL-1) present in the database. We report here the identification of cDNAs for a new SH2-domain-containing protein showing homology to the inositol 5-phosphatase SHIP and therefore referred to as SHIP2. SHIP2 differs at both N- and C-terminal ends with the sequence of INPPL-1. The translated sequence of SHIP2 encodes a 1258 amino acid protein with a predicted molecular mass of 142 kDa. Particularly high levels of SHIP2 were found in human heart, skeletal muscle and placenta as shown by Northern blot analysis. SHIP2 was also expressed in dog thyroid cells in primary culture where the expression was enhanced in TSH and EGF-stimulated cells.     

Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes

The hepatocytes in the mature normal liver are tightly coupled through gap junctions, except during compensatory hyperplasia (regeneration) after partial hepatectomy when the gap junctions become down-regulated. The significance of this down-regulation has been a long-standing enigma. The present study of hepatocytes in primary culture and in the regenerating liver aimed at defining the relationship, if any, between hepatocyte gap junctional communication and proliferation. Gap junctional down-regulation in the regenerating liver appeared to be a specific phenomenon because desmosomes and the surface contact area between neighboring hepatocytes remained constant. All agents and conditions (dexamethasone in vivo; dexamethasone, cyclic adenosine monophosphate, serum, and high cell density in vitro) delaying gap junctional down-regulation also increased the lag before the cells reached competence to enter S phase. This raised the possibility that hepatocyte DNA replication was inhibited through preservation of gap junctions. However, we disproved this assumption by showing that the DNA replication (more specifically the G1/S transition rate constant) was inhibited even in hepatocytes completely devoid of gap junctional communication. The teleological advantage of linking gap junctional down-regulation to hepatocyte G1 progression therefore may not be to trigger DNA replication but to ensure that proliferating hepatocytes and hepatocytes responsible for liver-specific metabolic functions maintain separate pools of metabolites and signaling molecules.     

Piroxicam modifies the effects of ethanol on isolated rat hepatocytes

It has been reported that piroxicam prevents the hepatic increase of triacylglycerides and thiobarbituric acid-reactive substances observed after acute ethanol intoxication in rats and also causes a decrease in blood ethanol concentration. The aim of this study was to assess the effect of piroxicam on these 3 metabolic indicators, using isolated rat hepatocytes incubated with ethanol or lactate, supplemented or not with epinephrine. Epinephrine stimulated the consumption of lactate, but not of ethanol. In the isolated hepatocytes, and in a dose-dependent fashion, piroxicam alone raised the consumption of lactate and ethanol, increased the triacylglyceride pool in cells incubated with lactate or ethanol, and decreased the content of thiobarbituric acid-reactive substances in cells incubated with ethanol, but not with lactate. Epinephrine blocked these actions of piroxicam, except the lowering of the content of thiobarbituric acid-reactive substances. Thus, piroxicam helps to control the oxidative stress produced in isolated hepatocytes by ethanol.     

Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3' exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3' ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol

BACKGROUND & AIMS: We reported previously that patients with alcoholic liver disease (ALD) have circulating immunoglobulins reacting with cytochrome P4502E1 (CYP2E1) complexed with hydroxyethyl free radicals. The aim of this study was to investigate whether hydroxyethyl radical adducts are present on the plasma membranes of ethanol-treated hepatocytes and their role in antibody-dependent cytotoxicity. METHODS: Immunofluorescence confocal laser microscopy, Western blotting, and antibody-dependent cell-mediated cytotoxicity assay were used. RESULTS: Isolated rat hepatocytes incubated in vitro with ethanol or obtained from ethanol-treated animals showed strong surface fluorescence when exposed to rabbit anti-hydroxyethyl radical serum or sera from patients with ALD. No surface fluorescence was evident on control hepatocytes or after scavenging hydroxyethyl radicals with 4-pyridyl-1-oxide-t-butyl nitrone. The presence of CYP2E1-hydroxyethyl radical adducts on hepatocyte plasma membranes was shown by Western blot and by immunofluorescence using double staining for human and rabbit anti-CYP2E1 immunoglobulin G. Cytotoxicity was observed in ethanol-treated hepatocytes incubated with immunoglobulin G from patients with ALD and normal human blood mononuclear cells. This effect was blocked by preabsorbing the sera with human albumin complexed with hydroxyethyl radicals, which also eliminated the antibody reaction with the plasma membranes. CONCLUSIONS: Hydroxyethyl radicals bound to CYP2E1 on hepatocyte plasma membranes can target immune reactions triggered by alcohol abuse.     

Diisopropyl fluorophosphate inhibits receptor-activated Ca2+ influx in isolated rat hepatocytes

The influence of diisopropyl fluorophosphate (DFP) on receptor-activated increases in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes was monitored by measuring phosphorylase a activity and the fluorescence ratio of the Ca2+ sensitive dye Indo-1. Pretreatment (2 min) of hepatocytes with DFP (1 mM) inhibited maximal increases in phosphorylase a activity stimulated by phenylephrine (1 microM), angiotensin II (5 nM), or vasopressin (10 nM) by 36, 35, and 17%, respectively, when the cells were incubated in Ca2+ (1 mM)-containing medium. In contrast, agonist-stimulated increases in phosphorylase a activity were similar in control and DFP-pretreated cells when cells were incubated in medium containing very low (10 nM) Ca2+. Addition of Ca2+ (1 mM) to hepatocytes maintained in the low Ca2+ buffer and exposed to agonists rapidly increased phosphorylase a activity in control cells; however, increases in DFP-pretreated cells were markedly attenuated. Changes in [Ca2+]i similar to those noted with phosphorylase a were observed using Indo-1. Addition of calcium ionophore A23187 to control or DFP-pretreated hepatocytes increased phosphorylase a activity to a similar extent in control and DFP-pretreated cells, demonstrating that DFP pretreatment did not alter the ability of the enzyme to respond to elevation in [Ca2+]i. Collectively, these data indicate that DFP pretreatment of hepatocytes irreversibly inhibits one or more components of the Ca2+ influx pathway.     

Migraine and mitochondrial dysfunction

Like many others neurological diseases, mitochondrial involvement, by means of abnormalities in cerebral oxidative metabolism, may play a role in migraine. This review summarizes clinical data, in vivo magnetic resonance spectroscopy studies, biochemical assays and therapeutic results that support the "mitochondrial hypothesis of migraine".     

Ethylmorphine O-deethylation in isolated rat hepatocytes. Involvement of codeine O-demethylation enzyme systems

Smoking cessation programs may be an important component in the implementation of worksite smoking policies. This study examines the impact of a smoke-free policy and the effectiveness of an accompanying hypnotherapy smoking cessation program. Participants in the 90-minute smoking cessation seminar were surveyed 12 months after the program was implemented (n = 2642; response rate = 76%). Seventy-one percent of the smokers participated in the hypnotherapy program. Fifteen percent of survey respondents quit and remained continuously abstinent. A survey to assess attitudes toward the policy was conducted 1 year after policy implementation (n = 1256; response rate = 64%). Satisfaction was especially high among those reporting high compliance with the policy. These results suggest that hypnotherapy may be an attractive alternative smoking cessation method, particularly when used in conjunction with a smoke-free worksite policy that offers added incentive for smokers to think about quitting.     

Stereochemical aspects of 1,3-butadiene metabolism and toxicity in rat and mouse liver microsomes and freshly isolated rat hepatocytes

In the last decade, silver staining of nucleolar organizer region-associated proteins (AgNORs) has been widely used in tumour pathology both for diagnostic and for prognostic purposes. However, a reliable and reproducible assessment of these proteins on routinely processed archival tissues has only become possible since the recent introduction of standardized staining method and computer-aided morphometric analysis. In the present study, the AgNOR content at the invasive front of 80 squamous cell carcinomas of the floor of the mouth/tongue was investigated using this novel approach, with regard to prognosis and a variety of clinico-pathological parameters. All standardized AgNOR parameters [mean of AgNOR number, mean of AgNOR area, coefficients of variation (CV) of both AgNOR number and area] were statistically significantly associated with the clinical course. The strongest correlation was found for the AgNOR-area univariate analysis (P = 0.006). In multivariate analysis, the mean of AgNOR number could independently predict both overall (P = 0.01) and disease-free survival (P = 0.001). It is concluded that standardized staining and computer-aided analysis of AgNORs are prerequisites for an objective and reproducible AgNOR assessment, which has potential as a supplementary diagnostic and prognostic tool in oral cancer.     

Regulation of Mg2+ homeostasis by insulin in perfused rat livers and isolated hepatocytes

Several recent studies demonstrate that adrenergic receptor stimulation evokes marked changes in Mg2+ homeostasis. As insulin counter-regulates many of the metabolic consequences of adrenergic receptor stimulation, we evaluated the potential influence of insulin on Mg2+ movements in response to adrenergic stimulation. The data demonstrate that insulin is able to block the Mg2+ efflux from perfused rat livers stimulated by isoproterenol or 8-Br-cAMP, but has little or no effect on epinephrine or phenylephrine induced Mg2+ efflux. Thus, evidence is provided demonstrating that there are redundant adrenergic pathways regulating Mg2+ efflux from liver tissue. One of these pathways, the beta-adrenergic component, is selectively blocked by insulin. Furthermore, these findings may provide a cellular explanation for hypomagnesemia associated with diabetes.