Immunocytochemical localization of angiotensinogen and cathepsins B, H, and L in rat hepatocytes, with special reference to degradation of angiotensinogen in lysosomes after colchicine

We examined the effects of bilateral nephrectomy and colchicine treatment on localization and content of angiotensinogen and cathepsins B, H, and L in rat liver using immunohistochemistry, radioimmunoassay, and enzyme assay. Angiotensinogen content increased in the liver of colchicine-treated rats, whereas a clear-cut increase was not detected in the liver of nephrectomized rats. This tendency was consistent with the immunocytochemical results; only perivenous hepatocytes in control and nephrectomized rats were diffusely immunostained by anti-angiotensinogen, whereas perivenous and periportal hepatocytes of colchicine-treated rats were strongly immunostained. Enzyme assay revealed no significant change in activities of cathepsins B, H, and L in liver extracts under these experimental conditions. Immunocytochemical localization of these cysteine proteinases in hepatocytes after colchicine treatment was more widespread in the cytoplasm than that in the control hepatocytes. By electron microscopy, angiotensinogen was localized in smaller vesicles and some larger vesicles (lysosomes) of hepatocytes after colchicine treatment. Double immunostaining demonstrated co-localization of cathepsins B, H, and L with angiotensinogen in lysosomes. These results suggest that cathepsins B, H, and L play a role in the degradation of excess angiotensinogen in hepatocytes of rats after colchicine treatment.     

Uncoupling protein-2 induction in rat hepatocytes after acute carbon tetrachloride liver injury

This study is focused on the role of UCP-2 in hepatic oxidative metabolism following acute CCl(4) administration to rats. UCP-2 mRNA, almost undetectable in the liver of controls, was significantly increased 24 h after CCl(4) administration, peaked at 72 h and then tended to disappear. UCP-2 protein, undetectable in controls, increased 48-72 h after CCl(4) treatment. Experiments with isolated liver cells indicated that in control rats UCP-2 was expressed in non-parenchymal cells and not in hepatocytes, whereas in CCl(4)-treated rats UCP-2 expression was induced in hepatocytes and was not affected in non-parenchymal cells. Addition of CCl(4) to the culture medium of hepatocytes from control rats failed to induce UCP-2 expression. Liver mitochondria from CCl(4)-treated rats showed an increase of H(2)O(2) release at 12-24 h, followed by a rise of TBARS. Vitamin E protected liver from CCl(4) injury and reduced the expression of UCP-2. Treatment with GdCl(3) prior to CCl(4), in order to inhibit Kupffer cells, reduced TBARS and UCP-2 mRNA increase in hepatic mitochondria. Our data indicate that CCl(4) induces the expression of UCP-2 in hepatocytes with a redox-dependent mechanism involving Kupffer cells. A role of UCP-2 in moderating CCl(4)-induced oxidative stress during tissue regeneration after injury is suggested.     

Binding and distribution of three prototype calcium channel blockers in perfused rat liver

This work represents a study of the binding and distribution of three different calcium channel blockers in the Sprague-Dawley rat liver, using an in situ perfusion technique. For this purpose, [3H] desmethoxyverapamil, [3H] PN200-110 (isradipine) and [3H] azidopine were used as binding probes interacting with calcium channels. The perfusion steps of the liver involved both portal vein and thoracic inferior vena cava cannulations as inlet and outlet respectively. The subhepatic inferior vena cava was ligated to prevent leakage of the perfusate. Buffer, containing the tracer drug, was administered via the portal vein at a rate of l mL/min and perfusate collected at the same rate within specified time intervals during 50 min. The concentration of the tracer solutes in the perfusate's outlet increased with time, and steady state was observed for all tracers at 40 min. The effect of adding cold isradipine to tracer desmethoxyverapamil, or cold verapamil to tracer PN200-110 were also assessed. First order rate constants for hepatocellular influx, efflux and calcium channel binding of the tracer substances were obtained using a simplified model from Goresky et al. [25]. These constants were mathematically manipulated and changed into permeability constants, second order binding constants, and residency times.Tracer solute influx across hepatocellular membranes is solubility-diffusion controlled, is inversely related to the molecular weights and is different in value from the efflux constants. Cold isradipine reduced the binding constant of desmethoxyverapamil by 36%, while cold verapamil reduced the binding constant of PN200-110 by 23%. Azidopine cellular distribution was low, however, binding to its receptor was analogous to desmethoxyverapamil and PN200-110. Moreover, PN200-110 had the highest residency time with no effect of cold verapamil on its receptor binding, while desmethoxyverapamil had the lowest residency time which significantly increased in the presence of cold isradipine.     

The effects of in vivo administration of endotoxin on the functions and interaction of hepatocytes and Kupffer cells

It was the purpose of this study to determine the effects of the in vivo administration of endotoxin on certain in vitro hepatocyte and Kupffer cell functions. An Alzet osmotic pump that contained endotoxin (LPS, 2.5 mg/100g) was implanted into the peritoneal cavity of 300g guinea pigs and delivered the endotoxin over a period of four days. In vivo administration of LPS did not cause a change in the in vitro release of albumin by isolated hepatocytes. However, when hepatocytes were co-cultured with Kupffer cells there was a significant decrease in albumin release for both control and LPS-treated animals. There was no difference between control and LPS-treated animals in the release of C3 by hepatocytes. However, there was a significant increase over the control group in C3 release by Kupffer cells from LPS-treated animals. When hepatocytes and Kupffer cells were cultured together, their release of C3 was not additive. Kupffer cells from LPS-treated animals released significantly greater amounts of PGE2 than control animals when stimulated in vitro with LPS. Thus, these Kupffer cells appeared to be primed to respond to an in vitro challenge of LPS. Kupffer cells from LPS-treated animals had significantly depressed antibody dependent cellular cytotoxicity (ADCC). This endotoxin model is useful for determining the in vivo effects of endotoxin on cellular function and gives some indirect evidence for the detrimental effects of LPS on the immune system and host defense.     

Differential effects of gadolinium chloride on Kupffer cells in vivo and in vitro

Gadolinium chloride (GdCl) is commonly used to study the role of Kupffer cells in liver disease in vivo. The in vitro effects of GdCl on cultured Kupffer cells are poorly characterised. The aim of this study was to characterise rat Kupffer cell TNFalpha production, phagocytic function, and ED1 and ED2 antigen expression following the administration of GdCl. For in vivo experiments, rats received 10mg/kg GdCl IV or sterile saline. Lipopolysaccharide 3mg/kg IP (LPS) was administered 4h prior to sacrifice on Days 1-3, 5 or 8 following GdCl injection. Hepatic ED1 and ED2 positive macrophage numbers and TNFalpha mRNA levels were determined. For in vitro experiments, Kupffer cells were cultured in the presence of 0-270 microM GdCl for 24h following which viability, TNFalpha protein production in response to LPS (10 ng/ml), phagocytosis, and ED1 and ED2 staining were evaluated. In vivo, the proportion of ED1 positive cells which were ED2 positive was reduced from 87 to 3% and hepatic TNFalpha mRNA levels following LPS declined by 60% over Days 1-5 after injection of GdCl (P<0.01). In vitro, phagocytosis declined with increasing concentrations of GdCl. GdCl (0-27 microM) did not effect cultured Kupffer cell viability, TNFalpha production, ED1 or ED2 staining. We conclude that GdCl significantly reduces ED2 expression by Kupffer cells in vivo. In vitro, GdCl has a dose dependent effect on phagocytosis but only effects viability and TNFalpha production at high concentrations. ED2 expression of cultured Kupffer cells is not affected by GdCl.     

Mannose-binding lectin augments the uptake of lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer cells through increased cell surface expression of scavenger receptor A

We investigated roles of scavenger receptor A (SR-A) and mannose-binding lectin (MBL) in the uptake of endotoxin and bacteria by Kupffer cells. When [3H]lipid A was injected into retro-orbital plexus of mice, significantly less accumulation of lipid A in the liver was observed in SR-A-deficient mice and wild-type mice coinjected with fucoidan or acetylated low-density lipoprotein, which are known ligands for SR-A. Isolated Kupffer cells were able to take up [3H]lipid A in a time-dependent manner. The amount of lipid A associated with nonadherent Kupffer cells derived from SR-A-deficient mice was reduced by approximately 80% when compared with wild-type cells, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. The lipid A uptake by Kupffer cells was significantly enhanced in the presence of rMBL. Coincubation of fucoidan with [3H]lipid A significantly inhibited the basal and the MBL-stimulated uptake of lipid A by Kupffer cells. Preincubation of MBL with Kupffer cells also increased the uptake of lipid A. These results indicate that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells. Consistently, the exposure of MBL to Kupffer cells increased cell surface SR-A expression. The phagocytosis of Staphylococcus aureus and Escherichia coli by Kupffer cells was also enhanced by preincubation of MBL with the cells. In addition, MBL bound to lipid A, LPS, and S. aureus, and precipitated S. aureus. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.     

D-galactosamine-induced cell damage enhances specific binding of prostaglandin E1 to rat hepatocytes.

Specific bindings of [3H]prostaglandin E1 ([3H]PGE1), 125I-glucagon and [3H]norepinephrine to D-galactosamine (GalN)-treated rat hepatocytes in primary culture were investigated. After a two hour-treatment with GalN (1 and 10mg/ml), hepatocytes showed an enhanced specific binding to [3H]PGE1, whereas 125I-glucagon binding was little affected and [3H]norepinephrine binding was strongly diminished. Scatchard plot analysis indicated an increase of binding sites of [3H]PGE1. This unusual manner of [3H]PGE1 binding is suggested to indicate a special property of PGE1 receptor and may be associated with the cytoprotective effect of prostaglandins.     

The uptake and metabolism of chylomicron-remnant lipids by rat liver parenchymal and non-parenchymal cells in vitro.

The uptake and metabolism of chylomicron-remnant lipids by individual liver cell types was examined by incubating remnants with monolayer cultures of hepatocytes, Kupffer cells, and endothelial cells from rat liver. Remnants were prepared in vitro from radiolabelled mesenteric-lymph chylomicra, utilizing either purified lipoprotein lipase from bovine milk, or plasma isolated from heparinized rats. The resulting particles contained [3H]phosphatidylcholine and cholesterol, and [14C]oleate in the acylglycerol, phospholipid, fatty-acid and cholesterol-ester fractions. The capacities of the three cell types for uptake of both [3H]lipids and [14C]lipids were determined to be, on a per-cell basis, in the order: Kupffer greater than hepatocytes greater than endothelial. The relative proportions of [3H]phospholipid and total [3H]cholesterol taken up by hepatocytes and non-parenchymal cells remained constant with time. The uptake of [14C]oleoyl lipids by all three cell types was slightly greater than that of the total [3H]cholesterol and [3H]phospholipid components. There was evidence of cholesterol-ester hydrolysis and turnover of [14C]oleate in the phospholipid fraction in hepatocytes and Kupffer cells, but not endothelial cells, over the first 2 h. With both remnant preparations, these observations indicate that significant differences exist between the three major liver cell types with respect to the uptake and metabolism of remnant lipid components.     

Gadolinium chloride-induced Kupffer cell blockade increases uptake of oxidized low-density lipoproteins by rat heart and aorta.

Oxidized low-density lipoproteins (LDL) play a key role in the formation of atherosclerotic lesions of arteries. We analyzed the effect of hepatic resident macrophage (Kupffer cell) blockade on oxidized [125I]LDL accumulation in different organs and tissues of the rat. Kupffer cell blockade was induced by gadolinium chloride (GdCl3) which was injected intravenously 24 h prior to injection of oxidized [125I]LDL into the rats. Ten minutes after administration to intact animals, oxidized [125I]LDL was accumulated in the liver (86.8% of the dose administered), muscles (4.7%), spleen (2.1%), lungs (0.8%), kidney (0.6%), adrenal glands (0.2%), heart (0.15%), and thymus (0.04%). Kupffer cell blockade significantly decreased the clearance rate of oxidized [125I]LDL from the blood. Specific radioactivity (per g tissue) decreased in the liver (1.3-fold compared to control), but increased in the aorta (2.5-fold), heart (2-fold), lungs (1.6-fold), and kidney (1.3-fold). The results indicate that the accumulation of oxidized LDL in heart and aorta significantly depends on the functional state of the mononuclear phagocyte system in the liver.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by flow cytometry.

BACKGROUND: The triggering of cellular responses during endotoxic shock is initiated for the binding of endotoxin (lipopolysaccharide; LPS) to the cell surface. Kupffer and endothelial liver cells, involved in the removal of endotoxin from blood circulation, show in vitro a rapid response to LPS in the absence of serum. METHODS: A double-labeling fluorescent assay was designed to evaluate the binding properties of Escherichia coli O111:B4 LPS to individual endothelial and Kupffer cells in suspension, where both populations occurred in the same relative proportion as in liver. After immunolabeling of the Kupffer cell population with the monoclonal antibody ED1 conjugated to R. phycoerythrin, the binding characteristics of LPS labeled with fluorescein to both endothelial and Kupffer cells were simultaneously studied by flow cytometry in serum-free conditions. RESULTS: Specific and saturable binding of endotoxin was observed with both populations, showing properties of a receptor-mediated process. The Kupffer cell population showed a faster capacity and a higher affinity for LPS binding. The Hill coefficients indicated positive cooperativity in the LPS interaction with both populations. CONCLUSIONS: Specific endotoxin binding to liver sinusoidal cells occurs in a serum-independent manner, particularly at high LPS concentrations. Flow cytometry is a fast, precise, and efficient technique to evaluate the simultaneous interaction of a ligand with two different cell types.     

Kupffer cell-derived prostaglandin E2 is involved in regulation of lipid synthesis in rat liver tissue

Our recent studies suggest that Kupffer cells play a role in the physiological regulation of lipid metabolism of the adjacent hepatocytes. In the present study, we have tested the hypothesis that inhibition of Kupffer cells decreases prostaglandin E(2) (PGE(2)) release inside liver tissue, a phenomenon contributing to lipid accumulation in hepatocytes. PGE(2) secretion as well as lipid synthesis were assessed in precision-cut liver slices (PCLS) from rats previously treated with Kupffer cell inhibitors (GdCl(3) 10 mg kg(-1) body wt, i.v. injection and glycine 5% in diet). In addition, lipid synthesis was assessed in primary rat hepatocytes cultured in the absence or presence of PGE(2) (0.01, 1 and 10 microM). Inhibition of Kupffer cell activity by GdCl(3) decreases PGE(2) secretion by PCLS and resulted in a higher lipid synthesis. Since incubation with PGE(2) over 48 h decreases lipid synthesis from acetate in cultured hepatocytes, we propose that the lower PGE(2) secretion linked to Kupffer cell inhibition, partly explains a higher rate of synthesis of lipids with a subsequent accumulation in liver tissue, as previously shown in fasted rats.     

Effects of hyperthyroidism on stimulation of [1-14C]oleate oxidation to14CO2 in isolated hepatocytes from fed rats by the catecholamines,vasopressin, and angiotensin II

Possible effects of adrenaline, noradrenaline, vasopressin, and angiotensin II to increase 14CO2 production from [1-14C]oleate were examined in hepatocytes from fed L-triiodothyronine (T3)-treated or control rats. Rates of 14CO2 production were decreased and rates of ketogenesis increased in hepatocytes from T3-treated rats. These changes were accompanied by a marked shift of the 3-hydroxybutyrate:acetoacetate concentration ratio towards acetoacetate. Rates of glucose and lactate release were decreased. Whereas the Ca2+-mobilizing hormones increased 14CO2 production from [1-14C]oleate by 64-84% with hepatocytes from control rats, they increased 14CO2 production from [1-14C]oleate by on 24-32% with hepatocytes from T3-treated rats. The magnitude of the response to the Ca2+-mobilizing hormones in hepatocytes from T3-treated rats was increased by the addition of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to the incubation medium (increases of 52-88%). In the presence of 3-mercaptopicolinate, the 3-hydroxybutyrate:acetoacetate concentration ratio in hepatocytes from fed, T3-treated rats was similar to that in hepatocytes from control rats in the absence of 3-mercaptopicolinate. The results demonstrate that hyperthyroidism per se does not lead to a loss of sensitivity, in terms of oleate oxidation, either to the catecholamines or to vasopressin and angiotensin II. The impaired ability of hepatocytes from T3-treated rats to respond to these hormones is a consequence of decreased net glycolytic flux or a more oxidized mitochondrial redox state.     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.     

Distensibility characteristics of caval veins and empirical exponential formulae

The stress-strain curves of the vena cava have been measured of vena cava superior, and the intrathoracic and abdominal portions of vena cava inferior excised from dogs. The stress sigma is expressed by the exponential function of the strain gamma as follows: sigma = sigma 0[exp (gamma/gamma 0) - 1] in the longitudinal direction, and sigma = sigma 1[exp(gamma/gamma 1) - 1] + sigma 2 [exp(gamma/gamma 2) - 1] in the circumferential one for all the three kinds of veins. The constants sigma 0, gamma 0, sigma 1, gamma 1, sigma 2 and gamma 2 are determined by a nonlinear least squares method.     

Metabolic profiling of serum from gadolinium chloride-treated rats by 1H NMR spectroscopy

Metabolic profiling of serum from gadolinium chloride (GdCl(3), 10 and 50 mg/kg body weight, intraperitoneal [i.p.])-treated rats was investigated by the NMR spectroscopic-based metabonomic strategy. Serum samples were collected at 48, 96, and 168h postdose (p.d.) after exposure to GdCl(3). (1)H NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The studies showed that there was a dose-related biochemical effect of GdCl(3) treatment on the levels of a range of low-molecular weight compounds in serum. The liver damage induced by GdCl(3) was characterized by the elevation of lactate, pyruvate, and creatine as well as the decrease of branched-chain amino acids (valine and isoleucine), alanine, glucose, and trimethylamine-N-oxide concentration in serum samples. The biochemical effects of GdCl(3) in rats could be consulted when evaluating the biochemical profile of gadolinium-containing compounds that are being developed for nuclear magnetic resonance imaging.     

Thromboxane A2 from Kupffer cells contributes to the hyperresponsiveness of hepatic portal circulation to endothelin-1 in endotoxemic rats

We examined the role of thromboxane A2 (TXA2) in LPS-induced hyperresponsiveness of hepatic portal circulation to endothelins (ETs) and whether Kupffer cells are the primary source of TXA2 release in response to ET-1 in endotoxemia. After 6 h of LPS (1 mg/kg body wt ip) or saline (control), liver was isolated and perfused with recirculating Krebs-Henseleit bicarbonate buffer at a constant flow rate (100 ml.min(-1).kg body wt(-1)). ET-1 (10 pmol/min) was infused for 10 min. Portal pressure (PP) was continuously monitored during perfusion. Perfusate was sampled for enzyme immunoassay of thromboxane B2 (TXB2; the stable metabolite of TXA2) and lactate dehydrogenase (LDH) assay. ET-1 infusion resulted in a significantly greater increase of PP in the LPS group than in controls. Both TXA2 synthase inhibitor furegrelate (Fureg) and TXA2 receptor antagonist SQ-29548 (SQ) substantially blocked enhanced increase of PP in the LPS group (4.9 +/- 0.4 vs. 3.6 +/- 0.5 vs. 2.6 +/- 0.6 mmHg for LPS alone, LPS + Fureg, and LPS + SQ, respectively; P < 0.05) while having no significant effect on controls. GdCl3 for inhibition of Kupffer cells had similar effects (4.9 +/- 0.4 mmHg vs. 2.9 +/- 0.4 mmHg for LPS alone and GdCl3 + LPS, respectively; P < 0.05). In addition, the attenuated PP after ET-1 was found concomitantly with significantly decreased releases of TXB2 and LDH in LPS rats treated with Fureg, SQ, and GdCl3 (886.6 +/- 73.4 vs. 110.8 +/- 0.8 vs. 114.8 +/- 54.7 vs. 135.2 +/- 45.2 pg/ml, respectively; P < 0.05). After 6 h of LPS, Kupffer cells in isolated cell preparations released a significant amount of TXA2 in response to ET-1. These results clearly indicate that hyperresponsiveness of hepatic portal circulation to ET-1 in endotoxemia is mediated at least in part by TXA2-induced receptor activation, and Kupffer cells are likely the primary source of increased TXA2 release.     

Oxidative products from alcohol metabolism differentially modulate pro-inflammatory cytokine expression in Kupffer cells and hepatocytes

Pro-inflammatory cytokines play a vital role in the pathogenesis of alcoholic steatohepatitis. The present study was to determine the role of alcohol-induced oxidative stress in modulating cytokine production. A rat model of alcohol consumption was used to determine alcohol-induced hepatic cytokine expression. Chronic alcohol exposure caused lipid accumulation, oxidative stress, and inflammation in the livers of Wistar rats. The role of oxidative stress in regulating cell type-specific cytokine production was further dissected in vitro. Lipopolysaccharide (LPS) dose-dependently upregulated TNF-α, MIP-1α, MCP-1, and CINC-1 in Kupffer cells-SV40, whereas TNF-α dose-dependently induced CINC-1, IP-10, and MIP-2 expression in H4IIEC3 hepatoma cells. An additive effect on cytokine production was observed in both Kupffer cells-SV40 and hepatocytes when combined hydrogen peroxide with LPS or TNF-α, respectively, which was associated with NF-κB activation and histone H3 hyper-acetylation. Unexpectedly, an inhibitory effect of 4-hydroxynonenal on cytokine production was revealed in LPS-treated Kupffer cells-SV40. Mechanistic study showed that 4-hydroxynonenal significantly enhanced mRNA degradation of TNF-α, MCP-1, and MIP-1α, and decreased the protein levels of MCP-1 in LPS-stimulated Kupffer cells-SV40 through reducing the phosphorylation of mRNA binding proteins. This study suggests that Kupffer cells and hepatocytes express distinct pro-inflammatory cytokines/chemokines in response to alcohol intoxication, and oxidative products (4-hydroxynonenal) differentially modulate pro-inflammatory cytokine/chemokine production via NF-κB signaling, histone acetylation, and mRNA stability.     

Aflatoxin inhibition of glucocorticoid finding capacity of rat liver nuclei.

The effect of aflatoxin B1 on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [3H]desamethasone-cytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B1. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 26 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B1 dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant. Administration of [3H]dexamethasone to alfatoxin B1 -treated rats produced a similar pattern of glucocorticoid binding distribution in vivo to that observed in vitro. No differences in [3H]dexamethasone-cytoplasmic receptor binding between control and alfatoxin B1 -treated rats were found, whereas nuclear [3H]dexanthasone binding was reduced 34% by alfatoxin B1 -treatment.