Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes.

Nitric oxide (NO) is a potent inhibitor of apoptosis in many cell types, including hepatocytes. We and others have described NO-dependent decreases in caspase activity in cells undergoing apoptosis. However, previous work has not determined whether NO disrupts the proteolytic processing and thus the activation of pro-caspases. Here we report that NO suppresses proteolytic processing and activation of multiple pro-caspases in intact cells, including caspase-3 and caspase-8. We found that both exogenous NO as well as endogenously produced NO via adenoviral inducible NO synthase gene transfer protected hepatocytes from tumor necrosid factor (TNF) alpha plus actinomycin D (TNFalpha/ActD)-induced apoptosis. Affinity labeling with biotin-VAD-fmk of all active caspase species in TNFalpha-mediated apoptosis identified four newly labeled spots (activated caspases) present exclusively in TNFalpha/ActD-treated cells. Both NO and the caspase inhibitor, Ac-DEVD-CHO, prevented the appearance of the four newly labeled spots or active caspases. Immunoanalysis of affinity labeled caspases demonstrated that caspase-3 was the major effector caspase. Western blot analysis also identified the activation of caspase-8 in the TNFalpha/ActD-treated cells, and the activation was suppressed by NO. Furthermore, NO inhibited several other events associated with caspase activation in cells, including release of cytochrome c from mitochondria, decrease in mitochondrial transmembrane potential, and cleavage of poly(ADP-ribose) polymerase in TNFalpha/ActD-treated cells. These findings indicate the involvement of multiple caspases in TNFalpha-mediated apoptosis in hepatocytes and establish the capacity of NO to inhibit not only active caspases but also caspase activation.     

Acetaldehyde-induced mitochondrial dysfunction sensitizes hepatocytes to oxidative damage

Acetaldehyde (Ac), the main metabolite of ethanol oxidation, is a very reactive compound involved in alcohol-induced liver damage. In the present work, we studied the effect of Ac in mitochondria functionality. Mitochondria from Wistar rats were isolated and treated with Ac. Ac decreased respiratory control by 50% which was associated with a decrease in adenosine triphosphate content (28.5%). These results suggested that Ac could be inducing changes in cell redox status. We determined protein oxidation, superoxide dismutase (SOD) activity, and glutathione ratio, indicating that Ac induced an enhanced oxidation of proteins and a decrease in SOD activity (90%) and glutathione/oxidized GSH ratio (36%). The data suggested that Ac-induced oxidative stress mediated by mitochondria dysfunction can lead to cell sensitization and to a second oxidative challenge. We pretreated hepatocytes with Ac followed by treatment with antimycin A, and this experiment revealed a noticeable decrease in cell viability, determined by neutral red assay, in comparison with cells treated with Ac alone. Our data demonstrate that Ac impairs mitochondria functionality generating oxidative stress that sensitizes cells to a second damaging signal contributing to the development of alcoholic liver disease.     

Mitochondrial dysfunction in choline deficiency-induced apoptosis in cultured rat hepatocytes

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.     

Insights into amyloid-β-induced mitochondrial dysfunction in Alzheimer disease

Amyloid-β has long been implicated in the pathogenesis of Alzheimer disease. The focus was initially on the extracellular fibrillar deposits of amyloid-β but more recently has shifted to intracellular oligomeric forms of amyloid-β. Unfortunately, the mechanism(s) by which either extracellular or intracellular amyloid-β induces neuronal toxicity remains unclear. That said, a number of recent studies indicate that mitochondria might be an important target of amyloid-β. Neurons rely heavily on mitochondria for energy and it is well established that mitochondrial dysfunction might be an important target of amyloid-β. Mechanistically, amyloid-β aggregates in mitochondria to impair function, leading to energy hypometabolism and elevated reactive oxygen species production. Additionally, amyloid-β affects the balance of mitochondrial fission/fusion and mitochondrial transport, negatively impacting a host of cellular functions of neurons. Here, we review the role that amyloid-β plays in mitochondrial structure and function of neurons and the importance of this in the pathogenesis of Alzheimer disease.     

Glutathione-S transferase activity in cultured human hepatocytes

Glutathione S-transferase (GST) activity was measured in human hepatocytes just after isolation and during culture in various media. Individual variations in enzyme activity, using 1-chloro-2,4-dinitrobenzene as substrate, were observed for freshly isolated human hepatocytes. When the hepatocytes were cultured, GST activity decreased dramatically during the first two days, to be stabilized around 30% of the initial value. Even factors known to favour maintenance of liver functions in vitro, such as nicotinamide and dimethylsulfoxide (DMSO), did not prevent this decline. In contrast to rat hepatocytes, no increase of GST activity was observed following the early decrease.     

Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes

Chronic alcohol consumption results in hepatotoxicity, steatosis, hypoxia, increased expression of inducible nitric oxide synthase (iNOS) and decreased activities of mitochondrial respiratory enzymes. The impact of these changes on cellular respiration and their interaction in a cellular setting is not well understood. In the present study we tested the hypothesis that nitric oxide (NO)-dependent modulation of cellular respiration and the sensitivity to hypoxic stress is increased following chronic alcohol consumption. This is important since NO has been shown to regulate mitochondrial function through its interaction with cytochrome c oxidase, although at higher concentrations, and in combination with reactive oxygen species, can result in mitochondrial dysfunction. We found that hepatocytes isolated from alcohol-fed rats had decreased mitochondrial bioenergetic reserve capacity and were more sensitive to NO-dependent inhibition of respiration under room air and hypoxic conditions. We reasoned that this would result in greater hypoxic stress in vivo, and to test this, wild-type and iNOS(-/-) mice were administered alcohol-containing diets. Chronic alcohol consumption resulted in liver hypoxia in the wild-type mice and increased levels of hypoxia-inducible factor 1 α in the peri-venular region of the liver lobule. These effects were attenuated in the alcohol-fed iNOS(-/-) mice suggesting that increased mitochondrial sensitivity to NO and reactive nitrogen species in hepatocytes and iNOS plays a critical role in determining the response to hypoxic stress in vivo. These data support the concept that the combined effects of NO and ethanol contribute to an increased susceptibility to hypoxia and the deleterious effects of alcohol consumption on liver.     

MPP(+)-induced mitochondrial dysfunction is potentiated by dopamine

MPP(+), the major metabolite of the Parkinsonism-inducing compound MPTP, responsible for the destruction of the nigrostriatal pathway in primates and rodents, has been assayed in isolated rat liver mitochondria in the presence of physiological concentrations of dopamine or analogous concentrations of melanin-dopamine. 5 microM MPP(+) in the presence of 70 microM dopamine or melanin-dopamine, but not alone, decreased the heat production and oxygen consumption of a mitochondrial suspension activated with succinate and ADP. Both dopamine and oxidized dopamine plus MPP(+) also decreased the mitochondrial reductive power measured with MTT. Mitochondrial swelling was observed, associated with an increase in membrane mitochondrial potential, as a synergistic effect between low concentrations of MPP(+) and dopamine. It is suggested that cytosolic dopamine, by itself or via its autooxidation products, may play a relevant role in the mitochondrial toxicity of MPP(+). A failure in the regulation of the storage/release of dopamine could aggravate a mitochondrial damage and trigger the neurodegenerative process underlying MPTP toxicity and Parkinson's disease.     

Aluminum-induced mitochondrial dysfunction leads to lipid accumulation in human hepatocytes: a link to obesity.

Mitochondrial dysfunction is the cause of a variety of pathologies associated with high energy-requiring tissues like the brain and muscles. Here we show that aluminum (Al) perturbs oxidative-ATP production in human hepatocytes (HepG2 cells). This Al-induced mitochondrial dysfunction promotes enhanced lipogenesis and the accumulation of the very low density lipoprotein (VLDL). Al-stressed HepG2 cells secreted more cholesterol, lipids and proteins than control cells. The level of apolipoprotein B-100 (ApoB-100) was markedly increased in the culture medium of the cells exposed to Al. (13)C-NMR and HPLC studies revealed a metabolic profile favouring lipid production and lowered ATP synthesis in Al-treated cells. Electrophoretic and immunoblot analyses pointed to increased activities and expression of lipogenic enzymes such as glycerol 3-phosphate dehydrogenase (G3PDH), acetyl CoA carboxylase (ACC) and ATP-citrate lyase (CL) in the hepatocytes exposed to Al, and a sharp diminution of enzymes mediating oxidative phosphorylation. D-Fructose elicited the maximal secretion of VLDL in the Al-challenged cells. These results suggest that the Al-evoked metabolic shift favours the accumulation of lipids at the expense of oxidative energy production in hepatocytes.     

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

Amyloid beta peptide 1-40 enhances the action of Toll-like receptor-2 and -4 agonists but antagonizes Toll-like receptor-9-induced inflammation in primary mouse microglial cell cultures

The interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells and macrophages after application of defined Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) (TLR4), the synthetic lipopeptide Pam3Cys-Ser-Lys4 (Pam3Cys) (TLR2) and single-stranded unmethylated CpG-DNA (CpG) (TLR9)] alone and in combination with amyloid beta peptide (Abeta) 1-40. Abeta1-40 stimulated microglial cells and macrophages primed by interferon-gamma in a dose-dependent manner. Co-administration of Abeta1-40 with LPS or Pam3Cys led to an additive release of nitric oxide (NO) and tumour necrosis factor alpha (TNF-alpha). This may be one reason for the clinical deterioration frequently observed in patients with Alzheimer's disease during infections. In contrast, co-application of Abeta1-40 with CpG led to a substantial decrease of NO and TNF-alpha release compared with stimulation with CpG alone. Abeta1-40 and CpG did not co-localize within the same subcellular compartment, making a direct physicochemical interaction as the cause of the observed antagonism very unlikely. This suggests that not all TLR agonists enhance the stimulatory effect of A beta on innate immunity.     

Attenuating effects of heat shock against TGF-beta1-induced apoptosis in cultured rat hepatocytes

Heat shock proteins (HSPs) induction confers protection against diverse forms of cellular injury. However, the mechanism by which HSPs exert cytoprotective effects remains unclear. Treatment of rat hepatocyte with transforming growth factor-beta1 (TGF-beta1) induces growth arrest followed by extensive cell death by apoptosis. In this study, the effects of preexposure to heat on TGF-beta1-induced apoptosis of cultured hepatocytes were examined. Treatment of hepatocytes for 24 h with TGF-beta1 resulted in significant apoptotic cell death, as demonstrated by DNA fragmentation, caspase activation, and hypodiploid DNA peak. Moreover, TGF-beta1-induced cell death was accompanied by an enhanced generation of reactive oxygen species and a loss of the mitochondrial membrane potential. These effects were attenuated when the hepatocytes were subjected to 43 degrees C for 20 min prior to the cytokine stimulation. The enhancement in HSP70 expression at mRNA and protein levels induced by heat preexposure was accompanied by an increase in mRNA levels of intracellular antioxidant enzymes. Heat treatment also prevented TGF-beta1-induced activation of nuclear factor kappa B (NF-kappaB) by preventing the degradation of the inhibitory protein kappa Balpha (IkappaBalpha). In conclusion, these data indicate that in the mechanism by which a mild heat pretreatment increases the resistance of hepatocytes to TGF-beta1-induced apoptotic cell death, the upregulation of catalase expression and a decrease in ROS generation are involved.     

Synthesis of transcobalamin II by cultured human hepatocytes

Cultured HepG2 cells, derived from a human hepatoma synthesized and released unsaturated, immunoreactive transcobalamin II. Synthesis was confirmed by the blocking with inhibitors of protein synthesis and by incorporation of tritiated leucine into transcobalamin II.     

3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPARα) signaling. Furthermore, using PPARα agonists and antagonists, we also analyzed the effect of PPARα signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.     

Effect of Salvia miltiorrhiza on aflatoxin B1-induced oxidative stress in cultured rat hepatocytes

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B₁ (AFB₁). Salvia miltiorrhiza (Sm), a herbal plant that has been used extensively in traditional Chinese medicine for treating cardiovascular and liver diseases, is believed to have some antioxidative capabilities. In this study, the protective effect of Sm against AFB₁-induced cytotoxicity was investigated in cultured primary rat hepatocytes. AFB₁-induced cytotoxicity and lipid peroxidation (LPO) were estimated by determination of lactate dehydrogenase (LDH) leakage and thiobarbituric acid reactive substances (TBARS) formation, respectively. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). In addition, changes of intracellular glutathione (GSH) content were also studied. Results showed that Sm was able to suppress the LDH leakage induced by AFB₁ in a dose-dependent manner. A dose-dependent inhibitory effect of Sm on AFB₁-induced LPO was also found in hepatocytes treated with Sm. It was further observed that Sm produced an inhibitory effect on ROS formation caused by AFB₁. Concomitantly, the GSH content in Sm-treated groups increased substantially compared to those without Sm treatment. These findings suggest that Sm can inhibit the cytotoxicity of AFB₁ through decreasing ROS formation, inhibiting LPO and preventing GSH depletion. The major component of the aqueous extract of Sm was identified by using high performance liquid chromatography (HPLC), proton magnetic resonance (¹H-NMR) and mass spectrum (MS). Analytical results suggested that D(+)β3,4-dihydroxyphenol lactic acid (DA) is the main compound of the aqueous extract of Sm.     

Cu-induced mitochondrial dysfunction is mediated by abnormal mitochondrial fission through oxidative stress in primary chicken embryo hepatocytes

BackgroundExcess copper (Cu) is an oxidative stress factor which associates with a variety of diseases. The aim of this study was to evaluate the effect of Cu in primary chicken embryo hepatocytes (CEHs).MethodsCEHs were isolated from 13 days old chicken embryos and followed by different concentration Cu (0, 10, 100, 200 μM) and/or ALC treatment (0.3 mg/mL) for 12 or 24 h. The effects of Cu exposure in CEHs were determined by detecting reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (MMP), and ATP levels. The expression of mitochondrial dynamics-related genes and proteins were also detected.ResultsResults showed that Cu treatment (100 or 200 μM) significantly decreased CEHs viability, MMP and ATP levels, increased ROS and MDA levels in 12 or 24 h. The up-regulated mitochondrial fission genes and protein in 100 and 200 μM Cu groups suggested Cu promoted mitochondrial division but not fusion. However, the co-treatment of ALC and Cu alleviated those changes compared with the 100 or 200 μM Cu groups.ConclusionIn conclusion, we speculated that Cu increased the oxidative stress and induced mitochondria dysfunction via disturbing mitochondrial dynamic balance in CEHs, and this process was not completely reversible.     

Effect of Salvia miltiorrhiza on aflatoxin B1-induced oxidative stress in cultured rat hepatocytes

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B₁ (AFB₁). Salvia miltiorrhiza (Sm), a herbal plant that has been used extensively in traditional Chinese medicine for treating cardiovascular and liver diseases, is believed to have some antioxidative capabilities. In this study, the protective effect of Sm against AFB₁-induced cytotoxicity was investigated in cultured primary rat hepatocytes. AFB₁-induced cytotoxicity and lipid peroxidation (LPO) were estimated by determination of lactate dehydrogenase (LDH) leakage and thiobarbituric acid reactive substances (TBARS) formation, respectively. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). In addition, changes of intracellular glutathione (GSH) content were also studied. Results showed that Sm was able to suppress the LDH leakage induced by AFB₁ in a dose-dependent manner. A dose-dependent inhibitory effect of Sm on AFB₁-induced LPO was also found in hepatocytes treated with Sm. It was further observed that Sm produced an inhibitory effect on ROS formation caused by AFB₁. Concomitantly, the GSH content in Sm-treated groups increased substantially compared to those without Sm treatment. These findings suggest that Sm can inhibit the cytotoxicity of AFB₁ through decreasing ROS formation, inhibiting LPO and preventing GSH depletion. The major component of the aqueous extract of Sm was identified by using high performance liquid chromatography (HPLC), proton magnetic resonance (¹H-NMR) and mass spectrum (MS). Analytical results suggested that D(+)β3,4-dihydroxyphenol lactic acid (DA) is the main compound of the aqueous extract of Sm.     

Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts

Protease-activated receptors (PARs) have been implicated in the development of acute and chronic inflammatory responses. We have examined the expression of mRNA for PARs and their regulation by growth factors and cytokines in synovial fibroblasts derived from patients with rheumatoid arthritis (RA). Messenger RNA for PAR-1, -2 and -3 was detected in these cells, but not that for PAR-4. Expression of mRNA for PAR-2 was up-regulated by bFGF in a concentration-dependent manner, whereas expression of mRNA for PAR-1 and PAR-3 was not affected. Levels of mRNA encoding PAR-1, PAR-2 and PAR-3 did not increase in response to IL-1beta and TNF-alpha. Expression of mRNA for PAR-2 was maximal 12 h after addition of bFGF, and maximal levels of immunoreactive PAR-2 were reached after 24 h. Furthermore, PAR-2 agonist peptide (SLIGKV-NH(2)), but not the inactive reverse peptide (VKGILS-NH(2)), induced transitory cytosolic Ca(2+) mobilization in cells, and its response was increased by pretreatment with bFGF. An important role could be played by bFGF in the regulation of functional PAR-2 expression in cultured RA synovial fibroblasts.