Lysosomal Injury Research Articles

Galectins Control mTOR in Response to Endomembrane Damage

The Ser/Thr protein kinase mTOR controls metabolic pathways, including the catabolic process of autophagy. Autophagy plays additional, catabolism-independent roles in homeostasis of cytoplasmic endomembranes and whole organelles. How signals from endomembrane damage are transmitted to mTOR to orchestrate autophagic responses is not known. Here we show that mTOR is inhibited by lysosomal damage. Lysosomal damage, recognized by galectins, leads to association of galectin-8 (Gal8) with the mTOR apparatus on the lysosome. Gal8 inhibits mTOR activity through its Ragulator-Rag signaling machinery, whereas galectin-9 activates AMPK in response to lysosomal injury. Both systems converge upon downstream effectors including autophagy and defense against Mycobacterium tuberculosis. Thus, a novel galectin-based signal-transduction system, termed here GALTOR, intersects with the known regulators of mTOR on the lysosome and controls them in response to lysosomal damage. VIDEO ABSTRACT.

Xylene Induces Oxidative Stress and Mitochondria Damage in Isolated Human Lymphocytes

Xylene is a cyclic hydrocarbon and an environmental pollutant. It is also used in medical technology, paints, dyes, polishes and in many industries as a solvent; therefore, an understanding of the interaction between xylene and human lymphocytes is of significant interest. Biochemical assessment was used to demonstrate that exposure of lymphocytes to xylene induces cytotoxicity (at 6 hr), generates intracellular reactive oxygen species, collapse of mitochondrial membrane potential, lysosomal injury, lipid peroxidation and depletion of glutathione (at 3 hr). The findings show that xylene triggers oxidative stress and organelle damage in lymphocytes. The results of our study suggest that the use of antioxidant, mitochondrial and lysosomal protective agents can be helpful for individuals subject to chronic exposure to xylene.

Protective effect of antioxidants contained in milk-based fruit beverages against sterol oxidation products

Sterol oxidation products (SOPs) have shown cytotoxic effect in human intestinal cells; however, their effect within a food matrix has not been assayed yet. This study evaluated the possible cytotoxic effect of SOPs within bioaccessible fractions (BFs) of two milk-based fruit beverages with (BFA)/without (BFB) plant sterols in differentiated Caco-2 cells and if the BFs counteracted the cytotoxic effect induced by COPs mixture (30 and 60μM). BFs did not evoke cytotoxic effect in any of the tests carried out and they protected against the loss of intestinal cohesion, mitochondrial depolarization and necrosis induced by COPs mixture. Moreover, BFB sample protected from cell cycle arrest, lessened lysosomal injury and overproduction of ROS at 30μM. Therefore, the formation of SOPs within milk-based fruit beverages with/without plant sterols does not contribute to intestinal epithelium damage and these types of food matrices confer additional protection against COPs at dietary concentrations.

PALLADIUM INDUCED OXIDATIVE STRESS AND CELL DEATH IN NORMAL HEPATOCYTES

Palladium (Pd) accumulates in many organs and renders many deleterious effects. Although the Pd toxicity has been documented, the precise mechanism of Pd toxicity still needs to be elucidated. In the current research, a hepatotoxicity mechanism of Pd has been investigated. Our findings clearly indicate that Pd induces reactive oxygen species (ROS) formation and oxidative stress, mitochondrial and lysosomal injury and finally cell death. These effects are reversed by antioxidants and ROS scavengers, mitochondrial permeability transmission [1] pore sealing agent, ATP progenitor, and lysosomotropic agent. Pretreatment of hepatocytes with ROS scavengers and MPT pore sealing agents reduced cell death which explains the role of oxidative stress and mitochondrial pathway of ROS formation in Pd hepatocytes cell toxicity. Overall, the results have distinctly determined the mechanism by which Pd-induced toxicity in the isolated rat hepatocytes. Keywords : Palladium; rat hepatocytes; reactive oxygen species; mitochondria; lysosome.

Surfactant-free poly(lactide-co-glycolide) nanoparticles for improving in vitro anticancer efficacy of tetrandrine

The objective of this study was to improve the efficacy of a natural compound tetrandrine against cancer by designing surfactant-free poly(lactic-co-glycolic acid) (PLGA) nanoparticles as drug carriers for tetrandrine. Nanoparticles were prepared from PLGA via the nano-precipitation method with or without the presence of surfactant poly(vinyl alcohol) (PVA) to encapsulate tetrandrine. Tetrandrine-loaded surfactant-free PLGA nanoparticles had an average particle size of 169.3 nm and morphology similar to the PLGA nanoparticles prepared using PVA as the surfactant. Tetrandrine-loaded surfactant-free PLGA nanoparticles could retard drug release in phosphate buffered saline (PBS) at pH 7.4 and the cumulative release of tetrandrine reached up to 68.33% over a period of 120 h. A549 cell line was used as the model cancer cells to investigate anticancer capability of tetrandrine-loaded surfactant-free PLGA nanoparticles via apoptosis assay, cytotoxicity and lysosome injury studies. The results showed that tetrandrine-loaded surfactant-free PLGA nanoparticles could effectively reduce cell viability and synergistically enhance tetrandrine-induced cell apoptosis.

Enhancing the imaging and biosafety of upconversion nanoparticles through phosphonate coating.

Upconversion nanoparticles (UCNPs), which are generated by doping with rare earth (RE) metals, are increasingly used for bioimaging because of the advantages they hold over conventional fluorophores. However, because pristine RE nanoparticles (NPs) are unstable in acidic physiological fluids (e.g., lysosomes), leading to intracellular phosphate complexation with the possibility of lysosomal injury, it is important to ensure that UCNPs are safely designed. In this study, we used commercially available NaYF4:Er/Yb UCNPs to study their stability in lysosomes and simulated lysosomal fluid. We demonstrate that phosphate complexation leads to REPO4 deposition on the particle surfaces and morphological transformation. This leads to a decline in upconversion fluorescence efficiency as well as inducing pro-inflammatory effects at the cellular level and in the intact lung. In order to preserve the imaging properties of the UCNPs as well as improve their safety, we experimented with a series of phosphonate chemical moieties to passivate particle surfaces through the strong coordination of the organophosphates with RE atoms. Particle screening and physicochemical characterization revealed that ethylenediamine tetra(methylenephosphonic acid) (EDTMP) surface coating provides the most stable UCNPs, which maintain their imaging intensity and do not induce pro-inflammatory effects in vitro and in vivo. In summary, phosphonate coating presents a safer design method that preserves and improves the bioimaging properties of UCNPs, thereby enhancing their biological use.

Endocytosis of Multiwalled Carbon Nanotubes in Bronchial Epithelial and Mesothelial Cells.

Bronchial epithelial cells and mesothelial cells are crucial targets for the safety assessment of inhalation of carbon nanotubes (CNTs), which resemble asbestos particles in shape. Intrinsic properties of multiwalled CNTs (MWCNTs) are known to cause potentially hazardous effects on intracellular and extracellular pathways. These interactions alter cellular signaling and affect major cell functions, resulting in cell death, lysosome injury, reactive oxygen species production, apoptosis, and cytokine release. Furthermore, CNTs are emerging as a novel class of autophagy inducers. Thus, in this study, we focused on the mechanisms of MWCNT uptake into the human bronchial epithelial cells (HBECs) and human mesothelial cells (HMCs). We verified that MWCNTs are actively internalized into HBECs and HMCs and were accumulated in the lysosomes of the cells after 24-hour treatment. Next, we determined which endocytosis pathways (clathrin-mediated, caveolae-mediated, and macropinocytosis) were associated with MWCNT internalization by using corresponding endocytosis inhibitors, in two nonphagocytic cell lines derived from bronchial epithelial cells and mesothelioma cells. Clathrin-mediated endocytosis inhibitors significantly suppressed MWCNT uptake, whereas caveolae-mediated endocytosis and macropinocytosis were also found to be involved in MWCNT uptake. Thus, MWCNTs were positively taken up by nonphagocytic cells, and their cytotoxicity was closely related to these three endocytosis pathways.

A lysosome-targeted fluorescent chemodosimeter for monitoring endogenous and exogenous hydrogen sulfide by in vivo imaging.

A lysosome-targeted fluorescent chemodosimeter, 1, was developed for monitoring endogenous and exogenous H2S by in vivo imaging of HeLa cells, D. melanogaster and C. elegans. In the tests of mutated C. elegans (SRP-6 nulls), chemodosimeter 1 could trace the accumulation of lysosome and lysosomal injury with a high resolution.

Comparison of cellular and molecular cytotoxic mechanisms of Cochlodinium polykrikoides in isolated trout and rat hepatocytes

Harmful algal blooms produced by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish globally. This study compared the cytotoxic mechanisms of C. polykrikoides total extract on both trout and rat liver hepatocytes. Trout hepatocytes were more sensitive than rat hepatocytes against C. polykrikoides extract. The effective concentration 50 after 3 hour incubation (EC503hr) concentrations found for C. polykrikoides extract in trout and rat hepatocytes (i.e., 50% membrane lysis in 3 hr) were Eq. 1 cell/ml and Eq. 240 cell/ml, respectively. C. polykrikoides extract exposure in both isolated trout and rat hepatocytes resulted in membrane lysis, reactive oxygen species formation, glutathione depletion, collapse of mitochondrial membrane potential, ATP depletion, increase in adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio, cytochrome c release into the hepatocyte cytosol, and activation of caspases cascade. Trout hepatocyte toxicity was also associated with lysosomal membrane injury. Mitochondrial permeability transition in both trout and rat hepatocytes produced cytochrome c release from the mitochondrial intramembrane space into the cytosol. Thus, the cytochrome c release triggered activation of caspase-3 and apoptosis. Finally, data demonstrated that C. polykrikoides extract may induce more apoptotic phenotype in rat than trout hepatocytes, which in the latter favored predominantly necrotic mode of cell death.

Hepcidin knockout mice fed with iron-rich diet develop chronic liver injury and liver fibrosis due to lysosomal iron overload

Hepcidin is the central regulator of iron homeostasis and altered hepcidin signalling results in both hereditary and acquired iron overload. While the association between iron overload and development of end-stage liver disease is well established, the underlying mechanisms are largely unknown. To improve that, we analysed hepcidin knockout (KO) mice as a model of iron overload-associated liver disease. Hepcidin wild type (WT) and KO mice fed with 3% carbonyl iron-containing diet starting at one month of age were compared to age-matched animals kept on standard chow. Liver histology and serum parameters were used to assess the extent of liver injury and fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy. Among mice kept on iron-rich diet, 6 months old hepcidin KO mice (vs. WT) displayed profound hepatic iron overload (3,186 ± 411 vs. 1,045 ± 159 μg/mg tissue, p<0.005), elevated liver enzymes (ALT: KO 128 ± 6, WT 56 ± 5 IU/L, p<0.05), mild hepatic inflammation and hepatocellular apoptosis. Twelve, but not six months old KO mice fed with iron-rich diet developed moderate liver fibrosis. The liver injury was accompanied by a marked lysosomal iron overload and lysosomal fragility with release of cathepsin B into the cytoplasm. Increased p62 levels and autofluorescent iron complexes suggested impaired protein degradation. As a mechanism leading to lysosomal iron overload, the autophagy (lysosomal influx) was increased. Hepcidin KO mice represent a novel model of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.

Accelerated atherosclerosis in individuals with the haptoglobin 2-2 genotype and diabetes is mediated by macrophage lysosomal injury and apoptosis

Background: There exists a common functional allelic polymorphism in the Haptoglobin (Hp) gene which has been associated with the risk of cardiovascular disease in individuals with diabetes mellitus (DM). In the atherosclerotic plaque, the CD163 macrophage Hp-hemoglobin (Hb) receptor is responsible for clearing Hb released after intraplaque hemorrhage. In DM, the Hp 2 allele protein product is defective in its Hb scavenging properties and has been associated with increased iron in the atherosclerotic plaque.

Genotoxic effects induced by zearalenone in a human embryonic kidney cell line

Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEA) is a hepatotoxic mycotoxin with estrogenic and anabolic activity found in cereal grains worldwide. ZEA affects hematological and immunological parameters in humans and rodents. The compound can induce cell death, cause lipid peroxidation, inhibit protein and DNA synthesis, and exert genotoxic effects. ZEA may cause increased phagolysosomal fragility in the kidney. Our research showed that exposure of human embryonic kidney (HEK293) cells to ZEA (10 or 20μM) resulted in a concentration-dependent increase in DNA strand breaks measured with the comet assay. Damage was reduced in cells pretreated with NH4Cl, pepstatin A, or desipramine for 1h. Production of reactive oxygen species (ROS) was increased in cells exposed to ZEA, but DNA strand break induction could not be inhibited by the antioxidant hydroxytyrosol (HT). These results suggest that oxidative stress does not play a key role in DNA strand breaks induced by ZEA, that lysosomal injury precedes DNA strand breaks, and that the lysosome may be a primary target for ZEA in HEK293 cells.

Hepcidin knockout mice develop chronic liver injury and liver fibrosis as a consequence of lysosomal iron overload

Background and Aims: Hepcidin is the central regulator of iron homeostasis. Disrupted hepcidin signaling results in hereditary hemochromatosis and iron overload seen in chronic liver disorders. While the association between iron overload and development of end-stage liver disease is well established, the understanding of the underlying mechanisms is hampered by the lack of a suitable animal model. To circumvent this problem, we analyzed hepcidin-knockout (KO) mice as a model of iron-overload associated liver disease. Methods: Hepcidin wild-type (WT) and KO animals fed 3% carbonyl iron-containing chow were compared to mice kept on standard diet. Liver histology and serum parameters were employed to assess the extent of liver injury/fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy. Results: Among mice kept on iron-rich diet, 6 month old hepcidin KOs (vs. WTs) displayed profound hepatic iron overload (2543±114 vs. 1493±136p<0,005), elevated liver enzyme (AST: KO 261±15, WT 142±34p<0,05) and serum iron levels, mild hepatocellular inflammation and apoptosis. 12, but not 6 month old KOs fed iron-rich diet developed moderate liver fibrosis as determined by Sirius red staining and increased hydroxyproline levels. The liver injury was likely due to a marked lysosomal iron overload and lysosomal fragility with release of cathepsins into the cytoplasm, while no increased lipid peroxidation or protein carbonylation was noted. As a potential mechanism, the expression of DMT1 and STEAP3, i.e. the molecules needed for lysosomal iron export, was greatly reduced. Conclusions: Hepcidin KOs represent a unique tool to study the mechanism of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.

Abstract P5-04-09: Squamous Cell Carcinoma Antigen 1 (SCCA1) Upregulation Causes Epithelial to Mesenchymal Transition (EMT) and Promotes Mammary Tumorigenesis

Abstract SCCA1, a member of the family of serine protease inhibitors, was first identified as a serological marker for cervical cancer but has since been shown to be upregulated in several other cancers including lung, head and neck, liver, skin, and breast. SCCA1 is believed to protect cells from unscheduled cathepsin release and activation upon lysosomal injury. In order to understand the molecular role of SCCA in cancer, we ectopically expressed it in the immortalized yet non-tumorigenic mammary epithelial cell line MCF10A. Expression of SCCA1 impairs the efficiency of lysosomal as well as proteasomal protein degradation pathways. Transcriptional profiling of SCCA expressing cells by microarray demonstrates an altered gene expression pattern that resembles EMT. Phenotypically, SCCA1 expression leads to spindle shaped cell morphology, increased migratory potential, resistance to anoikis, and the ability to form colonies in soft agar. SCCA1-expressing MCF10A cells can form tumors when injected into the mammary fat pad of immune-compromised mice. Correlative to the transforming activity, SCCA1 expressing cells have enhanced Erk and Akt signaling pathways, and have bypassed the requirement of growth factors and hormones that are essential for proliferation of MCF10A cells. In a transgenic mouse model, where SCCA1 is conditionally expressed in mammary epithelial cells under the control of MMTV promoter, increased mammary gland ductal branching and hyperplasia are observed. These findings indicate a previously unknown role of SCCA1 in promoting EMT and tumorigenesis, possibly by inducing chronic misfolded protein stress that contributes to enhance tumor-promoting signaling. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-09.

Polydatin – a new mitochondria protector for acute severe hemorrhagic shock treatment

Objective: The aim of the study was find out whether neuronal mitochondrial injury does take place in severe shock and to explore effective therapy for severe shock.Research design and methods: Rats were divided in the following group: sham, shock + normal saline (NS), shock + cyclosporine A (CsA), shock + resveratrol (Res) and shock + polydatin (PD). Rats were subjected to shock for 2 h, followed by administration of NS, CsA, Res and PD, and infusion of shed blood. Morphology, metabolism and function of mitochondria were measured.Results: Increased lipid peroxides (LPO) levels, lysosomal injury and mitochondrial permeability transition pore opening took place in neurons, resulting in swollen mitochondria with poorly defined cristae, decreased mitochondrial membrane potential (ΔΨ) and reduced ATP content in shock + NS group, indicating mitochondrial dysfunction. Mitochondrial protectors, such as CsA, Res and PD, partially inhibited these alterations, especially following PD protection, ATP level increased from 44.14 ± 13.81% in shock + NS group to 89.57 ± 9.21% and the survival time was prolonged from 6.3 ± 5.9 h in the shock + NS group to 31.6 ± 13.7 h in shock + PD group.Conclusions: The study shows that neuronal mitochondrial injury is involved in the genesis of severe shock and PD may be the best choice for protection of neuron against mitochondrial injury in severe shock.

Rejuvenating Bi(d)ology

The BH3-only Bid protein is a critical sentinel of cellular stress in the liver and the hematopoietic system. Bid's initial 'claim to fame' came from its ability-as a caspase-truncated product-to trigger the mitochondrial apoptotic program following death receptor activation. Today we know that Bid can response to multiple types of proteases, which are activated under different conditions such as T-cell activation, ischemical reperfusion injury and lysosomal injury. Activation of the mitochondrial apoptotic program by Bid-via its recently identified receptor mitochondrial carrier homolog 2-involves multiple mechanisms, including release of cytochrome c and second mitochondria-derived activator of caspase (Smac), alteration of mitochondrial cristae organization, generation of reactive oxygen species and engagement of the permeability transition pore. Bid is also emerging-in its full-length form-as a pivotal sentinel of DNA damage in the bone marrow regulated by the ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR) kinases. The ATM/ATR-Bid pathway is critically involved in preserving the quiescence and survival of hematopoietic stem cells both in the absence and presence of external stress, and a large part of this review will be dedicated to recent advances in this area of research.

Pluronic F108 coating decreases the lung fibrosis potential of multiwall carbon nanotubes by reducing lysosomal injury.

We compared the use of bovine serum albumin (BSA) and pluronic F108 (PF108) as dispersants for multiwalled carbon nanotubes (MWCNTs) in terms of tube stability as well as profibrogenic effects in vitro and in vivo. While BSA-dispersed tubes were a potent inducer of pulmonary fibrosis, PF108 coating protected the tubes from damaging the lysosomal membrane and initiating a sequence of cooperative cellular events that play a role in the pathogenesis of pulmonary fibrosis. Our results suggest that PF108 coating could serve as a safer design approach for MWCNTs.

Accidental potassium dichromate poisoning. Toxicokinetics of chromium by ICP-MS-CRC in biological fluids and in hair

Intoxications by chromium (Cr) compounds are very life threatening and often lethal. After oral ingestion of 2 or 3g of hexavalent Cr (Cr(VI)), gastrointestinal injury, but also hepatic and renal failure, often occurs which each leads to a fatal outcome in most patients. Cellular toxicity is associated with mitochondrial and lysosomal injury by biologically Cr(VI) reactive intermediates and reactive oxygen species. After Cr(VI) has been absorbed, there is not much that can be done except to control the main complications as the treatment is only symptomatic. The biotransformation of Cr(VI) to Cr(III) reduces the toxicity because the trivalent form does not cross cellular membranes as rapidly. In fact, more than 80% of Cr(VI) is cleared in urine as Cr(III). We report the case of a 58-year-old male patient who was admitted to hospital after accidental oral ingestion of a 30 g/L potassium dichromate (the estimated amount of ingested Cr is about 3g). ICP-MS equipped with a collision/reaction cell (CRC) and validated methods were used to monitor plasma (P), red blood cells (RBCs), urine (U) and hair chromium. For urine the results were expressed per gram of creatinine. After 7 days in the intensive care unit, the patient was discharged without renal or liver failure. P, RBC and U were monitored during 49 days. During this period Cr decreased respectively from 2088 μg/L to 5 μg/L, 631 μg/L to 129 μg/L and 3512 μg/g to 10 μg/g. The half-life was much shorter in P than in RBC as the poison was more quickly cleared from the P than from the RBC, suggesting a cellular trapping of the metal. Hair was collected 2 months after the intoxication. We report a very rare case of survival after accidental Cr poisoning which has an extremely poor prognosis and usually leads to rapid death. For the first time, this toxicokinetic study highlights a sequestration of chromium in the RBC and probably in all the cells.

Squamous Cell Carcinoma Antigen 1 Promotes Caspase-8-Mediated Apoptosis in Response to Endoplasmic Reticulum Stress While Inhibiting Necrosis Induced by Lysosomal Injury

Squamous cell carcinoma antigen 1 (SCCA1) is a member of the serine protease inhibitor (serpin) family of proteins, whose target proteases include the cathepsins. Initially identified as a serological marker for advanced squamous cell carcinomas of the cervix, SCCA1 has also been found to be associated with other cancer types of epithelial or endodermal origins such as lung cancer, head and neck cancer, melanoma, and hepatocellular carcinoma. While the biological function of SCCA1 remains largely unclear, it is believed to limit cellular damage resulting from lysosomal cathepsin release. Here, we show that SCCA1 acts as a molecular switch that inhibits cell death induced by lysosomal injury resulting from DNA alkylating agents and hypotonic shock, whereas it promotes a caspase-8-mediated apoptosis in response to endoplasmic reticulum (ER) stress. In response to ER stress, SCCA1 blocks both lysosomal and proteasomal protein degradation pathways and enhances the interaction between sequestosome 1/p62 and caspase-8, which leads to the aggregation of intracellular caspase-8 and its subsequent cleavage and activation. Hence, on one hand, SCCA1 inhibits cell death induced by lysosomal injury while, on the other hand, it sensitizes cells to ER stress by activating caspase-8 independently of the death receptor apoptotic pathway.

Elucidation mechanism of different biological responses to multi-walled carbon nanotubes using four cell lines

We examined differences in cellular responses to multi-walled carbon nanotubes (MWCNTs) using malignant pleural mesothelioma cells (MESO-1), bronchial epithelial cells (BEAS-2B), neuroblastoma cells (IMR-32), and monoblastic cells (THP-1), before and after differentiation. MESO-1, BEAS-2B and differentiated THP-1 cells actively endocytosed MWCNTs, resulting in cytotoxicity with lysosomal injury. However, cytotoxicity did not occur in IMR-32 or undifferentiated THP-1 cells. Both differentiated and undifferentiated THP-1 cells exhibited an inflammatory response. Carbon blacks were endocytosed by the same cell types without lysosomal damage and caused cytokine secretion, but they did not cause cytotoxicity. These results indicate that the cytotoxicity of MWCNTs requires not only cellular uptake but also lysosomal injury. Furthermore, it seems that membrane permeability or cytokine secretion without cytotoxicity results from several active mechanisms. Clarification of the cellular recognition mechanism for MWCNTs is important for developing safer MWCNTs.