Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

Esterification promotes the intracellular accumulation of roxadustat, an activator of hypoxia-inducible factors, to extend its effective duration.

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.

Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells.

Tributyltin (TBT) is an environmental pollutant that remains in marine sediments and is toxic to mammals. For example, TBT elicits neurotoxic and immunosuppressive effects on rats. However, it is not entirely understood how TBT causes toxicity. Autophagy plays a pivotal role in protein quality control and eliminates aggregated proteins and damaged organelles. We previously reported that TBT dephosphorylates mammalian target of rapamycin (mTOR), which may be involved in enhancement of autophagosome synthesis, in primary cultures of cortical neurons. Autophagosomes can accumulate due to enhancement of autophagosome synthesis or inhibition of autophagic degradation, and we did not clarify whether TBT alters autophagic flux. Here, we investigated the mechanism by which TBT causes accumulation of autophagosomes in SH-SY5Y cells. TBT inhibited autophagy without affecting autophagosome-lysosome fusion before it caused cell death. TBT dramatically decreased the acidity of lysosomes without affecting lysosomal membrane integrity. TBT decreased the mature protein level of cathepsin B, and this may be related to the decrease in lysosomal acidity. These results suggest that TBT inhibits autophagic degradation by decreasing lysosomal acidity. Autophagy impairment may be involved in the mechanism underlying neuronal death and/or T-cell-dependent thymus atrophy induced by TBT.

Methylmercury induces lysosomal membrane permeabilization through JNK-activated Bax lysosomal translocation in neuronal cells.

MeHg, an environmental toxicant, is highly toxic to the central nervous system. Recent studies have reported that LMP is an important way in the lysosomal damage. However, the role and molecular mechanism of LMP in MeHg-induced neurotoxicity remain unknown. To study MeHg-induced LMP, we used 10µM MeHg to treat SH-SY5Y cells and 2µM MeHg to treat rat cerebral cortical neurons. Acridine orange (AO) staining and analysis of cathepsin B (CTSB) release were used to determine LMP. We found that MeHg reduced red AO fluorescence and induced CTSB release from lysosomes to the cytoplasm in a time-dependent manner. Moreover, pretreatment with the CTSB inhibitor alleviated cytotoxicity in neuronal cells. These results indicate MeHg induces LMP and subsequent CTSB-dependent cytotoxicity in neuronal cells. Bax is a pore-forming protein, which is involved in mitochondrial outer membrane permeabilization. Intriguingly, we demonstrated that MeHg induced Bax to translocate to lysosomes by using immunofluorescence and Western blot analysis of subcellular fractions. Furthermore, downregulating Bax expression suppressed MeHg-induced LMP. Bax subcellular localization is regulated by protein interaction with the cytoplasmic 14-3-3. Our previous study demonstrated that JNK participated in neurotoxicity through regulating protein interaction. In the current study, we showed that JNK dissociated Bax-14-3-3 complex to facilitate Bax lysosomal translocation. Finally, inhibition of the JNK/Bax pathway could alleviate MeHg-induced cytotoxicity in neuronal cells. The present study implies that inhibiting lysosomal damage (LMP)-related signaling might alleviate MeHg neurotoxicity.

Harnessing autophagy to fight SARS-CoV-2: An update in view of recent drug development efforts.

Drug repurposing is an attractive option for identifying new treatment strategies, in particular in extraordinary situations of urgent need such as the current coronavirus disease 2019 (Covid-19) pandemic. Recently, the World Health Organization announced testing of three drugs as potential Covid-19 therapeutics that are known for their dampening effect on the immune system. Thus, the underlying concept of selecting these drugs is to temper the potentially life-threatening overshooting of the immune system reacting to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This viewpoint discusses the possibility that the impact of these and other drugs on autophagy contributes to their therapeutic effect by hampering the SARS-CoV-2 life cycle.

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1-A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy.

An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum.

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.

Understanding the interactions between inorganic-based nanomaterials and biological membranes.

The interactions between inorganic-based nanomaterials (NMs) and biological membranes are among the most important phenomena for developing NM-based therapeutics and resolving nanotoxicology. Herein, we introduce the structural and functional effects of inorganic-based NMs on biological membranes, mainly the plasma membrane and the endomembrane system, with an emphasis on the interface, which involves highly complex networks between NMs and biomolecules (such as membrane proteins and lipids). Significant efforts have been devoted to categorizing and analyzing the interaction mechanisms in terms of the physicochemical characteristics and biological effects of NMs, which can directly or indirectly influence the effects of NMs on membranes. Importantly, we summarize that the biological membranes act as platforms and thereby mediate NMs-immune system contacts. In this overview, the existing challenges and potential applications in the areas are addressed. A strong understanding of the discussed concepts will promote therapeutic NM designs for drug delivery systems by leveraging the NMs-membrane interactions and their functions.

Betaine Delayed Muscle Loss by Attenuating Samtor Complex Inhibition for mTORC1 Signaling Via Increasing SAM Level.

SCOPE: The muscle loss during aging results from the blunt of protein synthesis and poses threat to the elderly health. This study aims to investigate whether betaine affects muscle loss by improving protein synthesis. METHODS AND RESULTS: Male C57BL/6J mice are raised from age 12 or 15 months. Mice are fed with AIN-93M diet without or with 2% w/v betaine in distilled water as control group or betaine intervention group (Bet), respectively. Betaine supplementation to mice demonstrates better body composition, grip strength, and motor function. Muscle morphology upregulates expression of myogenic regulate factors, and elevates myosin heavy chain and also improves in Bet group. Betaine promotes muscle protein synthesis via tethering mammalian target of rapamycin complex1 protein kinase (mTORC1) on the lysosomal membrane thereby activating mTORC1 signaling. All these effects aforementioned are time-dependent (p < 0.05). Ultrahigh-performance liquid chromatography results show that betaine increases S-adenosyl-l-methionine (SAM) via methionine cycle. SAM sensor-Samtor-overexpression in C2C12 cells could displace mTORC1 from lysosome thereby inhibiting the mTORC1 signaling. Addition of betaine attenuates this inhibition by increasing SAM level and then disrupting interaction of Samtor complex. CONCLUSIONS: These observations indicate that betaine could promisingly promote protein synthesis to delay age-related muscle loss.

Caspase-8 deficiency induces a switch from TLR3 induced apoptosis to lysosomal cell death in neuroblastoma.

In cancer cells only, TLR3 acquires death receptor properties by efficiently triggering the extrinsic pathway of apoptosis with Caspase-8 as apical protease. Here, we demonstrate that in the absence of Caspase-8, activation of TLR3 can trigger a form of programmed cell death, which is distinct from classical apoptosis. When TLR3 was activated in the Caspase-8 negative neuroblastoma cell line SH-SY5Y, cell death was accompanied by lysosomal permeabilization. Despite caspases being activated, lysosomal permeabilization as well as cell death were not affected by blocking caspase-activity, positioning lysosomal membrane permeabilization (LMP) upstream of caspase activation. Taken together, our data suggest that LMP with its deadly consequences represents a "default" death mechanism in cancer cells, when Caspase-8 is absent and apoptosis cannot be induced.

Clofibric acid increases molecular species of phosphatidylethanolamine containing arachidonic acid for biogenesis of peroxisomal membranes in peroxisome proliferation in the liver.

The biogenesis of peroxisomes in relation to the trafficking of proteins to peroxisomes has been extensively examined. However, the supply of phospholipids, which is needed to generate peroxisomal membranes in mammals, remains unclear. Therefore, we herein investigated metabolic alterations induced by clofibric acid, a peroxisome proliferator, in the synthesis of phospholipids, particularly phosphatidylethanolamine (PE) molecular species, and their relationship with the biogenesis of peroxisomal membranes. The subcutaneous administration of clofibric acid to rats at a relatively low dose (130 mg/kg) once a day time-dependently and gradually increased the integrated perimeter of peroxisomes per 100 µm2 hepatocyte cytoplasm (PA). A strong correlation was observed between the content (µmol/mg DNA) of PE containing arachidonic acid (20:4) and PA (r2 = 0.9168). Moreover, the content of PE containing octadecenoic acid (18:1) positively correlated with PA (r2 = 0.8094). The treatment with clofibric acid markedly accelerated the formation of 16:0-20:4 PE by increasing the production of 20:4 and the activity of acyl chain remodeling of pre-existing PE molecular species. Increases in the acyl chain remodeling of PE by clofibric acid were mainly linked to the up-regulated expression of the Lpcat3 gene. On the other hand, clofibric acid markedly increased the formation of palmitic acid (16:0)-18:1 PE through de novo synthesis. These results suggest that the enhanced formation of particular PE molecular species is related to increases in the mass of peroxisomal membranes in peroxisome proliferation in the liver.

FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis.

FUN14 domain-containing protein 1 (FUNDC1) is an integral mitochondrial outer-membrane protein, and mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs). This study aims to determine the contributions of FUNDC1-mediated MAMs to angiogenesis in vitro and in vivo. In cultured endothelial cells, VEGF significantly increases the formation of MAMs and MAM-related proteins, including FUNDC1. Endothelial cell-specific deletion of FUNDC1, which disrupts MAM formation in endothelial cells, lowers VEGFR2 expression and reduces tube formation, spheroid-sprouting, and functional blood vessel formation in vitro and in vivo. Conversely, increased MAM formation using MAM linkers mimics the effects of VEGF and promotes endothelial angiogenesis. Mechanistically, increased MAMs formation led to increased levels of Ca2+ in cytosol, promoted the phosphorylation of serum response factor (SRF) and enhanced the binding of SRF to VEGFR2 promoter, resulting in increased VEGFR2 production, with consequent angiogenesis. Moreover, blocking FUNDC1-related MAM formation with a cell-penetrating inhibitory peptide significantly suppresses the expressions of downstream angiogenic genes and inhibits tumor angiogenesis. We conclude that decreased MAMs formation by silencing FUNDC1 can inhibit angiogenesis by decreasing VEGFR2 expression, and targeting FUNDC1-dependent MAMs might be a promising approach for treating human disorders characterized by defective angiogenesis.

Contribution of Particle-Induced Lysosomal Membrane Hyperpolarization to Lysosomal Membrane Permeabilization.

Lysosomal membrane permeabilization (LMP) has been proposed to precede nanoparticle-induced macrophage injury and NLRP3 inflammasome activation; however, the underlying mechanism(s) of LMP is unknown. We propose that nanoparticle-induced lysosomal hyperpolarization triggers LMP. In this study, a rapid non-invasive method was used to measure changes in lysosomal membrane potential of murine alveolar macrophages (AM) in response to a series of nanoparticles (ZnO, TiO2, and CeO2). Crystalline SiO2 (micron-sized) was used as a positive control. Changes in cytosolic potassium were measured using Asante potassium green 2. The results demonstrated that ZnO or SiO2 hyperpolarized the lysosomal membrane and decreased cytosolic potassium, suggesting increased lysosome permeability to potassium. Time-course experiments revealed that lysosomal hyperpolarization was an early event leading to LMP, NLRP3 activation, and cell death. In contrast, TiO2- or valinomycin-treated AM did not cause LMP unless high doses led to lysosomal hyperpolarization. Neither lysosomal hyperpolarization nor LMP was observed in CeO2-treated AM. These results suggested that a threshold of lysosomal membrane potential must be exceeded to cause LMP. Furthermore, inhibition of lysosomal hyperpolarization with Bafilomycin A1 blocked LMP and NLRP3 activation, suggesting a causal relation between lysosomal hyperpolarization and LMP.

Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma.

Changes in zinc content and dysregulated zinc homeostatic mechanisms have been recognized in several solid malignancies such as prostate cancer, breast cancer, or pancreatic cancer. Moreover, it has been shown that zinc serum and/or tissue levels are altered in melanoma with varying effects on melanoma development and biology. This study was conducted to explore the effects of acute increases of intracellular zinc in a set of melanoma tissue explants obtained from clinical samples. Measurements of their zinc content showed an extant heterogeneity in total and free intracellular zinc pools associated with varying biological behavior of individual cells, e.g., autophagy levels and propensity to cell death. Use of zinc pyrithione elevated intracellular zinc in a short time frame which resulted in marked changes in mitochondrial activity and lysosomes. These alterations were accompanied by significantly enhanced autophagy flux and subsequent cell demise in the absence of typical apoptotic cell death markers. The present results show for the first time that acutely increased intracellular zinc in melanoma cells specifically enhances their autophagic activity via mitochondria and lysosomes which leads to autophagic cell death. While biologically relevant, this discovery may contribute to our understanding and exploration of zinc in relation to autophagy as a means of controlling melanoma growth and survival.

Carbon tetrachloride suppresses ER-Golgi transport by inhibiting COPII vesicle formation on the ER membrane in the RLC-16 hepatocyte cell line.

Carbon tetrachloride (CCl4 ) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl4 inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl4 on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl4 suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl4 treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl4 -induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl4 inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.

Macrolide antibiotics enhance the antitumor effect of lansoprazole resulting in lysosomal membrane permeabilization­associated cell death.

The proton pump inhibitor lansoprazole (LPZ) inhibits the growth of several cancer cell lines, including A549 and CAL 27. We previously reported that macrolide antibiotics such as azithromycin (AZM) and clarithromycin (CAM) potently inhibit autophagic flux and that combining AZM or CAM with the epidermal growth factor receptor inhibitors enhanced their antitumor effect against various cancer cells. In the present study, we conducted the combination treatment with LPZ and macrolide antibiotics against A549 and CAL 27 cells and evaluated cytotoxicity and morphological changes using cell proliferation and viability assays, flow cytometric analysis, immunoblotting, and morphological assessment. Combination therapy with LPZ and AZM greatly enhanced LPZ­induced cell death, whereas treatment with AZM alone exhibited negligible cytotoxicity. The observed cytotoxic effect was not mediated through apoptosis or necroptosis. Transmission electron microscopy of A549 cells treated with the LPZ + AZM combination revealed morphological changes associated with necrosis and accumulated autolysosomes with undigested contents. Furthermore, the A549 cell line with ATG5 knockout exhibited complete inhibition of autophagosome formation, which did not affect LPZ + AZM treatment­induced cytotoxicity, thus excluding the involvement of autophagy­dependent cell death in LPZ + AZM treatment­induced cell death. A549 cells treated with LPZ + AZM combination therapy retained the endosomal Alexa­dextran for extended duration as compared to untreated control cells, thus indicating impairment of lysosomal digestion. Notably, lysosomal galectin­3 puncta expression induced due to lysosomal membrane permeabilization was increased in cells treated with LPZ + AZM combination as compared to the treatment by either agent alone. Collectively, the present results revealed AZM­induced autolysosome accumulation, potentiated LPZ­mediated necrosis, and lysosomal membrane permeabilization, thus suggesting the potential clinical application of LPZ + AZM combination therapy for cancer treatment.

Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death.

Mitochondrial oxidative stress and dysfunction play an important role of atrial remodeling and atrial fibrillation (AF) in diabetes mellitus. Endoplasmic reticulum (ER) stress has been linked to both physiological and pathological states including diabetes. The aim of this project is to explore the roles of ER stress in hyperglycemia-induced mitochondrial dysfunction and cell death of atrial cardiomyocytes. High glucose upregulated ER stress, mitochondrial oxidative stress, and mitochondria-associated ER membrane (MAM)- enriched proteins (such as glucose-regulated protein 75 (GRP75) and mitofusin-2 (Mfn2)) of primary cardiomyocytes in vitro. Sodium phenylbutyrate (4-PBA) prevented the above changes. Silencing of Mfn2 in HL-1 cells decreased the Ca2+ transfer from ER to mitochondria under ER stress conditions, which were induced by the ER stress agonist, tunicamycin (TM). Electron microscopy data suggested that Mfn2 siRNA significantly disrupted ER-mitochondria tethering in ER stress-injured HL-1 cells. Mfn2 silencing attenuated mitochondrial oxidative stress and Ca2+ overload, increased mitochondrial membrane potential and mitochondrial oxygen consumption, and protected cells from TM-induced apoptosis. In summary, Mfn2 plays an important role in high glucose-induced ER stress in atrial cardiomyocytes, and Mfn2 silencing prevents mitochondrial Ca2+ overload-mediated mitochondrial dysfunction, thereby decreasing ER stress-mediated cardiomyocyte cell death.

Interactions of the Lysosomotropic Detergent O-Methyl-Serine Dodecylamide Hydrochloride (MSDH) with Lipid Bilayer Membranes-Implications for Cell Toxicity.

O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

Benzotriazole alleviates copper mediated lysosomal membrane damage and antioxidant defense system responses in earthworms (Eisenia fetida).

Benzotriazole (BTR) is a common corrosion inhibitor used to protect copper (Cu) and Cu alloys. To reveal the combined subacute toxicity of BTR and Cu at environmental levels on terrestrial animals, the activity of antioxidative enzymes and the glutathione levels in earthworms (Eisenia fetida) of the single or co-exposure treatments were determined. The activity of both antioxidant enzymes and non-enzymatic antioxidants was affected by BTR in earthworms. Moreover, the analyses of lysosomal neutral red retention time and total antioxidant capacity indicated a detoxification effect of BTR on Cu-induced impairments of the antioxidant defense capacity in earthworms. The apoptotic rate of coelomocytes in earthworms of the co-exposure treatment was lower than that in earthworms treated with Cu only, indicating that BTR alleviates Cu mediated lysosomal membrane damage and antioxidant defense system responses in earthworms.

Enniatin B1-induced lysosomal membrane permeabilization in mouse embryonic fibroblasts.

The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24 h exposure to ENN B1 at levels of 2.5-10 µmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7 µmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4 h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.