Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Receptor mediated endocytosis and lysosomal overload leading to kidney toxicity||KeyEvent|
|lysosomal uptake induced liver fibrosis||KeyEvent|
|All life stages|
Key Event Description
Lysosomes were first described in 1955 (de Duve et al., 1955). They are acidic, single-membrane bound organelles that are present in all eukaryotic cells and are filled with more than 50 acid hydrolases to serve their purpose of degrading macromolecules (Johansson et al., 2010).
Lysosomes are the terminal organelle of the endocytic pathway, but are also involved in membrane repair and other cellular processes, such as immune responses (Repnik and Turk, 2010). There are numerous substances that can provoke increased permeability of lysosomal membrane or total lysosomal rupture, and as a consequence release of lysosomal enzymes. Among lysosomal enzymes, one of the major roles has cathepins. There are 11 cysteine cathepins in humans, B, C, F, H, K, L, O, S, V, W and X. Activation of proenzymes usually occurs within the lysosomes (Ishidoh and Kominami, 2002), therefore, the enzymes escaping from the lysosomes are in their active form. The amount of lysosomal enzymes that are released into the cytosol regulates the cell death pathway which is initiated by lysosomal damage: controlled increased permeability of lysosomal membrane, caused by limited level of stress, plays a vital role in the induction of apoptosis, whereas massive lysosomal rupture, caused by high stress levels, leads to necrosis (Bursch, 2001; Guicciardi et al., 2004). Lysosomes are known to be involved in external as well as internal apoptotic pathways. The external pathway triggers lysosomal destabilization by hydroxyl radicals, p53, and caspase 8, through activation of Bax or by ceramide which is converted into sphingosine (Terman et al., 2004). The internal apoptotic pathway on the contrary is activated through mitochondrial damage, for example via activation of Bax or Bid, phospholipases, or lysosomal enzymes (Terman et al., 2004). It has been shown that lack of cathepsin B prevents increased lysosomal membrane permeability in hepatocytes treated with TNF or sphingosine (Werneburg et al., 2002). This indicates that cathepsins can also have a role in initiation of increased lysosomal membrane permeabilization.
The lysosome contains redox-active labile irons which are suggested to be involved in local ROS production via a Fenton-type reaction (Kubota et al., 2010). It has been shown that lysosomal membrane disruption induced by lysosomotropic detergents causes early induction of lysosomal cathepsin B and D and induction of ferritin, together with an increase of cellular ROS and concomitant reduction of the antioxidants MnSOD (manganese superoxide dismutase) and GSH (glutathione), possibly due to the release of free iron into the cytosol (Ghosh et al., 2011; Hamacher-Brady et al., 2011).
The list of agents able to destabilize lysosomal membrane includes L-Leucyl-L-leucinemethyl ester (LLOMe) (Goldman and Kaplan, 1973; Uchimoto et al. 1999; Droga-Mazovec et al., 2008), N-dodecylimidazole (NDI) (Wilson et al., 1987), sphingosine (Kagedal et al., 2001), detergent MSDH (Li et al., 2000), siramesine (Ostenfeld et al., 2005), the quinolone antibiotics ciprofloxacin and norfloxacin (Boya et al., 2003a), hydroxychloroquine (Boya et al., 2003b) and NPs (Wang et al., 2018).
Cirman et al. showed that the lysosomotropic agent LLOMe inducing the disruption of lysosomes results in translocation of lysosomal proteases to the cytosol and induce apoptosis through a caspase dependent mechanism (Cirman et al., 2004). However, it has been proven that partially increased permeabilization of lysosome leads to apoptosis, while complete breakdown of the lysosome with release of high concentrations of the enzymes into the cytosol results in necrosis (Bursch, 2001; Kurz et al., 2008).
The short-term exposure to low concentrations of H2O2 induce lysosomal rupture by activation of phospholipase A2, which cause a progressive destabilization of the membranes of intracellular organelles degrading the membrane phospholipids (Zhao et al., 2001). Sumoza-Toledo and Penner showed that ROS activate lysosomal Ca2+ channels and contribute to increased lysosomal permeability (Sumoza-Toledo and Penner, 2011).
Considering nanomaterials (NMs) as a trigger for lysosomal damage, recent studies underpinned the importance of lysosomal NM uptake for NM-induced toxicity. Once the material is taken up by a cell and transported to the lysosome by autophagy, the acidic milieu herein can either enhance solubility of a NM, or the material remains in its initial nano form. Both situations can induce toxicity, causing lysosomal swelling, followed by lysosomal disruption and the release of pro-apoptotic proteins (Wang et al., 2013; Cho et al., 2011; Cho et al., 2012). Wang et al. showed that the exposure of cells to NH2-PS NPs results in increased lysosomal membrane permeability and release of lysosomal proteasis (cathepsin B and cathepsin D) into cytosol (Wang et al., 2018).
How It Is Measured or Detected
Lysosomes are typically analysed microscopically, usually with fluorescence microscopy (Kagedal et al., 2001).
Changes in morphology can be observed by using acridine orange (AO), a weak base that accumulates in the acidic compartment of the cell mainly composed of lysosomes. Red fluorescence is exhibited when it is highly concentrated in acidic vesicles, while green fluorescence is exhibited when it's less concentrated in other parts of the cell (Li et al., 2000; Reiners et al., 2002; Kroemer and Jäättelä, 2005). This is followed by flow cytometry (Zhao et al., 2001), static cytofluometry or flow cytofluometry (Antunes et al., 2001).
Lysotracker green (200 nM) is regularly used to assess lysosomal acidification; Anguissola and colleagues reported that it was excited through a 475+/240 nm band pass filter and fluorescence emission was collected through a 515+/220 nm band pass filter. Analysis is performed using microscopical methods such as High Content Analysis (Anguissola et al., 2014). This method as well as use of LysoSensor probes has been reported repeatedly elsewhere, for example (Wang et al., 2013; Kroemer and Jäättelä, 2005).
More specific staining can be achieved by staining with antibodies against lysosomal membrane proteins (Kroemer and Jäättelä, 2005).
Lysosomal membrane permeabilization can be visualized by immunostaining of lysosomal enzymes such as cathepsin B (Boya et al. 2003a).
Domain of Applicability
Typically, human or murine cell lines are used to assess this event. Examples are
murine (Reiners et al., 2002)
murine, human (Li et al., 2000)
murine, human (Ghosh et al., 2011)
human (Loos et al., 2014)
human, murine (Anguissola et al., 2014)
human (Hamacher-Brady et al., 2011)
Anguissola S, Garry D, Salvati A, O'Brien PJ, Dawson KA. High content analysis provides mechanistic insights on the pathways of toxicity induced by amine-modified polystyrene nanoparticles. PLoS One. (2014) 9(9):e108025.
Antunes F, Cadenas E, Brunk UT. Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture. Biochemical Journal. (2001) 356(Pt 2):549-555.
Boya P, Andreau K, Poncet D, Zamzami N, Perfettini JL, Metivier D, Ojcius DM, Jäättelä M, Kroemer G. Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion, J. Exp. Med. (2003) 197:1323–1334.
Boya P, Gonzalez-Polo RA, Poncet D, Andreau K, Vieira HL, Roumier T, Perfettini JL, Kroemer G. Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine, Oncogene (2003) 22:3927–3936.
Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. (2001) 8(6):569-81.
Cho W-S, Duffin R, Howie SEM, Scotton CJ, Wallace WAH, Macnee W, Bradley M, Megson IL, Donaldson K. Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes. Part Fibre Toxicol (2011) 8:27.
Cho W-S, Duffin R, Thielbeer F, Bradley M, Megson IL, MacNee W, Poland CA, Tran CL, Donaldson K. Zeta potential and solubility to toxic ions as mechanisms of lung inflammation caused by metal/metal oxide nanoparticles. Toxicol Sci (2012) 126:469–477.
Cirman T, Oresić K, Droga-Mazovec G, Turk V, Reed JC, Myers RM, Salvesen GS, Turk B. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem. (2004) 279(5): 3578–3587.
de Duve C, Pressman BC, Gianetto R, Wattiaux R, Applemans F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. (1955) 60(4):604-17.
Droga-Mazovec G, Bojič L, Petelin A, Ivanova S, Romih R, Repnik U, Salvesen GS, Stoka V, Turk V, Turk B. Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues, J. Biol. Chem. (2008) 283:19140–19150.
Ghosh M, Carlsson F, Laskar A, Yuan XM, Li W. Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages. FEBS Lett. (2011) 585(4):623-9.
Goldman R, Kaplan A. Rupture of rat liver lysosomes mediated by L-amino acid esters, Biochim. Biophys. Acta 1973, 318: 205–216.
Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene. (2004) 23(16):2881-90.
Hamacher-Brady A, Stein HA, Turschner S, Toegel I, Mora R, Jennewein N, Efferth T, Eils R, Brady NR. Artesunate activates mitochondrial apoptosis in breast cancer cells via iron-catalyzed lysosomal reactive oxygen species production. J Biol Chem. (2011) 286(8):6587-601.
Ishidoh K, Kominami E. Processing and activation of lysosomal proteinases. Biol Chem. (2002) 383(12): 1827–1831.
Johansson AC, Appelqvist H, Nilsson C, Kågedal K, Roberg K, Ollinger K. Regulation of apoptosis-associated lysosomal membrane permeabilization. Apoptosis. (2010) 15(5):527-40.
Kagedal K, Zhao M, Svensson I, Brunk UT. Sphingosine-induced apoptosis is dependent on lysosomal proteases. The Biochemical journal. (2001) 359:335-43.
Kroemer G, Jäättelä M. Lysosomes and autophagy in cell death control. Nat Rev Cancer. (2005) 5(11):886-97.
Kubota C, Torii S, Hou N, Saito N, Yoshimoto Y, Imai H, Takeuchi T. Constitutive reactive oxygen species generation from autophagosome/lysosome in neuronal oxidative toxicity. J Biol Chem. (2010) 285(1):667-74.
Kurz T, Terman A, Gustafsson B, Brunk UT. Lysosomes in iron metabolism, ageing and apoptosis. Histochem. Cell Biol. (2008) 129: 389-406.
Li W, Yuan X, Nordgren G, Dalen H, Dubowchik GM, Firestone RA, Brunk UT. Induction of cell death by the lysosomotropic detergent MSDH, FEBS Lett. (2000) 470:35–39.
Loos C, Syrovets T, Musyanovych A, Mailänder V, Landfester K, Nienhaus GU, Simmet T. Functionalized polystyrene nanoparticles as a platform for studying bio-nano interactions. Beilstein J Nanotechnol. (2014) 5:2403-12.
Ostenfeld MS, Fehrenbacher N, Høyer-Hansen M, Thomsen C, Farkas T, Jäättelä M. Effective tumor cell death by sigma-2 receptor ligand siramesine involves lysosomal leakage and oxidative stress, Cancer Res. (2005) 65:8975–8983.
Reiners J, Caruso J, Mathieu P, Chelladurai B, Yin X-M, Kessel D. Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves bid cleavage. Cell death and differentiation. (2002) 9(9):934-944.
Repnik U, Turk B. Lysosomal-mitochondrial cross-talk during cell death. Mitochondrion. (2010) 10(6):662-9.
Sumoza-Toledo A, Penner R. TRPM2: a multifunctional ion channel for calcium signalling. J. Physiol. (2011) 589:1515-1525.
Terman A, Kurz T, Navratil M, Arriaga EA, Brunk UT. Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging. Antioxid Redox Signal. (2010) 12(4):503-35.
Uchimoto T, Nohara H, Kamehara R, Iwamura M, Watanabe N, Kobayashi Y. Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester, Apoptosis (1999) 4:357–362.
Wang F, Bexiga MG, Anguissola S, Boya P, Simpson JC, Salvati A, Dawson KA: Time resolved study of cell death mechanisms induced by amine-modified polystyrene nanoparticles. Nanoscale (2013) 5:10868–76.
Wang F, Salvati A, Boya P. Lysosome-dependent cell death and deregulated autophagy induced by amine-modified polystyrene nanoparticles. Open Biol. (2018) 8(4): 170271.
Werneburg NW, Guicciardi ME, Bronk SF, Gores GJ. Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent. Am J Physiol Gastrointest Liver Physiol. (2002) 283:G947–G956.
Wilson PD, Firestone RA, Lenard J. The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole, J. Cell. Biol. (1987) 104:1223–1229.
Zhao M, Brunk UT, Eaton JW. Delayed oxidant-induced cell death involves activation of phospholipase A2. FEBS Lett. (2001) 509:399–404.
Zhao M, Eaton JW, Brunk UT. Bcl-2 phosphorylation is required for inhibition of oxidative stress induced lysosomal leak and ensuing apoptosis. FEBS Lett. (2001) 509: 405–412.