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|
|lysosomal uptake induced liver fibrosis||MolecularInitiatingEvent|
|o-methyl-serine dodecylamide hydrochloride (MSDH)|
|All life stages|
Key Event Description
Endocytosis was discovered by the Belgian Nobel laureate Christian De Duve in 1963.
Endocytosis is a form of active transport in which molecules are transported into the cell by engulfing them in an energy-using process. In endocytosis, the material to be internalized is surrounded by an area of plasma membrane, which then buds off inside the cell to form a vesicle containing the ingested material. The ingestion of large particles (generally >250 nm in diameter) is termed phagocytosis (cell eating); phagocytosis is actin-dependent and restricted to professional phagocytes. The non-specific receptor-independent process to internalize fluids and solutes is called pinocytosis (cell drinking; via small vesicles of about 100 nm in diameter) and found in all cells (Cooper, 2000; Alberts et al., 2002; Oh and Park, 2014).
Receptor-mediated endocytosis can be clathrin-, and caveolin-dependent; these proteins mediate the invagination of the cell membrane.
The clathrin-mediated endocytotic pathway produces small (approx. 100 nm in diameter) vesicles coated with the cytosolic protein clathrin, forming clathrin-coated pits in the plasma membrane.
Caveolae-mediated endocytosis produces small (approximately. 50 nm in diameter) caveolae, flask-shape pits in the membrane coated with the protein caveolin, derived from lipid rafts (rigid membrane microdomains enriched with phospholipids, sphingolipids, and cholesterol). Clathrin- and caveolae-independent endocytosis is further sub-classified as Arf6- dependent, flotillin-dependent, Cdc42-dependent and RhoA-dependent endocytosis (Cleal et al., 2013; Villamil Giraldo et al., 2014; Iversen et al., 2011; Sahay et al., 2010; Kirkham and Parton, 2005; Mailaender and Landfester, 2009).
Vesicles rapidly lose their coats and fuse to form larger compartments, known as endosomes.
Early endosomes are the first compartment of the endocytic pathway and receive most types of vesicles coming from the cell surface. Endocytosed material is transferred to late endosomes and further to lysosomes, vacuoles of 1-2 µm in diameter containing hydrolytic enzymes in an acid milieu. Their main task is the degradation of ingested material (Villamil Giraldo et al., 2014).
Substances that are taken up selectively into lysosomes are called lysosomotropic agents (De Duve et al., 1974). These agents tend to have both lipophilic or amphiphilic compounds with basic moieties and accumulate in the acidic intracellular compartment, where they become protonated, therefore cannot diffuse back into the cytosol and accumulate within lysosomes, a phenomenon called "acid trapping" (Villamil Giraldo et al., 2014).
Although structurally and pharmacologically diverse, lysosomotropic compounds share certain
physicochemical properties, namely a ClogP > 2 (partition coefficient of the neutral species of a compound between octanol and water) and a basic pKa (the negative base-10 logarithm of the acid dissociation constant of a solution; the lower the pKa value, the stronger the acid) between 6.5 and 11 (Nadanaciva et al., 2010; Lu et al., 2017).
How It Is Measured or Detected
- Lysosomal Trapping assay: A fluorescent dye, LysoTracker Red DND-99, which accumulates in lysosomes, is used for detecting lysosomotropism. There is a gradual decrease in the LysoTracker staining with increasing concentrations of lysosomotropic compounds. This assay is commercially available (Nadanaciva et al., 2011).
- The most widespread method for studying intracellular trafficking involves attaching different fluorescent probes to the protein and nanomaterial that allows analyzing distribution of colour or fluorescence resonance energy transfer (FRET) throughout the cell compartments. The advantage of such approach is that it allows using live cell imaging by confocal microscopy (Sahay et al., 2010).
- Cells can be transfected with constructs containing proteins that reside in specific endocytosis vesicles or intracellular organelles, which are fused with fluorescent proteins, such as the Green Fluorescent Protein (GFP) (Sahay et al., 2010).
- Apart from confocal microscopy, the electron microscopy is also highly useful as it allows visualizing nanomaterials coupled with electron dense labels in different vesicular structures under very high resolution. Atomic Force Microscopy (AFM) has also been used recently to demonstrate the interactions of nanomaterials with the cell membrane (Sahay et al., 2010).
- Exclusion of specific endocytosis mechanisms is a distinct and powerful technique to elucidate endocytosis. This can be achieved, for example, using various pharmacologic inhibitors of endocytosis that include chemical or biological agents or cell mutants (Sahay et al., 2010).
- Co-localization studies of nanomaterials with specific endocytosis markers and structures “pulse-chase” design: proteins, such as transferrin or cholera toxin B (CTB), with known trafficking pathways are exposed to cells simultaneously or before the nanomaterial (“pulse”) and their inclusion or exclusion from the same vesicles is detected at different time points (“chase”) (Sahay et al., 2010).
- Another way of studying co-localization is immunocytochemistry applied to fixed cells. This method allows employing specific antibodies to different proteins present along the endocytic vesicles and organelles (Sahay et al., 2010).
- Transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. TEM is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image (Brandenberger et al., 2010; Villamil Giraldo et al., 2016).
- Fluorescence Correlation Spectroscopy is a correlation analysis of fluctuation of the fluorescence intensity (Villamil Giraldo et al., 2016).
- Evaluation of NP cellular uptake by confocal laser scanning microscopy (CLSM). CLSM combines high-resolution optical imaging with depth selectivity which allows optical sectioning. The CLSM works by passing a laser beam through a light source aperture which is focused by an objective lens into a small area on the surface of the sample and an image is built up pixel-by-pixel by collecting the emitted photons from the fluorophores in the sample (Harush-Frenkel et al., 2006; Zhang and Monteiro-Riviere, 2009).
- Fluorescence-activated cell sorter (FACS) analysis of NP cellular uptake. FACS is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological based upon the specific light scattering and fluorescent characteristics of each cell (Harush-Frenkel et al., 2006; Zhang and Monteiro-Riviere, 2009).
- Quantitative cellular uptake of AuNPs was performed by inductively coupled plasma mass spectrometry (ICPMS), a type of mass spectrometry capable of detecting metals and several non-metals at very low concentrations. This is achieved by ionizing the sample with inductively coupled plasma and then using a mass spectrometer to separate and quantify those ions (Ng et al., 2015).
- We describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics for an understanding of intracellular trafficking pathways (Manunta et al., 2007).
Domain of Applicability
Endocytosis is an universal internalization route in eukaryotes (Dergai et al., 2016). In most animal cells, clathrin-coated pits and vesicles provide an efficient pathway for taking up specific macromolecules from the extracellular fluid (Cooper, 2000) and it is also a mechanism for uptaking extracellular molecules in plant cells (Fan et al., 2015).
The experimental studies have been done on mice in vivo, primary human and rat cells, and human and animal (rat, mouse)-derived cell lines (Harusch Fraenkel et al., 2007; Ng et al., 2015; Nadanaciva et al., 2011; Lu et al., 2017; Xie et al., 2007; Verma and Stellacci, 2010; Zhang and Monteiro-Riviere, 2009).
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Several well-known drugs have lysosomotropic abilities including chloroquine, the antipsychotics chlorpromazine, thioridazine, aripiprazole, the antidepressants desipramine, imipramine, and clomipramine, as well as fluoroquinolone antibiotics; another substance group are lysosomotropic detergents (Villamil Giraldo et al., 2014; Ouedraogo et al., 2000).
Fluoroquinolones such as lomefloxacin, norfloxacin, BAYy 3118 and ciprofloxacin are lysosomotropic substances because of their Lewis acid–base properties characterized by pKa nearby neutrality (Ouedraogo et al. 2000). The anti-malarial and anti-inflammatory agent chloroquine is a basic lipophilic and therefore lysosomotropic compound that accumulates in lysosomes via pH partitioning (Ashoor et al. 2013). 3-aminopropanal has the structure of a weak lysosomotropic base, concentrates within the acidic vacuolar compartment and causes lysosomal rupture (Yu et al., 2003). Artesunate preferably accumulates in the lysosomes (Yang et al., 2014)
Most nanoscale macromolecules and molecular assemblies are internalized through endocytosis upon contact with the cell membrane. Intracellular trafficking of NPs following endocytosis has been reported to be mediated via the endosomal pathway through early endosomes, late endosomes and then lysosomes (Gilleron et al., 2013; Yang et al., 2013; Ng et al., 2015). Verma and Stellacci showed that 3.4-nm gold NPs were taken up into macrophages via pinocytosis and 24 h after internalization they were found in lysosomes. This endocytic fate has also been observed for iron oxide NPs and fullerenes (Verma and Stellacci, 2010).
Jin et al. investigated the cytotoxicity of Nanotitanium dioxide TiO2 (an industrial material used as an additive in cosmetics, pharmaceuticals, and food colorants and able to penetrate the skin) in mouse fibroblast (L929) cells. They saw that TiO2 NPs were phagocytosed and encapsulated in the lysosomes (Jin et al. 2008).
The rate and mechanism of NP uptake are dependent on physiochemical causes related to the properties of the NPs and the cells, but also the local microenvironment (Zhang, 2015). Nanomaterial shape and size contribute significantly to their interaction with cells (Verma and Stellacci, 2010; Oh, 2014). Several reports showing that NPs of 20—50nm are taken up more rapidly than smaller or larger particles (Lu et al., 2009; Iversen et al., 2011; Dykman and Khlebtsov, 2014).
Other variables that could influence the uptake of a NP cargo include orientation, density and steric freedom of targeting ligands and surface groups (Cleal et al., 2013). Most NPs are first coated with serum proteins before they reach cell plasma membranes; endocytosis patterns of aggregated or agglomerated NPs differ from the one of individual NPs (Oh and Park, 2014).
Harush-Frenkel et al. compared the endocytosis into HeLa cells of NPs exposing either a negative or positive charge on their surface and found that the exposed charge significantly affected their ability to internalize as well as the cellular endocytosis mechanism utilized. Negatively charged NPs showed an inferior rate of endocytosis and did not utilize the clathrin-mediated endocytosis pathway, while positively charged NPs internalize rapidly primarily via clathrin-mediated pathways as well as macropinocytosis. When the clathrin-mediated endocytosis pathway is blocked positively charged NPs activate a compensatory endocytosis pathway that results in enhanced accumulation of NPs (Harush-Frenkel et al., 2007). In contrast, a higher uptake of negatively charged quantum dot NPs has been reported in HEK cells by Zhang and Monteiro-Riviere (2009).
Schuetz et al. demonstrated that positively charged SiNPs enter cells largely via dynamin 2-dependent caveolar internalization rather than clathrin-mediated endocytosis and accumulate in lysosomes (Schuetz et al., 2016).
Ng et al. have shown that the uptake of 20 nm size AuNPs in MRC5 lung fibroblasts and Chang liver cells was dependent upon clathrin-mediated endocytosis (Ng et al., 2014). Yang et al. studied the cellular uptake of ultra-small fluorescent gold nanoclusters (AuNCs) by HeLa cells and found that this energy-dependent process involved multiple mechanisms, with clathrin-mediated endocytosis and macropinocytosis appearing to play a significant role, whereas the caveolin-mediated pathway contributing only to a lesser extent (Yang et al., 2013).
Gilleron et al. monitored the uptake of lipid NPs (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver and found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis (Gilleron et al., 2013).
Saw et al. showed that the major uptake of confeito Au NP of 30 nm was both clathrin and caveolin mediated endocytosis, while for 60, 80 and 100 nm NP was via clathrin mediated pathway. Internalization by both clathrin and caveolin pathways explains higher cellular uptake of 30 nm Au NP compared to other ones (Saw et al., 2018). However, Brandenberger et al. showed that uptake of 30 nm citrate-cappedsphere Au NP was by a macroopinocytosis mechanism (Brandenberger et al., 2010).
It is obvious that not only size of NP, but also the surface property and shape are also important factors in the type of cellular uptake of NPs. As demonstrated earlier cationic NPs favor clathrin mediated endocytosis possibly due to electrostatic interaction with the cell surface receptors. However, not all the studies confirmed this preference of cationic NPs for clathrin mediated internalization. In contrast, caveolin mediated endocytosis occurs by interaction between hydrophobic group of NP with lipid raft on cell surface (Chakraborty and Jana, 2015). There is a connection between the endocytosis mechanism and the subcellular localization of the NPs. Clathrin mediated endocytosis leads to localization of NPs to the lysosome. Also, it has been found that if uptake of NPs occurs via both clathrin and lipid raft- mediated endocytosis, subcellular localization will also be mainly lysosomal. Caveolin mediated uptake of NPs is followed with formation of vesicles with neutral pH and transport to endoplasmic reticulum, Golgi apparatus or even nucleus (Rejman et al., 2005)
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