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Event: 1539

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Endocytotic lysosomal uptake

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
endocytosis

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
eukaryotic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
endocytosis lysosomal membrane increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
lysosomal uptake induced liver fibrosis MolecularInitiatingEvent Allie Always (send email) Under development: Not open for comment. Do not cite EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens NCBI
rat Rattus norvegicus NCBI
mouse Mus musculus NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Unspecific

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?
  • 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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

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)

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help
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  • Chakraborty A, Jana NR. Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle,  J. Phys.  Chem.  Lett. (2015) 6(18): 3688-3697
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