SNAPSHOT

How this Key Event Works

Lysosomes were first described by de Duve and colleagues in 1955 ^[1]. 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 ^[2].

Lysosomes are the terminal organelle of the endocytic pathway, but are also involved in membrane repair and other cellular processes, such as immune responses ^[3]. The amount of lysosomal enzymes that are released into the cytosol regulates the cell death pathway which is initiated by lysosomal damage: it plays a vital role in the induction of apoptosis, whereas massive lysosomal rupture leads to necrosis ^{[4] [5]}. Lysosomes are known to be involved in external as well as internal apoptotic pathways. The external pathway triggers lysosomal destabilization by hydroxyl radicals, p53, caspase 8,through activation of Bax or by ceramide which is converted into sphingosine ^[6]. The internal apoptotic pathway on the contrary is activated through mitochondrial damage, for example via activation of Bax or Bid, phospholipases, or lysosomal enzymes ^[6].

The lysosome contains redox-active labile irons which are suggested to be involved in local ROS production via a Fenton-type reaction ^[7]. 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 ^{[8] [9]}. Released enzymes such as phospholipases can further damage the outer membrane of the mitochondrion, leading to a further increased and uncontrolled ROS production, the release of cytochrome c, the activation of the caspase cascade, and subsequent apoptotic cell death ^[10].

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 ^{[11] [12] [13]}.

How it is Measured or Detected

Lysosomes are typically analysed microscopically.

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 ^{[14] [15] [16]}.

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 ^[17]. This method as well as use of LysoSensor probes has been reported repeatedly elsewhere, for example ^{[9] [16]}.

More specific staining can be achieved by staining with antibodies against lysosomal membrane proteins ^[16].

Lysosomal membrane permeabilization can be visualized by immunostaining of lysosomal enzymes such as cathepsin B ^[18].

References

1. ↑ 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 Aug;60(4):604-17
2. ↑ Johansson AC, Appelqvist H, Nilsson C, Kågedal K, Roberg K, Ollinger K. Regulation of apoptosis-associated lysosomal membrane permeabilization. Apoptosis. 2010 May;15(5):527-40
3. ↑ Repnik U, Turk B. Lysosomal-mitochondrial cross-talk during cell death. Mitochondrion. 2010 Nov;10(6):662-9
4. ↑ Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 2001 Jun;8(6):569-81
5. ↑ Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene. 2004 Apr 12;23(16):2881-90
6. ↑ ^{6.0 6.1} 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 Apr;12(4):503-35
7. ↑ 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 Jan 1;285(1):667-74
8. ↑ Ghosh M, Carlsson F, Laskar A, Yuan XM, Li W. Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages. FEBS Lett. 2011 Feb 18;585(4):623-9
9. ↑ ^{9.0 9.1 9.2} 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 Feb 25;286(8):6587-601
10. ↑ ^{10.0 10.1} 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 Dec 15;5:2403-12
11. ↑ 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
12. ↑ 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
13. ↑ 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
14. ↑ Cite error: Invalid <ref> tag; no text was provided for refs named Reiners2002
15. ↑ Cite error: Invalid <ref> tag; no text was provided for refs named Li2000
16. ↑ ^{16.0 16.1 16.2} Kroemer G, Jäättelä M. Lysosomes and autophagy in cell death control. Nat Rev Cancer. 2005 Nov;5(11):886-97
17. ↑ ^{17.0 17.1} 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 Sep 19;9(9):e108025
18. ↑ 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 May 19;197(10):1323-34
19. ↑ Ghosh M, Carlsson F, Laskar A, Yuan XM, Li W. Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages. FEBS Lett. 2011 Feb 18;585(4):623-9

How this Key Event Works

Oxidative stress corresponds to an imbalance between the rate of oxidant production and that of their degradation. The term oxidative stress indicates the outcome of oxidative damage to biologically relevant macromolecules such as nucleic acids, proteins, lipids and carbohydrates. This occurs when oxidative stress-related molecules, generated in the extracellular environment or within the cell, exceed cellular antioxidant defenses. Major reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anion, as well as 4-hydroxy- 2,3-nonenal (HNE) and related 4-hydroxy-2,3-alkenals (HAKs), major aldehydic end-products of lipid peroxidation, can act as potential mediators able to affect signal transduction pathways as well as the proliferative and functional response of target cells. H2O2 and superoxide anion may be also generated as molecular messengers within the cell as part of the cellular response to defined growth factors, cytokines and other mediators. The final consequence at tissue, cellular and molecular level is primarily affected by the steady state concentration of oxidative stress-related molecules. The main biological targets of free radicals are proteins, lipids and DNA.

Major consequences of reaction of ROS, HAKs and NO with biologically relevant macromolecules that can mediate pathophysiological effects:

ROS: DNA: oxidation, strand breaks, genotoxicity Proteins: oxidation, fragmentation, formation of carbonyls Lipids: lipid peroxidation and degradation

HAKs: DNA: adducts (low doses), strand breaks, genotoxicity (high doses) Proteins: adducts (Michael type reactions on Lys, Cys and His residues)

NO: DNA: oxidation, strand breaks Proteins: oxidation, nitrosation, nitration (nytrosylation of tyrosine) Lipids: lipid peroxidation and degradation

Continued oxidative stress can lead to chronic inflammation. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF-1α, PPAR-γ, β-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines and chemokines, which can activate inflammatory pathways. ^{[1] [2] [3]}

How it is Measured or Detected

measuring oxidative stress

Agents for ROS detection are primarily fluorescence based, but recently luminescent based detections have been introduced. The biggest difficulty reported with much of the cellular ROS research has been with the lack of reporter agents specific for discrete molecules. ROS moieties by their nature are reactive with a number of different molecules; as such designing reporter agents has been difficult. With more specific chemistries, particularly for hydrogen peroxide, the specific mechanisms for regulation will be elucidated.

Reduced glutathione (GSH) is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase GSSH + NADPH + H+ à 2 GSH + NADP+ Due to the rapid nature of the reduction of GSSH relative to its synthesis or secretion, the ratio of GSH to GSSH is a good indicator of oxidative stress within cells. GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically in microplates. Several different assays have been designed to measure glutathione in samples. By using a luciferin derivative in conjunction with glutathione S-transferase enzyme the amount of GSH would be proportional to the luminescent signal generated when luciferase is added in a subsequent step. Total glutathione can be determined colorimetrically by reacting GSH with DTNB (Ellman’s reagent) in the presence of glutathione reductase. Glutathione reductase reduces GSSH to GSH, which then reacts with DTNB to produce a yellow colored 5-thio-2-nitrobenzoic acid (TNB), which absorbs at 412 nm.

Lipid peroxidation is one of the most widely used indicators of free radical formation, a key indicator of oxidative stress. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA) reactive compounds such as malondialdehyde generated from the decomposition of lipid peroxidation products. While this method is controversial in that it is quite sensitive, but not necessarily specific to MDA, it remains the most widely used means to determine lipid peroxidation. This reaction, which takes place under acidic conditions at 90-100ºC, results in an adduct that can be measured colorimetrically at 532 nm or by fluorescence using a 530 nm excitation wavelength and a 550 nm emission wavelength. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be specific for lipid peroxidation. Unlike the TBA assay, measurement of IsoP appears to be specific to lipid peroxides, they are stable and are not produced by any enzymatic pathway making interpretation easier. There have been a number of commercial ELISA kits developed for IsoPs, but interfering agents in samples requires partial purification of samples prior to running the assay. The only reliable means for detection is through the use of GC/MS, which makes it expensive and limits throughput.

Superoxide detection is based on the interaction of superoxide with some other compound to create a measurable result. The reduction of ferricytochrome c to ferrocytochrome c has been used in a number of situations to assess the rate of superoxide formation. While not completely specific for superoxide this reaction can be monitored colorimetrically at 550 nm. Chemiluminescent reactions have been used for their potential increase in sensitivity over absorbance-based detection methods. The most widely used chemiluminescent substrate is Lucigenin, but this compound has a propensity for redox cycling, which has raised doubts about its use in determining quantitative rates of superoxide production. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical. These dyes are synthesized by reducing the iminium cation of the cyanine (Cy) dyes with sodium borohydride. While weakly fluorescent, upon oxidation their fluorescence intensity increases 100 fold. In addition to being fluorescent, oxidation also converts the molecule from being membrane permeable to an ionic impermeable moiety. The most characterized of these probes are Hydro-Cy3 and Hydro-Cy5.

Hydrogen peroxide (H2O2) is the most important ROS in regards to mitogenic stimulation or cell cycle regulation. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products. The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H2O2 form increasing amounts of fluorescent product.

Nitric Oxide The free radical nitric oxide (•NO) is produced by a number of different cell types with a variety of biological functions. Regardless of the source or role, the free radical •NO has a very short half life (t½= 4 seconds), reacting with several different molecules normally present to form either nitrate (NO3-) or nitrite (NO2-) A commonly used method for the indirect determination of •NO is the determination of its composition products nitrate and nitrite colorimetrically. This reaction requires that nitrate (NO3) first be reduced to nitrite (NO2), typically by the action of nitrate reductase. Subsequent determination of nitrite by a two step process provides information on the “total” of nitrate and nitrite. In the presence of hydrogen ions nitrite forms nitrous acid, which reacts with sulfanilamide to produce a diazonium ion. This then coupled to N-(1-napthyl) ethylenediamine to form the chromophore which absorbs at 543 nm. Nitrite only determinations can then be made in a parallel assay where the samples were not reduced prior to the colorimetric assay. Actual nitrate levels are then calculated by the subtraction of nitrite levels from the total. ^[4]

References

1. ↑ Parola, M. and Robino, G. (2001). Oxidative stress-related molecules and liver fibrosis. J Hepatol. 35, 297-306
2. ↑ Sánchez-Valle V. et al., (2012) Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem. 19, 4850-4860
3. ↑ Reuter S. et al., (2010) Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med.49, 1603-1616
4. ↑ Held P., 2010 Biotek, Measurement of ROS in Cells, http://www.biotek.com/assets/tech_resources/ROS%20Application%20Guide.pdf

How this Key Event Works

Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.

Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006).

Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation.

A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM)are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (MPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010).

Summing up: Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.

How it is Measured or Detected

Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail.

I. Mitochondrial dysfunction assays assessing a loss-of function.

1. Cellular oxygen consumption

See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005).

2. Mitochondrial membrane potential (Δψm )

The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. The classical, and still most quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode on suspensions of isolated mitochondria. The Δψm can also be measured in live cells by fluorimetric methods. These are based on dyes which accumulate in mitochochondria because of Δψm. Frequently used are tetramethylrhodamineethylester (TMRE), tetramethylrhodamine, methyl ester (TMRM) (Petronilli et al., 1999) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1). Mitochondria with intact membrane potential concentrate JC-1, so that it forms red fluorescent aggregates, whereas de-energized mitochondria cannot concentrate JC-1 and the dilute dye fluoresces green (Barrientos et al., 1999). Assays using TMRE or TMRM measure only at one wavelength (red fluorescence), and depending on the assay setup, de-energized mitochondria become either less fluorescent (loss of the dye) or more fluorescent (attenuated dye quenching).

3. Enzymatic activity of the electron transport system (ETS)

Determination of ETS activity can be determined following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990).

4. ATP content

For the evaluation of ATP levels, various commercially-available ATP assay kits are offered (e.g. Sigma, http://www.abcam.com/atp-assay-kit-colorimetricfluorometric-ab83355.html), based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005).

II. Mitochondrial dysfunction assays assessing a gain-of function.

1. Mitochondrial permeability transition pore opening (PTP)

The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999).

2. mtDNA damage as a biomarker of mitochondrial dysfunction

Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b).

3. Generation of ROS and resultant oxidative stress

a. general approach Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine.

b. Measurement of the cellular glutathione (GSH) status GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential).. GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress.

c. Quantification of lipid peroxidation Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific.. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method.

d. Detection of superoxide production Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (http://www.biotek.com/resources/articles/reactive-oxygen-species.html). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in teh absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at sit of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria.

e. Detection of hydrogen peroxide (H2O2) production There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H2O2 form increasing amounts of fluorescent product (Tarpley et al., 2004).

 

Summing up mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (MPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion)(Promega assay, TB369; Radkowsky et al., 1986 • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE Oxidative Stress, Increase

References

 

Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal. 2005, 7(9-10):1140-1149.

Bal-Price A. and Guy C. Brown. Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria. J. Neurochemistry, 2000, 75: 1455-1464.

Bal-Price A, Matthias A, Brown GC., Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J. Neurochem. 2002, 80: 73-80.

Brand MD, Nicholls DG. Assessing mitochondrial dysfunction in cells. Biochem J. 2011 Apr 15;435(2):297-312.

Braun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182.

Barrientos A., and Moraes C.T. (1999) Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. Vol. 274, No. 23, pp. 16188–16197.

Cammen M. Corwin, Susannah Christensen. John P. (1990) Electron transport system (ETS) activity as a measure of benthic macrofaunal metabolism MARINE ECOLOGY PROGRESS SERIES- (65) : 171-182.

Ciapaite, Lolita Van Eikenhorst, Gerco Bakker, Stephan J.L. Diamant, Michaela. Heine, Robert J Wagner, Marijke J. V. Westerhoff, Hans and Klaas Krab (2005) Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria Diabetes 54:4 944-951.

Correia SC, Santos RX, Perry G, Zhu X, Moreira PI, Smith MA. (2012). Mitochondrial importance in Alzheimer’s, Huntington’s and Parkinson’s diseases. Adv Exp Med Biol 724:205 – 221.

Cozzolino M, Ferri A, Valle C, Carri MT. (2013). Mitochondria and ALS: implications from novel genes and pathways. Mol Cell Neurosci 55:44 – 49.

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How this Key Event Works

Two types of cell death can be distinguished by morphological features, although it is likely that these are two ends of a spectrum with possible intermediate forms. Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of the cell into discrete bodies with intact plasma membranes. These are rapidly phagocytosed by neighbouring cells. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. In contrast, necrotic cell death is characterized by cell swelling and lysis. This is usually a consequence of profound loss of mitochondrial function and resultant ATP depletion, leading to loss of ion homeostasis, including volume regulation, and increased Ca2+. The latter activates a number of nonspecific hydrolases (i.e., proteases, nucleases, and phospholipases) as well as calcium dependent kinases. Activation of calpain I, the Ca2+-dependent cysteine protease cleaves the death-promoting Bcl-2 family members Bid and Bax which translocate to mitochondrial membranes, resulting in release of truncated apoptosis-inducing factor (tAIF), cytochrome c and endonuclease in the case of Bid and cytocrome c in the case of Bax. tAIF translocates to cell nuclei, and together with cyclophilin A and phosphorylated histone H2AX (γH2AX) is responsible for DNA cleavage, a feature of programmed necrosis. Activated calpain I has also been shown to cleave the plasma membrane Na+–Ca2+ exchanger, which leads to build-up of intracellular Ca2+, which is the source of additional increased intracellular Ca2+. Cytochrome c in cellular apoptosis is a component of the apoptosome.

DNA damage activates nuclear poly(ADP-ribose) polymerase-1(PARP-1), a DNA repair enzyme. PARP-1 forms poly(ADP-ribose) polymers, to repair DNA, but when DNA damage is extensive, PAR accumulates, exits cell nuclei and travels to mitochondrial membranes, where it, like calpain I, is involved in AIF release from mitochondria. A fundamental distinction between necrosis and apoptosis is the loss of plasma membrane integrity; this is integral to the former but not the latter. As a consequence, lytic release of cellular constituents promotes a local inflammatory reaction, whereas the rapid removal of apoptotic bodies minimizes such a reaction. The distinction between the two modes of death is easily accomplished in vitro but not in vivo. Thus, although claims that certain drugs induce apoptosis have been made, these are relatively unconvincing. DNA fragmentation can occur in necrosis, leading to positive TUNEL staining. Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis.

Two alternative pathways - either extrinsic (receptor-mediated) or intrinsic (mitochondria-mediated) - lead to apoptotic cell death. The initiation of cell death begins either at the plasma membrane with the binding of TNF or FasL to their cognate receptors or within the cell. The latter is due to the occurrence of intracellular stress in the form of biochemical events such as oxidative stress, redox changes, covalent binding, lipid peroxidation, and consequent functional effects on mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, or DNA. The intrinsic mitochondrial pathway involves the initiator, caspase-9, which, when activated, forms an “apoptosome” in the cytosol, together with cytochrome c, which translocates from mitochondria, Apaf-1 and dATP. The apoptosome activates caspase-3, the central effector caspase, which in turn activates downstream factors that are responsible for the apoptotic death of a cell ^[1]. Intracellular stress either directly affects mitochondria or can lead to effects on other organelles, which then send signals to the mitochondria to recruit participation in the death process ^[1][2]Constitutively expressed nitric oxide synthase (nNOS) is a Ca2+-dependent cytosolic enzyme that forms nitric oxide (NO) from L-arginine, and NO reacts with the free radical such as superoxide (O2−) to form the very toxic free radical peroxynitrite (ONOO−). Free radicals such as ONOO−, O2 − and hydroxyl radical (OH−) damage cellular membranes and intracellular proteins, enzymes and DNA ^[1], ^{[2] [3][4]}

How it is Measured or Detected

Necrosis:

LDH is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor. The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample. ^[5]

The MTT assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light ^[6].

Propidium iodide (PI) is an intercalating agent and a fluorescent molecule used to stain necrotic cells. It is cell membrane impermeant so it stains only those cells where the cell membrane is destroyed. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm ^[7] .

Apoptosis:

TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage.

Caspase activity assays measured by fluorescence. During apoptosis, mainly caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. Antibodies to the 85 kDa fragment of cleaved PARP or to caspase-3 both serve as markers for apoptotic cells that can be monitored using immunofluorescence ^[8].

Hoechst 33342 staining: Hoechst dyes are cell-permeable and bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. The stained, condensed or fragmented DNA is a marker of apoptosis. ^{[9] [10]}

References

1. ↑ ^{1.0 1.1 1.2} Fujikawa, D.G. (2015), The role of excitotoxic programmed necrosis in acute brain injury, Comput Struct Biotechnol J, vol. 13, pp. 212-221.
2. ↑ ^{2.0 2.1} Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.
3. ↑ Kaplowitz, N. (2002), Biochemical and Cellular Mechanisms of Toxic Liver Injury, Semin Liver Dis, vol. 22, no. 2, http://www.medscape.com/viewarticle/433631 (accessed on 20 January 2016).
4. ↑ Kroemer, G. et al., (2009), Classification of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death Differ, vol. 16, no. 1, pp. 3-11.
5. ↑ Chan, F.K., K. Moriwaki and M.J. De Rosa (2013), Detection of necrosis by release of lactate dehydrogenase (LDH) activity, Methods Mol Biol, vol. 979, pp. 65–70.
6. ↑ Berridge, M.V., P.M. Herst and A.S. Tan (2005), Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, vol. 11, pp 127-152.
7. ↑ Moore, A, et al.(1998), Simultaneous measurement of cell cycle and apoptotic cell death,Methods Cell Biol, vol. 57, pp. 265–278.
8. ↑ Li, Peng et al. (2004), Mitochondrial activation of apoptosis, Cell, vol. 116, no. 2 Suppl,pp. S57-59, 2 p following S59.
9. ↑ Loo, D.T. (2002), TUNEL Assay an overview of techniques, Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage, chapter 2, Didenko VV (ed.), Humana Press Inc.
10. ↑ Kubbies, M. and P.S. Rabinovitch (1983), Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes, Cytometry, vol. 3, no. 4, pp. 276–281.
11. ↑ Fink, S.L. and B.T. Cookson (2005), Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells, Infect Immun, vol. 73, no. 4, pp.1907-1916.

How this Key Event Works

Cytokines are small, soluble molecules secreted by cells to enable intercellular communication. Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), as well as on distant cells (endocrine action). Cytokines can act synergistically or antagonistically, and secretion from one cell can trigger upregulation of a further range of cytokines from the same cell or others ^[1]. Most cells in the body are able to secrete them, and several subfamilies belong to the group of cytokines, such as chemokines, interferons, interleukins, tumor necrosis factors (TNF), transforming growth factors (TGF) and colony-stimulating factors. They are important players in modulating fundamental biological processes, including body growth, adiposity, lactation, hematopoiesis, and also inflammation and immunity^[2]. Damaged cells, such as apoptotic cells, can trigger the upregulation and release of cytokines to induce the inflammatory response. An important receptor responsible for cell death-related cytokine regulation is Fas, a cell surface glycoprotein which belongs to the tumor necrosis factor (TNF) receptor family. The role of Fas in the onset of inflammation by upregulating inflammatory cytokines is increasingly discussed. Fas-activation can trigger the production of MCP-1 and IL-8 and its associated chemotaxis of phagocytes toward apoptotic cells^[3].

TNF-α is an inflammatory mediator that can be secreted by many cell types, including hepatocytes and Kupffer cells. TNF-induced cytokines and chemokines, such as IL-6, IL-8, GMCSF, CXCL1, and RANTES, can trigger immune responses by producing acute phase proteins and recruitment of inflammatory cells such as neutrophils, macrophages, and basophils to the site of inflammation. Moreover, an increased production of monocytes/macrophages from bone marrow is triggered^[3].

On the other hand, inflammation can be suppressed by cytokines and mediators such as IL-10 and TGF-β. In the liver, TGF-β1 is the most abundant isoform and is secreted by immune cells, stellate cells, and epithelial cells. IL-10 inhibits T cell-, monocyte-, and macrophage-mediated functions and has been detected in several liver cells, in¬cluding hepatocytes, stellate cells, and Kupffer cells ^[2].

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

mRNA expression levels of inflammatory cytokines can be determined by using real-time PCR as described in ^[4]. Equally, In Situ Hybridization of mRNA in liver tissue can be used ^[5].

Plasma levels of pro-inflammatory cytokines, or levels in cell supernatants can be analysed by enzyme linked immunosorbent assay (ELISA) using commercial kits ^[6][3]. A more advanced system was described recently by using a multiplex immunoassay platform. In a 96 well plate format the authors describe the analysis of blood, urine and breath samples of human volunteers in a Meso Scale Discovery (MSD) multiplex electrochemiluminescent immunoassay system ^[7].

References

1. ↑ Zhang JM, An J. Cytokines, inflammation, and pain. Int Anesthesiol Clin. 2007 Spring;45(2):27-37
2. ↑ ^{2.0 2.1} Braunersreuther V, Viviani GL, Mach F, Montecucco F. Role of cytokines and chemokines in non-alcoholic fatty liver disease. World J Gastroenterol. 2012 Feb 28;18(8):727-35
3. ↑ ^{3.0 3.1 3.2 3.3} Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, Martin SJ. Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells. Mol Cell. 2013 Mar 28;49(6):1034-48
4. ↑ ^{4.0 4.1} Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, Hu R, Hong M, Hong F. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. 2011; J. Biomed. Mater. Res. - Part A 96 A:221–229
5. ↑ ^{5.0 5.1} Faouzi S, Burckhardt BE, Hanson JC, Campe CB, Schrum LW, Rippe RA, Maher JJ. Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway. J Biol Chem. 2001 Dec 28;276(52):49077-82
6. ↑ ^{6.0 6.1} Ma L, Zhao J, Wang J, Liu J, Duan Y, Liu H, Li N, Yan J, Ruan J, Wang H, Hong F. The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2. Nanoscale Res Lett. 2009 Aug 1;4(11):1275-85
7. ↑ ^{7.0 7.1} Stiegel MA, Pleil JD, Sobus JR, Morgan MK, Madden MC. Analysis of inflammatory cytokines in human blood, breath condensate, and urine using a multiplex immunoassay platform. Biomarkers. 2015 Feb;20(1):35-46

How this Key Event Works

TNF-induced cytokines and chemokines, such as IL-6, IL-8, GMCSF, CXCL1, and RANTES, can instigate and amplify immune responses through triggering the production of acute phase proteins and the recruitment of neutrophils, macrophages, and basophils to the site of inflammation, and by triggering increased production of monocytes/macrophages from bone marrow^[1]. Monocytes are the precursors of macrophages and dendritic cells and circulate in the blood for 1-3 days. Upon secretion of chemokines such as CCL2 which is also referred to as monocyte chemoattractant protein 1 (MCP1), they can migrate towards affected tissue. This was nicely demonstrated when depletion of MCP-1 in supernatants of Fas-stimulated cells was sufficient to block almost all THP-1 monocyte chemotaxis. Using an in vivo mouse model, the authors found that Fas stimulation could trigger phagocyte migration by administration of anti-Fas (Jo2) antibody into C57BL/6 mice within 10 h of anti-Fas administration. This correlated with extensive cell death in the thymus and a dramatic increase of CD11b-positive macrophages in the same tissue^[1].

Neutrophils, on the other hand, account for about 50 70 % of all blood leukocytes in the human body ^[2][3]. Upon an inflammatory event, neutrophil production is upregulated, and its lifetime increases as a response to platelet activating factor (PAF), granulocyte-colony stimulating factor (G-CSF) or various pro-inflammatory cytokines, such as interleukin 1ß (IL-1ß) ^[3]. The crucial role of PMN in the human immune system is long known. In 1968, Baehner and Karnovsky described a link between a reduced PMN activity and the development of chronic granulomatous disease (CGD) ^[4]. The important peroxidase-mediated bactericidal role of PMN and the formation of superoxide radicals as one of the main bactericial mechanisms was already described more than 30 years ago ^[5][6]. A strong negative correlation between the chemotactic ability of PMN and patients with increased bacterial sepsis was demonstrated ^[7], and clinical morbidity from infections is clearly increased with a reduced number of circulating PMN in the blood ^[8]. The neutrophilic cytosol contains granules that are filled with a variety of proteins, such as defensins, bactericidal-permeability-increasing protein, proteases (e.g. elastase, cathepsins), and myeloperoxidase (MPO) that consumes hydrogen peroxide (H2O2) and generates hypochlorous acid (HOCl), the most bactericidal oxidant that is produced by PMN ^[8][2]. Activated neutrophils are capable of producing a variety of pro-inflammatory cytokines, e.g. IL-1ß, IL-6, IL-12 and IL-23, and transport internalised pathogens to lymph nodes to support macrophages and dendritic cells in antigen presentation^[9]. Also, contact with pathogens results not only in phagocytosis, but also in the so-called oxidative burst, marked by an increased consumption of molecular oxygen and resulting production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) ^[10].

Deregulation of this response by constant stimulation of PMNs, as could be shown for nanoparticles for example, ultimately leads to the establishment of a (chronic) inflammation. Here, also macrophages play a vital role. Resident alveolar macrophages, such as Kupffer cells in the liver, that usually phagocyte microorgansims or particles will be activated when overwhelmed by the amount of invading pathogens and in turn release inflammatory cytokines and chemokines. Consequently, neutrophils are recruited and activated as described above ^[11][12].

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Chemotaxis assays can be performed in vitro/ex vivo by using Chemotaxis Chambers (for example Neuro Probe Chambers). Supernatants can be added to the bottom well of the chamber and 3–8 mm nitrocellulose filters are placed on top, while the top chamber contains the inflammatory cells (for example neutrophils). After a certain time period, the number of migrated cells towards the lower chamber can be determined by staining of the cells^[1].

Influx of inflammatory cells (mainly neutrophils) can be analysed by tissue staining by using Haematoxylin and eosin ^[13].

In mice, neutrophil influx can be analysed using a mouse MPO ELISA kit for lysed tissue ^[14].

References

1. ↑ ^{1.0 1.1 1.2 1.3} Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, Martin SJ. Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells. Mol Cell. 2013 Mar 28;49(6):1034-48
2. ↑ ^{2.0 2.1} Freitas M, Lima JL, Fernandes E. Optical probes for detection and quantification of neutrophils' oxidative burst. A review. Anal Chim Acta 2009;649(1):8-23
3. ↑ ^{3.0 3.1} Wessels I, Jansen J, Rink L, Uciechowski P. Immunosenescence of polymorphonuclear neutrophils. ScientificWorldJournal 2010;10:145-60
4. ↑ Baehner RL, Karnovsky ML. Deficiency of reduced nicotinamide-adenine dinucleotide oxidase in chronic granulomatous disease. Science 1968;162(859):1277-9
5. ↑ Klebanoff SJ. Iodination of bacteria: a bactericidal mechanism. J Exp Med 1967;126(6):1063-78
6. ↑ Klebanoff SJ, Rosen H. The role of myeloperoxidase in the microbicidal activity of polymorphonuclear leukocytes. Ciba Found Symp 1978;(65):263-84
7. ↑ Christou NV, Meakins JL. Neutrophil function in surgical patients: Two inhibitors of granulocyte chemotaxis associated with sepsis. J Surg Res 1979;26(4):355-364
8. ↑ ^{8.0 8.1} Nauseef WM. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 2007;219:88-102
9. ↑ Silva MT. Neutrophils and macrophages work in concert as inducers and effectors of adaptive immunity against extracellular and intracellular microbial pathogens. J Leukoc Biol 2010;87(5):805-13
10. ↑ Babior BM. Phagocytes and oxidative stress. Am J Med 2000;109(1):33-44
11. ↑ Driscoll KE, Deyo LC, Carter JM, Howard BW, Hassenbein DG, Bertram TA. Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells. Carcinogenesis 1997;18(2):423-30
12. ↑ Knaapen AM, Seiler F, Schilderman PA, Nehls P, Bruch J, Schins RP, Borm PJ. Neutrophils cause oxidative DNA damage in alveolar epithelial cells. Free Radic Biol Med 1999;27(1-2):234-40
13. ↑ ^{13.0 13.1} Huebscher SG. Histological assessment of non-alcoholic fatty liver disease. Histopathol. 2006;49:450–465
14. ↑ ^{14.0 14.1} Kermanizadeh A, Brown DM, Hutchison GR, Stone V. Engineered Nanomaterial Impact in the Liver following Exposure via an Intravenous Route–The Role of Polymorphonuclear Leukocytes and Gene Expression in the Organ. Journal of Nanomed & Nanotechnol 2012;04(01):1–7
15. ↑ Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, Hu R, Hong M, Hong F. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. 2011; J. Biomed. Mater. Res. - Part A 96 A:221–229
16. ↑ Ma L, Zhao J, Wang J, Liu J, Duan Y, Liu H, Li N, Yan J, Ruan J, Wang H, Hong F. The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2. Nanoscale Res Lett. 2009 Aug 1;4(11):1275-85

How this Key Event Works

Approximately 29 million people in the European Union suffer from a chronic liver condition ^[1]. Inflammation is a crucial link that is related to many of these conditions, with the potential for the development of cirrhosis or primary liver cancer which represent the end-stage of liver pathology and are often associated with mortality: chronic hepatitis (A-E), non-alcoholic steatohepatitis (NASH) which is the progressive form of non-alcoholic fatty liver disease (NAFLD), primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) ^[1]. Drug-induced liver injury (DILI) still is a major problem in drug development as its early detection is problematic, and acute liver inflammation is the most common symptom. DILI is the main cause for withdrawal of drugs from the pharmaceutical market ^[2]. Liver inflammation is marked by an increased influx of neutrophils, following the secretion of signaling factors such as CXC chemokines and macrophage inflammatory protein 2 (MIP-2) from damaged cells ^[3]. Kupffer cells (KCs), the resident macrophages of the liver and accounting for about 15-20% of total cell numbers in a healthy liver. They are the gatekeepers in the liver, as they monitor the blood that enters this organ ^[4][5]. Activation of KCs by activation of toll like receptors, for example, leads to the recruitment of further inflammatory cells as well as amplified KC activation. This, in turn, activates Hepatic stellate cells (HSCs) ^[5] which can link liver inflammation to further severe outcomes such as development of fibrosis

A list of drugs generally known to induce DILI can be found here ^[6].

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Liver inflammation is usually confirmed by analysis of histological features, marked by influx of inflammatory cells (mainly neutrophils) which can be stained by using Haematoxylin and eosin ^[7].

In mice, neutrophil influx can be analysed using a mouse MPO ELISA kit for lysed tissue ^[4].

mRNA expression levels of inflammatory cytokines in tissue samples can be determined by using real-time PCR as described in ^[8].

Plasma levels of pro-inflammatory cytokines can be analysed by enzyme linked immunosorbent assay) ELISA using commercial kits ^[9].

References

1. ↑ ^{1.0 1.1} Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol. 2013 Mar;58(3):593-608
2. ↑ Larrey D. Epidemiology and individual susceptibility to adverse drug reactions affecting the liver. Semin Liver Dis. 2002;22(2):145-55
3. ↑ Jaeschke H. Inflammation in response to hepatocellular apoptosis. Hepatology. 2002 Apr;35(4):964-6
4. ↑ ^{4.0 4.1 4.2} Kermanizadeh A, Brown DM, Hutchison GR, Stone V. Engineered Nanomaterial Impact in the Liver following Exposure via an Intravenous Route–The Role of Polymorphonuclear Leukocytes and Gene Expression in the Organ. Journal of Nanomed & Nanotechnol 2012;04(01):1–7
5. ↑ ^{5.0 5.1} Arrese M, Cabrera D, Kalergis AM, Feldstein AE. Innate Immunity and Inflammation in NAFLD/NASH. Dig Dis Sci. 2016 May;61(5):1294-303
6. ↑ Ortega-Alonso A, Stephens C, Lucena MI, Andrade RJ. Case Characterization, Clinical Features and Risk Factors in Drug-Induced Liver Injury. Int J Mol Sci. 2016 May 12;17(5)
7. ↑ ^{7.0 7.1} Huebscher SG. Histological assessment of non-alcoholic fatty liver disease. Histopathol. 2006;49:450–465
8. ↑ ^{8.0 8.1} Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, Hu R, Hong M, Hong F. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. 2011; J. Biomed. Mater. Res. - Part A 96 A:221–229
9. ↑ ^{9.0 9.1} Ma L, Zhao J, Wang J, Liu J, Duan Y, Liu H, Li N, Yan J, Ruan J, Wang H, Hong F. The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2. Nanoscale Res Lett. 2009 Aug 1;4(11):1275-85
10. ↑ Alarifi S, Ali ., Al-Doaiss AA, Ali BA, Ahmed M, Al-Khedhairy AA. Histologic and apoptotic changes induced by titanium dioxide nanoparticles in the livers of rats. Intern J Nanomed. 2013;8:3937–3943

Relationship ID and Title:

144: Increase, Oxidative Stress leads to N/A, Mitochondrial dysfunction 1

How Does This Key Event Relationship Work

The mitochondrion consist of a plethora of antioxidant enzymes to defend against oxidative stress, such as catalases, which has been found in the liver, glutathione peroxidase, and thioredoxin peroxidase ^{[1] [2]}. At the same time, the mitochondrion itself is one of the main sources of intracellular ROS formation ^[3].

NAD(P)H plays a central role in the redox state of the mitochondrion: NADP is reduced, in part, by the activity of the NADH/NADP transhydrogenase that functions as a proton pump ^[1] and has a reductive effect on glutathione and thioredoxin. This directly links mitochondrial coupling and the membrane potential to the redox potential. As a consequence, an imbalance in the NAD(P) redox status can lead to mitochondrial permeability transition (MPT), a nonselective permeabilization of the inner mitochondrial membrane ^[4]. An imbalance of the redox state of these pyridine nucleotides and thus condition of oxidative stress can lead to an increased influx of Ca2+, which in turn facilitates activation of the mitochondrial permeability transition pore, leading to apoptosis ^{[5] [6]}.

Weight of Evidence

Biological Plausibility

Overwhelming the mitochondrial antioxidant defence system and subsequent uncoupling of the respiratory chain leads to MPT, resulting in loss of matrix components, impairment of mitochondrial functionality and substantial induction of apoptosis ^[4].

Empirical Support for Linkage

Include consideration of temporal concordance here

A direct effect of oxidative stress induction (by using t-butylhydroperoxide TBH) on the opening of the mitochondrial permeability transition pore has been reported using rat liver mitochondria ^[7]. This was found to lead to an increase in the mitochondrial membrane potential, which could be partly inhibited by addition of the antioxidant GSH ^[8]. Cell treatment with a lysosomal inhibitor was found to delay the production of ROS that act on mitochondria, thus mitochondria-related cell death was delayed ^[9]. Superoxide-radical-triggered increase in Ca2+ uptake to the mitochondrion was found to precede loss of mitochondrial membrane potential, which was independent of other oxidants and mitochondrially derived ROS, as determined by using respective inhibitors. This work shows the specific effects of external and not mitochondrially derived ROS on mitochondrial damage ^[10].

Quatitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Quantitative understanding of this KER is low. Inhibition of the ROS source could delay mitochondrial damage, and treatment with an antioxidant could partly inhibit the effect on the mitochondrion.

Evidence Supporting Taxonomic Applicability

^[10]: rat

^[7]: rat

^[8]: rat

^[9]: mouse

References

1. ↑ ^{1.0 1.1} Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE. Mitochondria and reactive oxygen species. Free Radic Biol Med. 2009 Aug 15;47(4):333-43
2. ↑ M. Salvi, V. Battaglia, A.M. Brunati, N. La Rocca, E. Tibaldi, P. Pietrangeli, L. Marcocci, B. Mondovì, C.A. Rossi, A. Toninello. Catalase takes part in rat liver mitochondria oxidative stress defense. J. Biol. Chem., 282 (2007), pp. 24407–24415
3. ↑ Kim I, Rodriguez-Enriquez S, Lemasters JJ. Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys. 2007 Jun 15;462(2):245-53
4. ↑ ^{4.0 4.1} Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeability transition and oxidative stress. FEBS Lett. 2001 Apr 20;495(1-2):12-5
5. ↑ Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999 Jul 15;341 (Pt 2):233-49
6. ↑ Hüser J, Rechenmacher CE, Blatter LA. Imaging the permeability pore transition in single mitochondria. Biophys J. 1998 Apr;74(4):2129-37
7. ↑ ^{7.0 7.1} Halestrap AP, Woodfield KY, Connern CP. Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase. J Biol Chem. 1997 Feb 7;272(6):3346-54
8. ↑ ^{8.0 8.1} Hüser J, Rechenmacher CE, Blatter LA. Imaging the permeability pore transition in single mitochondria. Biophys J. 1998 Apr;74(4):2129-37
9. ↑ ^{9.0 9.1} 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 Jan 1;285(1):667-74
10. ↑ ^{10.0 10.1} Madesh M, Hawkins BJ, Milovanova T, Bhanumathy CD, Joseph SK, Ramachandrarao SP, Sharma K, Kurosaki T, Fisher AB. Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis. J Cell Biol. 2005 Sep 26;170(7):1079-90

Relationship ID and Title:

144: N/A, Mitochondrial dysfunction 1 leads to N/A, Cell injury/death

How Does This Key Event Relationship Work

ROS generation is known to activate different pathways leading to apoptosis, whereas depletion of energy production induces necrotic cell death.

Weight of Evidence

Biological Plausibility

There is functional mechanistic understanding supporting this relationship between KE3 and KE4.

ROS are known to stimulate a number of events and pathways that lead to apoptosis, triggered by ROS-induced ER stress signalling pathway (Lu et al., 2014), caspase-dependent and -independent apoptosis (Zhou et al., 2015), mitogen-activated protein kinase (MAPK) signal transduction pathways (reviewed in Cuadrado and Nebreda, 2010, Harper and LoGrasso, 2001).

Depletion of cellular ATP is known to cause switching from apoptotic cell death triggered by a variety of stimuli to necrotic cell death (Leist et al., 1997) suggesting that the level of intracellular ATP determines whether the cell dies by apoptosis or necrosis (Nicotera et al., 1998). There is strong proof that apoptosis requires energy, as it is a highly regulated process involving a number of ATP-dependent steps such as caspase activation, enzymatic hydrolysis of macromolecules, chromatin condensation, bleb formation and apoptotic body formation (Richter et al., 1996).

Empirical Support for Linkage

Include consideration of temporal concordance here

In the case of DomA, in vitro studies have shown that oxidative stress and oxidative stress-induced activation of the stress-activated protein kinase/c-jun-N-terminal kinase (SAPK/JNK) pathway is implicated in DomA-mediated apoptosis (Giordano et al., 2007; 2008; 2009; Lu et al., 2010). In vivo findings also show that ROS-mediated cognitive deficits are associated with apoptosis induced by activation of the JNK pathway (Lu et al., 2010; 2011).

• Mice injected intraperitoneally (i.p.) with DomA at a dose of 2 mg/kg once a day for 4 weeks have shown increase (6 fold) of the TUNEL positive cells in the hippocampus . In the same study they have found that indicators of mitochondria function are markedly decreased (1.5-2 fold) and ROS levels are elevated (3.2 fold) (Lu et al., 2012). DomA treatment also significantly decreases the levels of bcl-2, procaspase-3 and procaspase-12 and increases the activation of caspase-3 and caspase-12 in the mouse hippocampus (Lu et al., 2012). The same research group using similar dose but longer exposure (4 weeks), has shown increase of ROS (3 fold) and NOX (2 fold) and elevated (8 fold) mean value of TUNEL-positive cells in the hippocampal CA1 sections as well as increase in the activation of caspase-8 and caspase-3 (Wu et al., 2012). These two in vivo studies (Lu et al., 2012; Wu et al., 2012) suggest that both KEs are affected in response to the same dose of DomA and exposure paradigm and that the incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction).

• The cell viability has been measured by the MTT reduction assay in mouse cerebellar granule neurons (CGNs) and showed that the IC50 values for DomA are 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons (Giordano et al., 2006). This reduction in cell viability has been demonstrated to be concentration dependent after studying a range of concentrations of DomA (0.01 and 10 µM). Giordano et al. 2007 have shown that 100 nM DomA induce apoptotic cell death in mouse CGNs. In a follow-up study, the same research group has performed a dose response evaluation and showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs derived from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione are more sensitive as 10 nM DomA induces a significant increase in apoptotic cell number (Giordano et al., 2009). The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes has been caused by 100 nM DomA. Interestingly, 1 and 10 µM DomA still cause significant apoptosis in both cell types but to a lesser extent compared to 100 nM DomA. ROS have been measured only at the dose of 100 nM DomA, 30 min after treatment and showed 2.5 fold increase compared to controls in CGNs from Gclm (+/+) mice (Giordano et al., 2009). Caspase 3 activity has also been measured after 12 h with prior 1 h exposure to 100 nM DomA and found to be increased (2.2 fold). In the same study, DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h exposure. Again these in vitro studies (Giordano et al., 2007; 2009) suggest that both KEs are affected by the same dose of DomA and that the incidence of KE down (cell death) is higher than the incidence of KE up (mitochondrial dysfunction). Furthermore, KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that takes place 12-24 h later.

• Mixed cortical cultures have been treated with 3, 5, 10, or 50 μM DomA for a variety of exposure durations (10 min, 30 min, 1 h, or 2 h), after which DomA is washed out and the culture medium is replaced with conditioned medium from unexposed sister cultures (Qiu et al., 2006). In all cases neuronal death has been measured 24 h following the beginning of exposure. The results show that DomA-induced neuronal death is determined by both concentration and duration of exposure. After a 10-min exposure, 50 μM DomA produces marked neuronal death of 47.4 %, whereas by 1 h of treatment, the same concentration produces near maximal neuronal death but longer exposures do not increase neuronal death further (Qiu et al., 2006). Regarding time dependence, this study shows that low concentrations of DomA produces more neuronal death if this is measured 22 h after the washout than if measured immediately after DomA treatment, while higher concentrations of DomA (20–100 μM) produces equivalent degrees of neuronal death when measured at these two time points (Qiu et al., 2006). Based on these findings, three EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration of exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) (Qiu et al., 2006).

• The mean concentration of DomA in rat brain samples obtained at 30 min after intraperitoneal (i.p.) administration of 1 mg/kg DA is 7.2 ng/g (Tsunekawa et al., 2013). These animals have been examined and revealed after histopathological analysis neuronal shrinkage and cell death, including an increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and after 5 days (19.0 %) compared to the controls (1.7 %) (Tsunekawa et al., 2013). In the same study, indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone (Tsunekawa et al., 2013).

• Brain slices from 8-day-old pups have been treated after 2 weeks with 10 μM DomA and assessed with propidium iodine (PI) stain to determine cellular damage (Erin and Billingsley, 2004). A time course has been carried out and viable cultures have been visualized 12, 24, 48 and 92 h after DomA treatment. Changes in PI uptake has been detected after 24 h post-treatment and at 4h the average fold-increase of PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively (Erin and Billingsley, 2004). In the same study, incubation of brain slices with DomA induces degradation of α-spectrin to the 120-kDa product after 18 h of treatment but no change has been noted after 12 h incubation, whereas caspase 3 activity results have not been conclusive (Erin and Billingsley, 2004).

• Using observations of neuronal viability and morphology, exposure of cultured murine cortical neurones to DomA for 24 h have shown to induce concentration-dependent neuronal cell death and the EC50 determined to be 75 µM (Larm et al., 1997).

Stressor	Experimental Model	Tested concentrations	Exposure route	Exposure duration	Mitochondrial dysfunction (KE up) (measurements, quantitative if available)	Cell death (KE down) (measurements, quantitative if available)	References	Temporal Relationship	Dose-response relationship	Incidence	Comments
DomA	16-month-old male ICR mice	2 mg/kg	Intraperitoneally (i.p.)	Once a day for 4 weeks	Indicators of mitochondrial function were markedly decreased (1.5-2 fold) and ROS levels were elevated (3.2 fold).	The mean of TUNEL positive cells in the hippocampus was increased (6 fold). The levels of bcl-2, procaspase-3 and procaspase-12 were significantly decreased and the activation of caspase-3 and caspase-12 in the mouse hippocampus were increased.	Lu et al., 2012		Same dose	Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)
DomA	16-month-old male ICR mice	2 mg/kg	i.p.	Once a day for 4 weeks	ROS levels were increased (3 fold) and NOX (2 fold).	The mean value of TUNEL-positive cells in the hippocampal CA1 sections was elevated (8 fold) and the activation of caspase-8 and caspase-3 was increased.	Wu et al., 2012		Same dose	Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)
DomA	Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice	0.01 to 10 µM		Time course (15 to 120 min)	DomA caused a significant time- and concentration-dependent increase in ROS production. The higher ROS production (2.5 fold increase) was recorded after 1 h of exposure.	IC50 values for DomA were 3.4 μM in Gclm (+/+) neurons and 0.39 μM in Gclm (-/-) neurons based on MTT assay after 24 h of exposure.	Giordano et al., 2006	KE up (mitochondrial dysfunction) happens earlier than KE down (cell death)	Same doses
DomA	CGNs from Gclm (+/+) and Gclm (−/−) mice	0.01 to 10 µM		Time course (0 to 180 min)	DomA (0.1μM) caused a 3 fold increase in DHR fluorescence, which accumulates in mitochondria and fluoresces when oxidized by ROS or reactive nitrogen species. This occurred between 1 and 2 h and was higher in CGNs from Gclm (−/−) mice.	0.1μM DomA was maximally effective in inducing apoptosis, while a concentration causing high toxicity (10μM) induced very limited apoptosis, 24 h after exposure.	Giordano et al., 2007	KE up (mitochondrial dysfunction) happens earlier (1-2 h) than KE down (cell death) that occurs after 24 h	Same doses
DomA	CGNs from Gclm (+/+) and Gclm (−/−) mice	0.01 to 10 µM		For ROS: 30min, Apoptosis: 12-24 h.	ROS levels were measured only at the dose of 100 nM DomA 30 min after treatment in CGNs from Gclm (+/+) mice and showed 2.5 fold increase compared to controls .	A dose response study that showed that even 50 nM DomA exposure for 1 h (after washout and additional 23 h incubation) can induce apoptosis in CGNs from Gclm (+/+) mice, whereas neurons from Gclm (−/−) mice that have very low levels of glutathione were more sensitive as 10 nM DomA induced a significant increase in apoptotic cells number .The maximal apoptosis (5 fold compared to controls) in CGNs from both genotypes was caused by 100 nM DomA. 1 and 10 µM DA caused significant apoptosis in both cell types but to less extend compared to 100 nM DomA. Caspase 3 activity after 12 h with prior 1 h exposure to 100 nM DomA found to be increased (2.2 fold). DomA (100 nM) caused a significant decrease (25%) of Bcl-2 protein levels after 6 h from exposure.	Giordano et al., 2009	KE up (mitochondrial dysfunction) happens earlier (30 min) than KE down (cell death) that take place 12-24 h later	Same dose	Incidence of downstream KE (cell death) is higher than the incidence of upstream KE (mitochondrial dysfunction)
DomA	Mixed cortical cultures obtained from pregnant Holtzman rats on embryonic day (ED) 16–18	3, 5, 10, or 50 μM		10 min, 30 min, 1 h or 2 h, after which DomA was washed out and the culture medium replaced with conditioned medium from unexposed sister cultures .		EC50 exposure paradigms have been established, which represent weak/prolonged exposure (3 μM/24 h), moderate concentration and duration exposure (10 μM/2 h), and strong/brief exposure (50 μM/10 min) .	Qiu et al., 2006
DomA	Rat	1 mg/kg DA	i.p.		Indirectly it has been shown that ROS production is associated with these histopathological findings by using the radical scavenger edaravone .	Neuronal shrinkage and cell drop out as well as increase in the percentage of TUNEL positive cells at 24 hours (8.3 %) and 5 days (19.0 %) has been found compared with that of controls (1.7 %) .	Tsunekawa et al., 2013
DomA	Rat rain slices from 8-day-old pups	10 μM		Time course (12, 24, 48 and 92 h) after DomA treatment.		PI uptake (DomA/control) was 14.5 and 34.5 in cortex and hippocampus, respectively . Degradation of α-spectrin to the 120-kDa product after 18 h of DomA treatment was noted but no change was noted after 12 h incubation, whereas caspase 3 activity results were not conclusive.	Erin and Billingsley, 2004
DomA	Cultured murine cortical neurones					DomA induces concentration-dependent neuronal cell death and the EC50 determined to be 75 µM .	Larm et al., 1997

Gap of knowledge: there are no studies showing that GLF induces neuronal cell death through mitochondrial dysfunction.

Uncertainties or Inconsistencies

Rats have been administered with DA at the dose of 1.0 mg/kg for 15 days. The histochemical analysis of hippocampus from these animals has revealed no presence of apoptotic bodies and no Fluoro-Jade B positive cells (Schwarz et al., 2014).

Quatitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

The experiments describing semi-quantitative effects for this KER is described in the table above.

References

Cuadrado A, Nebreda AR., Mechanisms and functions of p38 MAPK signalling. Biochem J., 2010, 429(3): 403–417.

Erin N, Billingsley ML., Domoic acid enhances Bcl-2-calcineurin-inositol-1,4,5-trisphosphate receptor interactions and delayed neuronal death in rat brain slices. Brain Res., 2004, 1014: 45-52.

Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG., Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol., 2006, 70: 2116-2126.

Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG., Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.

Giordano G, Klintworth HM, Kavanagh TJ, Costa LG., Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases. Neurochem Int., 2008, 52: 1100-1105.

Giordano G, Li L, White CC, Farin FM, Wilkerson HW, Kavanagh TJ, Costa LG., Muscarinic receptors prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells. J Neurochem., 2009, 109: 525-538.

Harper SJ, LoGrasso P., Signalling for survival and death in neurones: the role of stress-activated kinases. JNK and p38. Cell Signal., 2001, 13(5): 299–310.

Larm JA, Beart PM, Cheung NS., Neurotoxin domoic acid produces cytotoxicity via kainate- and AMPA-sensitive receptors in cultured cortical neurones. Neurochem Int., 1997, 31: 677-682.

Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P., Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med., 1997, 185: 1481−1486.

Lu J, Wu DM, Zheng YL, Hu B, Zhang ZF., Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis. Brain Pathol., 2010, 20: 598-612.

Lu J, Wu DM, Zheng ZH, Zheng YL, Hu B, Zhang ZF., Troxerutin protects against high cholesterol-induced cognitive deficits in mice. Brain., 2011, 134: 783-797.

Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF., Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52(3): 646-59.

Lu TH, Su CC, Tang FC, Chen CH, Yen CC, Fang KM, Lee KL, Hung DZ, Chen YW., Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway. Chem Biol Interact., 2014, 225: 1-12.

Nicotera P, Leist M, Ferrando-May E., Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicol Lett., 1998, 102-103: 139-142.

Qiu S, Pak CW, Currás-Collazo MC., Sequential involvement of distinct glutamate receptors in domoic acid-induced neurotoxicity in rat mixed cortical cultures: effect of multiple dose/duration paradigms, chronological age, and repeated exposure. Toxicol Sci., 2006, 89: 243-256.

Richter C, Schweizer M, Cossarizza A, Franceschi C. Control of apoptosis by the cellular ATP level. FEBS Lett., 1996, 378: 107-110.

Schwarz M, Jandová K, Struk I, Marešová D, Pokorný J, Riljak V. Low dose domoic acid influences spontaneous behavior in adult rats. Physiol Res., 2014, 63: 369-76.

Silvagni PA, Lowenstine LJ, Spraker T, Lipscomb TP, Gulland FMD., Pathology of Domoic Acid Toxicity in California Sea Lions (Zalophus californianus). Vet Path., 2005, 42: 184-191.

Tryphonas L, Truelove J, Iverson F, Todd EC, Nera EA. Neuropathology of experimental domoic acid poisoning in non-human primates and rats. Can Dis Wkly Rep. 1990 Sep;16 Suppl 1E:75-81.

Tsunekawa K, Kondo F, Okada T, Feng GG, Huang L, Ishikawa N, Okada S., Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation. BMC Neurosci., 2013, 14: 4-16.

Wu DM, Lu J, Zheng YL, Zhang YQ, Hu B, Cheng W, Zhang ZF, Li MQ., Small interfering RNA-mediated knockdown of protein kinase C zeta attenuates domoic acid-induced cognitive deficits in mice. Toxicol Sci., 2012, 128: 209-222.

Zhou Q, Liu C, Liu W, Zhang H, Zhang R, Liu J, Zhang J, Xu C, Liu L, Huang S, Chen L., Rotenone induction of hydrogen peroxide inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, leading to neuronal apoptosis. Toxicol Sci., 2015, 143: 81-96.

Relationship ID and Title:

144: N/A, Cell injury/death leads to Release, Cytokine

How Does This Key Event Relationship Work

Apoptosis is a complex process that regulates whether cell death leads to the induction of inflammation or quiet removal of a damaged cell, for example during development or normal tissue turnover. This most likely depends on the severity of the effect^[1]. Additional cell death mechanisms are known to be potent inducers of inflammation, such as necrosis (including secondary necrosis which can follow apoptosis if the removal of apoptotic cells by phagocytic cells fails). Necrosis leads to the release of intracellular contents into the extracellular milieu, thus inducing an inflammatory response^[2].

The induction of inflammation by apoptosis is usually linked to infiltration of neutrophils, which are recruited by secreted CXC chemokines. Dying hepatocytes can release intracellular molecules known as damage-associated molecular patterns (DAMPs), which, if persistent, can induce the so-called sterile inflammation. This occurs in the absence of pathogens and is a key factor for the development of (liver) inflammation ^[3][4]. Mitochondrial DNA (mtDNA) and mitochondria-derived formyl peptides are examples of mitochondria-derived DAMPs which bind to pattern recognition receptors (PPRs) such as toll-like receptors (TLRs). TLRs are found expressed in most liver cells, including hepatocytes, Kupffer cells (KCs) or hepatic stellate cells (HPCs) ^[3]. Specifically, mtDNA-activated TLR9 has recently been described to play a role in the development of liver inflammation and accompanied induction of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-a) ^[5].

Fas is a cell surface glycoprotein that belongs to the tumor necrosis factor receptor family. It is known that ligation of the Fas receptor promotes the proteolytic cleavage of intracellular caspases and thus leads to the induction of apoptosis^[6]. Activation of Fas directly leads to the activation of caspase-3 and induction of a variety of cytokines such as macrophage inflammatory protein-2 (MIP-2)/IL-8, KC, IL-6, MCP-1/CCL2 and sICAM-1. However, when caspase-3 was inhibited, the chemokine-induction was significantly reduced. Faouzi and coworkers could also show that the transcription factor AP-1, and not NF-kB, was involved in the onset of apoptosis-induced liver inflammation^[6][7].

Weight of Evidence

Biological Plausibility

The severity of cell death activation determines the outcome for the cell: inflammation is part of the tissue regeneration process, and intermediate apoptotic stimuli are able to trigger this response. Recruitment of inflammatory cells such as neutrophils is meant as a beneficial process, as for example apoptotic bodies of bacteria-infected cells can be removed. Thus the apoptotic cells can secrete soluble "find-me" factors that trigger infiltration of immune cells. However, if this becomes chronic it has the potential to enhance tissue damage and ultimately induce fibrosis^[1][7].

Empirical Support for Linkage

Include consideration of temporal concordance here

A high fat diet increases the amount of plasma mtDNA levels, which were shown to induce TLR9, accompanied by the induction of TNF-a. TLR9 knock-out mice were shown to show less severe symptoms for developing liver inflammation when put on a high fat diet compared to control mice ^[5].

Induction of apoptosis by using an anti-Fas antibody was found to lead to upregulation and secretion of KC and MIP-2 in liver tissue, while inhibition of caspase-3 significantly reduced chemokine-induction^[6].

Uncertainties or Inconsistencies

No dose-response or time dependency is described; proof is presented mainly by using respective inhibitors.

Quatitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Currently, there is no quantitative understanding of this KER. The use of Fas-inhibitors has led to understanding this mechanism. Interestingly, an increase of the stimulus (increased concentrations of anti-Fas) has led to decreased cytokine formation which is explained by a potential caspase-3-dependent block in chemokine translation^[6].

Evidence Supporting Taxonomic Applicability

^[6][5][7]: mouse ^[7]: human

References

1. ↑ ^{1.0 1.1} Jaeschke H. Inflammation in response to hepatocellular apoptosis. Hepatology. 2002 Apr;35(4):964-6
2. ↑ Hirsova P, Gores GJ. Death Receptor-Mediated Cell Death and Proinflammatory Signaling in Nonalcoholic Steatohepatitis. Cell Mol Gastroenterol Hepatol. 2015 Jan;1(1):17-27
3. ↑ ^{3.0 3.1} Arrese M, Cabrera D, Kalergis AM, Feldstein AE. Innate Immunity and Inflammation in NAFLD/NASH. Dig Dis Sci. 2016 May;61(5):1294-303
4. ↑ Miura K, Yang L, van Rooijen N, Brenner DA, Ohnishi H, Seki E. Toll-like receptor 2 and palmitic acid cooperatively contribute to the development of nonalcoholic steatohepatitis through inflammasome activation in mice. Hepatology. 2013 Feb;57(2):577-89
5. ↑ ^{5.0 5.1 5.2} Garcia-Martinez I, Santoro N, Chen Y, Hoque R, Ouyang X, Caprio S, Shlomchik MJ, Coffman RL, Candia A, Mehal WZ. Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9. J Clin Invest. 2016 Mar 1;126(3):859-64
6. ↑ ^{6.0 6.1 6.2 6.3 6.4} Faouzi S, Burckhardt BE, Hanson JC, Campe CB, Schrum LW, Rippe RA, Maher JJ. Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway. J Biol Chem. 2001 Dec 28;276(52):49077-82
7. ↑ ^{7.0 7.1 7.2 7.3} Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, Martin SJ. Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells. Mol Cell. 2013 Mar 28;49(6):1034-48

Relationship ID and Title:

144: Disruption, Lysosome leads to Increase, Oxidative Stress

How Does This Key Event Relationship Work

The lysosome contains redox-active labile irons which are suggested to be involved in local reactive oxygen species (ROS) formation via a Fenton-type reaction ^[1]. Many iron containing metallo-proteins are degraded within the lysosomes, leading to an enrichment of this transition metal within this organelle. Iron which is released inside lysosomes due to degradation processes is transported to the cytoplasm and then stored in ferritin, a ubiquitous and highly conserved iron-binding protein ^[2]. Induction of lysosomal membrane disruption by lysosomotropic detergents has been found to cause an induction of ferritin, together with an increase of cellular ROS and concomitant reduction of the antioxidants MnSOD (manganese superoxide dismutase) and GSH (glutathione). A suggested explanation for this is the release of free iron into the cytosol ^[2].

Weight of Evidence

Biological Plausibility

The main lysosomal function is the degradation of macromolecules. To this end, they are filled with more than 50 acid hydrolases ^[3] and are additionally enriched with iron, as explained above. Via the Fenton-type reaction, iron can catalyse formation of ROS. Thus, damage of the lysosomal membrane can induce cell death mechanisms such as necrosis and apoptosis, depending on the severity of lysosomal damage ^[4].

Empirical Support for Linkage

Include consideration of temporal concordance here

^[5]: By using galactosyl dextran-retinal (GDR) nanogels, the authors demonstrated a negative correlation between ROS production and lysosome function in dendritic cells. Neutralizing the lysosomal pH with NH4Cl partially recovered lysosomal fluorescence but dramatically attenuated GDR-induced ROS after 4h of incubation.

^[6]: The authors found that active autophagy is related to basal ROS generation in neuronal cells. Using relevant fluorescent probes, localisation of ROS at lysosomes was found. The decrease of lysosomal ROS by treatment of cells with lysosomal inhibitors delayed the mitochondrial ROS burst and thus cell toxicity.

^[2]: The authors suggest that ROS is initially produced due to LMP and release of lysosomal contents, which further promotes mitochondrial membrane permeabilisation (MMP) in apoptosis. This was further supported by experiments with NH4Cl pre-exposure, in which intra-lysosomal trapped NH4+ reduced cellular oxidative stress and apoptotic cell death by blocking lysosomal accumulation of the trigger (O-methyl-serine dodecylamide hydrochloride, MSDH). ROS production was found as early as 3 h and clear reduction of antioxidant enzymes took place from 6 h following exposure, prior to alteration of MMP.

^[7]: Using positively charged polystyrene nanoparticles (PS-NH2) as initiators, the authors performed a time-resolved experiment where lysosomal damage was found as the first adverse effect, followed by an increase in reactive oxygen species and subsequent loss in mitochondrial membrane potential. They could show that KEup occurred at earlier time points (3-6 hours) than KEdown (starting after 8 hours).

Uncertainties or Inconsistencies

All studies were performed in varying cell types, either immune or brain cells. Applicability in other cell types such as hepatocytes needs to be determined.

Quatitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

To date, there are no quantitative studies to determine this KER. However, when lysosomal damage is reduced by neutralizing the lysosomal pH with NH4Cl, ROS-induction is strongly decreased ^[5].

Evidence Supporting Taxonomic Applicability

^[5]: murine

^[6]: murine

^[2]: murine, human

^[7]: human

References

1. ↑ 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 Jan 1;285(1):667-74
2. ↑ ^{2.0 2.1 2.2 2.3} Ghosh M, Carlsson F, Laskar A, Yuan XM, Li W. Lysosomal membrane permeabilization (LMP) causes oxidative stress and ferritin induction in macrophages. FEBS Lett. 2011 Feb 18;585(4):623-9
3. ↑ Johansson AC, Appelqvist H, Nilsson C, Kågedal K, Roberg K, Ollinger K. Regulation of apoptosis-associated LMP. Apoptosis. 2010 May;15(5):527-40
4. ↑ Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 2001 Jun;8(6):569-81
5. ↑ ^{5.0 5.1 5.2} Wang C, Li P, Liu L, Pan H, Li H, Cai L, Ma Y. Self-adjuvanted nanovaccine for cancer immunotherapy: Role of lysosomal rupture-induced ROS in MHC class I antigen presentation. Biomaterials. 2016 Feb;79:88-100
6. ↑ ^{6.0 6.1} 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 Jan 1;285(1):667-74
7. ↑ ^{7.0 7.1} 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

Relationship ID and Title:

144: Disruption, Lysosome leads to N/A, Mitochondrial dysfunction 1

How Does This Key Event Relationship Work

Lysosomes were first described by de Duve and colleagues in 1955 ^[1]. 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 ^[2]. Amongst these are cathepsins that are released into the cytosol once lysosomal membrane permeabilisation (LMP) occurs.

The major substrate of cathepsins are Bid, Bcl-2 and Bax. This initiates the subsequent activation of caspase-9 and 3/7, leading to mitochondrial membrane permeabilisation (MMP) and mitochondria-induced apoptosis ^[3]. The role of cathepsins, especially cathepsin D on mitochondria has been studied in detail: microinjection of cathepsin D into the cytosol induced a subsequent release of cytochrome c, caspase activation and induction of apoptosis. This could be prevented by using inactivated cathepsin D or the cathepsin D inhibitor pepstatin A ^[4]. The use of this inhibitor and its effect on preventing cytochrome c release or caspase-3 activation, together with an inhibition of apoptosis, has also been reported previously ^[5].

Moreover, translocation of tBid was observed following LMP induced by Au-ZnO hybrid nanoparticles, which was accompanied by a time-dependent release of cytochrome c and activation of caspase 3 ^[6].

Another study applied high content imaging to show the occurrence of LMP and MMP at different concentrations in a variety of different cell lines. When determining the IC50/EC50 value, by using positively charged polystyrene particles (PS-NH2) it could be shown that lysosomal damage appeared at lower concentrations than mitochondrial damage in all cells ^[7].

The importance of MMP in LMP-induced cell death was furthermore confirmed by work of Boya and colleagues: by using the quinolone antibiotics ciprofloxacin (CPX) or norfloxacin (NFX) (with or without UV light) which are known inducers of LMP, treatment of cells resulted in caspase-independent cell death, with hallmarks of apoptosis such as chromatin condensation and phosphatidylserine exposure on the plasma membrane. However, inhibition of the lysosomal accumulation of CPX or NFX suppressed their capacity to induce LMP and to kill cells. Moreover, using Bax/Bak double deficient cells, MMP and subsequent cell death were completely abolished, showing that mitochondria are indispensable for cell death initiated by lysosomal destabilization ^[8].

Weight of Evidence

Biological Plausibility

Lysosomal permeabilisation has long been known to play a role in necrotic and autophagic cell death. More recently, its role in apoptosis as well as regulation of immune responses has been additionally acknowledged. Release of proteolytic enzymes such as cathepsin D from compromised lysosomes contributes to signalling pathways for apoptosis induction. Thus lysosomes are not only important in mediating cell death, but also play a key role in the induction and regulation of inflammation ^{[9] [10]}.

Empirical Support for Linkage

Include consideration of temporal concordance here

^[6]: Inhibition of cathepsin B decreased subsequent tBid translocation and downstream caspase 3 activation.

^{[5] [4]}: Inactivated cathepsin D or the cathepsin D inhibitor pepstatin A prevented the release of cytochrome c, caspase activation and induction of apoptosis.

^[7]: By using high content imaging to show the occurrence of LMP and MMP at different concentrations in a variety of different cell lines, it could be shown that the IC50/EC50 values for the induction of lysosomal damage were lower than those for mitochondrial damage in all tested cells.

^[8]: Cathepsin B release occurred before caspace c release (1 vs 6-15h); Lysotracker positive stain was present already after 1 hour, whereas MMP staining was positive only after 6 hours and later.

Uncertainties or Inconsistencies

The available quantitative information relates to LMP and MMP only, without providing information on the mechanism between (such as cathepsin D release). Therefore, involvement of for example ROS as intermediate MMP inducer cannot be ruled out (see KER 921).

Quatitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

The quantitative understanding of this KER is low. However, as described above, the absence of important modulators such as Bax/Bak completely prevents induction of MMP after LMP. Moreover, inhibition of cathepsin D prevented the subsequent effects on the mitochondrion, such as the release of cytochrome c, caspase activation and induction of apoptosis.Also, by using nanoparticles, LMP was induced at lower concentrations as MMP.

Evidence Supporting Taxonomic Applicability

^[6]: human

^[4]: human

^[7]: human, murine

^[8]: human, murine

References

1. ↑ 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 Aug;60(4):604-17
2. ↑ Johansson AC, Appelqvist H, Nilsson C, Kågedal K, Roberg K, Ollinger K. Regulation of apoptosis-associated lysosomal membrane permeabilization. Apoptosis. 2010 May;15(5):527-40
3. ↑ Repnik U, Stoka V, Turk V, Turk B. Lysosomes and lysosomal cathepsins in cell death. Biochim Biophys Acta. 2012 Jan;1824(1):22-33
4. ↑ ^{4.0 4.1 4.2} Roberg K, Kågedal K, Ollinger K. Microinjection of cathepsin d induces caspase-dependent apoptosis in fibroblasts. Am J Pathol. 2002 Jul;161(1):89-96
5. ↑ ^{5.0 5.1} Roberg K, Johansson U, Ollinger K: Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress. Free Radic Biol Med 1999, 27:1228–1237
6. ↑ ^{6.0 6.1 6.2} Gao W, Cao W, Zhang H, Li P, Xu K, Tang B. Targeting lysosomal membrane permeabilization to induce and image apoptosis in cancer cells by multifunctional Au-ZnO hybrid nanoparticles. Chem Commun (Camb). 2014 Aug 4;50(60):8117-20
7. ↑ ^{7.0 7.1 7.2} 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 Sep 19;9(9):e108025
8. ↑ ^{8.0 8.1 8.2} 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 May 19;197(10):1323-34
9. ↑ Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene. 2004 Apr 12;23(16):2881-90
10. ↑ Repnik U, Turk B. Lysosomal-mitochondrial cross-talk during cell death. Mitochondrion. 2010 Nov;10(6):662-9

Relationship ID and Title:

144: Release, Cytokine leads to Infiltration, Inflammatory cells

How Does This Key Event Relationship Work

Binding of damage- or pathogen-associated molecular patterns (DAMPs or PAMPs) to pattern recognition receptors (PPRs) such as toll-like receptors (TLRs) can lead to the activation of, amongst others, nuclear factor-κB (NF-κB) or the transcription factor AP-1. This leads to an upregulation of chemokines and inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukins or proteases ^[1][2]. TLRs are found expressed in most cells, including liver cells such as hepatocytes, Kupffer cells (KCs) or hepatic stellate cells (HPCs) ^[2]. Upon cytokine secretion, polymorphonuclear neutrophils (PMNs) that, amongst others, circulate in the blood, can become attracted. PMNs are potent phagocytes, but they also lead to pathogen destruction upon oxidative bursting and are for their part capable of pro-inflammatory cytokine production as well. Various endothelial adhesion molecules, such as the intercellular adhesion molecule 1 (ICAM-1), mediate neutrophil adhesion to endothelial cells. ICAM-1 expression on the luminal surface of the capillary is increased during inflammation, and interacts with ß2 integrin, which is expressed on the surface of PMN. Subsequent to adhesion, neutrophils begin to migrate across the endothelium and towards the center of inflammation ^[3][4]. Interleukin-8 (IL-8) is known to be one of the most potent chemoattractants for the recruitment and activation of neutrophils into various organs (e.g. lung, intestine), and binds to the human CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2) on the surface of the PMN ^[5][6][7]. But not only IL-8, also macrophage inflammatory protein-2 (MIP-2), growth-regulated oncogenes-α, -β, and -γ, as well as the rodent peptides cytokine-induced neutrophil chemoattractant and KC are all members of the CXC subfamily of chemokines and chemoattractants for inflammatory cells^[8]

Cullen and co-workers could further confirm the importance of specific chemokines for chemotaxis of different inflammatory cells. They depleted certain chemokines by using respective antibodies in supernatants of Fas-stimulated HeLa cells (see Relationship:924 for explanation on Fas and its role on cytokine induction) and subsequently assessed the chemotactic activity of immune cells. Only depletion of MCP-1 was sufficient to block almost all THP-1 monocyte chemotaxis. On the other hand, chemotaxis of primary human peripheral blood neutrophils was depending mainly on secreted IL-8. Using an in vivo mouse model, the authors found that Fas stimulation could trigger phagocyte migration by administration of anti-Fas (Jo2) antibody into C57BL/6 mice within 10 h of anti-Fas administration. This correlated with extensive cell death in the thymus and a dramatic increase of CD11b-positive macrophages in the same tissue^[9].

Weight of Evidence

Biological Plausibility

Secreted chemokines are signalling proteins that attract immune cells to migrate to the infected or damaged tissue, in order to trigger tissue repair, removal of cell bodies or bacteria.

Empirical Support for Linkage

Include consideration of temporal concordance here

Infiltration of the hepatic parenchyma by neutrophils was found coinciding with chemokine induction. When chemokines have been neutralized by the addition of neutralizing monoclonal antibodies, a study found KC mainly responsible for abrogating an inflammatory response to Fas-induced hepatic inflammation. In this study, chemokine induction in the livers of anti-Fas-treated mice was not associated with activation of NF-kB, but it coincided with nuclear translocation of activator protein-1 (AP-1). AP-1 activation in the liver was detected shortly (1–2 h) after anti-Fas treatment, suggesting a connection to the onset of apoptosis. When apoptosis was prevented by pre-treatment of the mice with a caspase-3 inhibitor, AP-1 activation and hepatic chemokine production were both significantly reduced. Moreover the authors report a reduction of the hepatic inflammation by 70%^[8].

Addition of 200ng/ml anti-Fas antibody to HeLa cells resulted in the secretion of about 0.7ng/ml IL-8 into the supernatant. In the same study, they could show that supernatants of cells treated with 250ng/ml anti-Fas induced the strongest infiltration of neutrophils (which was almost abolished when the supernatants were treated with an anti-IL-8 antibody). This infiltration was strongly decreased upon dilution of the supernatants. Thus, this allows for a rough quantification of the IL-8 concentrations that are needed for potent chemoattraction of neutrophils^[9].

A further study on quantification of neutrophil migration in dependence of IL-8 concentration found a biphasic exhibition of migration, with an optimum random neutrophil motility at 3 nM of IL-8 ^[10].

Uncertainties or Inconsistencies

Studies exist that report an optimal IL-8 concentration for strongest neutrophil motility, so this KER can actually be quantified. However, this is usually performed ex vivo. Isolated neutrophils are very sensitive towards manual handling and need to be treated with care and within a very short time frame. Therefore, results give an indication on necessary concentrations, but need to be carefully considered with regards to direct transferability to the in vivo situation. Moreover, not only IL-8 is responsible for the recruitment of neutrophils, but also other chemokines can contribute to attraction of inflammatory cells. As they play a minor role, they are usually not considered and included in ex vivo studies.

Quatitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

This KER can be described quantitatively, as useful chemotaxis-assays are available that make use of isolated immune cells ex vivo. Those results give an indication on concentrations necessary for cell migration, but need to be carefully considered with regards to direct transferability to the in vivo situation. Moreover, not only IL-8 is responsible for the recruitment of neutrophils, but also other chemokines can contribute to attraction of inflammatory cells. However, additional proof for this KER is provided by the neutralization of chemokines, which prevented a further onset of inflammation.

References

1. ↑ Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388(6640):394-7
2. ↑ ^{2.0 2.1} Arrese M, Cabrera D, Kalergis AM, Feldstein AE. Innate Immunity and Inflammation in NAFLD/NASH. Dig Dis Sci. 2016 May;61(5):1294-303
3. ↑ Drost EM, MacNee W. Potential role of IL-8, platelet-activating factor and TNF-alpha in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion receptor expression, and chemotaxis. Eur J Immunol 2002;32(2):393-403
4. ↑ Wang Q, Doerschuk CM, Mizgerd JP. Neutrophils in innate immunity. Semin Respir Crit Care Med 2004;25(1):33-41
5. ↑ Kunkel SL, Standiford T, Kasahara K, Strieter RM. Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung. Exp Lung Res 1991;17(1):17-23
6. ↑ Mitsuyama K, Toyonaga A, Sasaki E, Watanabe K, Tateishi H, Nishiyama T, Saiki T, Ikeda H, Tsuruta O, Tanikawa K. IL-8 as an important chemoattractant for neutrophils in ulcerative colitis and Crohn's disease. Clin Exp Immunol 1994;96(3):432-6
7. ↑ Buanne P, Di Carlo E, Caputi L, Brandolini L, Mosca M, Cattani F, Pellegrini L, Biordi L, Coletti G, Sorrentino C, Fedele G, Colotta F, Melillo G, Bertini R. Crucial pathophysiological role of CXCR2 in experimental ulcerative colitis in mice. J Leukoc Biol 2007;82(5):1239-46
8. ↑ ^{8.0 8.1 8.2} Faouzi S, Burckhardt BE, Hanson JC, Campe CB, Schrum LW, Rippe RA, Maher JJ. Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway. J Biol Chem. 2001 Dec 28;276(52):49077-82
9. ↑ ^{9.0 9.1 9.2} Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, Martin SJ. Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells. Mol Cell. 2013 Mar 28;49(6):1034-48
10. ↑ ^{10.0 10.1} Lin F, Nguyen CM, Wang SJ, Saadi W, Gross SP, Jeon NL. Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration. Biochem Biophys Res Commun. 2004 Jun 25;319(2):576-81

Relationship ID and Title:

144: Infiltration, Inflammatory cells leads to Inflammation, Liver

How Does This Key Event Relationship Work

Immune cells such as polymorphonuclear neutrophils (PMNs) or monocytes, circulate in the blood and become attracted towards a gradient of secreted pro-inflammatory cytokines. PMNs have a life span of only 7-12 hours. Therefore, around 1-2 x 1011 PMN are produced daily in the human body. They account for about 50-70 % of all blood leukocytes in the human body ^[1][2]. Upon an inflammatory event, neutrophil production is upregulated, and its lifetime increases as a response to platelet activating factor (PAF), granulocyte-colony stimulating factor (G-CSF) or various pro-inflammatory cytokines, such as interleukin 1ß (IL-1ß) ^[2]. In sterile tissue injury, for example as the result of apoptosis, there is no need for PMNs to function as antimicrobial effectors; instead, they clear debris and initiate the wound-healing process. Released damage-associated molecular patterns (see Relationship:924) stimulate Kupffer cells to produce IL-1ß which leads to intercellular adhesion molecular-1 (ICAM-1) upregulation on sinusoidal endothelial cells ^[3]. ICAM-1 in turn mediates neutrophil adhesion to endothelial cells, as it interacts with ß2 integrin, which is expressed on the surface of PMNs. Subsequent to adhesion, neutrophils begin to migrate across the endothelium and towards the affected tissue ^[4][5]. The transition of neutrophils from a resting state, as during circulation in the blood, to an activated state at the site of infection is triggered by an ordered sequence of signals from cytokines^[3].

The aberrant activation of neutrophils and their extended lifespan upon an inflammatory stimulus can increase the probability of extracellular damage. PMNs are potent phagocytes, but they also lead to pathogen destruction upon oxidative bursting. The oxidative burst is marked by an increased consumption of molecular oxygen, resulting in the production of reactive oxygen species (ROS) such as H2O2 and OH•, and reactive nitrogen species (RNS)^[6]. In general, the acute inflammatory response, as in the liver, is bi-phasic. The initial phase is characterised by a macrophage (Kupffer cell)-mediated phase, with the generation of reactive oxygen species aggravating the organ damage. The activated macrophages and subsequent infiltrating lymphocytes produce additional cytokines that further promote the inflammatory response, leading to a second phase, during which neutrophils become fully activated and secrete ROS, complement components, proteases, CXCL-1 and CXCL-2^[3]. The role of IL-1 and IL-17A in neutrophil activation and subsequent induction of inflammation has been confirmed by the use of knock-down models, showing that the absence of these mediators prevent neutrophil infiltration and subsequent onset of inflammation, inhibition of the latter also being shown by direct depletion of neutrophils^[7].

Weight of Evidence

Biological Plausibility

The infiltration of immune cells to the infected or damaged tissue is initiated in order to repair the tissue or remove cell bodies or bacteria. However, if the trigger persists, an overstimulation of immune cells such as neutrophils, and the corresponding secretion of ROS can enhance the tissue damage, in turn leading to further infiltration of inflammatory cells and eventually manifest a chronic inflammation.

Empirical Support for Linkage

Include consideration of temporal concordance here

Infiltration of the hepatic parenchyma by neutrophils was found coinciding with chemokine induction. When chemokines have been neutralized by the addition of neutralizing monoclonal antibodies, a study found the chemokine KC mainly responsible for abrogating an inflammatory response to Fas-induced hepatic inflammation. When apoptosis was prevented by pre-treatment of the mice with a caspase-3 inhibitor, AP-1 activation and hepatic chemokine production were both significantly reduced, directly resulting in a reduction of the hepatic inflammation by 70%^[8].

Neutrophils infiltration and subsequent liver inflammation and are drastically attenuated in IL-1R1 deficient mice or by using a neutralizing antibody, and also in the absence of IL-17RA signalling. The same study demonstrated that increased IL-17A was mainly expressed by CD4+ T cells, but also by neutrophils themselves, in the damaged liver, showing that these cells are critical for the further recruitment of circulating immune cells into the tissue. Depletion of neutrophils (by using the neutrophil depleting antibody NIMP-R14) directly resulted in a drastic reduction of the inflammation^[7].

A general proof of the importance of infiltrated neutrophils is the fact that liver inflammation is usually clinically confirmed by analysis of histological features, marked by the influx of neutrophils (which can be stained by using Haematoxylin and eosin) ^[9].

Quatitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Inhibition of messengers for the infiltration of inflammatory cells leads to a strong reduction of these. Furthermore, direct inhibition of neutrophils prevents the onset of liver inflammation.

Evidence Supporting Taxonomic Applicability

^[8][7]: mouse ^[9]: human

References

1. ↑ Freitas M, Lima JL, Fernandes E. Optical probes for detection and quantification of neutrophils' oxidative burst. A review. Anal Chim Acta 2009;649(1):8-23
2. ↑ ^{2.0 2.1} Wessels I, Jansen J, Rink L, Uciechowski P. Immunosenescence of polymorphonuclear neutrophils. ScientificWorldJournal 2010;10:145-60
3. ↑ ^{3.0 3.1 3.2} Xu R, Huang H, Zhang Z, Wang FS. The role of neutrophils in the development of liver diseases. Cell Mol Immunol. 2014 May;11(3):224-31
4. ↑ Drost EM, MacNee W. Potential role of IL-8, platelet-activating factor and TNF-alpha in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion receptor expression, and chemotaxis. Eur J Immunol 2002;32(2):393-403
5. ↑ Wang Q, Doerschuk CM, Mizgerd JP. Neutrophils in innate immunity. Semin Respir Crit Care Med 2004;25(1):33-41
6. ↑ Babior BM. Phagocytes and oxidative stress. Am J Med 2000;109(1):33-44
7. ↑ ^{7.0 7.1 7.2} Tan Z, Jiang R, Wang X, Wang Y, Lu L, Liu Q, Zheng SG, Sun B, Ryffel B. RORγt+IL-17+ neutrophils play a critical role in hepatic ischemia-reperfusion injury. J Mol Cell Biol. 2013 Apr;5(2):143-6
8. ↑ ^{8.0 8.1} Faouzi S, Burckhardt BE, Hanson JC, Campe CB, Schrum LW, Rippe RA, Maher JJ. Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway. J Biol Chem. 2001 Dec 28;276(52):49077-82
9. ↑ ^{9.0 9.1} Huebscher SG. Histological assessment of non-alcoholic fatty liver disease. Histopathol. 2006;49:450–465