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Event: 887

Key Event Title

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

Inhibition, NADH-ubiquinone oxidoreductase (complex I)

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Inhibition, NADH-ubiquinone oxidoreductase (complex I)

Biological Context

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

Cell term

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

Organ term

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

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
NADH dehydrogenase (ubiquinone) activity NADH-ubiquinone oxidoreductase chain 1 decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Mitochondrial dysfunction and Neurotoxicity KeyEvent Andrea Terron (send email) Open for citation & comment TFHA/WNT Endorsed
Complex I inhibition leads to Fanconi syndrome KeyEvent Marvin Martens (send email) Under development: Not open for comment. Do not cite
Mitochondrial complex inhibition leading to liver injury MolecularInitiatingEvent Wanda van der Stel (send email) Under development: Not open for comment. Do not cite

Stressors

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

Taxonomic Applicability

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

Life Stages

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

Sex Applicability

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

Key Event Description

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

Under physiological conditions complex I (CI) couples the oxidation of NADH to NAD+ by reducing flavin mononucleotide (FMN) to FMNH2. FMNH2 is then oxidized through a semiquinone intermediate. Each electron moves from the FMNH2 to Fe-S clusters, and from the Fe-S clusters to ubiquinone (Q). Transfer of the first electron results in the formation of the free-radical (semiquinone) form of Q, and transfer of the second electron reduces the semiquinone form to the ubiquinol form (CoQH2). Altogether, four protons are translocated from the mitochondrial matrix to the inter-membrane space for each molecule of NADH oxidized at CI. This leads to the establishment of the electrochemical potential difference (proton-motive force) that may be used to produce ATP (Garrett and Grisham, 2010). Binding of an inhibitor attenuates or completely blocks the activity of CI, i.e. the oxidation of NADH is impaired and protons are not moved. This causes two major consequences: first, electrons are channelled toward oxygen instead Q. This impairs normal oxygen reduction into water at complex IV and leads to the formation of the ROS superoxide at other sites of the respiratory chain. Superoxide may cause damage of proteins, lipid and DNA of the cell, or damage components of the mitochondria after transformation into e.g. hydrogen peroxide. These processes result in mitochondrial dysfunction (Voet and Voet., 2008). The second consequence is the increase of the NADH/NAD+ ratio in mitochondria. This affects the function of key dehydrogenase enzymes in the citric acid cycle and can lead to its block, resulting in an inhibition of mitochondrial ATP production and mitochondrial respiration. Prolonged treatment with an inhibitor results in a severe, progressive and irreversible inhibition of complex I, most likely by indirect mechanisms involving oxidative damage (Cleeter et al., 1992). The functional consequences of CI inhibition have been titrated in a time- and dose-dependent manner (Barrientos and Moraes, 1999), with mitochondrial dysfunction measured by a range of different assays (Barrientos and Moraes, 1999; Greenamyre et al., 2001). These included quantification of ROS derived from mitochondria, and of cellular respiration (see KE2: Mitochondrial dysfunction).

How It Is Measured or Detected

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

As CI has an enzymatic function as such, but also contributes to the overall function of oxidative phosphorylation, there are two fundamental approaches to assess CI inhibition. The first approach measures the enzymatic activity of the complex itself; the second one assesses the overall activity of oxidative phosphorylation of entire mitochondria, and indirectly infers from this a potential dysfunction of CI.

I. Direct detection of complex I activity. This type of assay is always performed in homogenates of cells or tissues, and requires at least a partial purification of mitochondria or respiratory chain components. In order to focus on CI activity, the activities of Complexes III (e.g. antimycin A) and complex IV (e.g. cyanide) need to be blocked by pharmacological inhibitors in these setups.

1. Forward Electron Transfer. Submitochondrial particles or intact isolated mitochondria are incubated with NADH as electron donor and with an electron acceptor to measure the flow of electrons from NADH, through CI to the acceptor. As readout, either the consumption of NADH, or the reduction of the electron acceptor is followed photometrically or fluorometrically (Lenaz et al. 2004; Spinazzi et al. 2012; Long et al. 2009; Kirby et al. 2007). The physiological electron acceptor of CI is Coenzyme Q10 (CoQ10). Due to its hydrophobicity, it is not suitable for use in an experimental in vitro setup. Short-chain analogs of CoQ10, such as CoQ1 or decylubiquinone (DB) with a 10 carbon-atom linear saturated side chain are hence applied as alternatives. With these non-physiological electron acceptors, it is important to consider that the activity of CI can easily be underestimated. As water-soluble electron acceptors, either ferricyanide or 2,6-dichlorophenolidophenol (DCIP) are used. However the reduction of such compounds is not strictly coupled to the transduction of energy. To identify the portion of rotenone-inhibitable CI activity, all samples investigated are assayed in parallel following treatment with rotenone. In contrast to the autoradiography assays, direct CI activity detection allows the identification also of CI inhibitors that bind to sites of CI different from the rotenone binding site.

2. Reverse Electron Transfer. An alternative setup for the direct measurement of CI activity with minimal interference by the activities of complex III and complex IV make use of the observation of a general reversibility of oxidative phosphorylation and electron flow across the mitochondrial respiratory chain (Ernster et al. 1967). With this method, electrons enter the respiratory chain via complex II. Based on the reverse flux, this method allows the complete circumvention of complexes III and IV. As electron donor, succinate is applied, together with NAD+ as electron acceptor. Formation of NADH from NAD+ can be determined photometrically. The succinate-linked NAD+ reduction can be performed either with intact isolated mitochondria or with submitochondrial particles. For the direct assessment of CI activity, submitochondrial particles are used. For assays with intact mitochondria, the succinate-linked reduction of NAD+ is performed in the presence of ATP as energy source. Potassium cyanide (KCN) is added for inhibition of forward electron transport towards complex IV.

3. Complex I activity dipstick assay. To assess CI activity and its inhibition in cell or tissue homogenates without interference by other components of the respiratory chain, CI-selective antibodies attached to a matrix (e.g. multiwell plates) are used (Willis et al., 2009). Homogenized tissue can directly be added for capturing of CI, the unbound supernatant is washed away and leaves a complex of the antibody and mitochondrial CI. For activity determination, NADH as electron donor and nitroblue tetrazolium (NBT) as acceptor are added. Reduced NBT forms a colored precipitate, its signal intensity is proportional to the amount of CI bound to the antibody. CI inhibitors can directly be added for an assessment of their inhibitory potential. This method, when applied in e.g. 96-well or 384-well plates, allows screening of large sets of potential CI inhibitors without any interference by other elements of the mitochondrial respiratory chain.

II. Indirect measurements of complex I activity. Such assays mostly require / allow the use of live cells.

1. Oxygen consumption. Electrons, fed into the mitochondrial respiratory chain either by CI or complex II, ultimately reduce molecular oxygen to water at complex IV. In a closed system, this consumption of oxygen leads to a drop of the overall O2 concentration, and this can serve as parameter for mitochondrial respiratory activity. Measurements are traditionally done with a Clark electrode, or with more sophisticated optical methods. At the cathode of a Clark electrode, oxygen is electrolytically reduced, which initiates a current in the electrode, causing a potential difference that is ultimately recorded. Clark electrodes however have the disadvantage that oxygen is consumed. Furthermore, interferences with nitrogen oxides, ozone, or chlorine are observed (Stetter et al., 2008). To circumvent these limitations, optical sensors have been developed that have the advantage that no oxygen is consumed, combined with a high accuracy and reversibility. Optical oxygen sensors work according to the principle of dynamic fluorescence quenching. The response of the respective fluorescence dye is proportional to the amount of oxygen in the sample investigated (Wang and Wolfbeis, 2014). In a model of isolated mitochondria in the absence of complex II substrates, oxygen consumption can serve as surrogate readout for the assessment of the degree of CI inhibition. It is however essential to realize that also complex III and complex IV activities are involved and their inhibition also results in a decline in O2 consumption. In addition to that, CI inhibitors can lead to a one-electron reduction of molecular oxygen at the site of CI to yield superoxide. The amount of superoxide formed hence contributes to the consumption of oxygen, but this must not be interpreted as oxygen consumption as a result of controlled and coupled electron flux through the complexes of the mitochondrial respiratory chain. A modern convenient method to measure oxygen consumption is provided by the Seahorse technology of extracellular flux (XF) analysis, in which cells are kept in a very small volume, so that changes of oxygen levels can be detected very sensitively by an oxygen sensor. To allow manipulation of the mitochondria in cells, the cell membrane can be permeabilized with saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), to allow addition of specific substrates to measure activity of different respiratory chain complexes, including CI. (Salabei et al., 2014).

2. Intracellular ATP levels. Intracellular ATP levels originate both from mitochondria and from glycolysis. If glycolytic ATP production is impaired or inhibited, the cellular production of ATP is a measure of mitochondrial function. If it is assumed that the ATP consumption remains constant, then the steady state ATP levels can serve as indirect readout for mitochondrial activity, and the latter depends on the functioning of CI. Inhibitors of CI reduce cellular ATP levels, but it has to be remembered that intracellular ATP levels are also affected by inhibitors of other parts of the respiratory chain, of the citric acid cycle or of the transport of energy substrates. For a proper interpretation of assay results, it has to be ascertained in each particular test system, that ATP production from other sources is excluded and that the cellular ATP consumption remains constant. ATP levels can be easily measured from lysates of in vitro cell cultures or from tissues by a luminometric luciferase/luciferin assay. The amount of light emitted is proportional to the amount of ATP in the sample (Nguyen et al. 1988, Leist et al., 1997).

3. Other approaches. As mitochondrial activity is coupled to many cellular functions, there is a multitude of other indirect assays that are sensitive to inhibitors of CI. Some of these tests may indeed be very sensitive, while they have a low specificity. Thus, their application requires usually a good control of the experimental system and care with the interpretation of the data. One exemplary approach is the measurement of NADH/NAD+ ratios in mitochondria by imaging methods. This provides resolution on the level of individual mitochondria within a living cell (van Vliet et al., 2014).

Domain of Applicability

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

The CI is well-conserved across species from lower organisms to mammals. The central subunits of CI harboring the bioenergetic core functions are conserved from bacteria to humans. CI from bacteria and from mitochondria of Yarrowia lipolytica, a yeast genetic model for the study of eukaryotic CI (Kerscher et al., 2002) was analyzed by x-ray crystallography (Zickermann et al., 2015, Hofhaus et al., 1991; Baradaran et al., 2013). The CI of the mitochondria of eukaryotes and in the plasma membranes of purple photosynthetic bacteria are closely related to respiratory bacteria and the close homology of sequences, function, and prosthetic groups shows a common ancestry (Friedrich et al., 1995).

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

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

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

Baradaran R., John M. Berrisford, Gurdeep S. Minhas , Leonid A. Sazanov. Crystal structure of the entire respiratory complex I. Nature , 2013,| 494,443–448.

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.

Cleeter MW, Cooper JM, Schapira AH. Irreversible inhibition of mitochondrial complex I by 1-methyl-4-phenylpyridinium: evidence for free radical involvement. J Neurochem. 1992 Feb;58(2):786-9.

Degli Esposti (1998) Inhibitors of NADH-ubiquinone reductase: an overview Biochimica et Biophysica Acta 1364-222-235.

Ernster L, Lee C (1967) Energy-linked reduction of NAD+ by succinate. Methods Enzym. 10:729-738.

Friedrich, T., Steinmüller, K. & Weiss, H. (1995) The proton-pumping respiratory complex I of bacteria and mitochondria and is homologue of chloroplasts. FEBS Lett. (Minireview), 367, 107-111.

Garrett and Grisham, Biochemistry, Brooks/Cole, 2010, pp 598-611.

Greenamyre, J T., Sherer, T.B., Betarbet, R., and Panov A.V. (2001) Critical Review Complex I and Parkinson’s Disease Life, 52: 135–141.

Hofhaus, G., Weiss, H. and Leonard, K. (1991): Electron microscopic analysis of the peripheral and the membrane parts of mitochondrial NADH dehydrogenase (Complex I). J. Mol. Biol. 221, 1027-1043.

Kerscher, S. Dröse, K. Zwicker, V. Zickermann, U. Brandt Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I. Biochim. Biophys. Acta 1555, 83–91 (2002).

Kirby DM, Thorburn DR, Turnbull DM, Taylor RW (2007) Biochemical assays of respiratory chain complex activity. Methods Cell Biol. 80:93-119.

Leist M, Single B, Castoldi AF, Kühnle S, Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med. 185:1481-6.

Leist M. Current approaches and future role of high content imaging in safety sciences and drug discovery. ALTEX. 2014;31(4):479-93.

Lenaz G, Fato R, Baracca A, Genova ML (2004) Mitochondrial quinone reductases: complex I. Methods Enzymol. 382:3-20.

Long J, Ma J, Luo C, Mo X, Sun L, Zang W, Liu J (2009) Comparison of two methods for assaying complex I activity in mitochondria isolated from rat liver, brain and heart. Life Sci. 85(7-8):276-80.

Nguyen VT, Morange M, Bensaude O. (1988) Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells. Anal Biochem. 171(2):404-8.

van Vliet E, Daneshian M, Beilmann M, Davies A, Fava E, Fleck R, Julé Y, Kansy M, Kustermann S, Macko P, Mundy WR, Roth A, Shah I, Uteng M, van de Water B, Hartung T, Spinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C (2012) Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc. 7(6):1235-46.

Salabei J.K., Gibb A.A. and Hill BG. (2014) Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nature Protocols, 9, 421–438.

Stetter JR, Li J (2008) Amperometric gas sensors--a review. Chem Rev. 108(2):352-66.

Wang XD, Wolfbeis OS (2014) Optical methods for sensing and imaging oxygen: materials, spectroscopies and applications. Chem Soc Rev. 43(10):3666-761.

Voet DJ and Voet JG; Pratt CW (2008). Chapter 18, Mitochondrial ATP synthesis. Principles of Biochemistry, 3rd Edition. Wiley. p. 608. ISBN 978-0-470-23396-2.

Willis JH, Capaldi RA, Huigsloot M, Rodenburg RJ, Smeitink J, Marusich MF (2009) Isolated deficiencies of OXPHOS complexes I and IV are identified accurately and quickly by simple enzyme activity immunocapture assays. Biochim Biophys Acta. 1787(5):533-8.

Zickermann V., Christophe Wirth, Hamid Nasiri, Karin Siegmund, Harald Schwalbe, Carola Hunte, Ulrich Brandt. Mechanistic insight from the crystal structure of mitochondrial complex I. Science 2 January 2015: Vol. 347 no. 6217 pp. 44-49.