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Event: 888

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)

Short name
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Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)
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Biological Context

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Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term
eukaryotic cell

Organ term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  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 signaling 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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
mitochondrial electron transport, NADH to ubiquinone 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 MolecularInitiatingEvent Andrea Terron (send email) Open for citation & comment WPHA/WNT Endorsed
Complex I inhibition leads to Fanconi syndrome MolecularInitiatingEvent 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

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.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
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI

Life Stages

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Sex Applicability

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Key Event Description

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Electron transport through the mitochondrial respiratory chain (oxidative phosphorylation) is mediated by five multimeric complexes (I–V) that are embedded in the mitochondrial inner membrane (Fig. 1). NADH-ubiquinone oxidoreductase is the Complex I (CI) of electron transport chain (ETC). It is a large assembly of proteins that spans the inner mitochondrial membrane. In mammals, it is composed of about 45-47 protein subunits (human 45) of which 7 are encoded by the mitochondrial genome (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6) and the remainder by the nuclear genome (Greenamyre, 2001). CI oxidizes NADH elevating the NAD+/NADH ratio by transferring electrons via a flavin mononucleotide (FMN) cofactor and several iron-sulfur centers to ubiquinone (Friedrich et al., 1994) (Fig. 1). Binding of an inhibitor to CI inhibits the NADH–ubiquinone oxido-reductase activity, i.e. blocks the electron transfer. Recent studies suggest that a wide variety of CI inhibitors share a common binding domain at or close to the ubiquinone reduction site (Ino et al., 2003). Furthermore, the structural factors required for inhibitory actions have been characterized on the basis of structure-activity relationships (Miyoshi, 1998, Hideto, 1998). Based on molecular docking simulations, in silico models mimicking the binding of chemicals to the pocket of NADH ubiquinone oxidoreductase have been created according to the crystal structure of mitochondrial CI. To investigate the ability of chemicals to bind to the active pocket, around 100 individual docking simulations have been performed. These confirmed the possible site of interaction between the chemical and the pocket of CI. In particular, Miao YJ and coworkers recently investigated the IC50 values of 24 chemicals (annonaceous acetogenins) for inhibition of mitochondrial CI (Miao et al., 2014).

Based on their binding sites, CI inhibitors are classified as follows (Degli Esposti, 1998) (Fig. 2): (i) type A inhibitors are antagonists of fully oxidized ubiquinone binding; (ii) type B inhibitors displace the partially reduced ubisemiquinone intermediate; (iii) type C inhibitors are antagonists of the fully reduced ubiquinol product. The affinity of the different types of CI inhibitors to their diverse CI binding sites is described in the paragraph Evidence for Chemical Initiation of this Molecular Initiating Event (see below) in the context of a specific type of inhibitor.

AOP-003-Figure1-smaller.JPG

Fig. 1. The electron transport chain in the mitochondrion. CI (NADH-coenzyme Q reductase or NADH dehydrogenase) accepts electrons from NADH and serves as the link between glycolysis, the citric acid cycle, fatty acid oxidation and the electron transport chain. Complex II also known as succinate-coenzyme Q reductase or succinate dehydrogenase, includes succinate dehydrogenase and serves as a direct link between the citric acid cycle and the electron transport chain. The coenzyme Q reductase or Complex III transfers the electrons from CoQH2 to reduce cytochrome c which is the substrate for Complex IV (cytochrome c reductase). Complex IV transfers the electrons from cytochrome c to reduce molecular oxygen into water. Finally, this gradient is used by the ATP synthase complex (Complex V) to make ATP via oxidative phosphorylation. mtDNA: mitochondrial DNA; nDNA: nuclear DNA.

MIE Fig. 2.jpg

Fig. 2. Schematic representation of CI and proposed inhibition binding sites by inhibitors of class A, B and C. Nicotinamide adenine dinucleotide (NADH, reduced and NAD, oxidized), flavin mononucleotide (FMN) and Ubiquinone (Q) (taken from Haefeli, 2012).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.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). Do not provide detailed protocols. More help

Two different types of approaches have been used. The first is to measure binding as such, and the corresponding assays are described below; the second is to infer binding indirectly from assays that quantify e.g. CI activity and to assume that the activity can only be altered upon binding. The second type of approach is dealt with in the chapter entitled KE1: Inhibition of NADH ubiquinone oxidoreductase (complex I). However, it has to be noted here that indirect assays can lead to wrong conclusions. For instance, some compounds may trigger oxidative stress without actually binding to CI. Such compounds, by triggering the generation of reactive oxygen species (ROS), may damage CI protein components, thus causing a reduction of CI activity.

Measurement of binding by quantitative autoradiography

To assess binding of an inhibitor at the rotenone binding site of CI in tissues (e.g. in the substantia nigra or in the striatum), the standard approach is to quantify the displacement of a radioactively labelled ligand of this binding site by the toxicant under evaluation. Most commonly, binding of [3H]-labeled dihydrorotenone (DHR) is measured and compared in control tissue and treated tissue. Binding of this rotenone-derivative is detected by autoradiography. Unselective binding is determined by measurement of [3H]-DHR binding in the presence of an excess of unlabeled rotenone. Since a rotenone-derivative is used for the assay, only CI inhibitors that bind to the rotenone-binding site in CI are detected. This was observed for e.g., meperdine, amobarbital, or MPP+. This method allows a spatial resolution of CI expression and the mapping of the binding of a competitive inhibitor on CI.

The method can be used for (a) in vitro measurements and for (b) ex vivo measurements:

a) In vitro measurements. Tissues are embedded in a matrix for cutting by a cryostat. The tissue slices are then mounted onto slides. For the binding experiment, they are incubated with the test compound in the presence of labeled [3H]-DHR. Then the tissue slices are washed and prepared for autoradiographic detection (Greenamyre et al. 1992; Higgins and Greenamyre, 1996). b) Ex vivo measurements. As rotenone can pass the blood brain barrier, the in vitro method was further extended for in vivo labeling of CI in the brains of living animals, and detection of binding after preparation of the tissue from such animals. Animals are exposed to test compounds and [3H]-DHR is applied intraventricularly for 2-6 h before the brain is dissected and arranged for the preparation of tissue slices (Talpade et al. 2000). In untreated animals, this method allows a precise spatial resolution of the expression pattern of CI. In animals with impaired CI activity, either as a result of CI deficiencies, or upon treatment with CI inhibitors, the assay allows an assessment of the degree of CI inhibition.

Complex I Enzyme Activity (Colorimetric)

The analysis of mitochondrial OXPHOS CI enzyme activity can be performed using human, rat, mouse and bovine cell and tissue extracts (abcam: http://www.abcam.com/complex-i-enzyme-activity-microplate-assay-kit-colorimetric-ab109721). Capture antibodies specific for CI subunits are pre-coated in the microplate wells. Samples are added to the microplate wells which have been pre-coated with a specific capture antibody. After the target has been immobilized in the well, CI activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be evaluated.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

CI has a highly conserved subunit composition across species, from lower organisms to mammals (Cardol, 2011). Fourteen subunits are considered to be the minimal structural requirement for physiological functionality of the enzyme. These units are well conserved among bacterial (E. coli), human (H. sapiens), and Bovine (B. taurus) (Vogel et al., 2007b; Ferguson, 1994). However, the complete structure of CI is reported to contain between 40 to 46 subunits and the number of subunits differs, depending on the species (Gabaldon 2005; Choi et al., 2008). In vertebrates CI consists of at least 46 subunits (Hassinen, 2007), particularly, in humans 45 subunits have been described (Vogel et al, 2007b). Moreover, enzymatic and immunochemical evidence indicate a high degree of similarity between mammalian and fungal counterparts (Lummen, 1998). Mammalian CI structure and activity have been characterized in detail (Vogel et al., 2007a; Vogel et al., 2007b), referring to different human organs including the brain. There is also a substantial amount of studies describing CI in human muscles, brain, liver, as well as bovine heart (Janssen et al., 2006; Mimaki et al. 2012) (Okun et al., 1999).

References

List of the literature that was cited for this KE description. More help

Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. (2000). Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat Neurosci 3:1301-1306.

Cardol, P., (2011) Mitochondrial NADH:ubiquinone oxidoreductase (complex I) in eukaryotes: A highly conserved subunit composition highlighted by mining of protein databases Biochimica et Biophysica Acta 1807, 1390–1397.

Choi WS., Kruse S.E., Palmiter R, Xia Z., (2008) Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP, or paraquat. PNAS, 105, 39, 15136-15141.

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

Desplats P, Patel P, Kosberg K, Mante M, Patrick C, Rockenstein E, Fujita M, Hashimoto M, Masliah E. (2012). Combined exposure to Maneb and Paraquat alters transcriptional regulation of neurogenesis-related genes in mice models of Parkinson’s disease. Mol Neurodegener 7:49.

Fendel UTocilescu MAKerscher SBrandt U. (2008) Exploring the inhibitor binding pocket of respiratory complex I.Biochim Biophys Acta. 2008, 1777(7-8):660-5.

Ferguson SJ. Similarities between mitochondrial and bacterial electron transport with particular reference to the action of inhibitors. Biochem Soc Trans. 1994 Feb;22(1):181-3.

Friedrich T, van Heek P, Leif H, Ohnishi T, Forche E, Kunze B, Jansen R, TrowitzschKienast W, Hofle G & Reichenbach H (1994) Two binding sites of inhibitors in NADH: ubiquinone oxidoreductase (complex I). Relationship of one site with the ubiquinone-binding site of bacterial glucose:ubiquinone oxidoreductase. Eur J Biochem 219(1–2): 691–698.

Gabaldon, T., Rainey, D., Huynen, M.A. (2005) Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (Complex I), J. Mol. Biol. 348; 857–870.

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.

Greenamyre JT, Higgins DS, Eller RV (1992) Quantitative autoradiography of dihydrorotenone binding to complex I of the electron transport chain. J Neurochem. 59(2):746-9.

Grivennikova, V.G., Maklashina, E.O., E.V. Gavrikova, A.D. Vinogradov (1997) Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition Biochim. Biophys. Acta, 1319 (1997), pp. 223–232.

Haefeli, RH (2012) Molecular Effects of Idebenone. Doctoral thesis http://edoc.unibas.ch/19016/1/Molecular_Effects_of_Idebenone_Roman_Haefeli.pdf.

Hassinen I (2007) Regulation of Mitochondrial Respiration in Heart Muscle. In Mitochondria – The Dynamic Organelle Edited by Schaffer & Suleiman. Springer ISBN-13: 978-0-387-69944-8.

Hideto M. Structure–activity relationships of some complex I inhibitors. Biochimica et Biophysica Acta 1364 _1998. 236–244.

Higgins DS Jr1, Greenamyre JT. (1996). [3H]dihydrorotenone binding to NADH: ubiquinone reductase (complex I) of the electron transport chain: an autoradiographic study. J Neurosci. 1996 Jun 15;16(12):3807-16.

Ichimaru, N., Murai, M., Kakutani, N., Kako, J., Ishihara, A., Nakagawa, Y., … Miyoshi, H. (2008). Synthesis and Characterization of New Piperazine-Type Inhibitors for Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I). Biochemistry, 47(40), 10816–10826.

Ino T, Takaaki N, Hideto M. Characterization of inhibitor binding sites of mitochondrial complex I using fluorescent inhibitor. Biochimica et Biophysica Acta 1605 (2003) 15– 20.

Janssen RJ, Nijtmans LG, van den Heuvel LP, Smeitink JA. Mitochondrial complex I: structure, function and pathology. J Inherit Metab Dis. 2006 Aug;29(4):499-515.

Keeney PM, Xie J,Capaldi RA,Bennett JP Jr. (2006) Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci. 10;26(19):5256-64.

Lin CJ, Lee CC, Shih YL, Lin CH, Wang SH, Chen TH, Shih CM. (2012). Inhibition of Mitochondria- and Endoplasmic Reticulum Stress-Mediated Autophagy Augments Temozolomide-Induced Apoptosis in Glioma Cells. PLoS ONE 7:e38706.

Lümmen, P., (1998) Complex I inhibitors as insecticides and acaricides1, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1364, Issue 2, Pages 287-296.

Majander A, Finel M, Wikstrom M. (1994) Diphenyleneiodonium inhibits reduction of iron–sulfur clusters in the mitochondrial NADH–ubiquinone oxidoreductase (complex I) J Biol Chem. 269:21037–21042.

Miao YJ, Xu XF, Xu F, Chen Y, Chen JW, Li X. (2014) The structure-activity relationships of mono-THF ACGs on mitochondrial complex I with a molecular modelling study. Nat Prod Res.28(21):1929-35.

Mimaki M, Wang X, McKenzie M, Thorburn DR, Ryan MT. Understanding mitochondrial complex I assembly in health and disease. Biochim Biophys Acta. 2012 Jun;1817(6):851-62. doi: 10.1016/j.bbabio.2011.08.010.

Miyoshi H. Structure-activity relationships of some complex I inhibitors. Biochim Biophys Acta. 1998, 6:236-244.

Okun, J.G, Lümmen, P and Brandt U., (1999) Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) J. Biol. Chem. 274: 2625-2630. doi:10.1074/jbc.274.5.2625.

Ramsay R., Krueger MJ., Youngster SK., Gluck MR., Casida J.E. and Singer T.P. Interaction of 1-Methyl-4-Phenylpyridinium Ion (MPP+) and Its Analogs with the Rotenone/Piericidin Binding Site of NADH Dehydrogenase. Journal of Neurochemistry, 1991, 56: 4, 1184–1190.

Sava V, Velasquez A, Song S, Sanchez-Ramos J. (2007). Dieldrin elicits a widespread DNA repair and antioxidative response in mouse brain. J Biochem Mol Toxicol 21:125-135.

Schuler, F. and Casida, JE. (2001) Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1506, Issue 1, 2 July 2001, Pages 79-87, ISSN 0005-2728, https://doi.org/10.1016/S0005-2728(01)00183-9.

Talpade DJ, Greene JG, Higgins DS Jr, Greenamyre JT (2000) In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone. J Neurochem. 75(6):2611-21.

Vogel R.O., van den Brand M.A., Rodenburg R.J., van den Heuvel L.P., Tsuneoka M., Smeitink J.A., Nijtmans L.G. (2007a). Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients. Mol. Genet. Metab. 91:176–182.

Vogel, R.O. Smeitink, J.A. Nijtmans L.G. (2007b) Human mitochondrial complex I assembly: a dynamic and versatile process Biochim. Biophys. Acta, 1767-. 1215–1227.

Zharova, TV, and Vinogradov, A.(1997) A competitive inhibition of the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) by ADP-ribose. Biochimica et Biophysica Acta, 1320:256-64.