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Event: 888

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

Binding of inhibitor, 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
Binding of inhibitor, 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
Molecular

Cell term

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

Organ term

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

Key Event Components

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

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

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). ?

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

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

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).

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.

Numerous hydrophobic, amphipathic compounds are known to inhibit the proton pumping NADH:ubiquinone oxidoreductase, also known as the ubiquinone reductase reaction of respiratory chain complex I (Fendel et al., 2008). However, the most studied examples of chemicals that inhibit CI are: rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Desplats et al., 2012; Lin et al., 2012; Sava et al., 2007). Both, rotenone (pesticide) and MPP+ (the active metabolite of MPTP) are well known to reproduce the anatomical, neurochemical, behavioural and neuropathological features of PD-like syndrome (Betarbet et al., 200; Greenamyre et al., 2001). Indeed, an overwhelming evidence has accumulated in the existing literature suggesting such a link and therefore these two inhibitors of CI will be discussed in the context of all KE identified in this AOP.

1. Rotenone affinity to complex I binding sites.

Rotenone (a flavonoid, extracted from the several plants e.g. Derris scandens)is one of the most powerful, an irreversible inhibitor of CI, binding with high affinity to CI and is typically used to define the specific activity of this complex. Rotenone is extremely lipophilic, it crosses biological membrane easily and it gets into brain very rapidly. Rotenone inhibits 20 kDa subunit of complex I (PSST) labeling without effect on 36 kDa subunit of complex I (ND1) (Schuler and Casida, 2001). The interaction of rotenone with active ('pulsed') and thermally de-activated ('resting') membrane-bound Complex I as revealed by inhibition of NADH-ubiquinone- and ubiquinol-NAD+ reductase activities was studied. Ki = 1 x 10(-9) M, k(on) = 5 x 10(7) M-1 min-1 and k(off) = 0.02 min-1 (inhibitory effect of rotenone on NADH oxidation) and Ki = 2 x 10(-8) M (inhibition of reverse electron transfer) were determined for pulsed enzyme. The equilibrium between de-activated and active enzyme is reached (K approximately 100) after the slow strongly temperature-dependent de-activation process has completed. Rotenone partially prevents and reverses the enzyme de-activation. About two order of magnitude difference in affinity of rotenone to the active and de-activated forms of the enzyme was demonstrated (Grivennikova et al., 1997). Dose-dependent relative affinities of rotenone to the inhibitory site of CI is shown in Fig. 3B (for more detail Grivennikova et al., 1997).

Most of the studies suggest that hydrophobic inhibitors like rotenone or Piericidin A most likely disrupt the electron transfer between the terminal Fe-S cluster N2 and ubiquinone (Fig. 3A).

MIE Fig. 3A.jpg

Fig. 3A. Rotenone structure and a schematic representation of its binding site (and other Rotenone-like compounds) to CI. IMS: inter-membrane space (based on Lummen, 1998)

MIE Fig. 3B.jpg

Fig. 3B. Fig. 2. Relative affinities of rotenone to the inhibitory site(s) of Complex I. Panel (A): activated submitochondrial particles (SMP) (2.8 mg/ml, approx. 0.4 microM Complex I) were incubated in the standard reaction mixture for 20 min at 25oC and residual initial rate of NADH oxidation was measured. 100% correspond to the specific activity of 1.0 micromol/min per mg of protein. Panel (B): curve 1 (o), SMP (48 microg/ml, approx. 8 nM Complex I) were activated in the assay cuvette and pre-incubated with rotenone in the presence of gramicidin and 10 mM malonate for 20 min at 25oC and the residual NADH oxidase activity was then measured; black circle: the same as (o), except that pre-incubation with rotenone was made in the presence of 10 mM succinate (no gramicidin and malonate), 10 mM malonate and gramicidin were added simultaneously with 100 microM NADH to measure the residual activity. Curve 2, presents the reverse electron transfer activity and curve 3, de-activated SMP were preincubated with rotenone as described for curve 1(o) (for further details see Grivennikova et al., 1997). Panel (C): The same as Panel B, curve 3, except for enzyme concentration was 0.5 mg/ml and rotenone concentration range which was increased to show interaction of the inhibitor with de-activated enzyme. The activity was measured after 200-fold dilution into the assay mixture. All the continues lines corresponds to the theoretical titration curves for the reversible single site inhibition with Ki values of 1 nM, 20 nM and 80 nM for the curves 1, 2 and 3, respectively (for further details see Grivennikova et al., 1997).

2. MPTP affinity to complex I binding sites. MPTP is not directly binding to CI and it is therefore non-toxic to DA neurons. MPTP exerts its toxicity after it is metabolized by mono-amino-oxidase, type B (MAO B), in astrocytes to 1-methyl-4-phenylpyridinium (MPP+). This metabolite binds to CI, and is toxic. MPP+ is a good substrate for dopamine transporters (DAT), expressed selectively by DA neurons (Greenamyre et al (2001). Due to both a positive charge and an amphoteric character, MPP+ specifically accumulates in mitochondria, where despite a lower affinity to the binding site of complex I than rotenone, it reaches high enough intra-mitochondrial concentrations to inhibit CI activity (Ramsay et al., 1991). The binding affinity of MPP+ is low (mM range), and it can be totally reversed by washing out.  Competitive binding experiments with rotenone and MPP+ suggest that the two compounds bind to the same site of the CI (Ramasay et al., 1991). Schuler and Casida (2001) reported that MPP+ inhibits PSST and elevates ND1 labelling subunits of the mitochondrial complex I.

3. General characteristics of other complex I inhibitors. There is a variety of CI inhibitors, both naturally occurring besides rotenone such as Piericidin A (from Streptomyces mobaraensis), acetogenins (from various Annonaceae species) as well as their derivatives, and synthetically manufactured compounds like pyridaben and various piperazin derivatives (Ichimaru et al. 2008). They have been used to probe the catalytic activity of complex I especially in order to clarify its ubiquinone binding site and indeed, most of these compounds inhibit the electron transfer step from the Fe-S clusters to ubiquinone (Friedrich et al. 1994). Therefore, classification of CI inhibitors is based on their types of action. Type A inhibitors, like piericidin A, 2-decyl-4-quinazolinyl amine (DQA), annonin VI and rolliniastatin-1 and -2, are considered to be antagonists of the ubiquinone substrate. For piericidin A, it has been shown that it inhibits NADH:Q2 activity in a partially competitive manner. Contrary to type A, type B inhibitors, like the commonly used rotenone, have hydrogen-bonding acceptors only in the cyclic head of the molecule and are non-competitive towards UQ (ubiquinone), but are believed to displace the semiquinone intermediate during the catalysis (Fig. 2). Finally, inhibitors classified as type C, like stigmatellin and capsaicin, form a third group of hydrophobic CI inhibitors that are believed to act as antagonists of reduced ubiquinone (Degli Esposti 1998, Friedrich et al. 1994, Haefeli 2012) (Fig. 2). Competition studies with representatives of all three different types of inhibitors revealed that type A and B and type B and C, but not type A and C, compete with each other for binding. This led to a suggestion that all CI inhibitors acting at the ubiquinone binding pocket share a common binding domain with partially overlapping sites (Okun et al. 1999).

Some inhibitors bind to the outside of the ubiquinone reduction site and do not fit the preceding classification. Examples of such compounds are ADP-ribose, which competes for substrate binding at the NADH site (Zharova and Vinogradov, 1997), and diphenyleneiodonium (DPI) that covalently binds to reduced flavin mononucleotide (FMN) in the hydrophilic part of the enzyme blocking the electron transfer to the Fe-S clusters (Majander et al., 1994). There are also new, commercially available insecticides/acaricides with potential to inhibit mitochondrial respiration such as benzimidazole, bullatacin, 6-chlorobenzothiadiazole, cyhalothrin, Fenazaquin Fenpyroximate, Hoe 110779, Pyridaben, Pyrimidifen, Sandoz 547A, Tebufenpyrad and Thiangazole (Greenamyre et al., 2001). It is clear that they are capable of inhibiting the mammalian CI of mitochondrial respiratory chain, by binding to and blocking ubiquinone-dependent NADH oxidation with high efficacy (Lummen, 1998).

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

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.

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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.

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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.

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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.

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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.

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