Key Event Title
|Level of Biological Organization|
Key Event Components
|mitochondrial electron transport, NADH to ubiquinone||NADH-ubiquinone oxidoreductase chain 1||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Mitochondrial dysfunction and Neurotoxicity||MolecularInitiatingEvent|
|Complex I inhibition leads to Fanconi syndrome||MolecularInitiatingEvent|
Key Event Description
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.
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.
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
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
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
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).
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)
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).
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.
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.