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Event: 105

Key Event Title

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Inhibition, Mitochondrial Electron Transport Chain Complexes

Short name

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Inhibition, ECT complexes of the respiratory chain

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Biological Context

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Level of Biological Organization
Molecular

Cell term

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Organ term

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

Key Event Overview

AOPs Including This Key Event

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AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Inhibition of Mt-ETC complexes leading to kidney toxicity	MolecularInitiatingEvent	Agnes Aggy (send email)	Under development: Not open for comment. Do not cite
Kidney failure induced by inhibition of mitochondrial ETC	KeyEvent	Brendan Ferreri-Hanberry (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

Life Stages

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Sex Applicability

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Key Event Description

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The electron transport chain, otherwise known as the respiratory chain, is composed of large protein complexes (CI, CII, CIII, CIV, CV) and two freely mobile electron transfer carriers, ubiquinone and cytochrome c, which are embedded in the inner membrane cristae of the mitochondria (Zhao et al., 2019). Three of these complexes (CI, CIII, CIV; NADH dehydrogenase, cytochrome c reductase, and cytochrome c oxidase, respectively) act as proton pumps and contribute to the formation of an electrochemical proton gradient across the inner mitochondrial membrane, which then drives ATP synthesis by complex V (ATP synthase) (Alberts et al., 2014). In eukaryotes, the electron transport chain is the major site of ATP production via oxidative phosphorylation. Superoxides (O₂^‑) are generated in low quantities as by-products of oxidative phosphorylation during electron transfer. The O₂^‑ released into the inter-membrane space (IMS) by CIII can be converted into H₂O₂ in a reaction catalyzed by superoxide dismutase 1 and H₂O₂ then may diffuse into the cytoplasm (Zhao et al., 2019). Superoxides behave as signalling molecules important in cell proliferation, hypoxia adaption, and cell fate determination but when present in excess or unregulated, induce cell damage and death.

While it is well known that heavy metals target the mitochondria, the exact mechanism of this targeting and inhibition is poorly understood (Belyaeva et al., 2012; Gobe & Crane, 2010). Respiratory complexes CI and CIII are shown to be particularly susceptible to perturbation by heavy metals such as chromium and cadmium (Adiele et al., 2012; Santos et al., 2007). In addition, Uranyl Acetate (UA) induced nephrotoxicity has been linked to the impairment of CII and CIII leading to inhibition of the mitochondrial electron transport chain (Shaki et al., 2012; Shaki & Pourahmad, 2013).

Several studies have been conducted in order to understand the exact mechanisms of inhibition by heavy metals. They show that these divalent cations bind to electron transport chain enzyme complexes and modify them, disturbing electron transfer and redox reactions (Blajszczak & Bonini, 2017). For example, rotenone blocks Complex I (Li et al., 2003) and cadmium has the capability to noncompetitively inhibit CIII (Wang et al., 2004). This blocking and inhibition interrupts the transport of electrons through the respiratory chain, specifically resulting in the increase of semiubiquinone formation and subsequently the generation of mitochondrial superoxides (Li et al., 2003). Shaki et al. (2012) have shown, as well, that uranyl acetate (UA) interferes with CII and CIII activity. Function of the electron transport chain can also be suppressed by indirect effects of heavy metals: cisplatin causes oxidative damage of mitochondrial membrane lipids such as cardiolipin, impacting mitochondrial membrane potential (MMP). This lipid is responsible for maintaining the inner mitochondrial membrane structure and linking CIII and CIV in a super complex through which protons and electrons move, producing ATP (Santos et al., 2007). Cardiolipin function is therefore vital and its disruption results in inhibition of mitochondrial integrity and function.

The inhibition of the electron transport chain initiates a sequence of events in the mitochondria, including: overproduction of reactive oxygen species (ROS); a reduced ability for oxidative phosphorylation and therefore decreased ATP synthesis; a lowered ATP/ADP ratio; the release of cytochrome c from the mitochondrial cristae; and the collapse of mitochondrial membrane potential (MMP) (Shaki et al., 2012; Adiele et al., 2012). All of these occurrences contribute to overall mitochondrial dysfunction and more adverse outcomes.

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

Assay Type & Measured Content	Description	Dose Range Studied	Assay Characteristics (Length / Ease of use/Accuracy)
MTT assay Measuring enzymatic activity of the electron transport system (Thiebault et al., 2007; Shaki et al., 2012)	CII and CIII, transmembrane electrical potential change was measured. The metabolic activity of mitochondrial complex II was assayed by measuring the reduction of MTT to a blue formazan compound. Mitochondrial suspensions were incubated with different concentrations of uranyl acetate prior to addition of MTT. The product of formazan crystals were dissolved in DMSO and the absorbance at 570nm was measured with an ELISA reader.	50, 100 and 500 μM of uranyl acetate; 0-1000µM U	Long Easy/Difficult High accuracy (mathematical measurement) Medium Precision
Cell Respiration Assay Measuring cellular oxygen consumption and uptake (Belyaeva et al., 2012)	Cell respiration is determined polarographically with the help of a Clark oxygen electrode in a thermostatic water-jacketed vessel with magnetic stirring at 37°C. PC12 cells (10⁷ cells) were incubated in 10 mL of the complete DMEM medium (with serum) in Petri dishes for different lengths of time with various concentrations of the corresponding heavy metal, then collected by centrifugation and transferred to the DMEM medium without serum.	10, 50, 100, or 500 μM	Long Difficult Medium accuracy (estimated spectrophotometrically)
Luciferin-luciferase assay (ATP determination) Measuring ATP content of the cell (Li et al., 2003)	For ATP measurement, a commercially available luciferin-luciferase assay kit was used. Briefly, HL-60 cells were treated with various concentrations of rotenone for 24 h and then collected. After a single wash with ice-cold PBS, cells were lysed with the somatic cell ATP-releasing reagent provided by the kit. Luciferin substrate and luciferase enzyme were added and bioluminescence was assessed on a spectroflurometer. Whole-cell ATP content was determined by running an internal standard.	0-1000nM of rotenone	Short Easy High accuracy and precision
Cytochrome c binding domain determination Measuring identification of the inhibitory site of Cd in CIII (Wang et al., 2004)	Cytochrome c binding domain determination was performed in 2 ml of an assay mixture containing 30 mM phosphate, 100 mM KCl, 2 mM KCN, and 0.1% DM, pH 7.0. The final concentration of the electron donor DBH2 ranged from 20 to 400 µM. The final concentration of the mitochondrial protein was 13.7 mg/ml. The reaction was started with addition of cytochrome c. DBH2 binding determination was done in the same reaction system as described above. The final concentration of DBH2 was 20 µM. The reaction was started with addition of DBH2.	5-40µM Cytochrome c	Short Easy High accuracy and precision
Enzyme Activity Determination (Kruiderig et al., 1997)	“Enzymatic activities of the complexes I to IV were determined by dual wavelength spectrophotometry with an Aminco Dual Wavelength 2 ATM UV-VIS spectrophotometer (Silver Spring, MD). All concentrations below are final concentrations. Complex I (NADH:ubiquinone oxidoreductase) activity was determined at 340 nm with 380 nm as reference wavelength, with a slit width of 3.0 nm according to Estornell et al. (1993). The assay was performed with 10 to 30 mg protein in a final volume of 1 ml of buffer, pH 7.4, containing 10 mM Tris-HCl, 50 mM KCl, 1 mM EDTA and 2 mM KCN. After addition of 75 ml of 1 mM NADH and stabilization of the signal, the reaction was started by addition of 100 ml of 1 mM ubiquinone-10. The activity was calculated from the rate of decrease of NADH (e 5 5.5 mM21 cm21) per mg protein. Complex II (succinate dehydrogenase) activity was determined by the difference in absorbency between 270 and 330 nm according to Estornell et al. (1993). The assay was performed with 10 to 30 mg protein in a final volume of 1 ml of 50 mM potassium phosphate buffer, pH 7.4, containing 100 mM EDTA, 1 mM KCN and 0.1% (w/v) BSA. After addition of 80 ml of 1 mM ubiquinone-0 and stabilization of the signal, the reaction was started by addition of 100 ml of 0.1 M sodium succinate. The activity was calculated from the rate of decrease in ubiquinone (e 5 9.6 mM21 cm21). Complex III (Ubiquinol-cytochrome c reductase) activity was determined by the difference in absorbency between 550 and 580 nm according to Birch-Machin et al. (1993b). The assay was performed with 10 to 30 mg protein in a final volume of 1 ml of 25 mM potassium phosphate buffer, pH 7.2, containing 5 mM MgCl2, 2 mM KCN, 2.5 mg/ml BSA, 2 mg/ml rotenone and 0.5 mM N-D-maltoside. After addition of 10 ml of 3.5 mM ubiquinol and stabilization of the signal, the reaction was started by the addition of 10 ml of 1.5 mM cytochrome cIII. The activity was calculated from the rate of reduction of cytochrome cIII (e 5 19 mM21 cm21). Complex IV (cytochrome c oxidase) activity was determined by the 640 Kruidering et al. Vol. 280 Downloaded from jpet.aspetjournals.org at ASPET Journals on June 28, 2019 difference in absorbency between 550 and 580 nm according to BirchMachin et al. (1993a). The assay was performed with 10 to 30 mg protein in a final volume of 1 ml of 25 mM potassium phosphate buffer, pH 7.0, containing 0.5 mM N-D-maltoside. After addition of 10 ml of 1.5 mM cytochrome cII and stabilization of the signal, the reaction was started by the addition of 10 to 30 mg cells. The activity was calculated from the rate of increase in absorbency caused by oxidation of cytochrome cII to cytochrome cIII (e 5 19 mM21 cm21). All activities were expressed per microgram of protein, which was determined according to Lowry et al. (1951)”

Domain of Applicability

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The inhibition of mitochondrial electron transport chain can occur in any eukaryotic cell.

References

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

Adiele, R. C., Stevens, D., & Kamunde, C. (2012). Differential inhibition of electron transport chain enzyme complexes by cadmium and calcium in isolated rainbow trout (oncorhynchus mykiss) hepatic mitochondria. Toxicological Sciences, 127(1), 110-119. doi:10.1093/toxsci/kfs091

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2014). Molecular biology of the cell. New York: Garland Science. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK21054/

Belyaeva, E. A., Sokolova, T. V., Emelyanova, L. V., & Zakharova, I. O. (2012). Mitochondrial electron transport chain in heavy metal-induced neurotoxicity : Effects of cadmium , mercury , and copper. Thescientificworld, 2012, 1-14. doi:10.1100/2012/136063

Blajszczak, C., & Bonini, M. G. (2017). Mitochondria targeting by environmental stressors : Implications for redox cellular signaling. Toxicology, 391, 84-89. doi:10.1016/j.tox.2017.07.013

Gobe, G., & Crane, D. (2010). Mitochondria, reactive oxygen species and cadmium toxicity in the kidney. Toxicology Letters, 198(1), 49-55. doi:https://doi.org/10.1016/j.toxlet.2010.04.013

Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J. A., & Robinson, J. P. (2003). Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. The Journal of Biological Chemistry, 278(10), 8516-8525. doi:M210432200 [pii]

Ma, L., Liu, J., Dong, J., Xiao, Q., Zhao, J., & Jiang, F. (2017). Toxicity of Pb2+ on rat liver mitochondria induced by oxidative stress and mitochondrial permeability transition. Toxicol.Res., 6, 822. doi:10.1039/c7tx00204a

Prakash, C., Soni, M., & Kumar, V. (2015). Biochemical and molecular alterations following arsenic-induced oxidative stress and mitochondrial dysfunction in rat brain. Biol.Trace Elem.Res., 167, 121-129. doi:10.1007/s12011-015-0284-9

Santos, N. A. G., Catão, C. S., Martins, N. M., Curti, C., Bianchi, M. L. P., & Santos, A. C. (2007). Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Archives of Toxicology, 81(7), 495-504. doi:10.1007/s00204-006-0173-2

Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015

Shaki, F., Hosseini, M., Ghazi-Khansari, M., & Pourahmad, J. (2013). Depleted uranium induces disruption of energy homeostasis and oxidative stress in isolated rat brain mitochondria. Metallomics, 5(6), 736-744. doi:10.1039/c3mt00019b

Thiébault, C., Carrière, M., Milgram, S., Simon, A., Avoscan, L., & Gouget, B. (2007). Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells. Toxicological Sciences : An Official Journal of the Society of Toxicology, 98(2), 479-487. doi:kfm130 [pii]

Wang, Y., Fang, J., Leonard, S. S., & Krishna Rao, K. M. (2004). Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radical Biology and Medicine, 36(11), 1434-1443. doi:10.1016/j.freeradbiomed.2004.03.010

Yu, L., Li, W., Chu, J., Chen, C., Li, X., Tang, W., . . . Xiong, Z. (2021). Uranium inhibits mammalian mitochondrial cytochrome c oxidase and ATP synthase. Environmental Pollution, 271, 116377. doi:10.1016/j.envpol.2020.116377

Zhao, R., Jiang, S., Zhang, L., & Yu, Z. (2019). Mitochondrial electron transport chain, ROS generation and uncoupling (review). International Journal of Molecular Medicine, 44(1), 3-15. doi:10.3892/ijmm.2019.4188