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Key Event Title
Inhibition, Mitochondrial Electron Transport Chain Complexes
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Inhibition of Mt-ETC complexes leading to kidney toxicity||MolecularInitiatingEvent||Baki Sadi (send email)||Under development: Not open for comment. Do not cite|
|Kidney failure induced by inhibition of mitochondrial ETC||KeyEvent||Yann GUEGUEN (send email)||Under development: Not open for comment. Do not cite|
Key Event Description
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.
How It Is Measured or Detected
|Assay Type & Measured Content||Description||Dose Range Studied||
Assay Characteristics(Length / Ease of use/Accuracy)
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
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
DifficultMedium 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 wereadded and bioluminescence was assessed on a spectroflurometer. Whole-cell ATP content was determined by running an internal standard.
|0-1000nM of rotenone||
EasyHigh 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 withaddition of DBH2.
|5-40µM Cytochrome c||
EasyHigh 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
The inhibition of mitochondrial electron transport chain can occur in any eukaryotic cell.
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