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Event: 105
Key Event Title
Inhibition, Mitochondrial Electron Transport Chain Complexes
Short name
Biological Context
Level of Biological Organization |
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Molecular |
Cell term
Organ term
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 | 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 Do not cite | |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 Do not cite |
Stressors
Taxonomic Applicability
Life Stages
Sex Applicability
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) |
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
The inhibition of mitochondrial electron transport chain can occur in any eukaryotic cell.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Rotenone
Rotenone acts as a mitochondrial respiratory chain complex I inhibitor, and its mechanism for mitochondrial toxicity is through induction of mitochondrial complex I substrate-supported mitochondrial ROS production, leading to a general decrease in ATP synthesis (Li et al., 2003). By inhibiting complex I, rotenone increases the formation of semiubiquinone, which is the primary electron donor in mitochondrial superoxide generation.
Li et al. (2003) demonstrated that rotenone impaired electron transport chain function through measurement of respiration and cellular ATP level of HL-60 cells. At rotenone levels as low as 10nM, as well as at 500nM, cell respiration was inhibited by over 96%. Increasing concentrations of rotenone below 100nM resulted in a sharp decrease of cellular ATP levels, and at 500nM, rotenone decreased cellular ATP level to 64% of control. Mitochondria treated with 200nM of rotenone showed significant levels of ROS production.
Cadmium
Mitochondrial electron transport chain impairment by cadmium occurs through its binding or its transport through the selectively permeable inner mitochondrial membrane to harm structures involved in oxidative phosphorylation (Adiele et al., 2012). Specifically, Wang et al. (2004) showed that CIII inhibition by cadmium is not related to the binding of Cd to the substrate binding sites, but rather to it binding allosterically to the Qo site of CIII. The binding of Cd prevents electron delivery from semiubiquinone to heme b566 and promotes the accumulation of semiubiquinone at the Qo site. The accumulated semiubiquinone is unstable and prone to donation of electrons to molecular oxygen, thus forming superoxide anion (Wang et al., 2004).
Cd-dependent inhibition of CIII was determined in the presence of excess zinc, which has been shown to bind to the Qo site of CIII and inhibit electron transfer activity (Wang et al., 2004). Double reciprocal plots for Zn inhibition of cytochrome c reductase (CIII) activity in the absence and presence of Cd showed that the Vmax values were not changed, indicating competition between Cd and Zn for the same binding site in CIII (Qo) (Wang et al., 2004).
Wang et al. (2004) measured the effects of Cd on the enzyme activity of CIII in the mitochondria of liver, brain, and heart, and determined that maximum inhibition was 30-77%. Adiele et al. (2012) found a maximum of 65% inhibition of CIII enzyme activity by Cd during state 3 mitochondrial respiration, and 75% inhibition of ATP production by Cd compared to the control in rainbow trout liver mitochondria.
Uranium
Yu et al. (2021) found that uranyl nitrate solution was capable of inhibiting electron transport chain complexes within a HK-2 cell line. This article shows inhibition of complex IV and ATP synthase at very low concentrations (>1 µM and <0.1 µM) (Yu et al., 2021). The IC50 values of complex IV and V were determined to be 3.8 mM (0.9 mg/L) and 4.8 (1.1 mg/L), respectively (Yu et al., 2021), from which it can be inferred that uranium exposure is more toxic to CIV than CV. However, within this experiment none of the other complexes were inhibited, despite the much higher concentrations being used for the treatment (50, 100, 250, and 500 µM) (Yu et al., 2021).
Silver
In Miyayama et al.’s (2013) study on the impact of silver treatment on rat livers they found silver dose-dependant inhibition of electron transport chain complexes at low concentrations. Complexes I to IV all showed inhibition, with CIII showing the most inhibition, followed by CI, CIV, and complex II when treated with 0.01, 0.1, 1.0, and 10 µM, respectively (Miyayama et al., 2013).
Gold nanoparticles
Ferreira et al. (2015) found that rat kidney mitochondria treated with varied sizes of gold nanoparticles (GNPs-10 and GNPs-30) had some complex inhibition as well as some complex activation. Acute exposure to GNPs-30 resulted in the inhibition of CIII, whereas GNPs-10 were able to significantly inhibit complex I activity (Ferreira et al., 2015). Chronic exposure to the GNPs-10 resulted in CII inhibition, while both GNPs-10 and 30 caused CII and CII-III activation when chronically exposed (Ferreira et al., 2015). The activation of CII and CII-III by GNPs-30 were likely a compensation mechanism for decreased activity from CIV (Ferreira et al., 2015).
Lead
In Ma et al.’s (2017) study on the effects of lead on rat liver mitochondria, they found significant dose-dependant inhibition of all four complexes. They found that CIII was the most inhibited complex, followed by CII (Ma et al., 2017). CI and CIV had very minimal inhibition when using 10, 20, 40, and 80 µM doses (Ma et al., 2017). It was suggested that the high level of ETC complex impairment induced by lead was a result of increased electron leakage which resulted in increased ROS generation, and would eventually trigger mitochondrial dysfunction (Ma et al., 2017).
Arsenic
Prakash, Soni, and Kumar (2015) measured the level of inhibition of mitochondrial electron transport chain complexes in rat brains after exposure to sodium arsenite in their drinking water for 12 weeks. They found that CII (32%), CIV (35%), and CI (42%) were all significantly inhibited (Prakash, Soni, and Kumar, 2015).
Chromium
García-Niño et al. (2013) investigated the effects of chromium treatment on mitochondria from rat liver. Their results showed that chromium treatment significantly inhibited complex I activity when compared to the control (García-Niño et al., 2013).
Other heavy metals
Belyaeva et al. (2012) measured basal, resting, and uncoupled respiration rates in the mitochondria after different times of exposure to mercury, cadmium, and copper. They demonstrated a complete inhibition of respiration after 3 hours of incubation with 50µM Hg2+ or 500µM Cd2+, and 50% inhibition of respiration after 48 hours of exposure to 500µM Cu2+ (Belyavea et al., 2012). All three measurements of respiration rates were strongly decreased, indicating a potent inhibitory effect on the respiratory chain (Belyaeva et al., 2012).
Cisplatin
In Santos et al.’s (2007) study on the nephrotoxicity induced by platinum-based cisplatin, ATP synthesis was assessed by measuringkidney ATP content, and was found to be significantly decreased in the cisplatin-treated group (75% of control). They also demonstrated a decrease in mitochondrial lipid levels due to their oxidation by cisplatin, which further inhibited the mitochondrial electron transport chain. Cardiolipin levels in particular were decreased to 73% of the control group (Santos et al., 2007).
References
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