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Event: 1968

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

Increase, Mitochondrial Dysfunction

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
Increase, Mt Dysfunction

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
Cellular

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

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

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
Inhibition of Mt-ETC complexes leading to kidney toxicity KeyEvent 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

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

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

Mitochondria are organelles found in all eukaryotic cells, crucial to the cellular consumption of oxygen, production of energy through the generation of ATP during oxidative phosphorylation, and regulation of cell death pathways (Alberts et al., 2014). The mitochondria are responsible for reduction of oxygen into water via the action of cytochrome c oxidase and other redox enzymes which transfer single electrons to oxygen and partially reduce it. The electron transfer is coupled with H+ ion transport across a membrane, producing the ion gradient that powers ATP synthesis (Alberts et al., 2014; Adiele et al., 2012). Under normal metabolic function, approximately 1-2% of the oxygen reduced by mitochondria converts into reactive oxygen species (ROS; such as superoxide, hydrogen peroxide, or hydroxyl radicals) at intermediate steps of the respiratory chain, as a result of electron transport (Kowaltowski and Vercesi, 1998; Volka et al., 2005; Li et al., 2003). This consistent and regular production of ROS and their signalling functionality at regulated levels contrasted with their harmful effects at high concentrations, justify the presence of antioxidant systems to regulate these processes.

Mitochondrial dysfunction, the loss of function or efficiency of oxidative phosphorylation, can be caused by a variety of factors and be apparent in a number of measurable ways. Some pathways of mitochondrial damage include: direct inhibition of mitochondrial proteins, indirect inhibition in upstream processes that affect mitochondrial metabolism, and indirect metabolic inhibition by ROS and physical damage to mitochondria. Dysfunction can be characterized through indicators of proton gradient loss, complex inhibition, or respiratory impairment such as mitochondrial permeability transition increase, mitochondrial membrane potential decrease, and ATP production (Shaki et al., 2013; Kruiderig et al., 1997). Any mitochondrial dysfunction impairs electron transfer and ATP production, which leads to deviation of electrons from their normal pathway in the electron transport chain (ETC), and increased ROS production. This, in turn, results in oxidative stress, mitochondrial permeability transition, and deregulation of cellular Ca2+ homeostasis (Nicholson, 2014; Shaki et al., 2013). Calcium, an imperative divalent cation to mitochondrial function, can be present at unsustainable levels due to increasing Ca2+ uptake, related to ROS generation and oxidative stress (reviewed Mei et al., 2013; Wang and Qin, 2010). Ca2+ accumulation and oxidative stress due additional ROS can trigger the opening of mitochondrial permeability transition pore (MPTP) by perturbing the osmolarity of mitochondria, disturbing Calcium homeostasis (Orrenius et al., 2015; Roos et al., 2012). The opening of the MPTP is a Ca2+-dependent process, that along with free proton movement collapses the mitochondrial membrane potential (MPP), halting ATP synthesis (Orrenius et al., 2015). ROS produced by the mitochondria can oxidize proteins and induce lipid peroxidation, compromising the barrier properties of the mitochondrial membrane (Orrenius et al., 2015) and therefore the proton gradient and ATP production. Respiration can also be impaired through mitochondrial DNA damage and increased permeability transition of the membrane as the mitochondrial inner membrane loses its impermeability to ions and other small molecules (up to a molecular weight of approximately 2kDa), this is loss of MPP and therefore proton gradient loss (Nicotera et al., 1998). Cytochrome c release is a major indicator of mitochondrial dysfunction as a combined result of a compromised mitochondrial membrane due to lipid peroxidation and the opening of the MPTP, and is commonly seen as an endpoint to mitochondrial toxicity (Chen et al., 2000). Mitochondrial damage can also be defined by loss of protein import and biosynthesis, as well as loss of mitochondrial motility as a result of failure to re-localize to sites with increased energy demands.

Metal-induced Mitochondrial Dysfunction

Mitochondria are an important site of Ca2+ regulation and storage, taking up Ca2+ ions electrophoretically from the cytosol through a Ca2+ uniporter, which can then accumulate in the mitochondria (Roos et al., 2012; Orrenius et al., 2015). Similarities between calcium and metals, such as cadmium and lead, makes the entrance and accumulation of these metals into the mitochondria via calcium metals possible by mode of molecular mimicry (Mathews et al., 2013; Adiele et al., 2012). The outer mitochondrial membrane also contains the divalent metal transporter (DMT1), which allows for mitochondrial uptake of divalent metals such as Fe and Mn. When cells are under heavy metal-induced stress, DMT has been shown to be overexpressed in the mitochondrial membrane, making the mitochondria targets of metal toxicity and accumulation.

Heavy metal exposure in aerobic organisms increases ROS formation through redox cycling, where metals with different valence states (Fe, Cu, Cr, etc.) directly produce ROS as they are reduced by cellular antioxidants and then react with oxygen (Shaki et al., 2012; Shaki et al., 2013; Pourahmad et al., 2006; Santos et al., 2007). The production of highly reactive hydroxyl radicals under mitochondrial oxidative stress and in the presence of transition metals occurs via the Fenton reaction or Haber-Weiss reaction (Hancock et al., 2001; Valko et al., 2005; Adam-Vizi et al., 2010). Metals and ROS are capable of damaging mitochondrial DNA as well as mechanisms of DNA repair and proliferation arrest (Valko et al., 2005). Metals and ROS have the potential to directly damage mitochondrial membranes and structure by binding to and oxidizing membrane lipids and proteins. This structural damage can collapse the MMP and lead to the opening of the MPTP (Orrenius et al., 2015; Roos et al., 2012; Pourahmad et al., 2006). Uranium and mercury, for example, have both been shown to directly inhibit the mitochondrial electron transport chain and interfere with ATP production (Shaki et al., 2012; Roos et al., 2012). Furthermore, as previously mentioned, metals have been shown to inhibit ROS-detoxifying enzymes. By binding to these enzymes, metals can inhibit their antioxidant functions, and cause an accumulation of ROS and increased synthesis of more antioxidant enzymes in order to combat the oxidative stress (Blajszczak and Bonini, 2017).

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). ?
Assay - What is being Measured Description Dose Range Studied Assay Length / Ease of use, accuracy
Rhodamine 123 Assay

Measuring Mitochondrial membrane potential (MMP) and its collapse 

(Shaki et al., 2012)

Mitochondrial uptake of cationic fluorescent dye, rhodamine 123, is used for estimation of mitochondrial membrane potential. The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively.
50, 100 and 500 μM of uranyl acetate

Short / easy

Medium accurancy

TMRE fluorescence Assay

Measuring Mitochondrial permeability transition pore (MPTP) opening

(Huser et al., 1998)

Laser scanning confocal microscopy in combination with the potentiometric fluorescence dye tetramethylrhodamine ethyl ester to monitor relative changes in membrane potential in single isolated cardiac mitochondria. The cationic dye distributes across the membrane in a voltage-dependent manner. Therefore, the large potential gradient across the inner mitochondrial membrane results in the accumulation of the fluorescent dye within the matrix compartment. Rapid depolarizations are caused by the opening of the transition pore. 1 µM cyclosporin A

Short / easy

Low accurancy

GSH / GSSG Determination Assay

Measuring  cellular glutathione (GSH) status; ratio of GSH/GSSG

(Owen & Butterfield, 2010; Shaki et al., 2013)

GSH and GSSG levels are determinted biochemically with DTNB (Ellman’s reagent). The developed yellow color was read at 412 nm on a spectrophotometer. 100 µM uranyl acetate

Short / easy

Low accurancy

TBARS Assay

Quantification of lipid peroxidation

(Yuan et al., 2016)

MDA content, a product of lipid peroxidation, was measured using a thiobarbituric acid reactive substances (TBARS) assay. Briefly, the kidney cells were collected in 1 ml PBS buffer solution (pH 7.4) and sonicated. MDA reacts with thiobarbituric acid forming a colored product which can be measured at an absorbance of 532 nm. 200, 400, 800 µM uranyl acetate

Medium / medium

High accurancy

Aequorin-based bioluminescence assay

Increase in mitochondrial Ca2+ influx

(Pozzan & Rudolf, 2009)

Together with GFP, the aequorin moiety acts as Ca2+ sensor in vivo, which delivers emission energy to the GFP acceptor molecule in a BRET (Bioluminescence Resonance Energy Transfer) process; the Ca2+ can then be visualized with fluorescence microscopy.  

Short / easy

Low accurancy

Western blot & immunostaining analyses

Measuring cytochrome c release

(Chen et al., 2000)
Examining the redistribution of Cyto c in cytosolic and mitochondrial cellular fractions. Cells are homogenized and centrifuged, then prepared for immunoblots. Cellular fractions were washed in PBS and lysed in 1% NP-40 buffer. Cellular proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes, probed using immunoblot analyses with antibodies specific to cyto c (6581A for Western and 65971A for immunostaining; Pharmingen)  

Short / easy

Medium accurancy

Quantikine Rat/Mouse Cytochrome c Immunoassay

Measuring cytochrome c release

(Shaki et al., 2012)

Cytochrome C release was measured a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing mono- clonal antibody specific for cytochrome c conjugated to horseradish peroxidase). After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm.  

Short / easy

Low accurancy

Membrane potential and cell viability – Flow Cytometry
Measuring cytochrome c release

(Kruiderig et al., 1997)

Dc and viability were determined by analyzing the R123 and propidium iodide fluorescence intensity with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser, with the Lysis software program (Becton Dickinson). R123 is a cationic dye that accumulates in the negatively charged inner side of the mitochondria. When the potential drops, less R123 accumulates in the mitochondria, which results in a lower fluorescence signal. The potential was measured as follows: at the indicated times, a 500-ml sample of the cell suspension was taken and transferred to an Eppendorf minivial. To this sample, 100 ml of 6 mM R123 in buffer D was added. After incubation for 10 min at 37°C, the cell suspension was centrifuged for 5 min at 80 3 g. The cell pellet was resuspended in 200 ml of buffer D, containing 0.2 mM R123 and 10 mM propidium iodide, to prevent loss of R123 and to stain nonviable cells, respectively. The samples were transferred to FACScan tubes and analyzed immediately. Analysis was performed at a flow rate of 60 ml/min. R123 fluorescence was detected by the FL1 detector with an emission detection limit below 560 nm. Propidium iodide fluorescence was detected by the FL3 detector, with emission detection above 620 nm. Per sample 3,000 to 5,000 cells were counted (Van de Water et al., 1993)”  

Short / easy

Medium accurancy

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

Mitochondrial dysfunction can occur in any eukaryotic cell.

Evidence for Perturbation by Stressor

Uranium

Shaki et al. (2012) found that uranyl acetate (UA) exposure in isolated rat kidney mitochondria decreased the ATP production levels and ATP/ADP ratio in a concentration-dependent manner, through inhibition of complexes II and III of the ETC. Both of these levels were significantly changed at UA concentrations of 100 µM and 200 µM. In addition, a concentration-dependent decrease in activity of complex II with exposure to U was observed (Shaki et al., 2012). They also found that mitochondrial membrane potential damage and mitochondrial swelling both increased significantly time- and dose-dependently in the treated rat kidneys (Shaki et al., 2012). ATP/ADP ratios were also decreased significantly by treatment with 100 µM or more uranium (Shaki et al., 2012). Mitochondrial outer membrane damage was significantly decreased by treatment with 200 µM of uranium (Shaki et al., 2012).

Shaki et al. (2013) also investigated the effects of uranium on rat kidneys. They found that mitochondrial permeability transition was also impacted by uranium treatment, causing increased mitochondrial swelling and increased disruption of energy homeostasis (Shaki et al., 2013).

Hao et al. (2014) assessed the changes in mitochondrial potential in human kidney proximal tubular cells treated with uranium and found that the group treated with 500 µM of depleted uranium for 24 hours showed a significant decreased mitochondrial membrane potential.

In their study of the effects of depleted uranium treatment on human embryonic kidney cells, Hao et al. (2016) found that ETHE1, a mitochondrial protein involved in mitochondrial homeostasis and mitochondrial diseases, had significant dose- and time-dependant decreases in gene expression when treated with 125 µM or more depleted uranium (DU) for 2 hours or more.

Nanoparticles and Micrometer Particles

Karlsson et al. (2009) conducted experiments to examine the effects of micrometer and nanoparticle treatments of copper and iron on human alveolar type-II epithelial cells. Their results showed that copper oxide micrometer and nanoparticle treatments were able to cause dose-dependant mitochondrial depolarization with doses as low as 5 µg/cm2 (Karlsson et al., 2009). Iron(III) oxide nanoparticles and micrometer particles were both able to cause similar amounts of mitochondrial depolarization, along with iron (IV) oxide micrometer particles, however they were all much less toxic than copper oxide nanoparticles or micrometer particles (Karlsson et al., 2009).

The effects of gold nanoparticle (Au1.4MS) treatment on human cervical cancer cells were assessed by Pan et al. (2009), who found that the treated cells experienced a significant increase in permeability transition.

Huerta-García et al. (2014) studied the effects of titanium oxide nanoparticle treatment on glial tumor rat neuronal cells and cancerous human brain cells. Their results showed that in the treated rat and human cells there was a clear time-dependant increase in depolarization (Huerta-García et al., 2014). They also found that both the human and rat cells showed time-dependant decreases in mitochondrial membrane potential, with the TiO2 nanoparticles being more toxic to the human cells, which showed significant decrease as early as 2 hours post-treatment, while the rat cells did not show significant decrease until 6 hours post-treatment (Huerta-García et al., 2014).

Zhang et al. (2018) investigated the effects of copper nanoparticles on mitochondrial membrane potential in pig kidney cells and found that the treated cells showed a dose-dependant increase in the rate of mitochondrial membrane potential change from 40 µg/mL to 80 µg/mL when treated for 12 hours.

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

Adam-Vizi, V., & Starkov, A. A. (2010). Calcium and mitochondrial reactive oxygen species generation: How to read the facts. Journal of Alzheimer's Disease : JAD, 20 Suppl 2, S413-S426. doi:10.3233/JAD-2010-100465

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

Buelna-Chontal, M., Franco, M., Hernandez-Esquivel, L., Pavon, N., Rodriguez-Zalvala, J. S.,

Correa, F., . . . Chavez, E. (2017). CDP-choline circumvents mercury-induced mitochondrial damage and renal dysfunction. Cell Biology International, 41, 1356-1366. doi:10.1002/cbin.10871

Chen, Q., Gong, B., & Almasan, A. (2000). Distinct stages of cytochrome c release from mitochondria: Evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death and Differentiation, 7(2), 227-233. doi:10.1038/sj.cdd.4400629

Görlach, A., Bertram, K., Hudecova, S., & Krizanova, O. (2015). Calcium and ROS: A mutual interplay. Redox Biology, 6, 260-271. doi:doi:10.1016/j.redox.2015.08.010

Hancock, J. T., Desikan, R., & Neill, S. J. (2001). Role of reactive oxygen species in cell signalling pathways. Biochemical Society Transactions, 29(Pt 2), 345-350. doi:10.1042/0300-5127:0290345 [doi]

Hao, Y., Huang, J., Liu, C., Li, H., Liu, J., Zeng, Y., . . . Li, R. (2016). Differential protein

expression in metallothionein protection from depleted uranium-induced nephrotoxicity. Scientific Reports, doi:10.1038/srep38942

Hao, Y., Ren, J., Liu, C., Li, H., Liu, J., Yang, Z., . . . Su, Y. (2014). Zinc protects human kidney cells from depleted uranium induced apoptosis. Basic & Clinical Pharmacology & Toxicology, 114, 271-280. doi:10.1111/bcpt.12167

Huerta-García, E., Perez-Arizti, J. A., Marquez-Ramirez, S. G., Delgado-Buenrostro, N. L.,

Chirino, Y. I., Iglesias, G. G., & Lopez-Marure, R. (2014). Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells. Free Radical Biology and Medicine, 73, 84-94. doi:10.1016/j.freeradbiomed.2014.04.026

Hüser, J., Rechenmacher, C. E., & Blatter, L. A. (1998). Imaging the permeability pore transition in single mitochondria. Biophysical Journal, 74(4), 2129-2137. doi:10.1016/S0006-3495(98)77920-2

Karlsson, H. L., Gustafsson, J., Cronholm, P., & Möller, L. (2009). Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size. Toxicology Letters, 188(2), 112-118. doi:10.1016/j.toxlet.2009.03.014

Kowaltowski, A. J., & Vercesi, A. E. (1999). Mitochondrial damage induced by conditions of oxidative stress. Free Radical Biology and Medicine, 26(3), 463-471. doi:10.1016/S0891-5849(98)00216-0

Kruidering, M., Van De Water, B., De Heer, E., Mulder, G. J., & Nagelkerke, J. F. (1997). Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: Mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. The Journal of Pharmacology and Experimental Therapeutics, 280(2), 638-649.

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

Mathews, C. K., Holde, K. E. van, Appling, D. R., & Anthony-Cahill, S. J. (2013). Biochemistry (4th ed.). Toronto: Pearson.

Mei, Y., Thompson, M. D., Cohen, R. A., & Tong, X. (2013). Endoplasmic reticulum stress and related pathological processes. Journal of Pharmacological & Biomedical Analysis, 1(2), 1000107-1000107.

Miccadei, S., & Floridi, A. (1993). Sites of inhibition of mitochondrial electron transport by cadmium. Elsevier Scientific Publishers Ireland Ltd., 89, 159-167.Xu, X. M., & Møller, S. G. (2010). ROS removal by DJ-1: Arabidopsis as a new model to understand Parkinson's Disease. Plant signaling & behavior5(8), 1034–1036. doi:10.4161/psb.5.8.12298

Nicolson, G. L. (2014). Mitochondrial dysfunction and chronic disease: Treatment with natural supplements. Integrative Medicine (Encinitas, Calif.), 13(4), 35-43.

Nicotera, P., Leist, M., & Ferrando-May, E. (1998). Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicology Letters, 102-103, 139-142. doi:https://doi.org/10.1016/S0378-4274(98)00298-7

Orrenius, S., Gogvadze, V., & Zhivotovsky, B. (2015). Calcium and mitochondria in the regulation of cell death. Biochemical and Biophysical Research Communications, 460(1), 72-81. doi:10.1016/j.bbrc.2015.01.137

Owen, J. B., & Butterfield, D. A. (2010). Measurement of oxidized/reduced glutathione ratio. Methods in Molecular Biology (Clifton, N.J.), 648, 269-277. doi:10.1007/978-1-60761-756-3_18 [doi]

Pan, Y., Leifer, A., Ruau, D., Neuss, S., Bonrnemann, J., Schmid, G., . . . Jahnen-Dechent, W. (2009). Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small, 5(8), 2067-2076. doi:10.1002/smll.200900466

Pourahmad, J., Ghashang, M., Ettehadi, H. A., & Ghalandari, R. (2006). A search for cellular and molecular mechanisms involved in depleted uranium (DU) toxicity. Environmental Toxicology, 21(4), 349-354. doi:10.1002/tox.20196

Pozzan, T., & Rudolf, R. (2009). Measurements of mitochondrial calcium in vivo. Biochimica Et Biophysica Acta (BBA) - Bioenergetics, 1787(11), 1317-1323. doi:https://doi.org/10.1016/j.bbabio.2008.11.012

Roos, D., Seeger, R., Puntel, R., & Vargas Barbosa, N. (2012). Role of calcium and mitochondria in MeHg-mediated cytotoxicity. Journal of Biomedicine and Biotechnology, 2012, 1-15. doi:10.1155/2012/248764

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

Turrens, J. F. (2003). Mitochondrial formation of reactive oxygen species. The Journal of Physiology, 552(Pt 2), 335-344. doi:jphysiol.2003.049478 [pii]

Valko, M., Morris, H., & Cronin, M. T. (2005). Metals, toxicity and oxidative stress. Current Medicinal Chemistry, 12(10), 1161-1208. doi:10.2174/0929867053764635 [doi]

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