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Created at: 2020-11-29 22:43

AOP ID and Title:

AOP 263: Uncoupling of oxidative phosphorylation leading to growth inhibition (1)
Short Title: Uncoupling of OXPHOS leading to growth inhibition (1)

Graphical Representation

Authors

You Songa and Daniel L. Villeneuveb

a Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo, Norway

b U.S. Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, Minnesota 55804, USA

 

Acknowledgement

This project was funded by the Research Council of Norway (RCN), grant no. 301397 “RiskAOP - Quantitative Adverse Outcome Pathway assisted risk assessment of mitochondrial toxicants” (https://www.niva.no/en/projectweb/riskaop), and supported by the NIVA Computational Toxicology Program, NCTP (www.niva.no/nctp).


Status

Author status OECD status OECD project SAAOP status
Open for comment. Do not cite Under Development

Abstract

Uncoupling of oxidative phosphorylation (OXPHOS) is a well-known mechanism of action of many chemicals. Mitochondrial uncoupler-mediated energetic dysfunction is considered to affect growth, a common physiological process in most organisms and a highly regulatory relevant chronic toxicity endpoint widely included in the OECD test guidelines. This adverse outcome pathway (AOP) causally links uncoupling of OXPHOS to growth inhibition, through ATP depletion and reduced cell proliferation as the intermediate key events (KEs), with strong weight of evidence support. The AOP is highly generalized with the intention of being applicable to a broad range of species groups, ranging from microalga to human. Three out of four KEs can be quantified using high-throughput methods, making this AOP particularly useful for screening, prioritization and hazard assessment of potential mitochondrial uncouplers and growth inhibiting chemicals. The AOP is also a core part of a larger network addressing uncoupling of OXPHOS mediated growth inhibition (AOP 263-268).


Background

The mitochondrial OXPHOS machinery is a key physiological process responsible for producing the primary cellular energy, adenosine triphosphate (ATP). During OXPHOS, a series of redox reactions (oxidation) are mediated by protein complexes in an electron transport chain to create a protonmotive force across the inner mitochondrial membrane (Liberman 1969). The PMF acts as a driving force of ATP synthesis through phosphorylation of adenosine diphosphate (ADP). The mitochondrial oxidation and phosphorylation are coupled to ensure continuous ATP supply for various physiological processes. A number of chemicals can bind to the inner mitochondrial membrane and dissipate the PMF, thus leading to uncoupling of OXPHOS and reduction in ATP synthetic efficiency. Classical “couplers” are normally protonophores with major characteristics of bulky hydrophobic moiety, an acid dissociable group and a strong electron-withdrawing group (Terada 1990). With the rapid development of in silico (Escher 2002; Attene-Ramos 2013; Attene-Ramos 2015; Xia 2018) approaches, more and more uncouplers have been identified. Their hazards and risks to the biota, however, remain to be assessed. Uncoupling of OXPHOS can affect many ATP-dependent biological functions. As a major process to achieve organismal growth, cell proliferation is positively correlated with the cellular ATP level and highly susceptible to energy depletion (Ramaiah 1964; Bonora 2012). Therefore, a link between uncoupling of OXPHOS and growth inhibition can be established with ATP depletion and reduced cell proliferation as the intermediate steps.


Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
MIE 1446 Decrease, Coupling of oxidative phosphorylation Decrease, Coupling of OXPHOS
KE 1771 Decrease, Adenosine triphosphate pool Decrease, ATP pool
KE 1821 Decrease, Cell proliferation Decrease, Cell proliferation
AO 1521 Decrease, Growth Decrease, Growth

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Decrease, Coupling of oxidative phosphorylation adjacent Decrease, Adenosine triphosphate pool High High
Decrease, Adenosine triphosphate pool adjacent Decrease, Cell proliferation Moderate Moderate
Decrease, Cell proliferation adjacent Decrease, Growth Moderate Moderate

Stressors


Name Evidence
2,4-Dinitrophenol High
Pentachlorophenol Moderate
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone High
Carbonyl cyanide m-chlorophenyl hydrazone High
Triclosan High
Dinoseb Moderate
3,5-Dichlorophenol Moderate
Emodin High

Overall Assessment of the AOP

The weight of evidence assessment of the AOP was conducted based on the evolved Bradford-Hill considerations (Becker 2015) and according to the criteria in OECD’s Guidance Document for Developing and Assessing AOPs (OECD 2018). Overall, the MIE and KE1 are scored as high due to good evidence to support their essentiality in the AOP, whereas KE2 is scored as moderate due to a lack of solid evidence to support its essentiality. The overall WoE of KER1 is considered high, as strong biological plausibility, empirical evidence and fairly good quantitative understanding were evidenced from multiple studies. The overall WoE of KER2 is considered moderate, due to high biological plausibility, acceptable empirical concordance and some biological understanding. The overall WoE of KER3 is scored as moderate, mainly due to good biological plausibility, whereas a lack of sufficient empirical evidence and quantitative understanding to further support the causality. The AOP is considered applicable to a wide range of species as well as a broad domain of chemicals. The rationales for making these judgements will be discussed in detail in the following sections.

Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Embryo High
Juvenile Not Specified
Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio High NCBI
Lemna minor Lemna minor Moderate NCBI
human Homo sapiens Moderate NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI
Sex Applicability
Sex Evidence
Unspecific High

The taxonomic application domain of the AOP potential covers all animals, plants and some microorganisms such as fungus and protists, as mitochondrial OXPHOS is highly conserved in eukaryotes (Roger 2017).

The life stage applicability domain mainly contains embryos and juveniles, as growth is more relevant to developing organisms. It should be noted that fully grown adults are also susceptible to uncouplers, as tissue/organ (e.g., adipose tissue) growth and regeneration still occur in adults (Yun 2015) and (Demine 2019). Classical uncoupler such as 2,4-DNP have been reported to cause weight loss in adult humans (Grundlingh 2011), suggesting that adults are partially in the applicability domain of this AOP.

The sex applicability domain is unspecific, as the AOP is mainly targeting growth effects in sextual immature organisms and the KEs are therefore harmonized between male and females. However, male and females may have different sensitivities to OXPHOS uncoupling, as the strategies for allocating energy for developmental processes may be gender specific (Demarest 2015). Follow-up studies may consider the inclusion of comparative analysis on gender susceptibility. 

Chemical applicability domain: Weak acids, such as phenols, benzimidazoles, N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylic acids, acyldithiocarbazates, cumarines, and aromatic amines are well-known protonophoric uncouplers. Classical uncouplers, such as 2,4-dinitrophenol (2,4-DNP), carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), pentachlorophenol (PCP), 3,5-dichlorophenol (3,5-DCP), 6-sec-butyl-2,4-dinitrophenol (dinoseb), SF 6847 (3,5-di-t-butyl-4-hydroxybenzylidinemalononitrile) have been widely used as positive controls in (eco)toxicological tests, whereas the hazards of “new” uncouplers, such as triclosan, emodin and metabolites of polybrominated diphenyl ethers (PBDEs) are also under extensive assessments. A number of potential uncouplers have been identified by in silico (Escher 2002; Attene-Ramos 2013; Attene-Ramos 2015; Xia 2018) approaches, and are considered in the chemical applicability domain of the AOP.

Essentiality of the Key Events

 

Support for Essentiality of KEs

Defining Question

What is the impact on downstream KEs and/or the AO if an upstream KE is modified or prevented?

High

Direct evidence from specifically designed experimental studies illustrating prevention or impact on downstream KEs and/or the AO if upstream KEs are blocked or modified.

Moderate

Indirect evidence that modification of one or more upstream KEs is associated with a corresponding (increase or decrease) in the magnitude or frequency of downstream KEs.

Low

No or contradictory experimental evidence of the essentiality of any of the KEs.

MIE:

(Decrease, Coupling of OXPHOS)

Essentiality of the MIE is high.

Rationale: There is direct evidence from several specifically designed studies showing that removal of an uncoupler from exposure, or addition of a “recoupler” can lead to recovery of the mitochondrial membrane potential (MMP) and total ATP caused by the uncoupler.

Evidence:

  • Removal of the classical uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) led to recovery of both MMP and ATP in rat cerebellar granule cells (Weisová 2012).
  • In the red abalone (Haliotis rufescens) larvae, removal of the uncoupler pentachlorophenol also led to recovery of the ATP level (Shofer 2002).
  • Addition of the recoupler GDP led to a rapid increase in ATP/ADP ratio in isolated guinea pig brown-adipose-tissue mitochondria where high activities of natural coupling by the UCPs were expected (Rafael 1976).
  • Addition of octanoate to 2,4-DNP exposed rat hepatocytes mitigated the uncoupling effect and partial restored the ATP/ADP ratio (Sibille 1995).
  • Removal of FCCP led to recovery from FCCP-mediated MMP and ATP reduction in Swiss mouse embryos (Zander-Fox 2015).

KE1:

(Decrease, ATP pool)

 

Essentiality of KE1 is moderate.

Rationale: There is limited direct evidence from specifically designed studies. However, multiple lines of indirect evidence show that modulation of ATP levels by uncouplers can also lead to corresponding changes in cell proliferation.

Evidence:

  • One inhibition-rescue type of study clearly showed that addition of ATP re-stimulated proliferation in human lung adenocarcinoma cells pretreated with the uncoupler emodin (Wang 2017), hence providing direct evidence to support the essentiality of this KE.
  • Positive relationships between uncoupler-mediated ATP depletion and reduced cell proliferation have been documented by multiple studies(Sweet 1999; Fine 2009; Guimarães 2012; Sugiyama 2019).

KE2:

(Decrease, Cell proliferation)

Essentiality of KE2 is moderate.

Rationale: There is no direct evidence from specifically designed studies to support this KE. However, there are multiple lines of indirect evidence showing positive relationships between cell proliferation and growth.

Evidence:

  • Indirect evidence can be obtained from a limited number of relevant studies showing a positive role of cell proliferation in mammalian tumor (Figarola 2018) zebrafish embryo growth(Bestman 2015).

Inconsistencies & uncertainties

There is currently no inconsistencies and uncertainties identified by the authors.

Weight of Evidence Summary

Biological plausibility

Support for Biological Plausibility of KERs

Defining Question

Is there a mechanistic (i.e., structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?

High

Extensive understanding based on extensive previous documentation and broad acceptance -Established mechanistic basis.

Moderate

The KER is plausible based on analogy to accepted biological relationships but scientific understanding is not completely established.

Low

There is empirical support for a statistical association between KEs, but the structural or functional relationship between them is not understood.

MIE => KE1:

(Decrease, Coupling of OXPHOS leads to Decrease, ATP pool)

Biological Plausibility of MIE => KE1 is high.

Rationale: In eukaryotic cells, the major metabolic pathways responsible for ATP production are OXPHOS, citric acid (TCA) cycle, glycolysis and photosynthesis. Oxidative phosphorylation is much (theoretically 15-18 times) more efficient than the rest due to high energy derived from oxygen during aerobic respiration (Schmidt-Rohr 2020). As the ATP level is relatively balanced between production and consumption (Bonora 2012), ATP depletion is a plausible consequence of reduced ATP synthetic efficiency following uncoupling of OXPHOS.

KE1 => KE2:

(Decrease, ATP pool leads to Decrease, Cell proliferation)

Biological Plausibility of KE1 => KE2 is high.

Rationale: Cell proliferation is a well-known ATP-dependent process. Cell division processes, such as the mitotic cell cycle uses ATP for chromosome movements and DNA replication (Kingston 1999). The synthetic processes of major cellular components that are necessary for cell structure and growth, such as proteins and lipids, also require sufficient ATP supply (Bonora 2012). Depletion of ATP therefore has a negative impact on these processes.

KE2 => AO:

(Decrease, Cell proliferation leads to Decrease, Growth)

Biological Plausibility of KE2 => AO is high.

Rationale: The biological causality between cell proliferation and growth has also been well established. It is commonly accepted that the size of an organism, organ or tissue is dependent on the total number and volume of the cells it contains, and the amount of extracellular matrix and fluids (Conlon 1999). Impairment to cell proliferation can logically affect tissue and organismal growth.

Inconsistencies & uncertainties

There is currently no inconsistencies and uncertainties identified by the authors.

 

Empirical support

Empirical Support for KERs

Defining Question

Does KEup occur at lower doses and earlier time points than KE down and at the same dose of stressor, is the incidence of KEup >than that for KEdown? Are there inconsistencies in empirical support across taxa, species and stressors that don’t align with expected pattern for hypothesized AOP?

High

Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors. (Extensive evidence for temporal, dose- response and incidence concordance) and no or few critical data gaps or conflicting data.

Moderate

Demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.

Low

Limited or no studies reporting dependent change in both events following exposure to a specific stressor (i.e., endpoints never measured in the same study or not at all); and/or significant inconsistencies in empirical support across taxa and species that don’t align with expected pattern for hypothesised AOP.

MIE => KE1:

(Decrease, Coupling of OXPHOS leads to Decrease, ATP pool)

Empirical support of MIE => KE1 is high.

Rationale: The majority of the supporting studies show good incidence, temporal and/or dose concordance in different organisms and cell types after exposure to known uncouplers, with few cases of exceptions.

  • Exposure of zebrafish (Danio rerio) embryos to 0.5 µM of the classical uncoupler 2,4-DNP led to significantly uncoupling of OXPHOS after 21h, whereas significant reduction in ATP was only observed after 45h  (Bestman 2015).
  • In human colon cancer cells (SW480), exposure to 150 µM of the uncoupler flavanoid morin caused 60% reduction in MMP, whereas only around 35% decrease in ATP (Sithara 2017).

KE1 => KE2:

(Decrease, ATP pool leads to Decrease, Cell proliferation)

Empirical support of KE1 => KE2 is moderate.

Rationale: Although only a few studies were found to be relevant, good temporal and incidence concordances were reported in zebrafish (Bestman 2015) and mammalian cells (Sithara 2017).

KE2 => AO:

(Decrease, Cell proliferation leads to Decrease, Growth)

Empirical support of KE2 => AO is low.

Rationale: This KER was included in a very limited number of studies, as it addresses effects occurring at the apical level that in vitro studies cannot cover. There is one zebrafish study reporting concordant relationship between reduced cell proliferation and embryo growth with some inconsistencies (Bestman 2015).

Inconsistencies & uncertainties

There are some inconsistencies regarding temporal and dose concordance:

  • A significant decrease followed by a significant increase of total ATP (KE1) was observed in human RD cells during a 48h exposure to the uncoupler FCCP(Kuruvilla 2003), possibly due to the enhancement of other ATP synthetic pathways (e.g., glycolysis) as a compensatory action to impaired OXPHOS (Jose 2011).
  • In zebrafish embryos exposed to 2,4-DNP, significant growth inhibition (AO) was identified after 21h, whereas non-significant reductions in ATP (KE1) and cell proliferation (KE2) were reported(Bestman 2015).
  • Chronic exposure of the plant L. minor to 3,5-DCP also led to more sensitive response of growth inhibition (AO) compared to uncoupling of OXPHOS (MIE).

Quantitative Consideration

Quantitative understanding of the KERs

High

 

Change in KEdownstream can be precisely predicted based on a relevant measure of KEupstream. Uncertainty in the quantitative prediction can be precisely estimated from the variability in the relevant measure of KEupstream. Known modulating factors and feedback/feedforward mechanisms are accounted for in the quantitative description. There is evidence that the quantitative relationship between the KEs generalises across the relevant applicability domain of the KER.

Moderate

Change in KEdownstream can be precisely predicted based on a relevant measure of KEupstream. Uncertainty in the quantitative prediction is influenced by factors other than the variability in the relevant measure of KEupstream. Quantitative description does not account for all known modulating factors and/or known feedback/feedforward mechanisms. The quantitative relationship has only been demonstrated for a subset of the overall applicability domain of the KER (e.g., based on a single species).

Low

Only a qualitative or semi-quantitative prediction of the change in KEdownstream can be determined from a measure of KEupstream. Known modulating factors and/or known feedback/feedforward mechanisms are not accounted for. The quantitative relationship has only been demonstrated for a narrow subset of the overall applicability domain of the KER (e.g., based on a single species).

MIE => KE1:

(Decrease, Coupling of OXPHOS leads to Decrease, ATP pool)

Quantitative understanding of MIE => KE1 is high.

Rationale: The theoretical quantitative relationship between OXPHOS and ATP yield has been well established. There are also published computational/mathematical models in which all possible modulation factors affecting OXPHOS and ATP synthesis are considered.

Evidence:

  • A biophysical computational model developed for mitochondrial respiration and OXPHOS (Beard 2005).
  • Continuous development of the mitochondrial energy transduction models since 1967 (Schmitz 2011).
  • A comprehensive mathematical model developed for OXPHOS and ATP production under different physiological and pathological conditions (Heiske 2017).
  • A comprehensive analysis of the quantitative relationships between protonmotive force, ATP synthase rotation, ATP synthesis and hydrolysis (Kubo 2020).
  • A regression based response-response relationship for uncoupling of OXPHOS and ATP depletion (Song 2020).

KE1 => KE2:

(Decrease, ATP pool leads to Decrease, Cell proliferation)

Quantitative understanding of KE1 => KE2 is moderate.

Rationale: The total ATP level has been used as an indicator of cell proliferation. Several studies have reported the quantitative relationships between the two events, as well as a threshold value for KE1 to trigger KE2. However, not all modulating factors have been accounted and no well-established computational/mathematical models are found.

Evidence:

  • Quantitative understanding of ATP level, cell viability and colony growth (Ahmann 1987).
  • Quantitative relationship between ATP level and cell proliferation (Crouch 1993).
  • Thresholds for ATP depletion (85-90% reduction) to determine cell cycle arrest (<85-90%) or cell death (>85-90%) (Nieminen 1994).

KE2 => AO:

(Decrease, Cell proliferation leads to Decrease, Growth)

Quantitative understanding of KE2 => AO is moderate.

Rationale: Multiple mathematical models describing the quantitative relationships between cell proliferation and tissue growth exist for both animals (Binder 2008) and plants (Mosca 2018). There are also numerous models that are specifically developed for predicting tumor growth based on the proliferation rate (Jarrett 2018). However, there is currently a lack of quantitative model to link cell proliferation and individual growth in the presence of uncouplers.

Evidence:

  • A mathematical model developed for describing the quantitative relationship between cell proliferation and tissue growth (Binder 2008).
  • A mathematical model developed for cell division and plant tissue growth (Mosca 2018).
  • Multiple mathematical models developed for cell proliferation and tumor growth (Jarrett 2018).

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Sweet S, Singh G. 1999. Changes in mitochondrial mass, membrane potential, and cellular adenosine triphosphate content during the cell cycle of human leukemic (HL-60) cells. Journal of Cellular Physiology 180:91-96. DOI: https://doi.org/10.1002/(SICI)1097-4652(199907)180:1<91::AID-JCP10>3.0.CO;2-6.

Terada H. 1990. Uncouplers of oxidative phosphorylation. Environ Health Perspect 87:213-218. DOI: 10.1289/ehp.9087213.

Troger F, Delp J, Funke M, van der Stel W, Colas C, Leist M, van de Water B, Ecker GF. 2020. Identification of mitochondrial toxicants by combined in silico and in vitro studies – A structure-based view on the adverse outcome pathway. Computational Toxicology 14:100123. DOI: https://doi.org/10.1016/j.comtox.2020.100123.

Wang X, Li L, Guan R, Zhu D, Song N, Shen L. 2017. Emodin inhibits ATP-induced proliferation and migration by suppressing P2Y receptors in human lung adenocarcinoma cells. Cellular Physiology and Biochemistry 44:1337-1351. DOI: 10.1159/000485495.

Weisová P, Anilkumar U, Ryan C, Concannon CG, Prehn JHM, Ward MW. 2012. ‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817:744-753. DOI: https://doi.org/10.1016/j.bbabio.2012.01.016.

Xia M, Huang R, Shi Q, Boyd WA, Zhao J, Sun N, Rice JR, Dunlap PE, Hackstadt AJ, Bridge MF, Smith MV, Dai S, Zheng W, Chu PH, Gerhold D, Witt KL, DeVito M, Freedman JH, Austin CP, Houck KA, Thomas RS, Paules RS, Tice RR, Simeonov A. 2018. Comprehensive analyses and prioritization of Tox21 10K chemicals affecting mitochondrial function by in-depth mechanistic studies. Environ Health Perspect 126:077010. DOI: 10.1289/EHP2589.

Yun MH. 2015. Changes in regenerative capacity through lifespan. International Journal of Molecular Sciences 16:25392-25432.

Zander-Fox DL, Fullston T, McPherson NO, Sandeman L, Kang WX, Good SB, Spillane M, Lane M. 2015. Reduction of mitochondrial function by FCCP during mouse cleavage stage embryo culture reduces birth weight and impairs the netabolic health of offspring1. Biology of Reproduction 92. DOI: 10.1095/biolreprod.114.123489.


Appendix 1

List of MIEs in this AOP

Event: 1446: Decrease, Coupling of oxidative phosphorylation

Short Name: Decrease, Coupling of OXPHOS

Key Event Component

Process Object Action
oxidative phosphorylation uncoupler activity increased

Stressors

Name
2,4-Dinitrophenol
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
Carbonyl cyanide m-chlorophenyl hydrazone
Pentachlorophenol
Triclosan
Emodin
Malonoben

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
cell

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

Decreased coupling of oxidative phosphorylation can be directly triggered by “uncouplers” as a molecular initiating event. Most of the chemical uncouplers are protonophores, a type of proton binders that can translocate protons across membranes. These protonophores several common structural characteristics, such as bulky hydrophobic moiety, an acid dissociable group and a strong electron-withdrawing group (Terada 1990). Weak acids such as phenols, benzimidazoles and salicylic acids are considered potential protonophores. Classical uncouplers, such as carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP), pentachlorophenol (PCP) and SF-6847 (Terada 1990), as well as new uncouplers, such as triclosan (Shim 2016; Weatherly 2016), emodin (Sugiyama 2019), and hydroxylated polybrominated diphenyl ethers (PBDEs) (Legradi 2014) have been widely investigated in vertebrates. Computational predictions based on quantitative structure-activity relationships (Russom 1997; Schultz 1997; Naven 2012; Dreier 2019; Troger 2020) and in vitro high-throughput screening (Escher 2002; Attene-Ramos 2013; Attene-Ramos 2015; Xia 2018) have facilitated the identification and classification of potential uncouplers from a massive list of chemicals.   



Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio High NCBI
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI
Lemna minor Lemna minor High NCBI
Life Stage Applicability
Life Stage Evidence
Embryo High
Juvenile High
Adult, reproductively mature Moderate
Sex Applicability
Sex Evidence
Unspecific High

Taxonomic applicability domain

This key event is in general considered applicable to most eukaryotes, as the mitochondrion and oxidative phosphorylation are highly conserved (Roger 2017).

 

Life stage applicability domain

This key event is considered applicable to all life stages, as ATP synthesis by oxidative phosphorylation is an essential biological process for most living organisms.

 

Sex applicability domain

This key event is considered sex-unspecific, as both males and females use oxidative phosphorylation as a main process to generate ATP.


Key Event Description

Decreased coupling of oxidative phosphorylation (OXPHOS), or uncoupling of OXPHOS, describes dissipation of protonmotive force (PMF) across the inner mitochondrial membrane (IMM) by environmental stressors. In eukaryotes, the mitochondrial electron transport chain mediates a series of redox reactions to create a PMF across the IMM. The PMF is used as energy to drive adenosine triphosphate (ATP) synthesis through phosphorylation of adenosine diphosphate (ADP). These processes are coupled and referred to as OXPHOS. A number of chemicals can dissipate the PMF, leading to uncoupling of OXPHOS. This key event describes the main outcome of the interactions between an uncoupler and the transmembrane PMF. An uncoupler can bind to a proton in the mitochondrial inter membrane space, transport the proton to the matrix side of the IMM, release the proton and move back to the inter membrane space. These processes are repeated until the transmembrane PMF is dissipated. This KE is therefore a lumped term of these processes and represents the final consequence of the interactions.


How it is Measured or Detected

Uncoupling of oxidative phosphorylation can be indicated by reduced mitochondrial membrane potential, increased proton leak and/or increased oxygen consumption rate. Mitochondrial membrane potential can be measured using fluorescent probes such as TMRM (tetramethylrhodamine, methyl ester, perchlorate), TMRE (tetramethylrhodamine, ethyl ester, perchlorate) and JC-1 (Perry 2011). Proton leak and oxygen consumption rate can be measured using a high-resolution respirometry (Affourtit 2018) or a Seahorse XF analyzer (Divakaruni 2014).


References

Affourtit C, Wong H-S, Brand MD. 2018. Measurement of proton leak in isolated mitochondria. In Palmeira CM, Moreno AJ, eds, Mitochondrial Bioenergetics: Methods and Protocols. Springer New York, New York, NY, pp 157-170.

Attene-Ramos MS, Huang R, Sakamuru S, Witt KL, Beeson GC, Shou L, Schnellmann RG, Beeson CC, Tice RR, Austin CP, Xia M. 2013. Systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening. Chemical Research in Toxicology 26:1323-1332. DOI: 10.1021/tx4001754.

Attene-Ramos MS, Huang RL, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia MH. 2015. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Persp 123:49-56. DOI: 10.1289/ehp.1408642.

Divakaruni AS, Paradyse A, Ferrick DA, Murphy AN, Jastroch M. 2014. Chapter Sixteen - Analysis and Interpretation of Microplate-Based Oxygen Consumption and pH Data. In Murphy AN, Chan DC, eds, Methods in Enzymology. Vol 547. Academic Press, pp 309-354.

Dreier DA, Denslow ND, Martyniuk CJ. 2019. Computational in vitro toxicology uncovers chemical structures impairing mitochondrial membrane potential. J Chem Inf Model 59:702-712. DOI: 10.1021/acs.jcim.8b00433.

Escher BI, Schwarzenbach RP. 2002. Mechanistic studies on baseline toxicity and uncoupling of organic compounds as a basis for modeling effective membrane concentrations in aquatic organisms. Aquatic Sciences 64:20-35. DOI: 10.1007/s00027-002-8052-2.

Legradi J, Dahlberg A-K, Cenijn P, Marsh G, Asplund L, Bergman Å, Legler J. 2014. Disruption of Oxidative Phosphorylation (OXPHOS) by Hydroxylated Polybrominated Diphenyl Ethers (OH-PBDEs) Present in the Marine Environment. Environmental Science & Technology 48:14703-14711. DOI: 10.1021/es5039744.

Naven RT, Swiss R, Klug-Mcleod J, Will Y, Greene N. 2012. The development of structure-activity relationships for mitochondrial dysfunction: Uncoupling of oxidative phosphorylation. Toxicol Sci 131:271-278. DOI: 10.1093/toxsci/kfs279.

Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA. 2011. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. BioTechniques 50:98-115. DOI: 10.2144/000113610.

Roger AJ, Munoz-Gomez SA, Kamikawa R. 2017. The origin and diversification of mitochondria. Curr Biol 27:R1177-R1192. DOI: 10.1016/j.cub.2017.09.015.

Russom CL, Bradbury SP, Broderius SJ, Hammermeister DE, Drummond RA. 1997. Predicting modes of toxic action from chemical structure: Acute toxicity in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 16:948-967. DOI: https://doi.org/10.1002/etc.5620160514.

Schultz TW, Cronin MTD. 1997. Quantitative structure-activity relationships for weak acid respiratory uncouplers to Vibrio fisheri. Environ Toxicol Chem 16:357-360. DOI: https://doi.org/10.1002/etc.5620160235.

Shim J, Weatherly LM, Luc RH, Dorman MT, Neilson A, Ng R, Kim CH, Millard PJ, Gosse JA. 2016. Triclosan is a mitochondrial uncoupler in live zebrafish. J Appl Toxicol 36:1662-1667. DOI: 10.1002/jat.3311.

Sugiyama Y, Shudo T, Hosokawa S, Watanabe A, Nakano M, Kakizuka A. 2019. Emodin, as a mitochondrial uncoupler, induces strong decreases in adenosine triphosphate (ATP) levels and proliferation of B16F10 cells, owing to their poor glycolytic reserve. Genes to Cells 24:569-584. DOI: https://doi.org/10.1111/gtc.12712.

Terada H. 1990. Uncouplers of oxidative phosphorylation. Environ Health Perspect 87:213-218. DOI: 10.1289/ehp.9087213.

Troger F, Delp J, Funke M, van der Stel W, Colas C, Leist M, van de Water B, Ecker GF. 2020. Identification of mitochondrial toxicants by combined in silico and in vitro studies – A structure-based view on the adverse outcome pathway. Computational Toxicology 14:100123. DOI: https://doi.org/10.1016/j.comtox.2020.100123.

Weatherly LM, Shim J, Hashmi HN, Kennedy RH, Hess ST, Gosse JA. 2016. Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes. Journal of Applied Toxicology 36:777-789. DOI: https://doi.org/10.1002/jat.3209.

Xia M, Huang R, Shi Q, Boyd WA, Zhao J, Sun N, Rice JR, Dunlap PE, Hackstadt AJ, Bridge MF, Smith MV, Dai S, Zheng W, Chu PH, Gerhold D, Witt KL, DeVito M, Freedman JH, Austin CP, Houck KA, Thomas RS, Paules RS, Tice RR, Simeonov A. 2018. Comprehensive analyses and prioritization of Tox21 10K chemicals affecting mitochondrial function by in-depth mechanistic studies. Environ Health Perspect 126:077010. DOI: 10.1289/EHP2589.


List of Key Events in the AOP

Key Event Component

Process Object Action
ATP biosynthetic process ATP decreased

Stressors

Name
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
Carbonyl cyanide m-chlorophenyl hydrazone
2,4-Dinitrophenol
Malonoben
Pentachlorophenol
Triclosan
Emodin

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio High NCBI
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
Life Stage Applicability
Life Stage Evidence
Embryo High
Juvenile High
Adult, reproductively mature Moderate
Sex Applicability
Sex Evidence
Unspecific High

Taxonomic applicability domain

This key event is in general considered applicable to all eukaryotes utilizing ATP as a direct source of energy and signaling molecule.

 

Life stage applicability domain

This key event is considered applicable to all life stages, as all developmental stages require energy supply to maintain necessary physiological processes.

 

Sex applicability domain

This key event is considered sex-unspecific, as both males and females use ATP as an essential energy molecule.


Key Event Description

Decreased adenosine triphosphate (ATP) pool describes the loss of balance between ATP synthesis and ATP consumption, leading to reduced total ATP. As a primary form of biological energy, ATP is used by many biological processes (Bonora 2012). Decrease in ATP level normally attributes to metabolic disorders in major ATP synthetic pathways, such as mitochondrial oxidative phosphorylation, fatty acid β-oxidation, glycolysis and plant photophosphorylation.


How it is Measured or Detected

The ATP pool in cells or tissue can be quantified using a well-established ATP bioluminescent assay (Lemasters 1978; Wibom 1990). Assay principles: ATP can react with luciferase and luciferin from firefly and the luminescence emitted from the reaction is proportional to the ATP concentration:

ATP + D-Luciferin + O2 è Oxyluciferin + AMP + PPi + CO2 + Light


References

Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F, Wieckowski MR, Pinton P. 2012. ATP synthesis and storage. Purinergic Signalling 8:343-357. DOI: 10.1007/s11302-012-9305-8.

Lemasters JJ, Hackenbrock CR. 1978. [4] Firefly luciferase assay for ATP production by mitochondria. Methods in Enzymology. Vol 57. Academic Press, pp 36-50.

Wibom R, Lundin A, Hultman E. 1990. A sensitive method for measuring ATP-formation in rat muscle mitochondria. Scandinavian Journal of Clinical and Laboratory Investigation 50:143-152. DOI: 10.1080/00365519009089146.


Event: 1821: Decrease, Cell proliferation

Short Name: Decrease, Cell proliferation

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:263 - Uncoupling of oxidative phosphorylation leading to growth inhibition (1) KeyEvent

Stressors

Name
2,4-Dinitrophenol
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
Carbonyl cyanide m-chlorophenyl hydrazone
Pentachlorophenol
Triclosan
Emodin
Malonoben

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
cell

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
zebrafish Danio rerio High NCBI
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
Life Stage Applicability
Life Stage Evidence
Embryo High
Juvenile High
Sex Applicability
Sex Evidence
Unspecific High

Taxonomic applicability domain

This key event is in general applicable to all eukaryotes.

 

Life stage applicability domain

This key event is in general applicable to all life stages.

 

Sex applicability domain

This key event is sex-unspecific.


Key Event Description

Decreased cell proliferation describes the outcome of reduced cell division and cell growth. Cell proliferation is considered the main mechanism of tissue and organismal growth (Conlon 1999). Decreased cell proliferation has been associated with abnormal growth-factor signaling and cellular energy depletion (DeBerardinis 2008).


How it is Measured or Detected

Cell proliferation can be measured using the well-established 5-bromo-2'-deoxyuridine (BrdU) (Raza 1985; Muir 1990) or 5-ethynyl-2´-deoxyuridine (EdU) assay. Both methods measure DNA synthesis in dividing cells to indicate proliferation status. The assay kits are commercially available and have high-throughput formats.


References

Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.

DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. 2008. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metabolism 7:11-20. DOI: https://doi.org/10.1016/j.cmet.2007.10.002.

Muir D, Varon S, Manthorpe M. 1990. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. Analytical Biochemistry 185:377-382. DOI: https://doi.org/10.1016/0003-2697(90)90310-6.

Raza A, Spiridonidis C, Ucar K, Mayers G, Bankert R, Preisler HD. 1985. Double labeling of S-phase murine cells with bromodeoxyuridine and a second DNA-specific probe. Cancer Research 45:2283-2287.


List of Adverse Outcomes in this AOP

Event: 1521: Decrease, Growth

Short Name: Decrease, Growth

Stressors

Name
2,4-Dinitrophenol
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
Carbonyl cyanide m-chlorophenyl hydrazone
Pentachlorophenol
Triclosan
Emodin
Malonoben

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Moderate NCBI
rat Rattus norvegicus Moderate NCBI
mouse Mus musculus Moderate NCBI
zebrafish Danio rerio High NCBI
fathead minnow Pimephales promelas High NCBI
Lemna minor Lemna minor High NCBI
Daphnia magna Daphnia magna Moderate NCBI
Life Stage Applicability
Life Stage Evidence
Embryo High
Juvenile High
Sex Applicability
Sex Evidence
Unspecific High

Taxonomic applicability domain

This key event is in general applicable to all eukaryotes.

 

Life stage applicability domain

This key event is applicable to early life stages such as embryo and juvenile.

 

Sex applicability domain

This key event is sex-unspecific.


Key Event Description

Decreased growth refers to a reduction in size and/or weight of a tissue, organ or individual organism. Growth is normally controlled by growth factors and mainly achieved through cell proliferation (Conlon 1999).


How it is Measured or Detected

Growth can be indicated by measuring weight, length, total volume, and/or total area of a tissue, organ or individual organism.  


Regulatory Significance of the AO

Growth is a regulatory relevant chronic toxicity endpoint for almost all organisms. Multiple OECD test guidelines have included growth either as a main endpoint of concern, or as an additional endpoint to be considered in the toxicity assessments. Relevant test guidelines include, but not only limited to:

 

-Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test

-Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages

-Test No. 215: Fish, Juvenile Growth Test

-Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA)

-Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test

-Test No. 221: Lemna sp. Growth Inhibition Test

-Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies (Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae))

-Test No. 211: Daphnia magna Reproduction Test


References

Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.


Appendix 2

List of Key Event Relationships in the AOP