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Aop: 263

AOP Title

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Short name:

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Uncoupling of OXPHOS leading to growth inhibition (1)

Graphical Representation

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Click to download graphical representation template

Authors

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You Song^a and Daniel L. Villeneuve^b

^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).

Point of Contact

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You Song   (email point of contact)

Contributors

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• You Song
• Dan Villeneuve

Status

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This AOP was last modified on November 29, 2020 17:41

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Revision dates for related pages

Page	Revision Date/Time
Decrease, Coupling of oxidative phosphorylation	November 27, 2020 14:49
Decrease, Growth	November 28, 2020 15:07
Decrease, Adenosine triphosphate pool	November 29, 2020 16:12
Decrease, Cell proliferation	November 28, 2020 14:39
Decrease, Coupling of OXPHOS leads to Decrease, ATP pool	November 28, 2020 16:28
Decrease, ATP pool leads to Decrease, Cell proliferation	November 28, 2020 17:09
Decrease, Cell proliferation leads to Decrease, Growth	November 28, 2020 17:05
2,4-Dinitrophenol	November 29, 2016 18:42
Pentachlorophenol	November 12, 2020 17:59
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone	November 12, 2020 17:59
Carbonyl cyanide m-chlorophenyl hydrazone	November 12, 2020 17:59
Triclosan	November 12, 2020 18:00
Dinoseb	November 12, 2020 18:00
3,5-Dichlorophenol	October 10, 2017 07:47
Emodin	November 20, 2020 13:48

Abstract

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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 (optional)

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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

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Events: Molecular Initiating Events (MIE)

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Key Events (KE)

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Adverse Outcomes (AO)

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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

Relationships Between Two Key Events
(Including MIEs and AOs)

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Title	Adjacency	Evidence	Quantitative Understanding

Decrease, Coupling of OXPHOS leads to Decrease, ATP pool	adjacent	High	High
Decrease, ATP pool leads to Decrease, Cell proliferation	adjacent	Moderate	Moderate
Decrease, Cell proliferation leads to Decrease, Growth	adjacent	Moderate	Moderate

Network View

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Stressors

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Life Stage Applicability

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Taxonomic Applicability

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Sex Applicability

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Overall Assessment of the AOP

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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

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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

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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.

Evidence Assessment

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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 Understanding

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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).

Considerations for Potential Applications of the AOP (optional)

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References

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