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Relationship: 3637

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Inhibition, Mitochondrial complex III leads to Mitochondrial dysfunction

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Inhibition, Mitochondrial complex III

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Mitochondrial dysfunction

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Inhibition of the mitochondrial complex III of nigro-striatal neurons leads to parkinsonian motor deficits	adjacent			Barbara Viviani (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	High	NCBI
rat	Rattus norvegicus	High	NCBI
mice	Mus sp.	High	NCBI
Dictyostelium discoideum	Dictyostelium discoideum	High	NCBI
Honey bee	Honey bee	High	NCBI
earthworms	earthworms	High	NCBI
zebrafish	Danio rerio	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	High
Female	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Mitochondrial complex III, also known as ubiquinol-cytochrome c reductase complex or cytochrome bc1 complex, is an essential component of the electron transport chain (ETC). It couples i) the transfer of electrons from reduced coenzyme Q (ubiquinol) to cytochrome c to ii) the pumping of protons from the mitochondrial matrix to the intermembrane space, thus contributing to the proton gradient. In turn, the proton gradient across the inner mitochondrial membrane drives ATP production through ATP synthase (complex V) (Crofts, 2004; Crofts, 2021). Reduced activity of complex III is casually and directly linked to impairment of mitochondrial functions such as, oxidative phosphorylation (i.e. ATP synthesis coupled to respiration), alteration of the mitochondrial membrane potential, generation of reactive oxygen species, alteration of metabolic pathways linked to ETC, e.g. Krebs cycle (Rugolo et al, 2021).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The implementation of AOP3 is based on a negotiated procedure with EFSA (reference NP/EFSA/PREV/2024/02). This procedure is intended to update AOP3 by adding more evidence to the AOP Wiki, considering the contribution of mitochondrial complex III inhibition to degeneration of dopaminergic neurons and occurrence of parkinsonian motor deficits. The starting conceptual model for this project is based on the key scientific sources, including EFSA (2017), Delp et al. (2019 and 2021), Van der Stel et al. (2020 and 2022), ENV/JM/MONO(2020)22. These publications provided the initial basis for this project and contributed to the Empirical Evidence.

Additional literature was identified through a structured, non-systematic search using a stressor-based search strategy as described in the “AOP development strategy” section.

The relationship between inhibition or deficiency of CIII and mitochondrial dysfunction is a well-established KER. Thus, evidence to support biological plausibility was retrieved from seminal publications recommended by domain experts and supplemented by expert knowledge.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

The weight of evidence supporting the relationship between inhibition or deficiency of CIII and mitochondrial dysfunction is strong. The mechanisms behind this KER have been elucidated by using chemical (Georgakopoulos et al, 2017) and genetic approaches (Čunátová & Fernández-Vizarra, 2024)

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

There is strong biological plausibility that inhibiting cIII activity triggers mitochondrial dysfunction. Inhibition or deficiency of CIII function may lead to (Rugolo et al, 2021):

- Decreased proton gradient, which affects membrane potential and ATP production by the ATP synthase. At the cellular level, reduced ATP production by mitochondria due to CIII inhibition or deficiency may be partially compensated by other means, i.e. increasing glycolysis.

- Inhibition of electron flow from CI and CII to ubiquinone, resulting in compromised NADH reoxidation at CI and inhibition of the TCA cycle. Inhibition of TCA may dampen anaplerosis. Reduced regeneration of NAD+ would affect many NAD+-dependent processes, such as redox reactions, protein ADP-ribosylation, and sirtuin-mediated protein deacetylation.

- Increased or decreased production of reactive oxygen species (ROS) via electron leak or reverse electron transfer due to an overreduced CoQ pool, which may cause oxidative damage to the electron transport system and other factors (proteins, lipids, nucleic acids), or alter cellular redox balance and compromise ROS signaling, respectively. Altered ROS production may be compensated by modulating multiple cellular ROS detoxification and defense mechanisms (see “Known modulating factors” section).

Human mutations and proof of concept in cellular and animal models.

The biological plausibility that inhibition of CIII leads to mitochondrial dysfunction can also be inferred from the severe phenotype associated with genetic alterations compromising complex III functionality. Diseases caused by mutations in genes encoding CIII subunits or its assembly factors are collectively called CIII deficiencies. Mutations at CIII subunits are very rare and associated with severe phenotypes (Banerjee et al., 2022). For example, a two-exon deletion in the human UQCRH gene (Ubiquinol-Cytochrome C Reductase Hinge Protein) has been identified as the cause of a rare familial mitochondrial disorder (Vidali et al., 2021). Although this gene is widely expressed in different tissue of a given organism, its function seems to be particularly important for organs with high-energy metabolism. Deletion of the corresponding gene in the mouse (Uqcrh-KO) resulted in striking biochemical and clinical similarities including impairment of CIII, failure to thrive, elevated blood glucose levels, and early death (Vidali et al., 2021). The following table provides examples of mutations that have been described in PD patients or that lead to motor impairment. Further details, including any uncertainties and inconsistencies, can be found in the overall assessment of AOP 587.

Mutation	Protein/function	Impact	Reference
Loss of function in Ttc19*	TTC19 is involved in the removal of N-terminal proteolytic fragments of the Rieske protein. It allows the physiological turnover of the Rieske protein and the preservation of complex III function.	Decreased cIII activity and increased ROS production in brain, liver and skeletal muscle from Ttc19 -/- mice (3 and 6 months of age).	Bottani et al. 2017
Four-base-pair deletion in the mitochondrial gene that encodes cytochrome b described in patients with early-onset parkinsonism	electron transport	High levels of this mutation in a cell line homoplastic for the patient’s wild-type mtDNA were associated with a marked defect in the assembly and function of complex III with the Rieske protein and subunit VI, reduced UQCRC1 levels and increased ROS formation	De Coo et al., 1999 Rana et al., 2000
UQCRC1° p.Tyr314ser or p.lle311Leu coding substitutions, low frequency mutation co-segregate with autosomal dominant Parkinsonism and neuropathy	Subunit required for oxidative phosphorylation and ATP production	mutant UQCRC1 knock-in SH-SY5Y cell lines show reduced oxygen consumption of cIII activity (no effect cI, cII or cIV activity) and ATP production, and increased ROS production	Lin et al. 2020

*TTC19: tetra-tricopep-repeat domain 19

° UQCRC1: ubiquinol-cytochrome c reductase core protein 1

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

The empirical evidence is based on Delp 2019, Delp 2021, Bennekou (2020), van der Stel, W. (2021) and Van der Stel et al., 2022.

Data were retrieved from assays measuring endpoint(s) relevant for AOP3 in relevant cell types.

KE1542Complex III inhibition.

Assays

For KE1542, only two assays were employed in the abovementioned publications, both using the Seahorse technology.

• The “LUHMES MitoComplexes” assay (Delp 2019, Delp 2021) used proliferating LUHMES cells.

• The “HepG2 MitoComplexes” assay (van der Stel 2020) uses HepG2 cells.

In both assays, the cells are permeabilized and treated acutely with the test chemical. Oxygen consumption rate (OCR) is then measured under different conditions of active or blocked mitochondrial complexes. Of note, the two assays are setup slightly differently, so that in the LUHMES assay activity on c1 – c4 can be distinguished separately, while in the HepG2 assay, activity on c2 and c3 is measured concurrently.

Cell Models

Despite HepG2 being a hepatic cell line, the assay was included in the evaluation. It was assumed that because the exposure is acute, in permeabilized cells, that the test chemical would have immediate access to the mitochondria. Other mechanisms such as transport into the cells or an indirect effect via other signaling pathways were considered negligible under these assay conditions.

Effect thresholds

The “LUHMES MitoComplexes” assay has an established prediction model (Delp 2019); a chemical is considered active, if OCR is reduced by more than 25% compared to the solvent control.

For the “HepG2 MitoComplexes”, no prediction model has been established. But van der Stel 2020 reported EC50 values, which was used also in the current evaluation.

Table 2. KE1542: criteria for data analysis

LUHMES	HepG2	Conclusion
Active* on complex cIII	Active* on complex cIII	strong evidence for KE1542
Active* on complex cIII	not measured	evidence for KE1542, with uncertainty.
Active* on complex cIII	inactive or active* on complex other than cIII	evidence for KE1542, with uncertainty. Unless a rationale why there is cell-type specific effects exist
Inactive*	Active* on complex cIII	Additional evidence needed (update according to OC/EFSA/PREV/2023/01#)
Inactive*	not measured	Excluded from the analysis
not measured	Active* on complex cIII	low evidence for KE1542. Additional evidence needed (update according to OC/EFSA/PREV/2023/01#)
not measured	Inactive*	Excluded from the analysis

*based on Effect threshold, # Environmental Neurotoxicants – Advancing Understanding on the Impact of Chemical Exposure on Brain Health and Disease: Parkinsonian NeurodegenerationRapid Assessment using NAMs (PANDORA)

KE177 Mitochondrial dysfunction

Assays

The following assay endpoints were included:

1. Oxygen consumption rate (in intact cells). This was considered the most reliable method to measure a direct effect on mitochondrial respiration.

2. Image-based measurement of mitochondrial membrane potential (MMP). These methods use dyes such as TMRE or rhodamine123.

3. Measurement of ATP levels. This was considered a more indirect measurement of mitochondrial function, as many cell types can generate ATP via glycolysis, which is independent of mitochondrial function.

The following assay endpoints were deemed not suitable for KE177:

• Resazurin: Indirect measure for KE177, the bioreduction of the dye depends on different sources among which mitochondria

• Lactate dehydrogenase release: Indirect measure for KE177, linked to cell viability

• Extracellular acidification rate: acidification/elevation of glycolysis can also be the consequence of an elevated energy demand that can no longer be met by mitochondria. Under these conditions, an elevation of acidification does not correlate with dysfunction of mitochondria

Cell Models

Only neuronal cell models were considered, which included the LUHMES and the SH-SY5Y cells, as relevant for AOP3.

Stressors identification.

Chemicals listed in EFSA 2017, Delp 2019 and Delp 2021 were considered for inclusion. In these publications, 46 of 61 chemicals were referred to or measured to be a MRC inhibitor. For these chemicals data were extracted from Delp et al. (2019, 2021), Bennekou- ENV/JM/MONO(2020)23, van Der Stel et al. (2020), Tebby et al. (2022). Data were carefully evaluated by subject-experts to determine whether the chemical affects KE1542, KE177, according to the criteria described in the previous section, and KE890. 4 cIII inhibitors with evidence of interference with KE1542, KE177 and KE890 were included as stressors (Table 3) in the assessment and subsequently described. An overview of these data across AOPs and KEs, summarising the percentage effect on each KE, is presented in the “Evidence assessment” and "Quantitative understanding" sections of AOP 587 (cIII inhibitors).

Table 3. cIII inhibitors

	KE1542	KE177	KE890	Conclusion
Antimycin A	Yes	Yes	Yes	Included
Azoxystrobin	Yes	Yes	Borderline	Included
Picoxystrobin	Yes	Yes	Yes	Included
Pyraclostrobin	Yes	Yes	Yes	Included

Antimycin A

KE1542: Three studies conducted in two cell lines (HepG2, LUHMES) measured inhibition of c3 immediately after treatment of cells with antimycin A. Antimycin A was tested in proliferating LUHMES cells at a single concentration of 50 µM (Delp 2019, 2021). In HepG2 cells, antimycin was tested in a range of concentrations, with an EC50 of 0,019 µM (van der Stel 2020). Of note, all experiments were conducted in permeabilized cells.

KE177: In intact LUHMES cells, 50 µM antimycin A lead to an immediate reduction in OCR by about 50% (relative to the solvent control) (Delp 2021, Bennekou 2020), whereby the number of independent biological replicates in each study was unclear. After 24 h of exposure, ATP content was affected at 25 µM in differentiating LUHMES (Day of Differentiation: DoD3) (Delp 2021). Differentiated SH-SY5Y cells were sensitive to antimycin A as measured by the mitochondrial membrane potential via rhodamine123 starting from 0,063 µM when treated for 24 h (Delp 2021, Bennekou 2020). ATP levels were decreased at 1 µM when cells were treated for 120 h (Bennekou 2020).

Additional studies retrieved from the literature search reported the activation of the KER3637 in synaptosomes obtained from brain of female Wistar rats. The inhibition of complex III activity by over than 70% with antimycin A 20 nM was necessary to induce a change in MMP within minutes (Killbride et al. 2021)

Data from different laboratories using SHSY5Y cells demonstrate concordance, exhibiting an impact on the KER between 50 and 80 nM according to the endpoint measured after an exposure of 24h (Bennekou et al 2020; Delp 2021; Bir et al. 2014).

KE	cell type	assay	exposure	effect level	effect conc	reference	notes
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2019, Figure 7 + Suppl. Item 5	1
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2021, Figure S8	1
KE 1542	HepG2	Mito Complexes, inhibition of c1-c3	immediate	EC50	0,019 µM	van der Stel 2020, Figure 4 + Table 2	1
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	50 µM (single conc)	Delp_2021, Figure 8ABC	2
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	50 µM (single conc)	ENV/JM/MONO(2020)23 Bennekou, Fig 5.4 / Annex 1.2	3
KE 177	LUHMES DoD3	ATP content	DoD2 – DoD3 (24h)	EC25	25 µM	Delp_2021, Fig S7 + Figure 4
KE 177	SH-SY5Y DoD7	MMP (rhodamine123)	DoD6 – DoD7 (24 h)	EC25 EC50	0,063 µM 0,079 µM	Delp_2021, Figure 7 ENV/JM/MONO(2020)23 Bennekou Fig 5.6 / Annex 1.6
KE 177	SH-SY5Y DoD8	ATP content	DoD3+DoD6 until DoD8 (120 h)	EC50	1 µM	Bennekou, Fig 5.11 / Annex 1.14
Additional literature data
KE 1542	SynaptosomeFemale Wistar rat	Oxydation of decylubiquinone	7-8 min	80%	20 nM	Killbride et al. 2021 doi: 10.1007/s11064-020-02990-8	n=9
KE 177	SH-SY5Y DoD na	MMP (JC1) Sodium pyruvate	24h	30%	50 nM	Bir et al. 2014 doi: 10.1111/jnc.12966 Fig. 7.a	n=6
KE 177	SH-SY5Y DoD na	ATP content Sodium pyruvate	24h	35%	50 nM	Bir et al. 2014 doi: 10.1111/jnc.12966 Fig. 7.a	n=6
KE 177	SynaptosomeFemale Wistar rat	MMP (JC1)	10 min	60%	20 nM	Killbride et al. 2021 doi: 10.1007/s11064-020-02990-8	n=9

¹Permeabilized cells.

²Only n=2

³No indication on the number of biological replicates.

Azoxystrobin

KE1542: Three studies conducted in two cell lines (HepG2, LUHMES) measured inhibition of c3 immediately after treatment of cells with azoxystrobin. Effective concentrations were in the range of 17 – 50 µM. Of note, these experiments were conducted in permeabilized cells.

KE177: In intact LUHMES cells, 50 µM azoxystrobin lead to an immediate reduction in OCR by about 50% (relative to the solvent control) (Delp 2021, Bennekou 2020), whereby the number of independent biological replicates in each study was unclear. However, 50 µM azoxystrobin did not affect ATP content after 24 h of treatment (Delp 2021). In differentiated SH-SY5Y cells, treatment with azoxystrobin impacted the mitochondrial potential, as measured with the rhodamine123 dye (EC25 = 3 µM) (Delp 2021, Bennokou 2020 ). When exposed for 120 h, ATP levels were decreased by about 50% at 10 µM (Bennekou 2020).

KE	cell type	assay	exposure	effect level	effect conc	reference	notes
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2019, Suppl. Item 5	1
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2021, Figure S8	1
KE 1542	HepG2	Mito Complexes, inhibition of c1-c3	immediate	EC50	17µM	van der Stel 2020, Figure 4 + Table 2	1
KE 1542	HepG2	Mito Complexes, inhibition of c4	immediate	50%	>100 µM (borderline)	van der Stel 2020, Figure 4 + Table 2	1
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	50 µM (single conc)	Delp_2021,Figure 8ABC	2
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	50 µM (single conc) partial effect	ENV/JM/MONO(2020)23 Bennekou, Fig 5.4 / Annex 1.2	3
KE 177	LUHMES DoD3	ATP content	DoD2 – DoD3 (24h)	EC25	>50 µM	Delp_2021, Fig S7 + Figure 4
KE 177	SH-SY5Y DoD7	MMP (rhodamine123)	DoD6 – DoD7 (24 h)	EC25	3 µM	Delp_2021, Figure 7 ENV/JM/MONO(2020)23 Bennekou, Fig 5.6 / Annex 1.6
KE 177	SH-SY5Y DoD8	ATP content	DoD3+DoD6 until DoD8 (120 h)	EC50	10 µM (borderline)	Bennekou, Fig 5.11

¹Permeabilized cells.

²Only n=2

³No indication on the number of biological replicates.

Picoxystrobin

KE1542: Three studies conducted in two cell lines (HepG2, LUHMES) measured inhibition of c3 immediately after treatment of cells with picoxystrobin. Effective concentrations were in the range of 4– 50 µM. Of note, these experiments were conducted in permeabilized cells.

KE177: In intact LUHMES cells, 50 µM picoxystrobin lead to an immediate reduction in OCR by about 80% (relative to the solvent control) (Delp 2021, Bennekou 2020), whereby the number of independent biological replicates in each study was unclear. ATP content was only borderline affected after treatment for 24 h with 50 µM picoxystrobin (Delp 2021).

In differentiated SH-SY5Y cells, treatment with picoxystrobin impacted the mitochondrial potential, as measured with the rhodamine123 dye (EC25 = 2 µM) (Delp 2021, Bennekou 2020). When exposed for 120 h, ATP levels were decreased by about 50% at 5 µM (Bennekou 2020).

KE	cell type	assay	exposure	effect level	effect conc	reference	notes
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2019, Suppl. Item 5	1
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2021, Figure S8	1
KE 1542	HepG2	Mito Complexes, inhibition of c1-c3	immediate	EC50	0,4 µM	van der Stel 2020, Figure 4 + Table 2	1
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	20 µM (single conc)	Delp_2021,Figure 8B	2
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	20 µM (single conc)	ENV/JM/MONO(2020)23 Bennekou, Fig 5.4 / Annex 1.2	3
KE 177	LUHMES DoD3	ATP content	DoD2 – DoD3 (24h)	EC25	13 µM	Delp_2021, Fig S7 + Figure 4
KE 177	SH-SY5Y DoD7	MMP (rhodamine123)	DoD6 – DoD7 (24 h)	EC25	0,31 µM	Delp_2021, Figure 7 ENV/JM/MONO(2020)23 Bennekou, Fig 5.6 / Annex 1.6
KE 177	SH-SY5Y DoD8	ATP content	DoD3+DoD6 until DoD8 (120 h)	EC50	1 µM	Bennekou, Fig 5.11

¹Permeabilized cells.

²Only n=2

³No indication on the number of biological replicates.

Pyraclostrobin

KE1542: Three studies conducted in two cell lines (HepG2, LUHMES) measured inhibition of c3 immediately after treatment of cells with pyraclosrobin. HepG2 cells were affected at 0.4 µM pyraclostrobin. Experiments in proliferating LUHMES cells were only conducted at a single concentration; at 50 µM c3 was inhibited. Of note, these experiments were conducted in permeabilized cells.

KE177: In intact LUHMES cells, 20 µM pyraclostrobin lead to an immediate reduction in OCR by about 90% (relative to the solvent control) (Delp 2021, Bennekou 2020), whereby the number of independent biological replicates in each study was unclear. After 24 h of treatment, ATP content was affected at concentrations starting from 13 µM.

In differentiated SH-SY5Y cells, treatment with pyraclostrobin impacted the mitochondrial potential, as measured with the rhodamine123 dye (EC25 = 0,31 µM) (Delp 2021, Bennekou 2020). When exposed for 120 h, ATP levels were decreased by about 50% at 1 µM (Bennekou 2020).

KE	cell type	assay	exposure	effect level	effect conc	reference	notes
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2019, Suppl. Item 5	1
KE 1542	LUHMES (proliferating)	Mito Complexes, inhibition of c1-c3	immediate	25%	50 µM (single conc)	Delp_2021, Figure S8	1
KE 1542	HepG2	Mito Complexes, inhibition of c1-c3	immediate	EC50	0,4 µM	van der Stel 2020, Figure 4 + Table 2	1
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	20 µM (single conc)	Delp_2021,Figure 8B	2
KE 177	LUHMES DoD3	MitoStress OCR	immediate	25%	20 µM (single conc)	ENV/JM/MONO(2020)23 Bennekou, Fig 5.4 / Annex 1.2	3
KE 177	LUHMES DoD3	ATP content	DoD2 – DoD3 (24h)	EC25	13 µM	Delp_2021, Fig S7 + Figure 4
KE 177	SH-SY5Y DoD7	MMP (rhodamine123)	DoD6 – DoD7 (24 h)	EC25	0,31 µM	Delp_2021, Figure 7 ENV/JM/MONO(2020)23 Bennekou, Fig 5.6 / Annex 1.6
KE 177	SH-SY5Y DoD8	ATP content	DoD3+DoD6 until DoD8 (120 h)	EC50	1 µM	Bennekou, Fig 5.11
Additional literature
KE 177	Primary cortical neurons	MMP (rhodamine123)	DoD 7 (24h)	EC50	5 µM	Regueiro et al. 2015 Text dx.doi.org/10.1016/j.envres.2015.03.013

¹Permeabilized cells.

²Only n=2

³No indication on the number of biological replicate

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Uncertainties and inconsistencies table

Uncertainty	Impact	Reason
KE 1542 measured in permeabilised cells		Permeabilisation provides direct access for the tested compounds and substrates to the mitochondria and respiratory chain components. The physicochemical properties of the tested compound may reduce its ability to permeate the plasma membrane of intact cells, thus reducing or preventing its uptake which could affect the concentration and the time required to impact the downstream KEs.
Lack of data in galactose condition	High	In vitro cell models in general are characterized by an unphysiological reliance on glycolysis. In the presence of glucose any KE is influenced by the contribution of oxidative phosphorylation in addition to glycolisis to meet the cellular need for ATP. Thus, the KEs are influenced by the glycolisis rate. Glucose concentrations in culture medium higher than the physiological level enhances cellular resistance to mitochondrial dysfunction. Application of galactose instead of glucose in the medium allows a shift towards mitochondrial ATP generation. Even under these conditions, glycolysis significantly contributes to ATP production.
Use of HepG2 concentration response curves related to the measurement of oxygen consumption upon inhibition of cIII as a surrogate to represent inhibition of cIII in LUHMES cells, due to the lack of concentration response data for LUHMES cells	Low	It is assumed that since the exposure is acute and in permeabilized cells, the test chemical would have immediate access to the mitochondria. Other mechanisms such as transport into the cells, ADME considerations or an indirect effect via other signaling pathways were considered negligible under these assay conditions. It should be noted that OCR was measured in the presence of glucose, which introduces an influence from the glycolitic rate. This factor may differ between hepatocytes and neurons.
no concentration-response data for OCR in LUHMES	High	Increase the uncertainty in the concordance concentration response relationship across the KEs
Methodological limits	Medium-low	•For some assays and chemicals, only two biological replicates were performed (instead of 3), therefore results should be considered with caution. •In certain studies (i.e., Bennekou 2020), concentration-response data were sometimes “re-normalized”. For many assays, results are normalized to an untreated control, which is set at 100%. But if the results of low concentrations of the solvent chemical are not sufficiently close to 100%, it is assumed that the solvent control was measured imprecise and the curve is re-normalized (i.e., shifted) to reach a 100%. In this situation, some information about the variability in the assay is lost. •Different assays have a different effect concentration (i.e. EC25 and EC50). Occasionally, also the same assay can have different effect levels depending on the publication, which reduces overall comparability. However, in most cases the EC25 and EC50 are within a factor of 3 of each other, thus limiting the uncertainty.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Modulating Factor (MF)	MF Specification	Effect(s) on the KER	Reference(s)
Paraoxonase 2	Antioxidant enzyme	Prevents mitochondrial dysfunction	Altenhöfer et al., 2010; Devarajan et al., 2011
Mitochondrial Cu superoxide dismutase	Antioxidant enzyme	Prevents mitochondrial dysfunction	Okado-Matsumoto and Fridovich, 2001
Mitochondrial Zn superoxide dismutase	Antioxidant enzyme	Prevents mitochondrial dysfunction	Okado-Matsumoto and Fridovich, 2001

Paraoxonase 2 (PON2), a member of the paraoxonase gene family, has been identified as a key intracellular antioxidant enzyme, playing a role in preserving mitochondrial function and integrity. It is broadly expressed across tissues and predominantly localized to the inner mitochondrial membrane (IMM) (Devarajan et al., 2011) where it prevents generation of superoxide (O₂⁻•) (Altenhöfer et al., 2010). Both studies highlight PON2’s involvement in regulating the ETC, particularly complexes I and III. Evidence suggests that PON2 interacts with coenzyme Q10 (CoQ10), stabilizing it and thereby preventing the formation of ubisemiquinone, which contributes to reactive oxygen species (ROS) production. Supporting this mechanism, Devarajan et al. (2011) observed elevated mitochondrial superoxide (O₂⁻•) levels in the liver and peritoneal macrophages of PON2-deficient mice administered an atherogenic diet. In contrast, overexpression of PON2 in HeLa cells or in isolated mitochondria, significantly lower O₂⁻• induced by antimycin in the mitochondria (Altenhöfer et al., 2010; Devarajan et al., 2011). Antimycin is a well-established inhibitor of complex III within the mitochondrial electron transport chain (ETC). By obstructing electron transfer at this site, it leads to the accumulation of ubisemiquinone—a reactive intermediate capable of donating electrons to molecular oxygen, thereby generating superoxide (O₂⁻•). This confirms that PON2 suppresses O₂⁻• generation at complex III (Altenhöfer et al., 2010; Devarajan et al., 2011). Notably, however, PON2 neither neutralises O₂⁻• once formed nor influences superoxide dismutase (SOD) activity (Altenhöfer et al., 2010). Instead, its protective effect is due to its ability to prevent O₂⁻• production, which is probably achieved by modulating CoQ10 prior to electron leakage.

Other antioxidant enzymes that protect mitochondria include Cu superoxide dismutase (CuSOD) and ZnSOD, which are found in both the cytoplasm and the intermembrane space, as well as MnSOD, which is found in the matrix and on the inner membrane (Okado-Matsumoto and Fridovich, 2001).

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The quantitative understanding of the KERs was gained by modelling the KERs within the qAOP framework and methods that were developed in Tebby et al., (2022). Data and uncertainties are reported in "quantitative understanding" section of AOP 587 (cIII inhibitors).

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

An overview of these data across AOPs and KEs, summarising the percentage effect on each KE, is presented in the “Evidence assessment” section of AOP 587 (cIII inhibitors).

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

The KER becomes active within seconds when OCR is considered a sign of mitochondrial dysfunction, and changes in MMP can be detected within minutes.

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

There are no sex or age restiction for the applicability of this KER and mitochondrial are essential for most of eukariotyc cells.

Taxonomic - The catalytic core of cIII (cytochrome b, cytochrome c₁, and the iron-sulfur protein) is structurally and functionally conserved across species (Xia et al. 2013). This KER is plausibly applicable across vertebrates and invertebrates with supporting data from experimental models and human cells. Complex III inhibitors (e.g. antimycin A, pyraclostrobin, azoxystrobin, picoxystrobin) impair key mitochondrial processes, including oxygen consumption, ATP synthesis, and membrane potential, through conserved mechanisms targeting the electron transport chain. These effects are observed in both invertebrates e.g., worms (Nicodemo et al. 2018; Zhao et al., 2025), honey bees (Martinović-Weigelt et al 2024; Nicodemo et al., 2020), Dictyostelium discoideum (Downs et al. 2021) and vertebrates e.g., zebrafish embryos (Li et al. 2021; Yang et al., 2020; Kumar et al. 2020 ), suggesting that mitochondrial dysfunction is not species-specific but rather a generalizable outcome of complex III inhibition. The consistency of endpoints such as reduced oxygen consumption rate (OCR), increased reactive oxygen species (ROS), and altered bioenergetic parameters across studies supports the taxonomic applicability of these inhibitors.

Sex/Life stages - This KER is plausibly applicable to both sexes and any life stage. However, sex differences have been observed in oxidative stress generation, which is one of the consequences of mitochondrial dysfunction due to cIII inhibition. For example, using high content respirometry in tissue homogenates from control mice, Khalifa et al. found that female brain exhibited enhanced respiration and higher reserve capacity associated with lower hydrogen peroxide production (Khalifa et al, 2017).

References

List of the literature that was cited for this KER description. More help

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Zhao W, Wang K, Mu X, Jiang J, Yang Y, Wang C. Picoxystrobin causes mitochondrial dysfunction in earthworms by interfering with complex enzyme activity and binding to the electron carrier cytochrome c protein. Environ Pollut. 2025 Mar 1;368:125732. doi: 10.1016/j.envpol.2025.125732. Epub 2025 Jan 21. PMID: 39842493.