11β-hydroxylase inhibition leads to Decreased, 11KT

Many studies show that the pathophysiological consequences of a partial or total CI inhibition are linked to mitochondrial dysfunction. In many of these experiments the cellular damage caused by mitochondrial dysfunction is reduced (or entirely prevented) by treatment with antioxidants. Different degrees of CI inhibition by rotenone have been studied in the human osteosarcoma-derived cell line (143B). A quantitative correlation between increasing inhibition of CI and mitochondrial dysfunction (as shown by inhibition of mitochondrial respiration, reduced ATP production, increased ROS release and lipid peroxidation, as well as decreased Δψm) was established (Fig. 1 and Table 1 based on Barrientos and Moraes, 1999). Based on the existing literature it is suggested that rotenone exerts toxicity via oxidative stress, rather than via decrease of ATP synthesis (bioenergetics effects).

A few examples illustrating mitochondrial damage and oxidative stress in animal model of PD and human cells induced by:

Rotenone

• Rotenone administered subcutaneously for 5 weeks (2.5 mg/kg/d) caused a selective increase (by ~2 folds) in oxidative damage in the striatum, as compared to the hippocampus and cortex, accompanied by massive degeneration of DA neurons (~80% decrease) in the substantia nigra. Rotenone reduced intracellular ATP levels in the striatum (by >40%), increases malondialdehyde (MDA, indicative of lipid peroxidation, by ~60%), reduced GSH levels (by ~20%), thioredoxin (by ~70%), and manganese superoxide dismutase (SOD, by ~15%) (all parameters significantly changed in the striatum). Antioxidant polydatin (Piceid) treatment significantly prevented the rotenone-induced changes by restoring the above parameters to control levels, confirming that rotenone- induced mitochondrial dysfunction resulted in oxidative stress (Chen et al., 2015).
• Rotenone was administered 2.5 mg/kg body weight to male Wistar rats for 4 weeks in the presence or absence of ferulic acid (FA, at the dose of 50 mg/kg) that has antioxidant and anti-inflammatory properties. Rotenone administration caused DA neuronal cell death (~50%), significant reduction in endogenous antioxidants, such as superoxide dismutase (~75%), catalase (~40%), and glutathione (~50%), and induced lipid peroxidation evidenced by increased MDA formation (~2 folds). Treatment with FA rescued DA neurons in substantia nigra pars compacta area and nerve terminals in the striatum, as well as restored antioxidant enzymes, prevented depletion of glutathione, and inhibited lipid peroxidation induced by rotenone (Ojha et al., 2015).

• Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration of DA neurons. In this study, rotenone (100 nM, 24 hr) in both SH-SY5Y cells and primary cultured substantia nigra (SN) DA neurons, was shown to reduce DA cell viability (~40%), reduce Δψm (~40%, as shown by TMRM), induce mitochondrial ROS production (~30%, as shown by increase of MitoSox Red), and increased cytosolic protein levels of proteins related to the mitochondrial apoptotic pathway (i.e. Bax, cytochrome c, active caspase-9 and active caspase-3) (~ 2 folds for all proteins).

The neuroprotective mechanism of ALDH2 was observed as overexpression of wild-type ALDH2 gene (but not the enzymatically deficient mutant ALDH2*2 (E504K)) reduced rotenone-induced DA neuronal cell death, prevented rotenone-induced reduction in TMRM signal (95.7±1.6% v.s. 67±3.5%), and prevented rotenone-induced increase in MitoSox Red intensity (103.1±1% v.s. 133.4±0.8%). Additionally, pre-treatment of cells with Alda-1 (activator of ALDH2) (1–10 μM, for 24 hr) prevented rotenone-induced loss of Δψm and ROS production in a dose-dependent manner. These results were confirmed by in vivo studies. Rotenone (50 mg/kg/day, oral administration for 14 days) or MPTP (40 mg/kg/day, i.p. for 14 days) both administered to C57BL/6 mice caused significant SN TH+ DA neuronal cell apoptosis (~50%). Alda-1 attenuated rotenone-induced apoptosis by decreasing ROS accumulation, reversing Δψm depolarization, and inhibiting the activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that rotenone or MPP+ induces DA neurotoxicity through oxidative stress. Moreover, Alda-1 is effective in ameliorating mitochondrial dysfunction by inhibiting rotenone or MPP+ induced mitochondria-mediated oxidative stress that leads to apoptosis (Chiu et al., 2015).

• Rotenone-induced mitochondrial dysfunction was observed in human neuroblastoma cells exposed to 5 nM rotenone for 1-4 weeks. After 3-4 weeks of treatment, rotenone-treated cells showed evidence of oxidative stress, including loss of GSH (by 5%) and increased oxidative DNA (qualitative, measured by using antibodies to 8-oxo-dG) and protein damage (223 ± 29% of control, as shown by the large increase in protein carbonyls in the insoluble fraction) (Sherer et al. 2002). This chronic rotenone treatment markedly sensitized cells to further oxidative challenge since in response to H2O2 cytochrome c release from mitochondria and caspase-3 activation occurred earlier and to a greater extent in rotenone-treated cells vs Ctr (1.44 ± 0.02% vs 0.38 ± 0.07% apoptosis/hr). This study indicates that chronic, low-level CI inhibition by rotenone induces progressive oxidative damage, and caspase-dependent neuronal cell death (Sherer et al., 2002).
• By using anti-oxidant, kaempferol (6 μM, 1 hr prior addition of rotenone) and rotenone (50 nM, max up to 24 hr) on SH-SY5Y cells, kaempferol was found to counteract rotenone-induced ROS production (especially superoxide: with kaempferol, ethidium fluorescence decreased below the control (Ctr) levels), rotenone-induced mitochondrial oxidative dysfunction (protein carbonyls values: 2.5 in Ctr, 6.2 with rotenone, 2.7 with kaempferol + rotenone), rotenone-induced oxygen respiration (values of nmol of atomic oxygen/minute/mg protein: 5.89 Ctr, 0.45 with rotenone, 2.47 with kaempferol + rotenone), rotenone-induced Δѱm decrease (~70% cells of with rotenone only vs ~30% with kaempferol + rotenone) (Filomeni et al., 2012).

• To model the systemic mitochondrial impairment, rats were exposed to rotenone. A single rotenone dose (10 nM, for 24 hr) induced mtDNA damage in midbrain neurons (>0.4 lesions/10kb vs 0 lesions/10kb in vehicle), but not in cortical neurons; similar results were obtained in vitro in cultured neurons. Importantly, these results indicate that mtDNA damage is detectable prior to any signs of neuronal degeneration and is produced selectively in midbrain neurons. The selective vulnerability of midbrain neurons to mtDNA damage was not due to differential effects of rotenone on CI since rotenone suppressed respiration equally (~60%) in midbrain and cortical neurons compared to vehicle. However, in response to CI inhibition, midbrain neurons produced more mitochondrial H2O2 (5 min of rotenone increased MitoPY1 fluorescence of ~10% in midbrain mitochondria vs vehicle, and progressively for the duration of measurement), than cortical neurons. The selective mtDNA damage in midbrain could serve as a molecular marker of vulnerable nigral neurons in PD. Oxidative damage to cell macromolecules in human PD and the rotenone model have been recently reviewed (Sanders et al., 2014).
• Adult male Sprague–Dawley rats were intranigrally infused with rotenone (6 μg in 1 μl) alone or in the presence of L-deprenyl (0.1, 1, 5 and 10 mg/kg; i.p.) at 12 h intervals for 4 days. Rotenone alone (100 μM, 30 min) increased the levels of hydroxyl radials in the mitochondrial P2 fraction 2,3-DHBA (122.90 ± 5.4 pmol/mg protein) and 2,5-DHBA (146.21 ± 6.3 pmol/mg protein). L-deprenyl (100 nM–1 mM) dose-dependently attenuated rotenone-induced ·OH generation in the mitochondrial P2 fraction. L-deprenyl-induced attenuation in the rotenone-mediated 2,3-DHBA generation was from 17 ± 1.1% to 67 ± 4.3%, respectively, for 100 nM–1 mM of the MAO-B inhibitor. Also, rotenone caused about 51 ± 3.3% reduction in GSH levels in the cell body region, SN and 34 ± 1.1% decrease in the nerve terminal region, NCP (nucleus caudatus putamen). L-deprenyl alone did not cause any significant difference in the GSH content in either region. L-deprenyl treatment dose-dependently attenuated the rotenone-induced GSH depletion in SN from 51 ± 3.1% to 44 ± 2.1%, 32 ± 1.7% and 9 ± 1.0%, respectively, for doses of 1, 5 and 10 mg/kg. Additionally, SOD activity was assayed in rotenone-lesioned animals, which were treated with l-deprenyl at different doses (1–10 mg/kg). SN exhibited 2- and 3-fold activity of Cu/Zn-SOD (i.e. cytosolic SOD fraction) and Mn-SOD (i.e. particulate SOD fraction), respectively, compared to the nerve terminal region, NCP. L-deprenyl (5 and 10 mg/kg) in rotenone-lesioned animals caused a significant increase in the cytosolic Cu/Zn SOD activity in SN of both the sides. Intranigral infusion of rotenone alone caused a significant increase in the enzyme activity in SN of the side of infusion as compared to the non-infused side (~20%). L-deprenyl (5 and 10 mg/kg) further increased catalase activity in both ipsilateral SN and striatum, as compared to the contralateral side of infusion. Finally, rotenone caused a 74% reduction in the striatal TH staining intensity, which was partially recovered by L-deprenyl. These results showed that oxidative stress is one of the major causative factors underlying DA neurodegeneration induced by rotenone and they support the view that L-deprenyl is a potent free radical scavenger and an antioxidant (Saravanan et al., 2006). Similar results were obtained after exposure to MPP+ (Wu et al., 1994).

• Antioxidant (Piperaceae; PLL) with some anti-inflammatory activities demonstrated in preclinical studies protective effects in PD animal models. Rats treated with rotenone and PLL-derived alkaloids showed decreased ROS, stabilized Δψm, and the opening of the mitochondrial PTP - which is triggered by ROS production - was inhibited. In addition, rotenone-induced apoptosis was abrogated in the presence of these alkaloids (Wang H. et al., 2015).
• In SK-N-MC human neuroblastoma cells, rotenone (10 nM - 1µM, 48 hr) caused dose-dependent ATP depletion (~35% reduction by 100 nM rotenone vs Ctr), oxidative damage (100% increase of carbonyls levels upon 100 nM rotenone), and death (100 nM rotenone after 48 hr caused 1.1 AU (arbitrary units) increase of cell death vs untreated – 0.00 AU -). α-Tocopherol pre-treatment (62.5 or 125 μM 24 hr before rotenone (10 nm)) attenuated rotenone toxicity (Sherer et al., 2003).

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) or MPP⁺ (1-methyl-4-phenyl-pyridinium ion)

• MPTP converted into MPP⁺ inhibits mitochondrial CI activity, resulting in excessive intracellular ROS production followed by further mitochondrial dysfunction leading to mitochondrial-dependent apoptosis. Lutein, a carotenoid of xanthophyll family (antioxidant) reversed MPTP-induced mitochondrial dysfunction, oxidative stress, apoptotic cell death and motor abnormalities. These results revealed that antioxidant protected DA neurons and diminished mitochondrial dysfunction and apoptotic death (Nataraj et al., 2015).
• Antioxidant (salidroside; Sal) pre-treatment protected DA neurons against MPTP/MPP⁺ induced toxicity in a dose-dependent manner by: (1) reducing the production of ROS, (2) regulating the ratio of Bcl-2/Bax, (3) decreasing cytochrome-c and Smac release, and inhibiting caspase-3, caspase-6, and caspase-9 activation, which are known to trigger apoptosis following mitochondrial dysfunction. Sal acted as an effective neuroprotective agent through modulation of the ROS-induced mitochondrial dysfunction in vitro and in vivo (Wang S. et al., 2015).
• In an in vitro study, MPP⁺ (1 mM, 24 hr) was found to elicit production of ROS (by 2 fold vs Ctr) and reduce by 50% SOD (by about 50%) and catalase (by about 65%) activity in SH-SY5Y human neuroblastoma cells. Pre-treatment with the antioxidant astaxanthin (AST; 50 μM, 24 hr) inhibited MPP⁺- induced production of ROS and attenuated both SOD and catalase activity decrease. Furthermore, MPP⁺ (1 mM, 48 hr) increased caspase-3 activity to 243% of the Ctr and also increased cleaved caspase-3 in the cells (qualitative). Addition of 50 μM AST attenuated MPP⁺-induced caspase-3 activation (57% suppression). MPP⁺ induced also a 70% reduction of Δψm and cytochrome c release (qualitative), while AST prevented both these effects. The protective effects of AST on MPP⁺ induced mitochondrial dysfunction was due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of SOD and catalase (as shown above) and regulating the expression of Bcl-2 and Bax (Bax/Bcl-2 ratio increased to 1.6-fold vs Ctr upon treatment with MPP+, while AST prevented the MPP⁺-induced increase of the Bax/Bcl-2 ratio). These results were confirmed by in vivo studies (Lee et al., 2011).

• DA neurons in primary mesencephalic cultures treated with MPP⁺ (100 μМ, for 48 hr) underwent reduction of cell viability (~55% MTT reduction), LDH release (~90%), about 60% reduction of TH+ cells, disruption of Δψm (~45% decline) and ROS production (~60% increase), upregulation of Nox2 (~45%) and Nox4 (~60%), while promoting a decrease of both SOD (~45%) and GSH activity (~85%). Additionally, MPP induced apoptosis via mitochondrial dysfunction, as shown by induction of cytochrome c (~55%), cleaved-caspase-3 (~75%), upregulation of Bax expression (~55%), and downregulation of Bcl2 (~60%). Liuwei dihuang (LWDH), a widely used traditional Chinese medicine (TCM), has antioxidant characteristics. LWDH-WH, derivative of LWDH (0.01-10 μg/ml, added 1 hr prior to MPP⁺ addition) reduced oxidative damage via increasing antioxidant defence (SOD, GSH), decreasing ROS production, and down-regulating NADPH oxidases (Nox2 and Nox4). LWDH-WH also inhibited neuronal apoptosis by increasing anti-apoptotic protein Bcl-2 expression, and down-regulating apoptotic signalling (Bax, cytochrome c, cleaved-caspase-3) in MPP⁺-treated neurons. All these protective effects were induced in a dose-dependent manner (Tseng et al., 2014).

• PC12 cells treated with MPP⁺ (500 μM, for 24 hr) underwent reduction of viability (~55% MTT reduction), oxidative stress (~160% increase in ROS production) and downregulation of heme oxygenase-1 expression (~ 2 folds). Pre-treatment with edaravone, a novel free radical scavenger, (25, 50, 75, 100 μM, for 1 h prior MPP+ treatment) protected PC12 cells against MPP+-cytoxicity via inhibiting oxidative stress and up-regulating heme oxygenase-1 expression in a dose-dependent manner (Cheng et al., 2014).

• The protective effects of antioxidant, apigenin (AP), naturally occurring plant flavonoids were observed on the MPP+ induced cytotoxicity in cultured rat adrenal pheochromocytoma cells (PC12 cells). The PC12 cells were pre-treated with various concentrations of the test compound for 4 h, followed by the challenge with 1,000 µM MPP⁺ for 48 h. Pre-treatment with AP (3 - 6 - 12 µM) before MPP⁺ significantly reduced the level of intracellular ROS and elevated Δψm in the MPP⁺ treated PC12 cells. In addition, AP markedly suppressed the increased rate of apoptosis and the reduced Bcl 2/Bax ratio induced by MPP⁺ in the PC12 cells. The findings demonstrated that AP exerts neuroprotective effects against MPP⁺ induced neurotoxicity in PC12 cells, at least in part, through the inhibition of oxidative damage and the suppression of apoptosis through the mitochondrial pathway (Liu et al., 2015).

• Brain mitochondria isolated from ventral midbrain of mitochondrial matrix protein cyclophilin D (CYPD) knockout mice were significantly less sensitive to acute MPP⁺ (20 µM) -induced effects. CYPD ablation attenuated in vitro Ca2+-induced mitochondrial dysfunction and ROS generation upon Ca2+ loading, both in the absence and in the presence of MPP⁺, compared to wild-type mice. CYPD ablation conferred a protection to mitochondrial functions upon in vivo treatment with MPTP.

Ventral midbrain mitochondria (that constitutes < 5% of SNpc DA neurons) isolated from brains of wild type (wt) mice acutely treated with MPTP (single MPTP 20 mg/kg injection, analysis done after 4 hr), as compared with saline-treated mice, showed a reduction of CI (by 53%), a reduced rate of phosphorylating respiration (by 38%), a reduced respiratory control index (by 37%), and a decreased ADP/O ratio (by 18%). Ventral midbrain mitochondria isolated from brains of CYPD knockout mice acutely treated with MPTP, as compared with MPTP-treated wt mice, exhibited higher activity of CI (~80%, vs 53% wt), higher rate of phosphorylating respiration (~82%, vs 62% wt), a better respiratory control index (~79%, vs 63% wt), and a higher ADP/O ratio (~90% vs 82% wt) (Thomas et al., 2012). CYP plays as a regulatory component of a calcium-dependent permeability transition pores (PTP), and the data suggest that PTP is involved in MPP⁺-induced mitochondrial damage. Under oxidative stress, the prolonged opening of the PTP results in calcium overload and with time mitochondrial dysfunction as they get de-energized, depolarized, triggering apoptotic or necrotic cell death (Bernardi, 1999).

There are many other studies showing that MPP⁺ induces NADH-dependent SOD formation and enhances NADH-dependent lipid peroxidation in submitochondrial particles, confirming that oxidative stress is induced by MPP+ (e.g. Takeshige, 1994; Ramsay and Singer, 1992).

Based on the human post mortem studies of PD brains it is well established that oxidative stress and mitochondrial dysfunction accompany the pathophysiology of PD (e.g. Dias et al., 2013; Zhu and Chu, 2010; Hartman et al., 2004; Fujita et al., 2014).

Examples of human data confirming the presence oxidative stress and mitochondrial dysfunction in PD post mortem brains:

• A significant decrease in CI activity has been identified in a large study of post-mortem PD brains, specifically in substantia nigra compared with age matched controls. In idiopathic PD all 10 patients studied had significant reductions of CI activity (Parker et al., 1989). It is hypothesize that the CI dysfunction may have an etiological role in the pathogenesis of PD (Greenamyre et al., 2001; Sherer et al., 2003, Schapira et al., 1989).
• The structure and function of mitochondrial respiratory-chain enzyme proteins were studied post-mortem in the substantia nigra of nine patients with PD and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. CI and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of CI activity in the substantia nigra of patients with PD (Schapira et al., 1990).
• Post mortem human studies show that CI deficiency in PD is anatomically specific for the substantia nigra, and they are not present in another neurodegenerative disorder involving the substantia nigra. These results suggest that CI deficiency may be the underlying cause of DA cell death in PD (Schapira et al., 1990; Schapira, 1994).
• The mitochondrial respiratory chain function was studied in various brain regions as well as in skeletal muscle and in blood platelets from patients with idiopathic PD and from matched controls. The evidence suggests that the CI deficiency in PD is limited to the brain and that this defect is specific for the substantia nigra (Mann et al., 1992).
• Immunoblotting studies on mitochondria prepared from the striata of patients who died of PD were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with PD, the 30-, 25- and 24-kDa subunits of CI were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and PD. The authors claim that deficiencies in CI subunits seem to be one of the most important clues to elucidate pathogenesis of PD (Mizuno et al., 1989).
• Redox markers have been found unchanged in PD patient-derived vs Ctr-derived fibroblasts at baseline. Basal mitochondrial respiration and glycolytic capacity resulted similar at baseline between PD and Ctr fibroblasts, while rotenone-sensitive respiration (analysed by using 0.5 μM rotenone) resulted lower in PD fibroblasts vs Ctr (174.74 ± 48.71 vs 264.68 ± 114.84) (Ambrosi et al., 2014).
• Augmented oxidative metabolism has been detected in PD brains by magnetic resonance studies, in conjunction with energy unbalance. Decreased glucose consumption (22% mean reduction), likely reflecting a decrease in neuronal activity, has been reported in the nigrostriatal system of PD patients (Piert et al., 1996). These symptoms were hypothesized to be indicative of mitochondrial dysfunction as early markers, present in the brain of patients with PD even in the absence of overt clinical manifestations (Rango et al., 2006). In particular, by using high temporal and spatial resolution 31P magnetic resonance spectroscopy (31P MRS) technique authors studied mitochondrial function by observing high-energy phosphates (HEPs) and intracellular pH in the visual cortex of 20 PD patients and 20 normal subjects at rest, during, and after visual activation. In normal subjects, HEPs remained unchanged during activation, but rose significantly (by 16%) during recovery, and pH increased during visual activation with a slow return to rest values. In PD patients, HEPs were within the normal range at rest and did not change during activation, but fell significantly (by 36%) in the recovery period; pH did not reveal a homogeneous pattern with a wide spread of values. Energy unbalance under increased oxidative metabolism requirements, that is, the post-activation phase, discloses a mitochondrial dysfunction that is present in the brain of patients with PD even in the absence of overt clinical manifestations,(Rango et al., 2006).

There are many other studies providing evidence that oxidative stress and mitochondrial dysfunction play an important role in PD pathophysiology (see indirect KER Mitochondrial dysfunction induced DA neuronal cell death of nigrastriatal pathway).

- Revision of AOP3 (Project: NP/EFSA/PREV/2024/02):

Data were retrieved from assays measuring endpoints relevant for AOP3 in relevant cell types.    

KE 887 Complex I inhibition

Assays 

For KE 887, 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 1. KE 887: criteria for data 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 

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 in glucose medium, 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 data were extracted from Delp et al. (2019, 2021), Bennekou- ENV/JM/MONO(2020)23, van der Stel- ENVJMMONO(2020)22, 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 and KE890. 5 cI and 4 cIII inhibitors with evidence of interference with KE 887, KE177, according to the criteria described in the previous section, and KE890 were included as stressors 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 3 (cI inhibitors). 

cI inhibitors 

Deguelin 

KE 887: Four studies conducted in two cell lines (HepG2, LUHMES) measured inhibition of cI immediately after treatment of cells with deguelin. Two studies in proliferating LUHMES cells were conducted only at a single, high concentration ( 50 µM). Effective concentrations were in the range of 60 – 100 nM for LUHMES and HepG2 cells (Tebby 2022). Of note, these experiments were conducted in permeabilized cells. 

KE 177: Multiple studies measured the OCR in differentiating LUHMES immediately after treatment with deguelin. The effective concentrations range from 0,016 µM to 10 µM (Delp 2021, Alimohammadi 2023, Tebby 2022). When cells were treated for 24 h, ATP content was affected in 8-10 µM when cells were cultured in glucose containing medium (ENVJMMONO(2020)22, Delp 2021). This value dropped to 0,0045 µM (2000-fold) when cells were cultured in galactose containing medium (ENVJMMONO(2020)22,). 

In differentiated SH-SY5Y cells, 24 h exposure to deguelin lead to a decrease in mitochondrial membrane potential as measured via rhodamine 123 with an EC25 of 0,31 µM, but ATP content was not affected up to 10 µM, which was the highest tested concentration. A prolonged exposure to deguelin (120 h) however, affected the ATP content with an EC50 of 1 µM. 

¹Permeabilized cells. 

²EC estimated visually from graph. 

³Likely same underlying data reprinted 

⁴Only n=2 

⁵Number of biological replicates unclear 

Rotenone 

KE 887: Five studies conducted in two cell lines (HepG2, LUHMES) measured inhibition of c1 immediately after treatment of cells with rotenone. Two studies in proliferating LUHMES cells were conducted only at a single, high concentration (10 and 100 µM). The few concentration-response experiments measured c1 inhibition at 10-30 nM for both LUHMES and HepG2 cells. Of note, all experiments were conducted in permeabilized cells. Some experiments consisted of only two biological experiments. 

KE 177: Multiple studies measured the OCR in differentiating LUHMES immediately after treatment with rotenone. The lowest effective concentration was at 10 nM. When cells were treated for 24 h, OCR was affected at 100 – 500 nM. In the same exposure scenario, MMP was 24 nM, but ATP levels only decreased at 5-40 µM, when cells were cultured with glucose-containing medium. When fed with galactose, cells were more sensitive, with MMP and ATP affected at 0.2 nM and 0.01-6 nM, respectively. This corresponds to a shift of 200 to 1000-fold. 

In differentiated SH-SY5Y cells, 24 h exposure to rotenone lead to a decrease in mitochondrial membrane potential at 40-100 nM as measured via rhodamine 123, but ATP content was not affected up to 10 µM, which was the highest tested concentration. A prolonged exposure to rotenone (120 h) however, affected the ATP content at 100 nM. To summarize, rotenone is potent in disturbing the mitochondrial membrane potential, but an effect on ATP and viability can only be observed upon prolonged exposure. 

¹ Permeabilized cells. 

² EC estimated visually from graph. 

³ Likely same underlying data reprinted 

⁴ Only n=2 

⁵ Number of biological replicates unclear 

Fenpyroximate 

KE 887: Multiple studies measured that 50 µM fenpyroximate inhibited c1 by abou 80% in proliferating LUHMES cells. The only concentration-response study was conducted in HepG2, which estimated an EC50 of 0.019 µM. 

KE 177: In intact LUHMES exposed in glucose medium, OCR was reduced with 20 µM fenpyroximate (only 2 biological replicates of a single concentration). MMP, as measured via TMRE, was affected at 26 µM and ATP content at 13-15 µM. : In galactose-containing medium, MMP was affected at 0.2 nM, and ATP was affected at 0.7 nM. These values correspond to a shift of 10.000 fold. 

Only one study investigated the effect of fenpyroximate on K177 in SH-SY5Y cels. MMP (measured via rhodamine123) was affected at 0.79 µM.