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Quantitative assessment of KEs in AOP464 Parkinson's disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that has been associated with exposure to pesticides. Nevertheless, neurodegeneration is not currently an endpoint that is covered in pesticide safety assessment. Adverse Outcome Pathways (AOPs) are increasingly being developed for risk assessment purposes. To make practical use of these AOPs, quantifying Key Event Relationships (KERs) is a critical next step. Here, we demonstrate one approach of quantifying an AOP (AOP464) for pesticide-induced PD. Dopaminergic cell lines were used to generate data for KEs ‘oxidative stress’, ‘mitochondrial damage’, and ‘cell death’. Effects on oxidative stress were determined with the H2DCFDA-AM probe after 1, 24, and 72 hour(s). Mitochondrial damage and cell death were determined after 24, and 72 hour(s) by Alamar blue and CFDA-AM assay, respectively. Six pesticides (dinoseb, endosulfan, folpet, mancozeb, pyraclostrobin, and rotenone) were used for all experiments. Preliminary data shows that the pesticides tested do not necessarily trigger the KEs in the same linear manner. For instance, mancozeb and folpet induce ROS after 1 hour of exposure, but do not significantly affect mitochondrial activity at 24 hours. In contrast, dinoseb and pyraclostrobin do not induce ROS at 1 hour, but do cause mitochondrial dysfunction at 24 hours. As such, a common linear response-response model is not applicable in this case study. Instead, a probabilistic model that combines effects of early KEs to predict cell death provides a more fitting approach. Here we show that although qualitative AOPs are linear by design, this linearity is not mirrored in vitro. For practical application of qAOPs, it is necessary that this is taken into consideration, for instance by applying a probabilistic modelling approach. Moreover, to support mechanistic understanding and increase confidence in the model, measuring KEs at multiple time points can be useful. Finally, this case study highlights that developing AOPs for use in safety assessment should be an iterative process in which KEs and KERs are drawn and redrawn based on new data.

Details
Type
study
Internal ID
S-VHPS28
Release date
2026-03-18
Version
—
License
CC-BY
URL
https://www.ebi.ac.uk/biostudies/studies/S-VHPS28
DOI
10.6019/S-VHPS28
Publications

No linked publications available.

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Authors
Funding
  • Dutch Research Agenda (NWA)
    1292.19.272
LUHMES
RRID: CVCL_B056 · Supplier: Leist lab, Konstanz University
Homo sapiens · Brain · Substantia nigra pars compacta · Dopaminergic neurons
Structure for CID 6950
Dinoseb
CAS: 88-85-7
PubChem: CID 6950
CompoundCloud: Q4723
Structure for CID 3224
Endosulfan
CAS: 115-29-7
PubChem: CID 3224
CompoundCloud: Q4727
Structure for CID 8607
Folpet
CAS: 133-07-3
PubChem: CID 8607
CompoundCloud: Q4771
Structure for CID 3034368
Mancozeb
CAS: 8018-01-7
PubChem: CID 3034368
CompoundCloud: Q4734
Structure for CID 6422843
Pyraclostrobin
CAS: 175013-18-0
PubChem: CID 6422843
CompoundCloud: Q5155
Structure for CID 6758
Rotenone
CAS: 83-79-4
PubChem: CID 6758
CompoundCloud: Q38
Hydrogen peroxide
CAS: 7722-84-1
Structure for CID 7048594
Staurosporine
CAS: 62996-74-1
PubChem: CID 7048594
CompoundCloud: Q2054
Exposure duration: 24 hours · Exposure duration: 72 hours
Exposure concentration: [DRAFT] µM · Exposure-measurement interval: 0 hours
Bioassays: Alamar blue, 5-CFDA
Exposure duration: 1 hours · Exposure duration: 4 hours · Exposure duration: 24 hours
Exposure concentration: [DRAFT] µM · Exposure-measurement interval: 0 hours
Bioassays: H2DCFDA-AM
Exposure duration: 8 hours
Exposure concentration: [DRAFT] µM · Exposure-measurement interval: 0 hours
Bioassays: CellTiter-Glo 2.0
Bioassays
  • H2DCFDA-AM BAO
    Increase, Oxidative Stress
    Measured: Fluorescence intensity · Readout: Reactive oxygen species production (Increase in fluorescence over 1 hour in comparison to startvalue (%)) · Replicates: 3-4 · Technical replicates: 6 · Positive control: Hydrogen peroxide
  • Alamar blue ENM
    Mitochondrial Injury
    Measured: Fluorescence intensity · Readout: Mitochondrial metabolic activity (% of solvent control) · Replicates: 3-4 · Technical replicates: 6 · Positive control: Rotenone
  • 5-CFDA BAO
    Increase, Cell death
    Measured: Fluorescence intensity · Readout: Cell death (% of solvent control) · Replicates: 3-4 · Technical replicates: 6 · Positive control: Staurosporine
  • CellTiter-Glo 2.0
    Increase, Mitochondrial dysfunction
    Measured: Luminescence · Readout: Mitochondrial metabolic activity (% of solvent control) · Replicates: 4 · Technical replicates: 6 · Positive control: Rotenone
AOP Linkage

Coming soon: normalization, model fitting, statistics, QC, etc.

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Parkinson Question 1: Hazard Question 2: Risk Toxicodynamics Adverse Outcome Chemical Characteristics and Hazard Identification