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Respiratory uptake of inhaled dinoseb and rotenone using primary bronchial epithelial cells

Inhaled substances can be taken up via the respiratory tract and reach the systemic circulation. To improve estimates of respiratory uptake with in vitro models of the respiratory epithelium, normal human primary bronchial epithelial cells (NHBE, three donors; a donor mix in the 2024 pilot) were differentiated at the air-liquid interface and exposed to dinoseb or rotenone by applying a small droplet on the apical side of the culture (quasi-ALI). Samples of the basolateral medium were taken during the exposure period; at the start and end of the exposure an apical wash, a cell lysate and an extract of the insert membrane were taken for chemical analysis by LC-MS. Cytotoxicity was measured by LDH release and WST-1, barrier integrity by transepithelial electrical resistance (TEER) and cytokine production with a LEGENDplex panel. Reference compounds: caffeine, antipyrine and fluorescein.

Details
Type
study
Internal ID
S-VHPS29
Release date
2026-10-29
Version
—
License
CC-BY
URL
https://www.ebi.ac.uk/biostudies/studies/S-VHPS29
DOI
10.6019/S-VHPS29
Publications

No linked publications available.

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Authors
  • Yvonne Staal
    National Institute for Public Health and the Environment (RIVM)
    ORCID iD 0000-0002-7187-0121
  • Véronique de Bruijn
    National Institute for Public Health and the Environment (RIVM)
    ORCID iD 0000-0003-0248-2309
  • Jolanda Vermeulen
    National Institute for Public Health and the Environment (RIVM)
  • Eric Gremmer
    National Institute for Public Health and the Environment (RIVM)
  • Theo Sinnige
    Institute for Risk Assessment Sciences (IRAS), Utrecht University
  • Edwin Zwart
    National Institute for Public Health and the Environment (RIVM)
  • Eline Groeneveld
    National Institute for Public Health and the Environment (RIVM)
  • Chiel Jonker
    Institute for Risk Assessment Sciences (IRAS), Utrecht University
Funding
  • Dutch Research Council (NWO)
    NWA 1292.19.272
NHBE (normal human bronchial epithelial cells, primary)
RRID: N/A · Supplier: Lonza
Homo sapiens · Lung · Bronchial epithelium · Bronchial epithelial cell

Primary normal human bronchial epithelial cells from three female donors (24, 62 and 73 years), differentiated at the air-liquid interface on cell culture inserts; the 2024 pilot used a mix of donors.

Structure for CID 6950
Dinoseb
CAS: 88-85-7
PubChem: CID 6950
CompoundCloud: Q4723
Structure for CID 6758
Rotenone
CAS: 83-79-4
PubChem: CID 6758
CompoundCloud: Q38
Structure for CID 2519
Caffeine
CAS: 58-08-2
PubChem: CID 2519
CompoundCloud: Q2026
Structure for CID 2206
Antipyrine
CAS: 60-80-0
PubChem: CID 2206
Structure for CID 16850
Fluorescein
CAS: 2321-07-5
PubChem: CID 16850
Exposure concentration: dinoseb 50, 150, 500, 1000; rotenone 0.04, 0.2, 1, 2, 4, 8; caffeine 40 µM · Exposure duration: 120 hours · Exposure-measurement interval: 0.33, 0.67, 1, 1.33, 2, 6, 24, 48, 120 hours
Exposure type: single
Bioassays: Respiratory uptake (LC-MS chemical analysis)
Exposure concentration: dinoseb 50, 150, 500, 1000; rotenone 0.04, 0.2, 1, 2, 4, 8; caffeine 40 µM · Exposure duration: 120 hours · Exposure-measurement interval: 0, 48, 120 hours
Exposure type: single
Bioassays: LDH release assay, WST-1 cell viability assay, Transepithelial electrical resistance (TEER), Cytokine secretion (LEGENDplex)
Exposure concentration: dinoseb 1000; rotenone 0.04, 0.2, 1; caffeine 40; antipyrine 50; fluorescein 20 (2024 pilot, 37 °C and 4 °C) µM · Exposure duration: 6; 24 hours · Exposure-measurement interval: 1 hours
Exposure type: single
Bioassays: Respiratory uptake (LC-MS chemical analysis), Fluorescein permeability (pilot), LDH release assay, WST-1 cell viability assay, Transepithelial electrical resistance (TEER), Cytokine secretion (LEGENDplex)
Bioassays
  • Respiratory uptake (LC-MS chemical analysis) OBI
    Measured: Peak area (LC-MS) · Readout: Respiratory uptake (amount translocated) (nM) · Replicates: 3 · Technical replicates: 3
  • LDH release assay BAO
    Measured: Absorbance · Readout: Cytotoxicity (LDH release) · Time point: 0, 120 hours · Replicates: 3 · Technical replicates: 1
    Assay supplier: Roche
    Assay catalogue number: 11644793001
    Assay incubation time: 20 minutes
    Absorbance wavelength: 490/600 nm
  • WST-1 cell viability assay BAO
    Measured: Absorbance · Readout: Relative metabolic activity · Time point: 48 hours · Replicates: 3 · Technical replicates: 2
    Assay supplier: Roche
    Assay catalogue number: 11644807001
    Assay incubation time: 1 hours
  • Transepithelial electrical resistance (TEER) OBI
    Measured: Resistance · Readout: Barrier integrity (TEER) (ohm·cm²) · Time point: 0, 120 hours · Replicates: 3 · Technical replicates: 1
  • Cytokine secretion (LEGENDplex) BAO
    Measured: Fluorescence intensity · Readout: Cytokine concentration (ng/mL) · Replicates: 3
    Assay supplier: BioLegend
  • Fluorescein permeability (pilot) BAO
    Measured: Fluorescence intensity · Readout: Fluorescein translocation (µM) · Time point: 0, 1, 2, 3, 4, 5 hours · Replicates: 3 · Technical replicates: 1
    Excitation wavelength: 485 nm
    Emission wavelength: 535 nm

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

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Toxicokinetics