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Intestinal absorption of pesticides across Caco-2 cell monolayers

A monolayer of Caco-2 cells was cultured in transwells, maintaining a stable TEER for at least one week. On the apical side, cells were exposed to pesticides in HBSS (without additives) for a maximum of 5 hours. Samples were collected over time from both the basolateral and apical compartments: either 50 µL or 100 µL immediately after exposure (time point 0.01), and at 30 minutes, 1 hour, 2 hours, and 5 hours. Each sample was diluted with an equal volume (50 µL or 100 µL) of LC-MS/MS grade methanol (cyclohexane for endosulfan). Pesticide concentrations were quantified by LC-MS/MS and used to determine apical-to-basolateral transport (absorption) across the intestinal epithelial barrier. In parallel, the cytotoxicity of the pesticides towards Caco-2 cells was assessed with an alamarBlue (resazurin) mitochondrial-activity assay, to confirm that the tested concentrations did not compromise monolayer viability. The Transwell permeability procedure was adapted from Hubatsch et al., Nature Protocols 2007 (doi:10.1038/nprot.2007.303).

Details
Type
study
Internal ID
S-VHPS18
Release date
2026-06-01
Version
—
License
CC0
URL
https://www.ebi.ac.uk/biostudies/studies/S-VHPS18
DOI
10.6019/S-VHPS18
Publications

No linked publications available.

Files

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Authors
  • Alex KleinJan
    HU University of Applied Sciences Utrecht, Institute for Life Sciences & Chemistry, Research Group Innovative Testing in Life Sciences & Chemistry
    ORCID iD 0000-0003-0226-1362
Funding
  • Dutch Research Agenda (NWA)
    1292.19.272
Caco-2
RRID: CVCL_0025 · Supplier: ATCC (HTB-37)
Homo sapiens · Intestine · Intestinal epithelium · Enterocyte

Human colorectal adenocarcinoma cell line that spontaneously differentiates into a polarized, enterocyte-like monolayer with tight junctions, widely used as an in vitro model of the intestinal epithelial barrier for absorption/permeability studies.

Structure for CID 6950
Dinoseb
CAS: 88-85-7
PubChem: CID 6950
CompoundCloud: Q4723
Structure for CID 3017
Diazinon
CAS: 333-41-5
PubChem: CID 3017
CompoundCloud: Q4726
Structure for CID 6758
Rotenone
CAS: 83-79-4
PubChem: CID 6758
CompoundCloud: Q38
Structure for CID 3224
Endosulfan
CAS: 115-29-7
PubChem: CID 3224
CompoundCloud: Q4727
Structure for CID 3034368
Mancozeb
CAS: 8018-01-7
PubChem: CID 3034368
CompoundCloud: Q4734
Structure for CID 2730
Chlorpyrifos
CAS: 2921-88-2
PubChem: CID 2730
CompoundCloud: Q4722
Structure for CID 2249
Atenolol
CAS: 29122-68-7
PubChem: CID 2249
Structure for CID 2519
Caffeine
CAS: 58-08-2
PubChem: CID 2519
CompoundCloud: Q2026
Exposure type: single · Exposure concentration: 0.1, 1, 10 µM · Exposure duration: 0.01 hours · Exposure-measurement interval: 0 hours
Bioassays: Caco-2 monolayer permeability assay
Exposure type: continuous · Exposure concentration: 0.1, 1, 10 µM · Exposure duration: 5 hours · Exposure-measurement interval: 0 hours
Bioassays: Caco-2 monolayer permeability assay
Exposure type: continuous · Exposure concentration: 0.1–10000 µM · Exposure duration: 24 hours · Exposure-measurement interval: 2 hours
Bioassays: Alamar blue
Bioassays
  • Caco-2 monolayer permeability assay BAO
    Measured: Pesticide concentration · Readout: Apparent permeability (Papp) (cm/s) · Replicates: 3 · Technical replicates: 2
  • Alamar blue ENM
    Measured: Fluorescence intensity · Readout: Relative mitochondrial metabolic activity (%) · Replicates: 3 · Positive control: Triton X-100 (1)
    Assay format: Cell-based format
    Detection agent: alamarBlue
    Assay supplier: MedChemExpress / Gibco
    Reagent concentration: 1 µg/mL
    Assay incubation time: 1–4 hours

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

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Toxicokinetics