Study Summary for:
TEX-VAL-TOX-CC-2024-11-25-PFAS Renal Clearance_TERT-OAT1_Transwell

Description

This study assessed the transport and accumulation of 23 compounds in TERT1-OAT1 RPTECs using a 96-well Transwell model, which allows for the evaluation of both apical-to-basolateral (A→B) and basolateral-to-apical (B→A) transport. Cells were cultured until stable TEER was reached (2 days) before exposure to the test compounds for 24 hours. Media samples were collected at 4 and 24 hours from both compartments, with additional cell lysates collected at 24 hours. LDH assays measured cytotoxicity, TEER assessed barrier integrity, and LC-MS/MS quantified compound accumulation and transport.

Treatments:
Tobramycin (1mM)
Streptomycin (1mM)
Gentamicin (1mM)
Rifampin (10uM)
Indomethacin (10uM)
Tenofovir (TFV, 10uM)
Colchicine (10uM)
PFHxA (5uM)
PFBA (5uM)
PFBS (5uM)
PFHpA (5uM)
PFNA (5uM)
PFOS (5uM)
PFOA (5uM)
PFHxS (5uM)
PFUnDA (5uM)
PFDA (5uM)
4:2 FTS (5uM)
8:2 FTS (5uM)
PFPeA (5uM)
6:2 FTS (5uM)
TAB (50uM)
BP4 (10uM)

After exposure, media was sampled from both sides at 4 and 24 hours, and cell lysates were collected at 24 hours. LDH testing was performed on cell culture media, and TEER (cell barrier function) to determine cytotoxic effects on the RPTECs. LCMS/MS testing was performed to determine the fate of compounds added to the cells (cellular accumulation of compounds).

Conclusions:
--Testing concentrations of the compounds were sub-cytotoxic (which is good as we were not trying to cause cell stress, only investigate transport). LDH was not elevated, and TEER did not decrease with the exception of the positive control (TAB).
--Cells accumulated PFNA, PFOS, PFDA, 8-2FTS, and PFUnDA in large quantities. Other faster clearing compounds showed less cellular uptake.
--Cell uptake (lysate) results of this study were similar to the previously tested 96-well plate 2D study, however the Transwell platform provides additional information on cell transport between the “blood” (basolateral) and “urine” (apical) compartments.
--Results closely mirrored those from the previous TERT1-parental RPTEC study, except for tenofovir, which showed significantly higher accumulation in TERT1-OAT1 cells. This aligns with tenofovir being a known substrate for the OAT1 transporter.

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