Mechanisms of pH-Dependent Uptake of Ionizable Organic Chemicals by Fish from Oil Sands Process-Affected Water (OSPW)

Abstract

Uptake and effects of ionizable organic chemicals (IOCs) that are weak acids in aqueous solution by fish can differ as a function of pH. While the pH-dependent behavior of select IOCs is well-understood, complex mixtures of IOCs, e.g., from oil sands process-affected water (OSPW), have not yet been studied systematically. Here, we established an in vitro screening method using the rainbow trout gill cell line, RTgill-W1, to investigate pH-dependent cytotoxicity and permeation of IOCs across cultured epithelia using ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UPLC-HRMS). The assay was benchmarked using model chemicals and technical mixtures, and then used to characterize fractions and reconstituted extracts of field-collected OSPW. Significant pH-dependent cytotoxicity of individual IOCs, acidic fractions, and reconstituted extracts of OSPW was observed. In vitro data were in good agreement with data from a 96 h in vivo exposure experiment with juvenile rainbow trout. Permeation of some IOCs from OSPW was mediated by active transport, as revealed by studies in which inhibitors of these active transport mechanisms were applied. We conclude that the RTgill-W1 in vitro assay is useful for the screening of pH-dependent uptake of IOCs in fish, and has applications for in vitro–in vivo extrapolation, and prioritization of chemicals in nontarget screenings.

Effect of Water pH on the Uptake of Acidic (Ibuprofen) and Basic (Propranolol) Drugs in a Fish Gill Cell Culture Model

“…Cyclosporine A, an inhibitor of the solute carrier and ATP-binding cassette transport family of proteins, reduced the appearance of chemical species in the basolateral compartment . This is of interest because the TEER of RT-gill W1 grown on the inserts is ∼25 Ω cm 2 , indicating a relatively leaky epithelium that would presumably allow paracellular movement of chemicals down a concentration gradient . In a previous study, numerous SLC and ABC transporter blockers including cyclosporine A, cimetidine, MK571, and quinidine reduced the propranolol transport across the FIGCS epithelium into the basolateral compartment .…”

“…Further support for the involvement of carrier-mediated transport of ionizable compounds across the fish gill comes from a recent study accessing the pH-dependent uptake of chemicals from oil sands process-affected water using the rainbow trout gill cell line RT-gill W1 grown on inserts . Cyclosporine A, an inhibitor of the solute carrier and ATP-binding cassette transport family of proteins, reduced the appearance of chemical species in the basolateral compartment . This is of interest because the TEER of RT-gill W1 grown on the inserts is ∼25 Ω cm 2 , indicating a relatively leaky epithelium that would presumably allow paracellular movement of chemicals down a concentration gradient .…”

“…A number of rainbow trout cell lines, derived from different tissues including the gill, and intact gills express transcripts for ABC transporters . Further support for the involvement of carrier-mediated transport of ionizable compounds across the fish gill comes from a recent study accessing the pH-dependent uptake of chemicals from oil sands process-affected water using the rainbow trout gill cell line RT-gill W1 grown on inserts . Cyclosporine A, an inhibitor of the solute carrier and ATP-binding cassette transport family of proteins, reduced the appearance of chemical species in the basolateral compartment .…”

Inflammation of Gill Epithelia in Fish Causes Increased Permeation of Petrogenic Polar Organic Chemicals via Disruption of Tight Junctions

“…To assess the effects of LPS on the permeability of simulated gill epithelia, RTgill-W1 cells were seeded to full confluency into 24-well plates with permeable ThinCert cell culture inserts (Greiner Bio-One, Frickenhausen, Germany) using previously established methods. 30 Briefly, cells were grown in these inserts under symmetric conditions (supplemented L-15 culture media in both apical and basolateral chamber) for 2 weeks, with one exchange of media after 7 days of incubation. Thereafter, cells were adapted to L-15/ex media in the apical chamber.…”

“…This study was conducted in accordance with Canadian Council on Animal Care regulations (University of Saskatchewan Animal Use Protocol 20170103), following previously published protocols. 30 LPS-spiked fish food was prepared by thoroughly mixing 10 mL of a 1.5-mg mL −1 solution of LPS in ultrapure water with 50 g of dry fish food (size #0 Biovita starter food, Bio-Oregon, Vancouver, Canada). The mixture was dried at 45 °C for 6 h and subsequently stored at 4 °C.…”