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In vitro screening model for compound interactions with human and dairy animal BCRP orthologs
Lérica Le Roux-Pullen1, Jeroen J M W Van den Heuvel2, Noraly B Jonis2
1Faculty of Veterinary Medicine, Institute of Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands.
Abstract:
Orthologs of breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) efflux transmembrane transporter, are present in several species. The list of compounds known to interact with BCRP is growing, and many questions remain concerning species-specific variations in substrate specificity and affinity and the potency of inhibitors. As the most abundant efflux transporter known to be present in the blood-milk barrier, BCRP can increase the elimination of certain xenobiotics to milk, posing a risk for suckling offspring and dairy product consumers. Here we developed a model that can be employed to investigate species-specific differences between BCRP substrates and inhibitors. Membrane vesicles were isolated from transiently transduced human embryonic kidney (HEK) 293 cells, overexpressing BCRP, with human, bovine, caprine, and ovine cDNA sequences. To confirm BCRP transport activity in the transduced cells, D-luciferin efflux was measured and to confirm transport activity in the membrane vesicles, [3H] estrone-3-sulfate ([3H]E1S) influx was measured. We also determined the Michaelis-Menten constant (Km) and Vmax of [3H]E1S for each species. We have developed an in vitro transport model to study differences in compound interactions with BCRP orthologs from milk-producing animal species and humans. BCRP transport activity was demonstrated in the species-specific transduced cells by a reduced accumulation of D-luciferin compared with the control cells, indicating BCRP-mediated efflux of D-luciferin. Functionality of the membrane vesicle model was demonstrated by confirming ATP-dependent transport and by quantifying the kinetic parameters, Km and Vmax for the model substrate [3H]E1S. The values were not significantly different between species for the model substrates tested. This model can be insightful for appropriate inter-species extrapolations and risk assessments of xenobiotics in lactating woman and dairy animals.
Insights
Researchers developed a new in vitro model to study species-specific differences in breast cancer resistance protein (BCRP) interactions. This model aids in assessing risks of xenobiotics in humans and dairy animals.
Area of Science:
- Pharmacology
- Biochemistry
- Toxicology
Background:
- Breast cancer resistance protein (BCRP/ABCG2) is a key efflux transporter found across species.
- BCRP in the blood-milk barrier influences xenobiotic elimination into milk, posing risks to offspring and consumers.
- Understanding species-specific BCRP variations is crucial for safety assessments.
Purpose of the Study:
- To develop an in vitro model for investigating species-specific differences in BCRP substrates and inhibitors.
- To compare BCRP orthologs from human, bovine, caprine, and ovine species.
- To facilitate inter-species extrapolations for risk assessments of xenobiotics.
Main Methods:
- Overexpressed human, bovine, caprine, and ovine BCRP in HEK293 cells.
- Isolated membrane vesicles for in vitro transport assays.
- Measured D-luciferin efflux and [3H]estrone-3-sulfate influx to confirm BCRP activity and kinetics (Km, Vmax).
Main Results:
- Demonstrated BCRP-mediated D-luciferin efflux in species-specific cell lines.
- Confirmed functionality of the membrane vesicle model with ATP-dependent transport.
- Found no significant species-specific differences in kinetic parameters (Km, Vmax) for the model substrate [3H]E1S.
Conclusions:
- An in vitro model was successfully developed to study species-specific BCRP interactions.
- The model allows for the investigation of xenobiotic transport differences across species.
- This tool is valuable for inter-species risk assessments concerning BCRP and xenobiotics in lactating mammals.
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