Lérica Le Roux-Pullen, Jeroen J.M.W. Van den Heuvel, Noraly B. Jonis, Tom Scheer-Weijers, Ilse R. Dubbelboer, Jan B. Koenderink, Frans G.M. Russel, Ronette Gehring
{"title":"化合物与人类和乳用动物 BCRP 同源物相互作用的体外筛选模型。","authors":"Lérica Le Roux-Pullen, Jeroen J.M.W. Van den Heuvel, Noraly B. Jonis, Tom Scheer-Weijers, Ilse R. Dubbelboer, Jan B. Koenderink, Frans G.M. Russel, Ronette Gehring","doi":"10.1111/jvp.13460","DOIUrl":null,"url":null,"abstract":"<p>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, [<sup>3</sup>H] estrone-3-sulfate ([<sup>3</sup>H]E<sub>1</sub>S) influx was measured. We also determined the Michaelis–Menten constant (Km) and Vmax of [<sup>3</sup>H]E<sub>1</sub>S 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 [<sup>3</sup>H]E<sub>1</sub>S. 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.</p>","PeriodicalId":17596,"journal":{"name":"Journal of veterinary pharmacology and therapeutics","volume":"47 5","pages":"437-441"},"PeriodicalIF":1.5000,"publicationDate":"2024-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/jvp.13460","citationCount":"0","resultStr":"{\"title\":\"In vitro screening model for compound interactions with human and dairy animal BCRP orthologs\",\"authors\":\"Lérica Le Roux-Pullen, Jeroen J.M.W. 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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, [<sup>3</sup>H] estrone-3-sulfate ([<sup>3</sup>H]E<sub>1</sub>S) influx was measured. We also determined the Michaelis–Menten constant (Km) and Vmax of [<sup>3</sup>H]E<sub>1</sub>S 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 [<sup>3</sup>H]E<sub>1</sub>S. The values were not significantly different between species for the model substrates tested. 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In vitro screening model for compound interactions with human and dairy animal BCRP orthologs
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.
期刊介绍:
The Journal of Veterinary Pharmacology and Therapeutics (JVPT) is an international journal devoted to the publication of scientific papers in the basic and clinical aspects of veterinary pharmacology and toxicology, whether the study is in vitro, in vivo, ex vivo or in silico. The Journal is a forum for recent scientific information and developments in the discipline of veterinary pharmacology, including toxicology and therapeutics. Studies that are entirely in vitro will not be considered within the scope of JVPT unless the study has direct relevance to the use of the drug (including toxicants and feed additives) in veterinary species, or that it can be clearly demonstrated that a similar outcome would be expected in vivo. These studies should consider approved or widely used veterinary drugs and/or drugs with broad applicability to veterinary species.