In vitro screening of environmental chemicals identifies zearalenone as a novel substrate of the placental BCRP/ABCG2 transporter

Abstract

The BCRP (ABCG2) transporter is responsible for the efflux of chemicals from the placenta to the maternal circulation. Inhibition of BCRP activity could enhance exposure of offspring to environmental chemicals leading to altered reproductive, endocrine, and metabolic development. The purpose of this study was to characterize environmental chemicals as potential substrates and inhibitors of the human placental BCRP transporter. The interaction of BCRP with a panel of environmental chemicals was assessed using the ATPase and inverted plasma membrane vesicle assays as well as a cell-based fluorescent substrate competition assay. Human HEK cells transfected with wild-type BCRP or the Q141K genetic variant, as well as BeWo placental cells that endogenously express BCRP were used to further test inhibitor and substrate interactions. To varying degrees, the eleven chemicals inhibited BCRP activity in activated ATPase membranes and inverted membrane vesicles. Further, genistein, zearalenone, and tributyltin increased the retention of the fluorescent BCRP substrate, Hoechst 33342, between 50–100% in BeWo cells. Additional experiments characterized the mycotoxin and environmental estrogen, zearalenone, as a novel substrate and inhibitor of BCRP in WT-BCRP and BeWo cells. Interestingly, the BCRP genetic variant Q141K exhibited reduced efflux of zearalenone compared to the wild-type protein. Taken together, screening assays and direct quantification experiments identified zearalenone as a novel human BCRP substrate. Additional in vivo studies are needed to directly determine whether placental BCRP prevents fetal exposure to zearalenone.

Localization of the placental BCRP/ ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity

“…Hoechst 33342 is a fluorescent substrate often used for measuring BCRP efflux activity [31], while zearalenone is an estrogenic mycotoxin produced by fungi that occur naturally on cereal crops. We previously identified zearalenone as a novel substrate for BCRP [9]. In utero exposure to zearalenone has been shown to cause developmental toxicities including feminization, mammary epithelial proliferation and precocious puberty [40].…”

“…The transport activity of BCRP in BeWo cells was measured by analyzing the cellular retention of two BCRP transporter substrates, Hoescht 33342 and zearalenone, an estrogenic mycotoxin [9, 31]. …”

“…After 30 min at 37°C, cells were washed, collected in lysis buffer and stored at −80°C. Quantification of intracellular ZLN was performed using an ELISA kit (Abnova, Taipei City, Taiwan), and standards were prepared in lysis buffer [9]. …”

Down-regulation of ABCG2 and ABCB4 transporters in the placenta of rats exposed to cadmium

“…They have ATP-binding sites which play a fundamental role in the transportation of substrates out of cells or into subcellular compartments against gradient fueled by ATP hydrolysis [27, 28]. ABCG2, also named the breast cancer resistance protein (BCRP), was a drug efflux transmembrane protein [29].…”

Buprenorphine, Norbuprenorphine, R-Methadone, and S-Methadone Upregulate BCRP/ABCG2 Expression by Activating Aryl Hydrocarbon Receptor in Human Placental Trophoblasts

“…Efflux activities of BCRP before and after drug treatment were measured by quantifying intracellular accumulation of Hoechst33342 as previously described for Jar and BeWo cells (Mason et al, 2014;Xiao et al, 2015) with slight modifications. Briefly, a serum-starved confluent monolayer of JEG3 or BeWo cells grown in 96-well plates was treated with a drug or 3-MC at the concentrations indicated for 24 hours.…”