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.

Keywords: ABCG2; BCRP; BeWo; Q141K; genistein; placenta; transporter; tributyltin; zearalenone.

Figure 1. Inhibition and Activation of Human BCRP ATPase by Environmental Chemicals

BCRP membranes were incubated with ATP and increasing concentrations of environmental chemicals in the (A) presence and (B) absence of sulfasalazine (10 μM) for 30 min at 37°C. Additional reactions included sodium orthovanadate in order to determine the ATPase activity attributed to transport. The amount of inorganic phosphate released was quantified by spectrophotometry following addition of a colorimetric reagent. Data are presented as the vanadate-sensitive mean ATPase activity ± SE of 3 replicates from one experiment. Following non-linear regression analysis, R² values ranged between 0.87–0.99 with the exception of genistein (in the presence of sulfasalazine) (R² = 0.13).

Figure 2. Inhibition of Human BCRP Transport in Inverted Vesicles by Environmental Chemicals

Inverted BCRP-expressing plasma membrane vesicles (20 μg) were incubated with Lucifer yellow (50 μM) for 2 min in the presence and absence of ATP and increasing concentrations of test chemical at 37°C. Data are presented as mean ATP-dependent BCRP activity ± SE of 3–4 replicates from one experiment. Non-linear regression analysis yielded R² values between 0.98–0.99.

Figure 3. Characterization of BCRP Expression and Function in Human BeWo Placental Cells

(A) Western blot of BCRP protein (~72 kDa) in BeWo cell lysates. GAPDH was used as a loading control. (B) Representative images of Hoechst 33342 accumulation (blue) in BeWo cells in the absence and presence of Ko143 (1 μM) collected using the Nexcelom Cellometer Vision. (C) Bar graphs represent the mean relative fluorescence ± SE (n = 4) adjusted for cell size and normalized to control (no Ko143). Asterisks (*) represent statistically significant differences (p < 0.05) compared with control.

Figure 4. Inhibition of BCRP Transport by Environmental Chemicals in Human BeWo Placental Cells

(A) Line graphs represent the distribution of individual cell Hoechst 33342 fluorescence in the absence and presence of the various environmental chemicals (50 μM). Each point represents the mean percentage of cells ± SE (n = 3–4) exhibiting a quantity of fluorescence. (B) Bar graphs represent the mean relative fluorescence ± SE (samples run in triplicate from three to four independent experiments). Data were adjusted for cell size and normalized to control (0 μM). Asterisks (*) represent statistically significant differences (p < 0.05) compared with control.

Figure 5. Interaction of Human BCRP Transporter with Zearalenone in BCRP-Overexpressing Cells

(A) Western blot of BCRP protein (~72 kDa) in cell lysates from HEK cells overexpressing empty vector (EV), wild-type BCRP (WT) and the Q141K BCRP variant (Q141K). GAPDH was used as a loading control. BCRP protein expression was semi-quantified and presented as a bar graph. (B) Accumulation of Hoechst 33342 (7 μM) was quantified using the Nexcelom Cellometer Vision. Bar graphs represent the mean relative fluorescence ± SE (n = 4) adjusted for cell size. (C) Cell viability was assessed using the alamar Blue Assay following exposure to mitoxantrone (5 μM) and zearalenone (100 μM) for 72 h. Data were normalized to untreated control cells and presented as mean ± SE. (D) Accumulation of Hoechst 33342 (7 μM) was quantified in WT cells exposed to increasing concentrations of zearalenone using the Nexcelom Cellometer Vision. Bar graphs represent the mean relative fluorescence (normalized to 0 μM) ± SE (n = 4 from one experiment) adjusted for cell size. Asterisks (*) represent statistically significant differences (p < 0.05) compared with control (0 μM or EV). Daggers (†) represent statistically significant differences (p < 0.05) compared with WT.

Figure 6. Transport of Zearalenone by the Human BCRP Transporter

(A) HEK cells overexpressing empty vector (EV), wild-type BCRP (WT) and the Q141K BCRP variant (Q141K) were treated with zearalenone (50 μM) for 30 min (uptake period), washed, and then incubated in fresh culture media for 60 min (efflux period). (B) Human BeWo placental cells were treated with zearalenone (10 μM) in the presence and absence of the BCRP inhibitor, Ko143 (1 μM) for 30 min (uptake period), washed, and then incubated in fresh culture media with or without Ko143 (1 μM) for 60 min (efflux period). Intracellular zearalenone accumulation was quantified by ELISA and adjusted to protein concentrations of the cellular lysates. Values were normalized to control (EV or no Ko143). Data are presented as mean ± SE (n = 12–15). (C) Western blot of BCRP protein (~72 kDa) in cell lysates from BeWo cells treated with 0, 1 or 10 μM zearalenone for 48 h. β-actin was used as a loading control. BCRP protein expression was semi-quantified and presented as a bar graph. Asterisks (*) represent statistically significant differences (p < 0.05) compared to EV cells. Daggers (†) represent statistically significant differences (p < 0.05) compared to no Ko143.