Placental BCRP/ABCG2 Transporter Prevents Fetal Exposure to the Estrogenic Mycotoxin Zearalenone

Abstract

In the placenta, the breast cancer resistance protein (BCRP)/ABCG2 efflux transporter limits the maternal-to-fetal transfer of drugs and chemicals. Previous research has pointed to the estrogenic mycotoxin zearalenone as a potential substrate for BCRP. Here, we sought to assess the role of the BCRP transporter in the transplacental disposition of zearalenone during pregnancy. In vitro transwell transport assays employing BCRP/Bcrp-transfected Madine-Darby canine kidney cells and BeWo trophoblasts with reduced BCRP expression were used to characterize the impact of BCRP on the bidirectional transport of zearalenone. In both models, the presence of BCRP protein increased the basolateral-to-apical transport and reduced the apical-to-basolateral transport of zearalenone over a 2-h period. In vivo pharmacokinetic analyses were then performed using pregnant wild-type and Bcrp-/- mice after a single tail vein injection of zearalenone. Zearalenone and its metabolite α-zearalenol were detectable in serum, placentas, and fetuses from all animals, and β-zearalenol was detected in serum and fetuses, but not placentas. There were no significant differences in the maternal serum concentrations of any analytes between the two genotypes. In Bcrp-/- mice, the free fetal concentrations of zearalenone, α-zearalenol, and β-zearalenol were increased by 115%, 84%, and 150%, respectively, when compared with wild-type mice. Concentrations of free zearalenone and α-zearalenol were elevated 145% and 78% in Bcrp-/- placentas, respectively, when compared with wild-type placentas. Taken together, these data indicate that the placental BCRP transporter functions to reduce the fetal accumulation of zearalenone, which may impact susceptibility to developmental toxicities associated with in utero zearalenone exposure.

Keywords: ABCG2; BCRP; mycotoxin; placenta; zearalenone.

Figure 1.

Characterization of BCRP expression and activity in hBCRP- and mBcrp-transfected MDCK cells. A, Western blot of transfected and control MDCK cells analyzing BCRP/Bcrp protein expression (72 kDa). β-actin (42 kDa) was used as a loading control. B, Retention of H33342 (10 μM) in empty vector control and transfected MDCK cells. Ko143 (1 μM) was used as a pharmacological inhibitor of BCRP/Bcrp (n = 6). Asterisks (*) represent statistically significant differences (p < .05) compared with control/vehicle. Daggers (†) represent statistically significant differences (p < .05) compared with vehicle within treatment group. Data represent the mean ± SD.

Figure 2.

Transport of glyburide and zearalenone by MDCK cells in transwell cultures. MDCK cells grown on transwell inserts were assessed for translocation of (A) BODIPY-glyburide (1 μM) and (B) zearalenone (50 μM) across cell monolayers for 2 h as described in the Materials and Methods. Data represent the mean pmol detected in the receiver compartment ± SD (n = 3 independent experiments). Asterisks (*) represent statistically significant differences (p < .05) compared with control cells.

Figure 3.

Basolateral-to-apical transport of zearalenone and Rhodamine 123 in MDCK cells in the presence of a MDR1 inhibitor. Control MDCK cells grown on transwell inserts were assessed for translocation of (A) the MDR1 substrate Rhodamine 123 (10 μM) and (B) zearalenone (50 μM) across cell monolayers for 2 h as described in the Materials and Methods. PSC833 (5 μM) was used as a pharmacological inhibitor of MDR1. Data represent the mean pmol detected in the receiver compartment ± SD (n = 3–4). Asterisks (*) represent statistically significant differences (p < .05) compared with control cells.

Figure 4.

Characterization of BCRP protein and activity in BeWo shBCRP cells. A, Western blot of BCRP protein (72 kDa) in lysates from BeWo cells after treatment with control or shBCRP lentiviral particles. GAPDH (37 kDa) was used as a loading control. B, Retention of H33342 (5 μM) in control and shBCRP BeWo cells. Ko143 (1 μM) was used as a pharmacological inhibitor of BCRP (n = 6). Asterisks (*) represent statistically significant differences (p < .05) compared with control. Data represent the mean ± SD.

Figure 5.

Transport of glyburide and zearalenone by BeWo cells in transwell cultures. BeWo cells grown on transwell inserts were assessed for translocation of (A) BODIPY-glyburide (1 μM) and (B) zearalenone (50 μM) across cell monolayers for 2 h as described in the Materials and Methods. Data represent the mean pmol detected in the receiver compartment ± SD (n = 3 independent experiments). Asterisks (*) represent statistically significant differences (p < .05) compared with control cells.

Figure 6.

Quantification of zearalenone and its metabolites in pregnant wild-type and Bcrp^−/− mice. A, Western blot of placental homogenates from pregnant wild-type and Bcrp^−/− mice analyzing BCRP protein expression (72 kDa) on gestation day 14. GAPDH (37 kDa) was used as a loading control. B–D, Concentration of zearalenone and its metabolites detected in serum (B), fetuses (C), and matched placentas (D) 1 h after tail vein injection as determined by LC-MS. Total concentration was determined after incubation of samples with β-glucuronidase overnight at 37°C. Data represent the mean ± SD (n = 4–5). Asterisks (*) represent statistically significant differences (p < .05) compared with wild-type mice.