Folate transfer in placental choriocarcinoma cells treated with valproic acid: Insights gained through comparisons with the amide derivative sec-butylpropylacetamide and its individual stereoisomers

Abstract

Objective: We have previously shown that valproic acid (VPA) alters the expression of placental carriers of essential nutrients, including folates. Here, we exposed a placental cell line to VPA, its central nervous system-active amide derivative sec-butylpropylacetamide (SPD), and its individual stereoisomers to address the question of whether folate carrier expression can predict the teratogenicity of VPA-related compounds. We additionally conducted a pilot analysis of folate transfer across placental cell monolayers to estimate the translation of altered carrier expression to carrier activity.

Methods: BeWo cells were incubated for 2 or 5 days with racemic SPD, its stereoisomers (2S,3S)-SPD, (2R,3S)-SPD, and (2R,3R)-SPD (previously found to be teratogenic at high doses in mice; .5 or 1 mmol·L^-1), VPA (1 mmol·L^-1 = 144 mg/L), or their vehicle. Expression of FOLR1 (folate receptor alpha), SLC19A1 (reduced folate carrier), and ABCG2 (breast cancer resistance protein) was measured by real-time polymerase chain reaction. Folate transfer across monolayers of BeWo b30 cells exposed to .5 mmol·L^-1 (2R,3R)-SPD or 1 mmol·L^-1 VPA was quantified by liquid chromatography-mass spectrometry analysis.

Results: At 1 mmol·L^-1, racemic SPD, (2S,3S)-SPD, and (2R,3S)-SPD reduced by twofold SLC19A1 expression, similar to VPA (p < .001). SPD and its enantiomers induced FOLR1 expression by up to twofold (p < .05) or did not significantly affect it, and racemic SPD increased ABCG2 expression (p < .01). After 5 days, VPA, but not (2R,3R)-SPD, enhanced both maternal-to-fetal and fetal-to-maternal folate transfer (p < .01), resulting in a 15% increase in net transfer in the fetal direction.

Significance: Altered expression of the studied carriers could not explain the folate transfer kinetics across placental cell monolayers. Future studies should assess the effects of VPA and other antiseizure medications on transplacental transfer of essential compounds in vivo and the ability to predict it by functional in vitro assays.

Keywords: BeWo cells; antiseizure medications; folate carriers; pregnancy; teratogenicity.

FIGURE 1

Chemical structures of valproic acid (VPA), sec‐butylpropylacetamide (SPD), and its individual stereoisomers (2S,3S)‐SPD, (2R,3S)‐SPD, and (2R,3R)‐SPD.

FIGURE 2

Folate carrier expression after incubation with racemic sec‐butylpropylacetamide (SPD), (2S,3S)‐SPD, (2R,3S)‐SPD, and (2R,3R)‐SPD compared with the vehicle and 1 mmol·L⁻¹ valproic acid (VPA). (A, B) FOLR1 (folate receptor alpha) expression after 2 (a) and 5 (B) incubation days. (C, D) SLC19A1 (reduced folate carrier) expression after 2 (C) and 5 (D) days. (E, F) ABCG2 (breast cancer resistance protein) expression after 2 (E) and 5 (F) days. Data are presented as median and interquartile range. *p < .05, **p < .01, ***p < .001; Kruskal–Wallis test followed by Dunn posttest.

FIGURE 3

Transepithelial electrical resistance (TEER) values before (day −1) and along the 5‐day treatment with the tested compounds (beginning immediately after the day 0 measurement; Ω*cm²). BCRP, breast cancer resistance protein; FRα, folate receptor α; RFC, reduced folate carrier; SPD, sec‐butylpropylacetamide; VPA, valproic acid.

FIGURE 4

Effects of (2R,3R)‐sec‐butylpropylacetamide (SPD) and valproic acid (VPA) on folic acid (FA) transfer across BeWo cell monolayers. FA transfer was measured after 2 (A, B, E, F) or 5 (C, D, G, H) incubation days with the vehicle, (2R,3R)‐SPD, and VPA. (A, C) Concentrations (ng/mL) at the basolateral side after 4‐h incubation, apical‐to‐basolateral transfer. (B, D) Concentrations at the apical side, basolateral‐to‐apical transfer. (E, G) Papp of apical‐to‐basolateral transfer. (F, H) Papp of basolateral‐to‐apical transfer. FA concentrations at the donor side were not affected by the treatments (not shown). **p < .01; Kruskal–Wallis test followed by Dunn posttest.

FIGURE 5

Boron‐dipyrromethene prazosin (BP) kinetics in (2R,2S)‐sec‐butylpropylacetamide (SPD)‐ and valproic acid (VPA)‐treated BeWo cell monolayers after 5 incubation days. (A, B) Signal intensity of the receiver compartment medium. (A) Apical‐to‐basolateral transfer experiment. (B) Basolateral‐to apical transfer. (C, D) Intracellular BP intensity normalized to cell count. (C) Apical‐to‐basolateral transfer. (D) Basolateral‐to apical transfer. (E–G) Representative images of BeWo b30 cell monolayers exposed for 1 h to BP in the presence of the vehicle (E), (2R,2S)‐SPD (F), or VPA (G).