Prenatal Choline Supplementation Alters One Carbon Metabolites in a Rat Model of Periconceptional Alcohol Exposure

Abstract

Prenatal alcohol exposure disturbs fetal and placental growth and can alter DNA methylation (DNAm). Supplementation with the methyl donor choline can increase fetal and placental growth and restore DNAm, suggesting converging effects on one-carbon metabolism (1CM). We investigated the impact of periconceptional ethanol (PCE) exposure and prenatal choline supplementation on 1CM in maternal, placental, and fetal compartments. Female Sprague Dawley rats were given a liquid diet containing 12.5% ethanol (PCE) or 0% ethanol (control) for 4 days before and 4 days after conception. Dams were then placed on chow with different concentrations of choline (1.6 g, 2.6 g, or 7.2 g choline/kg chow). Plasma and tissues were collected in late gestation for the analysis of 1CM components by means of mass spectrometry and real-time PCR. PCE reduced placental components of 1CM, particularly those relating to folate metabolism, resulting in a 3−7.5-fold reduction in the ratio of s-adenosylmethionine:s-adenosylhomocysteine (SAM:SAH) (p < 0.0001). Choline supplementation increased placental 1CM components and the SAM:SAH ratio (3.5−14.5-fold, p < 0.0001). In the maternal and fetal compartments, PCE had little effect, whereas choline increased components of 1CM. This suggests that PCE impairs fetal development via altered placental 1CM, highlighting its role in modulating nutritional inputs to optimize fetal development.

Keywords: DNA methylation; mass spectrometry; maternal nutrition; methyl group; one carbon metabolism; placenta; prenatal alcohol.

Figure 1

Illustration of the one-carbon metabolism (1CM) pathway and choline derivatives. The 1CM pathway is divided into the folate cycle, methionine cycle, and the choline pathway. The choline derivatives acetylcholine (Ach) and trimethylamine oxide (TMAO), and the endogenous choline synthesis pathway, catalyzed by phosphatidylethanolamine methyltransferase (PEMT), are also shown. THF = tetrahydrofolate; 5MTHF = 5-methyl-tetrahydrofolate; MTHFR = methylenetetrahydrofolate reductase; MTR = methionine synthase; MTRR = methionine synthase reductase; SAM = s-adenosylmethionine; SAH = s-adenosylhomocysteine; Hcy = homocysteine; DMG = dimethylglycine; BHMT = betaine homocysteine methyltransferase.

Figure 2

The effects of periconceptional ethanol and choline supplementation on the one-carbon metabolism (1CM) pathway in late gestation maternal plasma. (A) Principal components analysis (PCA) of 12 molecules from the 1CM pathway across the 6 experimental groups. Molecules were measured using mass spectrometry. Standard chow (1.6 choline/kg chow, Std) with control (0% EtOH, Con) liquid diet (Std-Con, red); Std with periconceptional ethanol (12.5% v/v EtOH, PCE) liquid diet (Std-PCE, green); intermediate chow (2.6 g choline/kg chow, Int) with Con (Int-Con, dark blue); Int with PCE (Int-PCE, light blue); supplemented chow (7.2 g choline/kg chow, Supp) with Con (Supp-Con, pink); Supp with PCE (Supp-PCE, yellow). Individual analysis of (B) 5-methyltetrahydrofolate (5MTHF) and (C) vitamin B2. Open bars are Con and grey bars are PCE (n = 5–9 per group). Data expressed as fold-change relative to the Std-Con group (mean ± SEM). Significant differences were identified via two-way ANOVA and Tukey’s post hoc analysis. * p < 0.05, *** p < 0.001 for differences between Con and PCE within each chow group; chow effects between Con groups are shown by lower-case letters, and between PCE groups by upper-case letters.

Figure 3

The effects of periconceptional ethanol and choline supplementation on the one-carbon metabolism (1CM) pathway in the placenta. Principal components analysis (PCA) of 12 molecules from the 1CM pathway across the 6 experimental groups in placentas of (A) male and (B) female fetuses. Molecules were measured using mass spectrometry. Standard chow (1.6 g choline/kg chow, Std) with control (0% EtOH, Con) liquid diet (Std-Con, red); Std with periconceptional ethanol (12.5% v/v EtOH, PCE) liquid diet (Std-PCE, green); intermediate chow (2.6 g choline/kg chow, Int) with Con (Int-Con, dark blue); Int with PCE (Int-PCE, light blue); supplemented chow (7.2 g choline/kg chow, Supp) with Con (Supp-Con, pink); Supp with PCE (Supp-PCE, yellow). Individual analysis of the s-adenosylmethionine:s-adenosylhomocysteine ratio (SAM:SAH) in placentas of (C) male and (D) female fetuses. Open bars are Con and grey bars are PCE (n = 6–9 per group). Data expressed as fold-change relative to the male Std-Con group (mean ± SEM). Kruskal–Wallis test for non-parametric data was used to identify significant differences across all 6 groups, and Dunn’s post hoc analysis for differences between Con and PCE within each chow group; * p < 0.05; differences between Con groups are shown by lower-case letters, and between PCE groups by upper-case letters.

Figure 4

The effects of periconceptional ethanol and maternal choline supplementation on specific components of the folate cycle and choline in placentas of male (A–D) and female (E–H) fetuses. (A,E) folic acid; (B,F) vitamin B12; (C,G) 5-methyl tetrahydrofolate (5MTHF); and (D,H) choline. During the periconceptional period, dams received a control liquid diet (Con; open bars) or a liquid diet containing 12.5% v/v ethanol (PCE; grey bars). For the remainder of pregnancy, dams received either standard chow (Std), intermediate chow (Int), or supplemented chow (Supp), with increasing levels of choline across these groups (1.6 g, 2.6 g, or 7.2 g choline/kg chow), n = 6–9 per group. Molecules were measured using mass spectrometry and data are expressed as fold-change relative to the average of the male Std-Con group (mean ± SEM). Significant differences due to alcohol exposure or chow group were identified by two-way ANOVA and Tukey’s post hoc analysis. ** p < 0.01; *** p < 0.001, and **** p < 0.0001 for differences between Con and PCE within each chow group; chow effects between Con groups are shown by lower-case letters, and between PCE groups by upper-case letters.

Figure 5

The effects of periconceptional ethanol and maternal choline supplementation on expression of key enzymes of one-carbon metabolism in placentas of male (A–D) and female (E–H) fetuses. Mtrr (methionine synthase reductase); (B,F) Mtr (methionine synthase); (C,G) Mthfr (5–10 methylenetetrahydrofolate reductase); (D,H) Bhmt (betaine homocysteine methyl transferase). During the periconceptional period, dams received a control liquid diet (Con; open bars) or a liquid diet containing 12.5% v/v ethanol (PCE; grey bars). For the remainder of pregnancy, dams received either standard chow (Std), intermediate chow (Int), or supplemented chow (Supp), with increasing levels of choline across these groups (1.6 g, 2.6 g, or 7.2 g choline/kg chow), n = 7–10 per group. Gene expression was analyzed relative to the geometric mean of Actb and RPL19, with fold-change relative to the male Std-Con group. All data are presented as mean ± SEM. Significant differences due to alcohol exposure or chow group were identified via two-way ANOVA and Tukey’s post hoc analysis for parametric data, and via the Kruskal–Wallis test across all 6 groups, and Dunn’s post hoc analysis for non-parametric data. Results of post hoc analysis of chow effects are shown by lower case letters for control groups; upper-case letters for alcohol exposure groups.

Figure 6

The effects of periconceptional ethanol and choline supplementation on the one-carbon metabolism (1CM) pathway in late-gestation fetal plasma. Principal components analysis (PCA) of 10 molecules from the 1CM pathway across the 6 experimental groups in fetal plasma of (A) males and (B) females. Molecules were measured using mass spectrometry. Standard chow (1.6 choline/kg chow, Std) with control (0% EtOH, Con) liquid diet (Std-Con, red); Std with periconceptional ethanol (12.5% v/v EtOH, PCE) liquid diet (Std-PCE, green); intermediate chow (2.6 g choline/kg chow, Int) with Con (Int-Con, dark blue); Int with PCE (Int-PCE, light blue); supplemented chow (7.2 g choline/kg chow, Supp) with Con (Supp-Con, pink); Supp with PCE (Supp-PCE, yellow).

Figure 7

The effects of periconceptional ethanol and choline supplementation on acetylcholine (Ach) levels in placentas of male and female fetuses (A,C) and male and female fetal brain (B,D). During the periconceptional period, dams received a control liquid diet (Con; open bars) or a liquid diet containing 12.5% v/v ethanol (PCE; grey bars). For the remainder of pregnancy, dams received either standard chow (Std), intermediate chow (Int), or supplemented chow (Supp), with increasing levels of choline across these groups (1.6 g, 2.6 g, or 7.2 g choline/kg chow), n = 6–9 per group. Molecules were measured using mass spectrometry and data expressed as fold-change relative to the average of the male Std-Con group (mean ± SEM). Significant differences due to alcohol exposure or chow group were identified via two-way ANOVA and Tukey’s post hoc analysis. **** p < 0.0001 for differences between Con and PCE within each chow group; chow effects between Con groups are shown by lower-case letters, and between PCE groups by upper-case letters.

Figure 8

The effects of periconceptional ethanol and choline supplementation on trimethylamine oxide (TMAO) levels in placentas of male and female fetuses (A,E), male and female fetal plasma (B,F), male and female fetal liver (C,G), and male and female fetal brain (D,H). During the periconceptional period, dams received a control liquid diet (Con; open bars) or a liquid diet containing 12.5% v/v ethanol (PCE; grey bars). For the remainder of pregnancy, dams received either standard chow (Std), intermediate chow (Int), or supplemented chow (Supp), with increasing levels of choline across these groups (1.6 g, 2.6 g, or 7.2 g choline/kg chow), n = 4–9 per group. Molecules were measured using mass spectrometry and data expressed as fold-change relative to the average of the male Std-Con group (mean ± SEM). Significant differences due to alcohol exposure or chow group were identified via two-way ANOVA and Tukey’s post hoc analysis. **** p < 0.0001 for differences between Con and PCE within each chow group; chow effects between Con groups are shown by lower-case letters, and between PCE groups by upper-case letters.