Excess Folic Acid Supplementation before and during Pregnancy and Lactation Alters Behaviors and Brain Gene Expression in Female Mouse Offspring

Abstract

Use of folic acid (FA) during early pregnancy protects against birth defects. However, excess FA has shown gender-specific neurodevelopmental toxicity. Previously, we fed the mice with 2.5 times the recommended amount of FA one week prior to mating and during the pregnancy and lactation periods, and detected the activated expression of Fos and related genes in the brains of weaning male offspring, as well as behavioral abnormalities in the adults. Here, we studied whether female offspring were affected by the same dosage of FA. An open field test, three-chamber social approach and social novelty test, an elevated plus-maze, rotarod test and the Morris water maze task were used to evaluate their behaviors. RNA sequencing was performed to identify differentially expressed genes in the brains. Quantitative real time-PCR (qRT-PCR) and Western blots were applied to verify the changes in gene expression. We found increased anxiety and impaired exploratory behavior, motor coordination and spatial memory in FA-exposed females. The brain transcriptome revealed 36 up-regulated and 79 down-regulated genes in their brains at weaning. The increase of Tlr1; Sult1a1; Tph2; Acacb; Etnppl; Angptl4 and Apold1, as well as a decrease of Ppara mRNA were confirmed by qRT-PCR. Among these genes; the mRNA levels of Etnppl; Angptl4andApold1 were increased in the both FA-exposed female and male brains. The elevation of Sult1a1 protein was confirmed by Western blots. Our data suggest that excess FA alteres brain gene expression and behaviors in female offspring, of which certain genes show apparent gender specificity.

Oxidative damage contributes to bisphenol S-induced development block at 2-cell stage preimplantation embryos in mice through inhibiting of embryonic genome activation

“…The experimental results showed that 1200 U/mL SOD can effectively relieve 2-cell block, but CoQ10 and FA did not. This may be due to the low toxicity of CoQ10 31 and FA 32 , and the fact that SOD, as an antioxidant enzyme, has a direct effect on oxidative damage. The following experiments were carried out to test whether adding 1200 U/mL SOD in a culture medium containing 10 –4 mol/L BPS can also improve other phenotypes.…”

Risk of Excess Maternal Folic Acid Supplementation in Offspring

“…Maternal FA supplementation typically comprises three phases: pre-mating, gestation, and lactation. The pre-mating period varies in duration, from as short as one week [ 59 , 60 , 61 ] to as long as six weeks [ 62 ], as in the study by Henzel et al, where female mice received FA diets for six weeks before mating and then returned to a control diet [ 62 ]. In contrast, some studies applied FA diets during all three phases to mimic the FA supplementation scenario in the human population [ 60 , 61 , 63 ].…”

“…In contrast, subsequent researchers reached a consensus that 2 mg FA/kg diet is the recommended level for rodents, and this criteria was applied to the most widely used with an AIN-93 diet [ 67 ]. As such, most recent studies used a control diet containing 2 mg/kg FA [ 56 , 57 , 58 , 60 , 61 , 63 ], with the exception of Henzel et al, who used a slightly different diet containing 2.3 mg/kg FA as the control [ 62 ]. For the FA-supplemented diet, Barua et al, who used 0.4 mg/kg FA for the control diet (CD), utilized 4 mg/kg FA, a tenfold higher level [ 59 ].…”

“…Given that 2 mg/kg FA is considered to be the conventional level for rodents, a 10 times higher level of FA (20 mg/kg) is considered to be the high FA dosage [ 56 , 60 , 63 ]. Additionally, a few studies explored the influence of moderate FA supplementation in offspring and used either 2.5 times higher (5 mg/kg FA) [ 60 , 61 ] or 5 times higher (10 mg/kg FA) [ 57 , 58 ] levels of FA compared to the control diet. In this review, we excluded references that used 0.4 mg/kg FA as the control diet, as it falls outside the scope.…”

“…Most studies supplemented the desired concentration of FA in the diet provided by the manufacturers. However, Chu, D. et al and Yang, X. et al from the same research group used the control diet containing 2 mg/kg FA for all mice and supplemented FA in the drinking water at the levels of 0 mg/L (control), 3.75 mg/L (MFA), or 22.5 mg/L (HFA) [ 60 , 61 ]. With an estimated daily consumption of 5 g diet and 4~8 mL water, the MFA and HFA groups received at least 2.5 times and 10 times more FA, respectively.…”