Maternal folic acid consumption during gestation and its long-term effects on offspring ’ s liver : a systematic review

Objectives: describing the effects of maternal supplementation with folic acid (FA) exclusively during gestation on offspring’s liver at later stages in life. Supplementation with FA during gestation has been recommended by the medical society worldwide. The liver has a central role on the substances of metabolism and homeostasis and some studies have shown that a high intake of FA at other periods in life may cause hepatic damage. Methods: a systematic review through which the following databases were consulted: Medline, through platforms of Pubmed, Lilacs and Scielo. The research was performed by keywords such as: “Folic acid”, “Gestation”, “Rat”, “Offspring” and “Liver”. Articles which evaluate the effect of FA consumption during both gestation and lactation were


Introduction
Supplementation with folic acid (FA) during gestation has been recommended by the medical society worldwide.According to the World Health Organization, even in adequate diets, the amount of folic acid is not enough to supply the daily necessities which are getting higher in pregnant women.This can cause anemia, especially in young women. 1 Suitable diet consumption during gestation is worthwhile for normal developing of offsprings and may determine their metabolic and hormonal status at adulthood. 2,3It was stated that nutritional imbalance during critical periods, such as gestation, may predispose to diseases at adulthood.
The liver has been shown to be an important target organ for metabolic programming in animal models used to demonstrate diseases.Inadquate maternal diet influences this offspring's organ homeostasis and plays a central role on foetal programming. 4igh folic acid consumption during gestation can trigger an increase of body weight and metabolic syndrome of the offspring in later stages of life. 5It was demonstrated that maternal supplementation with high dose of this vitamin exacerbates the detrimental effects of high fat diets, such as glucose intolerance and insulin resistance, 3 besides changing the peroxisome proliferator activated receptors (PPARs) levels in some foetal tissues when consumed by them during gestation and/or lactation periods. 6he intake of a high folic acid diet at other periods in life besides gestational, may alter lipid metabolism and cause hepatic damage through hepatocytes degeneration. 7Marsillach et al. 8 have demonstrated that when rats received high dose folic acid supplementation for eight to ten weeks, they presented higher amount of collagen, stellate and apoptotic cells on the liver, along with changes in albumin and bilirubin serum levels.
The benefical effects of folic acid consumption by pregnant women are well documented with regards to some parameters, such as offspring's neural tube closing and better learning.In relation to the other organs and systems, there is no consensus in the literature about its benefits.Due to the liver having a central role on the substances of metabolism and homeostasis, it becomes a valuable organ to study effects of several diets and comprehend the metabolic programming.
Therefore, the goal of this study is to determine through a systematic review the effects of maternal supplementation with folic acid exclusively during gestation on the offspring's liver.This study has intended to contribute to the knowledge about the effects of this vitamin on offsprings whose dams have ingested, through previously published studies.

Chemistry
The folic acid vitamin (pteroylmonoglutamate -PteGlu) contains a 2-amino-4-hydroxy-pteridine (pterin, which contains a pyrazine ring) moiety linked to a p-aminobenzoylglutamate moiety through a methylene group.Folate reactions in mammalian cells involve the reduction of its pyrazine ring to the active form called tetrahydrofolate.Additionally, the glutamate chain of folate may be elongated by the addition of glutamate residues.Finally, folate may acquire one-carbon units at N-5 and/or N-10 positions. 9,10Indeed, folate is a coenzyme that participates of reactions involving the transfer of one-carbon moieties in nucleotide and amino acid metabolism. 10,11In such reactions, folate is found in the folylpolyglutamate form and the polyglutamate chain serves to chanelling the substrates in the active site of the enzyme.Furthermore, such reactions avoid the escape of intermediate products from the protein complex.The tissues retain folylpolyglutamates instead of monoglutamates, which are a form of transport in aqueous systems. 9,12olate is not produced by mammalian cells and may be acquired from diet.Microorganisms and plants synthesize the dihydrofolate (DHF) derivative.When reduced folates undergo oxidation, its vitamin activity is affected.Ascorbate (a reducing agent) or absence of oxygen may protect folates from oxidation and inactivation.Actually, ascorbate found in plasma and urine helps to maintain folate in a reduced form. 9

Transport across membranes
Folates uptake depends on transmembrane transporters to be effective.There are five proteins involved in folate transport across membranes.Two transporters are found in organelles (lysosomes and mitochondria). 13,14he Reduced Folate Carrier 1 (RFC1) is enconded by the RFC1 gene and is a transmembrane anionic exchanger whose activity is higher at physiological pH.RFC1 is a facilitative carrier and its activity is up-regulated by intracellular organic anions.RFC1 is expressed in fetal and adult tissues.Additionally, it is found in cultured cells and some

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Long-term effects of folic acid on offspring's liver tumor cells.RFC is characterized as saturable and has a low affinity for reduced folates (Kt is approximately 3 µM). 15he Proton Coupled Folate Transporter (PCFT) functions in acidic pH and its activity is decreased at physiological pH.Its affinity for folates is higher than that observed on RFC1. 16PCFT is found at high amounts in the small intestine, liver, placenta, brain, and among other tissues. 17he Multidrug Resistance-Associated Protein (MRP) also serves as folate exporters from different tissues.However, its affinity for folate is very low (Kt values 0.2-2 mM). 18itochondria and lysosomes also possess folate transporters.Mitochondrial transporter is specific for reduced folates (folic acid, for example, is not transported) and the role of lysosomal transporters are not completely understood regarding folates metabolism. 14,19olate may also be transported across membranes by three specific receptors (the folate receptors -FR α, β, γ), which are attached to the plasma membrane through a glycosylphosphatidylinositol anchor. 9,20FR-α is found in the apical membrane facing the circulation in choroid plexus, kidney proximal tubules, ovaries, lung alveolar cells, retinal pigment epithelial cells, and among others. 21R-β is expressed in placenta, spleen, and thymus.Moreover, FR-β is found in fetal tissue.FR-γ is expressed in other types of epithelial cells. 22FRs transport folates at slower rates than transmembrane transporters.However, deletion of a gene equivalent to FR-α in a mouse resulted in embryonic death. 20,23Rs also mediates binding of folates to intracellular proteins responsible for carrying folates in the cytosol. 24

Intestinal absorption
Polyglutamate folates obtained from diet are hydrolyzed to monoglutamate forms in the gut and then such mono forms are absorbed by the intestinal mucosa.The enzyme responsible for folylpolyglutamate hydrolysis is a brush-border membrane called γ-glutamyl hydrolase (γ-GH, or glutamate carboxypeptidase II).After hydrolysis, folate crosses basolateral membrane of mucosal cell and is released into the portal circulation. 9Intestinal mucosa contains both PCFT and RCF1.Due to the acidic pH bathing the mucosal cells, folic acid is transported at higher rates as reduced folate monoglutamates by PCFT, which is active at acidic pH (about 5.5). 25In such pH, RCF1 would not be effective in transporting folates, as mentioned above.The release of folates to the portal circulation is believed to occur through the low-affinity, high-capacity MRP3 transporters, since mucosal cells do not express RCF1 or PCFT. 13,25

Tissue uptake
In the mammalian plasma, the major forms of folates circulating are pteroylmonoglutamates (mainly 5-methyl-THF). 26Nonetheless, mammalian cells are not able to uptake long chain length of polyglutamates (three and above). 27The liver is able to absorb much of the folate that entered the portal circulation.Hepatic cells metabolize folate to polyglutamate derivatives retaining it or releasing it into the blood or bile. 27,28The folate form that predominates in the plasma (5-methyl-THF) may reflect that this is the major folate form in the cytosol of mammalian cells.In the plasma, folate may be found associated to proteins, as for instance albumin and a soluble form of FR.Such proteins are characterized to bind folates through a low-affinity association. 29he concentrations of folates are higher in red blood cells than in plasma. 30Folates may be retained in red blood cells due to its binding to hemoglobin.In the hepatocytes, the transport of folate is ATPdependent.Moreover, basolateral membranes of hepatic cells contain PCFT at high levels in addition to an electroneutral folate-H+ cotransporter that binds reduced and oxidized folates. 28Peripheral tissues primarily utilize RFC1 to transport folates inside cells. 29In the kidney proximal tubules, folate is reabsorbed and virtually no folate is found in the urine at normal folate ingestion conditions. 31nside mammalian cells, the major folate derivatives are found as folylpolyglutamate. 9The concentration of polyglutamates derivatives is about 10 to 30 µM in hepatic cells. 32In plasma of humans, folate concentration is found in the range of 10 to 30 nM. 27hus, total folate content in human body has been estimated to be around 10 to 100 mg (3-16 mg in liver). 9,26,27The generation of polyglutamate derivatives occurs in both cytosol and mitochondria and is required to folate accumulation inside cells. 19,33olate monoglutamate obtained from plasma may be converted to folylpolyglutamate by the cytosolic enzyme folylpoly-γ-glutamate synthetase or be transported to mitochondria by the folate transporter found in such organelle. 33Up to 50% of cellular folate is transported to mitochondria in some tissues. 33Mitochondria utilize folate derivatives in the synthesis and cleavage of glycine and to the degradation of choline. 33,34A folate pool is also detected in the nucleus and it serves to the thymidylate synthesis. 19,35Folate homeostasis is mainly maintained by the action of folylpoly-γ-glutamate synthetase and γ-glutamyl hydrolase (γ-GH). 36The former is required to generate polyglutamate derivatives leading to the accumulation of such derivatives inside cells (this enzyme is found in both cytosol and mitochondria). 35The later hydrolyzes polyglutamate chain to folates and is found in the lysosome. 37ther enzymes utilize folate derivatives in the onecarbon metabolism, as for instance glycine Nmethyltransferase, 10-formyl-THF dehydrogenase, and serine hydroxymethyltransferase.Such enzymes are found at high amounts in liver cells and are called intracellular folate-binding proteins. 38olate excretion occurs mainly through bile (100 µg/day). 27,39However, folate is reabsorbed in the small intestine.Furthermore, folate is filtered at the glomerulus and reabsorbed in the proximal renal tubule.No folate is found in urine.Instead, folate cleavage products are observed in the urine. 40

Methods
We conducted a systematic review, which has been performed by the utilization of different databases from November 2015.In order to obtain more knowledge about the effects of maternal folic acid consumption during gestation on the offspring's liver, scientific articles were published on the following databases: Medline, through the platforms of Pubmed, Lilacs, and Scielo.
In these databases, the research was performed through keywords such as: "Folic acid", "Gestation", and "Liver".These descriptors just were used jointly and with the filter AND for advanced searches.116 articles were obtained, distributed as follows: 34 on Lilacs, 82 on Medline and none on Scielo database.
The study goal was to determine the effects of maternal folic acid consumption specifically on the offspring's liver.The keyword "offspring" to others was added in order to direct papers researched which address the effects upon the offspring.At this phase, 26 articles were obtained, 5 on Lilacs, 21 on Medline and none on the Scielo database.
The following inclusion criteria were applied: (1) only studies with rats or mice, (2) only studies that have shown the effects of maternal consumption of folic acid on the offspring's liver.On the other hand, exclusion criteria were as follows: (1) studies which evaluate the effect of folic acid consumption during gestation associated to any disease; (2)  studies that evaluate maternal folic acid consumption during gestation and lactation or only during lactation and (3) articles that assess the folic acid consumption by their own offspring.
Applying such criteria, 5 articles were excluded because they appeared repeatedly in different databases, 4 were eliminated because evaluated intake of folic acid in different phases in life or was associated to another experimental model and 1 which assessed the effects on dams instead of on the offspring.2 studies were excluded because evaluated folic acid with the purpose of determing the effect on gestational hypertension and the last one assessed the folic acid intake by their own offspring.Finally, 3 papers were not regarded because of the lack of ability to verify the effect of folic acid on different parameters or tissues.Therefore, 11 articles were selected for this study, all being experimental works and whereby we extracted our results.

Results
The papers that have composed the sample of our study relate several effects of maternal supplementation with folic acid only during gestational period.Such effects are arranged by categories.

Effects of folic acid on the offspring's liver at molecular parameters
Maloney et al. 41 have studied the effects of a folic acid-deficient diet during early gestation and have demonstrated that it did not alter mRNA levels to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peroxisome proliferator-activated receptor alpha (PPARα), PPAR-gamma and glucocorticoid receptor (GccR) on male offsprings at six months of age.
According to those authors, it seems that event programming hepatic fat metabolism through PPAR isoforms, at least, occur later in foetal development and maternal deprivation of folic acid at early gestation does not change those proteins.
Recently, some studies have also investigated if folic acid supplementation is able to protect against detrimental effects of others nutritional disorders, such as under nutrition.For this purpose, Lillycrop et al. 42 evaluated whether the effects of the maternal protein-restricted diet can be ameliorated by supplementing it with folic acid exclusively during gestation in the offspring's liver.These authors found that supplementation of the protein restricted diet with folic acid prevented hypomethylation of GR and PPARα, and the associated increase in the expression of GR and PPARα.
Besides this, PPARα and GR receptor expression did not differ between the offspring of the control group and protein restricted with folic acid dams.It seems that folic acid consumption protects against harmful effects of deficient protein diet, avoiding disorders on expression of these receptors and metabolic alterations.
Meher et al. 43 evaluated the expression of several transcription factors and levels of global methylation on the offsprings' liver of dams fed with folic acid deficient diet during gestation.There was a reduction on the absolute weight of the liver along with changes on hepatic transcription factors and higher global DNA methylation.
These same authors comment that folate, as well as vitamin B12 regulate S-adenosyl methionine (SAM) generation, a molecule which is the universal methyl donor and crucial for many reactions and any change on it may promote disturbances on essential mechanisms, for instance, cell proliferation.

Effects of folic acid on offspring's liver at enzimatic/protein levels
Maloney et al. 41 have demonstrated that the level of proteins classified into three main groups, such as proteins associated with energy metabolism, antioxidant defence and amino acid and peptide metabolism were changed in the livers of the male offspring whose dams received folic acid deficient diet at 6 and 12 months of age.
was also alteration on GAPDH levels on the offspring's liver whose dams received FA deficient diet at 6 months of age.Although GAPDH is mainly known for its role in glycolysis, there is evidence to suggest that this protein is involved in the control of proliferation, apoptosis and transcription. 44n the other hand, Maloney et al. 41 demonstrated that enzyme activity of male offspring's liver from dams fed with FA deficient diet during the first five days of gestation was unchanged in these animals at 6 months.This paper suggests that the lack of FA on maternal diet at early gestation does not change this parameter in adulthood.

Effects of folic acid on offspring's liver at mineral amount and lipid composition
Król et al. 45 have shown that supplementation with FA during gestation did not change iron hepatic levels of the offspring.It seems that folic acid maternal supplementation was not able to change the mineral status of the young offspring.
In regards to hepatic lipid composition, maternal FA intake did not alter in the adult offspring.Burdge et al. 46 suggest that gender can determine differences in liver phospholipid fatty acid composition, but not maternal folic acid consumption.

Protective and antioxidant effect of folic acid on the offspring's liver
Ojeda et al. 47 demonstrated that a supplemented diet with selenuim (Se) plus folic acid to ethanol-exposed dams is able to restore Se pancreas concentration to a control status and displaces Se to the liver and brain.These authors related that exposure to ethanol during gestation decreases the levels of these nutrients and it disrupts oxidative balance. 47he chronic use of ethanol during pregnancy causes oxidative stress and liver disorder in the mother and her progeny by increasing the specific activity of glutathinone redutase (GR).This damage can be prevented by treatment of the pregnant mother with FA associated to selenium that reduces GR activity and increases glutathione levels. 48,49

Discussion
According to the results of this review, folic acid can protect against harmful effects of maternal protein restriction during gestation.When dams submitted to protein restriction during gestation receive higher doses of folic acid, hypomethyilation of dinucleotides in the PPARα promoter is prevented.Such hypomethylation occurs on liver offspring whose mothers were fed on low protein diet and lower doses of folic acid.Hypomethylation and the resulting increase in PPARα expression was prevented, reinforcing protective effect of folic acid. 50PPAR activities are central to lipid and carbohydrate homeostasis. 51owever, two other specific dinucleotides were hypermethylated in the offspring whose dams received higher doses of folic acid, which suggests that increased maternal folic acid intake can cause subtle effects, but may still alter gene function. 50hese results confirm that alterations in the amount of specific nutrients in the maternal diet during pregnancy modify the metabolic phenotype of the offspring.
Hyper or hypomethylation processes may lead to alterations on gene transcription and as consequence, change the cellular metabolism.As peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor protein expressed in the liver and through the ability of FA to increase glutathione levels on tissues, since glutathione is the most important scavenger-endogenous of the free radicals.Folic acid can reverse apoptosis and cell damage caused by toxic agents. 58sing rats with streptozotocin-induced diabetes, Zal et al. 59 have shown that systemic administration of FA, when associated to vitamin E (VE), can prevent oxidative damage and apoptosis in the uterus of these animals.The authors suggested that pharmacology effects associated to FA and VE are related with the increase of levels of the glutathione and with decreased of the malondialdehyde, 59 reinforcing the protective effect of folic acid.

Final Considerations
The studies analyzed here have shown that maternal diet supplementation with folic acid during gestation brings benefits in adverse conditions, such as alcohol consumption and protein deprivation.It exerts a protective effect, such as avoiding deleterious epigenetic changes on the offspring's liver which could initiate many diseases in Furthermore, folic acid enhanced defenses in mammalian cells.
Besides being needed during pregnancy, supplementation with FA did not cause toxic effects at recommended doses.Moreover, FA and its analogs can prevent the damages caused by potentially toxic drugs such as methotrexate, 5fluorouracil, and other antifolate drugs used for treatment of several diseases. 60ecause folic acid is widely used during gestation and even before it, more studies are needed in order to clarify the impact of maternal folic acid consumption on offspring's metabolism, not only on the liver, but in other crucial organs, such as the kidney.Studies that report morphological alterations caused by this vitamin, for example, are scarce and might be worth analyzing to reveal its effects on several tissues.
Due to relevance, our group has focused on knowing the effects of total absence, normal and overdose consumption of folic acid during gestation on adult offspring by morphologic changes in the structures related to metabolism as cited above, since unsuitable maternal nutrition can trigger several impairments to the offspring's health.other tissues that play essential roles in the regulation of cellular differentiation, development and metabolism of carbohydrate, protein and lipid, besides tumorigenesis of higher organisms, 52 any change involving such receptor can be harmful and determine long-term diseases by changing the metabolic profile.
Any change in gene expression is critical for normal cell differentiation and embryogenesis. 53NA methylation patterns are largely established in the uterus, and any change on fetal environment may alter DNA methylation, inducing stable changes in gene expression that can be sustained throughout the life of an individual and constitute the origin of diseases.42 Some studies have proposed that methyl donor deficiency (folate) changes DNA methylation models and initiates hypomethylation in the liver and other tissues.43 Gong et al. 54 analyzed insulin-like growth factor II (Igf2) expression on the male offspring's liver.Igf2 works as a mediator in the liver and brain to respond to prenatal dietary manipulations, stress such as choline deficiency, or supplementation.55 These authors found that in the group of dams fed on low protein diet with folic acid supplement, the Igf2 mRNA expression was similar to the level of the control group.In the same way, epigenetic modification was avoided in the offspring's liver following maternal supplementation with folic acid.These results suggest that maternal folic acid supplementation can reverse the effects from maternal low protein intake during pregnancy.
Several proteins, including GAPDH can be changed by maternal FA deficiency.These results reinforce that the absence of this vitamin in the maternal diet at the early period of gestation or any nutritional disorder are critical and programming is altered in several cellular processes that remain long term, extending until adulthood.In this regard, the availability of folic acid at early phases of embryogenesis seems important.
Regarding minerals, FA supplementation during gestation did not change iron hepatic levels of the offspring.This fact is favorable, due to oral supplementation with elemental iron (60 mg/day), alone or with folic acid, is useful to treat irondeficiency anemia in pregnant women 56 and can reduce the rate of preterm birth. 57inally, the examined papers have showed that supplementation of folic acid plus Se can reduce the oxidative stress and has a protective effect. 47FA prevents peroxidation protein products and neutralizes the damage of ethanol consumption during gestation. 48,49This defense property is given