Adverse fetal environment due to maternal undernutrition or exposure to environmental chemicals alters glucocorticoid (GC) metabolism increasing the risk of metabolic disorders in adulthood. In this study, we investigated the effects of maternal exposure to cadmium (Cd, 50 ppm) during pregnancy in the methylation of fetal hepatic glucocorticoid receptor promoter (GR) and the correlation with its expression and that of the DNA methyltransferases (DNMT1a and 3a). We also studied the expression of liver phosphoenolpyruvate carboxykinase (PEPCK) and acyl-CoA oxidase (AOX), two enzymes involved in the metabolism of carbohydrates and lipids respectively. The methylation of the rat GR gene exon 1₁₀ (GR1₁₀) in nucleotides -2536 to -2361 was analyzed by pyrosequencing. Quantitative real time PCR was used to assess hepatic GR, PEPCK and AOX mRNA, and their protein levels using Western blotting analysis. Differential methylation was noted across groups at all CpG sites in the GR exon 1₁₀ in a sex-dependent manner. In males, CpG were more methylated than the controls (185±21%, p<0.001) but only CpG sites 1,6,7 and 9 showed a significantly different extent of methylation. In addition, a lower expression of GR (mRNA and protein) was found. On the contrary, in females, CpG were less methylated than the controls (62±11%, p<0.05) and overexpressed, affecting PEPCK and AOX expression, which did not change in males. The GR methylation profile correlates with DNMT3a expression which may explain epigenetic sex-dependent changes on GR1₁₀ promoter induced by Cd treatment. In conclusion, Cd exposure during pregnancy affects fetal liver DNMT3a resulting in sex-dependent changes in methylation and expression of GR1₁₀. Although these effects do not seem to be directly involved in the low birth weight and height, they may have relevant implications for long-term health.

Changes in DNA methylation, one of the most studied epigenetic mechanisms, are considered an initial marker for early cancer detection. We evaluated how availability of dietary factors (folates and vitamin B12) involved in one-carbon metabolism may contribute to DNA methylation changes of cancer-related genes in human subjects. We studied, by pyrosequencing, the methylation of tumor suppressor gene p16, DNA repair genes MLH1 and MGMT, and the repetitive element LINE-1 (as a surrogate for global DNA methylation), in blood of elderly individuals (n = 249) who had been exposed to folic acid (FA) through FA-fortified wheat flour during the last 12 years. We found that serum folate and to a lesser extent, vitamin B12 concentrations, were significantly correlated with DNA methylation of p16, MLH1, and MGMT, but not with LINE-1. High serum folate concentrations (>45.3 nmol/L) were present in 31.1% of the participants. Although the methylated fraction of CpG sites in p16, MLH1, and MGMT was low (1.17–3.8%), high folate concentrations were significantly associated with methylation at the 3rd tertile of specific CpG sites in all genes with OR between 1.97 and 4.17. This study shows that a public policy, like food fortification with FA that increases circulating serum folate levels, could affect methylation levels of specific genes linked to cancer risk. Our present results deserve additional studies to clarify the real impact of high FA levels for risk of cancer in a whole population chronically exposed to a fortified food such as wheat flour. ISRCTN 48153354 and ISRCTN 02694183

Folate deficiency during pregnancy has been related to low birth weight, preterm (PT) birth and other health risks in the offspring; however, it is unknown whether prematurity is related to low folate transport through the placenta due to altered expression of specific folate transporters. We determined placental expression (mRNA and protein concentrations by RT-qPCR and WB respectively) of specific folate transporters: RFC, PCFT/HCP1 and FOLR1 in chorionic (fetal) and basal (maternal) plates of placentas of PT pregnancies (PT, 32–36 weeks, n = 51). Term placentas were used as controls (T, 37–41 weeks, n = 47). Folates and vitamin B12 levels were measured by electrochemiluminescence in umbilical cord blood of newborns. FOLR1 mRNA expression was lower and protein concentration higher in PT placentas (both plates) relative to the control group (p <0.05). In addition, gestational age was positively correlated with mRNA expression (Rho = 0.7), and negatively with protein concentration (Rho = -0.7 for chorionic and -0.43 for basal plate). PCFT/HCP1 mRNA was lower in PT placentas, without changes in protein levels. RFC did not differ in PT placentas compared to controls. PT newborns presented higher cord blood folate level (p = 0.049) along with lower vitamin B12 concentration compared to controls (p = 0.037).In conclusion, placental FOLR1 mRNA was positively associated with gestational age. Conversely, FOLR1 protein concentrations along with folate/vitamin B12 ratio in cord blood were negatively associated with gestational age. Placental FOLR1 is likely the main placental folate transporter to the fetus in newborns.

In the placenta, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (−378 to −275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11β-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.