Fatty acid and lipid metabolism in liver of pregnant mice and their offspring is influenced by unbalanced folates/vitamin B12 diets.

Micronutrients (folates and vitamin B12) and long chain polyunsaturated fatty acids (LC-PUFAs) are linked through the one carbon cycle. We studied the effects of pre and postnatal high FA/low B12 diets (HFLB12) on hepatic fatty acid metabolism. Pregnant C57BL/6 mice were divided in two groups: control (2 mg folic acid: FA/25 µg vitamin B12/Kg food) and HFLB12 diets (8 mg FA/5 µg vitamin B12/Kg food). Offspring continued on the same diets until 60 days old. We determined hepatic fatty acid profile in dams and offspring and the expression of PPARα, Cpt-1, Acox-1 and Fas and the enzymatic activity of desaturases, all involved in lipid metabolism. In liver of dams, the HFHB12 diet decreased total fatty acids and desaturase activities; in offspring, effects were opposite, being more noticeable in females. Prenatal and postnatal unbalanced folic acid/B12 diets play a crucial role in regulating genes and enzymes involved in lipid metabolism in liver of dams and their offspring in adulthood.

Impact of cadmium exposure during pregnancy on hepatic glucocorticoid receptor methylation and expression in rat fetus.

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 1(10) (GR1(10)) 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 1(10) 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 GR1(10) 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 GR1(10). 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.

Cardiac tissue injury resistance during myocardial infarction at adulthood by developmental exposure to cadmium.

It has been suggested that prenatal exposure to cadmium may alter the cardiovascular function during adulthood. Using the left coronary artery ligation model of acute myocardial infarction, we studied the cardiac function of female adult offspring rats exposed to cadmium (30 ppm) during gestation. The cardiac ischemic zone in the control and cadmium-exposed groups was measured 72 h post-ligation using the TPT staining technique. Offspring from cadmium-treated dams showed a significantly smaller infarcted area compared with the control group (7.1 ± 1.5 vs. 19.6 ± 2.8%, P ≤ 0.05). We also performed echocardiographic and biochemical studies, which positively correlated with the differences observed previously. To evaluate whether the effects were associated to pre-infarct tissue damage and/or angiogenic molecules, we performed histological studies and measured the expression of vascular endothelial growth factor (VEGF), and platelet endothelial cellular adhesion molecule-1 (PECAM-1). Results revealed a higher heart vascularization in the exposed offspring that was associated with an increase in PECAM and a decrease in VEGF expression. We conclude that prenatal exposure to cadmium induces fetal adaptive responses involving changes in the expression of some cardiac angiogenic molecules resulting in long-term resistance to infarction.

Maternal exposure to cadmium during gestation perturbs the vascular system of the adult rat offspring.

Several cardiovascular diseases (CVD) observed in adulthood have been associated with environmental influences during fetal growth. Here, we show that maternal exposure to cadmium, a ubiquitously distributed heavy metal and main component of cigarette smoke is able to induce cardiovascular morpho-functional changes in the offspring at adult age. Heart morphology and vascular reactivity were evaluated in the adult offspring of rats exposed to 30ppm of cadmium during pregnancy. Echocardiographic examination shows altered heart morphology characterized by a concentric left ventricular hypertrophy. Also, we observed a reduced endothelium-dependent reactivity in isolated aortic rings of adult offspring, while endothelium-independent reactivity remained unaltered. These effects were associated with an increase of hem-oxygenase 1 (HO-1) expression in the aortas of adult offspring. The expression of HO-1 was higher in females than males, a finding likely related to the sex-dependent expression of the vascular cell adhesion molecule 1 (VCAM-1), which was lower in the adult female. All these long-term consequences were observed along with normal birth weights and absence of detectable levels of cadmium in fetal and adult tissues of the offspring. In placental tissues however, cadmium levels were detected and correlated with increased NF-κB expression--a transcription factor sensitive to inflammation and oxidative stress--suggesting a placentary mechanism that affect genes related to the development of the cardiovascular system. Our results provide, for the first time, direct experimental evidence supporting that exposure to cadmium during pregnancy reprograms cardiovascular development of the offspring which in turn may conduce to a long term increased risk of CVD.

Effect of cadmium on cortisol production and 11beta-hydroxysteroid dehydrogenase 2 expression by cultured human choriocarcinoma cells (JEG-3).

Cadmium is a toxicant with known carcinogenic and endocrine disruptor effects. We have previously reported that prenatal exposure to cadmium may induce low birth weight which is associated to increased foetal exposure to glucocorticoids; both signals constitute "hallmarks" of developmental programming. Since the effect of cadmium on the glucocorticoid system of placental carcinogenic cells is unknown, in the present work, we studied the effect of acute low dose of cadmium on cortisol production and 11beta-HSD2 expression and activity by cultured human choriocarcinoma cells (JEG-3). In addition, it was also evaluated whether those effects were related to the methylation index of the HSD11B2 gene, which can be regulated by epigenetic mechanisms. Cells were incubated with low cadmium dose (0.5 and 1 microM) for 24h and viability (MTT), cortisol production (EIA), 11beta-HSD2 expression (qRT-PCR) and activity (radioassay), and methylation index of the HSD11B2 gene were determined. Results show lower cortisol concentrations in the incubation media of exposed cells, which were associated to increased 11beta-HSD2 expression and activity and to a lower methylation index of the gene. These results suggest that cadmium-induced endocrine disruptor effects on JEG-3 cells could be mediated by changes in the methylation status of some target genes.

Fetal growth restriction is related to placental levels of cadmium, lead and arsenic but not with antioxidant activities.

The objectives of this study were: to measure some essential metals and toxicants in placentas of mothers delivering neonates with fetal growth restriction, and to establish potential associations between environmental adverse stimulus and antioxidant protective mechanisms. Placentas of 20 mothers delivering neonates with low birth weight (<2500g) and normal birth weight (>3000g) at term were collected. Placental concentration of zinc, mercury, selenium and arsenic were measured by Instrumental Neutron Activation Analysis (INAA), and iron, copper, cadmium and lead by Atomic Absorption Spectrometry (AAS). Total glutathione, lipid peroxidation, total antioxidant activity and antioxidant enzyme activities (superoxide dismutase and glutathione peroxidase) were determined spectrophotometrically. Results showed reduced iron levels and increased concentrations of cadmium, lead and arsenic in placentas of mothers delivering low birth weight neonates, but not differences in oxidative stress parameters or antioxidant enzymatic activities, suggesting a relationship between low birth weight and placental concentration of cadmium, arsenic and lead.

Smoking specifically induces metallothionein-2 isoform in human placenta at term.

Recently, we reported the presence of higher levels of metallothionein (MT) in placentas of smokers compared to non-smokers. In the present study, we designed experiments to separate and evaluate two isoforms of MT (MT-1 and MT-2) in placentas of smokers and non-smokers. Metallothionein was extracted and separated by ion-exchange high performance liquid chromatography (HPLC), previous saturation with cadmium chloride. Two peaks eluting at 6 and 12.5 min, corresponding to MT-1 and MT-2, respectively, were obtained. Metallothionein present in both peaks was identified by Western blot analysis using a monoclonal antibody directed against MT-1 and MT-2. Each isoform concentration was calculated after measuring its cadmium content by atomic absorption spectrometry with inductively coupled-plasma. In placentas of smokers, MT-2 levels increased by seven-fold compared to non-smokers, whereas MT-1 was not changed. Total placental cadmium and zinc concentrations, determined by atomic absorption spectrometry and neutron activation analysis, respectively, were higher in smokers. Metallothioneins levels were clearly in excess to bind all cadmium ions present in placentas. However, most of placental zinc remains unbound to MTs, although as much as twice zinc ions could be bound to MT in smokers. In conclusion, MT-2 is the main isoform induced by smoking, suggesting that this isoform could be involved in placental cadmium and zinc retention. This fact, which could contribute to reduce the transference of zinc to the fetus, may be associated to detrimental effects on fetal growth and development.

Increased levels of metallothionein in placenta of smokers.

Experiments were designed to evaluate and compare metallothionein (MT), zinc and cadmium levels in human placentas of smoking and non-smoking women. Smoking was assessed by self-reported cigarette consumption and urine cotinine levels before delivery. Smoking pregnant women with urine cotinine levels higher than 130 ng/ml were included in the smoking group. Determination of placental MT was performed by western blot analysis after tissue homogenization and saturation with cadmium chloride (1000 ppm). Metallothionein was analyzed with a monoclonal antibody raised against MT-1 and MT-2 and with a second anti mouse antibody conjugated to alkaline phosphatase. Zinc and cadmium were determined by neutron activation analysis and atomic absorption spectrometry respectively. Smokers showed higher placental MT and cadmium levels, together with decreased newborn birth weights, as compared to non-smokers. The semi-quantitative analysis of western blots by band densitometry indicated that darker bands corresponded to MT present in smokers' samples. This study confirms that cigarette smoking increases cadmium accumulation in placental tissue and suggests that this element has a stimulatory effect on placental MT production.