The impact of micronutrient supplementation in alcohol-exposed pregnancies on reaction time responses of preschoolers in Ukraine.

The potential of micronutrients to ameliorate the impact of prenatal alcohol exposure (PAE) on attentional regulation skills was explored in a randomized clinical trial conducted in Ukraine. Women who differed in prenatal alcohol use were recruited during pregnancy and assigned to one of three groups [No study-provided supplements, Multivitamin/Mineral Supplement (MVM), or MVM plus Choline]. Their offspring were seen in the preschool period and a reaction time task was administered. Participants were asked to press a response button as quickly as possible as 30 stimuli from the same category (animals) were presented consecutively and then followed by six stimuli from a novel category (vehicles). Number correct, mean latency of the response over trials, and variability in the latency were analyzed separately by sex. During the initial animal trials, boys whose mothers received MVM during pregnancy had more correct responses and reduced response latency compared to boys whose mothers had no MVM treatment. During vehicle trials, maternal choline supplementation was associated with increased response speed in males without a PAE history. Females receiving supplements did not show the same benefits from micronutrient supplementation and were more adversely impacted by prenatal alcohol exposure. Relationships between maternal levels of choline, betaine, and dimethylglycine (DMG) and task performance were also assessed. Although no effects were found for choline after adjusting for multiple comparisons, lower baseline DMG level was associated with greater accuracy and shorter latency of responses in the initial animal trials and shorter latency in the vehicle trials in female preschoolers. Level of betaine in Trimester 3 was associated with reduced variability in the latency of male responses during the animal trials. Maternal micronutrient supplementation in pregnancy appears to improve preschool reaction time performance, but the effects varied as a function of sex and PAE exposure status.

Implications of altered maternal cytokine concentrations on infant outcomes in children with prenatal alcohol exposure.

Excessive alcohol consumption has been shown to increase serum plasma levels of numerous immune cytokines. Maternal immune activation and elevated cytokines have been implicated in certain neurological disorders (e.g., autism and schizophrenia) in the offspring. We investigated the hypothesis that elevated cytokines during pregnancy are a risk factor in women who gave birth to a child with Fetal Alcohol Spectrum Disorder (FASD) or a child with neurobehavioral impairment, regardless of prenatal alcohol exposure. Moderate to heavy alcohol-exposed (AE) (N = 149) and low or no alcohol-exposed (LNA) (N = 92) women were recruited into the study during mid pregnancy (mean of 19.8 ± 5.8 weeks' gestation) in two regions of Ukraine: Khmelnytsky and Rivne. Maternal blood samples were obtained at enrollment into the study at early to mid-pregnancy and during a third-trimester follow-up visit and analyzed for plasma cytokines. Children were examined at 6 and/or 12 months of age and were classified as having FASD if their mothers reported alcohol use and if they had at least one standardized score (Bayley Scales of Infant Development II Mental Development Index [MDI], or Psychomotor Development Index [PDI]) below 85 with the presence or absence of physical features of FASD. In multivariate analyses of maternal cytokine levels in relation to infant MDI and PDI scores in the entire sample, increases in the ratio of TNF-α/IL-10 and IL-6/IL-10 were negatively associated with PDI scores at 6 months (p = 0.020 and p = 0.036, respectively) and 12 months (p = 0.043 and p = 0.029, respectively), and with MDI scores at 12 months (p = 0.013 and p = 0.050, respectively). A reduction in the odds ratio of having an FASD child was observed with increasing levels of IL-1ß, IL-2, IL-4, IL-6, and IL-10 in early to mid-pregnancy and IL-1ß and IL-10 during late pregnancy. However, women that failed to increase IL-10 levels in the third trimester in order to maintain the balance of pro- and anti-inflammatory cytokines had an elevated risk of having an FASD child, specifically a significant increase in the odds ratio of FASD with every one-unit log increase in late pregnancy TNF-α/IL-10 levels (aOR: 1.654, CI: 1.096-2.495, p = 0.017). These data support the concept that disruptions in the balance between pro- and anti-inflammatory cytokines may contribute to neurobehavioral impairment and alter the risk of FASD.

The impact of micronutrient supplementation in alcohol-exposed pregnancies on information processing skills in Ukrainian infants.

The potential of micronutrients to ameliorate the impact of prenatal alcohol exposure (PAE) was explored in a clinical trial conducted in Ukraine. Cardiac orienting responses (ORs) during a habituation/dishabituation learning paradigm were obtained from 6 to 12 month-olds to assess neurophysiological encoding and memory. Women who differed in prenatal alcohol use were recruited during pregnancy and assigned to a group (No study-provided supplements, multivitamin/mineral supplement, or multivitamin/mineral supplement plus choline supplement). Heart rate was collected for 30 s prior to stimulus onset and 12 s post-stimulus onset. Difference values (∆HR) for the first 3 trials of each condition were aggregated for analysis. Gestational blood samples were collected to assess maternal nutritional status and changes as a function of the intervention. Choline supplementation resulted in a greater ∆HR on the visual habituation trials for all infants and for the infants with no PAE on the dishabituation trials. The latency of the response was reduced in both conditions for all infants whose mothers received choline supplementation. Change in gestational choline level was positively related to ∆HR during habituation trials and levels of one choline metabolite, dimethylglycine (DMG), predicted ∆HR during habituation trials and latency of responses. A trend was found between DMG and ∆HR on the dishabituation trials and latency of the response. Supplementation did not affect ORs to auditory stimuli. Choline supplementation when administered together with routinely recommended multivitamin/mineral prenatal supplements during pregnancy may provide a beneficial impact to basic learning mechanisms involved in encoding and memory of environmental events in alcohol-exposed pregnancies as well as non- or low alcohol-exposed pregnancies. Changes in maternal nutrient status suggested that one mechanism by which choline supplementation may positively impact brain development is through prevention of fetal alcohol-related depletion of DMG, a metabolic nutrient that can protect against overproduction of glycine, during critical periods of neurogenesis.

Effect of butyl benzyl phthalate on reproduction and zinc metabolism.

Butyl benzyl phthalate (BBP) has been shown to be teratogenic. One mechanism contributing to the teratogenicity of several developmental toxicants, is chemical-induced changes in maternal zinc (Zn) metabolism which result in an increased synthesis of maternal liver metallothionein (Mt), and a subsequent reduction in Zn delivery to the conceptus. We investigated the effects of maternal BBP exposure on maternal-fetal Zn metabolism in Wistar rats. In study I, dams were gavaged with BBP (0,250,1000,1500 or 2000 mg/kg) on gestation days (GD) 11 through 13, and killed on GD 20. Maternal toxicity was evident in the three highest dose groups. Embryo/fetal death and small pup weights and lengths were noted in the 2000 mg BBP/kg group. Fetuses in the 1500 and 2000 mg/kg groups were characterized by poor skeletal ossification, and a high frequency of cleft palate. Rib anomalies were observed in the three highest dose groups. Maternal liver Mt concentrations were only slightly elevated in the 1500 and 2000 mg/kg groups. In study II, dams treated as above, were gavaged with 65Zn and killed 18 h later. While the 2000 mg/kg group had high percentages of 65Zn in some maternal tissues, sequestration of 65Zn in maternal liver was not evident. Thus, BBP is not a strong inducer of Mt, and the teratogenicity of BBP does not appear to be due to alterations in maternal and/or embryonic Zn metabolism.

Lysyl oxidase and P-ATPase-7A expression during embryonic development in the rat.

Lysyl oxidase activity is critical for the assembly and cross-linking of extracellular matrix proteins, such as collagen and elastin. Moreover, lysyl oxidase activity is sensitive to changes in copper status and genetic perturbations in copper transport, e.g., mutations in the P-type ATPase gene, ATP7A, associated with cellular copper transport. Lysyl oxidase may also serve as a vehicle for copper transport from extracellular matrix cells. Herein, we demonstrate that sufficient lysyl oxidase functional activity is present in the rat embryo at gestation day (GD) 9 to be detected in conventional enzyme assays. Estimation of embryonic lysyl oxidase functional activity, however, required partial purification in order to remove inhibitors. From GD 9 to GD 15, lysyl oxidase activity was relatively constant when expressed per unit of protein or DNA. In contrast, the steady-state levels of lysyl oxidase and ATP7A mRNA, measured by RT-PCR and expressed relative to total RNA and cyclophilin mRNA, increased approximately fourfold from GD 9 to 15. The pattern of temporal expression for ATP7A was consistent with its possible role in copper delivery to lysyl oxidase.

Activation of chick tendon lysyl oxidase in response to dietary copper.

Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10-30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 microg/g diet) to which copper was added from 0 to 16 microg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 microg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 microg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 microg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da +/- 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da +/- 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.