Maternal corticotropin-releasing hormone is associated with LEP DNA methylation at birth and in childhood: an epigenome-wide study in Project Viva

BackgroundCorticotropin-releasing hormone (CRH) plays a central role in regulating the secretion of cortisol which controls a wide range of biological processes. Fetuses overexposed to cortisol have increased risks of disease in later life. DNA methylation may be the underlying association between prenatal cortisol exposure and health effects. We investigated associations between maternal CRH levels and epigenome-wide DNA methylation of cord blood in offsprings and evaluated whether these associations persisted into mid-childhood.MethodsWe investigated mother-child pairs enrolled in the prospective Project Viva pre-birth cohort. We measured DNA methylation in 257 umbilical cord blood samples using the HumanMethylation450 Bead Chip. We tested associations of maternal CRH concentration with cord blood cells DNA methylation, adjusting the model for maternal age at enrollment, education, maternal race/ethnicity, maternal smoking status, pre-pregnancy body mass index, parity, gestational age at delivery, child sex, and cell-type composition in cord blood. We further examined the persistence of associations between maternal CRH levels and DNA methylation in children's blood cells collected at mid-childhood (n = 239, age: 6.7-10.3 years) additionally adjusting for the children's age at blood drawn.ResultsMaternal CRH levels are associated with DNA methylation variability in cord blood cells at 96 individual CpG sites (False Discovery Rate <0.05). Among the 96 CpG sites, we identified 3 CpGs located near the LEP gene. Regional analyses confirmed the association between maternal CRH and DNA methylation near LEP. Moreover, higher maternal CRH levels were associated with higher blood-cell DNA methylation of the promoter region of LEP in mid-childhood (P < 0.05, β = 0.64, SE = 0.30).ConclusionIn our cohort, maternal CRH was associated with DNA methylation levels in newborns at multiple loci, notably in the LEP gene promoter. The association between maternal CRH and LEP DNA methylation levels persisted into mid-childhood

Toxicity Testing in the 21st Century: Defining New Risk Assessment Approaches Based on Perturbation of Intracellular Toxicity Pathways

The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21st Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) “toxicity pathways” (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair

Structure and Dynamics of the G121V Dihydrofolate Reductase Mutant: Lessons from a Transition-State Inhibitor Complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on 15N and 2H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect