Effects of drinking water, sanitation, handwashing and nutritional interventions on stress physiology, oxidative stress, and epigenetic programming in young children living in rural Bangladesh: A randomized clinical trial

Importance: A regulated stress response is essential for healthy trajectories, but the integrated effects of early childhood environmental and nutritional interventions on stress physiology are unknown. Objective: To assess the effects of a combined nutritional, water, sanitation, and handwashing intervention on physiological stress response, oxidative stress, and DNA methylation. Design, Setting, and Participants: In a trial in rural Bangladesh, we randomized geographical clusters of pregnant women and their in-utero children into either the combined nutritional, water, sanitation, and handwashing intervention or the control group. Physiological stress response, oxidative stress, and methylation levels of 757 children were measured at ages one and two years. Analysis was intention-to-treat. Interventions: The intervention group received combined nutritional counseling and lipid-based nutrient supplements, chlorinated drinking water, upgraded sanitation, and handwashing with soap (N+WSH). The control group did not receive interventions. Main Outcomes and Measures: We measured four isomers of urinary F2-isoprostanes [iPF(2)-III; 2,3-dinor-iPF(2)-III; iPF(2)-VI; 8,12-iso-iPF(2)-VI] at year one. At year two, we measured pre- and post-stressor concentrations of salivary alpha-amylase and cortisol, overall methylation of the glucocorticoid receptor (NR3C1) exon 1F promoter including methylation levels at the nerve growth factor-inducible protein A (NGFI-A) binding site, mean arterial pressure, and resting heart rate. Results: Children in the N+WSH group had lower levels of F2-isoprostanes compared to controls (difference -0.16 to -0.19 log ng/mg of creatinine, P<0.01). Compared to the control group, post-stressor cortisol levels were elevated (0.24 log g/dl; 95% CI, 0.07 to 0.4; P<0.01) and the residualized gain score for cortisol was higher (0.06 g/dl; 95% CI, 0.01 to 0.12; P=0.023) in the N+WSH group. Children in the N+WSH group exhibited decreased methylation of the NGFI-A transcription factor binding site (-0.04 logit-transformed %; 95% CI, -0.08 to 0; P=0.037). Conclusions and Relevance: A nutritional, water, sanitation, and handwashing intervention reduced oxidative stress, enhanced hypothalamic-pituitary-adrenocortical axis activity, and reduced methylation levels in a transcription factor binding site of the glucocorticoid receptor gene. A targeted environmental and nutritional intervention affected the set point, reactivity, and regulation of the physiological stress system in early childhood, which may have implications for long-term health and developmental trajectories. Trial Registration: ClinicalTrials.gov NCT01590095