Use of metabolomics in refining the effect of an organic food intervention on biomarkers of exposure to pesticides and biomarkers of oxidative damage in primary school children in Cyprus: A cluster-randomized cross-over trial

Background: Exposure to pesticides has been associated with oxidative stress in animals and humans. Previously, we showed that an organic food intervention reduced pesticide exposure and oxidative damage (OD) biomarkers over time; however associated metabolic changes are not fully understood yet. Objectives: We assessed perturbations of the urine metabolome in response to an organic food intervention for children and its association with pesticides biomarkers [3-phenoxybenzoic acid (3-PBA) and 6-chloronicotinic acid (6-CN)]. We also evaluated the molecular signatures of metabolites associated with biomarkers of OD (8iso-PGF2a and 8-OHdG) and related biological pathways. Methods: We used data from the ORGANIKO LIFE + trial (NCT02998203), a cluster-randomized cross-over trial conducted among primary school children in Cyprus. Participants (n = 149) were asked to follow an organic food intervention for 40 days and their usual food habits for another 40 days, providing up to six first morning urine samples (>850 samples in total). Untargeted GC-MS metabolomics analysis was performed. Metabolites with RSD < 20% and D-ratio < 50% were retained for analysis. Associations were examined using mixed-effect regression models and corrected for false-discovery rate of 0.05. Pathway analysis followed. Results: Following strict quality checks, 156 features remained out of a total of 610. D-glucose was associated with the organic food intervention (13 = -0.23, 95% CI: -0.37,-0.10), aminomalonic acid showed a timedependent increase during the intervention period (13int = 0.012; 95% CI:0.002, 0.022) and was associated with the two OD biomarkers (13 = -0.27, 95% CI:-0.34,-0.20 for 8-iso-PGF2a and 13 = 0.19, 95% CI:0.11,0.28 for 8-OHdG) and uric acid with 8-OHdG (13 = 0.19, 95% CI:0.11,0.26). Metabolites were involved in pathways such as the starch and sucrose metabolism and pentose and glucuronate interconversions. Discussion: This is the first metabolomics study providing evidence of differential expression of metabolites by an organic food intervention, corroborating the reduction in biomarkers of OD. Further mechanistic evidence is warranted to better understand the biological plausibility of an organic food treatment on children's health outcomes.