Novel plasma cytokines identified and validated in children during lead exposure according to the new updated BLRV.

Lead is a pervasive environmental contaminant with significant health risks, particularly to children. It is known for its neurotoxic and immunotoxic effects, causing developmental, cognitive, and behavioral impairments. Despite extensive research, the mechanisms of lead toxicity remain unclear. Cytokines, which are critical in immune response and inflammation, have emerged as potential biomarkers for lead toxicity. The recent Centers for Disease Control and Prevention (CDC) update to the blood lead reference value (BLRV) to 3.5 µg/dL emphasizes the need to explore novel biomarkers and mechanisms. The study involved 100 healthy children aged 1 to 5 years, divided into two groups based on BLRV: elevated (≥ 3.5 µg/dL) and low (< 3.5 µg/dL). The research consisted of two phases: discovery and validation. Plasma samples were analyzed using RayBio Human Cytokine Antibody Arrays and Enzyme-linked immunosorbent assay (ELISA) for cytokine levels. Ethical approval was obtained, and statistical analyses included t-tests, chi-squared tests, pearson correlations, and multivariate logistic regression. Protein-protein interaction (PPI), Gene Ontology (GO) enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to explore the roles of significant differentially expressed proteins (DEPs). No significant differences in age, gender, or BMI between the two groups, but BLRV levels were significantly higher in the elevated BLRV group compared to the low BLRV group. In the discovery phase, significant changes in cytokine expression were identified, including increased levels of IL-6, IL-8, and IL-17, and decreased levels of BDNF, BMP-4, IGF-1, IL-7, IL-10, and Leptin. These findings were validated in the second phase using ELISA. Significant positive correlations were found between BLRV and IL-6, IL-8, and IL-17. Negative correlations were observed with BDNF, BMP-4, IGF-1, IL-7, IL-10, and Leptin. Multivariate regression confirmed that BLRV significantly affects these cytokine levels. PPI networks revealed that DEPs had strong interactions with multiple proteins, indicating their central role in lead toxicity. GO and KEGG analyses highlighted pathways related to neurotoxicity and inflammatory responses, including "negative regulation of myotube differentiation," "neurotrophin signaling pathway," and "alcoholism." This study provides insights into the role of cytokines as biomarkers for lead toxicity and offers a comprehensive analysis of the mechanisms involved. The findings underscore the importance of early detection and intervention based on updated BLRV thresholds.