Gene-specific DNA methylation responses to Pb²⁺ exposure in aged male zebrafish and associations with locomotor...

Lead (Pb²⁺) is a well-established neurotoxin that impairs motor, learning, and memory functions, particularly in children and younger adults. However, its impact on older adults remains less understood. Pb²⁺ toxicity involves disruption of DNA methyltransferase activity and associated epigenetic pathways, potentially altering the expression of specific genes relevant to neurological functions. As methylation patterns naturally shift during aging, Pb²⁺ exposure may induce additional neurological risks in aged populations. Using a zebrafish model, we investigated the combined effects of Pb²⁺ exposure and brain aging. Two-year-old male zebrafish were exposed to 1, 10, 100, 1000, 10 000 µg/l Pb²⁺ or fish water control for five days. Brain tissues were collected for DNA extraction and whole-genome bisulfite sequencing to assess global and gene-specific methylation changes. Our results found that Pb²⁺ exposures ≥ 100 μg/l significantly increased global methylation levels in the aged brain. Differentially methylated genes (DMGs) exhibited methylation changes within gene body regions and were mostly annotated with ion transportation and signal transduction pathways. Although only a limited number of DMGs showed corresponding changes in gene expression, several of them were associated with locomotor-related functions, including shank1 at 10 000 Pb²⁺ μg/l, and ptprsa, plxna2, and aopep at 100 μg/l Pb²⁺. These findings suggest that Pb²⁺ exposure during aging predominantly induces gene body-localized DNA methylation changes, and the role of such epigenetic regulation in Pb-associated neurobehavioral outcomes warrants further investigation.