Developmental exposure to cigarette smoke particulate matter induces cross-generational DNA methylation changes in zebrafish.

Prenatal exposure to cigarette smoke has been associated with adverse developmental outcomes and epigenetic alterations; however, the persistence of these effects across generations remains poorly understood. Using zebrafish (Danio rerio), we investigated whether developmental exposure to cigarette smoke total particulate matter (TPM) induces epigenetic changes in exposed larvae (F0) and their unexposed offspring (F1). Embryos were exposed to TPM (0.1, 0.2, or 0.3 μg/mL equi-nicotine units) from 6 to 96 h post fertilization (hpf). Transcript abundance of DNA methyl transferases, global methylation (5-methylcytosine content), and genome-wide methylation profiles were subsequently evaluated in F0 and F1 larvae. TPM exposure did not significantly alter transcript abundance of Dnmt1, Dnmt3a1, Dnmt3a2, Dnmt3b1, Dnmt3b2, or Dnmt3b3 in either generation. In contrast, global DNA methylation was significantly reduced in F0 TPM0.3 larvae, indicating concentration-dependent hypomethylation, whereas no significant differences were observed in F1 larvae. Genome-wide methylation analysis identified 5 and 4157 differentially methylated CpG sites in F0 TPM0.2 and TPM0.3 larvae, respectively, with the majority hypomethylated. In F1 larvae, only 6, 1, and 23 differentially methylated CpG sites were detected in TPM0.1, TPM0.2, and TPM0.3 groups, respectively, and all were hypermethylated relative to controls. Differentially methylated genes in F0 TPM0.3 larvae were associated with developmental, neuronal, metabolic, immune, and stress-response pathways. Fourteen genes exhibited altered methylation in both generations. To our knowledge, this is the first study demonstrating cross-generational DNA methylation changes following developmental exposure to TPM in zebrafish.