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Exposure to mixtures of toxic metals is known to cause adverse health effects through epigenetic alterations. Here we aimed to examine the unexplored area of aberrant DNA methylation in the H19/IGF2 domain following combined toxic metal exposure. An in vitro epigenotoxicity assay using the human normal liver epithelial cell line THLE-3 was conducted. When THLE-3 cells were exposed to specific concentrations of either organic arsenic or MeHgCl, an increase in the H19 lncRNA levels and a marked reduction in the IGF2 mRNA levels were observed. In contrast, combined exposures coupled with CdCl 2 resulted in the transcriptional repression of H19 and transcriptional activation of IGF2. It should be noted that the correlation between the dysregulated expression of H19/IGF2 and the hypermethylated CpG sites within the H19 differentially methylated region (DMR) was statistically significant. Furthermore, we performed transcriptomic analysis of the hepatocytes exposed to toxic metal combinations indicating enrichment of pro-inflammatory and anti-proliferative pathways compared to the unexposed cells. Our results suggest that hazardous metal mixtures may trigger epigenetic aberrations at the H19/IGF2 locus. We propose that altered CpG methylation in the H19 DMR could be a candidate biomarker for hepatic epigenotoxicity, in part, due to environmental exposure., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The H19/insulin-like growth factor 2 (Igf2) gene plays vital roles during development, but their expression and methylation status have not been elucidated in male cloned animals. In this study, we investigated the methylation status of imprinting control region (ICR) and differentially methylated region (DMR) of the H19/Igf2 locus in the sperm and testes of naturally reproduced Saanen dairy goats (NG, control), transgenic cloned dairy goats (CG), and the first generation of mating the CGs with the female NGs (FG). Bisulfite sequencing polymerase chain reaction revealed that the H19 ICR was highly methylated in the sperm of all testing groups (97.07% ± 0.47%, 93.73% ± 0.85%, and 96.67% ± 0.54%). Notably, the methylation level of H19 ICR was decreased in the testes of CG goats compared with that of NG goats (73.83% ± 3.28% vs. 97.27% ± 0.71%, p < 0.05), while the Igf2 DMR2 was highly methylated in the sperm of these three groups (89.57% ± 1.74%, 87.23% ± 1.11%, and 83.2% ± 1.27%). However, the Igf2 DMR2 was abnormally hypomethylated in the testes of CG goats (27.33% ± 1.005%, p < 0.001), and moderately methylated in FG goats (53.1% ± 1.47%, p < 0.01) compared with that in NG goats (82.67% ± 0.70%). Furthermore, we found significantly decreased expression of H19 (p < 0.01) and increased expression of Igf2 (p < 0.01) in CG goats. Thus, we infer that somatic cell nuclear transfer has clear effect on genomic imprinting in the testes, but not sperm of cloned goats.

Abstract The sexually dimorphic expression of H19/IGF2 is evolutionarily conserved. To investigate whether the expression of H19/IGF2 in the female porcine eye is sex-dependent, gene expression and methylation status were evaluated using quantitative real-time PCR (qPCR) and bisulfite sequencing PCR (BSP). We hypothesized that H19/IGF2 might exhibit a different DNA methylation status in the female eye. In order to evaluate our hypothesis, parthenogenetic (PA) cells were used for analysis by qPCR and BSP. Our results showed that H19 and IGF2 were over-expressed in the female eye compared with the male eye (3-fold and 2-fold, respectively). We observed a normal monoallelic methylation pattern for H19 differentially methylated regions (DMRs). Compared with H19 DMRs, IGF2 DMRs showed a different methylation pattern in the eye. Taken together, these results suggest that elevated expression of H19/IGF2 is caused by a specific chromatin structure that is regulated by the DNA methylation status of IGF2 DMRs in the female eye.