Background Environmentally induced epigenetic changes can lead to health problems or disease, but the mechanisms involved remain unclear. Morphine can pass through the placental barrier leading to abnormal embryo development. However, the mechanism by which morphine causes these effects and how they sometimes persist into adulthood is not well known. To unravel the morphine-induced chromatin alterations involved in aberrant embryo development, we explored the role of the H3K27me3/PRC2 repressive complex in gene expression and its transmission across cellular generations in response to morphine. Results Using mouse embryonic stem cells as a model system, we found that chronic morphine treatment induces a global downregulation of the histone modification H3K27me3. Conversely, ChIP-Seq showed a remarkable increase in H3K27me3 levels at specific genomic sites, particularly promoters, disrupting selective target genes related to embryo development, cell cycle and metabolism. Through a self-regulatory mechanism, morphine downregulated the transcription of PRC2 components responsible for H3K27me3 by enriching high H3K27me3 levels at the promoter region. Downregulation of PRC2 components persisted for at least 48 h (4 cell cycles) following morphine removal, though promoter H3K27me3 levels returned to control levels. Conclusions Morphine induces targeting of the PRC2 complex to selected promoters, including those of PRC2 components, leading to characteristic changes in gene expression and a global reduction in H3K27me3. Following morphine removal, enhanced promoter H3K27me3 levels revert to normal sooner than global H3K27me3 or PRC2 component transcript levels. We suggest that H3K27me3 is involved in initiating morphine-induced changes in gene expression, but not in their maintenance. Graphic abstract Model of Polycomb repressive complex 2 (PRC2) and H3K27me3 alterations induced by chronic morphine exposure. Morphine induces H3K27me3 enrichment at promoters of genes encoding core members of the PRC2 complex and is associated with their transcriptional downregulation.

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Study question Can morphine create changes in the mESC methylation pattern that are potentially related to the mismatches observed during embryonic development? Summary answer Morphine creates an even more hypomethylated state of the mESC genome, which results in a delay in mESC differentiation and, consequently, an increase in pluripotency. What is known already Epigenetic changes are essential for normal development. However, some environmental factors can cause epigenetic changes that leads to health problems or diseases. Morphine is known to pass through the placental barrier and impact normal embryo development by affecting the neural tube, frontal cortex and spinal cord development, and, as a consequence, delaying nervous system development. In fact, in-utero morphine exposure has shown alterations in anxiety-like behaviours, analgesic tolerance, synaptic plasticity and the neuronal structure of offspring. Nonetheless, how morphine leads to abnormal neurogenesis and other physiological consequences during embryo development is still unknown. Study design, size, duration Considering that DNA methylation is a key epigenetic factor crucial for embryo development, our aim is to elucidate the suitability of using this opioid in pregnant women, analyzing the role of methylation in response to morphine. To study morphine effects on embryo development, we used mouse embryonic stem cells (mESCs) culture, one of the most widely used models for studying embryo development in vitro. Participants/materials, setting, methods mESCs was treated with a chronic morphine treatment (24 h, 10 μM). DNA extraction was done using a classic phenol-chloroform/isoamyl methodology, and RNA extraction with commercial Nucleozol reagent according to the manufacturer’s instructions. Liquid chromatography–mass spectrometry (LC-MS/MS) measured global genome methylation/hydroxymethylation levels, Whole Genome Bisulfite Sequencing (WGBSeq) measured cytosines methylation, and Real Time quantitative Polymerase Chain Reaction (RT-qPCR) measured gene expression. The statistical analysis (LC-MS/MS + RT-qPCR) was made by t-student. Main results and the role of chance By MS/MS approaches, we observed a global methylation decrease and a global hydroxymethylation increase in mESCs, changes that occurred after a chronic morphine treatment (24 h, 10 μM). WGBSeq identified 13329 sensitive cytosines to morphine that are involved in embryo development, signalling pathways, metabolism and/or gene expression. This suggests that morphine might impact methylation levels at developmental genes. Integrative analyses between WGBSeq and RNASeq identified Tet Methylcytosine Dioxygenase 1 (Tet1) as sensitive morphine gene. Morphine increased the gene expression of Tet1, modifying the methylation levels at the promoter. Otherwise, RNASeq and qRT-PCR analyses revealed that DNA Methyltransferase 1 (Dnmt1) gene expression decreased after morphine treatment. In conclusion, morphine induces a global hypomethylation in mESC through different mechanisms that involves active and passive demethylation, and the first one has a self-regulating mechanism that significantly enhances the demethylation process. Limitations, reasons for caution Taking into account all the ethical problems caused by the use of hESCs, this study has been carried out with mESCs. Although the physiology of mice and humans is somewhat different, this is the most recommended model for this type of pilot study. Wider implications of the findings This hypomethylation can potentially lead to a significant delay in optimal embryonic development; The global hypomethylated state of the genome is one of the most significant characteristics of stem cells. Consequently, the possible use of this and other opioids in patients who are pregnant should be strongly discouraged. Trial registration number Not applicable